Aspirin-exacerbated respiratory diseases (AERD) are associated with the metabolism of arachidonic acid. FPR2 (formyl peptide receptor2) is a high-affinity ligand receptor for potent anti-inflammatory lipid metabolites: lipoxins. Thus, functional alterations of the FPR2 may contribute to AERD. We investigated the relationship between single-nucleotide polymorphisms (SNPs) in the FPR2 and AERD. Asthmatics were categorized into AERD <15% decreases in forced expiratory volume in one second (FEV1), and/or naso-ocular reactions after oral aspirin challenge (n=170) and aspirin-tolerant asthma (ATA, n=268). In all, 11 SNPs were genotyped. FPR2 protein expressions on CD14-positive monocytes in peripheral blood were measured using flow cytometric analysis. We performed RT-PCR of the FPR2 mRNA expressed by peripheral blood mononuclear cells. Logistic regression analysis showed that the minor allele frequency of FPR2 −4209T>G (rs1769490) in intron 2 was significantly lower in the AERD group (n=170) than in the ATA group (n=268) (P=0.006, Pcorr=0.04, recessive model). The decline of FEV1 after aspirin challenge was significantly lower in the subjects with GG homozygotes of FPR2 −4209T>G than those with the other genotypes (P=0.0002). Asthmatic homozygotes for FPR2 −4209T>G minor allele exhibited significantly higher FPR2 protein expression in CD14-positive monocytes than did those with the common allele of FPR2 −4209T>G allele (P=0.01). There was no difference in the expression of the wild form and the exon 2 deleted variant form of FPR2 gene according to the genotypes of FPR2 −4209T>G. The minor allele at FPR2 −4209T>G may have a protective role against the development of AERD, via increase of FPR2 protein expression in inflammatory cells.
Aspirin-exacerbated respiratory diseases (AERD) refer to the development of bronchoconstriction in asthmatic individuals following the ingestion of aspirin (acetylsalicylic acid) or other non-steroidal anti-inflammatory drugs. This syndrome is characterized by the ‘aspirin triad,’ which consists of aspirin hypersensitivity, bronchial asthma, nasal polyposis and chronic hyperplastic eosinophilic sinusitis.1, 2, 3 Similar to other asthmatic individuals, the airways of patients with AERD show signs of persistent inflammation with marked eosinophilia, epithelial disruption, cytokine production and upregulation of inflammatory molecules.4 Overproduction or underproduction of critical modulators important for the metabolism of arachidonic acids, including leukotrienes, lipoxins, thromboxane and prostaglandins, likely accounts for the observed susceptibility to aspirin. Variations within the genes of the arachidonate pathway are responsible for changes in the production and metabolism of these modulators. Polymorphisms in genes such as LTC4S,5 ALOX5,6 PTGER2,7 PTGER3,8 CYSLTR2,9 TBXA2R10 and CYSLTR111 have previously been shown to be associated with AERD. Among the proteins encoded by these genes, the cysteinyl leukotriene (CysLT) receptor is selectively antagonized by several currently available leukotriene modifiers, including montelukast, pranlukast and zafirlukast. However, clinical studies have demonstrated that the response to these medications is incomplete.12, 13 These results suggest that an alternative pathway that does not include CysLT or the CystLT receptor may lead to aspirin hypersensitivity in asthma.
Both LXA4 and aspirin- triggered 15-epi lipoxin 4 (ATL) serve as ‘stop’ signals for leukocyte trafficking, facilitating the resolution of inflammatory responses.14, 15 These responses are mediated by formyl peptide receptor 2 (FPR2, also known as FPRL1, ALXR, HM63, FMLPX, FPR2A, FPRH1 and FPRH2), which is the surface membrane seven-transmembrane G-protein-coupled receptors. FPR2 is expressed by various kinds of inflammatory cells, including macrophages, monocytes, neutrophils, T lymphocytes, bronchial epithelial cells and microvascular endothelial cells.16, 17 These inflammatory cells are involved in the pathogenesis of asthma and in asthmatic subphenotypes, including aspirin hypersensitivity. On the basis of these biological functions, genetic alterations in FPR2 may be postulated to contribute to the pathophysiological processes of AERD. However, a comprehensive genetic analysis of FPR2 in patients with AERD has not been performed to date. To investigate the genetic role of FPR2 in AERD, we identified single-nucleotide polymorphisms (SNPs) of the FPR2 and performed genetic association analyses and functional validation studies to determine the association between the alterations in FPR2 with the risk of AERD.
