University of Groningen

A search for missing pieces of the puzzle; the development of and atopy Reijmerink, Naomi Elizabeth

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Association of IL1RL1, IL18R1 and IL18RAP cluster polymorphisms with asthma and atopy

Naomi E. Reijmerink Dirkje S. Postma Marcel Bruinenberg Ilja M. Nolte Deborah A. Meyers Eugene R. Bleecker Gerard H. Koppelman

J Allergy Clin Immunol. 2008 Sep;122(3):651-4. 3 Several genome wide screens, including our own, have shown strong linkage with asthma, lung function and atopy on 2q. 1 The -1 receptor like-1 (IL1RL1) gene, also known as ST2 is a promising candidate gene for asthma and atopy. IL1RL1 is located on 2q12 and resides in a cluster of IL1 receptor genes (Interleukin-1 receptor 2 (IL1R2),Interleukin-1 receptor 1 (IL1R1), Interleukin-1 receptor like 2 (IL1RL2), IL1RL1, Interleukin-18 receptor 1 (IL18R1), Interleukin-18 receptor accessory (IL18RAP)). 2 IL1RL1, a member of the Toll-interleukin 1 receptor (TIR) superfamily, is a receptor located on mast cells, T-helper (Th) 2 cells, regulatory T cells and macrophages and is also present in serum in a soluble form. IL1RL1 binds IL-33 and exerts its role through TLR pathways. Various forms of IL1RL1 can either stimulate or inhibit Th-2 responses. 3-6 Interestingly, single nucleotide polymorphisms (SNPs) located in the IL1RL1 gene are associated with . 7 The adjacently located family members IL18 Receptor 1 (IL18R1) and IL18 receptor accessory protein (IL18RAP) may also be important in the development of asthma and atopy. The gene products of both IL18R1 and IL18RAP form the alpha and beta chain of the IL-18 receptor (IL-18R). 8 IL18R is a key regulator of Th-1 cells. Binding of IL-18 to IL-18R stimulates Th-1 cytokine release, but also Th-2 type cytokines, depending on its cytokine milieu. 9 A recent publication showed strong associations with SNPs located in IL18R1 and asthma and atopic phenotypes.10 Our aim is to investigate whether SNPs in the IL1RL1 gene are associated with asthma, atopy and . Given the strong Linkage Disequilibrium (LD) in this region, we also investigate the adjacently located family members IL18R1 and IL18RAP. We analysed 200 well characterized Dutch asthma families (n=1259) with an asthma proband and used 407 independent asthma trios and 226 rhinitis trios to replicate our results. Furthermore, we performed a combined analysis of asthma families and trios to increase power. The medical ethics committee of the University Medical Center Groningen approved all studies and all participants signed written informed (parental) consent. Families were assessed as described previously. 11 The trios were characterised using the standardized protocol similar as used in the family study. Bronchial hyperresponsiveness (BHR) to histamine was measured as described 11 previously. BHR was defined as a PC20 ≤ 32mg/ml (30 seconds inhalation). Intracutaneous skin testing was performed with 16 (for the families) and 12 (for the trios) common aeroallergens. Asthma was defined by a previously published algorithm. 11 Twenty one SNPs located in IL1RL1, IL18R1 and IL18RAP were genotyped. SNPs were selected from the Celera database (http://www.celeradiscoverysystem.com, Maryland USA) and primers were ordered through assay by design service (Applied Biosystems®, California USA).” SNP selection was based on minor allele frequency > 10 % and location. Whenever possible the SNPs were located in coding regions with ≈5kb distance between each other. Eleven selected SNPs are present in the Hapmap database (release 22, April ‘07) of which seven were haplotype tagging SNPs capturing ≈75% of SNPs located in the IL1RL1, IL18R1 and IL18RAP gene cluster.

52 The IL1RL1, IL18R1 and IL18RAP gene cluster, astma and atopy

PCR was performed using an ABI prism 9700 HT real-time thermal cycler, applied biosystems, California USA. In-house software was used to test for LD (D’and r2) between the SNPs. Family Based Association Tests (FBAT version 2.0.2) analyses were employed for investigation of preferential transmission of alleles, (www.biostat.harvard.edu). 12 Sliding window analysis with 3 consecutive SNPs were analysed using Haplotype Based Association Tests (HBAT). 12 Results were considered significant if p< 0.05.

