stroke is the leading cause of neurological disability and it is one of the leading causes of death worldwide , and ischemic stroke ( is ) is the major type of stroke in china . although genome wide association study ( gwas ) and genetic studies based on candidate genes have provided numerous probable susceptibility locus , a large part of genetic risks of is remains unexplained . several candidate genes were identified for is based on genome - wide linkage search and subsequent fine - mapping in the past decades , such as pde4d gene , alox5ap gene , and protein kinase c  ( prkch ) gene . besides , candidate genes were also proposed and demonstrated based on different pathways of pathogenic mechanisms , such as selenoprotein s ( seps1 ) gene which involved in the regulation of inflammatory response and protection from oxidative damage . gwas has been used in exploring genetic risk factors of is since 2007 , and has provided more replicable novel genetic markers , such as rs10757278 in 9p21   and rs11833579 encoded ninj2 . numerous replicate studies are needed to perform in various populations in order to provide more solid evidence of causality . to date , most studies aim at confirming previous susceptibility locus are population - based case control study , however , this kind of study design will cause misleading false positive results because of population stratification . therefore , we used a random non - is sibling of is patients as matched control , and carried out family - based case control study to confirm the association between single nucleotide polymorphisms ( snps ) in several novel genes and is . as with most complex diseases , is is likely to be influenced not only by genetic , but also by environmental components . traditional cardiovascular disease risk factors , such as cigarette smoking and alcohol drinking , may increase the risk of is [ 9 , 10 ] . some of these traditional risk factors may dramatically increase risk of disease together with risk alleles . identification of interactions between genetic and environment components will help to recognize individuals with high risk in developing is , and make cost - effective prevention strategies . thus , the joint effects of traditional environment risk factors and genes which have main effect in is were also investigated in the present study . following the protocol of fangshan / family - based ischemic stroke study in china ( fissic ) , the subjects were recruited in fangshan from june 2005 to june 2012 . fangshan located in southwest of beijing is a district with high prevalence of cardiovascular disease including is . is patients aged above 18  years old with computerized tomography ( ct ) or magnetic resonance imaging ( mri ) confirmed were recruited in the first hospital of fangshan district . patients are eligible for inclusion if they have at least one parents or a full sibling aged above 18  years participating in the study . demographic characters ( age , sex , etc . ) , lifestyle risk factors ( cigarette smoking etc . ) and medical history ( diagnose and treatment of stroke and other chronic disease ) were collected through a face - to - face interview by trained interviewers using uniform questionnaires . current smoker was defined as the one who smoked more than 100 cigarettes and still smoking during last 1  month , past smoker was defined as the one who had smoked that amount of cigarettes but have not smoke for at least 1  month , and non - smoker are those who has smoked < 100 cigarettes accumulatively . in the analysis , current smokers and blood pressure was measured three times at the right brachial artery using a sphygmomanometer after the participant has rested in a seated position for 5  min . concentrations of total cholesterol , triglycerides , and high - density lipoprotein cholesterol were measured with hitachi 7180 auto - analyzer ( hitachi high - technologies corp . , tokyo , japan ) in central clinical laboratory , central hospital . this study had been approved by the ethics committee of peking university health sciences center . each participant had been informed about the study and they had agreed for blood sampling and physical examination in this study . all subjects had signed an informed consent prior to participating in the study . in the end , we recruited 229 unrelated chinese patients with their 229 full sibling non - is controls . genomic dna was extracted from the peripheral blood with a dna extraction kit ( dp319 - 01 ; tiangen biotech , beijing , china ) . the pcr was performed on a thermal cycler ( abi geneamp 9700 384 dual ; abi , foster city , ca , usa ) . genotypes were analyzed with typer 4.0 software ( massarray compact system ; sequenom , san diego , ca , usa ) . all snps in this report had a genotyping success rate of > 98  % . the 199 singleton families contain only one