document: multiple sclerosis ( ms ) is a common autoimmune system disease which affects the central nervous system with network vascular inflammation , demyelination and axonal damage symptoms ( 1 , 2 ) . 
   it is a chronic disease of the central nervous system and causes severe cognitive and physical impairments ( 3 , 4 ) . 
   accordingly , it has been documented that white matter lesion may affect the cognition of ms patients ( 5 ) . among the various clinical forms , 
   relapsing - remitting is the most common type , found in approximately 85% to 90% of cases ( 6 ) . 
   although , it is difficult to differentiate primary progressive multiple sclerosis ( ppms ) from relapsing remitting multiple sclerosis ( rrms ) in the early phases , yet previous studies have demonstrated that mrs can be used as a potential way to diagnose ppms and rrms ( 2 ) . 
   in fact , after traumatic and rheumatic diseases , ms is the third factor for asthenic diseases , and has been the subject of extensive studies worldwide ( 7 ) . 
   multiple sclerosis is a disease of young adults and most of patients are diagnosed between the ages of 20 and 40 ( 9 ) . although the exact reason for this disease is unknown , it has been reported that several factors such as genetic and environmental factors ( especially viruses ) are involved in the pathogenesis of ms ( 1 , 10 , 11 ) . 
   accordingly , several investigators are trying to improve symptoms of ms by alteration in the behavior of ms patients including exercise ( 4 ) . in spite of such efforts 
   interestingly , researchers do not limit this disease just to a single gene and candidate several immune and non - immune genes in the pathogenesis of ms . 
   recently , scientists have investigated interleukin-25 ( il-25 ) including its effects on the immune system . 
   this cytokine has a similar structure to il-17 and plays a key role in stimulating and development of t helper 2 ( th2 ) responses ( 12 ) . 
   furthermore , il-25 is involved in the control function of endothelial cell , th1 and th17 , which are the main cells involved in the pathogenic activities of the immune system ( 12 ) . 
   therefore , any factor , which can regulate the expression of the cytokine , can be considered as a candidate for investigation in the ms disease process . 
   it has been reported that there is a known polymorphism ( c424c / a polymorphism ) within exon 2 of the il-25 gene ( 13 ) , which may be associated with immune system - related disease . 
   therefore , according to this explanation , it seems that the polymorphism may be associated with ms . 
   thus , the main aim of this study was to evaluate the association between c424c / a polymorphism within the il-25 gene with ms in an iranian population . 
   in this case - control study , according to the study of arababadi et al . 
   ( 1 ) and using sample - size estimation we compared two binomial independent proportions with the following equation : where significance level was 0.05 , the expected power was considered 90% , p1 = 1% ( frequency of aa genotype in ms patients ) , p2 = 17% ( frequency of aa genotype in healthy controls ) , k = 1 ( sample size was considered equal in both groups ) , and  = 15% ( the minimum difference in frequency of aa genotype in the two groups , which was considered clinically important ) . based on the above parameters , sample size was determined as 75 subjects in either group . in this study , 80 patients with ms were recruited for blood sampling yet according to the inclusion and exclusion criteria , 74 patients with relapse and remitting ms ( rrms ) and 75 healthy controls were finally enrolled during august 2013 to september 2014 . " 
   blood samples were collected from consecutively admitted patients to the sina hospital , the main referral hospital for ms patients from all provinces of iran . 
   age and gender matched control samples were also collected from the tehran blood transfusion services . 
   the occurrence of ms was confirmed based on mcdonald s criteria ( diplopia , blurry vision , finger pruritus and disability ) ( 14 ) and brain magnetic resonance imaging ( mri ) results by an expert neurologist , according to clinical and preclinical findings . 
   having at least one central nervous system ( cns ) demyelinating event and being at least 18 years old to give informed consent were considered as the main inclusion criteria . 
   the two groups were matched in terms of age and sex and subjects with genetic diseases , diabetes , autoimmune disease and smokers were excluded from the study . 
   informed consent was filled out by all participants and this study was approved by the ethics committee of rafsanjan university of medical sciences ( rums ) with number 9/1077 on the 15th of july 2013 . regarding ethical considerations , 
   patients data were collected by their nurses and the data and blood samples were trans - located to the laboratory without names . 
   dna was purified from peripheral blood immune cells of participants using a commercial kit ( cinnaclon , tehran , iran ) , according to the manufacturer s instructions . 