Materials and methods
Subjects and aspirin challenge
Subjects were recruited from the Asthma Genome Research Center, which is comprised of nine university hospitals in Korea. All subjects were Korean. All patients were diagnosed by physicians based on the definition of asthma by the Global Initiative for Asthma guidelines.18 All patients had a history of dyspnea and wheezing during the previous 12 months and also met one of the following criteria: (1) >15% increase in forced expiratory volume in one second (FEV1) or >12% increase plus 200 ml following inhalation of a short-acting bronchodilator, (2) <10 mg ml−1 PC20 methacholine and (3) >20% increase in FEV1 following 2 weeks of treatment with inhaled steroids and long-acting bronchodilators. A total of 24 common inhalant allergens were utilized for a skin prick test. Atopy was defined as having a wheal reaction equal to or greater than 3 mm in diameter or than that of histamine. Total IgE was measured using the CAP system (Pharmacia Diagnostics, Uppsala, Sweden).
Asthmatic patients had experienced no exacerbation of asthma or respiratory tract infection in the 6 weeks preceding oral aspirin challenge. Oral aspirin challenge was performed with increasing doses of aspirin using methods slightly modified from those described previously.2, 19 Briefly, the patient having history of aspirin hypersensitivity was given 30 mg and those having no history started 100 mg of aspirin orally. Respiratory and naso-ocular symptoms, blood pressure, external signs (urticaria and angio-oedema) and FEV1 were documented every 30 min for a period of 1.5 h. In the absence of any symptoms or signs suggestive of adverse reaction, 60 mg or 100 mg of aspirin was administered and the same measurements were repeated every 1 h, with a final dose of 450 mg or until the patient developed a reaction. If no reaction occurred 4 h after the final dose, the test was deemed negative. Aspirin-induced bronchospasms, reflected by decline (%) of FEV1, were calculated as the pre-challenge FEV1 minus the post-challenge FEV1 divided by the pre-challenge FEV1. Oral aspirin challenge reactions were categorized into two groups as follows: (1) 15% or greater decrease in FEV1 and/or naso-ocular reactions (AERD) or (2) less than 15% decrease in FEV1 without naso-ocular or cutaneous reactions (aspirin tolerant asthma (ATA)). The ethics committee at each hospital approved the protocols and a written informed consent was obtained from all patients.
Genotyping of FPR2 polymorphisms
In all, 11 polymorphisms found in the FPR2 in the National Center for Biotechnology Information (Build 36) were selected for this study, and amplification and extension primers were designed for genotyping each of the polymorphic sites by single-base extension.20 All primer extension reactions were performed using the SNaPshot ddNTP Primer Extension Kit (Applied Biosystems, Foster City, CA, USA). Following the extension reaction, the reaction mixtures were incubated at 37 °C for 1 h with 1 U of shrimp alkaline phosphatase, followed by incubation for 15 min at 72 °C to inactivate the enzyme. The extension products and Geescan 120 Liz size standard solution were then mixed with Hi-Di formamide according to the manufacturer’s instructions and incubated at 95 °C for 5 min, followed by 5 min on ice. The products of the reaction mixture were resolved using an ABI Prism 3100 Genetic Analyzer (Applied Biosystems). Results were analyzed using GeneScan and Genotyper (Applied Biosystems). Primer sequences are shown in Supplementary Table 1. For quality control for genotyping, 16 blank and 16 duplicate were used, and the concordance rate was 100% in this study.
Measurement of FPR2 protein expression on CD14-positive monocytes of asthmatics
Peripheral blood mononuclear cells (PBMC) were purified from the heparinized peripheral blood of the study subjects by using a density gradient reagent (Histopaque 1077). FITC-conjugated anti-rabbit antibody (BD Pharmingen, San Diego, CA, USA), FPR2 antibody (Abcam, Cambridge, UK) and phycoerythrin (PE)-conjugated anti-human CD14 antibody (R&D Systems, Minneapolis, MN, USA) were incubated with 5 × 105 cell ml−1 PBMC and then washed twice with PBS. The stained cells were analyzed by using a flow cytometer (FACScan; Becton-Dickinson, Mountain View, CA, USA). For isotype-matched controls, FITC-conjugated Rabbit IgG (Abcam) and PE-conjugated mouse IgG1 isotype (R&D Systems) were used with the same concentration of each antibody tested. The stained cells were analyzed by using a flow cytometer (FACScan; Becton-Dickinson) and Cell Quest Software (Counter Corporation, Miami, FL, USA). Levels of FPR2 protein expression are monocyte X-mean FPR2/Isotype. Percentage of CD14 (+) monocytes is monocyte gate% FPR2-Isotype.
