asthma is a complex and common disorder that is characterized by chronic inammation of the respiratory system , airow obstruction , and airway hyper - reactivity induced by certain stimulants , such as exercise , infection , allergens , and occupational exposures .
as one of the most prevalent chronic diseases , asthma affects more than 30 million people globally , especially children and young adults . in the united state alone , asthma affects more than 23 million adults , and women are more likely to be diagnosed with asthma that imposes greater morbidity than in men .
symptoms of asthma are multifaceted and include wheezing , coughing , hyperpnea , and feeling of suppression in the chest .
previously published evidence suggested that both environmental components and genetic variations may contribute to asthma susceptibility .
recently , the associations between asthma and gene polymorphisms have become an intensive area of research that has generated important insights into the pathogenesis of asthma .
interleukin-17 ( il-17 ) is a novel pro - inflammatory cytokine family containing 6 distinct isoforms ( il-17a , il-17b , il-17c , il-17d , il-17e , and il-17f ) and 5 receptors ( l-17ra , il-17rb , il-17rc , il-17rd , and il-17rsef ) .
produced by the t helper 17 ( th17 ) subsets of cd4 + t cells , il-17 plays an important role in the development and progression of inflammatory and autoimmune diseases .
il-17 can induce the production of pro - inflammatory cytokines and recruit neutrophils and monocytes , together resulting in an immune - mediated inflammatory reaction through the expression of il-17 receptors . from a genetic perspective
the polymorphisms of il-17 are shown to be associated with various autoimmune diseases , including asthma , rheumatoid arthritis and inflammatory bowel disease .
an increasing number of studies have demonstrated a close relationship between il-17 and neutrophilic inflammation , implicating il-17 as a potential candidate gene in predicting asthma susceptibility .
interestingly , an analysis using the tagger software implemented in the haploview software according to the han - chinese beijing data set revealed that rs763780 ( 7488a / g ) , rs2275913 ( 197g / a ) and rs8193036 ( 737c / t ) were found to be commonly associated with asthma [ 15 , 16 ] .
however , conflicting evidence has also been uncovered indicating that not all 3 snps were associated with asthma susceptibility .
therefore , we carried out the current case - control study supplemented with a meta - analysis to definitively evaluate the impact of rs763780 , rs2275913 , and rs8193036 of il-17 gene on clinical outcomes in asthma patients .
the study design was reviewed and approved by the ethics committee of huaihe hospital of henan university .
written informed consents were obtained from all subjects prior to the study . all procedures in this study were in compliance with the declaration of helsinki .
2014 , 125 asthma patients , comprising 64 males and 61 females in huaihe hospital of henan university , were enrolled as the case group .
the mean age for the case group was 39.498.61 years ( ranging from 25 to 55 years ) .
all asthma patients met the standards of asthma diagnosis as established by the chinese thoracic society , which include : ( 1 ) daytime paroxysmal cough or chronic persistent cough ; ( 2 ) postnasal drip syndrome or pharyngeal mucus ; ( 3 ) any history of rhinitis , sinusitis , nasal polyps , or chronic laryngopharyngitis ; ( 4 ) examinations revealed pharyngeal mucus in the posterior pharyngeal wall with formed cobble stone ; and ( 5 ) the cough was relieved after specific treatments .
the inclusion criteria were : ( 1 ) age : 24~55 years old ; ( 2 ) ethnicity : han ; ( 3 ) pulmonary function test : positive in bronchial dilation test ; and ( 4 ) no wheezing , dyspnea , chest distress , or cough symptoms caused by other diseases . the exclusion criteria were : ( 1 ) patients with severe complications ; ( 2 ) patients with heart diseases or complications with liver disease and nephropathy ; ( 3 ) patients with diseases of the hematopoietic system complicated with tumor diseases ; and ( 4 ) pregnant or lactating women .
in addition , 132 healthy controls ( 67 males and 65 females ) who underwent physical examinations in our hospital were randomly selected as the control group .
the age of the control group ranged from 24 to 52 years with a mean age of 38.238.45 years .
ten ml of venous blood were extracted from all subjects after fasting for more than 12 h. the blood samples ( 4 ml ) were anticoagulated with ethylenediaminetetraacetic acid ( edta ) and stored at 70c .
then the samples were incubated in an upright position for 1 h , followed by centrifuging at 3000 rpm for 10 min at room temperature to isolate the peripheral blood mononuclear cells .
afterwards , the genomic dna was isolated using a dna extraction kit ( cat no . : dp318 - 03 , tiangen biotech beijing co. ltd . ,
the remaining 6 ml blood samples were incubated in an upright position for 1 h and were centrifuged at 3000 rpm for 10 min at room temperature .
three snp sites , il-17 rs763780 , rs2275913 , and rs8193036 , were selected to conduct the current research .
restriction fragment length polymorphism polymerase chain reaction ( pcr - rflp ) was used to analyze the genotype and allele frequency in 3 sites between the case and control groups .
the pcr primers were designed using primer premier 5.0 software and synthesized by shanghai chemical company ( china ) .
the amplification sites , primer sequences , fragment length , annealing temperature , and cycle number for the pcr - rflp are presented in table 1 .
the total volume of pcr reaction included 20 l : 2 l of 10pcr reaction buffer , 2 l of deoxy - ribonucleoside triphosphate ( dntp ) ( 2.5 mmol / l for each ) , 0.5 l of each forward and reverse primer ( 10 pmol/l ) , 2.5 u of platinum taq dna polymerase ( invitrogen , shanghai , china ) , and 50 ng of genomic dna .
dna was amplified during 30 cycles with 5 min predegeneration at 95c , 30 s denaturation at 95c , 30 s annealing at 58c , and 40 s extension at 72c .
then the dna was further extended for 5 min at 72c and stored at 4c .
a total of 20 l pcr production was extracted to construct the enzyme reaction system .
then the pcr products were digested with restricted enzyme rsai and xmni ( new england biolabs ltd . ,
after incubation at 21c overnight , the pcr products were placed in a water bath at 4c for 15 min to terminate the reaction .
then the enzyme - digested products were electrophoresis - separated by 2% agarose gel ( containing ethidium bromide ) .
the gel imaging system ( bio - rad , usa ) was used for genotyping interpretation .
the electropherograms of enzyme digestion for il-17 rs763780 , rs2275913 , and rs8193036 snps are presented in figure 1 .
statistical analysis was conducted using spss 18.0 software ( ibm corporation , somers , ny , usa ) .
continuous data are expressed as   standard deviation ( sd ) , using t test or variance analysis for comparisons .
categorical data was presented with percentages and chi - square test was applied for comparisons between groups .
chi - square test was also used to verify whether the genotype distribution of the 3 snps met hardy - weinberg ( hw ) equilibrium . the genotype frequency and allele frequency between the case and control were calculated by or ( odds ratio ) with
the association between il-17 snps and asthma was assessed using or with 95%ci under a fixed - effects model or a random - effects model .
the study design was reviewed and approved by the ethics committee of huaihe hospital of henan university .
written informed consents were obtained from all subjects prior to the study . all procedures in this study were in compliance with the declaration of helsinki .
2014 , 125 asthma patients , comprising 64 males and 61 females in huaihe hospital of henan university , were enrolled as the case group .
the mean age for the case group was 39.498.61 years ( ranging from 25 to 55 years ) .
all asthma patients met the standards of asthma diagnosis as established by the chinese thoracic society , which include : ( 1 ) daytime paroxysmal cough or chronic persistent cough ; ( 2 ) postnasal drip syndrome or pharyngeal mucus ; ( 3 ) any history of rhinitis , sinusitis , nasal polyps , or chronic laryngopharyngitis ; ( 4 ) examinations revealed pharyngeal mucus in the posterior pharyngeal wall with formed cobble stone ; and ( 5 ) the cough was relieved after specific treatments .
the inclusion criteria were : ( 1 ) age : 24~55 years old ; ( 2 ) ethnicity : han ; ( 3 ) pulmonary function test : positive in bronchial dilation test ; and ( 4 ) no wheezing , dyspnea , chest distress , or cough symptoms caused by other diseases . the exclusion criteria were : ( 1 ) patients with severe complications ; ( 2 ) patients with heart diseases or complications with liver disease and nephropathy ; ( 3 ) patients with diseases of the hematopoietic system complicated with tumor diseases ; and ( 4 ) pregnant or lactating women .
in addition , 132 healthy controls ( 67 males and 65 females ) who underwent physical examinations in our hospital were randomly selected as the control group . the age of the control group ranged from 24 to 52 years with a mean age of 38.238.45 years .
ten ml of venous blood were extracted from all subjects after fasting for more than 12 h. the blood samples ( 4 ml ) were anticoagulated with ethylenediaminetetraacetic acid ( edta ) and stored at 70c .
then the samples were incubated in an upright position for 1 h , followed by centrifuging at 3000 rpm for 10 min at room temperature to isolate the peripheral blood mononuclear cells . afterwards , the genomic dna was isolated using a dna extraction kit ( cat no . : dp318 - 03 , tiangen biotech beijing co. ltd . , beijing , china ) according to the manufacturer s instructions .
the remaining 6 ml blood samples were incubated in an upright position for 1 h and were centrifuged at 3000 rpm for 10 min at room temperature .
three snp sites , il-17 rs763780 , rs2275913 , and rs8193036 , were selected to conduct the current research . restriction fragment length polymorphism polymerase chain reaction ( pcr - rflp )
was used to analyze the genotype and allele frequency in 3 sites between the case and control groups .
the pcr primers were designed using primer premier 5.0 software and synthesized by shanghai chemical company ( china ) .
the amplification sites , primer sequences , fragment length , annealing temperature , and cycle number for the pcr - rflp are presented in table 1 .
the total volume of pcr reaction included 20 l : 2 l of 10pcr reaction buffer , 2 l of deoxy - ribonucleoside triphosphate ( dntp ) ( 2.5 mmol / l for each ) , 0.5 l of each forward and reverse primer ( 10 pmol/l ) , 2.5 u of platinum taq dna polymerase ( invitrogen , shanghai , china ) , and 50 ng of genomic dna .
dna was amplified during 30 cycles with 5 min predegeneration at 95c , 30 s denaturation at 95c , 30 s annealing at 58c , and 40 s extension at 72c .
then the dna was further extended for 5 min at 72c and stored at 4c .
a total of 20 l pcr production was extracted to construct the enzyme reaction system . then the pcr products were digested with restricted enzyme rsai and xmni ( new england biolabs ltd . ,
after incubation at 21c overnight , the pcr products were placed in a water bath at 4c for 15 min to terminate the reaction .
then the enzyme - digested products were electrophoresis - separated by 2% agarose gel ( containing ethidium bromide ) .
the gel imaging system ( bio - rad , usa ) was used for genotyping interpretation .
the electropherograms of enzyme digestion for il-17 rs763780 , rs2275913 , and rs8193036 snps are presented in figure 1 .
statistical analysis was conducted using spss 18.0 software ( ibm corporation , somers , ny , usa ) .
continuous data are expressed as   standard deviation ( sd ) , using t test or variance analysis for comparisons .
categorical data was presented with percentages and chi - square test was applied for comparisons between groups .
chi - square test was also used to verify whether the genotype distribution of the 3 snps met hardy - weinberg ( hw ) equilibrium .
the genotype frequency and allele frequency between the case and control were calculated by or ( odds ratio ) with 95%ci ( confidence interval ) .
the association between il-17 snps and asthma was assessed using or with 95%ci under a fixed - effects model or a random - effects model .
no significant difference was found between the asthma patients and healthy controls in terms of age and sex ( both p>0.05 ) .
the case group had elevated percentages of patients with a smoking history , a family history of cancers , and a history of alcohol consumption compared with the control group , although no statistical significance was achieved ( all p>0.05 ) . however , the fev1 was significantly lower in the case group than the control group ( p<0.001 ) .
the genotype frequencies and allele frequencies of il-17 7488a / g ( rs763780 ) , 197g / a ( rs2275913 ) and 737c / t ( rs8193036 ) are presented in table 3 .
the genotypes constructed from the genotype frequencies and allele frequencies met hardy - weinberg equilibrium and were representative of the case that emerged .
our results revealed that the asthma patients presented with higher frequencies of the ga genotype in rs2275913 and the tt genotype in rs8193036 of il-17 compared to healthy controls ( both p<0.001 ) ; however , the genotype frequency of il-17 rs763780 between the asthma patients and healthy controls failed to achieve statistical significance ( p>0.05 ) .
the comparisons of the rs2275913 and rs8193036 frequencies between the asthma patients and healthy controls were statistically significant in both allele and additive models ( all p<0.05 ) .
the frequency of il-17 rs763780 between the asthma patients and healthy controls were statistically different in the allele model ( p<0.05 ) , but not in the additive model ( p>0.05 ) .
the present meta - analysis enrolled 12 eligible case and control studies [ 13,14,17,2028 ] , including 3575 asthma patients and 3671 healthy controls .
a total of 6 studies investigated the association between the il-17 7488a / g ( rs763780 ) snp and asthma , which demonstrated that il-17 7488a / g ( rs763780 ) snp may increase asthma risk in allele models ( or=0.667 , 95%ci=0.4890.908 , p=0.010 ) , but not additive models ( or=0.693 , 95%ci=0.4531.060 , p=0.091 ) ( figure 2 ) .
the association between il-17197g / a(rs2275913 ) snp and asthma was investigated in 4 studies , which revealed that the il-17197g / a ( rs2275913 ) polymorphism may elevate the risk of asthma in both allele and additive models ( allele model : or=0.764 , 95%ci=0.6070.961 , p=0.021 ; additive model : or=0.639 , 95%ci=0.4360.937 , p=0.022 ) ( figure 3 ) .
a total of 4 studies explored the association between il-17737c / t ( rs8193036 ) snp and asthma susceptibility .
the results in allele and additive model suggested that il-17737c / t ( rs8193036 ) snp may enhance the risk of asthma ( allele model : or=0.807 , 95%ci=0.7220.903 , p<0.001 ; additive model : or=0.751 , 95%ci=0.6480.870 , p<0.001 ) ( figure 4 ) .
no significant difference was found between the asthma patients and healthy controls in terms of age and sex ( both p>0.05 ) .
the case group had elevated percentages of patients with a smoking history , a family history of cancers , and a history of alcohol consumption compared with the control group , although no statistical significance was achieved ( all p>0.05 )
. however , the fev1 was significantly lower in the case group than the control group ( p<0.001 ) .
the genotype frequencies and allele frequencies of il-17 7488a / g ( rs763780 ) , 197g / a ( rs2275913 ) and 737c / t ( rs8193036 ) are presented in table 3 .
the genotypes constructed from the genotype frequencies and allele frequencies met hardy - weinberg equilibrium and were representative of the case that emerged .
our results revealed that the asthma patients presented with higher frequencies of the ga genotype in rs2275913 and the tt genotype in rs8193036 of il-17 compared to healthy controls ( both p<0.001 ) ; however , the genotype frequency of il-17 rs763780 between the asthma patients and healthy controls failed to achieve statistical significance ( p>0.05 ) .
the comparisons of the rs2275913 and rs8193036 frequencies between the asthma patients and healthy controls were statistically significant in both allele and additive models ( all p<0.05 ) .
the frequency of il-17 rs763780 between the asthma patients and healthy controls were statistically different in the allele model ( p<0.05 ) , but not in the additive model ( p>0.05 ) .
the present meta - analysis enrolled 12 eligible case and control studies [ 13,14,17,2028 ] , including 3575 asthma patients and 3671 healthy controls .
a total of 6 studies investigated the association between the il-17 7488a / g ( rs763780 ) snp and asthma , which demonstrated that il-17 7488a / g ( rs763780 ) snp may increase asthma risk in allele models ( or=0.667 , 95%ci=0.4890.908 , p=0.010 ) , but not additive models ( or=0.693 , 95%ci=0.4531.060 , p=0.091 ) ( figure 2 ) .
the association between il-17197g / a(rs2275913 ) snp and asthma was investigated in 4 studies , which revealed that the il-17197g / a ( rs2275913 ) polymorphism may elevate the risk of asthma in both allele and additive models ( allele model : or=0.764 , 95%ci=0.6070.961 , p=0.021 ; additive model : or=0.639 , 95%ci=0.4360.937 , p=0.022 ) ( figure 3 ) .
a total of 4 studies explored the association between il-17737c / t ( rs8193036 ) snp and asthma susceptibility .
the results in allele and additive model suggested that il-17737c / t ( rs8193036 ) snp may enhance the risk of asthma ( allele model : or=0.807 , 95%ci=0.7220.903 , p<0.001 ; additive model : or=0.751 , 95%ci=0.6480.870 , p<0.001 ) ( figure 4 ) .
our study interrogated the associations between rs763780 , rs2275913 and rs8193036 snps in il-17 gene and asthma susceptibility in an asian population .
asthma is an inflammatory and immune disease mediated by aberrant th2 immune responses induced by excessive stimulation of exogenous factors , which engages the ige receptor to elicit the secretion of pro - inflammatory cytokines , including il-17 , tnf- , il-1 , and il-6 .
emerging evidence supports that il-17 is closely involved in the pathologies of the airway asthmatics .
indeed , functional analysis demonstrated that overexpression of il-17f in a mouse model with increased numbers of neutrophils in the airways resulted in antigen - induced allergic inflammatory responses , whereas il-17f - deficient mice had defective airway neutrophilia responding to allergen challenge .
the ability of il-17a and il-17f to induce neutrophils migration suggests that they are involved in severe asthma , in which accumulation of neutrophils in the airways is a major defining hallmark .
this is consistent with the role of il-17 in enhancing the activation of bronchial fibroblasts , epithelial cells , and smooth muscle cells and in inducing the secretion of cytokines and chemokines , which in concert leads to the accumulation of neutrophils with proteolytic enzymes that may burden the airway .
the most important finding in our study was that rs763780 , rs2275913 and rs8193036 snps were closely associated with asthma susceptibility .
conducted a genetic analysis investigating the association between asthma and common variants il-17 and il-13 , indicating that il-17 may play a role in the etiology of asthma because the il-17a rs8193036 sequence variations significantly influence the risk of asthma .
a close association between rs8193036 in the il17a promoter region and pediatric bronchial asthma was also reported in a taiwanese population .
the snp rs2275913 , located at position 152 bp upstream of the starting site of il-17 mrna , has been reported to influence the susceptibility and pathophysiological features of ulcerative colitis .
in addition , chen et al . demonstrated that il-17 snp rs2275913 was implicated in several asthma - related traits that confer genetic susceptibility to childhood asthma .
our study also found that the frequencies of il-17 rs763780 in the asthma patients and healthy controls were statistically different in allele models .
intriguingly , a study conducted in saudi arabia suggested no significant association between il17f snps and asthma risk , except for the ag heterozygotes of rs17880588 in il17a .
however , qian f et al . indicated that the c allele of rs763780 in il17 was associated with an increased risk of asthma .
our study provides compelling evidence that il-17 snps ( rs763780 , rs2275913 , and rs8193036 ) may be associated with asthma susceptibility .
our study combined a carefully designed case - control study with a large sample size and carried out a meta - analysis using data from previously published studies .
however , our results must be interpreted with caution because more prospective studies with larger sample size and diverse populations are needed to validate the association between il-17 snps and the development of asthma .
 backgroundthe aim of this study was to examine the associations between the single - nucleotide polymorphisms ( snps ) of interleukin-17 ( il-17 ) , including rs763780 ( 7488a / g ) , rs2275913 ( 197g / a ) , and rs8193036 ( 737c / t ) , and asthma susceptibility in an asian population.material/methodsfrom oct 2013 to dec 2014 , 125 asthma patients enrolled in our hospital were selected as the case group . another 132 healthy controls undergoing physical examinations in our hospital were enrolled as the control group . 
 the genotype frequencies of il-17 rs763780 , rs2275913 and rs8193036 snps were detected using polymerase chain reaction - restriction fragment length polymorphism ( pcr - rflp ) . 
 comprehensive meta - analysis 2.0 ( cma 2.0 ) software was applied for meta-analysis.resultsour results demonstrated that asthma patients presented with higher frequencies of ga genotype in rs2275913 and tt genotype in rs8193036 of il-17 than healthy controls ( both p<0.001 ) . 
 the genotype frequencies of il-17 rs763780 between the asthma patients and healthy controls exhibited no significant differences ( p>0.05 ) . 
 the comparisons on the rs2275913 and rs8193036 frequencies between the asthma patients and healthy controls were statistically significant in both allele and addictive models ( all p<0.05 ) . 
 the frequency of il-17 rs763780 between the asthma patients and healthy controls were statistically different in allele models ( p<0.05 ) , but not in addictive models ( p>0.05 ) . 
 the overall results of our case - control study were further confirmed by meta-analysis.conclusionsour results revealed that , in an asian population , il-17 rs763780 , rs2275913 , and rs8193036 snps may be associated with asthma susceptibility , and ga genotype in rs2275913 and tt genotype in rs8193036 of il-17 may contribute to increased risk of asthma in asians . 
Background
Material and Methods
Ethical statements
Subjects
Sample collection
SNP detection
Statistical analysis
Results
Comparisons on baseline characteristics
Genotype distribution
Meta-analysis results
Discussion
Conclusions