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Erbek SS, Yurtcu E, Erbek S, Atac FB, Sahin FI, Cakmak O. Proinflammatory Cytokine Single Nucleotide Polymorphisms in Nasal Polyposis. Arch Otolaryngol Head Neck Surg. 2007;133(7):705–709. doi:10.1001/archotol.133.7.705
To investigate the association between nasal polyposis (NP) and single nucleotide polymorphisms of the proinflammatory cytokines IL (interleukin) 1α (the IL1A gene), IL-1β (the IL1B gene), and tumor necrosis factor α (the TNFA gene).
Prospective case-control trial.
Tertiary referral center.
Eighty-two patients with NP and 106 healthy volunteers without sinonasal disease.
Main Outcome Measures
Genotypes of IL1A (4845G, 4845T), IL1B (–511C, –511T) and TNFA (–238G, –238A and –308G, –308A) were identified by restriction fragment length polymorphism analyses after polymerase chain reaction.
The 4845 GT and 4845 TT genotypes of the IL1A gene were associated with NP (P < .05). The frequency of the –511 CC genotype of the IL1B gene was significantly higher in patients with NP than in controls (P = .01). The frequency of the –511 CT genotype of IL1B was significantly higher (P = .01) in the controls than in the patients with NP. The –238 AA genotype of the TNFA gene was higher in the patients with NP than in the controls (P = .05). There was a significantly high risk of susceptibility to NP in patients with the –308 GA genotype of TNFA (P = .001). None of the genotypes of the proinflammatory cytokines were related to sex, the presence of atopy, asthma, or aspirin intolerance (P > .05).
The IL1A (4845 GT and 4845 TT), IL1B (–511 CC), and TNFA (–238 AA and –308 GA) genotypes were associated with susceptibility to NP in our study population.
Nasal polyposis (NP), a chronic disease of the nasal and paranasal sinus mucosa, is characterized by proliferation of the epithelial layer, glandular hyperplasia, thickening of the basement membrane, edema, focal fibrosis, and cellular infiltration of the stromal layer.1 Nasal polyposis is frequently associated with asthma and aspirin intolerance. Recent studies of the underlying mechanism of NP strongly suggest that it is a multifactorial disease with several etiologic factors. Chronic persistent inflammation is undoubtedly a major factor in the development of NP, regardless of its underlying cause. Polyp tissue includes mixed inflammatory cells, of which eosinophils are the most dominant; they have the primary role in the perpetuation of chronic inflammation. However, polyp tissue eosinophilia is an entity independent of atopy. Wei et al2 suggested that eosinophils more often migrate to nasal tissue in patients with chronic sinusitis than in controls. They concluded that research should focus on eosinophil activation and chemotaxis pathways.2
Tumor necrosis factor α (TNF-α) and IL (interleukin) 1 are members of the proinflammatory cytokine gene family; they are produced by various cells, including epithelial cells and macrophages. Those cytokines act synergistically in the process of chronic inflammation. Tumor necrosis factor α and IL-1 regulate the extravasation of eosinophils into the lamina propria by up-regulating adhesion molecule expression in nasal polyps.3
The delicate balance between proinflammatory and anti-inflammatory cytokines may regulate the inflammatory reaction in NP as it does in other infectious diseases. It has been suggested that various genetic and epigenetic factors modify the severity of chronic inflammatory diseases.4 Among the genetic factors, single nucleotide polymorphisms (SNPs) or microsatellite polymorphisms (particularly those within the regulatory regions of genes that code for cytokines) often affect expression levels and can serve as disease modifiers.
Interleukin 1, a pleotrophic cytokine that is produced in response to pathogenic infection, is a well-established mediator of chronic inflammatory disease. In humans, the IL-1 cytokine gene family consists of 3 genes located on the long arm of chromosome 2 that encode for the IL-1α, IL-1β, and the IL-1 receptor antagonist. There are 2 variants in the IL1A gene (at sites −889 and 4845 [both C→T and in linkage disequilibrium]). Three SNPs in the IL1B gene have been described; 2 of them are located in the promoter region (−511 and −31 [both C→T]), and the other is in the coding region (3954C, 3954T).4,5 Those transitions affect the level of IL-1 expression in response to various stimuli, and their presence has been associated with a variety of immune and chronic inflammatory diseases. Karjalainen et al6 investigated the association of IL1A and IL1B genotypes with NP. However, their study was limited to patients with asthma.
