Evidence of association between interferon regulatory factor 5 gene polymorphisms and asthma

Gene 504 (2012) 220–225

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Evidence of association between interferon regulatory factor 5 gene polymorphisms and asthma Chuan Wang a,⁎, Matthew J. Rose-Zerilli b, 1, Gerard H. Koppelman c, 1, Johanna K. Sandling a, 1, 2, John W. Holloway b, Dirkje S. Postma d, Stephen T. Holgate b, Vincent Bours e, Ann-Christine Syvänen a, Vinciane Dideberg e a

Department of Medical Sciences, Uppsala University, Uppsala, Sweden Faculty of Medicine, University of Southampton, Southampton General Hospital, Southampton, UK c Department of Pediatric Pulmonology and Pediatric Allergology, Beatrix Children's Hospital, GRIAC Research Institute, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands d Department of Pulmonology, GRIAC Research Institute, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands e Department of Human Genetics, University Medical Center of Liège, University of Liège, Liège, Belgium b

a r t i c l e

i n f o

Article history: Accepted 10 May 2012 Available online 18 May 2012 Keywords: Interferon regulatory factor 5 Asthma Autoimmune disorder Genetic association study

a b s t r a c t Asthma is a heterogeneous disorder hallmarked by chronic inflammation in the respiratory system. Exacerbations of asthma are correlated with respiratory infections. Considering the implication of interferon regulatory factor 5 (IRF5) in innate and adaptive immunity, we investigated the preferential transmission patterns of ten IRF5 gene polymorphisms in two asthmatic family cohorts. A common IRF5 haplotype was found to be associated with asthma and the severity of asthmatic symptoms. Stratified analysis of subgroups of asthmatic individuals revealed that the associations were more pronounced in nonatopic asthmatic individuals. In addition, the risk alleles of IRF5 polymorphisms for asthma were almost completely opposite to those for autoimmune disorders. Our study provides the first evidence of association between IRF5 and asthma, and sheds light on the related but potentially distinct roles of IRF5 alleles in the pathogenesis of asthma and autoimmune disorders. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Asthma, characterized by airway inflammation, airflow obstruction and bronchial hyperresponsiveness (BHR), is one of the most common chronic inflammatory disorders in the world (Anandan et Abbreviations: BHR, bronchial hyperresponsiveness; CD, Crohn's disease; FBAT, family based association test; FEV1, forced expiratory volume in 1 second; FP, fluorescence polarization; IFN, interferon; IgE, immunoglobulin E; IL, interleukin; indel, insertion–deletion polymorphism; IRF5, interferon regulatory factor 5; LD, linkage disequilibrium; MS, multiple sclerosis; NA, not available; NS, not significant; PCR, polymerase chain reaction; pSS, primary Sjögren's syndrome; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; SNP, single nucleotide polymorphism; SSc, systemic sclerosis; TH, T helper cell; TLR, toll-like receptor; TNF, tumor necrosis factor; UC, ulcerative colitis; UTR, untranslated region. ⁎ Corresponding author at: Molecular Medicine, Department of Medical Sciences, Entrance 70, 3rd floor, Research Department II, University Hospital, SE-751 85, Uppsala, Sweden. Tel.: + 46 18 611 2524; fax: + 46 18 55 3601. E-mail addresses: [email protected] (C. Wang), [email protected] (M.J. Rose-Zerilli), [email protected] (G.H. Koppelman), [email protected] (J.K. Sandling), [email protected] (D.S. Postma), [email protected] (S.T. Holgate), [email protected] (V. Bours), [email protected] (A-C. Syvänen), [email protected] (V. Dideberg). 1 Authors contributed equally. 2 Present address: Genetics of Complex Traits in Humans, Wellcome Trust Sanger Institute, Hinxton, UK. 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2012.05.021

al., 2010). Asthma pathogenesis is the consequence of a combined effect of genetic predisposition and environmental factors such as viral infections and aeroallergens, which drive different disease subtypes (Holgate and Polosa, 2006). On the basis of whether or not sensitization to specific allergens occurs in association with the disease, asthma can be classified into atopic and nonatopic subtypes in broad clinical terms (Bradding and Green, 2010). Atopic asthma is more prevalent in childhood and young-adults, in which it is mainly driven by T helper (TH) 2 cell inflammatory responses (Hollams et al., 2009); whereas nonatopic asthma typically has a later onset, is dominated by neutrophils rather than eosinophils, is usually associated with lower circulating IgE levels and more severe, persistent and corticosteroid refractory symptoms (Douwes et al., 2002; Holgate and Polosa, 2006; Romanet-Manent et al., 2002). Experimental and clinical evidence indicates that nonatopic asthma is related with both TH1 and TH17 cell responses (Alcorn et al., 2010; Cho et al., 2005). Nonatopic asthma may thus share some properties with autoimmune disorders, such as TH1 and TH17 activity and higher disease risk in the female population (Nieves et al., 2005). Elevated levels of auto-antibodies, anti-nuclear antibodies and T lymphocytes activity have also been reported in nonatopic asthma (Rottem and Shoenfeld, 2003). Although the exact implication of viral infection in asthma is still unknown, pathogens, especially respiratory viruses, are strongly