Results

Families and trios characteristics are shown in the online repository, Table E1. All SNPs were in Hardy-Weinberg equilibrium (p>0.01). Minor allele frequencies within the asthma families, asthma trios and rhinitis trios were similar (Table E2). Genotyping of two out of 21 SNPs failed because of designing problems. Strong LD between the SNPs was present in all three populations (e.g. asthma families; figure 1).

IL18R1

IL1RL1 IL18RAP

1023864 102527543 80

D'' r2 rs1468791 rs1861246 rs10206753 rs12999364 rs1420099 rs1558627 rs2270297 rs1041973 rs873022 rs1420101 rs12905 rs1054096 rs1921622 rs1035130 rs1420096 rs3732127 rs1420106 rs887971 rs2058659 0.0 - 0.1 rss1041973 rss873022 0.1 - 0.2 rss1420101 0.2 - 0.3 rrs12905 rss1054096 0.3 - 0.4 rss1921622 0.4 - 0.5

r ss1861246 0.5 - 0.6 rss10206753 rss12999364 0.6 - 0.7 rss1420099 0.7 - 0.8 rss1558627 rss2270297 0.8 - 0.9 r ss1035130 0.9 - 1.0 rss1420096 rss3732127 rss1420106 rss887971 rss2058659 rss1468791

Figure 1 SNPs genotyped in IL1RL1, IL18R1 and IL18RAP genes. LD structure (r2 below and D’ above the diagonal) in the asthma families.

53 3 Asthma families: eight of 19 SNPs located in the IL1RL1, IL18R1 and IL18RAP gene were strongly associated with BHR, severity of BHR and total serum IgE (Table I and Tables E3, E4 and E6). SNPs located in IL18R1 and IL18RAP were also associated with serum eosinophils (Table E7). Haplotype analysis using sliding windows of three consecutive SNP in all three genes showed even stronger associations (asthma (p=0.003-0.05), BHR (p=0.005-0.05), severity of BHR (p=0.0003-0.05), IgE (p=0.03-0.05) and serum eosinophils (p=0.03-0.05)). Asthma trios: Significant associations were observed with total serum IgE, serum eosinophils and skin test positivity and 2 SNPs located in the IL1RL1 and IL18R1 gene (Table I and Table E6 to E8). Haplotype analysis showed the associations to be present with SNPs located in the IL1RL1 and IL18R1 gene with total IgE (p=0.03-0.05), serum eosinophils (p=0.04-0.05) and skin test positivity (p=0.02-0.05). Three haplotype combinations, [rs1921622, rs1861246 and rs10206753]; [rs10206753, rs12999364 and rs1420099] and [rs1420099, rs1558627 and rs2270297] strictly replicated results in the asthma families with similar (borderline) associations with BHR, asthma and total IgE (Table I) and serum eosinophils (p=0.03-0.06). Asthma families and asthma trios combined: Combination of asthma families and trios resulted in similar or more significant results (Table I and Table E3 to E8). Interestingly significant associations between asthma and SNPs located in IL1RL1 and IL18R1 were found that were not present when analysing the both cohorts separately (Tables I and E5). Also haplotype analysis showed a more significant association with asthma (p=0.0008-0.05), BHR (p=0.0009-0.05), BHR severity (p=0.0006-0.05), total IgE (p=0.007-0.05) and serum eosinophils (p=0.005-0.05) in all three genes, compared to the analysis of both cohorts separately. Rhinitis trios: No significant associations were found with SNPs located in the IL1RL1 and IL18R1 gene and atopic phenotypes in the rhinitis trios (data not shown).

This is the first study that provides suggestive evidence for associations of SNPs in the IL1RL1 gene and adjacently located family members IL18R1 and IL18RAP with asthma and atopy in two independent Dutch asthma populations, but not with rhinitis. This study thus shows the importance of analysing SNPs in adjacently located genes since it is not decisive whether the associations are caused by the IL1RL1, IL18R1 and/or IL18RAP gene due to strong LD in the region.