   the pcr amplification of exon 2 of the il-25 gene and rflp ( using bsrfi restriction enzyme ) was performed according to the study of buning and et al . 
   numerical variables are presented as mean  sd , while categorical variables are summarized by absolute frequencies and percentages . 
   continuous variables were compared using independent two - sample t - test , and categorical variables were compared using the chi - square test across the two study groups . 
   one - sample kolmogorov - smirnov test was applied to test for normal distribution in cases and controls . 
   the logistic regression model was established to estimate the odds ratio ( or ) and 95% confidence interval ( ci ) of ms development in patients with the ac and aa compared to patients with the cc genotype . for the statistical analysis , the spss version 18.0 software for windows ( spss inc . , chicago , il ) was used . 
   all p values were two - tailed with statistical significance defined by p  0.05 . 
   the results also revealed 1 to 2% missing values in the evaluated genotypes and alleles . 
   in this case - control study , according to the study of arababadi et al . 
   ( 1 ) and using sample - size estimation we compared two binomial independent proportions with the following equation : where significance level was 0.05 , the expected power was considered 90% , p1 = 1% ( frequency of aa genotype in ms patients ) , p2 = 17% ( frequency of aa genotype in healthy controls ) , k = 1 ( sample size was considered equal in both groups ) , and  = 15% ( the minimum difference in frequency of aa genotype in the two groups , which was considered clinically important ) . based on the above parameters , sample size was determined as 75 subjects in either group . in this study , 80 patients with ms were recruited for blood sampling yet according to the inclusion and exclusion criteria , 74 patients with relapse and remitting ms ( rrms ) and 75 healthy controls were finally enrolled during august 2013 to september 2014 . " 
   blood samples were collected from consecutively admitted patients to the sina hospital , the main referral hospital for ms patients from all provinces of iran . 
   age and gender matched control samples were also collected from the tehran blood transfusion services . 
   the occurrence of ms was confirmed based on mcdonald s criteria ( diplopia , blurry vision , finger pruritus and disability ) ( 14 ) and brain magnetic resonance imaging ( mri ) results by an expert neurologist , according to clinical and preclinical findings . 
   having at least one central nervous system ( cns ) demyelinating event and being at least 18 years old to give informed consent were considered as the main inclusion criteria . 
   the two groups were matched in terms of age and sex and subjects with genetic diseases , diabetes , autoimmune disease and smokers were excluded from the study . 
   informed consent was filled out by all participants and this study was approved by the ethics committee of rafsanjan university of medical sciences ( rums ) with number 9/1077 on the 15th of july 2013 . regarding ethical considerations , 
   patients data were collected by their nurses and the data and blood samples were trans - located to the laboratory without names . 
   dna was purified from peripheral blood immune cells of participants using a commercial kit ( cinnaclon , tehran , iran ) , according to the manufacturer s instructions . 
   the pcr amplification of exon 2 of the il-25 gene and rflp ( using bsrfi restriction enzyme ) was performed according to the study of buning and et al . 
   numerical variables are presented as mean  sd , while categorical variables are summarized by absolute frequencies and percentages . 
   continuous variables were compared using independent two - sample t - test , and categorical variables were compared using the chi - square test across the two study groups . 
   one - sample kolmogorov - smirnov test was applied to test for normal distribution in cases and controls . 
   the logistic regression model was established to estimate the odds ratio ( or ) and 95% confidence interval ( ci ) of ms development in patients with the ac and aa compared to patients with the cc genotype . for the statistical analysis , the spss version 18.0 software for windows ( spss inc . , chicago , il ) was used . 
   all p values were two - tailed with statistical significance defined by p  0.05 . 
   the results also revealed 1 to 2% missing values in the evaluated genotypes and alleles . 
   the results revealed that eight ( 11.3% ) out of 74 patients with ms had the aa genotype , while 16 ( 22.5% ) had ac and 47 ( 66.2% ) had cc genotypes ( table 2 ) . 
   the results also showed that 8 ( 11.4% ) , 18 ( 25.7% ) and 44 ( 62.9% ) of the healthy controls had the aa , ac and cc genotypes , respectively . 
   the chi - square test revealed that the frequency distribution of the genotypes was not statistically different between patients with ms and the controls ( p = 0.901 ) ( table 2 ) . 
   according to the logistic regression model , the or of ms development in patients with ac versus patients with cc genotypes was 0.832 ( 95% ic : 0.378 - 1.832 ) ( p = 0.648 ) . 
