The IL1B-511 Polymorphism (rs16944 AA Genotype) Is Increased in Aspirin-Exacerbated Respiratory Disease in Mexican Population

Aspirin exacerbated respiratory disease (AERD) is characterized by chronic hyperplastic rhinosinusitis, nasal polyposis, asthma, and aspirin sensitivity. The mechanisms which produce these manifestations of intolerance are not fully defined, current research focuses on cyclooxygenase 1 (COX-1) inhibition, metabolism of arachidonic acid, and the COX pathway to the lipoxygenase (LO) route, inducing increased synthesis of leukotrienes (LT). The biological plausibility of this model has led to the search for polymorphisms in genes responsible for proinflammatory cytokines synthesis, such as IL1B and IL8. We performed a genetic association study between IL8-251 (rs4073) and IL1B-511 (rs16944) polymorphisms in AERD, aspirin-tolerant asthma (ATA), and healthy control subjects. Using allelic discrimination by real-time PCR, we found statistically nonsignificant associations between AERD, ATA, and healthy control subjects for the GG and GA genotypes of IL1B (rs16944). Interestingly, the AA genotype showed an increased frequency in the AERD patients versus the ATA group (GF = 0.19 versus 0.07, p = 0.018, OR 2.98, and 95% CI 1.17–7.82). This is the first observation that IL1B polymorphisms are involved in AERD. Thus, future studies must investigate whether interleukin-1β is released in the airways of AERD patients and whether it relates to genetic polymorphisms in the IL1B gene.


Introduction
Aspirin-exacerbated respiratory disease (AERD) is a syndrome characterized by chronic hyperplastic rhinosinusitis, nasal polyposis, asthma, and aspirin sensitivity, as described in 1922 by SzcZeklik et al. [1,2]. The prevalence of AERD is variable; Stevenson and Szczeklik reported in 2006 that AERD occurs in 3% of adult patients with asthma in the United States, with the onset of symptoms during the third decade of life, and that it is more common in women than in men, with approximately 70% versus 30% in Europe [2] and 57% versus 43% in the USA [3]. The mechanisms underlying aspirin intolerance are not fully defined, with current research focusing on cyclooxygenase 1 (COX-1) inhibition by aspirin and other NSAIDs diverting arachidonic acid metabolism from COX pathways to the lipoxygenase (LO) pathway. This leads to increased synthesis of the cysteinylleukotrienes (LT), LTC 4 , LTD 4 , and LTE 4 , resulting in bronchoconstriction, mucus hypersecretion, and possibly the development of polyps and urticaria [1]. The biological plausibility of this hypothesis fact has led to the search for polymorphisms in genes responsible for LT synthesis, to explore associations between these polymorphisms and local tissue levels of the proteins.
Other factors such as polymorphisms in the genes for proinflammatory cytokines including TNF, IL1B, IL6, and IL8 are involved in chronic inflammatory and autoimmune diseases. Interleukin 1 (IL-1) is a cytokine associated with inflammatory responses and found in two forms, IL-1α (produced by the IL1A gene) and IL-1β (IL1B), with both genes located on chromosome 2. IL-1 is expressed in nasal polyps, nasal epithelium, macrophages, activated T lymphocytes, and monocytes; its expression is regulated by adhesion molecules, and others inflammatory cytokines [4]. IL1B polymorphisms have been associated with inflammatory bowel disease and gastric cancer among other diseases [5]. Recently, genetic polymorphisms in proinflammatory cytokines such as IL-1β have been recognized as key players in the pathogenesis of asthma [6]. Similarly, IL-8 has been implicated in the asthmatic inflammatory process, and genetic variation in this cytokine has been associated with both the susceptibility and the severity of this disease [7].
In the present study, we have investigated the frequencies of polymorphisms in the genes encoding these two cytokines in AERD patients.

Subjects.
Patients with aspirin-exacerbated respiratory disease (AERD) (n = 78) and aspirin-tolerant asthma (ATA) (n = 135) were recruited from the allergy and otolaryngology departments at the Instituto Nacional de Enfermedades Respiratorias (INER). Healthy control subjects (HCS) (n = 134) were invited to participate through AERD-screening campaigns. All participants underwent simple spirometry, inhaled methacholine challenge, and nasal challenge with lysine-aspirin (L-ASA) according to international guidelines to determine the degree of bronchial hyperresponsiveness and confirm AERD diagnosis. The AERD group had positive L-ASA and methacholine challenges, the asthmatic group had positive methacholine challenge, but negative L-ASA challenge, and the healthy control subjects were volunteers with negative L-ASA and methacholine challenges. A positive L-ASA challenge was defined as a decrease of at least 40% in total nasal airflow after L-ASA application compared with baseline measures; methacholine challenge was considered positive with a decrease of at least 20% in forced expiratory volume in one second (FEV 1 ) compared with baseline FEV 1 after the administration of different concentrations of methacholine (beginning with 0.03 mg/mL, increasing gradually the concentration twice every 2 minutes until the concentration of 32 mg/mL); in case it does not have it, it is considered negative. Blood samples were collected for genotyping studies in the HLA laboratory research. The study was approved by the science and bioethics committees of INER, and all participants gave their informed consent [8,9]. Patients and healthy control subjects had ancestry of at least two generations born in Mexico and were thus considered to be Mexican Mestizo in descent [10,11].

DNA Extraction.
Peripheral blood was drawn by venipuncture, and genomic DNA was obtained using the commercial BDtract DNA isolation kit (Maxim Biotech, San Francisco, Calif, USA). The DNA was quantified by absorption of ultraviolet light at 260 nm wavelength using an ACTGene spectrophotometer (ACTGene, Inc., NJ, USA).