54 The IL1RL1, IL18R1 and IL18RAP gene cluster, astma and atopy

Table I. Family-Based Association Test of IL1RL1, IL18R1 and IL18RAP with BHR, asthma and total IgE in the separate and combined analysis of Dutch asthma families and asthma trios. Significant SNP results and consistent haplotype results are shown. Asthma Asthma Asthma families and trios families trios combined Z Z Informative Z Gene Rs number Allele* (p-values) (p-values) families (p-values) BHR IL1RL1 rs1420101 2 1.311 (0.190) 1.474 (0.141) 211 1.927 (0.054) rs1921622 1 2.067 (0.039) 1.432 (0.152) 212 2.463 (0.014) rs1861246 1 1.969 (0.049) 1.292 (0.196) 175 2.314 (0.021) IL1RL1 / rs12999364 2 1.660 (0.097) 1.777 (0.076) 198 2.402 (0.016) IL18R1 IL18R1 rs1558627 1 2.291 (0.022) 0.526 (0.599) 180 1.973 (0.049) rs2270297 1 2.208 (0.027) 0.590 (0.555) 185 1.975 (0.048) rs1035130 2 1.496 (0.142) 1.273 (0.203) 174 1.981 (0.048) IL18RAP rs1420106 1 2.418 (0.016) 0.813 (0.416) 184 2.277 (0.023) Intergenic rs1468791 1 2.376 (0.018) 0.221 (0.825) 188 1.816 (0.069) Haplotype rs1921622, IL1RL1 rs1861246, 111 2.803 (0.005) 1.844 (0.065) 192 3.311 (0.0009) rs10206753 rs10206753 IL1RL1 rs12999364 121 2.125 (0.033) 1.800 (0.072) 179 2.770 (0.006) /IL18R1 rs1420099

Asthma§ Allele IL1RL1 rs1921622 1 1.508 (0.132) 1.451 (0.147) 193 2.074 (0.038) rs1861246 1 1.201 (0.230) 1.552 (0.121) 163 1.952 (0.050) IL1RL1 / rs12999364 2 1.702 (0.089) 1.622 (0.105) 185 2.312 (0.021) IL18R1 IL18R1 rs1035130 2 1.547 (0.122) 1.205 (0.228) 159 1.996 (0.046) Haplotype rs1921622, 3.354 IL1RL1 rs1861246, 111 3.020 (0.003) 1.766 (0.077) 170 (0.0008) rs10206753 rs1420099, IL18R1 rs1558627, 111 2.749 (0.006) 1.694 (0.090) 180 3.086 (0.002) rs2270297

Total IgE Allele IL1RL1 rs1054096 2 -0.971 (0.331) 1.998 (0.046) 127 0.246 (0.806) rs1921622 1 1.729 (0.084) 1.335 (0.182) 276 2.207 (0.027) rs1861246 1 2.069 (0.039) 1.070 (0.285) 230 2.320 (0.020) IL18R1 rs1558627 1 2.554 (0.011) 0.229 (0.819) 238 2.203 (0.028) rs2270297 1 2.142 (0.032) 0.364 (0.716) 239 1.930 (0.054) IL18RAP rs1420106 1 2.659 (0.008) 0.554 (0.579) 234 2.516 (0.012) Intergenic rs1468791 1 2.118 (0.034) 0.257 (0.797) 243 1.833 (0.067) Haplotype rs1921622, IL1RL1 rs1861246, 111 1.988 (0.046) 1.888 (0.059) 245 2.719 (0.007) rs10206753 * Allele 1=major allele, 2=minor allele. § Asthma based on algorithm. Z-score and (uncorrected) p-values are shown for the asthma families and asthma trios. Numbers of informative families, Z-score and p-values are shown for the combined analysis.