   the corresponding results for aa versus cc genotypes was 0.936 ( 95% ic : 0.323 - 2.710 ) ( p = 0.903 ) . 
   the results also indicated that the frequencies of a and c alleles were 110 ( 77.5% ) and 32 ( 22.5% ) , respectively , in the patients with ms and were 106 ( 75.7% ) and 34 ( 24.3% ) , respectively , in the healthy controls . 
   the statistical analysis found that the differences in distribution of a and c alleles between groups were not statistically significant ( p = 0.728 ) ( table 2 ) . 
   the or of ms development in patients with the a allele versus patients with the c allele was 0.907 ( 95% ic : 0.522 - 1.574 ) , which did not reach statistical significance level ( p = 0.729 ) . 
   multiple sclerosis is an autoimmune disease , with several immune - related genes involved in its pathogenesis ( 15 ) . 
   it has been confirmed that th1 and th17 responses are the main causes of progression of ms ( 16 ) . therefore , th2 responses , which regulate th1 and th17 , can improve the clinical and laboratory outcome of the disease ( 16 ) . 
   it is believed that il-25 , as a th2 cytokine , can participate in the regulation of immune responses during inflammatory diseases including ms . 
   accordingly , previous studies suggested that serum levels of il-25 were decreased in ms patients when compared with the healthy controls ( 17 ) . 
   it has been proposed that polymorphisms within cytokine genes are associated with their expression ( 18 , 19 ) . 
   the authors of this study hypothesized that the polymorphisms within the il-25 gene may be associated with ms disease ; hence , the c424c / a polymorphism within the il-25 gene was evaluated in this study . 
   the results of our study identified that neither aa , ac and cc genotypes nor a and c alleles were associated with ms in the iranian population . to the best of our knowledge , this is a unique study , which evaluated the c424c / a polymorphism within the il-25 gene in patients with ms , yet there is a study , which evaluated this polymorphism in inflammatory bowel disease ( ibd ) , as an inflammatory disease ( 13 ) . 
   parallel to our results , researchers have reported that the c424c / a polymorphism within the il-25 gene was not associated with ibd ( 13 ) . 
   interestingly , this study documented that the expression level of il-25 decreased in ibd patients ( 13 ) . again , as mentioned previously , it has also been revealed that expression of il-25 decreased in ms patients ( 17 ) . 
   based on our results and the mentioned study it may be concluded that although il-25 may play significant roles in the pathogenesis of inflammatory diseases such as ms and ibd , c424c / a polymorphism is not important in the pathogenesis of these diseases . on the other hand , another study on iranian patients with ms revealed that other polymorphisms , except c424c / a , within il-25 are associated with ms ( 17 ) . 
   thus , it may be concluded that other polymorphisms like 4076a > a , 3672 t > ta , 3712 g > ga , and 3463c > ca , but not the c424c / a polymorphism , may regulate il-25 expression . according to our and previous studies it appears that evaluation of the c424c / a polymorphism , as the first report , and lack of evaluation of other polymorphisms within the il-25 gene as well as lack of evaluation of serum levels of il-25 are the strong and weak points , respectively , of our study . 
   additionally , it can be proposed that using a large sample size may be better for understanding the roles of c424c / a polymorphism in the pathogenesis of ms . according to the earned results of our investigation 
   it can be hypothesized that c424c / a polymorphism within the il-25 gene is not associated with ms and can not be considered as a risk factor for ms development .
summary: backgroundmultiple sclerosis ( ms ) is a common autoimmune system disease which affects the central nervous system . 
   it has been documented that interleukin-25 ( il-25 ) plays key roles in suppressing th1 responses , which is increased during ms.objectivesthe aim of this study was to investigate the c424c / a polymorphism within the il-25 gene in ms patients in comparison to healthy controls.patients and methodsin this case - control study , 74 patients with ms and 75 healthy controls were selected . 
   polymerase chain reaction - restriction fragment length polymorphism ( pcr - rflp ) was used in order to determine c424c / a polymorphism within the il-25 gene.resultsthe results showed that there was no statistical significant difference in distribution of genotype ( aa , ac and cc ) and allele ( a and c ) frequencies between ms patients and healthy controls ( p = 0.901 and p = 0.728 , respectively).conclusionsin conclusion , it appears that the c424c / a polymorphism within the il-25 gene has no significant relationship with ms , and this polymorphism is probably not associated with ms complications , its onset and gender distribution .