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Genetic and epigenetic risk factors for
asthma
Manuel A R Ferreira
QUEENSLAND INSTITUTE OF
MEDICAL RESEARCH
Queensland Institute of
Medical Research
1. Genetic risk factors
Linkage studies in Australian samples
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5
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7
8
9
10 11
12 13 14 15
16 17
18
19 20
21 22
12q24
Ferreira et al. (2006) Eur J Hum Genet 14: 953
20q13
Ferreira et al. (2005) Am J Hum Genet 77: 1075
2q33
Evans et al. (2004) J Allergy Clin Immunol 114: 826
X
Y
Chromosome 2q33
CD28
CD28
1879877
3181096
3181098
1181388
0
10
CTLA4
CTLA-4
1181425
3116496
3181113
6435203
20
30
40
50
926169
231770
5742909
231779
3087243
231746
231735
60 120
130
ICOS
ICOS
140
150
160
170
1365965
180
3096851
190
200
3116505
3096859
2033171
1978594
210
220
230
4522587
6728120
1559931
4675379
3116534
4675389
933988
240
250
260
270
280
Distance (Kb)
28 SNPs (270 Kb)
933988
4675389 3116534
1559931, 4675379
6728120
4522587
1978594
3096859
1,946 individuals (663 families)
41% 1 offspring (23% : 7% : 11%)
59% >1 offspring (18% : 11% : 30%)
4 continuous traits:
FEV1
FEV1/FVC
Immunoglobulin E
Eosinophilia
1.0
2033171
3116505
3096851
1365965
D', r2
231779 3087243
5742909
231770
926169
231735
231746
1181425
6435203
3181113
3116496
0.0
1181388
1879877, 3181096, 3181098
0
50
100
150
Distance (Kb)
200
250
Chromosome 2q33
Univariate association analysis
Fulker et al. (1999), e.g. QTDT
Threshold for significance:
α = 0.05/(4 traits × 28 SNPs) = 0.0005
Power: < 30% (Locus explained up to 1.5% of the variance, p = 0.3, dominant model)
Multivariate association analysis
Lange et al. (2004), PBAT
Threshold for significance:
α = 0.05/(1 trait × 28 SNPs) = 0.0018
Chromosome 2q33
Chromosome 2q33
Position
(bp)
Sequence
204395500
204395550
204395600
204395650
204395700
204395750
204395800
204395850
CTCTCTNCAAAGGGCCTGGGAGTTGAAGAAGGGTGCAGTCGGGTGGTGGT
TATGAATCTCAGAAATCCTGCCACGGAGCCTCCTTTTGTGCCCTATTANT
TAACCTTGAGGGACATAGAGAGCATGAGACACCAAGGGGCTTTTGNTTGC
CTTACTGTCCCACTAAGAACATAGAATGTTGTTTTGACTTTCCCTTTGCT
TAGGGAACCTCCCCCAGATACTCAGCTGGCTGGCTGCTTGCACGTAGAAT
GGGTTTTGCAAAGTTCCTAGAAGTGAGTTGGAGGAGGCTTGACATAAATC
AAGCACTGTGTGCTAAATGCTCCAGAGGGCTACCTTATGTCCTACACAAA
TGTTACATTTCTAATATTTGTAACTCCTTTAAACNTTTATGCAGATGTTT
204396700
204396750
204396850
204396900
204396950
204397000
AGTCTAAAGTCATCAAAACAACGTTATATCCTGTGTGAAATGCTGCAGTC
AGGATGCCTTGTGGTTTGAGTGCCTTGATCATGTGCCCTAAGGGGATGGT
CGGTGGTGGTGGCCGTGGATGACGGAGACTCTCAGGCCTTGGCAGGTG
CGTCTTTCAGTTCCCCTCACACTTCGGGTTCCTCGGGGAGGAGGGGCTGG
AACCCTAGCCCATCGTCAGGACAAAGATGCTCAGGCTGCTCTTGGCTCTC
AACTTATTCCCTTCAATTCAAGTAACAGGTAAACAATGTTAATGTCTTTC
TTTCTGTAAATATTTTTTGAGGTCTTCCAATTGGCTTAGTTTATTTTAAA
204398200
204398250
204398300
204398350
204398400
204398450
204398500
204398550
AAAAGGCCCCCGCTTGGTTCAAAAACTGGACTGATAGGGGTCAACAGTCA
TGCTTAAATAAGGACAGTTATTTTTCCCTGAAAGATACATTGAAAAGCCA
GTATCCTCAATTTTCTTTCTTATTTTGGCAGTACAGAGACTGCATTATTT
GTTGTTATTCTTAAACATTAAGTGTACATAGCCCAAAGAGTATAATTTCC
CAATCTGCAGAGGTACAGTAGTTGCATATATACCCGTTTATTTTATGGTC
TGACGTACCAGTGAGCACAAATTGTGTATATTTATAAAACGTGTTGATAT
AATGAAAGACATGAGTTGGCAATGAGATCTGGTACCAAGCGTTTACAGCT
ACCAAATATATTCTACAAGAATCTTTAACATTTATTTTAAAAAGGTCAAA
2
0
4
3
9
6
8
0
0
G
G
Comments
rs3181096, E4BP4 TFS
VDR/RXR TFS,
PRE and IRF-2 TFS (-)
GRE TFS
rs3181098, MEF2 and MTATA TFS
Promoter ()
Promoter (β)
Vallejo (2005)
Exon 1
Proscan
Predicted
Promoter
Chromosome 2q33
Genotyped 3 more samples: Holland, Denmark and Tristan da Cunha Island
Genotyped more SNPs to increase LD coverage (ICOS and CD28)
Test for epistasis using a novel gene-based association method (Purcell et al. )