The TNFA gene is located on human chromosome 6 between HLA-B and HLA-DR within the class III region of the major histocompatibility complex.7 Among the reported polymorphisms defined in the TNFA gene, promoter polymorphisms at positions −238 and −308 are the best characterized and have been shown to influence the production of protein at the transcriptional level. As discussed by Haukim et al,4 there have been several recent studies that reported the association of TNFA (−238G, −238A and −308G, −308A) polymorphisms with certain chronic diseases, including asthma. Recently, the association of the TNFA genotype and simple NP without allergy or aspirin intolerance was reported by Fajardo-Dolci et al8; however, they could not define the association between simple NP and TNFA (−238G, −238A and −308G, −308A) polymorphisms in their study population. In view of the location and proposed biologic effect of TNF-α and IL-1, we thought that it would be prudent to further evaluate the association between TNFA (−238G, −238A and −308G, −308A), IL1A (4845G, 4845T), and IL1B (−511C, −511T) polymorphisms and NP. We also investigated whether the genotypes of the proinflammatory cytokines mentioned in this section were related to presence of allergy, asthma, or aspirin intolerance in patients with NP.
A prospective study was conducted with 82 consecutive patients with NP. The diagnosis of NP was based on each patient's medical history and on the results of nasal endoscopy and computed tomography. Diagnoses of asthma or the acetylsalicylic acid triad (aspirin intolerance, asthma, and nasal polyposis) were based on the patients' medical history and examinations in the department of pulmonology. The presence of an antrochoanal polyp, cystic fibrosis, or an inverted papilloma were the exclusion criteria. The control group consisted of 106 healthy volunteers (65 men, 41 women; mean [SD] age, 45.1 [12.6] years) without sinonasal disease. All participants provided written informed consent, and the ethics committee of Baskent University, Ankara, Turkey, approved the study protocol.
Polyp size was classified on a 0 to 3 scale as described by Lildholdt et al.9 The results of paranasal sinus computed tomography were staged according to the Lund and Mackay staging system.10 Skin prick tests were performed on all patients. Each patient was evaluated for sensitivity to 18 common allergen extracts (ALK Abello, Madrid, Spain) and to positive and negative control substances. A test result was considered positive for sensitivity when at least 1 of the induration diameters was 3 mm higher than that in the negative control. Serum total IgE values, which were determined by means of the chemiluminescent immunoassay method, and serum total eosinophil counts were also obtained.
Peripheral blood samples were drawn from all participants. Genomic DNA (hereinafter, DNA) was extracted from peripheral blood leukocytes by means of a high pure polymerase chain reaction (PCR) template preparation kit (Roche Diagnostics GmbH, Mannheim, Germany). Genotypes of IL1A (4845G, 4845T), IL1B (−5111C, −5111T), and TNFA (−238G, −238A and −308G, −308A) were determined by restriction fragment length polymorphism analyses after PCR with appropriate primers according to the slightly modified procedures previously described.8,11
The 4845G, 4845T polymorphism of the IL1A gene was identified after digestion with the restriction enzyme SatI, which yielded 124–, 76–, and 29–base pair (bp) bands in the presence of allele G, as well as 153- and 76-bp bands in the presence of allele A.
The IL1B (−511C, −511T) polymorphism was identified after digestion with AvaI, which yielded 305-bp bands in the presence of allele C, as well as 190- and 115-bp bands in the presence of allele T.
To detect the −238G, −238A polymorphism of the TNFA gene, a 152-bp PCR product was cut with MspI. The uncut product (152 bp) showed the presence of the A allele. If the PCR product was cut into 2 fragments (as 132 and 20 bp), it revealed the G allele.
A 220-bp PCR product was cut with NcoI to reveal the TNFA −308A, −308G polymorphism. The uncut product (220 bp) showed the presence of the A allele. If the PCR product was cut into 2 fragments (as 201 and 19 bp), it revealed the G allele.
Calculations were performed with SPSS (version 11.0; SSPS Inc, Chicago, Illinois) and MINITAB (version 13.0; Minitab Inc, State College, Pennsylvania) statistical software. Cytokine genotypes in the patients with NP and in the controls were compared by means of the Pearson χ2 test, χ2 test, and 2-proportion z score. The χ2 test was also used to evaluate association of atopy, sex, and polyp size with cytokine genotypes in patients with NP. The 1-way analysis of variance test was used to correlate the presence of blood eosinophilia, total IgE levels, and the results of computed tomography with cytokine genotypes in the patients with NP.
Of the 82 patients with NP, 53 (65%) were men and 29 (35%) were women (mean [SD] age, 45.23 [11.77] years; range, 19-78 years). The diagnoses were NP in 55 patients, NP with asthma in 15, and NP with aspirin-induced asthma in 12. The clinical characteristics of the patients are shown in Table 1.
The genotypes did not differ according to the sex of the subjects (P > .05). There were no differences in any of the genotypes of IL1A, IL1B, or TNFA when patients with only NP were compared with those who had concomitant asthma or aspirin-induced asthma (P > .05). The cytokine genotypes cited were not associated with atopy, blood eosinophilia, or total IgE level (P > .05).
