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   and 57% versus 43% in the usa .
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 cysteinyl - leukotrienes ( lt ) , ltc4 , ltd4 , and lte4 , resulting in bronchoconstriction , mucus hypersecretion , and possibly the development of polyps and urticaria .
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-1 ( produced by the il1a gene ) and il-1 ( 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 .
il1b polymorphisms have been associated with inflammatory bowel disease and gastric cancer among other diseases .
recently , genetic polymorphisms in proinflammatory cytokines such as il-1 have been recognized as key players in the pathogenesis of asthma .
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 . in the present study
, we have investigated the frequencies of polymorphisms in the genes encoding these two cytokines in aerd patients .
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 )
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 ( fev1 ) compared with baseline fev1 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 .
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 ] .
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 .
we selected two polymorphisms in two genes related to chronic inflammation : rs16944 in il1b and rs4073 in il8 .
allelic discrimination of snps rs16944 ( il1b ) and rs4073 ( il8 ) was performed by real - time pcr ( rt - pcr ) on a 7300 real time pcr system ( applied biosystems , calif , usa ) using taqman commercial probes ( applied biosystems , usa ) for each of the polymorphisms mentioned above and followed the cycling program : preread 50c , 1 minute ; absolute quantitation : 50c , 2 minutes , 1 cycle ; 95c , 10 minutes , 1 cycle ; 95c , 15 seconds , 60c 1  min , 40 cycles ; postread 50c , 1 minute .
the results were assessed taking into account the allelic discrimination and absolute quantitation in all samples ; additionally , we included four contamination controls per plate ( nontemplate controls ) .
the fluorescence signal detectors used were vic which was assigned to the b allele and fam assigned to the a allele for both snps .
statistical analysis was performed between groups of cases ( aerd and ata ) versus the healthy control subjects by  test with 3  2 tables , using spss ( v. 15.0 ) software for windows , to identify the difference between the allele and genotype frequencies of each polymorphism evaluated .
in addition , odds ratios and 95% confidence intervals were calculated with epi - info ( v. 6.04 ) software .
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 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 1.177.82 ) ( 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 ) .
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 .
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-1 has been reported to be involved in the genesis of both asthma [ 13 , 14 ] and chronic rhinosinusitis with nasal polyposis in a turkish population .
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 . in 2007 , erbek et al .
described a susceptibility for developing nasal polyps associated with the il1b-511 polymorphism ( rs16944 ) , but mfuna endam et al .
, in 2010 , failed to reproduce this finding in canadian patients with chronic rhinosinusitis . 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 ) . in parallel ,
karajalein et al . evaluated 245 patients with asthma and nasal polyposis and did not find any association between the polymorphism il1b-511 c / t and nasal polyps .
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 .
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-1 in bacterial infections , while mononuclear cells from subjects with the gg genotype showed an increased release of il-1 after stimulation with lipopolysaccharide .
recent studies suggest that the functional role of rs16944 may depend on il1b promoter region haplotypes including rs16944 [ 2326 ] .
although the findings are inconsistent , these previous studies suggest that rs16944 could affect the expression levels of il-1. 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 .
in fact , polymorphic il8 alleles ( 251 t and 781c ) have been associated with asthma in a european population [ 7 , 27 ] , but not in asthmatics of korean origin ; 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 . in the present study ,
we did not find a significant difference in the rs4073 a / t between the aerd group and ata patients .
 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  1.177.82 ) . 
 this is the first observation that il1b polymorphisms are involved in aerd . 
 thus , future studies must investigate whether interleukin-1 is released in the airways of aerd patients and whether it relates to genetic polymorphisms in the il1b gene . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion