despite the recent advancements in prevention and treatment of stroke , stroke remains the leading cause of disability and the fifth leading cause of death in the us . especially in those older than 65 years
, stroke is a leading cause of death and disability . in the future , due to rapid aging
therefore , to aid in prevention , the identification of markers of stroke risk is important .
although stroke has been believed to be a multifactorial disorder with minimal classical patterns of inheritance , accumulating evidence has shown the importance of genetic factors in stroke .
especially , it has been reported that a number of inflammatory mechanisms play a fundamental part in stroke ( 2 ) ; thus , various inflammatory genes showing an association with stroke have received attention as candidate genes ( 34567891011 ) . also , genetic factors could have an effect on stroke onset , infarct size , and prognosis ( 12,13 ) .
although several candidate genes have been studied as risk factors for stroke , there are few useful markers for the prevention , diagnosis , and treatment of stroke ( 14,15 ) .
interleukin-6 ( il-6 ) is a potent pleiotropic cytokine that regulates survival and differentiation of neuronal cells .
the counterpart , interleukin-6 receptor ( il-6r ) complex consists of il-6r and the interleukin 6 signal transducer ( il6st / gp130/il-6 beta ) , which is associated with many other cytokines .
if il-6 binds to il-6r , this complex binds to gp130 , leading to intracellular signaling cascades ( 16 ) .
il-6 performs as the main stimulator of c - reactive protein ( crp ) production , the levels of which are known to be associated with metabolic syndromes such as obesity and diabetes , as well as with vascular events ( 17 ) . as an extension of the study of il-6r , il-6 , and crp in stroke ,
, there is no study about the associations between il-6r polymorphisms and ischemic stroke ( is ) .
the national institutes of health stroke scale ( nihss ) is a neurologic impairment assessment tool that provides an objective quantified measurement in stroke patients ( 18,19 ) .
higher scores on nihss indicate greater stroke severity . in a previous il-6 polymorphism study , patients with severe
disability ( nihss  6 ) were associated with a specific genotype ( 20 ) .
stroke rehabilitation outcomes are usually assessed using the modified barthel index ( mbi ) , which is a validated tool scoring independent daily living ( 21 ) .
the aim of this study was to assess whether single nucleotide polymorphisms ( snps ) of the il-6r gene were associated with the development , neurologic status , and clinical features of ischemic stroke in the korean population .
all participants with stroke were patients who visited the departments of rehabilitation medicine at kyung hee medical center and kyung hee university hospital at gangdong .
is patients were diagnosed based on magnetic resonance ( mr ) imaging , computed tomography ( ct ) , or angiography .
exclusion criteria were ischemic heart disease or other causes of cerebrovascular events such as traumatic brain injury , transient ischemic attack , and vascular malformation , etc .
peripheral blood samples were collected from the subjects , and genomic dna extraction was performed using a qiaamp dna mini kit ( qiagen , valencia , ca , usa ) .
the genotypes of the two selected snps ware determined by direct sequencing ( macrogen , seoul , korea ) .
the following primers were used in the polymerase chain reaction ( pcr ) amplification : rs4845617 ( forward 5-ctgttctccccgctcaggtgcg-3 , reverse 5-agaggcggacaggctaatg-3 ) and rs2228144 ( forward 5-gtagcctgggccacttcatca t-3 , reverse 5-gacctctgaggcacaactcac-3 ) .
pcr consisted of 40 cycles at 94c for 30 sec , 58c for 30 sec , 72c for 30 sec , and 1 cycle at 72c for 5 min .
an abi prism 3730xl analyzer ( pe applied biosystems , foster city , ca , usa ) sequenced the pcr products , and seqmanii software ( dnastar , madison , wi , usa ) was used to analyze the sequencing data ( 22 ) . to obtain odds ratios ( ors ) , 95% confidence intervals ( cis ) , and p values adjusted for age and sex as covariates , snpanalyzer pro ( istech , goyang , korea ) , helixtree (
golden helix , bozeman , mt , usa ) , and snpstats ( http://bioinfo.iconcologia.net/index.php?module=snpstats ) were used .
the chi - square test was used to determine hardy - weinberg equilibrium ( hwe ) .
a multiple logistic regression analysis was adjusted for age and sex . in terms of increasing risk of disease
, an increased specific allele frequency was noted in is patients compared with the allele frequency of controls . to analyze the association between a polymorphism and disease ,
a contingency table and chi - square test were applied using a prespecified genetic model .
