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Keywords:

  • asthma;
  • IL17A;
  • single nucleotide polymorphism

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

Background:  The interleukin 17A (IL17A) gene, located on chromosome 6p and linked to asthma phenotype, is a highly potential candidate gene conferring asthma susceptibility. The purpose of this study was to investigate the genetic association between single nucleotide polymorphisms (SNPs) of IL17A and asthma in Taiwanese children.

Methods:  We selected and performed genotyping on nine SNPs that encompass the genomic region of IL17A in Taiwanese children with or without asthma. A total of 1939 subjects containing 1027 subjects in testing group and 931 subjects in validation group were recruited in this study.

Results:  After Bonferroni correction, SNP rs8193036 was found to have a weak association (P = 0.0074 × 9 = 0.066) in genotype frequency test. This association was confirmed by validation group. Logistic regression adjusted allergy comorbidity and gender showed a slightly weaker association.

Conclusions:  The results indicated an independent role of IL17A promoter polymorphism rs8193036 in the association with pediatric asthma in Taiwanese population.

Chronic asthma is a complex disease affecting nearly 300 million individuals worldwide (1). The rising incidence of asthma and atopic disorders over the past decades attests to the importance of environmental and lifestyle factors in disease risk assessment (2–4). Strong genetic components associated with asthma were supported by family and twin studies (5, 6), and many genes have been identified or suspected to be involved in the pathogenesis of asthma (7). In Taiwan, there are slight differences in the reported symptoms of allergic diseases; nevertheless, the prevalence of allergic diseases is rising (8–10).

Interleukin 17A (IL17A or known as IL-17), the key cytokine of TH17 cells, is known to induce pro-inflammatory cytokines, the hallmarks of acute inflammatory processes (11). Experimental models suggested that TH17 cells may be important for neutrophilic inflammation in acute airway inflammation (12–14). In obstructive airway diseases, such as bronchial asthma and chronic obstructive pulmonary disease, accumulation of neutrophils in the airways has been a major characteristic (15, 16). Elevation plasma IL17A level associated with asthma severity (17) suggests the potential role in airway remodeling. The accumulated evidence suggests that IL17A may play an important pathologic role in the development of allergies and asthma (18). However, there are no reports on the association of IL17A gene polymorphisms with asthma.

Chromosome 6p of which the genomic region has been reported to be linked to asthma and asthma-related phenotypes in multiple genome scans (19–21). IL17A is located on 6p12.1 the genomic region of which was reported to be associated with different types of asthma (21) but not on 6p21–23 region revealed by linkage studies on asthma (19, 20). Previously, researchers reported that IL17A is a good candidate gene for studying genetic susceptibility of asthma. In our study, we investigated the association between selected single nucleotide polymorphisms (SNPs) of IL17A and asthma and validated the results in another independent subject group. The results strongly indicate that polymorphism in IL17A promoter region is associated with pediatric bronchial asthma in Taiwanese population.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

Sample composition and clinical evaluation

Our study population consists of asthmatic children with their age ranging from 5 to 12 years. The study protocol was approved by the Ethical and Clinical Trial Committee of National Cheng-Kung University Hospital and Mackay Memorial Hospital. Informed consent form was required from all participants or their guardians after they answered a modified British Medical Society respiratory questionnaire, which is the same as that of the European Community Respiratory Health Survey. These surveys have the similar validity as ISAAC pertinent to the diagnosis and assessment of asthma (22, 23). Pulmonary function was tested using standard methods including spirometry before and after the administration of two puffs of inhaled salbutamol (200 μg/puff). The definition of asthma must meet the following criteria: (i) history of having wheezing and experiencing short of breath during or without concurrent respiratory infections, (ii) chronic coughing for more than 1 month and being diagnosed by physician of the presence of wheezing episode(s) and (iii) bronchodilator test has confirmed the positive response of increased 15% of Forced Expiratory Volume in the first second (FEV1). Nonasthma controls were defined as neither with asthma history as above criteria (i) nor diagnosed as criteria (ii). Other evaluations included skin prick tests for responsiveness to six common aeroallergens, a differential blood count (including total eosinophil count), and levels of total serum immunoglobulin E (IgE), as well as IgE specific to house dust and mixed pollens using Unicap system (Pharmacia Diagnostic, Uppsala, Sweden). A positive skin test was defined as the presence of ≥1 reaction with a wheal diameter ≥5 mm. Total serum IgE was measured by solid-phase immunoassay (Pharmacia IgE EIA; Pharmacia Diagnostics). Nonallergy subjects were defined as having a total serum IgE <200 and with negative skin test. The subjects group 1 (1027 subjects) were recruited from National Cheng-Kung University Hospital in the periods of 2002–2004. The 931 subjects in the validation group (group 2) were recruited from MacKay Memorial Hospital and National Cheng-Kung University Hospital during 2005–2006. All study subjects are Han-Taiwanese and living in Taiwan.