C. Wang et al. / Gene 504 (2012) 220–225

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associated with exacerbations of asthma in both atopic and nonatopic subtypes (Johnston, 2007; Le Souef, 2009). Interactions between pathogens and their pattern recognition receptors, especially the toll-like receptors (TLRs), may initiate a bifurcating signaling cascade. While in one direction sensitization and TH2 cell-mediated atopic reactions are facilitated (Schroder, 2009), in the other direction, timing, dose of pathogen exposure, and potential genetic susceptibility may invoke the polarization towards TH1 cell responses with the release of the cytokines interleukin (IL)-1, IL-6, IL-8, interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Their coordination can skew the TH1–TH2 balance away from atopy and towards TH1 cellmediated inflammation in the respiratory system, leading to risk for asthmatic symptoms (Hammad and Lambrecht, 2008; Holgate and Polosa, 2006). The IFN regulatory factor 5 (IRF5, GenBank ID: 3663) gene encodes a key transcription factor in the type I IFN pathway. IRF5 can be directly activated upon viral infection, and participates in the transcriptional regulation of type I IFN genes and the genes encoding proinflammatory cytokines/chemokines such as IL-6, IL-10, IL-12 and TNF-α (Ronnblom and Pascual, 2008). Previous studies have revealed the crucial roles of IRF5 in the functioning of immune cells (Krausgruber et al., 2010; Lien et al., 2010) and in the innate antiviral response (Barnes et al., 2004; Paun et al., 2008; Yanai et al., 2007). In addition, IRF5 contributes to macrophage polarization and promotes the TH1–TH17 cell responses (Krausgruber et al., 2011). In this study, we aimed to investigate the potential involvement of the IRF5 gene in the genetic susceptibility to asthma predisposition and to asthmatic symptoms.

asthma patients were excluded, which left 153 atopic and 53 nonatopic asthmatic families for further analysis. The recruitment of this cohort was approved by the Local Research Ethics Committees of Southampton & South-West Hampshire and of Portsmouth & South-East Hampshire. All participants had given their written informed consent. The second cohort consisted of 1198 individuals from 195 pedigrees, which were collected from the northern part of the Netherlands (Dutch cohort) and had sufficient DNA available for this study. Asthmatic probands were selected during a period between 1962 and 1975, following the criteria of characteristic asthma symptoms, positive status for BHR (PC20 FEV1 histamine b32 mg/ml, 30 s method), and age younger than 45 years. The original probands together with their spouses, children, children's spouses, and grandchildren older than 6 years were recruited and evaluated between 1990 and 1999. In the 195 pedigrees, 151 were of two-generation structure, 42 of three-generation, 2 of four-generation, and 1 extended family involving half-siblings. Examinations on total serum IgE, specific IgE levels, FEV1, BHR status, as well as skin prick tests were carried out as previously described (Koppelman et al., 2002). Due to the lower number of affected children per family (0.8 in the Dutch cohort vs. 2.0 in the UK cohort) and the complex structure of the pedigrees, we did not perform atopic/nonatopic stratification in the Dutch cohort. This study was approved by the Medical Ethics Committee of the University Hospital Groningen. Written informed consent for all adults and written parental consent for children was obtained from all participants. A summary of the characteristics of the two cohorts is listed in Table 1.

2. Materials and methods

2.2. Genotyping

2.1. Subjects

A panel of ten IRF5 gene polymorphisms was selected based on their association with major autoimmune disorders or functional potential in regulating IRF5 expression (Kristjansdottir et al., 2008). This panel included one 5-bp insertion–deletion polymorphism (CGGGG indel) and nine single nucleotide polymorphisms (SNPs). The CGGGG indel and five SNPs are spanning the 5′‐region of the IRF5 gene, from the promoter to the first intron. The other four SNPs are located within the 3′‐untranslated region (UTR) or downstream of the IRF5 gene. In both the UK and Dutch cohorts, the CGGGG indel was genotyped by polymerase chain reaction (PCR) amplification followed by 4% MetaPhor high resolution agarose gel electrophoresis (Cambrex Bio Science Rockland Inc, Maine, USA). Eight out of the nine SNPs (rs729302, rs3757385, rs2004640, rs3807306, rs10954213, rs11770589, rs2280714 and rs12539741) were genotyped with the GenomeLab SNPstream Genotyping System (Beckman Coulter Inc, Brea, USA). The SNP rs4728142 was genotyped with a fluorescence polarization assay (FP-TDI) (Molecular Devices, Sunnyvale, USA) in the UK cohort, whereas in the Dutch cohort it was integrated into the SNPstream panel with the other eight SNPs.