The frequency of the IL1A GG genotype was significantly higher in controls than in patients with NP (P < .001). The 4845 GT and 4845 TT genotypes of the IL1A gene were found to be highly associated with NP (P < .001 and P = .05, respectively), and the susceptibility to NP was markedly increased (common odds ratio, 2.743; P < .001) in these patients. These findings were associated with the higher frequency of the T allele in patients with NP vs controls (P < .001). On the one hand, the frequency of the –511 CC genotype of the IL1B gene was significantly higher in patients with NP than in controls (P = .01). On the other hand, the −511 CT genotype frequency of IL1B was significantly higher in the controls than in the patients with NP (P = .01) (Table 2). However, the IL1B −511 TT genotype was similar in both groups (P > .05).
The frequencies of the –238 GG and –238 GA genotypes of the TNFA gene were similar in patients with NP and in controls (Table 3). However, the –238 AA genotype of the TNFA gene was significantly higher in patients with NP than in controls (P = .05). The frequency of the –308 GG genotype of the TNFA gene did not differ in controls vs patients with NP (P > .05). The risk of susceptibility to NP was significantly higher in patients with NP who had a –308 GA genotype of TNFA than in controls (P < .001) (Table 3). However, distribution of the –308 AA genotype of TNFA was significantly higher in the control group than in patients with NP (P = .004).
Neither the polyp size nor the results of CT (P > .05) were associated with the studied proinflammatory cytokine gene polymorphisms.
Most of the changes in the inflammation can be triggered by the activities of TNF-α and IL-1; many of their functions are shared, especially those leading to the amplification of immunologic and inflammatory processes. In this preliminary study, we identified 4 functional polymorphisms (TNFA –238G, –238A and –308G, –308A; IL1A 4845G, 4845T; and IL1B –511C, –511T) in 2 proinflammatory cytokine genes. Those polymorphisms are thought to be related to the inflammatory pathway in NP. Therefore, we examined their potential association with the development of NP. In our series of patients, IL1A (4845 GT and 4845 TT), IL1B (–511 CC), and TNFA (−238 AA and –308 GA) genotypes were associated with susceptibility to NP. None of the genotypes of those cytokines were related to sex, the presence of atopy, asthma, or aspirin intolerance (P > .05). Moreover, the genotypes were not associated with polyp size, sinus opacification, blood eosinophilia, or total IgE level (P > .05).
Interleukin 1 is highly associated with chronic airway disease. The IL1A 4845 GT and 4845 TT genotypes were associated with NP in our series of patients. Moreover, the frequency of the IL1A 4845 GG genotype was significantly higher in our control subjects than that in the subjects with NP (P < .001). This finding contradicts those reported by Karjalainen et al,6 in which IL1A was defined as a specific gene locus and the 4845 GG genotype was identified as an important susceptibility factor for NP in patients with asthma. We found no association between cytokine genotypes and asthma or aspirin-induced asthma in patients with NP. Thus, we concluded that 4845T allele (either in heterozygote or homozygote) of the IL1A gene was solely associated with NP in our series of patients representing the Turkish population. In contrast to other SNPs reported in this study, the IL1A 4845G, 4845T polymorphism is found in the coding region of the gene, and it is in close proximity to a protease cleavage site wherein a calpainlike protease cleaves between amino acids 112 and 113 that convert pro–IL-1α to a mature cytokine. Therefore, the amino acid alteration at residue 114 from alanin to serine may exert an effect owing to the change in the hydrophobisity index of the protein, which is required for the enzymatic efficiency of the protease during the cleavage process. Therefore, it may cause resistance during the conversion of pro–IL-1α to IL-1α that may trigger the fibrotic cascade.