SNP Selection.
We selected two polymorphisms in two genes related to chronic inflammation: rs16944 in IL1B and rs4073 in IL8. Genetic data from each of the polymorphisms are described in Table 1

Results
Clinical data for the three groups were compared and are described in Table 2. We performed a genetic association study of the IL8 (rs4073) and IL1B (rs16944) gene polymorphisms in the three groups. Genetic data for the SNPs included in this study are shown in Table 1; minor allele frequency (MAF) of the polymorphisms tested in healthy control subjects had a similar distribution to that reported in international databases (Table 1). Gene frequencies for each genotype within the three subject groups are shown in AERD: Aspirin-exacerbated respiratory disease, ATA: Aspirin-tolerant asthma, HCS: Healthy control subjects, SD: Standard deviation, FEV 1 : Forced expiratory volume in one second, L-ASA: Lysine aspirin. AERD: Aspirin-exacerbated respiratory disease, ATA: aspirin-tolerant asthma, HCS: healthy control subjects, GF: genotype frequency. Table 3. The frequency of genotypes AA, AT, and TT of the IL8 rs4073 SNP was not statistically significant and different between the three studied groups. Analysis of GG and GA genotypes of the IL1B (rs16944) SNP for AERD and ATA patients versus the healthy control subjects showed nonstatistically significant associations. Interestingly, the AA genotype showed increased frequency in the AERD patients when compared to the ATA group (GF = 0.19 versus 0.07); this association was statistically significant (p = 0.018, OR 2.98, and CI 1.17-7.82) (Tables 3 and 4) and was not found when AERD or ATA groups were compared to healthy control subjects. There is no difference in AA versus AG + GG using contrast of healthy control subjects versus AERD patients (data not shown).

Discussion
The airways of aspirin-sensitive patients are characterized by chronic inflammation with cell infiltration even when they are not exposed to aspirin or other NSAIDs [12]. In addition to alterations in the metabolism of arachidonic acid, several proinflammatory cytokines have been associated with AERD. The role of IL-1, however, has not been investigated previously. Here, we report that AERD patients show an increased frequency of the IL1B-511 polymorphism (rs16944 AA genotype) compared to aspirin-tolerant asthmatics.
Interleukin-1β has been reported to be involved in the genesis of both asthma [13,14] and chronic rhinosinusitis with nasal polyposis in a Turkish population [15]. Most studies have attempted to establish the association of polymorphisms in the IL1B promoter gene, mainly at positions-511 G/A (rs16944) and -31 C/T. For example, Park et al., in 2004, did not find any association between these polymorphisms and either asthma or atopy in a Korean population [16]. In 2007, Erbek et al. described a susceptibility for developing nasal polyps associated with the IL1B-511 polymorphism (rs16944) [15], but Mfuna Endam et al., in 2010, failed to reproduce this finding in Canadian patients with chronic rhinosinusitis [17]. In 2003, Allen et al. did not find any association with IL1 gene polymorphisms in asthmatic families (n = 244), but they reported an association with the DNA microsatellite D2S308 in these asthma families (p = 0.00001) [18]. In parallel, Karajalein et al. evaluated 245 patients with asthma and nasal polyposis and did not find any association between the polymorphism IL1B-511 4 Journal of Allergy C/T and nasal polyps [19]. Evidence for the role of IL-1 in pulmonary immune responses has been gathered in murine models of allergic asthma using IL-1R1-deficient [IL-1R1 (−/−)] mice; changes observed in these mice include significant reduction of pulmonary eosinophilic inflammation, diminished goblet cell hyperplasia, and reduction of cell recruitment to the lungs, as compared to control BALB/c mice [20]. However, there are no studies linking gene promoter polymorphisms and levels of expression of the cytokine in the lung microenvironment.
In the present study, we have demonstrated that the frequency of the IL1B-511 polymorphism (rs16944 AA genotype) is three-fold higher in AERD (19.2%) than in ATA patients (7.4%), suggesting that patients carrying this polymorphism may exhibit genetic susceptibility to develop AERD.
Findings on the biological functionality of the rs16944 polymorphism have not been consistent across studies. The AA genotype has been associated with higher gastric mucosal levels of IL-1β in bacterial infections [21], while mononuclear cells from subjects with the GG genotype showed an increased release of IL-1β after stimulation with lipopolysaccharide [22]. Recent studies suggest that the functional role of rs16944 may depend on IL1B promoter region haplotypes including rs16944 [23][24][25][26]. Although the findings are inconsistent, these previous studies suggest that rs16944 could affect the expression levels of IL-1β. Our report is the first demonstration of the involvement of IL1B polymorphism in AERD. The sample size is relatively small, particularly in the AERD group, which may limit the statistical power, so it would be desirable to replicate our findings in an independent population.
Future studies must investigate whether this cytokine is released in the airways of AERD patients and whether its levels relate to genetic polymorphisms in the IL1B gene.
Interleukin-8 has been implicated in asthma and found in high concentrations in bronchoalveolar lavage fluid of patients with acute asthma exacerbations [26]. In fact, polymorphic IL8 alleles (−251T and 781C) have been associated with asthma in a European population [7,27], but not in asthmatics of Korean origin [7]; the differences could be explained by the different ethnic populations studied. In contrast, Korean asthmatics were found to show four nonsynonymous amino acid substitutions in the IL-8 receptor A (IL8RA) and an association of one synonymous variation in IL8RB [28]. In the present study, we did not find a significant difference in the rs4073 A/T between the AERD group and ATA patients.