The first cohort included 1467 individuals from 340 families, which were collected from the Southampton area of the United Kingdom (UK cohort). Asthmatic status was defined by physician's diagnosis and regular asthma medication. Except for seven families which were single-parental, all families had an intact first-degree structure with four members on average (two parents plus two children), and at least one child with asthma. Related clinical parameters including total serum IgE and specific IgE levels, results of skin prick test, forced expiratory volume in 1 second (FEV1) value, BHR status, atopy severity score and asthma severity score were available for this cohort (Sayers et al., 2003; Van Eerdewegh et al., 2002). The asthma severity score was estimated according to a detailed questionnaire regarding symptoms, treatment response and daily life qualities. Atopic and nonatopic asthma were stratified based on whether a patient was positive in a skin prick test (wheal diameter ≥3 mm) and/or had an elevated specific IgE level (>0.35 IU) against one or more common allergens. Families with confounding inheritance between atopic and nonatopic

Table 1 Characteristics of the UK and Dutch asthmatic family cohorts. UK

No. of individuals Age (year) Gender (male %) Total IgEa FEV1 (%) BHR (positive %) Skin prick test (positive %) Atopy severity score Asthma severity score

Dutch

Affected

(Atopic, nonatopic)

Healthy

Affected

Healthy

489 16.2 ± 11.0 51.9 1.92 ± 1.27 94.6 ± 14.7 75.6 76.2 1.5 ± 1.1 4.3 ± 0.8

(366, 123) (16.5 ± 11.1, 15.2 ± 10.9) (55.5, 41.5) (2.43 ± 0.94, 0.39 ± 0.78) (93.8 ± 15.3, 97.3 ± 12.7) (85.3, 48.3) (100, 25.4) (1.9 ± 0.9, 0.2 ± 0.3) (4.4 ± 0.8, 4.0 ± 0.7)

401 36.2 ± 12.2 51.4 0.62 ± 0.92 102.6 ± 12.9 20.9 51.9 0.5 ± 0.7 2.7 ± 0.4

451 34.9 ± 18.8 51.7 1.95 ± 0.73 80.3 ± 21.1 94.7 69.6 NA NA

705 32.0 ± 15.1 45.7 1.61 ± 0.67 97.1 ± 12.2 24.2 38.6 NA NA

BHR, bronchial hyperresponsiveness; FEV1, forced expiratory volume in 1 s; NA, not available. a Natural logarithm of age-corrected total serum IgE level.

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of the IRF5 gene (rs4728142 p = 0.0090; CGGGG indel p = 0.00085; rs2004640 p = 0.0031; and rs3807306 p = 0.0015). In attempt to replicate these findings, similar analyses were conducted in the Dutch cohort. Although no significant association signal was observed from these ten IRF5 polymorphisms in the Dutch cohort (Table 2 and Table A.1), most of the risk alleles or risk genotypes for asthma in the UK cohort were transmitted in the same direction in the Dutch cohort. When the UK cohort and the Dutch cohort were analyzed jointly, we observed marginal associations for the risk alleles of two polymorphisms (CGGGG indel and rs2004640) (Table 2). In addition, significant p values for preferential transmission were detected for the homozygous genotypes of four polymorphisms (rs4728142, CGGGG indel, rs2004640 and rs3807306) (Table A.1). The families in the UK cohort were stratified into atopic or nonatopic subgroups based on the presence/absence of specific IgE and/or skin prick tests to a panel of common allergens as previously described (Sayers et al., 2003). We found that most of the association signals were more pronounced in the nonatopic asthmatic families (Table 2). In contrast, for atopic asthma, there were only marginal association signals detected from the polymorphisms in the 5′‐region of the IRF5 gene.

2.3. Statistical analysis The genotype distributions of all ten polymorphisms were controlled with the Haploview 4.1 software (Barrett et al., 2005). One family with Mendelian inheritance errors was removed from further analysis. In both cohorts, each of the ten polymorphisms was in Hardy–Weinberg equilibrium (p > 0.2). Linkage disequilibrium (LD) plots based on r 2-values were drawn with the R package SNP plotter (Luna and Nicodemus, 2007). Possible associations between individual IRF5 polymorphisms or haplotypes and asthma affection, together with the related clinical parameters (FEV1 status and BHR status as dichotomous traits and natural logarithm of age-corrected total serum IgE level, atopy severity score and asthma severity score as continuous traits) were analyzed using the software Family Based Association Tests (FBAT) 2.03 (Laird et al., 2000). In order to adjust for the high prevalence of asthma, we applied a variance minimization approach (offset option) with an estimated prevalence value μ, which allows both affected and unaffected offspring in the test statistic (Lunetta et al., 2000). P values were calculated with two-sided Chisquare (χ 2) tests and a p ≤ 0.05 was considered statistically significant. The combined analysis of the UK and the Dutch cohorts was performed by pooling the data from both cohorts. This strategy was validated by χ 2-based homogeneity tests, which did not show any significant differences in the allele frequency of each of the ten polymorphisms between the cohorts. The statistical power was estimated with the software PBAT v3.61 (Lange et al., 2004), using a log-additive model, a two-sided type I error rate of 0.05 and a prevalence for asthma estimated at 10%.

