epigenetics asthma denmark koppelman 2014
TRANSCRIPT
Epigenetics of asthma
Groningen Research Institute of Asthma and COPD
Gerard KoppelmanGerard KoppelmanGerard KoppelmanGerard Koppelman
Groningen Research Institute of Asthma and COPD
Pediatric Pulmonology and Pediatric Allergology
Beatrix Children’s Hospital, University Medical Center Groningen
Epigenetics of asthma
Groningen Research Institute of Asthma and COPD
Gerard KoppelmanGerard KoppelmanGerard KoppelmanGerard Koppelman
Groningen Research Institute of Asthma and COPD
Pediatric Pulmonology and Pediatric Allergology
Beatrix Children’s Hospital, University Medical Center Groningen
This presentation
• From genetics to epigenetics?
• The promise of epigenetics
• What do we know so far?
• Novel insights in the epigenetics of asthma
This presentation
From genetics to epigenetics?
The promise of epigenetics
What do we know so far?
Novel insights in the epigenetics of asthma
Genetic variation
DNA3 billion basepairs
gene
Single nucleotide polymorphisms
About 90% of all human genetic variation
SNPs
30,000 genes
Genetic variation
ACCGTTCGGG
Single nucleotide polymorphisms
About 90% of all human genetic variation
From genetics to epigenetics
mRNA
DNA - Genetic variation
mRNA
Asthma
Protein
From genetics to epigenetics
Genetic assocation study
• Method: case
Reference allele
Genetic assocation study
control
Risk allele
Genetics (status 2012)Genetics (status 2012)
Contribution of genetics to understanding disease
• Mendelian disease• Complex disease
variants from 165 diseases
How much of heritability explained?• Type 1 diabetes• Crohn’s disease• Asthma
Contribution of genetics to understanding disease
2000 2010
<100 2.85012 1.100
variants from 165 diseases
How much of heritability explained?60 %20-25%< 5 % Lander ES, Nature 2010
GWAS of the GABRIEL consortium
/IL1RL1
GWAS of the GABRIEL consortium
Moffatt et al, NEJM 2010; 363: 13
Asthma risk loci overlap in EVE and Gabriel
Gene/Region EVE17q21(ORMDL3/GSDML)
1.2x10-14
IL1RL1/IL18R1 (chr. 2) 1.4x10-8
TSLP (chr. 5) 7.3x10-10
IL33 (chr. 9) 2.5x10-7IL33 (chr. 9) 2.5x10-7
PYHIN1 (chr. 1) 3.6x10-7
HLA-DQ (chr. 6) 0.014SMAD3 (chr. 15) 1.9x10-4
IL2RB (chr. 22) nsSLC22A5 (chr. 5) nsIL13 (chr. 5) nsRORA (chr. 15) 2.1x10-3
*childhood asthmaThe EVE Consortium – C. Ober
Asthma risk loci overlap in EVE and Gabriel
GABRIEL Race/Ethnicity
6.4x10-23* All
3.4x10-9 All7.5-8 All
9.2x10-10 All9.2x10-10 All-- African Ancestry
7.0x10-14 All3.9x10-9 Eur/EurAmer1.1x10-8 Europeans2.2x10-7 Europeans1.4x10-7 Europeans1.1x10-7 Eur/EurAmer
*childhood asthma
What have we learnt?
• 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
How many different basepairs are there?
• A, C, G, T• 5 mC (5 methyl cytosine)• 5 hmC (5 hydroxy methyl cytosine)• 5 hmC (5 hydroxy methyl cytosine)
How many different basepairs are there?
