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Functional Polymorphism in the Suppressor of Cytokine Signaling 1 Gene Associated with Adult Asthma

Laboratory for Genetics of Allergic Diseases, SNP Research Center, The Institute of Physical and Chemical Research (RIKEN), Kanagawa; Department of Health Promotion and Human Behavior, Kyoto University Graduate School of Public Health, Kyoto; Department of Microbiology and Immunology, Kagoshima University Dental School, Kagoshima; Department of Pediatric Allergy, Osaka Prefectural Medical Center for Respiratory and Allergic Diseases, Habikino, Osaka; School of Human Nursing, The University of Shiga Prefecture, Shiga; Department of Otolaryngology, Japanese Red Cross Society, Wakayama Medical Center, Wakayama; Department of Public Health, Graduate School of Medicine, Chiba University, Chiba; Department of Otorhinolaryngology, Jikei University School of Medicine; Department of Allergy and Immunology, National Research Institute for Child Health & Development; and Laboratory of Molecular Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

Correspondence and requests for reprints should be addressed to Mayumi Tamari, M.D., Ph.D., Laboratory for Genetics of Allergic Diseases, SNP Research Center, Institute of Physical and Chemical Research (RIKEN), 1-7-22 Suehiro, Tsurumi-ku, Yokohama, Kanagawa 230–0045, Japan. E-mail: tamari{at}src.riken.jp

	Abstract

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

 
Suppressor of cytokine signaling (SOCS) 1 is an essential physiologic regulator of the IFN- signaling that is crucial to lead appropriate immune responses, and impaired IFN- production is considered a hallmark of atopic diseases. Recent study has shown that SOCS1 is also crucial in attenuating type 1 IFN signaling and in limiting the host response to viral infection. Clinical and experimental evidence suggest an important role for respiratory viral infections in the development of asthma. To assess genetic functional variants of SOCS1 related to susceptibility and clinical phenotypes in adult asthma in a Japanese population, we conducted association and haplotype analyses of 462 subjects with adult asthma and 639 control subjects. After screening for polymorphisms, we identified a total of 13 variants and characterized the linkage disequilibrium (LD) mapping of the gene. Three variants were selected for genotyping with regard to the LD pattern, and we found a significant association between an SOCS1 promoter polymorphism –1478CA > del and adult asthma (P = 0.0063). The three-locus haplotype of SOCS1 using these three polymorphisms also showed a positive association with a haplotype T-C-del (–5388T, –3969C, and –1478 del; P = 0.0097). Furthermore, reporter gene analysis revealed that related promoter variant –1478 del enhanced the transcriptional level of SOCS1 in human lung epithelial cells, and induced higher levels of protein expression of SOCS1 and lower phosphorylation of STAT1 stimulated with IFN-. These findings suggest that the SOCS1 gene might be involved in the development of adult asthma through functional genetic polymorphism.

Key Words: SOCS1 • bronchial asthma • polymorphisms • haplotype • association study

CLINICAL RELEVANCE Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References   Viral infections influence both the development and the severity of asthma. Our data suggest that the susceptible variant might affect the increased sensitivity to viral infection through higher SOCS1 production and contribute to asthma etiology.

 
Bronchial asthma is a complex disease caused by a combination of genetic and environmental factors (1, 2). Cytokines play an important role in chronic inflammation of the airways in asthma (3), and suppressor of cytokine signaling (SOCS)1, a member of the SOCS family of proteins, is rapidly transcribed after exposure of cells to cytokines (4, 5). SOCS1 interacts directly with the Janus kinases (JAK), essential intracellular mediators of immune cytokine action, and inhibits their tyrosine-kinase activity (4, 5).

SOCS1 is an essential physiologic regulator of the IFN- signaling that is crucial to lead appropriate immune responses (6, 7). Asthma is thought to arise from an imbalance in T helper type 1 (Th1)-Th2 immune regulation, and IFN-–producing Th1 cells have been suggested to protect against allergic responses by attenuating the activity of Th2 effectors cells (3). These findings implicated SOCS1 as a candidate gene for involvement in asthma.