3.2. Association analysis for common IRF5 haplotypes Considering that only a few association signals from the individual IRF5 gene polymorphisms were retained after Bonferroni correction, and that these polymorphisms were correlated to an intermediate level in both cohorts (pairwise r 2-based LD pattern in Fig. 1), we conducted family based association tests for the common haplotypes with a frequency ≥5% in the studied cohorts. As shown in Table 3, the two most common haplotypes are associated with asthma in the UK cohort. The most common haplotype had a frequency about 31% and was composed of all the under-transmitted alleles of the IRF5 polymorphisms and had a protective effect against asthma (H1, p = 0.00013). In contrast, the second most common haplotype, with a frequency about 18%, was composed of almost all the overtransmitted alleles of IRF5 polymorphisms (except for rs12539741) and it appeared to be a risk haplotype for asthma (H2, p = 0.0041). The Dutch cohort shared the two most common haplotypes with the UK cohort. Although no significant association was observed, the

3. Results 3.1. Association analysis for individual IRF5 polymorphisms In the UK cohort, eight out of the ten polymorphisms were associated with asthma with p ≤ 0.05 under an allelic transmission model, as shown in Table 2. All the homozygous genotypes of the risk alleles were associated with asthma in a genotypic test except for the SNP rs729302 which showed a suggestively significant p value (Table A.1). The lowest p values were observed for the 5′‐polymorphisms

Table 2 FBAT analysis of allelic transmission of ten IRF5 polymorphisms in asthma.

Na Freq. z valueb p valuec Asthma Na Freq. z valueb p valuec Atopic asthma Na Freq. z valueb p valuec Nonatopic asthma Na Freq. z valueb p valuec Asthma Na Freq. z valueb p valuec

Combined Asthma

UK

Dutch

a b c

rs729302

rs4728142

rs3757385

rs3807306

rs10954213

rs11770589

rs2280714

C

G

T

CGGGG indel rs2004640 3×

G

G

G

G

C

T

279 0.34 1.23 0.22 125 0.33 2.33 0.02 89 0.34 1.45 0.15 36 0.33 1.89 0.058 154 0.34 0.00 1.00

307 0.58 1.48 0.14 136 0.57 1.94 0.053 98 0.58 0.71 0.48 38 0.55 2.23 0.026 171 0.59 0.59 0.55

294 0.39 1.26 0.21 138 0.40 3.02 0.0025 101 0.40 1.83 0.067 37 0.42 2.50 0.012 156 0.37 − 0.37 0.71

324 0.58 1.63 0.10 151 0.58 2.74 0.0061 110 0.58 1.87 0.062 41 0.56 2.09 0.037 173 0.58 0.69 0.49

314 0.50 1.93 0.053 144 0.50 2.70 0.0069 102 0.50 1.71 0.087 42 0.52 2.12 0.034 170 0.50 0.14 0.89

310 0.54 1.37 0.17 137 0.53 2.22 0.026 98 0.54 1.12 0.26 39 0.52 2.13 0.033 173 0.55 0.45 0.65

305 0.42 1.13 0.26 134 0.42 2.39 0.017 100 0.42 1.53 0.13 34 0.42 1.87 0.062 171 0.42 0.08 0.94

320 0.53 1.15 0.25 151 0.53 2.13 0.033 115 0.53 0.45 0.65 36 0.51 2.90 0.0038 169 0.53 0.15 0.88

301 0.36 0.91 0.36 139 0.37 2.13 0.033 104 0.37 1.34 0.18 35 0.40 1.59 0.11 162 0.35 − 0.30 0.76

118 0.90 0.44 0.66 51 0.91 0.38 0.70 36 0.91 − 0.89 0.37 15 0.91 1.83 0.067 67 0.89 0.03 0.98

Number of informative families. A positive (negative) z value indicates that the tested allele is over-transmitted (under-transmitted) to affected offspring. p value ≤ 0.05 is highlighted in bold.

rs12539741

C. Wang et al. / Gene 504 (2012) 220–225

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Fig. 1. LD between IRF5 gene polymorphisms in (A) UK and (B) Dutch family cohorts. Pair-wise r2 values of ten IRF5 gene variants were calculated based on the genotype data of the unrelated parents in both cohorts. Color scheme for the r2 values is provided at the bottom.

haplotype H1 was also under-transmitted to the affected offspring as in the UK cohort. In the combined analysis with both the UK and the Dutch cohort, the significant association signal from the haplotype H1 was retained (p = 0.0046). Similarly with the results from the analysis for individual polymorphisms in the UK cohort, the associations of these two haplotypes were exclusive in the nonatopic asthma subgroups, in which the p values for the protective (H1) and the risk haplotype (H2) were 0.00029 and 0.010, respectively (Table 3). Given the association signals from these two common haplotypes, we also examined their association with asthma-related clinical traits. Significant association signals were observed from the protective haplotype (H1) with asthma severity score in the complete UK cohort (p = 0.0037), as in the nonatopic subgroup (p = 0.0023).