5 mC (5 methyl cytosine)5 hmC (5 hydroxy methyl cytosine)5 hmC (5 hydroxy methyl cytosine)
Histone modification
Eu-chromatin
Hetero-chromatin
Histone modification
Ummethylation of CpG,
acetylation of histones:
Kabesch et al, ERJ 2010
histones:
EXPRESSION
Methylation of CpG and histones:
NO EXPRESSION
DNA Methylation and histone modifications
regulate gene expression
Kabesch and Adcock Biochimie 94 (2012) 2231 e2241
DNA Methylation and histone modifications
regulate gene expression
Kabesch and Adcock Biochimie 94 (2012) 2231 e2241
The synthesis and processing of microRNA
POL II
C T D
TFIIE
TFIID Complex
TAF II130 TAF II250
TBPTAF II31
TAF II80
TAF II20
TFIIF
TFIIH
Kin28
TFIIBTFIIA
RNaseIII(Drosha)
Transcription
Dicer
Argonaute 2(RNase H)
m7G
(Drosha)
TRBP
Capping, splicingand polyadenylation
mRNA degradation
Translation inhibition
The synthesis and processing of microRNA
RNase IIIRNase
(Drosha)
Pasha
RNaseIII
Pasha
Nucleus
Dicer
Argonaute 2(RNase H)
Dicer
AAAA
(Drosha) III(Drosha)
TRBP
Exportin 5
Ran
TRBP
What increases with more complex organisms (homo sapiens) (when
compared to bacteria, yeast)?
• The number of chromosomes?• The number of chromosomes?
• The number of base pairs?
• The number of genes?
• The percentage of non-coding DNA?
What increases with more complex organisms (homo sapiens) (when
compared to bacteria, yeast)?
The number of chromosomes?The number of chromosomes?
The number of base pairs?
coding DNA?
What correlates with more complex organisms (homo sapiens)?
What correlates with more complex organisms (homo sapiens)?
What correlates with more complex organisms (homo sapiens)?
What correlates with more complex organisms (homo sapiens)?
What correlates with more complex organisms (homo sapiens)?
What correlates with more complex organisms (homo sapiens)?
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
The promise of epigenetics of asthma
• Epigenetics may explain– Effect of the environment during critical time window
“programming”– Differences in end organ expression (tissue specific
effects) effects) – Changes in phenotypes over time– Transgenerational effects– Parent of offspring effects
Koppelman and Nawijn, Curr Opin Allergy & Clin Immunology 2011: 11 (5) 414
The promise of epigenetics of asthma
Epigenetics may explainEffect of the environment during critical time window
Differences in end organ expression (tissue specific
Changes in phenotypes over timeTransgenerational effectsParent of offspring effects
Koppelman and Nawijn, Curr Opin Allergy & Clin Immunology 2011: 11 (5) 414–419
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
• Keywords : asthma, allergy, methylation, epigenetics
• Comparison of reviews versus original research
• Genome wide versus candidate gene studies
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
IL13, IL4, STAT6, RUNX3, FOXP3, CHI3LIIL13, IL4, STAT6, RUNX3, FOXP3, CHI3LI– Questions:
– Change over time?– Effect of farm?– Relation with asthma?
Effects of farming
PASTURE study: rural birth cohortDNA from cord blood and whole blood age 4,5 y
10 candidate genes, including ORMDL3, RAD50, IL13, IL4, STAT6, RUNX3, FOXP3, CHI3LIIL13, IL4, STAT6, RUNX3, FOXP3, CHI3LI
Relation with asthma?
Michel et al, Allergy, 2013; 68 : 355-64
Effects of farming– PASTURE study– DNA from cord blood and – 46 samples– 10 candidate genes, including
IL13,IL4, STAT6, RUNX3, FOXP3, CHI3LIIL13,IL4, STAT6, RUNX3, FOXP3, CHI3LI– Questions:
– Change over time?– Effect farm?– Relation asthma?
