University of Groningen A search for missing pieces of the puzzle; the development of asthma and atopy Reijmerink, Naomi Elizabeth IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2009 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Reijmerink, N. E. (2009). A search for missing pieces of the puzzle; the development of asthma and atopy: innate immunity genes and environment. [s.n.]. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 26-09-2021 Chapter 3 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 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 chromosome 2q. 1 The Interleukin-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 protein (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 atopic dermatitis. 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 allergic rhinitis. 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