4. Discussion Although the influence of the IRF5 gene in inflammatory diseases has been widely confirmed by genetic association and functional studies, our findings that the IRF5 gene polymorphisms might also be implicated in asthma and asthma severity provide the first evidence linking IRF5 to this disease. Concerning the association patterns of the IRF5 polymorphisms, it is interesting that the risk alleles identified for asthma in this study, and the ones reported for autoimmune disorders are almost completely opposite in direction (Table 4). The functional potential of the risk alleles in the IRF5 gene for autoimmune disorders are in the direction of over-expression of IRF5. The 4× allele of the CGGGG indel, which may be the most promising functional polymorphism

Table 3 FBAT analysis of common IRF5 haplotypes in asthma. Haplotype composition

H1 H2 H3 H4 H5 H6

rs729302

rs4728142

rs3757385

CGGGG indel

rs2004640

rs3807306

rs10954213

rs11770589

rs2280714

rs12539741

A C A C A A

A G G G A G

G T T G G G

4× 3× 3× 3× 4× 3×

T G G G T T

T G G G T G

A G G A A G

A G G A G G

T G G T T T

C C C C T C

Combined

Asthma

UK

Asthma

Atopic asthma

Nonatopic asthma

Dutch

Asthma

Na Freq. z valueb p valuec Na Freq. z valueb p valuec Na Freq. z valueb p valuec Na Freq. z valueb p valuec Na Freq. z valueb p valuec

H1

H2

H3

H4

H5

H6

221 0.30 − 2.83 0.0046 106 0.31 − 3.82 0.00013 77 0.30 − 1.95 0.052 29 0.34 − 3.63 0.00029 115 0.29 − 1.11 0.27

160 0.17 1.35 0.18 80 0.18 2.87 0.0041 57 0.18 1.51 0.13 23 0.19 2.57 0.010 80 0.16 − 0.38 0.70

152 0.17 0.64 0.52 77 0.18 0.61 0.54 55 0.17 0.89 0.37 22 0.20 − 0.07 0.94 75 0.17 0.22 0.83

109 0.11 0.76 0.45 47 0.09 0.47 0.64 38 0.10 0.51 0.61 NA NA NA NA 62 0.12 0.28 0.78

96 0.09 0.09 0.93 41 0.08 1.02 0.31 29 0.09 − 0.29 0.77 12 0.07 2.15 0.031 55 0.10 0.30 0.76

71 0.06 − 0.51 0.61 29 0.05 − 1.48 0.14 26 0.06 − 1.52 0.13 NA NA NA NA 42 0.07 0.59 0.56

NA, not available. a Number of informative families. b A positive (negative) z value indicates that the tested haplotype is over-transmitted (under-transmitted) to affected offspring. c p value ≤ 0.05 is highlighted in bold.

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Table 4 Risk alleles of IRF5 gene polymorphisms in asthma and autoimmune disorders. IRF5 gene polymorphisms

References

rs729302

rs4728142

rs3757385

CGGGG indel

rs2004640

rs3807306

rs10954213

rs11770589

rs2280714

rs12539741

Asthma SLE

C A

G A

T Ga,b

3× 4×

G T

G T

G A

G NS

G T

T Ta

CD UC RA

NS A A

A A A

NS NS G

4× 4× 4×

NS T T

NS T T

NS NS A

NS NS

NS NS T

NS NS Ta

MS pSS

Ab

A

G

4× 4×

T T

T

A NS

NS

T Tb

NS Ta

A

G

T

Ta

SSc

T

A

This study Cunninghame Graham et al. (2007); Graham et al. (2006); Sigurdsson et al. (2008) Dideberg et al. (2007) Dideberg et al. (2007) Sigurdsson et al. (2007); Wang et al. (2011) Kristjansdottir et al. (2008) Miceli-Richard et al. (2007); Nordmark et al. (2009) Dieude et al. (2009); Dieude et al. (2010); Ito et al. (2009); Radstake et al. (2010)

NS, not significant; SLE, systemic lupus erythematosus; CD, Crohn's disease; UC, ulcerative colitis; RA, rheumatoid arthritis; MS, multiple sclerosis; pSS, primary Sjögren's syndrome; SSc, systemic sclerosis. a The risk allele of rs3757385 for SLE, and the risk allele of rs12539741 for SLE, RA, pSS and SSc, are deduced from the data of rs3757386, rs10488631, respectively (pairwise r2based LD ≥ 0.9 according to the data from the CEU panel in HapMap 3 (release 2) and the 1000 Genomes Pilot 1). b Trend for association, p value between 0.05 and 0.1.