affected individual and at least one unaffected siblings . the 44 multiplex families comprise families with at least 2 affected siblings , and any number of unaffected siblings . after excluding 14 multiplex families which have no unaffected siblings for this study , in affected group , one case from each family was selected . in unaffected group , one non - is siblings of is patient from each families was randomly selected . during data analysis process , means and standard deviations ( sds ) were reported for continuous variables , and percentages were reported for categorical variables . paired sample t test was used to examine the difference of continuous variables , and mcnemar s chi squared tests were used to test for the difference of categorical variables . each snp was tested separately for deviation from hardy  weinberg equilibrium ( hwe ) in control group using chi squared test . we used the sibship disequilibrium test ( sdt ) to test association between snps and is in the first step . conditional logistic regression served as a second method to confirm the sdt results and provided estimates of relative risks . we analyzed the family - based data , by using the procedure cox regression in ibm spss statistics 20.0 software , to fit the conditional logistic regression model and search for gene - environment interactions . all p values were two - tailed , with statistical significance defined as < 0.05 . following the protocol of fangshan / family - based ischemic stroke study in china ( fissic ) , the subjects were recruited in fangshan from june 2005 to june 2012 . fangshan located in southwest of beijing is a district with high prevalence of cardiovascular disease including is . is patients aged above 18  years old with computerized tomography ( ct ) or magnetic resonance imaging ( mri ) confirmed were recruited in the first hospital of fangshan district . patients are eligible for inclusion if they have at least one parents or a full sibling aged above 18  years participating in the study . demographic characters ( age , sex , etc . ) , lifestyle risk factors ( cigarette smoking etc . ) and medical history ( diagnose and treatment of stroke and other chronic disease ) were collected through a face - to - face interview by trained interviewers using uniform questionnaires . current smoker was defined as the one who smoked more than 100 cigarettes and still smoking during last 1  month , past smoker was defined as the one who had smoked that amount of cigarettes but have not smoke for at least 1  month , and non - smoker are those who has smoked < 100 cigarettes accumulatively . in the analysis , current smokers and blood pressure was measured three times at the right brachial artery using a sphygmomanometer after the participant has rested in a seated position for 5  min . concentrations of total cholesterol , triglycerides , and high - density lipoprotein cholesterol were measured with hitachi 7180 auto - analyzer ( hitachi high - technologies corp . , tokyo , japan ) in central clinical laboratory , central hospital . this study had been approved by the ethics committee of peking university health sciences center . each participant had been informed about the study and they had agreed for blood sampling and physical examination in this study . all subjects had signed an informed consent prior to participating in the study . in the end , we recruited 229 unrelated chinese patients with their 229 full sibling non - is controls . genomic dna was extracted from the peripheral blood with a dna extraction kit ( dp319 - 01 ; tiangen biotech , beijing , china ) . the pcr was performed on a thermal cycler ( abi geneamp 9700 384 dual ; abi , foster city , ca , usa ) . genotypes were analyzed with typer 4.0 software ( massarray compact system ; sequenom , san diego , ca , usa ) . all snps in this report had a genotyping success rate of > 98  % . the 199 singleton families contain only one affected individual and at least one unaffected siblings . the 44 multiplex families comprise families with at least 2 affected siblings , and any number of unaffected siblings . after excluding 14 multiplex families which have no unaffected siblings for this study , in affected group , one case from each family was selected . in unaffected group , one non - is siblings of is patient from each families was randomly selected . during data analysis process , means and standard deviations ( sds ) were reported for continuous variables , and percentages were reported for categorical variables . paired sample t test was used to examine the difference of continuous variables , and mcnemar s chi squared tests were used to test for the difference of categorical variables . each snp was tested separately for deviation from hardy  weinberg equilibrium ( hwe ) in control group using chi squared test . we used the