Clinical and experimental evidence suggests an important role for respiratory infections in the development of asthma (8, 9). Recent studies have shown that SOCS1 is an important in vivo inhibitor of type I interferon signaling (10). Cardiac myocyte-specific transgenic expression of SOCS1 inhibits enterovirus-induced signaling of JAK-STAT, with accompanying increases in viral replication, cardiomyopathy, and mortality in coxsackievirus-infected mice (11). Inappropriate overexpression of SOCS1 results in viral-mediated end-organ damage during the early stages of infection. Furthermore, SOCS1 inhibits expression of the antiviral proteins myxovirus resistance-A (MxA) and 2'-5'-oligoadenylate synthetase (2'-5'-OAS) (12). In addition, a recent study has shown that dsRNA induces SOCS1 expression, and SOCS1-negative feedback of STAT1 activation is a key pathway in the dsRNA-induced innate immune response (13). The interferons produced after viral infection or treatment with dsRNA stimulate Toll-like receptor (TLR)-3, interferon regulatory factor (IRF)-7, and monocyte inflammatory protein (MIP)-1 expression by activating the JAK-STAT pathway, which is inhibited by SOCS1 (13). These findings suggest that SOCS1 is a key protein regulating the antiviral response.

To discover genetic components in the pathogenesis of asthma, a large number of studies using polymorphic DNA markers, including linkage and association studies, have been performed (14–16). Although genetic studies have been conducted for the polymorphisms in IFN- and IFN-–related genes, the genetic influences of the polymorphisms and the haplotype of SOCS1 are unclear (17, 18). To test whether genetic variations of SOCS1 contribute to asthma susceptibility or asthma-related phenotypes, we first performed LD mapping of the gene and conducted an association study and haplotype analyses with regard to the LD pattern. Furthermore, we performed functional analysis of the associated promoter variant –1478CA > del.

	MATERIALS AND METHODS

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Study Subjects
All subjects with asthma were diagnosed according to the American Thoracic Society criteria as described (19–21). The diagnosis of atopic asthma was based on one or more positive skin prick test responses to seven common aeroallergens in the presence of a positive histamine control and a negative vehicle control (22). The seven aeroallergens were house dust, Felis domesticus dander (Feld), Canis familiaris dander, Dactylis glomerata, Ambrosia, Cryptomeria japonica, and Alternaria. We recruited 462 adults with asthma (mean age, 50.1 yr [range, 20–75 yr]; male:female ratio = 1.0:1.35; atopic asthma 91.2%) and recorded the age, sex, serum total IgE level, eosinophil count, and clinical severity. The clinical severity of adult asthma was classified according to the criteria of the National Institutes of Health/Global Initiative for Asthma by physicians who were experts in allergic diseases, and was defined by symptoms at the time of entry into the study. The distribution of subjects was as follows: step 1, mild intermittent 3% (12 individuals); step 2, mild persistent 54% (246 individuals); step 3, moderate persistent 26% (117 individuals); and step 4, severe persistent 18% (83 individuals) (23). The serum IgE level was log₁₀-transformed before analysis. In this study, "high IgE" and "high eosinophil count" levels were defined as those values in the 75th percentile or higher for total IgE and the eosinophil count (%). The mean of log₁₀ (total IgE [tIgE] [IU/ml]) of patients was 2.34 (= log₁₀ [218.8 IU/ml]). The 75th percentile value of log₁₀ (tIgE) in patients was 2.71 (= log₁₀ [512.9 IU/ml]), and that of eosinophils in patients was 8.0 (%). A total of 639 healthy individuals who had neither respiratory symptoms nor a history of asthma-related diseases (mean age, 43.5 yr [range, 20–75 yr]; male:female ratio = 2.67:1.0) were recruited by detailed physicians' interviews about whether they had been diagnosed with asthma, atopic dermatitis, and nasal allergies. No lung function or serum IgE data are available for this population. Estimation for population stratification in this study was done by genotyping 60 randomly selected single-nucleotide polymorphisms (SNPs), as suggested by Pritchard and Rosenberg (24). The global ² value was 51.6, with a corresponding P value of 0.77, given 60 degrees of freedom. Genomic DNA was prepared in accordance with standard protocols. All individuals were Japanese and gave written informed consent to participate in the study in accord with the rules of the process committee at the SNP Research Center, The Institute of Physical and Chemical Research (RIKEN).