of IRF5, carries an additional CGGGG repeat unit compared to the 3× allele. This allows more binding of the transcription factor SP1 than the 3× allele and results in an elevated IRF5 expression in peripheral blood cells (Kristjansdottir et al., 2008; Sigurdsson et al., 2008). The A allele of the SNP rs10954213 creates a functional polyadenylation site in the 3′‐UTR of IRF5, which is correlated with the enhanced expression of a shorter IRF5 transcript variant (Cunninghame Graham et al., 2007). In addition, the T alleles of both rs2004640 and rs2280714 have been reported to associate with increased IRF5 mRNA levels (Graham et al., 2006), although their effects may be due to their correlation with the CGGGG indel and the SNP rs10954213. It follows that the risk alleles of the IRF5 gene polymorphisms for asthma are related to lower IRF5 expression, whereas those risk alleles for autoimmune disorders linked to a greater IRF5 expression level appear protective for asthma, especially when considering the joint effects of these alleles (haplotype H1). Interestingly, a diminished level of IL-10 and IL-12 has been reported in patients with asthma (Borish et al., 1996; Message et al., 2008; Plummeridge et al., 2000). As IL-10 and IL-12 are directly regulated by IRF5, these observations corroborate our hypothesis that lower IRF5 expression is a risk factor for asthma. Stratified analysis in the atopic and nonatopic asthmatic families revealed that the association signals of the IRF5 gene polymorphisms are more pronounced in the nonatopic subgroup. Additional evidence has been reported from a survey of the TLR-related pathway genes, in which neither the IRF5 gene alone nor its interactions with other genes were found to be associated with total or specific circulating IgE levels (Reijmerink et al., 2010), which is a hallmark of atopy. Based on these observations, IRF5 is likely to harbor a more profound impact in the pathogenesis and severity of nonatopic asthma, whereas for atopic asthma and atopic symptoms, IRF5 might have only a subtle or no influence. Evidence on the involvement of the type I IFN system in the pathogenesis of asthma is accumulating. Reduced expression levels of type I IFN and IFN-regulated genes have been reported in asthma patients compared to healthy controls (Chung, 2001; Contoli et al., 2006; Wark et al., 2005). Moreover, clinical symptoms of asthma can be controlled using low doses of recombinant IFN-α (Gratzl et al., 2000). Genetic association studies have also identified several risk factors for asthma in the type I IFN system, such as variants of IFNA, IFNB and TLR gene super-families (Chan et al., 2006; Yang et al., 2006). Together with the observations in this study, we can postulate that nonatopic asthma and autoimmune disorders are closely

related, and they may well be two opposite outcomes downstream of a common pathway, in which IRF5 as well as the type I IFN system are involved. Accordingly, we suggest a model for the involvement of IRF5 in asthma. The risk alleles of IRF5 polymorphisms for asthma are associated with lower IRF5 expression, weaker activation of the type I IFN pathway and suppressed M1 macrophage activity (Martinez, 2011). The direct consequence could be an impaired microbial clearance by M1 macrophages, which potentially leads to greater damage to lung tissue during respiratory infection and subsequently a stronger/ sustained M2 macrophage activity for wound healing, resulting in increased fibrosis. In addition, M1 macrophages will still recruit neutrophils, the major effector cell of nonatopic asthma. Therefore, the prolonged anti-microbial response in a subset of individuals who carry the risk alleles of IRF5 polymorphisms and other genetic susceptibility factors may be more likely to develop a nonatopic asthma phenotype. On the other hand, over-expression of the IRF5 gene will activate M1 macrophages, which is believed to participate in varieties of autoimmune disorders (Krausgruber et al., 2011). We did not obtain solid support for most of the association signals from the Dutch cohort, which may be due to the lower number of asthmatic offspring in the informative families resulting in limited statistical power for association test. For detecting a risk allele with a hypothesized effect size of 1.3, which is comparable with the effect sizes of IRF5 risk alleles in autoimmune disorders, we had 55–60% statistical power in the UK cohort, compared to about 37–40% in the Dutch cohort. Another reason for the failure in replicating the findings in the UK cohort might be the age difference of asthmatic subjects between the two cohorts in study (Table 1). The asthma patients in the UK cohort are mainly children and younger adults, whereas the affected subjects in the Dutch cohort are mainly middleaged adults. It is possible that the accumulation effect of environmental factors during aging dilutes the impact from inherent genetic factors. Nevertheless, verification for association between asthma and the IRF5 gene in independent cohorts remains as an interesting task for the future. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.gene.2012.05.021.

Conflict of interest The authors have declared no conflict of interest.

