Abstract
Introduction:
Chronic rhinosinusitis is a multifactorial disease whose pathogenesis, influenced rhinosinusitis; by both genetic and environmental factors, is still unclear. Previous genetic studies have shown that patients with chronic rhinosinusitis have reduced expression of the Interleukin-22 (IL-22) gene.
Objective:
Identify and compare the frequency of polymorphisms in the IL22RA1 gene (IL22 alpha-1 subunit receptor) among chronic rhinosinusitis patients – either with or without nasal polyps.
Methods:
Peripheral blood samples were collected from 70 chronic rhinosinusitis with polyps patients, 14 chronic rhinosinusitis without polyps patients and 68 subjects without chronic rhinosinusitis, followed by DNA extraction and IL22RA1 gene sequence analysis.
Results:
Among ten polymorphisms identified in the IL22RA1 gene, three were not found in any of the genetic databases analyzed. Chronic rhinosinusitis patients displayed higher frequency of the c.113_114insA frameshift insertion, possibly pathogenic. Conversely, in the control group, polymorphism c.435A > C had a significant predominance of the mutated allele, perhaps related to a potential protection against the chronic rhinosinusitis phenotype. Polymorphism c.770C > T, characterized as a non-synonymous variant, was exclusively found in Black chronic rhinosinusitis with polyps patients.
Conclusions:
Although no direct causal relationship could be established between IL22RA1 gene polymorphisms and the pathophysiology of chronic rhinosinusitis, genetic variations such as c.113_114insA and c.435A > C may be involved in the susceptibility to or protection against the chronic rhinosinusitis phenotype, respectively. Testing this hypothesis will require studies with larger cohorts.
KEYWORDS
Nasal polyps; Chronic rhinosinusitis; Polymorphisms
Resumo
Introdução:
A rinossinusite crônica é uma doença multifatorial cuja patogênese, influenciada por fatores genéticos e ambientais, ainda é incerta. Estudos genéticos anteriores mostraram que pacientes com rinossinusite crônica têm expressão reduzida do gene da Interleucina-22 (IL-22).
Objetivo:
Identificar e comparar a frequência de polimorfismos no gene IL22RA1 (receptor subunidade alfa 1 da interleucina 22) entre os pacientes com rinossinusite crônica com ou sem pólipos nasais.
Método:
Amostras de sangue periférico foram coletadas de 70 pacientes com rinossinusite crônica com pólipos nasais, 14 pacientes com rinossinusite crônica sem pólipos nasais e 68 indivíduos sem rinossinusite crônica, seguido por extração de DNA e análise da sequência do gene IL22RA1.
Resultados:
Dos dez polimorfismos identificados no gene IL22RA1, três não foram encontrados em alguma banco de dados genético analisado. Pacientes com rinossinusite crônica apresentaram maior frequência da inserção frameshift c.113_114insA, possivelmente patogênica. Por outro lado, no grupo controle, o polimorfismo c.435A>C mostrou predominância significativa do alelo mutado, talvez relacionado a uma proteção potencial contra o fenótipo da rinossinusite crônica. O polimorfismo c.770C> T, caracterizado como uma variante não sinônima, foi encontrado exclusivamente em pacientes negros com rinossinusite crônica com pólipos nasais. Conclusões: Embora nenhuma relação causal direta possa ser estabelecida entre os polimorfismos do gene IL22RA1 e a fisiopatologia da rinossinusite crônica, variações genéticas como c.113_114insA e c.435A>C podem estar envolvidas na suscetibilidade ou proteção contra o fenótipo da rinossinusite crônica, respectivamente. Testar essa hipótese exigirá estudos com coortes maiores.
PALAVRAS-CHAVE
Pólipos nasais; Rinossinusite crônica; Polimorfismos
Introduction
Despite the identification of several factors as potentially involved in chronic rhinosinusitis (CRS) – including allergic responses, impaired mucociliary clearance, immune dysfunction, altered epithelial defense, microbial agents, and environmental exposure11 Zhang F, Shang D, Zhang Y, Tian Y. Interleukin-22 suppresses the growth of A498 renal cell carcinoma cells via regulation of STAT1 pathway. PLoS One. 2011;6:e20382. – CRS etiology remains unclear. A more assertive management of the patients requires further clarification on how these factors interact with each other, as well as with genetic factors, in the etiology of CRS.