the codominant model was the most general model  where the disease risk associated with ab individuals lies between that of aa and bb individuals .
in dominant model , with the hypothesis that carrying allele b increased risk of disease ( dominant model ) , the ab and bb genotypes are pooled giving a 2 * 2 table .
alternatively , under a recessive model for allele b , cells aa and ab would be pooled .
additive model is assumed that increased disease risk of  for ab genotypes and 2  for bb genotypes  ( 23 ) . in this study ,
codominant model 1 compared major allele homozygotes with heterozygotes , and codominant model 2 compared major allele homozygotes with minor allele homozygotes .
to determine the haplotypes between the two snps and the clinical features in patients with is , haploview version 4.2 ( daly lab , cambridge , ma , usa ) was used .
the study was approved by the institutional review board of medical research institute , school of medicine , kyung hee university and center , and kyung hee university hospital at gangdong ( irb no .
the study was approved by the institutional review board of medical research institute , school of medicine , kyung hee university and center , and kyung hee university hospital at gangdong ( irb no .
there were 121 is patients ( mean  sd , 65.7  12.1 years ) and 291 controls ( mean  sd , 63.0  9.3 years ) .
there was a difference in mean age between is patients and control subjects , but it was not significant ( p = 0.30 ) .
is patients were classified into two subgroups according to nihss score ( nihss 1 , nihss score < 6 ; and nihss 2 , nihss score  6 ) and mbi score ( mbi 1 , mbi  60 ; and mbi 2 , mbi score > 60 ) .
the numbers of is patients with nihss score < 6 and  6 were 56 and 57 , respectively .
the numbers of is patients with mbi score  60 and > 60 were 71 and 25 , respectively ( data not shown ) .
nihss 1 , nihss score < 6 ; nihss 2 , nihss score  6 .
the genotype and allele frequencies of the two examined snps are shown in tables 2 and 3 , respectively .
two snps ( rs4845617 , 5tur ; rs2228144 , ala31ala ) of the il-6r gene were in hwe in the is and control groups , respectively ( p > 0.05 , data not shown ) .
the genotype and allele frequencies of the two snps demonstrated no significant difference in is cases and controls ( table 2 ) .
as shown in table 3 , the snp rs4845617 was associated with the nihss score of is patients ( p = 0.011 , or = 0.24 , 95% ci = 0.08 - 0.72 in codominant model 2 , p = 0.006 , or = 0.30 , 95% ci = 0.12 - 0.74 in recessive model , and p = 0.008 , or = 0.48 , 95% ci = 0.28 - 0.84 in log - additive model ) . in snp rs4845617 ,
the frequencies of genotype according to nihss score was significantly different in the codominant model 2 , recessive model , and log - additive model , and the a allele frequency was significantly higher in the nihss 1 group of is patients .
however , in snp rs2228144 , the genotype and allele frequencies did not correlate with the nihss score of the is group ( table 3 ) .
furthermore , rs4845617 and rs2228144 had no correlation with mbi score in the is group ( data not shown ) .
nihss 1 , nihss score < 6 ; nihss 2 , nihss score  6 .
we investigated the associations between haplotypes of control subjects and is patients and nihss score .
the haplotype frequencies of the two snps were not significantly different between control subjects and is patients ( table 4 ) .
however , as shown in tables 5 and 6 , the ag and gg haplotypes differed between the nihss 1 and nihss 2 groups in is patients .
the frequency of the ag haplotype was higher in the nihss 1 group , and that of the gg haplotype was higher in the nihss 2 group of is patients ( p = 0.014 , p = 0.0024 , respectively ) .
however , the haplotype frequencies found no significant difference by mbi scores ( table 6 ) .
nihss 1 , nihss score < 6 ; nihss 2 , nihss score  6 .
ischemic stroke is known to be a heterogeneous multifactorial disease ( 24 ) . although conventional risk factors such as hypertension , diabetes , dyslipidemia and smoking account for a significant proportion of these stroke events , a considerable portion remains insufficiently explained by these factors ( 25,26 ) . as risk factors for stroke ,
several genetic factors have been studied ; however , there are few useful markers for prevention , diagnosis , and treatment of stroke ( 26 ) . therefore ,
increasing our understanding of risk factors including genetic components would allow us to predict the risk better , identification of novel stroke mechanisms , and new therapeutic approaches using genetic factors ( 27 ) .