DNA preparation

Genomic DNAs were extracted from blood samples of the study subjects using QIAamp DNA Blood kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The extracted genomic DNAs were analyzed by agarose gel electrophoresis, quantified by spectrophotometer, and stored at −80°C until use.

SNP selection and genotyping

The tagging SNPs (tSNPs) of IL17A genomic region and upstream 1500 base pairs were selected according to seattle SNPs website (http://pga.mbt.washington.edu/education.html). The Seattle SNPs database showed 12 polymorphisms [minor allele frequency (MAF) ≥ 0] in our target region. According to Han-Chinese Beijing data, nine tSNPs were selected (minimum R2 = 0.8) from the 12 polymorphisms.

All SNP genotypings were performed using the TaqmanR SNP genotyping assays (ABI: Applied Biosystems Inc., Foster City, CA, USA). The primers and probes of selected SNPs were from ABI assay on demand kit. Reactions were carried out according to the manufacturer’s protocol. The probe fluorescence signal detection was performed using the ABI Prism 7900 Real-Time PCR System.

Statistical analysis

Quality of the genotype data was evaluated by the Hardy-Weinberg equilibrium (HWE) proportion tests. Inter-marker linkage disequilibrium (LD) measures, r2 and D′, were estimated and haplotype blocks defined using the Haploview program (http://www.broad.mit.edu/mpg/haploview/). All single-point association analyses were carried out using the sas/genetics package (SAS Inc. Cary, NC, USA). Single nucleotide polymorphism(s) showing significant association (P ≤ 0.05) in the tests were further evaluated using logistic regressions adjusted with allergy in odds ratio (OR) analysis.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

Characteristics of study subjects

One thousand twenty-seven DNA samples extracted from 546 nonasthma children and 481 asthmatic children were collected as group 1 and genotypings were performed on all selected SNPs. Group 2 subjects (including 729 asthmatic children and 202 controls), as validation group, were recruited from two different hospitals and only asthma status and total serum IgE by fulfilling earlier described criteria were evaluated. In two study groups, the asthma subjects had a higher level of total serum IgE than nonasthma subjects [comparing ln(IgE), t-test, P < 0.0001]. According to allergy phenotype, 1027 subjects in group 1 were further divided into four subgroups, 380 allergic asthma, 101 nonallergic asthma, 250 allergy subjects without asthma, and 296 nonallergy and nonasthma subjects. In group 1, the proportion of allergy was significantly greater in asthma group than in the nonasthma group [380/481 vs 250/546; P < 0.0001; OR = 4.45; 95% confidence interval (CI) 3.38, 5.89].

The mean of ages of initial diagnosis from asthma children and control was 8.11±2.97 and 8.37±2.45, respectively. There was no significant difference between the two groups (P = 0.214, t-test). The ratio of male/female gender from asthma children and control was 321/160 and 254/289, respectively. There was a significant difference in gender between the two groups (P < 0.0001, chi-squared test).

The SNP rs8193036 in IL17A promoter region associated with asthma

Except rs1974226, genotype distributions of other polymorphic SNPs did not deviate from the HWE either in asthma subjects or in nonasthma subjects. Linkage disequilibrium of the polymorphic SNPs of the IL17A genes is listed in Fig. 1. One haplo-block including five SNPs was identified at the IL17A gene.

image

Figure 1.  Linkage disequilibrium plot in D′ demonstrating adjacent strength between SNP-pairs at the IL17A gene. D′ values are multiplied by 100, e.g. 77 in the square at the bottom means a D′ of 0.77. Square without a number has a value of 100 that equals to a D′ of 1.

Download figure to PowerPoint

Strengths of associations and genotype frequencies of all polymorphic SNPs with asthma are summarized in Table 1. One SNP rs8193036 was significantly associated with asthma (P = 0.0074, genotype test). Although there is no single SNP displaying statistically significant difference after Bonferroni correction (P < 0.05/9 = 0.0055), the SNP rs8193036 showed marginally significant association with asthma (P = 0.0074 × 9 = 0.067) after Bonferroni correction.