IL-22 is a cytokine of the IL-10 family that may be involved in acute phase inflammatory response, innate immunity system activation, induced cell migration, dendritic cell inhibition, and attenuation of allergic responses.11 Zhang F, Shang D, Zhang Y, Tian Y. Interleukin-22 suppresses the growth of A498 renal cell carcinoma cells via regulation of STAT1 pathway. PLoS One. 2011;6:e20382.,22 Chung Y, Yang X, Chang SH, Ma L, Tian Q, Dong C. Expression and regulation of IL-22 in the IL-17-producing CD4+ T lymphocytes. Cell Res. 2006;16:902–7. The IL22RA1 receptor may also play an essential role in tissue healing and reorganization. Kotenko et al.33 Kotenko SV, Izotova LS, Mirochnitchenko OV, Esterova E, Dickensheets H, Donnelly RP, et al. Identification of the functional interleukin-22 (IL-22) receptor complex: The IL-10R2 chain (IL-10Rbeta) is a common chain of both the IL-10 and IL-22 (IL-10-related T cell-derived inducible factor, IL-TIF) receptor complexes. J Biol Chem. 2001;276:2725–32. showed that IL22RA1 levels were significantly lower in the epithelial cells of CRS patients with NPs (CRSwNP) who did not respond to surgery. This could result in damage to the epithelial barrier, with a decrease in the production of Th1 pro-inflammatory cytokines, causing an inversion in the Th1/Th2 response pattern. Alternatively, changes in the heterodimer regulatory complex (IL22RA1-IL10R2, IL20, and IL24) could lead to decrease in the innate immune response (β-defensins, mucins and metalloproteinases), increased levels of pro-inflammatory cytokines (IL-6, IL-8, and TNF), and reduced apoptosis,33 Kotenko SV, Izotova LS, Mirochnitchenko OV, Esterova E, Dickensheets H, Donnelly RP, et al. Identification of the functional interleukin-22 (IL-22) receptor complex: The IL-10R2 chain (IL-10Rbeta) is a common chain of both the IL-10 and IL-22 (IL-10-related T cell-derived inducible factor, IL-TIF) receptor complexes. J Biol Chem. 2001;276:2725–32.,44 Dumoutier L, Louahed J, Renauld JC. Cloning and characterization of IL-10-related T cell-derived inducible factor (IL-TIF), a novel cytokine structurally related to IL-10 and inducible by IL-9. J Immunol. 2000;164:1814–9. providing other possible patho- physiological mechanisms involved in the development of CRS.
Although genetic variants might not be the origin of a given disease, they may confer susceptibility to its development. Therefore, the present study aimed to identify variants associated with the IL22RA1 gene in Brazilian patients diagnosed with chronic rhinosinusitis and in individuals without this diagnosis.
Methods
The choice of the IL22RA1 gene resulted from a previous Canadian DNA clustering survey (pool-based Genome-Wide Association Study)55 Fokkens WJ, Lund V, Bachert C, Clement P, Helllings P, Holmstrom M, et al. European position paper on rhinosinusitis and nasal polyps 2012. Rhinology Suppl. 2012;23:1–298. that selected five high-priority Single Nucleotide Polymorphisms (SNPs) and another 18 polymorphisms from the HapMap international project.66 Salgado D, Desvignes JP, Rai G, Blanchard A, Miltgen M, Pinard A, et al. UMD-Predictor: A High-Throughput Sequencing Compliant System for Pathogenicity Prediction of any Human cDNA Substitution. Hum Mutat. 2016;37:439–46. Amongst these 23 SNPs, the IL22RA1 gene was a strong candidate for the development of CRS.55 Fokkens WJ, Lund V, Bachert C, Clement P, Helllings P, Holmstrom M, et al. European position paper on rhinosinusitis and nasal polyps 2012. Rhinology Suppl. 2012;23:1–298.
The present study was approved by the Research Ethics Committee of the Clinical Hospital of the Ribeirão Preto Medical School of the University of São Paulo (HCFMRPUSP) and the National Commission for Research Ethics (CONEP), under Protocol no 5243/2011. All patients and con- trol subjects were invited to participate in the survey after clarification of study procedures and objectives, sample collection method, risks, and possible access to the results, as indicated in a Free and Informed Consent Form (ICF).