some of these snps include apolipoprotein e ( 3 ) , 5-methyltetrahydrofolate - homocysteine methyltransferase ( 6 ) , interleukin 4 ( 11 ) , interleukin-1 receptor antagonist , tumor necrosis factor , interleukin 1 beta ( 8) , neuropeptide y ( 5 ) , peroxisome proliferator - activated receptor gamma ( 9 ) , phosphodiesterase 4d camp - specific chemokine ( c - c motif ) ligand 5 ( 4 ) , thromboxanea2 receptor , and thromboxane a synthase 1 ( platelet ) ( 10 ) .
several snps have been identified in the il-6r gene , associated with carotid plaque ( 28 ) , crp ( 17,29 ) and obesity or diabetes ( 30313233 ) .
however , there has been no study of polymorphisms of the il-6r gene in relation to stroke .
carotid plaque is assumed to be associated with increased stroke risk . in a genetic study in a dominican population
, il-6r was suggested to be a related gene associated with carotid plaque ( 28 ) .
il-6 has been shown to have a positive correlation with crp and to predict the risk of vascular events .
the polymorphism of il-6r gene is linked with crp expression and plasma il-6 level ( 34 ) .
( 35 ) demonstrated that the il-6r variants ( rs6684439 , rs4845622 , rs8192284 , rs4329505 ) are significantly associated with plasma crp level , independent of il-6 level .
in addition , the il-6r variant interacts with crp in relation to diabetes risk . in another study , the il-6r gene ( rs4845617 )
was suggested to play an important role in the pathogenesis of dyslipidemia and atherosclerosis ( 36 ) .
we identified snps of il-6r , in coding regions near the promoter region , that had greater than 0.1 heterozygosity and greater than 0.1 minor allele frequency .
for rs4845617 , heterozygosity is 0.471 , and minor allele frequency is 0.3804 ; for rs2228144 , heterozygosity is 0.208 , and minor allele frequency is 0.1178 .
this is the first report to identify an association between snps of the il-6r gene associated with risk of stroke and the clinical features of stroke .
the two snps ( rs4845617 and rs2228144 ) were not associated with development or daily activities of is . however , our results revealed that snp rs4845617 contributes to the neurologic status of is patients .
the mechanisms remain of the effects of il-6r gene polymorphisms on stroke severity remain unclear . compared with previous snps studies , the sample size in this study was relatively small .
therefore , other stroke risk factors analyses could be included while sample size became smaller after dividing groups with regard to hypertension , diabetes , dyslipidemia , and smoking history in this study .
further studies with larger sample size are necessary to elucidate this result . in summary ,
two snps of the il-6r gene ( rs4845617 , rs2228144 ) were analyzed in korean ischemic stroke patients .
the frequencies of genotype and alleles of the two snps demonstrated no significant difference in is cases and controls .
. however , in snp rs4845617 , the frequencies of genotypes according to nihss score are statistically different , and the a allele frequency in the mild stroke patients is significantly higher than an allele frequency in the severe stroke patients .
this indicates that snp rs4845617 is associated with the neurologic status evaluated by nihss in patients with is .
in addition , the a allele of snp rs4845617 might have a protective effect against neurologic deficit in is patients .
 to investigate the contribution of the interleukin-6 receptor ( il-6r ) gene single nucleotide polymorphisms ( snps ) to the neurological status of korean patients with ischemic stroke ( is ) , two snps of the il-6r gene ( rs4845617 , 5 utr ; rs2228144 , ala31ala ) were selected . 
 is patients were classified into clinical phenotypes according to two well - defined scores : the national institutes of health stroke survey ( nihss ) and the modified barthel index scores . 
 there were 121 is patients and 291 control subjects . 
 the snp rs4845617 significantly contributed to the neurological status of patients with is ( p = 0.011 in codominant model 2 , p = 0.006 in recessive model , and p = 0.008 in log - additive model ) . 
 allele frequencies of rs4845617 and rs2228144 demonstrated no significant difference in is patients and controls . 
 the ag and gg haplotypes differed between the nihss 1 ( nihss scores < 6 ) group and the nihss 2 ( nihss scores  6 ) group in patients with is ( p = 0.014 , p = 0.0024 ) . 
 these results suggest that rs4845617 of the il-6r gene is associated with the neurologic status of korean patients with is . 