sibship disequilibrium test ( sdt ) to test association between snps and is in the first step . conditional logistic regression served as a second method to confirm the sdt results and provided estimates of relative risks . we analyzed the family - based data , by using the procedure cox regression in ibm spss statistics 20.0 software , to fit the conditional logistic regression model and search for gene - environment interactions . all p values were two - tailed , with statistical significance defined as < 0.05 . of the 229 is patients , 157 ( 57.9  % ) were male , which were significantly more than control group ( p  <  0.001 , table  1 ) . the percent of is cases that had ever smoked cigarettes was significantly higher than controls ( 58.6 vs. 41.4  % , p  =  0.001 ) . systolic and diastolic blood pressures were significantly higher in cases than controls ( p    0.001 ) . there was no significant difference of the concentration of total cholesterol between patients and controls ( p  =  0.366 ) , while the concentration of hdl - cholesterol ( hdlc ) was significantly higher in patients than controls ( p  <  0.001).table  1clinical characteristics of cases and their unaffected siblingsaffectedunaffected 
 t/ 

 p valueage ( years)61.08    10.88558.63    9.133.1030.002**male157 ( 57.9  % ) 114 ( 42.1  % ) 16.711<0.001***smoke139 ( 58.6  % ) 98 ( 41.4  % ) 14.8960.001**diabetes61 ( 67.0  % ) 30 ( 33.0  % ) 13.719<0.001***waist ( cm)92.16    8.85088.16    9.5364.899<0.001***bmi ( kg / m)26.86    3.98726.05    3.5982.4350.016*hypertension173 ( 61.3  % ) 109 ( 38.7  % ) 37.798<0.001***systolic blood pressure ( mmhg)144.72    18.344134.21    19.9216.172<0.001***diastolic blood pressure ( mmhg)90    10.94286.21    11.0593.4320.001**dyslipidemia166 ( 54.2  % ) 140 ( 45.8  % ) 6.6570.010*tc ( mmol / l)4.70    1.0594.63    1.0910.9070.366hdlc ( mmol / l)1.42    0.431.57    0.5074.400<0.001 * * * 
 bmi body mass index , sbp systolic blood pressure , dbp diastolic blood pressure , tc total cholesterol , hdlc high density lipoprotein cholesterol *  p  <  0.05 ; * *  p  <  0.01 ; * * *  p  <  0.001 clinical characteristics of cases and their unaffected siblings 
 bmi body mass index , sbp systolic blood pressure , dbp diastolic blood pressure , tc total cholesterol , hdlc high density lipoprotein cholesterol *  p  <  0.05 ; * *  p  <  0.01 ; * * *  p  <  0.001 all 13 snps showed no significant deviation from hardy  weinberg equilibrium in the control group ( p  >  0.05 ) . the results of sdt and allele frequencies for patients and controls were shown in table  2 . significant associations with is were found in ninj2 gene rs11833579 ( p  =  0.008 ) , prkch gene rs2230501 ( p  =  0.039 ) . the a allele frequency in rs11833579 was significantly higher in stroke patients ( 40.0  % ) than that of unaffected controls ( 33.6  % ) . the c allele of rs2230501 is significantly higher in stroke patients ( 22.9  % ) than that of unaffected controls ( 19.4  % ) .table  2sdt results and allele frequency for 14 snpsgene / locationalleleaffected%unaffected% 
 p valuehwe p valuers266729 
 adipoq 
 c34372.0635175.650.1440.164g13327.9411324.35rs520540 
 mmp3 
 g29361.5529062.50.6220.302a18338.4517437.5rs966221 
 pde4d 
 t36076.2735777.270.7450.319c11223.7310522.73rs1800796 
 alox5ap 
 c30764.531167.030.1030.144g16935.515332.97rs2200733intergenict24551.9123851.520.9670.297c22748.0922448.48rs2230501 
 prkch 
 a36777.137480.60.039 * 0.654c10922.99019.4rs7178239 
 sels 
 g24250.8424051.720.6220.215c23449.1622448.28rs7193343 
 zfhx3 
 t32468.0731567.890.5960.351c15231.9314932.11rs9536591intergenicc28960.7129964.440.0510.931a18739.2916535.56rs10204475 
 znf650 
 g40985.9239284.850.9980.971t6714.087015.15rs107572778intergenica24755.1322752.550.8350.136g20144.8720547.45rs11052413intergenicg42190.7341389.780.7460.089t439.274710.22rs11833579 
 ninj2 
 g2826030466.380.008**0.390a1884015433.62 *  p  <  0.05 ; * *  p  <  0.01 sdt results and allele frequency for 14 snps *  p  <  0.05 ; * *  p <  0.01 conditional logistic regression validated the significant association of rs11833579 and rs2230501 with is . table  3 showed that , before any adjustments , the a allele of rs11833579 was the risk allele of is in dominant model [ odds ratio ( or ) 2.17 , 95  % ci 1.014.76 , p  =  0.049 ] . the aa genotype of rs11833579 increased risk of is 3.04-fold relative to the gg genotype ( 95  % ci 1.148.16 , p  =  0.027 ) . after adjusting for age , sex , hypertension , diabetes , serum lipids and smoke , the a allele of rs11833579 could increase 2.69-fold risk of is ( 95  % ci 1.066.78 ; p the aa genotype of rs11833579 could increase 1.51-fold risk of is ( 95  % ci 1.043.46 , p  =  0.043 ) . for rs2230501 , after multiple adjustments , the ac genotype gave a 2.93-fold increased risk of is relative to the aa genotype ( 95  % ci 1.078.00 , p  =  0.036).table  3conditional logistic regression results of rs11833579 and rs2230501variableunadjustedadjusted 