55 3 Reference list

1. Koppelman GH, Stine OC, Xu J, Howard TD, Zheng SL, Kauffman HF et al. Genome-wide search for atopy susceptibility genes in Dutch families with asthma. J Allergy Clin Immunol 2002; 109(3):498- 506. 2. Dale M, Nicklin MJ. Interleukin-1 receptor cluster: gene organization of IL1R2, IL1R1, IL1RL2 (IL- 1Rrp2), IL1RL1 (T1/ST2), and IL18R1 (IL-1Rrp) on human chromosome 2q. Genomics 1999; 57(1):177-9. 3. Brint EK, Xu D, Liu H, Dunne A, McKenzie AN, O’Neill LA et al. ST2 is an inhibitor of interleukin 1 receptor and Toll-like receptor 4 signaling and maintains endotoxin tolerance. Nat Immunol 2004; 5(4):373-9. 4. Mangan NE, Dasvarma A, McKenzie AN, Fallon PG. T1/ST2 expression on Th2 cells negatively regulates allergic pulmonary inflammation. Eur J Immunol 2007; 37(5):1302-12. 5. Schmitz J, Owyang A, Oldham E, Song Y, Murphy E, McClanahan TK et al. IL-33, an interleukin- 1-like cytokine that signals via the IL-1 receptor-related protein ST2 and induces T helper type 2-associated cytokines. Immunity 2005; 23(5):479-90. 6. Hayakawa H, Hayakawa M, Kume A, Tominaga S. Soluble ST2 blocks interleukin-33 signaling in allergic airway inflammation. J Biol Chem 2007; 282(36):26369-80. 7. Shimizu M, Matsuda A, Yanagisawa K, Hirota T, Akahoshi M, Inomata N et al. Functional SNPs in the distal promoter of the ST2 gene are associated with atopic dermatitis. Hum Mol Genet 2005; 14(19):2919-27. 8. Torigoe K, Ushio S, Okura T, Kobayashi S, Taniai M, Kunikata T et al. Purification and characterization of the human interleukin-18 receptor. J Biol Chem 1997; 272(41):25737-42. 9. Nakanishi K, Yoshimoto T, Tsutsui H, Okamura H. Interleukin-18 is a unique cytokine that stimulates both Th1 and Th2 responses depending on its cytokine milieu. Cytokine Growth Factor Rev 2001; 12(1):53-72. 10. Zhu G, Whyte MK, Vestbo J, Carlsen K, Carlsen KH, Lenney W et al. receptor 1 gene polymorphisms are associated with asthma. Eur J Hum Genet 2008 ;16(9):1083-90. 11. Panhuysen CI, Bleecker ER, Koeter GH, Meyers DA, Postma DS. Characterization of obstructive airway disease in family members of probands with asthma. An algorithm for the diagnosis of asthma. Am J Respir Crit Care Med 1998; 157(6 Pt 1):1734-42. 12. Laird NM, Horvath S, Xu X. Implementing a unified approach to family-based tests of association. Genet Epidemiol 2000; 19 Suppl 1:S36-S42.

56 Chapter 3

Online repository

Association of IL1RL1, IL18R1 and IL18RAP gene cluster polymorphisms with asthma and atopy

Naomi E. Reijmerink Dirkje S. Postma Marcel Bruinenberg Ilja M. Nolte Deborah A. Meyers Eugene R. Bleecker Gerard H. Koppelman 3 39.6 59.8 32.8 194 (51) 20.5 (0-177) 12.7 (4.6-41.8) 63.8 (0.5-3710) Grandchildren 31.4 45.3 53.3 556 (54) Children 24.5(6.8-53.4) 15.8 (0-294.8) 63.5 (0.5-3360) 33 10.6 25.6 31.0 15.3 95.5 Spouses 201* (62) 226 (56.2) 9.4 (0-63.8) 16.3 (1.0-78) 32.5 (18-46.7) 115.7 (5-6915) 26.2 (0.5-1940) 51.5 (33.1-76.5) Rhinitis Probands 91 91 100 86.9 82.9 82.8 108.5 200 (38) 407 (63.1) Probands (0.5-12400) 16.5 (1.0-94) 15.8 (0-126.5) 92.9 (1.0-2880) 52.5 (37.8-75.3) 34.3 (18.1-64.7) Asthma Probands

(%)) Number (females median (range) Age in years, % Asthma, % hyperresponsiveness, Bronchial IgE in IU/ml, Total geometric mean (range) % skin tests, Positive mean (range) Blood eosinophils 10E7, Table E1. Table with asthma probands Clinical characteristics of Dutch families from (%)) Number (females median (range) Age in years, % Asthma, % hyperresponsiveness, Bronchial IgE in IU/ml, Total geometric mean (range) % skin tests, Positive mean (range) Blood eosinophils 10E7, of asthma and rhinitis trios Clinical characteristics of probands * one proband married twice, both spouses participated in the study twice, married * one proband