SNPs and Linkage Disequilibrium Mapping
To identify SNPs in the human SOCS1 gene, we sequenced all exons, including a minimum of 200 bases of the flanking intronic sequence, 2 kb of the 5' flanking region, and a 2 kb continuous 3' flanking region of the last exon except for regions with interspersed repeats from 24 subjects with asthma. Primer sets were designed on the basis of genomic sequences from the GenBank database (accession number AC003665) (Table 1). The PCR product was reacted with BigDye Terminator v3.1 (Applied Biosystems, Foster City, CA). Sequences were assembled and polymorphisms were identified by the SEQUENCHER program (Gene Codes Corporation, Ann Arbor, MI).

Statistical Analysis
Pairwise linkage disequilibrium (LD) was calculated as |D'| and r² by using the Haploview 3.2 program (http://www.broad.mit.edu/mpg/haploview/). We calculated allele frequencies and tested agreement with Hardy-Weinberg equilibrium using a ² goodness-of-fit test at each locus. To test the association between SOCS1 variants and adult asthma, we compared differences in allele frequency and genotype distribution of each polymorphism between case and control subjects by using a contingency ² test. Odds ratios (ORs) with 95% confidence intervals (95% CI) were also calculated. Haplotype frequencies for multiple loci were estimated, and haplotype association tests were performed using Haploview 3.2. We also performed permutation tests for our association results, and one thousand permutated data were generated for the three single markers and haplotypes in blocks using the Haploview 3.2 program. We investigated associations between asthma-related phenotypes (eosinophil count, serum total IgE, disease severity, and atopic asthma) and variants within patients with asthma (case-only association study). Serum total IgE levels and eosinophil counts were examined as a dichotomized phenotype using the 75th percentile as the cut point. In addition, serum total IgE and eosinophil counts were analyzed as quantitative levels by the Mann-Whitney U-test or the Kruskal-Wallis test. Comparisons in reporter assays, mRNA expression analysis, and protein expression analysis were performed with Student's t test. Statistical significance was defined at the standard 5% level.

Luciferase Assay
The human lung alveolar epithelial cell line A-549 (lung adenocarcinoma, ATCC CCL-185) was purchased from American Type Culture Collection (Manassas, VA). Three concatenated copies of the 29-bp DNA fragments were cloned into pGL3-basic vector (Promega, Madison, WI) in the 5'-3' orientation. The DNA fragments were: for –1478CA, 5'-CTAGCCCTCCCCAGCCTCAGTTTCTTCCGCAT-3'; and for –1478 del, 5'-CTAGCCCTCCCCAGCCTGTTTCTTCCGCAT-3'. We then transfected subconfluent A549 cells (5 x 10⁴) cultured in 24-well plates with 0.125 µg of each construct and 0.0025 µg of pRL-TK Renilla luciferase vector (Promega), an internal control for transfection efficiency, using 0.75 µl of FuGENE 6 transfection reagent (Roche Diagnostics, Basel, Switzerland). After 24 h, we lysed the cells and measured firefly and Renilla luciferase activities in a luminometer using the Dual-Luciferase Reporter Assay System (Promega). The relative luciferase activity of the mock and SOCS1 reporter constructs is represented as the ratio of the firefly luciferase activity to that of Renilla. Each experiment was repeated three times, and each sample was studied in triplicate as described (22). The mock-transfected average is represented as 1. Comparison in reporter assays was performed with Student's t test. A P value of < 0.05 was considered statistically significant.

Protein Expression of SOCS1 and Phospho-STAT1 in Nasal Fibroblasts
Nasal fibroblasts were established from nasal polyp biopsy specimens and maintained in DMEM/F-12 HAM medium containing 10% heat-inactivated fetal calf serum (FCS; Gibco-BRL, Grand Island, NY), 100 U/ml penicillin, 100 µg/ml streptomycin (Gibco-BRL), and 3 µg/ml amphotericin B (Sigma, St. Louis, MO) as described (25). Polyinosinic acid:polycytidylic acid (poly (I:C)) and IFN- were purchased from InvivoGen (San Diego, CA) and PeproTeck Inc. (Rocky Hill, NJ), respectively. Human NPF cells, homozygous for CA (n = 7) and heterozygous for CA/del (n = 7), were examined. We treated cultured NPF cells with the poly (I:C) (100 µg/ml) for 0, 1.5, 3, 6, and 12 h, and with IFN- (50 ng/ml) for 1 h. Cells were washed with ice-cold PBS and made soluble in 1x SDS sample buffer (62.5 mM Tris·HCl, pH 6.8/2% SDS/10% glycerol/50 mM DTT/0.01 wt/vol bromophenol). Cell lysates were collected, boiled for 5 min, and run on SDS-PAGE. Proteins were transferred to a PVDF membrane and then immunoblotting was performed with antibodies against SOCS1 (IMGENEX, San Diego, CA), -actin (Sigma), phospho-STAT1 (Cell Signaling Technology, Santa Cruz, CA) or STAT1 (Cell Signaling Technology). The intensity of each band was quantified using the by a Luminescent Image Analyzer LAS-3000 (Fujifilm, Tokyo, Japan). The relative levels of protein expression of SOCS1 and phospho-STAT1 were calculated by comparison with -actin protein and STAT1 protein, respectively.