Table 1.   Chi-squared test result of genotyping for IL17A testing SNPs
SNP IDLocationAlleleCaseControlP-value
1/211/12/2211/12/22
  1. SNPs, single nucleotide polymorphisms, UTR, untranslation region.

  2. *The distance from start position of IL-17A mRNA (NM_002190.2).

rs4711998−832*A/G275/173/30290/210/330.58
rs8193036−692*C/T285/151/36273/220/430.0074
rs3819024−399*A/G103/223/143115/263/1380.41
rs2275913−152*A/G110/234/129122/251/1410.98
rs3819025Intron_1A/G18/132/32815/154/3630.67
rs8193038Intron_1A/G379/95/6429/106/20.30
rs3804513Intron_2A/T370/103/6412/123/30.45
rs19742263′ UTRA/G11/48/4129/50/4660.73
rs37480673′ UTRA/G381/92/6404/118/50.43

To confirm and validate the results described above, genotyping on the association SNPs for the group 2 subjects was performed. Genotype distributions of rs8193036 in group 2 did not deviate from the HWE either in asthma subjects or in nonasthma subjects. It showed that SNP rs8193036 displayed statistical significance in group 2. The validation also showed that rs8193036 is statistically significant in the comparison with total subjects (Table 2). The OR analysis showed that the risk genotype of rs8193036 was CC and was consistent in group 1, group 2 and total subjects (Table 3). In group 1 subjects, effects of rs8193036 on asthma were further adjusted for the influence of allergy comorbidity and gender by logistic regression. As shown in Table 3, the strength of associations was slightly diluted by allergy comorbidity and gender. Group 2 subjects and total subjects were not further adjusted for allergy and gender because group 2 subjects were recruited from two different hospitals, which did not use consistent allergy evaluation and gender information was not recorded completely.

Table 2.   Genotype frequencies, proportions and statistical analyses of two independent subject groups for SNPs rs8193036
GenotypeGroup 1Group 2Total
CaseControlCaseControlCaseControl
  1. SNPs, single nucleotide polymorphisms.

CC (%)285 (60)273 (51)455 (62)107 (53)740 (62)380 (51)
CT (%)151 (32)220 (41)238 (33)78 (39)389 (32)298 (40)
TT (%)36 (8)43 (8)36 (5)17 (8)72 (6)60 (8)
χ29.6868 7.3560 23.5023 
P-value0.0074 0.0253 <0.0001 
Table 3.   Odds ratio analyses of two independent subject groups for SNPs rs8193036
GenotypeGroup 1Group 2Total
CaseControlCaseControlCaseControl
  1. SNPs, single nucleotide polymorphisms; case, asthma subjects; control, nonasthma subjects; OR, odds ratio; CI, confidence interval.

  2. *SNP was coded as a categorical variable, 1 for CC, and 0 for others.

CC285273455107740380
CT + TT (ref.)18726327495461358
OR (95% CI)1.47 (1.14, 1.89)1.47 (1.08, 2.02)1.55 (1.29, 1.87)
P-value0.00260.0152<0.0001
OR (95% CI) adjust with allergy*1.36 (1.04, 1.77)    
OR (95% CI) adjust with sex*1.36 (1.05, 1.76)    

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References

In the current study, we investigated the association between IL17A polymorphisms and pediatric asthma. We found that association between asthma phenotype and IL17A polymorphism rs8193036 was significant before Bonferroni correction in group 1 subjects. After Bonferroni correction, it revealed that rs8193036 is marginally significantly associated with asthma. The association between asthma phenotype and rs8193036 was further validated using group 2 subjects and still showed a significant association. The results of this study indicated a possibility that the polymorphisms of IL17A gene may confer risk for pediatric asthma in Taiwanese population.

The SNP rs8193036 is located at position −692 from the starting site of mRNA and −737 from the start codon of IL17A. Another IL17A promoter SNP rs2275913 is located at position of −197 from the start codon and associated with ulcerative colitis (24). The SNP 3804513, in IL17A intron 2, has been reported to be associated with radiographic progression in Japanese patients with early rheumatoid arthritis (25). No nonsynonymous SNP of IL17A was reported in NCBI SNP database suggesting that regulatory polymorphism(s) of IL17A may play a role in pathophysiological processes of related diseases.