58 The IL1RL1, IL18R1 and IL18RAP gene cluster, astma and atopy

Table E2. SNPs selected for IL1RL1, IL18R1 and IL18RAP in Dutch asthma family study, asthma trios and rhinitis trios Gene Rs number Function Allele* Asthma Asthma Rhinitis families trios trios

IL1RL1 rs1041973 missense C/A 0.15 0.18 0.19 IL1RL1 rs873022 intron G/T 0.33 0.29 0.29 IL1RL1 rs1420101 intron/silent G/A 0.38 0.35 0.36 IL1RL1 rs12905 3’UTR/intron G/A 0.32 0.29 0.29 IL1RL1 rs1054096 intron T/C 0.08 0.09 0.11 IL1RL1 rs1921622 intron A/G 0.46 0.46 0.46 IL1RL1 rs1861246 intron C/T 0.22 0.24 0.23 IL1RL1 rs10206753 missense T/C 0.38 0.39 0.38 IL1RL1 /IL18R1 rs12999364 3’UTR/promoter C/T 0.36 0.38 0.39 IL18R1 rs1420099 intron C/G 0.38 0.38 0.38 IL18R1 rs1558627 intron A/G 0.22 0.25 0.23 IL18R1 rs2270297 intron C/T 0.23 0.25 NG IL18R1 rs1035130 missense A/G 0.33 0.29 0.29 IL18R1 rs1420096 intron C/T 0.47 0.46 NG IL18R1 rs3732127 3’UTR G/C 0.13 0.13 NG IL18RAP rs1420106 promoter G/A 0.22 0.25 NG IL18RAP rs887971 intron T/C 0.32 0.33 NG IL18RAP rs2058659 intron G/A 0.48 0.45 NG intergenic rs1468791 intergenic G/A 0.22 0.25 NG

NG: not genotyped * Major allele first

59 3 Table E3. Family-Based Association Test of IL1RL1, IL18R1 and IL18RAP with BHR in the separate and combined analysis of Dutch asthma families and asthma trios. BHR Asthma families and trios Families Trios combined Z Z Informative Z Gene Rs number Allele (p-values) (p-values) families (p-values) IL1RL1 rs1041973 1 0.028 (0.978) 0.266 (0.790) 154 0.212 (0.831) rs873022 2 1.204 (0.229) 0.587 (0.557) 197 1.297 (0.194) rs1420101 2 1.311 (0.190) 1.474 (0.141) 211 1.927 (0.054) rs12905 2 1.513 (0.130) 0.899 (0.368) 190 1.741 (0.082) rs1054096 2 0.186 (0.852) 1.395 (0.163) 91 1.016 (0.310) rs1921622 1 2.067 (0.039) 1.432 (0.152) 212 2.463 (0.014) rs1861246 1 1.969 (0.049) 1.292 (0.196) 175 2.314 (0.021) rs10206753 1 0.337 (0.736) 1.237 (0.216) 214 1.044 (0.297) IL1RL1 / rs12999364 2 1.660 (0.097) 1.777 (0.076) 198 2.402 (0.016) IL18R1 IL18R1 rs1420099 1 0.335 (0.738) 1.504 (0.133) 214 1.210 (0.226) rs1558627 1 2.291 (0.022) 0.526 (0.599) 180 1.973 (0.049) rs2270297 1 2.208 (0.027) 0.590 (0.555) 185 1.975 (0.048) rs1035130 2 1.496 (0.142) 1.273 (0.203) 174 1.981 (0.048) rs1420096 1 -0.302 (0.763) 0.469 (0.639) 227 0.072 (0.942) rs3732127 1 -0.043 (0.966) 0.749 (0.454) 126 0.596 (0.551) IL18RAP rs1420106 1 2.418 (0.016) 0.813 (0.416) 184 2.277 (0.023) rs887971 2 0.795 (0.427) 0.840 (0.401) 205 1.157 (0.247) rs2058659 1 -0.217 (0.828) 0.668 (0.504) 233 0.281 (0.777) Intergenic rs1468791 1 2.376 (0.018) 0.221 (0.825) 188 1.816 (0.069)