	RESULTS

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

 
Association of SOCS1 Polymorphisms with Asthma Susceptibility
We performed screening of polymorphisms with genomic DNA from 24 randomly selected individuals with asthma. After extensive examination of SOCS1 by direct sequencing, we identified 13 polymorphisms (11 SNPs in the promoter region and one SNP within the transcript) (Table 2). Six polymorphisms were contained in the two available public databases: NCBI dbSNP (http://www.ncbi.nlm.nih.gov/SNP/) and IMS-JST JSNP DATABASE (http://snp.ims.u-tokyo.ac.jp/). To examine the linkage disequilibrium (LD) between identified SNPs, pairwise LD coefficients D' and r² were calculated using the Haploview program. Since four of the SNPs were quite rare, pairwise LD was measured by |D'| and r² among the nine SNPs with a frequency of greater than 5% (Table 3). –3969C > T was in complete LD (D' = 1.00 and r² = 1.00) with –5635C > T, –5485G > A, –5162G > A, –820G > T, and 1125G > C, and in strong LD (D' = 1.00 and r² = 0.90) with –1656A > G. However, –5388C > T was in marginal LD with other SNPs (maximum r² = 0.62), and it was the SNP with the highest minor allele frequency. –1478CA > del was not in strong LD with other SNPs (maximum r² = 0.19). We finally selected three polymorphisms, –5388C > T, –3969C > T, and –1478CA > del, for association studies (Figure 1).

View larger version (2K): [in this window] [in a new window]   Figure 1. A graphical overview of genotyped polymorphisms identified in relation to the exon/intron structure of the human SOCS1 gene. Two exons are shown by black boxes, and positions for polymorphisms are relative to the translation start site (+1).

View larger version (11K): [in this window] [in a new window]   Figure 2. Effect of –1478CA > deletion polymorphism on the transcriptional activity of the human SOCS1 promoter in a human lung epithelial cell line. Each experiment was conducted in triplicate for each sample, and the results are expressed as mean ± SD for three independent experiments (P < 0.001 by Student's t test).

View larger version (25K): [in this window] [in a new window]   Figure 3. The relative protein expression levels of SOCS1 and phosphorylated STAT1 according to the promoter genotype were measured in primary nasal fibroblasts. Data are presented as mean values ± SEM normalized to 1.0 for the -actin control (*P < 0.05). (A) Cells were stimulated with 100 µg/ml poly (I:C) for the indicated number of hours. Nasal fibroblasts with the CA/del allele exhibited significantly higher expression of SOCS1 protein under no stimulation and in response to poly (I:C) for 12 h. (B) Higher expression of SOCS1 in cells with the CA/del allele was observed with or without stimulation with 50 ng/ml IFN- for 1 h.

