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

2 4 53 7 86 10 119 12 13 14 15 16 17 18 19 201 21 22 X Y

Linkage studies in Australian samples

12q24

20q13Ferreira et al. (2005) Am J Hum Genet 77: 1075

Ferreira et al. (2006) Eur J Hum Genet 14: 953

2q33Evans et al. (2004) J Allergy Clin Immunol 114: 826

18798773181096

1181388

31164963181113

6435203

1181425 231746231735

926169231770

5742909231779

3087243

1365965 3096851 31165053096859

20331711978594

45225876728120

15599314675379

31165344675389

933988

3181098

CD28 CTLA-4 ICOS

120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 2800 10 20 30 40 50 60

Distance (Kb)

CD28 CTLA4 ICOS

Chromosome 2q33

1879877, 3181096, 31810981181388

3116496

9339884675389 31165341559931, 4675379

67281204522587

19785942033171

30968593116505

3096851

1365965

30872432317795742909 231770

926169

231735231746

1181425

64352033181113

500 150100 250200

Distance (Kb)

0.0

1.0

D', r2

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

28 SNPs (270 Kb)

Chromosome 2q33

Threshold for significance: α = 0.05/(4 traits × 28 SNPs) = 0.0005

Univariate association analysis

Power: < 30% (Locus explained up to 1.5% of the variance, p = 0.3, dominant model)

Threshold for significance: α = 0.05/(1 trait × 28 SNPs) = 0.0018

Multivariate association analysis

Fulker et al. (1999), e.g. QTDT

Lange et al. (2004), PBAT

Chromosome 2q33

Chromosome 2q33

Position

(bp) Sequence Comments

204395500 CTCTCTNCAAAGGGCCTGGGAGTTGAAGAAGGGTGCAGTCGGGTGGTGGT 204395550 TATGAATCTCAGAAATCCTGCCACGGAGCCTCCTTTTGTGCCCTATTANT rs3181096, E4BP4 TFS 204395600 TAACCTTGAGGGACATAGAGAGCATGAGACACCAAGGGGCTTTTGNTTGC VDR/RXR TFS, 204395650 CTTACTGTCCCACTAAGAACATAGAATGTTGTTTTGACTTTCCCTTTGCT PRE and IRF-2 TFS (-) 204395700 TAGGGAACCTCCCCCAGATACTCAGCTGGCTGGCTGCTTGCACGTAGAAT 204395750 GGGTTTTGCAAAGTTCCTAGAAGTGAGTTGGAGGAGGCTTGACATAAATC 204395800 AAGCACTGTGTGCTAAATGCTCCAGAGGGCTACCTTATGTCCTACACAAA GRE TFS 204395850 TGTTACATTTCTAATATTTGTAACTCCTTTAAACNTTTATGCAGATGTTT rs3181098, MEF2 and MTATA TFS 204396700 AGTCTAAAGTCATCAAAACAACGTTATATCCTGTGTGAAATGCTGCAGTC Promoter () 204396750 AGGATGCCTTGTGGTTTGAGTGCCTTGATCATGTGCCCTAAGGGGATGGT Promoter (β) 204396800 GGCGGTGGTGGTGGCCGTGGATGACGGAGACTCTCAGGCCTTGGCAGGTG Vallejo (2005) 204396850 CGTCTTTCAGTTCCCCTCACACTTCGGGTTCCTCGGGGAGGAGGGGCTGG 204396900 AACCCTAGCCCATCGTCAGGACAAAGATGCTCAGGCTGCTCTTGGCTCTC Exon 1 204396950 AACTTATTCCCTTCAATTCAAGTAACAGGTAAACAATGTTAATGTCTTTC 204397000 TTTCTGTAAATATTTTTTGAGGTCTTCCAATTGGCTTAGTTTATTTTAAA 204398200 AAAAGGCCCCCGCTTGGTTCAAAAACTGGACTGATAGGGGTCAACAGTCA 204398250 TGCTTAAATAAGGACAGTTATTTTTCCCTGAAAGATACATTGAAAAGCCA Proscan 204398300 GTATCCTCAATTTTCTTTCTTATTTTGGCAGTACAGAGACTGCATTATTT Predicted 204398350 GTTGTTATTCTTAAACATTAAGTGTACATAGCCCAAAGAGTATAATTTCC Promoter 204398400 CAATCTGCAGAGGTACAGTAGTTGCATATATACCCGTTTATTTTATGGTC 204398450 TGACGTACCAGTGAGCACAAATTGTGTATATTTATAAAACGTGTTGATAT 204398500 AATGAAAGACATGAGTTGGCAATGAGATCTGGTACCAAGCGTTTACAGCT 204398550 ACCAAATATATTCTACAAGAATCTTTAACATTTATTTTAAAAAGGTCAAA

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

MMMMMM

M

CpG island Gene

Methylated

Suppressed

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)

Meth

yla

tion

1 2 3 43’ UTR

1 Kb

Exon

IL4 (Interleukin 4)

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

1 2 3 4 5 6 7 3’ UTR

1 Kb

Exon

MS4A2 (FCER1B) M

eth

yla

tion

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.

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 between methylation and

asthma

-0.4

-0.3

-0.2

-0.1

0.0

0.1

0.2

0.3

0.4

Eosinophils IgE

*** *

IL4 MS4A2

***

CpG units CpG units

Cor

rela

tion

coe

ffic

ien

t

1 2 3 4 5 6 1-6 1 2 3 4 5 6 7 8 9 10 11 1-11

* P < 0.05, ** P < 0.01

Methylation state of asthma genes

ASTHMA

Genetic risk factors

Environmental risk factors

ASTHMA

Genetic risk factors

Environmental risk factors

Methylation state of asthma genes

BA

Summary

Potentially relevant transcription factors bind to this promoter region

Genetic risk factors

Extending our study to validate these results

Identified SNPs in the promoter of CD28 that are associated with asthma phenotypes

Mostly methylated in PBLs of asthmatic children

Role of epigenetics in asthma

Significant variation in methylation between CpG sites and between individuals

Measured the methylation state of IL4 and MS4A2

This variation is associated with the expression of asthma clinical phenotypes

Doctorate scholarship, Ministry of Science, PortugalNHMRC project grant 290274The Asthma Foundation of QueenslandNHMRC Sidney Sax post-doctoral fellowship

Acknowledgments

Funding

Peter Le SouëfPaul R. Burton

Brett G. Toelle

Colin Robertson

Nick MartinDavid DuffyEmma WhitelawGrant MontgomeryMegan CampbellLeanne McNeillSri ShekarZhen Zhen ZhaoRenee MayneLouise O’GormanNathan Oates

Queensland Institute of Medical Research

Princess Margaret Hospital for Children, Perth

Woolcock Institute of Medical Research, Sydney

Royal Children’s Hospital, Melbourne

Sequenom

Mathias EhrichJeff Bryant