Effects of farming
and whole blood age 4,5 y
including ORMDL3, RAD50, IL13,IL4, STAT6, RUNX3, FOXP3, CHI3LIIL13,IL4, STAT6, RUNX3, FOXP3, CHI3LI
IL4
Michel et al, Allergy, 2013; 68 : 355-64
Effects of farming
Results– Effect of farming on
STAT6, IL13, RAD50
– Effect of timing: 15 gene
– Asthma: one gene regionAsthma87.9
Michel et al, Allergy, 2013; 68 : 355
Effects of farming
4 genes (ORMDL1, STAT6, IL13, RAD50)
Effect of timing: 15 gene regions
region ORMDL3 Asthma Control87.9 85.8 p=0.03
Michel et al, Allergy, 2013; 68 : 355-64
Effects of materal smoking
Norwegian Mother and Child– DNA from cord blood– 1,062 samples– Smoking assessed by– Smoking assessed by
mothers– Epigenome wide analysis– Replication study in 36 samples
Joubert et al, Env Health Perspectives, 120 (10): 1425
Effects of materal smoking
Child Cohort Studyblood
by plasma cotinine in by plasma cotinine in
analysis using Illumina 450 Kin 36 samples from US
Joubert et al, Env Health Perspectives, 120 (10): 1425-31
Effects of materal smoking
Joubert et al, Env Health Perspectives, 120 (10): 1425
Effects of materal smoking
Joubert et al, Env Health Perspectives, 120 (10): 1425-31
Effects of materal smoking
- 26 CpG sites in 10 genes associated with maternal smoking
- AHRR (aryl hydrocarbon receptor repressor gene ) CYP1A1 and GFI1 replicatedCYP1A1 and GFI1 replicated
- Average methylation difference: 3- Biological plausible pathway: detoxification of cigarette
smoke components
Joubert et al, Env Health Perspectives, 120 (10): 1425
Effects of materal smoking
26 CpG sites in 10 genes associated with maternal
aryl hydrocarbon receptor repressor gene ) , replicatedreplicated
Average methylation difference: 3-10% Biological plausible pathway: detoxification of cigarette
Joubert et al, Env Health Perspectives, 120 (10): 1425-31
Effects of active smoking
- German KORA study- 1793 participants- Several EWAS hits, Including AHRRIncluding AHRRMethylation patterns of Former smokers more Like never smokers
Zeilinger et al, PLOS ONE 2013
Effects of active smoking
Zeilinger et al, PLOS ONE 2013
Environmental factors linked to asthma may induce epigenetic effects
• Folic acid (mouse) Hollingsworth, J Clin Invest. 2008;118(10):3462
• Plasma homocysteine, birth weight2011 6:1,86
• Maternal smoking Breton et al, AJRCCM 2009.1;180(5):462• Maternal smoking Breton et al, AJRCCM 2009.1;180(5):462
• Air pollution Perera et al, PLOS ONE 2009; 4(2): e4488)
• Maternal Stress Chen et al, AJRCCM 2013 (187): 584
– Important in asthma development?
Environmental factors linked to asthma may induce epigenetic effects
Hollingsworth, J Clin Invest. 2008;118(10):3462-9
Plasma homocysteine, birth weight Fryer et al, Epigenetics 2011 6:1,86-94
Breton et al, AJRCCM 2009.1;180(5):462-7.Breton et al, AJRCCM 2009.1;180(5):462-7.
Perera et al, PLOS ONE 2009; 4(2): e4488)
Chen et al, AJRCCM 2013 (187): 584-8
Important in asthma development?
Tissue specific effects on
Asthma
Asthma + BHR
Asthma and Total IgE
Asthma and specific IgE
Tissue specific effects on ADAM 33
Van Eerdewegh et al., Nature 2002
ADAM33 was the first asthma gene found by positional cloning
A D G J MC F I L NKHEB
A Disintegrin And Metalloprotease (
ADAM33, ADAM33, ADAM33, ADAM33, a gene for asthma and BHRa gene for asthma and BHRa gene for asthma and BHRa gene for asthma and BHR
A-VT-AY-H
proteolysis adhesion
Zn2+
site
activation
Expression of ADAM33 mRNA restricted to mesenchymal cells ; smooth muscle, fibroblasts & myofibroblasts
CpG CpG CpG CpG
P S VO R UTQ
etalloprotease ( ADAM)33
a gene for asthma and BHRa gene for asthma and BHRa gene for asthma and BHRa gene for asthma and BHR
adhesion fusion signalling
3’ UTR
Promotes efficient maturaton of ADAM33
Expression of ADAM33 mRNA restricted to mesenchymal cells ; smooth muscle, fibroblasts & myofibroblasts
Epigenetic effects are tissue specific
• ADAM33 mRNA is expressed in fibroblasts, but not in epithelial cells
Epithelial cells
Yang et al, JACI 2008; 121
Epithelial cells
Epigenetic effects are tissue specific
ADAM33 mRNA is expressed in fibroblasts, but not in epithelial cells
fibroblasts
Yang et al, JACI 2008; 121-1393-9
ADAM33
GAPDH
fibroblasts
Epigenetic effects are tissue specific
