Epigenetic Groningen Research Institute of Gerard Kop Gerard Kop Gerard Kop Gerard Kop Groningen Research Institute of Pediatric Pulmonology and Pedia Beatrix Children’s Hospital, Univ cs of asthma Asthma and COPD ppelman ppelman ppelman ppelman Asthma and COPD atric Allergology versity Medical Center Groningen
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Epigenetics Asthma Denmark Koppelman 2014 ... · • Role of DNA methylation in astma. T cell differentiation Gene Mechanism IL4 Demethylation of GATA4 binding site Demethylation
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• Multiple SNPs, small effects, so far explains less than 5 % of asthma
• Asthma heterogeneity has genetic basis• Asthma heterogeneity has genetic basis– Ethnic specific
• PYHIN1 (Pyrin and HIN domain family member 1interferon inducible nuclear factor 1
• Susceptibility SNPs in Afr Am are not present in subjects of European descent
– Phenotype specific • PDE4D as Asthma + Bronchial hyperresponsiveness gene• 17q21 confers susceptibility to childhood onset asthma
What have we learnt?
Multiple SNPs, small effects, so far explains less
Asthma heterogeneity has genetic basisAsthma heterogeneity has genetic basis
PYHIN1 (Pyrin and HIN domain family member 1, aka interferon inducible nuclear factor 1) is specific to Afr Am. Susceptibility SNPs in Afr Am are not present in subjects of
Phenotype specific as Asthma + Bronchial hyperresponsiveness gene
17q21 confers susceptibility to childhood onset asthma
Chr 17q21 SNPs and asthma onset
Age at onset asthma
Chr 17q21 SNPs and asthma onset
Bouzigon et al NEJM 2008
Age at onset asthma
From genetics to epigenetics
mRNA
Genetic variationCpG
MiRNA
Small non coding RNAmRNA
Asthma
Protein
Small non coding RNA
Histone modification
Chromatin remodelling
From genetics to epigenetics
CpG-M DNA methylation
MiRNA
Small non coding RNA Micro RNAsSmall non coding RNA
Histone modification
Chromatin remodellingRegulation of
DNA transcription
Epigenetics
• Heritable ? changes in gene expression that occur without directly changing the DNA sequencesequence
• Why? Adaptation to the environment, organ development, role in disease
Epigenetics
Heritable ? changes in gene expression that occur without directly changing the DNA
Why? Adaptation to the environment, organ development, role in disease
DNA Methylation
• Cytosine -> 5’-Methylcytosine
• CpG Islands (1-4 kB)
• CpG Islands in • CpG Islands in promoters and at transcription start sites (75 – 88%)
DNA Methylation
Kabesch et al, ERJ 2010
Methylation: DNA methyltransferases add
methylgroup to cytosine
MMMMMM
CpG island
Methylated
M
Methylated
Not methylated
Small differences in methylation (~15%)(~40%) in gene transcription Oates et al. (2006)
Methylation: DNA methyltransferases add
methylgroup to cytosine
Gene
SuppressedSuppressed
Active
(~15%) can result in large differences Oates et al. (2006) Am J Hum Genet 79: 155
Environmental factors may induce epigenetic effects
Environmental factors may induce epigenetic effects
Miller and Ho, AJRCCM 2008
Transgenerational effects of ETS exposure on asthma
Transgenerational effects of ETS exposure on asthma
Transgenerational effects of ETS exposure on asthma
• Risk of ETS exposure may be transmitted across two generationsacross two generations
• N = 338 children with asthma, age 5 • N = 570 controls, matched for in utero smoking• Risk of asthma – depending on smoking of
grandmother and / or mother
Transgenerational effects of ETS exposure on asthma
Risk of ETS exposure may be transmitted
N = 338 children with asthma, age 5 N = 570 controls, matched for in utero smoking
depending on smoking of grandmother and / or mother
Li et al, Chest 2005
Transgenerational effects of ETS exposure on asthma
I
II
III
Transgenerational effects of ETS exposure on asthma
grandchild
Transgenerational effects of ETS exposure on asthma
• Risk of asthma in grandchild
– Grandmaternal smoking– Grandmaternal smoking(independent from maternal smoking)
– Grandmaternal and maternal smoking
– This could be explained by epigenetics, but this remains to be shown!
Transgenerational effects of ETS exposure on asthma
Risk of asthma in grandchild
Grandmaternal smoking OR 1.8 [1.0 – 3.3]Grandmaternal smoking OR 1.8 [1.0 – 3.3](independent from maternal smoking)Grandmaternal and maternal smoking
OR 2.6 [1.6 – 4.5]This could be explained by epigenetics, but this
Li et al, Chest 2005
Transgenerational effects of ETS exposure on asthma
Transgenerational effects of ETS exposure on asthma
Grandmother exposes
- Daughter - Daughter
- Granddaughter
(in females, oogenesis starts in utero)
Maternal effects in asthma
• Risk of asthma higher in children of mother with asthma, compared to fathers with athma
• Example: • Example: – 200 consecutive cases of childhood asthma, – Approximately twice as many children had atopic
mothers than atopic fathers (Bray, J Allergy, 1931)• Could epigenetic inheritance be gender specific?