Patients and control subjects were recruited at the Otorhinolaryngology Outpatient Clinic of the HCFMRP-USP. According to the clinical criteria of the 2012 European position paper on rhinosinusitis and nasal polyps guideline (EPOS),77 Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, et al. A method and server for predicting damaging missense mutations. Nat Methods. 2010;7:248–9. patients were classified as having CRS with or with- out NPs. The control group comprised 68 volunteers with no nasal symptoms and with normal nasofibroscopy, unrelated to the participants in the other two groups. All participants filled out a questionnaire with their name, age, gender, current and remote origin, tobacco use, skin color and current disease history, including information on seasonal and perennial allergies, asthma and intolerance to acetylsalicylic acid. Patients with Churg-Strauss syndrome, primary ciliary dyskinesia, and cystic fibrosis were excluded from the study.
Plasma was extracted from the peripheral blood samples by centrifugation at 2500 RPM for 10 min at 4◦C. Afterwards, DNA was extracted from the ‘‘buffy coat’’. The seven exons of the IL22RA1 gene were amplified by Polymerase Chain Reaction (PCR), with the seventh exon divided into five sequences, due to its size, to simplify the analysis. The PCR protocol included denaturation at 95◦C, followed by annealing, at temperatures chosen according to the primer used and DNA extension at 72◦C, repeated for approximately 35 cycles until the plateau of the reaction (final phase) was reached. PCR products were analyzed in 1.5 % agarose gels to confirm the amplification and expected size of the fragment.
Each exon was sequenced in the 3500 XL Genetic Analyzer automated sequencer (Applied Biosystems), using the Sanger method. The sequencer output file consists of a chromatogram that contains the information of the sequence and quality of the nucleotides.
The pathogenicity of variants was investigated by UMD-predictor,88 Choi Y, Sims GE, Murphy S, Miller JR, Chan AP. Predicting the functional effect of amino acid substitutions and indels. PLoS One. 2012;7:e46688. which combines biochemical properties of mutated amino acids, impact on splicing signals, location of mutations in functional domains, variant frequency in the global population, conservation of mutated amino acids with the global BLOSUM62 substitution matrix, and conservation of amino acid sequences in 100 species. The predictors PolyPhen299 Kircher M, Witten DM, Jain P, O’Roak BJ, Cooper GM, Shendure J. A general framework for estimating the relative pathogenicity of human genetic variants. Nat Genet. 2014;46:310–5. and Sift/PROVEAN1010 Kopanos C, Tsiolkas V, Kouris A, Chapple CE, Aguilera MA, Meyer R, et al. VarSome: the human genomic variant search engine. Bioinformatics. 2019;35:1978–80. were used to aid the results by the UMD-predictor. Variants were also analyzed with the Combined Annotation Dependent Depletion (CADD) algorithm, which evaluates the degree of mutation pathogenicity, including small insertions and deletions (InDels).1111 Endam LM, Bosse Y, Filali-Mouhim A, Cormier C, Boisvert P, Boulet LP, et al. Polymorphisms in the interleukin-22 receptor alpha-1 gene are associated with severe chronic rhinosinusitis. Otolaryngol Head Neck Surg. 2009;140:741–7. The VarSome platform was used to consolidate all information regarding pathogenicity prediction.1212 International HapMap Consortium. A haplotype map of the human genome. Nature. 2005;437:1299–320.
Fisher’s exact test was used to assess possible associations between the analyzed variables. The polymorphisms analysis was performed in different steps: CRSwNP group versus control group, CRSsNP group versus control group, and CRSwNP group versus CRSsNP group (Tables 1–3).
Statistical analysis comparing the allelic frequencies in patients with chronic rhinosinusitis with nasal polyps and control subjects.
Results
Overall, 152 samples were analyzed: 70 from CRSwNP, 14 from CRSsNP, and 68 from control subjects. Women were 55 % of the study participants, and their mean age was 46.9 years. Regarding skin color, 80 % stated being White, 9 % Brown/Mulatto 16 %, Black 3 %, and 1 % Yellow. Among CRS patients, 13 % had asthma, with most of these having NPs (90 %). Tobacco use was reported by 3 % of participants, mostly in the CRSsNP group. Of the 19 % of participants who had positive allergy skin tests (Prick test), 86 % had NPs. Of the 9 % of participants intolerant to Aspirin, 92 % were CRSwNP patients (Table 4).