RNA extraction and RT-PCR of FPR2 mRNA
Total RNA of PBMC was isolated using the modified guanidium thiocyanate-phenol-chloroform extraction method. We quantified RNA and reverse transcribed cDNA from 3 μg of total RNA. DNase I (10 000 U ml−1; Stratagene, La Jolla, CA, USA)-treated RNA was reverse-transcribed by incubating with 0.5 mM dNTP, 2.5 mM MgCl2, 5 mM DTT, 1 μl of random hexamer (50 μg μl−1) and SuperScript II RT (200 U μl−1; Life Technologies, Grand Island, NY, USA) at 42 °C for 50 min, and heat inactivated at 70 °C for 15 min. Specific primer pairs for RT-PCR were as follows GAPDH forward; 5′-IndexTermACCCAGAAGACTGTGGATGG-3′, GAPDH reverse; 5′-IndexTermTTCTAGACGGCAGGTCAGGT-3′ and FPR2 forward; 5′-IndexTermGCACACAGGAAAAGGAGCTT-3′, FPR2 reverse; and 5′-IndexTermACAGATGGTGGTGACTGTGC-3′. Amplification was performed for 35 cycles (one cycle: 30 s at 94 °C, 30 s at 55 °C and 40 s at 72 °C) with initial denaturation at 94 °C for 5 min and a final extension at 72 °C for 10 min. The size and amount of the PCR products generated were determined by agarose gel electrophoresis in the presence of ethidium bromide and analyzed with the Kodak EDAS 1D analysis package. The sequence of RT-PCR products was determined using the BLAST search program after direct sequencing.
We applied two widely used measures of linkage disequilibrium to all pairs of biallelic loci: Lewontin's D′ (∣D′∣)21 and r2. Haplotypes for each individual were inferred using the PHASE algorithm (ver. 2.0), developed by Stephens et al.22 Genotype and haplotype distributions were analyzed using logistic models with age (continuous value), gender (male=0, female=1), smoking status (non-smoker=0, ex-smoker=1, smoker=2), atopy (absence=0, presence=1) and BMI as covariates. Differences in the decline of FEV1 following aspirin challenge among the genotypes were examined using independent t-test. Statistical data were managed and analyzed using SAS version 9.1 (SAS, Inc., Cary, NC, USA) and SPSS version 11.0 (SPSS, Inc., Chicago, IL, USA). To correct P-values, the effective number of independent markers in FPR2 was calculated using SNPSpD software (http://genepi.qimr.edu.au/general/daleN/SNPSpD).23 This program is based on the spectral decomposition of matrices of pairwise linkage disequilibrium between SNPs (P-value X 6.7767). Corrected P-values less than 0.05 were regarded as being statistically significant. Intergroup comparisons of FPR2 expression were assessed by the non-parametric Kruskal–Wallis H-test for continuous data. If differences were found to be statistically significant, Mann–Whitney U-test was applied to analyze differences between the two groups. The FPR2 protein expressions are expressed as the mean±s.e. of the mean (s.e.m.).
P-values less than 0.05 were regarded as being statistically significant.
Characteristics of the study subjects
A total of 438 subjects were recruited from the asthma cohort. The clinical characteristics of the study subjects are summarized in Table 1 . Significant differences in onset age of asthma, prevalence of smoking and BMI were found between the AERD and the ATA groups (P<0.05, Table 2 ). Aspirin-induced declines in FEV1 of −15 to 68% were observed.
Frequencies and haplotype construction of FPR2 polymorphisms
In all, 11 polymorphic SNPs in the FPR2 gene were selected in the National Center for Biotechnology Information SNP database (NCBI SNP DB; build 36). In build 36 of NCBI SNP DB, there were no SNPs in exon 1 and 2 (Figure 1a). The minor allele frequencies of the 11 SNPs in the study subjects are summarized in Supplementary Table 2. The distributions of all loci were in Hardy–Weinberg equilibrium (P>0.05). The linkage disequilibrium coefficients (∣D′∣) and r2 for the FPR2 polymorphisms were calculated for all study subjects (Figure 1c). Absolute linkage disequilibrium (∣D′∣=1 and r2=1) was present between −1750T>C and +1315A>G. Tight linkages were also observed between −4209T>G and −2940C>T (r2>0.99), and among −1750T>C, −1365G>A, −970T>C, −894C>A and −421A>G (r2>0.95). Ten haplotypes were constructed from the 11 SNPs. Among these haplotypes, FPR2-ht2 and FPR2-ht3 (frequency > 0.05) were used for analysis, because FPR2-ht1 and FPR2-ht4 were almost tagged with −1825C>G and −1750T>C, respectively.
