1. Accounting for age-specific sex-limitation in IgE QTL linkage analysis: example of 11p13 Manuel A R Ferreira, David Duffy & Nick Martin Queensland Institute of Medical Research Australia

2. Accounting for non-additive genetic effects in IgE QTL linkage analysis: example of 11p13 Manuel A R Ferreira, David Duffy & Nick Martin Queensland Institute of Medical Research Australia

3. 2 453786 101191213 14151617181920 12122XY Replicated regions @ p 1.2) – Ober et al. (2000) ADAM33 PHF11 DPP10 EHF, ELF5 (11p13) FCER1B GPRA Positionally cloned genes Genetics of asthma EHF and ELF5 (11p13) not replicated

4. Patent reports suggestive linkage (p = 0.0001) of asthma to D11S907 and association to D11S2008 (p < 0.0001) Brooks-Wilson et al. (1998) filed a US patent for ELF5 and EHF, asthma susceptibility genes 6/27 Linkage and association of asthma to 11p13 One extended pedigree from Tristan-da-Cunha island (n=282), with high prevalence of asthma. Own replication with a small sample of Canadian nuclear families with 1+ asthmatic children. Baron et al. (2002) failed to replicate association of asthma to ELF5 and EHF, small number of SNPs (4+3)

5. Problem: can we replicate linkage of asthma to 11p13? 7/27

6. 1995-1998 QIMR Asthma & Allergy study 8/27 Replication of Linkage to 11p13 – large Australian dataset 802 families, mean size 4 (range 1-10), mostly parents and children Each family with at least 1 asthmatic child 2500+ individuals with phenotypic and genotypic data at candidate regions Extensive respiratory questionnaire (American Thoracic Society) Total Immunoglobulin E, Eosinophils, BHR, atopy, FEV 1 /FVC Dataset includes 490 DZ twin pairs and 312 MZ pairs

7. Significant Replication EHF 100 Kb VC lod IgE Variance components (VC) Additive QTL effects only Multipoint IBD Chr11 ELF5 D11S2008 D11S907 Similar with other phenotypes 1 Mb

8. Have we failed to replicate linkage of asthma to 11p13? Can it be a false negative result? 10/27

9. 1. Hypothesis A:power? 11/27

10. α = 0.01 α = 0.0001 Size 3 (n = 128) Size 2 (n = 325) Size 4 (n = 38) Size 5+ (n = 6) Overall (n = 497) QTL magnitude (proportion total variance) NCP VC linkage test r = 0.25 Biallelic single QTL Additive effects only Perfect marker information θ = 0 Sham et al. (2000) 12/27

11. Comparable or better than most published asthma linkage studies Enough power QTL >25% variance 13/27

12. 2. Hypothesis B: sex-specific age-of-onset? 14/27

13. Cumulative incidence Age (years) n = 949 doctor diagnosed asthmatics Kaplan-Meier survival curves for age of first wheeze Similar results for atopy Mean age-of-onset. Males: 8.2 (7.2-9.2). Females: 11.8 (11-12.5) Males have earlier onset of asthma/atopy 15/27

14. Can this affect linkage analysis? yes, it if affects the expression of phenotypes, such as IgE 16/27

15. Same-sex sib-pairs Opposite-sex sib-pairs Young (age ≤ 20) Older (age > 20) 17/27 r = 0.08 (-0.07-0.23) r = 0.36 (0.24-0.47) r = 0.25 (0.12-0.37) r = 0.16 (0.04-0.28) Sib 2 IgE Sib 1 IgE n = 858 sib-pairs r = 0.23 Not observed with other phenotypes

16. When tested formally by maximum likelihood estimation, only 8/802 (1%) families were found to be significantly inconsistent with a model that assumed homogenous sib-correlation. Low IgE correlation young opposite-sex sib-pairs was unrelated to sex-specific age-of-onset, likely to be of stochastic nature 18/27 True heterogenous sib-pair correlation for IgE confounder linkage analysis Follow up of these families did not reveal any plausible epidemiological explanation for male/female discordance

17. Other issues could explain failure to replicate 11p13? 19/27

18. 3. Hypothesis C: non-additive genetic effects? 20/27

19. Monozygotic twins IgE Eosinophils 21/27 r = 0.17 (0.07-0.27) r = 0.68 (0.61-0.74) r = 0.13 (0.00-0.25) r = 0.56 (0.43-0.67) Dizygotic twins Suggestive of dominance, epistasis? Twin 2 Twin 1

20. Significant Replication VC lod ELF5 EHF 100 Kb Chr11 22/27 2-df test Fulker et al. (1999) linkage & association model implemented in QTDT (Abecasis et al. 2000). Similar results with SOLAR (Almasy & Blangero 1998) Chr11 IgE

21. Significant Replication ELF5 EHF 100 Kb Chr11 VC lod Eosinophils 23/27 Type 1 error? Chr11 D11S2008 774F D11S907

22. 120 453786 101191213 14151617181920 21222324 2526 Frequency Chi-square 16.91 (LOD = 2.98), p = 0.008 Marker 774F Eosinophils 24/27 Meets criteria for replication (p < 0.01) Simulated genotypic data for the 11p13 region (9 markers), unlinked to the trait 5,000 replicates 2-df test Replicates and IBDs obtained from Merlin (Abecasis et al. 2002) Analysis performed with QTDT (Abecasis et al. 2000) Empirical type 1 Error

23. Have we replicated linkage of asthma to 11p13? 25/27

24. 1. Initially failed to replicate linkage to 11p13 on an additive model 26/27 2. Enough power to detect a QTL >25% of total variance 3. Failure to replicate not a consequence of unaccounted sex differences 6. Statistical validity of dominance tests to be investigated Summary 4. Failure to replicate due to unmodelled non-additive genetic effects ? 5. Replication when modelling dominance

25. Acknowledgements 1. All participating families 2. Project team 3. Everyone @ Genetic Epidemiology, QIMR Nick Martin David Duffy Melbourne Colin Robertson Tim Newson Marita Dalton Perth Peter LeSouef Nigel Dore Sunalene Devadason Sydney Jennifer Peat Brett Toelle Janet Li [Sequana] Lon Cardon Brisbane Dixie Statham Barbara Haddon Olivia Zheng Louise Mattick 27/27 4. Scholarship SFRH/4824/2001, FCT - Portugal