Statistical analysis comparing the allelic frequencies in patients with chronic rhinosinusitis without nasal polyps and control subjects.
Statistical analysis comparing the allelic frequencies in patients with chronic rhinosinusitis with and without nasal polyps.
Characterization of the polymorphisms found in the IL22RA1 gene in the different study groups.
The sequencing procedure identified 10 polymorphisms in the IL22RA1 gene, located on chromosome 1 (Table 5). Information on the pathogenicity of the polymorphisms c.135 G > A, c.74 T > A, c.141C > A, c.435A > C, c.770C > T, c.936C > T, and c.1077C > T was not available, but c.613 G > A and c.1552C > G have been described as non-pathogenic. The variant c.113_114insA (Gln26Profs*11) is probably pathogenic, a typical characteristic of frameshift mutations (Table 6).
Variant c.113_114insA (Gln26Profs*11), which had not been previously described, was found mainly in the CRSwNP. This frameshift variant could result in a nonfunctional protein, possibly contributing to CRS development.
Regarding exon 4 in heterozygosity, only variant c.435A > C (p.Pro145=) presented a statistically significant predominance of the mutated allele in the control group (51.5 %) compared to the CRSwNP group, suggesting a possible protection against the CRS phenotype. However, information on the pathogenicity of this polymorphism is not available in the prediction databases.
Discussion
In an attempt to identify possible genetic mechanisms that trigger chronic rhinosinusitis, we compared the polymorphisms of the IL22RA1 gene in Brazilian CRS samples and in a control group, without CRS. We identified a novel mutation in the IL22RA1 gene, the c.113 114insA (Q26Pfs*11) frameshift, a potentially pathogenic mutation that appeared primarily in the patient groups, but there was no statistical significance when compared to the control group. Additionally, polymorphism c.435A > C, in heterozygosity, occurred more frequently in the control group than in the CRSwNP group, suggesting that this variant could confer protection against the development of CRS.
Three polymorphisms have never been described in the genetic databases analyzed (dbSNP, ExAC): Gln26PProfs*11, p.Leu25His, and p.Gly47 = . Variant p.Pro257Leu, detected in two Black patients with NPs, retained a Minor Allele Frequency (MAF) of less than 0.01. As this is a variant characterized as of uncertain significance and has a low frequency in the general population, it would be interesting to perform functional assays to confirm its pathogenicity.
The study conducted by Ramanathan et al.1313 Ramanathan M Jr, Spannhake EW, Lane AP. Chronic rhinosinusitis with nasal polyps is associated with decreased expression of mucosal interleukin 22 receptor. Laryngoscope. 2007;117:1839–43. was the first to demonstrate decreased IL22RA1 gene expression in subjects with CRSwNP compared to subjects without polyps and control group, but no explanation was proposed for this difference. A study with Canadian patients, proposed polymorphisms of the IL22RA1 gene as a potential cause for CRS.99 Kircher M, Witten DM, Jain P, O’Roak BJ, Cooper GM, Shendure J. A general framework for estimating the relative pathogenicity of human genetic variants. Nat Genet. 2014;46:310–5. However, the IL22RA1 polymorphisms described here are distinct from those described by Mfuna-Endam et al. (2009).55 Fokkens WJ, Lund V, Bachert C, Clement P, Helllings P, Holmstrom M, et al. European position paper on rhinosinusitis and nasal polyps 2012. Rhinology Suppl. 2012;23:1–298. This might be due to the distinct populations from which they were sampled, the different environmental factors to which they were exposed, or even to the smaller number of patients analyzed in our study.
We did not investigate how the mutations observed here could contribute to CRS development, with or without NPs. Patients with CRS are known to have lower IL-22 expression, potentially causing epithelial barrier dysfunction and changes in Th1 response, and the inflammatory imbalance between Th1 and Th2 could contribute to CRS development. Variations in the IL22RA1 gene might dysregulate the innate immunity mechanisms, leading to decreases in specific inflammatory mediators. Impairment of the heterodimer complex comprising IL-10R2, IL-20 and IL-24 is another mechanism that could hamper signal transduction and transcription activation (JAK/STAT pathway), leading to dysregulation of pro-inflammatory cytokine levels.
Despite resulting in alterations that may lead to changes in protein configuration, the incidence of the detected polymorphisms was low. Further studies are required, within larger cohorts, to decrease the likelihood that these findings were random, assuring their replicability, and to define the impact of these polymorphisms in the disease in question.
Conclusions
In this study, we identified a novel polymorphism positively associated with CRSwNP, variant c.113_114insA (Q26Pfs*11). Also, a polymorphism in exon 4 was significantly more frequent in the control group than in the CRSwNP group (c.435A > C in heterozygosity), suggesting a potential protection against the CRS phenotype.
Acknowledgements
We thank all the staff of the Genetics Department and Center for Medical Genomics at Clinics Hospital of the Ribeirão Preto Medical School, University of São Paulo, Brazil, who in some way contributed to this research.
References
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1Zhang F, Shang D, Zhang Y, Tian Y. Interleukin-22 suppresses the growth of A498 renal cell carcinoma cells via regulation of STAT1 pathway. PLoS One. 2011;6:e20382.
-
2Chung Y, Yang X, Chang SH, Ma L, Tian Q, Dong C. Expression and regulation of IL-22 in the IL-17-producing CD4+ T lymphocytes. Cell Res. 2006;16:902–7.
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3Kotenko SV, Izotova LS, Mirochnitchenko OV, Esterova E, Dickensheets H, Donnelly RP, et al. Identification of the functional interleukin-22 (IL-22) receptor complex: The IL-10R2 chain (IL-10Rbeta) is a common chain of both the IL-10 and IL-22 (IL-10-related T cell-derived inducible factor, IL-TIF) receptor complexes. J Biol Chem. 2001;276:2725–32.
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4Dumoutier L, Louahed J, Renauld JC. Cloning and characterization of IL-10-related T cell-derived inducible factor (IL-TIF), a novel cytokine structurally related to IL-10 and inducible by IL-9. J Immunol. 2000;164:1814–9.
-
5Fokkens WJ, Lund V, Bachert C, Clement P, Helllings P, Holmstrom M, et al. European position paper on rhinosinusitis and nasal polyps 2012. Rhinology Suppl. 2012;23:1–298.
-
6Salgado D, Desvignes JP, Rai G, Blanchard A, Miltgen M, Pinard A, et al. UMD-Predictor: A High-Throughput Sequencing Compliant System for Pathogenicity Prediction of any Human cDNA Substitution. Hum Mutat. 2016;37:439–46.
-
7Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, et al. A method and server for predicting damaging missense mutations. Nat Methods. 2010;7:248–9.
-
8Choi Y, Sims GE, Murphy S, Miller JR, Chan AP. Predicting the functional effect of amino acid substitutions and indels. PLoS One. 2012;7:e46688.
-
9Kircher M, Witten DM, Jain P, O’Roak BJ, Cooper GM, Shendure J. A general framework for estimating the relative pathogenicity of human genetic variants. Nat Genet. 2014;46:310–5.
-
10Kopanos C, Tsiolkas V, Kouris A, Chapple CE, Aguilera MA, Meyer R, et al. VarSome: the human genomic variant search engine. Bioinformatics. 2019;35:1978–80.
-
11Endam LM, Bosse Y, Filali-Mouhim A, Cormier C, Boisvert P, Boulet LP, et al. Polymorphisms in the interleukin-22 receptor alpha-1 gene are associated with severe chronic rhinosinusitis. Otolaryngol Head Neck Surg. 2009;140:741–7.
-
12International HapMap Consortium. A haplotype map of the human genome. Nature. 2005;437:1299–320.
-
13Ramanathan M Jr, Spannhake EW, Lane AP. Chronic rhinosinusitis with nasal polyps is associated with decreased expression of mucosal interleukin 22 receptor. Laryngoscope. 2007;117:1839–43.
Publication Dates
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Publication in this collection
29 Sept 2021 -
Date of issue
2021
History
-
Received
30 June 2019 -
Accepted
7 Oct 2019 -
Published
15 Nov 2019