 hazard ratiolower 95  % ciupper 95  % ci 
 p valuehazard ratiolower 95  % ciupper 95  % ci 
 p valuers11833579gg(ref)ag1.1090.7662.5910.2700.9870.4352.2370.975aa3.0471.1388.1620.027 * 1.5061.0393.4580.043*recessive1.5670.9042.7160.1101.1610.5882.2900.688dominant2.1711.0114.7550.049 * 2.6871.0646.7830.036*rs2230501aa(ref)ac1.6390.8113.3110.1692.9301.0737.9960.036 
 cc1.7940.4137.7830.4351.2100.1718.5640.849recessive1.8690.8294.2130.1321.8690.8294.2130.132dominant1.4310.4354.7070.5551.0310.2374.4930.967 *  compared with gg genotype , p  <  0.05 
 compared with aa genotype , p  <  0.05 
 adjusted for age , sex , hypertension , diabetes , tc , hdlc , smoke conditional logistic regression results of rs11833579 and rs2230501 *  compared with gg genotype , p  <  0.05 
 compared with aa genotype , p  <  0.05 
 adjusted for age , sex , hypertension , diabetes , tc , hdlc , smoke smoking had significant interaction with rs111833579 in is ( table  4 ) . the odds ratios ( or ) to is rise continuously as the increasing number of risk allele in rs11833579 and ors rise more distinct if risk allele carriers were also smokers . the aa genotype could increase 3.58 times of risk in smokers than gg genotype in non - smokers ( 95  % ci 1.548.31 , p  =  0.003 ) , and 3.17-fold is risk than gg / ga genotype non - smokers ( 95  % ci 1.466.88 , p  =  0.004 ) . after adjusting for age and sex , the aa genotype of smokers could increase 2.72-fold risk of is ( 95  % ci 1.096.78 , p  =  0.032 ) than the gg genotype of non - smokers , and increase 2.41-fold risk of is than gg / ga genotype non - smokers ( 95  % ci 1.045.61 , p  =  0.041).table  4interaction of rs11833579 in ninj2 gene with smoke in ischemic strokeunadjustedadjusted 
 orlower 95  % ciupper 95  % ci 
 p valueorlower 95  % ciupper 95  % ci 
 p valuesmoke  no  gg(ref)  ga1.2460.6892.2520.4671.2190.6742.2080.512  aa1.8520.7524.5590.1801.7810.6964.5630.229  yes  gg2.0851.1383.8190.017 * 1.5280.7543.0990.240  ga2.3941.3324.2970.003**1.6360.8203.2640.162  aa3.5751.5378.3140.003**2.7161.0876.7800.032*smoke  no  gg(ref) ga  +  aa1.3490.7662.3730.3001.3150.7462.3190.343  yes  gg2.0851.1383.8190.017 * 1.5420.7633.1140.227  ga  +  aa2.6251.5054.5810.001**1.8590.9623.5970.065smoke  no  gg  +  ga(ref)  aa1.6390.7103.7890.2471.5970.6593.8650.300  yes  gg  +  ga1.9881.3242.9860.001**1.4080.8292.3920.206  aa3.1651.4566.8760.004**2.4111.0375.6130.041 * *  p  <  0.05 ; * *  p  <  0.01 
 adjusted for age and sex interaction of rs11833579 in ninj2 gene with smoke in ischemic stroke *  p  <  0.05 ; * *  p  <  0.01 
 adjusted for age and sex our study described the association between novel snps and is using case - sibling matched case in addition , we also found a significant interaction of rs11833579 and smoking in is . the strength of our study include well characteristic is patients with imaging data , and the usage of non - is sibling as matched control , so as to reduce false positive rate due to population stratification . additionally , fangshan district , where our study was conducted , was located in the  stroke belt  of china similar to the  stroke belt  in the south - eastern united states , and its population was representative of the rural northern han chinese . besides , to our knowledge , it is the first study to examine the interaction of smoke and rs11833579 in is . ninjurin2 ( ninj2 ) , which is a cell surface adhesion molecule , plays a role in nerve regeneration via hemophilic interaction . the result of our study was consistent with the previous genome - wide association study which demonstrated that rs11833579 were associated with is in caucasians . similarly , some replicate study in asia also found the association between rs11833579 and is [ 14 , 15 ] . tong et al .   reported that the a allele of rs11833579 was significantly associated with is in the han population ( or 1.27 , 95  % ci 1.081.49 ) , and rs11833579 conferred susceptibility to is in recessive model ( p  =  0.004 ) . on the contrary , another chinese study showed that there was no statistically significant association between rs11833579 and is . besides , a study included 137 is patients and 66 controls from nanjing china , also found no association between rs11833579 and is . the difference may be explained by the several possibilities : ( 1 ) our study was conducted in a population with stable genetic background in north china , the studies provide different results recruited their samples in south china [ 16 , 17 ] , the different genetic background may confers to the difference ; ( 2 ) the living habits are different between north and south of china , since is is a complex disease affected by both genetic and environment , the complex interaction of different environmental risk factors