60 The IL1RL1, IL18R1 and IL18RAP gene cluster, astma and atopy

Table E4. Family-Based Association Test of IL1RL1, IL18R1 and IL18RAP with BHR severity in the separate and combined analysis of Dutch asthma families and asthma trios. BHR severity Asthma families and trios Families Trios combined Z Z Informative Z Gene Rs number Allele (p-values) (p-values) families (p-values) IL1RL1 rs1041973 2 0.703 (0.482) -0.184 (0.854) 160 0.446 (0.656) rs873022 2 1.637 (0.101) 0.106 (0.916) 198 1.413 (0.158) rs1420101 2 2.145 (0.032) -0.028 (0.978) 213 1.79 (0.073) rs12905 2 1.742 (0.082) 0.228 (0.819) 192 1.578 (0.114) rs1054096 2 1.693 (0.090) 0.032 (0.974) 101 1.512 (0.131) rs1921622 1 2.637 (0.008) -0.246 (0.806) 214 1.979 (0.048) rs1861246 1 2.235 (0.025) 1.113 (0.266) 176 2.420 (0.016) rs10206753 1 1.045 (0.296) 0.241 (0.810) 208 1.032 (0.301) IL1RL1 / rs12999364 2 3.150 (0.002) 0.142 (0.887) 203 2.640 (0.008) IL18R1 IL18R1 rs1420099 1 1.004 (0.315) -0.106 (0.916) 210 0.827 (0.409) rs1558627 1 2.631 (0.009) -0.199 (0.841) 180 1.881 (0.060) rs2270297 1 2.442 (0.015) 0.083 (0.934) 183 1.925 (0.054) rs1035130 2 1.836 (0.066) 0.376 (0.707) 176 1.747 (0.081) rs1420096 2 0.272 (0.785) -0.149 (0.881) 221 0.152 (0.880) rs3732127 2 0.926 (0.355) 0.227 (0.821) 127 0.851 (0.395) IL18RAP rs1420106 1 2.633 (0.008) -0.006 (0.995) 181 2.000 (0.045) rs887971 2 1.494 (0.135) -0.334 (0.739) 204 1.010 (0.312) rs2058659 2 0.196 (0.844) -0.028 (0.978) 226 0.151 (0.880) Intergenic rs1468791 1 2.673 (0.008) -0.224 (0.823) 184 1.887 (0.059)

61 3 Z (p-values) 0.083 (0.934) 1.201 (0.230) 2.063 (0.039) 1.540 (0.124) 1.212 (0.225) 2.074 (0.038) 1.952 (0.050) 1.575 (0.115) 2.312 (0.021) 1.823 (0.068) 1.527 (0.127) 1.536 (0.125) 1.996 (0.046) 0.266 (0.790) 1.019 (0.308) 1.796 (0.073) 1.556 (0.120) 0.762 (0.446) 1.169 (0.243) 79 139 178 196 171 193 163 190 185 190 164 171 159 201 122 170 185 203 173 families Informative Informative Asthma families and trios combined Z Trios (p-values) 1.451(0.147) 0.092 (0.926) 0.300 (0.764) 1.155 (0.248) 0.688 (0.491) 1.612 (0.107) 1.552 (0.121) 0.827 (0.408) 1.622 (0.105) 1.109 (0.268) 0.846 (0.397) 0.905 (0.366) 1.205 (0.228) 0.000 (1.000) 0.385 (0.700) 1.134 (0.257) 0.641 (0.521) 0.206 (0.837) 0.526 (0.599) § Z Asthma Families (p-values) 0.020 (0.984) 1.382 (0.167) 1.816 (0.069) 1.460 (0.144) 0.000 (1.000) 1.508 (0.132) 1.201 (0.230) 1.397 (0.162) 1.702 (0.089) 1.479 (0.139) 1.387 (0.166) 1.309 (0.191) 1.547 (0.122) 0.381 (0.703) 1.175 (0.240) 1.427 (0.154) 1.592 (0.111) 0.892 (0.372) 1.180 (0.238) with asthma in the separate and combined analysis of Dutch asthma families and trios. with asthma in the separate and combined analysis IL18RAP and 2 2 2 2 2 1 1 1 2 1 1 1 2 1 1 1 2 1 1 Allele IL1RL1, IL18R1 IL1RL1,