2. Role of epigenetics in asthma
Methylation of CpG dinucleotides
CpG island
Gene
MMMM MM
Methylated
Suppressed
M
Not methylated
Active
Methylation and asthma
1. What is the methylation state of known asthma genes?
2. Are there significant differences in methylation levels between individuals?
3. Do methylation levels correlate with clinical markers of asthma?
Selected 30 children aged 10-19 (70% asthmatic, 75% atopic)
Extracted DNA from peripheral blood leukocytes
Quantified methylation state of CpG islands using Sequenom MassSpectometry assay
(Ehrich et al. 2005 PNAS 102: 15785)
Two genes involved in asthma: IL4 and MS4A2 (beta subunit of the IgE high affinity receptor)
IL4 (Interleukin 4)
1 Kb
Exon 1
2
3
4
3’ UTR
Methylation
Mean methylation:
75%
Significant differences between CpG
sites (P < 0.0001)
Lower methylation in regulatory elements
Significant differences between
individuals (P < 0.0001)
e.g. 75% vs 40% (CpG 5)
No significant effects of age,
sex or steroid medication
MS4A2 (FCER1B)
1 Kb
Exon 1
2
3
4
5
6
7
3’ UTR
Methylation
Mean methylation:
90%
Significant differences between
CpG sites (P < 0.0001)
CpG 2 in regulatory element?
Significant differences between
individuals (P < 0.0001)
e.g. 75% vs 30% (CpG 2)
No significant effects of age,
sex or steroid medication
2006/12/10. CORRECTION: data for CpG2 was found to be
unreliable in the Sequenom assay. All other CpGs ok.
Correlation between methylation and asthma
Significant differences in methylation between individuals. Do these correlate with the
expression of asthma phenotypes?
Small differences in methylation (~15%) can result in large differences (~40%) in gene transcription
Oates et al. (2006) Am J Hum Genet 79: 155
Correlation coefficient
IL4
MS4A2
0.4
Eosinophils
0.3
IgE
0.2
0.1
0.0
-0.1
-0.2
-0.3
-0.4
*
**
1
2
3
4
5
CpG units
6
**
1-6
1
*
*
2
3
4
5
6
7
8
9
10 11 1-11
CpG units
* P < 0.05, ** P < 0.01
A
B
Genetic risk
factors
Environmental
risk factors
Methylation state of
asthma genes
Genetic risk
factors
Environmental
risk factors
Methylation state of
asthma genes
ASTHMA
ASTHMA
Summary
Genetic risk factors
Identified SNPs in the promoter of CD28 that are associated with asthma phenotypes
Potentially relevant transcription factors bind to this promoter region
Extending our study to validate these results
Role of epigenetics in asthma
Measured the methylation state of IL4 and MS4A2
Mostly methylated in PBLs of asthmatic children
Significant variation in methylation between CpG sites and between individuals
This variation is associated with the expression of asthma clinical phenotypes
Acknowledgments
Queensland Institute of Medical
Research
Princess Margaret Hospital for Children,
Perth
Nick Martin
David Duffy
Emma Whitelaw
Grant Montgomery
Megan Campbell
Leanne McNeill
Sri Shekar
Zhen Zhen Zhao
Renee Mayne
Louise O’Gorman
Nathan Oates
Peter Le Souëf
Paul R. Burton
Woolcock Institute of Medical Research,
Sydney
Brett G. Toelle
Royal Children’s Hospital, Melbourne
Colin Robertson
Sequenom
Funding
Mathias Ehrich
Jeff Bryant
Doctorate scholarship, Ministry of Science, Portugal
NHMRC project grant 290274
The Asthma Foundation of Queensland
NHMRC Sidney Sax post-doctoral fellowship