C. Wang et al. / Gene 504 (2012) 220–225

Funding This work was supported by grants from the “Fonds National de la recherche scientifique” and the “Fonds d'investissements de recherché scientifique” from Centre Hospitalier Universitaire de Liège, and by a grant from the Swedish Research Council for Medicine and Health (A0280001). The SNP&SEQ Technology Platform is supported by Uppsala University, Science for Life Laboratory in Uppsala, Uppsala University Hospital and the Swedish Council for Research Infrastructures (80576801). The Southampton asthma family cohort was originally recruited in collaboration with Genome Therapeutics Corporation and Schering-Plough. The Dutch Asthma Family study was supported by grants from the Netherlands Asthma Foundation (AF 95.09 and AF 98.48). Role of funding source The funding agencies played no role in the study design; in the collection, analysis and interpretation of data; in the writing of the manuscript; and in the decision to submit the manuscript for publication. Acknowledgments We thank the SNP&SEQ Technology Platform (http://www. genotyping.se) in Uppsala for assistance with genotyping. We also thank the study team in Beatrixoord, Haren, for their contribution to the clinical assessments of the Dutch Asthma Families. References Alcorn, J.F., Crowe, C.R., Kolls, J.K., 2010. TH17 cells in asthma and COPD. Annu. Rev. Physiol. 72, 495–516. Anandan, C., Nurmatov, U., van Schayck, O.C., Sheikh, A., 2010. Is the prevalence of asthma declining? Systematic review of epidemiological studies. Allergy 65, 152–167. Barnes, B.J., Richards, J., Mancl, M., Hanash, S., Beretta, L., Pitha, P.M., 2004. Global and distinct targets of IRF-5 and IRF-7 during innate response to viral infection. J. Biol. Chem. 279, 45194–45207. Barrett, J.C., Fry, B., Maller, J., Daly, M.J., 2005. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 21, 263–265. Borish, L., Aarons, A., Rumbyrt, J., Cvietusa, P., Negri, J., Wenzel, S., 1996. Interleukin-10 regulation in normal subjects and patients with asthma. J. Allergy Clin. Immunol. 97, 1288–1296. Bradding, P., Green, R.H., 2010. Subclinical phenotypes of asthma. Curr. Opin. Allergy Clin. Immunol. 10, 54–59. Chan, A., Newman, D.L., Shon, A.M., Schneider, D.H., Kuldanek, S., Ober, C., 2006. Variation in the type I interferon gene cluster on 9p21 influences susceptibility to asthma and atopy. Genes Immun. 7, 169–178. Cho, S.H., Stanciu, L.A., Holgate, S.T., Johnston, S.L., 2005. Increased interleukin-4, interleukin-5, and interferon-gamma in airway CD4 + and CD8 + T cells in atopic asthma. Am. J. Respir. Crit. Care Med. 171, 224–230. Chung, F., 2001. Anti-inflammatory cytokines in asthma and allergy: interleukin-10, interleukin-12, interferon-gamma. Mediators Inflamm. 10, 51–59. Contoli, M., et al., 2006. Role of deficient type III interferon-lambda production in asthma exacerbations. Nat. Med. 12, 1023–1026. Cunninghame Graham, D.S., et al., 2007. Association of IRF5 in UK SLE families identifies a variant involved in polyadenylation. Hum. Mol. Genet. 16, 579–591. Dideberg, V., et al., 2007. An insertion–deletion polymorphism in the interferon regulatory factor 5 (IRF5) gene confers risk of inflammatory bowel diseases. Hum. Mol. Genet. 16, 3008–3016. Dieude, P., et al., 2009. Association between the IRF5 rs2004640 functional polymorphism and systemic sclerosis: a new perspective for pulmonary fibrosis. Arthritis Rheum. 60, 225–233. Dieude, P., et al., 2010. Phenotype–haplotype correlation of IRF5 in systemic sclerosis: role of 2 haplotypes in disease severity. J. Rheumatol. 37, 987–992. Douwes, J., Gibson, P., Pekkanen, J., Pearce, N., 2002. Non-eosinophilic asthma: importance and possible mechanisms. Thorax 57, 643–648. Graham, R.R., et al., 2006. A common haplotype of interferon regulatory factor 5 (IRF5) regulates splicing and expression and is associated with increased risk of systemic lupus erythematosus. Nat. Genet. 38, 550–555. Gratzl, S., Palca, A., Schmitz, M., Simon, H.U., 2000. Treatment with IFN-alpha in corticosteroid-unresponsive asthma. J. Allergy Clin. Immunol. 105, 1035–1036.