(15) rs12905 rs873022 rs887971 rs1041973 rs1420101 rs1054096 rs1921622 rs1861246 rs1420099 rs1558627 rs2270297 rs1035130 rs1420096 rs3732127 rs1420106 rs2058659 rs1468791 rs10206753 rs12999364 Rs number Family-Based Association Test of Test Association Family-Based Gene IL1RL1 IL1RL1 /IL18R1 IL18R1 IL18RAP Intergenic Asthma based on algorithm Table E5. Table §

62 The IL1RL1, IL18R1 and IL18RAP gene cluster, astma and atopy Z (p-values) 0.364 (0.716) 0.952 (0.341) 1.469 (0.142) 1.406 (0.160) 0.246 (0.806) 2.207 (0.027) 2.320 (0.020) 0.638 (0.524) 1.212 (0.226) 0.824 (0.410) 2.203 (0.028) 1.930 (0.054) 1.109 (0.267) 0.598 (0.550) 0.872 (0.383) 2.516 (0.012) 0.324 (0.746) 0.057 (0.954) 1.833 (0.067) 197 265 276 255 127 276 230 274 266 274 238 239 240 289 158 234 274 296 243 Informative families Informative Asthma families and trios combined Z (p-values) 0.513 (0.879) 0.396 (0.692) 1.385 (0.166) 0.891 (0.373) 1.998 (0.046) 1.335 (0.182) 1.070 (0.285) 1.351 (0.179) 1.800 (0.072) 1.743 (0.081) 0.229 (0.819) 0.364 (0.716) 1.131 (0.258) 0.760 (0.447) 0.554 (0.579) 0.583 (0.560) 0.257 (0.797) -0.441 (0.659) -0.675 (0.499) Z Total IgE Total Trios (p-values) 0.343 (0.732) 0.872 (0.383) 0.840 (0.401) 1.091 (0.275) 1.729 (0.084) 2.069 (0.039) 0.100 (0.920) 2.554 (0.011) 2.142 (0.032) 0.582 (0.561) 1.062 (0.288) 0.432 (0.666) 2.659 (0.008) 0.568 (0.570) 2.118 (0.034) -0.971 (0.331) -0.124 (0.901) -0.181 (0.856) -0.018 (0.986) with total IgE in the separate and combined analysis of Dutch asthma families and with total IgE in the separate and combined analysis IL18RAP and 2 2 2 2 2 1 1 1 2 1 1 1 2 2 1 1 2 2 1 Allele Families IL1RL1, IL18R1 IL1RL1, rs12905 rs873022 rs887971 rs1041973 rs1420101 rs1054096 rs1921622 rs1861246 rs1420099 rs1558627 rs2270297 rs1035130 rs1420096 rs3732127 rs1420106 rs2058659 rs1468791 rs10206753 rs12999364 Rs number Family-Based Association Test of Test Association Family-Based Gene IL1RL1 IL1RL1 /IL18R1 IL18R1 IL18RAP Intergenic

Table E6. Table trios.