Association of FPR2 polymorphisms with the risk of aspirin intolerance in asthmatics
All of the single SNPs and two haplotypes (ht2 and ht3) were analyzed for association with the risk of AERD using logistic models (Table 2). Association analysis revealed that two SNPs, FPR2 −4209T>G and −2940C>T, were significantly associated with AERD in the recessive model (P=0.006 and 0.01). The other SNPs and haplotypes and haplotypes were not associated with the risk of AERD. After correction for multiple comparisons, the significant difference remained only for FPR2 −4209T>G (Pcorr=0.04). The frequency of the homozygous GG genotype for FPR2 −4209T>G was three times lower in AERD patients, a significant difference from ATA patients (4.4% and 13.5%, respectively).
Differences in the rates of FEV1 decline following aspirin challenge among the genotypes and haplotypes were examined using multiple regression models (Figure 2). Asthmatics homozygous for the minor allele of FPR2 − 4209T>G exhibited a lower decline in FEV1 induced by aspirin provocation than did heterozygotes and homozygotes carrying the common allele (11.66±0.76 % vs 6.24±1.19%, P=0.0002, Figure 2).
Comparison of FPR2 protein expression between FPR2 −4209T>G genotypes using flow cytometry
To determine whether the sequence variants of FPR2 −4209T>G affected expression of the FPR2 protein, we measured FPR2 expression in CD14-positive (CD14 (+)) monocytes by flow cytometry (Figure 3a and b). FPR2 protein expression was measured in those obtained from asthmatics with the following genotypes: FPR2 −4209TT (n=26), FPR2 −4209GG (n=6) and FPR2 −4209TG (n=27). FPR2 expression in CD14 (+) monocytes was significantly different among the three groups (P=0.021). The CD14 (+) monocytes from asthmatic patients homozygous for the minor allele of −4209T>G (GG) exhibited a twofolds higher intensity of fluorescence stain for FPR2 protein than did those from patients having the common allele, FPR2 −4209 T (69.41±22.16 vs 27.91±2.24, respectively; P=0.01, Figure 3c). More than 80% CD14 (+) monocytes expressed the FPR2 protein on their surface regardless of the genotypes of FPR2 −4209T>G (Figure 3d).
Comparison of FPR2 mRNA expression between FPR2 −4209T>G genotypes
To investigate the genetic effect of −4209T>G polymorphism on the alternative splicing of FPR2 on PBMC, RT-PCR for the FPR2 mRNA was performed. As FPR2 −4209T>G is located in the intron 2, we amplified mRNA containing FPR2 using primers to detect alternative splicing as described in methods and Figure 4a. We identified a wild-form (upper band, 360 bp) and an alternative form of FPR2 (lower band, 231 bp) (Figure 4b). The wild and the variant forms were similarly found in the RT-PCR products on mRNA from PBMC of the subjects having −4209GG or those having −4209TT. The sequence of RT-PCR products was determined using the BLAST search program after direct sequencing. The alternative form was a variant deleted of exon 2 of 129 bp. (Figure 4c).
In the present study, we are the first, to the best of our knowledge, to identify a SNP located in the intron of FPR2 that is related to aspirin hypersensitivity in asthmatics. This association was characterized using a case–control association study, followed by functional validation of protein expression. The minor allele frequency of FPR2 −4209T>G significantly differed between the AERD and ATA groups, even after correction for multiple comparisons. Few studies have explored the association of FPR2 polymorphisms with human disease. In a previous association study of FPR2 genetic variants, two SNPs (F110S and C126W), located in the open reading frame of FPR2, were found to be associated with juvenile periodontitis.24 These SNPs were not analyzed in our study, because the design of this study was based on the NCBI DB and these two SNPs were not in NCBI DB. These SNPs were not analyzed in the present study. We found that asthmatics homozygous for the FPR2 − 4209T>G minor allele exhibited a lower FEV1 decline after aspirin challenge than patients who were heterozygous or homozygous for the common allele. These data indicate that the minor allele of FPR2 −4209T>G may protect against the development of aspirin-induced bronchospasms in asthmatics.