Maternal effects in asthma
Risk of asthma higher in children of mother with asthma, compared to fathers with athma
200 consecutive cases of childhood asthma, Approximately twice as many children had atopic mothers than atopic fathers (Bray, J Allergy, 1931)
Could epigenetic inheritance be gender specific?
Maternal effects in asthma
• Several studies show differences in methylation patterns depending on paternal or maternal origin of the alleleles (imprinting, X chromosome origin of the alleleles (imprinting, X chromosome inactivation)
• No positive studies so far link this to asthma
Maternal effects in asthma
Several studies show differences in methylation patterns depending on paternal or maternal origin of the alleleles (imprinting, X chromosome origin of the alleleles (imprinting, X chromosome
No positive studies so far link this to asthma
This presentation
• What is epigenetics?
• The promise of epigenetics
• What do we know so far?
• Novel insights in the epigenetics of asthma
This presentation
The promise of epigenetics
What do we know so far?
Novel insights in the epigenetics of asthma
What do we know so far ?
• Literature review 2013 on epigenetics and asthma
DeVries and Vercelli Asian Pac J All Immunol 2013:31:183
What do we know so far ?
Literature review 2013 on epigenetics and
Keywords : asthma, allergy, methylation,
Comparison of reviews versus original
Genome wide versus candidate gene studies
DeVries and Vercelli Asian Pac J All Immunol 2013:31:183-9
What do we know so far ?
Comparison reviews versus original data
40
Number of manuscripts until 2013 Number of manuscripts until 2013 Number of manuscripts until 2013 Number of manuscripts until 2013
DeVries and Vercelli Asian Pac J All Immunol 2013:31:183
0
10
20
30
Reviews Original research
31
What do we know so far ?
Comparison reviews versus original dataNumber of manuscripts until 2013 Number of manuscripts until 2013 Number of manuscripts until 2013 Number of manuscripts until 2013
DeVries and Vercelli Asian Pac J All Immunol 2013:31:183-9Original research
25
Number
What do we know so far ?• Genome wide versus candidate gene studies
15
Number Number Number Number
DeVries and Vercelli Asian Pac J All Immunol 2013:31:183
0
5
10
Genome wide Candidate genes
7
What do we know so far ?Genome wide versus candidate gene studies
Number Number Number Number
DeVries and Vercelli Asian Pac J All Immunol 2013:31:183-9Candidate genes
14 Number
What do we know so far ?
• Role of methylation in allergy (Th1/Th2 skewing)
• Environmental effects on the DNA methylome• Tissue specific effects explained by
methylation• Role of DNA methylation in astma
What do we know so far ?
Role of methylation in allergy (Th1/Th2
Environmental effects on the DNA methylomeTissue specific effects explained by
Role of DNA methylation in astma
T cell differentiation
Gene MechanismIL4 Demethylation of GATA4 binding site
Demethylation of the 5’ flanking region of the IL4 promoterDemethylation of the 5’ flanking region of the IL4 promoterIncrease in histone acetylation
IFNG Methylation of AP1 binding site in promoterIL13, IL5 Increase in histone acetylation
This leads to Th2 deviationThis leads to Th2 deviationThis leads to Th2 deviationThis leads to Th2 deviation
T cell differentiation
Demethylation of GATA4 binding siteDemethylation of the 5’ flanking region of Demethylation of the 5’ flanking region of
Increase in histone acetylationMethylation of AP1 binding site in promoterIncrease in histone acetylation
S-M Ho, JACI 2010:126: 453-65
This leads to Th2 deviationThis leads to Th2 deviationThis leads to Th2 deviationThis leads to Th2 deviation
Epigenetics in T cell differentiation
Kabesch et al, ERJ 2010
Epigenetics in T cell differentiation
S-M Ho, JACI 2010:126: 453-65Kabesch et al, ERJ 2010
Effects of farming
– PASTURE study: rural birth cohort– DNA from cord blood and whole blood age 4,5 y– 46 samples– 10 candidate genes, including
• Aim– DNA methylation of cord blood cells– Transplacental exposure to PAH – Childhood asthma
Perera et al, PLOS ONE 2009; 4(2): e4488
Air Pollution, methylation and asthma
Inner city cohort of 700 Children in New YorkAsthma prevalence 25 % !Role of transplacental exposure to airborne polycyclic aromatic hydrocarbonspolycyclic aromatic hydrocarbons
DNA methylation of cord blood cellsTransplacental exposure to PAH
Perera et al, PLOS ONE 2009; 4(2): e4488
Air Pollution, methylation and asthma
• N = 20 children from New York cohort• Methylation sensitive restriction fingerprinting
of cord white blood cells• 31 loci were related to maternal PAH exposure• 31 loci were related to maternal PAH exposure• Of these, 6 genes with known CpG Island
– One gene whose methylation status correlated with best with gene expression:
(Acyl co-A synthethase long
Perera et al, PLOS ONE 2009; 4(2): e4488
Air Pollution, methylation and asthma
N = 20 children from New York cohortMethylation sensitive restriction fingerprinting of cord white blood cells31 loci were related to maternal PAH exposure31 loci were related to maternal PAH exposureOf these, 6 genes with known CpG Island
One gene whose methylation status correlated with best with gene expression: ACSL3
A synthethase long-chain family member 3)
Perera et al, PLOS ONE 2009; 4(2): e4488
Air Pollution, methylation and asthma
**
Perera et al, PLOS ONE 2009; 4(2): e4488
N= 56 children
Air Pollution, methylation and asthma
*
Perera et al, PLOS ONE 2009; 4(2): e4488
Air Pollution, methylation and asthma
• Comments
– Small pilot study– Incomplete coverage of the genome by initial assay– Incomplete coverage of the genome by initial assay– Needs replication
– Is one of the first study linking prenatal exposure, methylation patterns and asthma at age 5.