IL17A is significantly expressed in sputum samples from patients with asthma compared with control subjects (17, 26–28) and its level in sputum of patients with asthma correlated negatively with the provocative concentration of methacholine causing a 20% fall in FEV1 (27). IL17A is able to induce the expression of two mucin genes in bronchial epithelial cells (29), and an increased expression of IL17A is associated with enhanced mucin gene expression in vivo (30). The above observations suggest that IL17A expression level and the factors that influence IL17A expression are strong candidates for asthma susceptibility factors.

In group 1, the SNP rs8193036 only showed a marginally significant association with asthma (P = 0.067) after Bonferroni correction. The power to detect significant association was calculated by Power for Association With Errors (PAWE; http://linkage.rockefeller.edu/pawe/) (31, 32). For example, rs8193036 in group 1, given 472 cases and 536 controls, adjust P-value = 0.066 and data without error, the power for genotypic test was 0.65. Under the same condition of ratio and allele frequencies in case and control, 726 case subjects and 820 controls are necessary to detect P = 0.05 and power = 0.8. The SNP rs8193036 in group 2, given 729 cases and 202 controls, P = 0.0253, and data without error, the power for genotypic test was 0.59. The calculation data suggested that our sample size in group 1 and group 2 may not be large enough to detect an association for IL17A effect on asthma. In total subjects including 1201 cases and 738 controls, P < 0.0001, the power for genotypic test was 0.96. The sample size pooling two study groups should be large enough to detect the association for rs8193036 and asthma.

From the results in this study, it may be suggested that IL17A is a candidate gene that confers the genetic susceptibility for pediatric asthma in Taiwanese population. Furthermore, the results provided a genetic basis indicating that the expression regulation was involved in asthma pathological mechanism. To understand further the functions and mechanisms of the associated SNPs in regulating IL17A expression demands further investigations.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  • 1
    Masoli M, Fabian D, Holt S, Beasley R. The global burden of asthma: executive summary of GINA Dissemination Committee Report. Allergy 2004;59:469478.
  • 2
    Burr ML, Butland BK, King S, Vaughan-Williams E. Changes in asthma prevalence: two surveys 15 years apart. Arch Dis Child 1989;64:14521456.
  • 3
    Beasley R. The burden of asthma with specific reference to the United States. J Allergy Clin Immunol 2002;109:S482S489.
  • 4
    Bach JF. The effect of infections on susceptibility to autoimmune and allergic diseases. N Engl J Med 2002;347:911920.
  • 5
    Skadhauge LR, Christensen K, Kyvik KO, Sigsgaard T. Genetic and environmental influence on asthma: a population-based study of 11688 Danish twin pairs. Eur Respir J 1999;13:814.
  • 6
    Palmer LJ, Burton PR, James AL, Musk AW, Cookson WO. Familiar aggregation and heritability of asthma-associated quantitative traits in a population-base sample of nuclear families. Eur J Hum Genet 2000;8:853860.
  • 7
    Ober C, Hoffjan S. Asthma genetics 2006: the long and winding road to gene discovery. Genes and Immunity 2006;7:95100.
  • 8
    Beasley R, Keil U, Von Mutius E, Pearce N, ISACC Steering Committee. Worldwide variation in prevalence of symptoms of asthma, allergic rehinoconjunctivtis, and atopic eczema: ISAAC. Lancet 1998;351:12251232.
  • 9
    Hsieh KH, Shen JJ. Prevalence of childhood asthma in Taipei, Taiwan, and other Asian pacific countries. J Asthm 1988;25:7382.
  • 10
    Lee CS, Tang RB, Chung RL. The evaluation of allergens and allergic disease in children. J Microbiol Immunol Infect 2000;33:227232.
  • 11
    Schmidt-Weber CB, Akdis M, Akdis CA. TH17 cells in the big picture of immunology. J Allergy Clin Immunol 2007;120:247254.
  • 12
    Hellings PW, Kasran A, Liu Z, Vandekerckhove P, Wuyts A, Overbergh L et al. Interleukin-17 orchestrates the granulocyte influx into airways after allergen inhalation in a mouse model of allergic asthma. Am J Respir Cell Mol Biol 2003;28:4250.
  • 13
    Prause O, Bozinovski S, Anderson GP, Linden A. Increased matrix metalloproteinase-9 concentration and activity after stimulation with interleukin-17 in mouse airways. Thorax 2004;59:313317.
  • 14
    Hoshino H, Laan M, Sjostrand M, Lotvall J, Skoogh BE, Linden A. Increased elastase and myeloperoxidase activity associated with neutrophil recruitment by IL-17 in airways in vivo. J Allergy Clin Immunol 2000;105:143149.
  • 15
    Ordonez CL, Shaughnessy TE, Matthay MA, Fahy JV. Increased neutrophil numbers and IL-8 levels in airway secretions in acute severe asthma: clinical and biologic significance. Am J Respir Crit Care Med 2000;161:11851190.
  • 16
    O’Donnell RA, Peebles C, Ward JA, Daraker A, Angco G, Broberg P et al. Relationship between peripheral airway dysfunction, airway obstruction, and neutrophilic inflammation in COPD. Thorax 2004;59:837842.
  • 17
    Chakir J, Shannon J, Molet S, Fukakusa M, Elias J, Laviolette M et al. Airway remodeling-associated mediators in moderate to severe asthma: effect of steroids on TGF-beta, IL-11, IL-17, and type I and type III collagen expression. J Allergy Clin Immunol 2003;111:12931298.
  • 18
    Tesmer LA, Lundy SK, Sarkar S, Fox DA. Th17 cells in human disease. Immunol Rev 2008;223:87113.
  • 19
    Wjst M, Fischer G, Immervoll T, Jung M, Saar K, Rueschendorf F et al. A genome-wide search for linkage to asthma. German Asthma Genetics Group. Genomics 1999;58:18.
  • 20
    Haagerup A, Bjerke T, Schiotz PO, Binderup HG, Dahl R, Kruse TA. Asthma and atopy – a total genome scan for susceptibility genes. Allergy 2002;57:680686.
  • 21
    Wang JY, Lin CGJ, Bey MSJ, Wang LM, Lin FYF, Huang L et al. Discovery of genetic difference between asthmatic children with high IgE level and normal IgE level by whole genome linkage disequilibrium mapping using 763 autosomal STR markers. J Hum Genet 2005;50:249258.
  • 22
    Burney PG, Luczynska C, Chinn S, Jarvis D. The European Community Respiratory Health Survey. Eur Respir J 1994;5:954960.
  • 23
    Pearce N, Sunyer J, Cheng S, Chinn S, Björkstén B, Burr M et al. Comparison of asthma prevalence in the ISAAC and the ECRHS. ISAAC Steering Committee and the European Community Respiratory Health Survey. International Study of Asthma and Allergies in Childhood. Eur Respir J 2000;16:420426.
  • 24
    Arisawa T, Tahara T, Shibata T, Nagasaka M, Nakamura M, Kamiya Y et al. The influence of polymorphisms of interleukin-17A and interleukin-17F genes on the susceptibility to ulcerative colitis. J Clin Immunol 2007;28:4449.
  • 25
    Furuya T, Hakoda M, Ichikawa N, Higami K, Nanke Y, Yago T et al. Associations between HLA-DRB1, RANK, RANKL, OPG, and IL-17 genotypes and disease severity phenotypes in Japanese patients with early rheumatoid arthritis. Clin Rheumatol 2007;26:21272141.
  • 26
    Molet S, Hamid Q, Davoine F, Nutku E, Taha R, Page N et al. IL-17 is increased in asthmatic airways and induces human bronchial fibroblasts to produce cytokines. J Allergy Clin Immunol 2001;108:430438.
  • 27
    Barczyk A, Pierzchala W, Sozanska E. Interleukin-17 in sputum correlates with airway hyperresponsiveness to methacholine. Respir Med 2003;97:726733.
  • 28
    Bullens DM, Truyen E, Coteur L, Dilissen E, Hellings PW, Dupont LJ et al. IL-17 mRNA in sputum of asthmatic patients: linking T cell driven inflammation and granulocytic influx? Respir Res 2006;7:135.
  • 29
    Chen Y, Thai P, Zhao YH, Ho YS, DeSouza MM, Wu R. Stimulation of airway mucin gene expression by interleukin (IL)-17 through IL-6 paracrine/autocrine loop. J Biol Chem 2003;278:1703617043.
  • 30
    Hashimota K, Graham BS, Ho SB, Adler KB, Collins RD, Olson SJ et al. Respiratory syncytial virus in allergic lung inflammation increases Muc5ac and gob-5. Am J Respir Crit Care Med 2004;170:306312.
  • 31
    Gordon D, Finch SJ, Nothnagel M, Ott J. Power and sample size calculations for case-control genetic association tests when errors present: application to single nucleotide polymorphisms. Hum Hered 2002;54:2233.
  • 32
    Gordon D, Levenstien MA, Finch SJ, Ott J. Errors and linkage disequilibrium interact multiplicatively when computing sample sizes for genetic case–control association studies. Pac Symp Biocomput 2003:490501.