	DISCUSSION

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

Viral infections can influence both the development and the severity of asthma (26). An epidemiologic study showed that 50% of adult asthma attacks are associated with viral upper respiratory infections (27). IFNs play a central role in host defense against invasive viruses (28). A recent study has shown that SOCS1 is crucial in attenuating type I interferon signaling in vivo and in limiting the host response to viral infection (10). SOCS1 associates with and regulates IFNAR1-specific signals, abrogating tyrosine phosphorylation of transcription factor STAT1 and reducing the duration of antiviral gene 2'-5'-OAS expression (10). Another report showed that SOCS1 proteins inhibit IFN-–induced activation of the JAK-STAT pathway and expression of antiviral proteins, MxA and 2'-5'-OAS (12). MxA and 2'-5'-OAS play an important role in mediating at least some of the antiviral activities induced by type I interferons (12). In vivo, SOCS1 cardiac myocyte-specific transgenic expression during the early stages of viral infections decreases the antiviral defense by inhibiting IFNs and promotes tissue injury in the heart (11). Transgenic expression of SOCS1 inhibits viral-induced JAK signaling and STAT, with accompanying increases in viral replication, cardiomyopathy, and mortality in virus-infected mice (11). It has been previously reported that asthmatic bronchial epithelial cells have abnormal innate immune responses to viral infection characterized by impaired type I interferon production and impaired virus-induced apoptosis, resulting in increased viral replication (29). A recent study has shown that poly(I:C) induces SOCS1 and IFN-/ expression, and SOCS1 inhibits STAT1-dependent TLR3, IRF-7, and MIP-1 production by blocking the JAK-STAT1 pathway (13). In this study, we found that the –1478 promoter genotype affected the protein levels of SOCS1 and phosphorylated STAT1 under no stimulation and in response to poly (I:C) or IFN-. Dysfunction of SOCS1 molecules could contribute to the etiology of asthma by affecting the antiviral defense in the respiratory tract. The susceptibility allele might affect the increased sensitivity to viral infection through higher SOCS1 production.

A growing body of evidence suggests that two subsets of T helper (Th) cells, Th1 and Th2, may play an important role in allergic and autoimmune disorders (30). In the case-control study, an association of –1478CA > del on SOCS1 with adult atopic asthma was observed. The hallmark of allergic disease is infiltration by Th2 cells (1, 2). T cells from atopic subjects respond to allergens in vitro by inducing cytokines produced by Th2 cells, rather than cytokines produced by Th1 cells (31). A recent study has shown that the normal type 1–like antiviral response is defective in atopic asthma, both at the inducing (IL-12) and effector (IFN-) levels (32). IFN- is induced by IL-12, a cytokine regulated by SOCS1 (33). Exposure of PBMC to rhinoviruses resulted in time- and dose-dependent up-regulation of IFN- and IL-12 in both normal subjects and in subjects with atopic asthma. Deficient production of the type 1 cytokines IFN- and IL-12 in the atopic asthma group compared with the normal subjects was observed (32). Given the role of SOCS1 in attenuating Th1 cytokine signaling, the functional genetic variant in SOCS1 might contribute to the atopic phenotype in adult asthma.

In the case-only study, there was a positive association between –3969C > T of the SOCS1 gene and the most severe cases of adult asthma. Recent studies reported reduced production of IFN- by PBMC of patients with asthma, which correlates with disease severity (34). In another study, patients with severe ongoing asthma had significantly reduced house dust mite–induced IFN- production compared with control subjects and patients with resolved asthma (35). Furthermore, a cytokine imbalance with a deficient Th1 response, decreased generation of IFN-, to rhinovirus is associated with asthma severity (36). The SOCS1 variant might be involved in severe phenotypes of adult asthma through alteration of IFN- signaling. In this study, we did not analyze the functional effects of the –5635C > T, –5485G > A, –5162G > A, –1656A > G, –820G > T, and 1125G > C, which were in linkage disequilibrium with promoter variant –3969C > T. To validate the involvement of the SOCS1 gene in the severity of asthma, however, it will be necessary to elucidate the functional effects of the polymorphisms.

Our data strongly suggest the important role of SOCS1 in asthma, and further investigations of the connection between genotypes and the functional role of SOCS1 will be helpful to clarify the etiology of asthma.

	Acknowledgments

 
The authors thank all the participants in the study. They are grateful to the members of The Rotary Club of Osaka-Midosuji District 2660 Rotary International in Japan for supporting their study. They also thank Hiroshi Sekiguchi and Aya Ito for technical assistance and Chinatsu Fukushima for providing patients' data.

	Footnotes

 
^* These authors equally contributed to this work.

This work was supported by a research grant from the Japanese Government's Millennium Genome Project and by a grant from RIKEN SNP Research Center.

Originally Published in Press as DOI: 10.1165/rcmb.2006-0090OC on November 10, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form February 28, 2006

Accepted in final form September 22, 2006

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