• ADAM33 gene is methylated in epithelial cells, but not in fibroblasts
Epithelial cells,
Yang et al, JACI 2008; 121
Epithelial cells, n=3
Fibroblasts, n=3
Epigenetic effects are tissue specific
ADAM33 gene is methylated in epithelial cells, but not in fibroblasts
Yang et al, JACI 2008; 121-1393-9
Epigenetic effects are tissue specific
• Demethylation of CpG Islands of ADAM33 induces mRNA expression in epithelial cells
Yang et al, JACI 2008; 121
Epigenetic effects are tissue specific
Demethylation of CpG Islands of ADAM33 induces mRNA expression in epithelial cells
Yang et al, JACI 2008; 121-1393-9
Epigenetic effects are tissue specific
• Micro RNAs are regulatory small non coding RNAs• At least 700 miRNAs described• Micro RNA targets 3’UTR sequences of on average
100 genes
• Aims: – Role in asthma? – Effect of inhaled corticosteroids? – Tissue specific effects?
Williams et al, PLOS ONE 2009 4(6): e5889
Epigenetic effects are tissue specific
Micro RNAs are regulatory small non coding RNAsAt least 700 miRNAs describedMicro RNA targets 3’UTR sequences of on average
Effect of inhaled corticosteroids?
Williams et al, PLOS ONE 2009 4(6): e5889
Micro RNAs in asthma?
• Airway wall biopsies of 8 patients with mild asthma, and 8 controls
• 227 miRNAs investigated in airway wall biopsies
• 227 miRNAs investigated in airway wall biopsies
Williams et al, PLOS ONE 2009 4(6): e5889
Micro RNAs in asthma?
Airway wall biopsies of 8 patients with mild asthma, and 8 controls227 miRNAs investigated in airway wall 227 miRNAs investigated in airway wall
Williams et al, PLOS ONE 2009 4(6): e5889
No differences in micro RNA expression asthma vs healthy controls
Williams et al, PLOS ONE 2009 4(6): e5889
No differences in micro RNA expression asthma vs healthy controls
Williams et al, PLOS ONE 2009 4(6): e5889
Tissue specific expression of micro RNAs
Differences in micro RNA expression between epithelial cells, smooth muscle cells, alveolar macrophages
Williams et al, PLOS ONE 2009 4(6): e5889
Targets suggest role in Important cellular processes in the lung
Tissue specific expression of micro RNAs
Differences in micro RNA expression between epithelial cells, smooth muscle cells, alveolar
Williams et al, PLOS ONE 2009 4(6): e5889
Important cellular processes
Integration: Environment (E), methylation and asthma
- Model 1: Environmental effect is methylation mediated- E � Methylation � Disease
- Model 2: Reverse Causality- E � Disease, Disease �
- Model 3: Independent (Common causes)- E � Methylation- E � Disease
Integration: Environment (E), methylation and asthma
: Environmental effect is methylation
Disease
: Reverse Causality� Methylation
: Independent (Common causes)
After Liu et al, Nat Biotech 2013 (31): 142-7
Air Pollution, methylation and asthma
• Inner city cohort of 700 Children in New York• Asthma prevalence 25 % !• Role of transplacental exposure to airborne
polycyclic aromatic hydrocarbonspolycyclic aromatic hydrocarbons
• 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
Integration: SNPs and methylation
: Genetic effect is methylation mediatedDisease
: Reverse Causality: Reverse CausalityDisease, Disease � Methylation
: Independent (Common causes)
Liu et al, Nat Biotech 2013 (31): 142-7
Putting it together: integrative genomics
Genome
Transcriptome
Proteome
Putting it together: integrative genomics
Genome
Epigenome
Proteome
B. Raby, personal communication
Genome Wide Association in asthma
• SNPs on chromosome 17q12to asthma in children
Moffatt et al Nature 2007 26;448(7152):470
Genome Wide Association in asthma
SNPs on chromosome 17q12-21 contribute
Moffatt et al Nature 2007 26;448(7152):470-3
17q12-21 SNPs regulate with mRNA expression 21 SNPs regulate with mRNA expression
Verlaan et al, AJHG 2009
a 17q21 DNA variant regulates chromatin remodelling by binding CTCF
CTCF is a protein involved in chromatin looping andis known to reposition nucleosomes, and thus is able to regulate multiple transcripts
a 17q21 DNA variant regulates chromatin remodelling by binding CTCF
Verlaan et al, AJHG 2009
CTCF is a protein involved in chromatin looping andis known to reposition nucleosomes, and thus is able to regulate multiple transcripts
Dna, methylation, environment, disease? Dna, methylation, environment, disease?