Interleukin 1β is the primary secreted form of IL-1. The cellular effect of a high level of IL-1 may trigger the inflammatory process in NP via stimulation of the synthesis of other inflammatory proteins and/or adhesion molecules. One of the 2 polymorphisms defined in the promoter region of the IL1B gene is −511C, −511T. The increased local production of IL-1β is pronounced in –511 TT carriers.12 The activity of IL-1β has been proposed to play a role in the development of asthma and some other chronic airway diseases as well.13 Recently, Karjalainen et al6 reported the lack of association between IL1B −511C, −511T and NP. In our study, the −511 CC genotype was associated with the NP phenotype (P = .01). Hence, the existence of the heterozygote T allele at this position had a protective effect against NP (P = .01). This finding can be seen as contradictory to the cellular effect of the –511 TT genotype. There may be 2 possible explanations for this result. First, −31C, −31T is the second polymorphism located in the promoter region of IL1B where the T allele is the first base of the TATA box. The allelic interaction between the −511 and −31 polymorphic sites may determine the overall strength of the IL1B promoter. Second, the strong linkage disequilibrium defined among markers of the IL1A-IL1B-IL1-RN genes has led investigators to search for predisposing loci within the highly polymorphic IL1 gene cluster instead of a single loci.14 Therefore the IL-1 system may act in concert to determine an overall inflammatory phenotype, the identification of which may help to pinpoint molecular markers of both NP susceptibility and outcome.15-17
Tumor necrosis factor α stimulates the production of oxygen metabolites that cause toxic cell injury. Data implicate the role of TNF-α in airway remodeling and fibrosis.18 This evidence suggests that the TNF-α level may be a determinant of pathogenesis and disease progression in NP. Because the TNF response to infection is partly regulated at the transcriptional level, TNFA promoter polymorphisms have been the subject of intense interest as potential determinants of disease susceptibility. In a recent report, SNPs in the promoter regions of TNFA (−238 and −308) did not show statistically significant differences between the control group and patients with simple NP.8 We identified a statistically significant association between the −238 AA genotype of TNFA and NP (P < .05). This finding probably emphasizes the role of elevated TNF-α level in inflammation. As reported previously,18 the −238 A allele of the TNFA gene results in increased transcriptional activity. The TNFA repressor site has been identified between −254 and −230 in the TNFA promoter. The increased transcriptional activity might result from a G to A substitution by decreasing the binding of a transcriptional repressor or by enhancing the binding of an activator of transcription by changing the DNA conformation.
The more commonly studied promoter polymorphism of the TNFA gene is at the 308 nucleotide upstream of the transcription start site (G −308A). Tumor necrosis factor α promoter activity is higher when the −308 nucleotide is adenosine because transcription factors preferentially bind to this nucleotide.11 In our study, we could not define a statistically significant association between the −308 AA genotype of TNFA and NP. However, the risk of susceptibility to NP was significantly increased in patients with the −308 GA genotype of the TNFA gene (P < .001). Wilson et al19 reported that the −308 GA genotype of the TNFA gene showed an increased TNF-α level when compared with that of subjects with G homozygotes. All of these findings underline the importance of elevated TNF-α level in triggering the inflammation, but the TNFA gene itself may not be the only factor in determining TNF-α level. Evident data strongly indicate that a TNFA gene polymorphism might contribute to histocompatibility complex associations. It has been reported20 that the polymorphism of the TNFA gene is linked with the HLA haplotype. Moreover, a polymorphic site in the TNFB gene was reported as another molecular determinant of TNF-α production.21 As a result, taking into consideration all reports, the linkage of the 2 TNF genes (TNFA and TNFB) with histocompatibility complex polymorphisms may be responsible for interindividual differences in TNF-α production. Further molecular studies are required to test this hypothesis.
In conclusion, IL1A (4845 GT and 4845 TT), IL1B (−511 CC), and TNFA (−238 AA and −308 GA) genotypes were associated with susceptibility to NP in our series of patients representing the Turkish population. Despite this, it may be possible that other known, or as yet unknown, SNPs within these genes could still be important in the pathogenesis of NP (ie, haplotyping of all the SNPs in a candidate gene may be positive). With this in mind, further research is needed to explore the usefulness of cytokine gene polymorphisms as markers of disease susceptibility (and for risk stratification) and to define their precise role in the pathogenesis of NP. In doing so, areas for therapeutic intervention using population-based treatment strategies, such as use of monoclonal antibodies, might be successfully implemented.
Correspondence: Selim S. Erbek, MD, Department of Otolaryngology, Baskent University Konya Teaching and Research Center, Saray Caddesi No. 1, Selcuklu Konya, Turkey (email@example.com).
Submitted for Publication: December 14, 2006; final revision received March 12, 2007; accepted March 14, 2007.
Author Contributions: Drs S. S. Erbek, Yurtcu, S. Erbek, Atac, Sahin, and Cakmak had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: S. S. Erbek, S. Erbek, Sahin, and Cakmak. Acquisition of data: S. S. Erbek, Yurtcu, S. Erbek, and Atac. Analysis and interpretation of data: S. S. Erbek and Sahin. Drafting of the manuscript: S. S. Erbek, Yurtcu, S. Erbek, and Atac. Critical revision of the manuscript for important intellectual content: Atac, Sahin, and Cakmak. Statistical analysis: Yurtcu. Obtained funding: Yurtcu and Atac. Study supervision: Atac, Sahin, and Cakmak.
Financial Disclosure: None reported.
Funding/Support: This study was supported by the Baskent University Research Fund (project No. KA05/22).
Additional Contributions: Ayse Canan Yazici, PhD, contributed to the statistical analysis; Tendu Gozkaya, BSc, provided excellent technical assistance; and Mehri Demiratan assisted with blood sampling.