Genes and Immunity (2006) 7, 169–178 & 2006 Nature Publishing Group All rights reserved 1466-4879/06 $30.00 www.nature.com/gene ORIGINAL ARTICLE Variation in the type I interferon gene cluster on 9p21 influences susceptibility to asthma and atopy A Chan1,2, DL Newman1,3, AM Shon, DH Schneider, S Kuldanek and C Ober Department of Human Genetics, The University of Chicago, Chicago, IL, USA A genome-wide screen for asthma and atopy susceptibility alleles conducted in the Hutterites, a founder population of European descent, reported evidence of linkage with a short tandem repeat polymorphism (STRP) within the type I interferon (IFN) gene cluster on chromosome 9p21. The goal of this study was to identify variation within the IFN gene cluster that influences susceptibility to asthma and atopic phenotypes. We screened approximately 25 kb of sequence, including the flanking sequence of all 15 functional genes and the single coding exon in 12, in Hutterites representing different IFNA-STRP genotypes. We identified 78 polymorphisms, and genotyped 40 of these (in 14 genes) in a large Hutterite pedigree. Modest associations (0.003oPo0.05) with asthma, bronchial hyper-responsiveness (BHR), and atopy were observed with individual variants or genes, spanning the entire 400 kb region. However, pairwise combinations of haplotypes between genes showed highly significant associations with different phenotypes (Po10À5) that were localized to specific pairs of genes or regions of this cluster. These results suggest that variation in multiple genes in the type I IFN cluster on 9p22 contribute to asthma and atopy susceptibility, and that not all genes contribute equally to all phenotypes. Genes and Immunity (2006) 7, 169–178. doi:10.1038/sj.gene.6364287; published online 26 January 2006 Keywords: asthma; atopy; type I interferons Introduction (P ¼ 4.9 Â 10À3). Both tests indicated that the 150 and 156 base pair (bp) alleles were over-represented and the Asthma is a complex disease that affects nearly 300 154 and 158 bp alleles under-represented in affected million individuals worldwide.1 The rising incidence of individuals. The IFNA-STRP lies within the type I IFN asthma and atopic disorders over the past decades attests gene cluster, which spans approximately 400 kb on to the importance of environmental and lifestyle factors chromosome 9p22. The region consists of 15 functional in disease risk,2–4 although family and twin studies IFN genes, including 13 IFN-a genes, one IFN-o gene, support a strong genetic component to both.5,6 Allergic and one IFN-b gene, in addition to 11 IFN pseudogenes.14 sensitization to one or more allergens (atopy) is one of All IFN genes consist of a single exon and are highly the most important risk factors for asthma, as are family homologous to each other. On the whole, the coding history and early life environmental exposures.7–11 regions of the 13 IFN-a genes display greater than 80% To identify genes that might be involved in asthma nucleotide identity to each other, while the IFN-o gene and atopy pathogenesis, we conducted a genome-wide displays approximately 60% identity to any of the IFN-a screen in a founder population, the Hutterites.12 In this genes, and the IFN-b gene is about 40% identical to the earlier study, a short tandem repeat polymorphism rest of the cluster.15 (IFNA-STRP) on the short arm of chromosome 9 showed The type I interferons are pleiotropic cytokines with evidence of linkage to asthma using the transmission important roles in inflammation, immunoregulation, and disequilibrium test (TDT) in a large Hutterite pedigree T cell responses,16 making them outstanding candidates (P ¼ 2.5 Â 10À5). More recently, using a case–control test,13 for asthma and atopy susceptibility genes. The potent this STRP was also associated with asthma antiviral properties of type I interferons have long been recognized,17,18 and viral infections, such as respiratory syncytial virus (RSV) and rhinovirus (RV), may be early Correspondence: Professor C Ober, Department of Human Genetics, 19–22 The University of Chicago, 920 E. 58th Street, Room CLSC 507C, life triggers of asthma. More recently, however, Chicago, IL 60636, USA. additional roles for interferons have been described, E-mail: [email protected] including skewing the adaptive immune system toward 1These authors contributed equally. a Th1 profile23 and the induction of apoptosis.24 2Current address: Department of Pediatrics, Weill Medical College, Furthermore, reduced levels of IFN-a in patients with Cornell University, New York, NY, USA. 3 the Th2-associated diseases asthma and atopy have been Current address: Department of Biological Sciences, Rochester 25,26 Institute of Technology, Rochester, NY, USA. reported, and treatment with IFN-a therapy improves 27–30 Received 30 August 2005; revised 29 November 2005; accepted 20 clinical symptoms in some patients. Thus, we December 2005; published online 26 January 2006 hypothesized that variation in one or more of these Type I interferons and asthma and atopy A Chan et al 170 genes could modulate expression levels or function Evaluation of phenotypes of the type I interferons and, as a result, influence Details of our protocols have been described pre- the development or course of asthma and atopic viously;12,34,35 the clinical characteristics of the study diseases. population are shown in Table 1. Briefly, 660 subjects The goal of this study was to identify polymorphisms were phenotyped. Individuals were considered to have in the 15 IFN genes and their flanking sequences, asthma if they (1) were bronchial hyper-responsive genotype selected single-nucleotide polymorphisms (X20% decrease in baseline forced expiratory volume (SNPs) in a sample of 4700 Hutterites who are well in one second [FEV1] after inhalation of p25 mg characterized with respect to asthma and atopy pheno- methacholine/ml); (2) reported the presence of two or types, and test the hypothesis that variation in the type I three symptoms (wheeze, cough, shortness of breath); IFN gene cluster contributes to asthma and atopy and (3) had a doctor’s diagnosis of asthma. Eighty-three susceptibility. individuals met this definition of asthma, which was in general mild, as both oral steroid use and hospitaliza- tions were rare. Because during field trips to Hutterite Materials and methods colonies, we identified cases of undiagnosed asthma and because many Hutterites did not reliably recall past Subjects asthma symptoms (some did not understand the word The Hutterites are a founder population of European ‘wheeze’ because it is not in the German language), we descent. The 760 Hutterites in our study population also used a more relaxed diagnosis of asthma, referred to belong to a single 13-generation, 1623 member pedigree here as ‘possible asthma’ (N ¼ 236), that included and live on nine communal farms (colonies) in South additional individuals with bronchial hyper-responsive- Dakota.31 The Hutterites in our studies can be traced ness (BHR) (N ¼ 80) or at least two symptoms (N ¼ 73) or back to 62 Hutterite ancestors who lived in the early both (N ¼ 83). Four hundred twenty-four individuals 1700’s to early 1800’s.32 The relatively small number of who had neither symptoms nor BHR were considered as founders minimizes genetic heterogeneity in the popula- controls. tion, and their communal, farming lifestyle ensures that Atopy (allergic sensitization) was evaluated by mea- all members are exposed to a relatively uniform suring specific IgE response by skin prick test (SPT) to 14 environment. In particular, all meals are prepared and common allergens: house dust mites (HDM) (Dermato- eaten in a common kitchen and dining room, smoking is phagoides pteronyssinus, Dermatophagoides farinae), cock- prohibited, and exposures to allergens are relatively roaches (Blattela germanica, Periplaneta Americana), molds uniform among the colonies. Overall, the similar (Alternaria alternate, Cladosporium herbarum, Aspergillus environmental exposure should enhance the role of fumigatus), pollens (Lolium perenne, Ambrosia artemisifolia, genetic variation on disease risk in this population.33 Artemisia vulgaris, Quercus alba, Betula verrucosa), and We visited nine Hutterite colonies that were chosen animal danders (Felis domesticus, Canis familiaris). Be- based both on their location and to represent the cause few Hutterites were sensitized to animal danders different lines of Hutterite colony descent.32 Nearly all (Table 1), we did not analyze that as a separate category. Hutterites over the age of 5 who were present in the All tests included a negative (saline solution) and a colonies on the days of our visits participated in the positive (histamine) control. A positive ( þ ) SPT was study. The mean age of participants was 30.1 years (s.d. defined as equivalent to a 3 mm or greater wheal 18.2 years; range 6–89), and mean inbreeding coefficient diameter resulting from the corrected reaction area of the individuals in this sample is 0.034 (s.d. 0.015), (subtraction of saline control). Analyses were performed slightly greater than that of first cousins once removed.12 for five atopy categories: þ SPT to one or more allergen These studies were approved by the Institutional Review group (n ¼ 311), þ SPT to HDM (n ¼ 158), þ SPT to Boards at the University of Chicago and the University of cockroach (n ¼ 150), þ SPT to mold (n ¼ 75), and þ SPT South Dakota. to tree/pollen (n ¼ 158). Table 1 Clinical characteristics of the Hutterites by asthma status Unaffected Symptoms BHR Asthma Sample size 424 73 80 83 Mean age (years) (s.d.) 30.4 (15.7) 32.1 (14.7) 25.6 (19.4) 23.4 (16.6) Sex ratio (M/F) 0.77 0.92 0.90 1.18 Mean log IgE (IU) (s.d.) 1.3 (0.7) 1.5 (0.8) 1.5 (0.8) 1.8 (0.8) % +SPTX1 allergen 42.1 52.1 50.6 48.1 % +SPTX2 allergens 31.2 39.7 39.1 40.3 % +SPT HDM 21.1 21.9 32.9 27.3 % +SPT cockroach 20.3 23.3 29.1 18.2 % +SPT molds 9.9 11.0 11.4 15.6 % +SPT pollens/trees 19.9 28.8 19.0 33.8 % +SPT animal danders 6.5 8.2 11.4 11.7 HDM (D.