225

Hammad, H., Lambrecht, B.N., 2008. Dendritic cells and epithelial cells: linking innate and adaptive immunity in asthma. Nat. Rev. Immunol. 8, 193–204. Holgate, S.T., Polosa, R., 2006. The mechanisms, diagnosis, and management of severe asthma in adults. Lancet 368, 780–793. Hollams, E.M., et al., 2009. Elucidation of asthma phenotypes in atopic teenagers through parallel immunophenotypic and clinical profiling. J. Allergy Clin. Immunol. 124, 463–470 (470 e461-416). Ito, I., et al., 2009. Association of a functional polymorphism in the IRF5 region with systemic sclerosis in a Japanese population. Arthritis Rheum. 60, 1845–1850. Johnston, S.L., 2007. Innate immunity in the pathogenesis of virus-induced asthma exacerbations. Proc. Am. Thorac. Soc. 4, 267–270. Koppelman, G.H., et al., 2002. Genome-wide search for atopy susceptibility genes in Dutch families with asthma. J. Allergy Clin. Immunol. 109, 498–506. Krausgruber, T., Saliba, D., Ryzhakov, G., Lanfrancotti, A., Blazek, K., Udalova, I.A., 2010. IRF5 is required for late-phase TNF secretion by human dendritic cells. Blood 115, 4421–4430. Krausgruber, T., et al., 2011. IRF5 promotes inflammatory macrophage polarization and TH1–TH17 responses. Nat. Immunol. 12, 231–238. Kristjansdottir, G., et al., 2008. Interferon regulatory factor 5 (IRF5) gene variants are associated with multiple sclerosis in three distinct populations. J. Med. Genet. 45, 362–369. Laird, N.M., Horvath, S., Xu, X., 2000. Implementing a unified approach to family-based tests of association. Genet. Epidemiol. 19 (Suppl. 1), S36–S42. Lange, C., DeMeo, D., Silverman, E.K., Weiss, S.T., Laird, N.M., 2004. PBAT: tools for family-based association studies. Am. J. Hum. Genet. 74, 367–369. Le Souef, P.N., 2009. Gene–environmental interaction in the development of atopic asthma: new developments. Curr. Opin. Allergy Clin. Immunol. 9, 123–127. Lien, C., Fang, C.M., Huso, D., Livak, F., Lu, R., Pitha, P.M., 2010. Critical role of IRF-5 in regulation of B-cell differentiation. Proc. Natl. Acad. Sci. U.S.A. 107, 4664–4668. Luna, A., Nicodemus, K.K., 2007. snp.plotter: an R-based SNP/haplotype association and linkage disequilibrium plotting package. Bioinformatics 23, 774–776. Lunetta, K.L., Faraone, S.V., Biederman, J., Laird, N.M., 2000. Family-based tests of association and linkage that use unaffected sibs, covariates, and interactions. Am. J. Hum. Genet. 66, 605–614. Martinez, F.O., 2011. Regulators of macrophage activation. Eur. J. Immunol. 41, 1531–1534. Message, S.D., et al., 2008. Rhinovirus-induced lower respiratory illness is increased in asthma and related to virus load and Th1/2 cytokine and IL-10 production. Proc. Natl. Acad. Sci. U.S.A. 105, 13562–13567. Miceli-Richard, C., Comets, E., Loiseau, P., Puechal, X., Hachulla, E., Mariette, X., 2007. Association of an IRF5 gene functional polymorphism with Sjogren's syndrome. Arthritis Rheum. 56, 3989–3994. Nieves, A., et al., 2005. Phenotypes of asthma revisited upon the presence of atopy. Respir. Med. 99, 347–354. Nordmark, G., et al., 2009. Additive effects of the major risk alleles of IRF5 and STAT4 in primary Sjogren's syndrome. Genes Immun. 10, 68–76. Paun, A., et al., 2008. Functional characterization of murine interferon regulatory factor 5 (IRF-5) and its role in the innate antiviral response. J. Biol. Chem. 283, 14295–14308. Plummeridge, M.J., Armstrong, L., Birchall, M.A., Millar, A.B., 2000. Reduced production of interleukin 12 by interferon gamma primed alveolar macrophages from atopic asthmatic subjects. Thorax 55, 842–847. Radstake, T.R., et al., 2010. Genome-wide association study of systemic sclerosis identifies CD247 as a new susceptibility locus. Nat. Genet. 42, 426–429. Reijmerink, N.E., et al., 2010. TLR-related pathway analysis: novel gene-gene interactions in the development of asthma and atopy. Allergy 65, 199–207. Romanet-Manent, S., Charpin, D., Magnan, A., Lanteaume, A., Vervloet, D., 2002. Allergic vs nonallergic asthma: what makes the difference? Allergy 57, 607–613. Ronnblom, L., Pascual, V., 2008. The innate immune system in SLE: type I interferons and dendritic cells. Lupus 17, 394–399. Rottem, M., Shoenfeld, Y., 2003. Asthma as a paradigm for autoimmune disease. Int. Arch. Allergy Immunol. 132, 210–214. Sayers, I., et al., 2003. Allelic association and functional studies of promoter polymorphism in the leukotriene C4 synthase gene (LTC4S) in asthma. Thorax 58, 417–424. Schroder, N.W., 2009. The role of innate immunity in the pathogenesis of asthma. Curr. Opin. Allergy Clin. Immunol. 9, 38–43. Sigurdsson, S., et al., 2007. Association of a haplotype in the promoter region of the interferon regulatory factor 5 gene with rheumatoid arthritis. Arthritis Rheum. 56, 2202–2210. Sigurdsson, S., et al., 2008. Comprehensive evaluation of the genetic variants of interferon regulatory factor 5 (IRF5) reveals a novel 5 bp length polymorphism as strong risk factor for systemic lupus erythematosus. Hum. Mol. Genet. 17, 872–881. Van Eerdewegh, P., et al., 2002. Association of the ADAM33 gene with asthma and bronchial hyperresponsiveness. Nature 418, 426–430. Wang, C., et al., 2011. Preferential association of interferon regulatory factor 5 gene variants with seronegative rheumatoid arthritis in 2 Swedish case‐control studies. J. Rheumatol. 38, 2130–2132. Wark, P.A., et al., 2005. Asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus. J. Exp. Med. 201, 937–947. Yanai, H., et al., 2007. Role of IFN regulatory factor 5 transcription factor in antiviral immunity and tumor suppression. Proc. Natl. Acad. Sci. U.S.A. 104, 3402–3407. Yang, I.A., Fong, K.M., Holgate, S.T., Holloway, J.W., 2006. The role of toll-like receptors and related receptors of the innate immune system in asthma. Curr. Opin. Allergy Clin. Immunol. 6, 23–28.