As the FPR2 was initially cloned based on homology to the FPR cDNA,25, 26 the importance of FPR in the host defense against bacterial infections has been demonstrated by the increased susceptibility of Fpr2 knockout mice to Listeria monocytogenes.27 Additionally, the FPR2 has been demonstrated to have novel roles for lipoxin and aspirin-triggered lipoxin in the regulation of T-cell-mediated responses. The lipoxin and aspirin-triggered lipoxin inhibit TNF-alpha secretion from activated T cells via the FPR2.28 In previous functional studies, transcription of FPR2 was shown to be dramatically upregulated by IL-13, IFN-gamma, IL-4 and IL-6.29, 30 All of these modulators are involved in the development of allergic inflammation in asthmatics.28 In the present study, asthmatic patients homozygous for the minor −4209T>G allele exhibited higher FPR2 protein expression and less decrease of FEV1 fall rate after aspirin challenge. This data suggest that FPR2, a high-affinity ligand receptor for lipoxins and lipoxinA4 (LXA4) analogue, may exert protective effect against aspirin induced bronchospasm. Lipoxin A4 generation and lipoxin A4 receptor expression were found to be decreased in airway tissues of patients with severe asthma.31 Thus, downregulation of lipoxin and aspirin-triggered lipoxin may have additive effects on inflammatory reactions in the presence of low-level expression of the FPR2, as seen in AERD.
In the flow cytometry analysis, we found that asthmatic patients homozygous for the rare −4209T>G allele exhibited higher FPR2 protein expression than patients with the common allele. These findings demonstrate that FPR2 protein expression may be dependent on the genetic function of the −4209T>G allele. It is well known that intronic SNPs can affect transcript processing through alternative splicing or producing RNA secondary structure.32, 33, 34 The FPR2 −4209T>G located in the second intron. Therefore, we have checked the presence of alternatively spliced forms of FPR2 by RT-PCR over exon 1 to exon 3. A 231-bp wild form and a new alternative form of 360 bp were clearly visualized. The expression of the two forms of FPR2 are obvious in the both genotypes of FPR2 −4209T>G. This data indicate that the new alternative spliced variants may not be related with the genotypes of FPR2 −4209T>G. However, we did not measure the m-RNA synthesis of the two forms of FPR2 using real time PCR. Further investigation is needed to better understand the genetic mechanism of the intronic SNP of FPR2 on the generation of the wild and the new-spiced variants of this gene.
Another explanation for the differential protein expression of FPR2 may be different rate of mRNA production, which depends on the secondary structure of mRNA. The secondary structure can be predicted with using mfold web server version 3.1.35 The secondary structure containing the T allele of −4209T>G has a ΔG of −150.59 kcal mol−1 and that with G allele has a ΔG of −150.00 kcal mol−1. These data indicate that synthesis rate of FPR2 mRNA may be not different according to the genotypes of FPR2 −4209T>G. Further investigation is needed to better understand the genetic mechanism of the intronic SNP of FPR2 in aspirin induced asthma (AIA) and to clarify the molecular function of the new-spiced variants of this gene.
In conclusion, we genotyped 11 SNPs located within FPR2 and examined their associations with AERD. Logistic analysis showed that FPR2 −4209T>C is associated with AERD. This association was further validated by showing that patients homozygous for the minor allele of FPR2 −4209T>C exhibit higher levels of FPR2 protein expression in CD14 positive peripheral blood monocytes compared with those having the common allele. These data indicate that the minor allele of FPR2 −4209T>G may have a protective role in the development of aspirin hypersensitivity in asthma through an increase in FPR2 protein expression in inflammatory cells. These results may be useful for the development of new methods to diagnose AERD and new therapeutic strategies for the control of aspirin hypersensitivity in asthma.
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DNA samples were generously provided by the Soonchunhyang University Bucheon Hospital Biobank, a member of the National Biobank of Korea, supported by the Ministry of Health, Welfare and Family Affairs, Republic of Korea.
Supplementary Information accompanies the paper on Journal of Human Genetics website
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Kim, HJ., Cho, SH., Park, JS. et al. Association analysis of formyl peptide receptor 2 (FPR2) polymorphisms and Aspirin exacerbated respiratory diseases. J Hum Genet 57, 247–253 (2012). https://doi.org/10.1038/jhg.2012.12
- FPR2 (formyl peptide receptor 2)
- SNPs (single-nucleotide polymorphisms)
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