Perera et al, PLOS ONE 2009; 4(2): e4488
Air Pollution, methylation and asthma
Incomplete coverage of the genome by initial assayIncomplete coverage of the genome by initial assay
Is one of the first study linking prenatal exposure, methylation patterns and asthma at age 5.
Perera et al, PLOS ONE 2009; 4(2): e4488
Methylation and wheezing
- INMA project (Menorca and Sabadell)- Whole blood DNA at age 4- Wheezing phenotypes at age 4
- Stage 1: 27K Methylation array in 141 children - Stage 1: 27K Methylation array in 141 children from INMA Menorca - 61 never / 41 transient wheezing- 3 late onset / 17 persistent wheezing
- Stage 2: Replication in INMA Sabadell - Assessment of relation SNPs
Methylation and wheezing
INMA project (Menorca and Sabadell)Whole blood DNA at age 4Wheezing phenotypes at age 4-6 y
Stage 1: 27K Methylation array in 141 children Stage 1: 27K Methylation array in 141 children
61 never / 41 transient wheezing3 late onset / 17 persistent wheezing
Stage 2: Replication in INMA Sabadell Assessment of relation SNPs - methylation
Morales et al, AJRCCM 2012 (185): 937-43
Methylation and wheezing
Gene Chr Never Persistent
ZNF264 19 78.3 64.4
ALOX12 7 34.4 21.5
Morales et al, AJRCCM 2012 (185): 937
ALOX12 7 34.4 21.5
EPO 17 15.4 30.8
PDGFB 22 16.5 38
Methylation and wheezing
Persistent Difference P value
-13.7 1.4 x 10-5
-12.9 0.003
Morales et al, AJRCCM 2012 (185): 937-43
-12.9 0.003
15.3 0.006
21.4 0.007
Methylation and disease?
- ALOX12 : arachidonate 12 lipoxygenase- Involved in airway inflammation
- Array results confirmed with pyrosequencing, - Array results confirmed with pyrosequencing, although lower methylation levels were observed
- In Menorca Sabadell : 4 CpG Islands in p values 0.019 – 0.168
Morales et al, AJRCCM 2012 (185): 937
Methylation and disease?
arachidonate 12 lipoxygenaseInvolved in airway inflammation
Array results confirmed with pyrosequencing, Array results confirmed with pyrosequencing, although lower methylation levels were observed
In Menorca Sabadell : 4 CpG Islands in ALOX12,
Morales et al, AJRCCM 2012 (185): 937-43
SNP affects Methylation
- Association SNP and methylation status in
Morales et al, AJRCCM 2012 (185): 937
SNP affects Methylation
Association SNP and methylation status in ALOX12
Morales et al, AJRCCM 2012 (185): 937-43
SNP x M Interaction in asthma? - Interaction of SNP and methylation status in
IL4RA on asthma in the Isle of Wight study
SNP x M Interaction in asthma? Interaction of SNP and methylation status in
on asthma in the Isle of Wight studyRs3024685
CC vs TT
Soto-Ramirez et al, Clin Epigenetics 2013 (5): 1
CT vs TT
Integration: SNPs and methylation
- Model 1: Genetic effect is methylation mediated- SNP � Methylation � Disease
- Model 2: Reverse Causality- Model 2: Reverse Causality- SNP � Disease, Disease
- Model 3: Independent (Common causes)- SNP � Methylation- SNP � Disease