63 3 Z (p-values) 0.183 (0.855) 0.853 (0.394) 1.369 (0.171) 1.203 (0.229) 0.566 (0.572) 2.040 (0.041) 2.374 (0.018) 0.657 (0.511) 1.281 (0.200) 0.844 (0.399) 2.323 (0.020) 2.127 (0.033) 0.886 (0.376) 0.754 (0.451) 0.717 (0.473) 2.968 (0.003) 0.397 (0.691) 0.513 (0.608) 2.093 (0.036) 190 254 267 249 125 268 227 266 257 265 235 234 233 279 152 230 265 286 239 families Informative Informative Asthma families and trios combined Z Trios (p-values) 0.298 (0.766) 0.461 (0.645) 1.379 (0.168) 0.834 (0.404) 1.791 (0.073) 1.438 (0.150) 1.538 (0.124) 1.226 (0.220) 2.012 (0.044) 1.705 (0.088) 0.640 (0.522) 0.773 (0.440) 0.979 (0.328) 0.318 (0.750) 1.081 (0.280) 0.891 (0.373) 0.684 (0.494) -0.155 (0.877) -0.343 (0.731) Z Families (p-values) 0.010 (0.992) 0.720 (0.471) 0.784 (0.433) 0.904 (0.366) 1.512 (0.130) 1.861 (0.063) 0.012 (0.990) 0.147 (0.883) 2.411 (0.016) 2.086 (0.037) 0.444 (0.657) 1.007 (0.314) 0.661 (0.509) 2.879 (0.004) 0.841 (0.400) 2.123 (0.034) -0.330 (0.742) -0.074 (0.941) -0.105 (0.916) with serum eosinophils in the separate and combined analysis of Dutch asthma families and families asthma Dutch of analysis combined and separate the in eosinophils serum with Serum eosinophils IL18RAP and 2 2 2 2 2 1 1 1 2 1 1 1 2 2 1 1 2 2 1 Allele IL1RL1, IL18R1 IL1RL1, rs12905 rs873022 rs887971 rs1041973 rs1420101 rs1054096 rs1921622 rs1861246 rs1420099 rs1558627 rs2270297 rs1035130 rs1420096 rs3732127 rs1420106 rs2058659 rs1468791 rs10206753 rs12999364 Rs number Family-Based Association Test of Test Association Family-Based

Gene IL1RL1 IL1RL1 /IL18R1 IL18R1 IL18RAP Intergenic Table E7. Table asthma trios.

64 The IL1RL1, IL18R1 and IL18RAP gene cluster, astma and atopy Z (p-values) 0.249 (0.080) 0.096 (0.932) 0.584 (0.560) 0.244 (0.807) 0.796 (0.426) 0.968 (0.333) 1.273 (0.203) 0.751 (0.452) 0.321 (0.748) 0.798 (0.424) 1.030 (0.303) 0.676 (0.499) 0.102 (0.919) 0.578 (0.563) 0.699 (0.485) 1.626 (0.104) 0.211 (0.833) 0.077 (0.939) 0.724 (0.469) 88 137 192 202 184 205 169 203 197 205 180 182 172 217 120 174 194 218 183 families Informative Informative Asthma families and trios combined Z Trios (p-values) 2.151(0.031) 1.199 (0.230) 0.318 (0.750) 1.474 (0.141) 0.811 (0.417) 1.631 (0.103) 1.818 (0.069) 1.613 (0.107) 0.160 (0.873) 0.157 (0.875) 0.828 (0.408) 1.294 (0.196) 0.552 (0.581) -0.191 (0.849) -0.234 (0.815) -0.489 (0.625) -0.455 (0.649) -0.626 (0.532) -0.078 (0.938) Z

Families (p-values) 0.573 (0.567) 0.307 (0.759) 0.063 (0.950) 0.000 (1.000) 1.020 (0.308) 1.256 (0.209) 0.787 (0.431) 1.211 (0.226) 1.693 (0.090) 0.660 (0.509) 0.668 (0.504) 1.065 (0.287) -0.176 (0.860) -0.863 (0.388) -0.338 (0.735) -1.121 (0.262) -0.283 (0.777) -0.512 (0.609) -0.388 (0.698) with positive skintest in the separate and combined analysis of Dutch asthma families and Positive Skintest Positive IL18RAP and 2 1 2 2 2 1 1 1 2 1 1 1 2 2 1 1 1 2 1 Allele* IL1RL1, IL18R1 IL1RL1,

rs12905 rs873022 rs887971 rs1041973 rs1420101 rs1054096 rs1921622 rs1861246 rs1420099 rs1558627 rs2270297 rs1035130 rs1420096 rs3732127 rs1420106 rs2058659 rs1468791 rs10206753 rs12999364 Rs number Family-Based Association Test of Test Association Family-Based

Gene IL1RL1 IL1RL1 /IL18R1 IL18R1 IL18RAP Intergenic Table E8. Table asthma trios. 2=minor allele. Allele 1=major allele, * the asthma families and trios. for shown p-values are and (uncorrected) Z-score the combined analysis. for shown and p-values are Z-score families, Numbers of informative

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