So….
MethylationMethylation
So….
AsthmaAsthma
So….
Environment
Methylation
So….
Methylation Asthma
So….
Environment
Methylation
Genetics
So….
Methylation 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
EWAS in asthma: the first results
- MEDALL Project: collaboration in Europe- BAMSE, Sweden- PIAMA, the Netherlands- EDEN, France- EDEN, France- INMA, Spain
- Paired samples of cord blood or blood DNA0 and 4/5 years: EDEN, INMA4/5 and 8 years: BAMSE, PIAMA
- Illumina 450 k Array, including 2 controls
EWAS in asthma: the first results
MEDALL Project: collaboration in Europe
PIAMA, the Netherlands
Paired samples of cord blood or blood DNA0 and 4/5 years: EDEN, INMA4/5 and 8 years: BAMSE, PIAMA
Illumina 450 k Array, including 2 controls
Xu et al, in preparation, 2014
450K Methylation array
• Successor of 27K methylation array
• Similar to Infinium II technique, but some technique, but some differences:– Bisulphite conversion– 2 types of chemistries
on 1 beadchip• Sensitive to batch
effects
450K Methylation array
Future of epigenetics in asthma and allergy? Future of epigenetics in asthma and allergy?
Epigenetics in asthma and allergy
• Epigenetics regulates gene expression
• There are not 4 but 6 basepairs
• Epigenetic mechanisms may explain important observations in asthma
• Environment may change epigenetic signatures: smoking, air pollution
Epigenetics in asthma and allergy
Epigenetics regulates gene expression
There are not 4 but 6 basepairs
Epigenetic mechanisms may explain important observations in asthma
Environment may change epigenetic signatures: smoking, air pollution
Epigenetics in asthma and allergy
• Tissue specific studies are important, further analysis of mixed cell types (blood) is needed
• First EWAS in asthma is underway• First EWAS in asthma is underway
• Next step: Functionally important? How relevant is 5 % difference in gene methylation
Epigenetics in asthma and allergy
Tissue specific studies are important, further analysis of mixed cell types (blood)
First EWAS in asthma is underwayFirst EWAS in asthma is underway
Next step: Functionally important? How relevant is 5 % difference in gene
Epigenetics in asthma
• Need for integration: systems approach to genomics–To understand cause and effect–To understand disease mechanisms–To understand disease mechanisms
• The good news: –Epigenetic modifications can be changed.
New therapeutic options?
Epigenetics in asthma and allergy
Need for integration: systems approach to
To understand cause and effectTo understand disease mechanismsTo understand disease mechanisms
Epigenetic modifications can be changed. New therapeutic options?
Thank you !
MedALL Collaborators
BAMSEErik Melen, Magnus Wickman
EDENIsabelle Annesi -MaesanoIsabelle Annesi -Maesano
INMAJordy Sunyer
PIAMAJet Smit, Alet Wijga, Bert Brunekreef, Johan de Jongste
MEDALL CoordinatorsJean Bousquet and JosepAnto
Thank you !
Groningen Research Institute of Asthma and COPD
Cheng XuDirkje PostmaMarjan KerkhofCisca WijmengaCisca WijmengaSoesma MedemaPieter van der VliesColleagues, Pediatric
Pulmonology and Pediatric Allergology
Benjamin Raby, Harvard Medical School, Boston MA
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