with snps may explain the difference ; ( 3 ) some population based case  control studies with limited sample size may lack of power to detect associations with alleles with a small effect . although the results remain controversial , our results provide additionally evidence for the association between rs11833579 and is with relatively lower false positive rate . in addition , our study demonstrated that rs2230501 in prkch gene had significant association with is . protein kinase c mediates a wide variety of signaling pathways   and involves in the induction of inducible nitric oxide synthase ( inos ) which lead to vascular endothelial dysfunction . moreover , it also plays an important role in the development of atherosclerotic diseases . to our knowledge , this is the first study to replicate the association between rs2230501 and is . another snp rs2230500 , located near and almost in absolute linkage disequilibrium ( ld ) with rs2230501 ( d  =  0.99 ) , is the most frequently studied snp in prkch gene . a large - scale genetic epidemiological study   in japanese subjects first showed that a nonsynonymous snp rs2230500 in the prkch gene with cerebral infarction . chinese researchers also demonstrated that rs2230500 was significantly associated with is ( or 1.31 ; 95  % ci 1.081.60 ; p , the associations remained significant ( or 1.37 , 95  % ci 1.121.67 , p  =  0.0019 ) . our study showed the heterozygote of rs2230501 , which is in absolute ld with rs2230500 , could increase the risk of is compared with wild homozygote . more studies are still need to confirm this association with larger sample size and higher statistical power in chinese han population . in addition , our study found that smoking could increase the risk of is together with rs11833579 . it is known that smoking affects cell - mediated immune responses , causes oxidative damage and arterial endothelial dysfunction [ 21 , 22 ] . ninjurin2 is a hemophilic adhesion molecule , which is nerve injury - induced and expressed in many tissues including peripheral nerves . although the independent role of smoke and ninj2 gene is relatively clear , the mechanism involved their joint effect on is is unknown . it is possible that ninj2 affects is by influencing the reaction to brain cells to injury , which might be modified by the oxidative damage or inflammation due to smoke . more replicable studies based on large population are needed to confirm this result and reveal the underlying mechanism of the interaction . first , the sample size is relatively small , since it is difficult to recruit is patients together with their discordant siblings . one of the largest family - based is studies in the western countries is siblings with ischemic stroke study ( swiss ) , which recruited 223 probands and 84 stroke - unaffected siblings . although our sample size is not as large as some population - based case control studies , it is larger than most is family - based studies . second , associations of snps with subtypes of is were not examined in the present investigation due to the limited sample size . thus , studies are needed to examine the association of ninj2 gene variation in different is subtypes in the future so as to provide further evidence for the pathogenic mechanisms of is . in conclusion , we confirmed that ninj2 genetic variants were related to risk of is in the northern chinese population , and found the interaction between rs11833579 and smoking . further research is needed to confirm this interaction so that the pathogenic mechanisms of is can be featured and intervened .
novel susceptibility genes related to ischemic stroke ( is ) are proposed in recent literatures . 
 population - based replicate studies would cause false positive results due to population stratification . 
 229 recruit is patients and their 229 non - is siblings were used in this study to avoid population stratification . 
 the family - based study was conducted in beijing from june 2005 to june 2012 . 
 association between snps and is was found in the sibship discordant tests , and the conditional logistic regression was performed to identify effect size and explore gene  environment interactions . 
 significant allelic association was identified between ninj2 gene rs11833579 ( p  =  0.008 ) , protein kinase c  gene rs2230501 ( p  =  0.039 ) and is . 
 the aa genotype of rs11833579 increased 1.51-fold risk ( 95  % ci 1.043.46 ; p  =  0.043 ) of is , and it conferred susceptibility to is only in a dominant model ( or 2.69 ; 95  % ci 1.066.78 ; p  =  0.036 ] . 
 risk of is was higher ( hr 3.58 ; 95  % ci 1.548.31 ; p  =  0.003 ) especially when the carriers of rs11833579 aa genotype were smokers . 
 the present study suggests a allele of rs11833579 may play a role in mediating susceptibility to is and it may increase the risk of is together with smoking .