nitric oxide ( no ) is known to have several important vasculo - protective effects ( el - mas et al 1997 ; grdal et al 2005 ) . reduced production of no may lead to atherosclerosis ( lscher and vanhoutte 1990 ; harrison 1997 ; shimokawa 1999 ) .
the synthesis of no from the amino acid , l - arginine , is catalysed by no synthase ( nos ) .
no then plays a critical role in the relaxation of vascular smooth muscle cells ( vsmcs ) .
it is well known that no is involved in reducing vsmc proliferation , adhesion of platelets and leukocytes ( moncada and higgs 1993 ; draijer et al 1995 ; myers and tanner 1998 ; brocq et al 2008 ) .
no is likely , therefore , to be an important contributory factor in the pathogenesis of some forms of stroke .
there are 3 distinct isoforms of nos identified in humans : endothelial ( enos ) , neuronal nos ( nnos ) and inducible nos ( inos ) .
it synthesizes very low levels no under basal conditions ( lscher and vanhoutte 1990 ; harrison 1997 ; shimokawa 1999 ) . because enos plays a central role in the maintenance of vascular homeostasis , including regulation of the cerebral circulation ,
a number of studies have been conducted to investigate the genetic association between the enos gene and the incidence of ischemic stroke .
howard and colleagues ( 2005 ) detected 3 single nucleotide polymorphisms ( snps ) in the promoter region , 1 in exon 7 ( g894 t ) and 1 insertion / deletion polymorphism within intron 4 among 110 cases with ischemic stroke and 206 controls .
they found strong associations of both the 922 g - a and 786 t - c snps with ischemic stroke .
hou and colleagues ( 2001 ) were the first to report the genetic association between the enos gene and ischemic stroke in a chinese population and a recent work has confirmed the intial finding ( berger et al 2007 ) although the enos association failed to be replicated in a japanese population ( yahashi et al 1998 ) .
this study might have been hampered by poor replication , a common problem with genetic analysis .
linkage disequilibrium ( ld ) structure and sample power may be one of major reasons for poor replication of an initial finding .
therefore , the present study was designed to apply a large sample size to investigate genetic association between the enos gene and ischemic stroke .
the present study recruited 560 unrelated patients with ischemic stroke and 153 unrelated controls in order to undertake a genetic analysis for association between the nos3 gene and ischemic stroke .
patients with ischemic stroke were admitted to the first hospital of jilin university , changchun , china in the period between 2005 and 2006 .
they were diagnosed as having ischemic stroke independently by at least two well - trained neurologists based on strict neurological examination , computed tomography , or magnetic resonance imaging , which meet the international classification of diseases ( nchs 2007 ) .
detailed information regarding their medical history , medication and clinical presentation was taken through either their close relatives or themselves .
of these 560 patients ( 398 males and 162 females ) , 426 were suffering from simple lacunar infarction , 246 from large - vessel infarction and 112 from both .
the study was granted prior approval by the ethics committee of jilin university , changchun , china . to perform genetic analysis , we selected snp rs3918181 ( rsa i site ) as a genetic marker at the nos3 locus .
it is an a to g base change present in intron 14 of the gene .
polymerase chain reaction ( pcr)-based restriction fragment length polymorphism ( rflp ) analysis was performed to genotype rs3918181 .
genomic dna used for pcr amplification was extracted from the whole blood sample using a dna extraction kit ( promega , beijing , china ) .
pcr amplification was performed in a 25-l reaction volume containing 10 mm tris - hcl ( ph 8.3 ) , 50 mm kcl , 1.5 mm mgcl2 , 0.001% ( w / v ) gelatin , 200 m of each dntp , 0.4 m of each primer , 1.0 unit of taq dna polymerase ( promega , beijing , china ) , and 3050 ng of genomic dna .
the conditions used for pcr amplification included an initial denaturation at 94 c for 5 min , followed by 3540 cycles of 94 c for 45s , 5560 c for 1 min and 72 c for 1 min , and a final elongation at 72 c for 10 min .
a 15-l aliquot of the pcr products was completely digested with 68 units of restriction enzymes .
the hardy - weinberg equilibrium for the genotypic distributions of rs3918181 was tested using the chi - square (  ) goodness - of - fit test .
applied to test sample power for individual alleles was tested using the spss samplepower 2.0 software package ( spss inc . ,
allelic association was tested by the cocaphase module of the unphased program ( dudbridge 2003 ) and genotypic association was tested by the spss program for windows 15.0 ( spss inc . ) .
the  goodness - of - fit test showed that the genotypic distributions of rs3918181 were not deviated from hardy - weinberg equilibrium in both the patient group (  = 0.166 , p = 0.684 ) and the control group (  = 0.421 , p = 0.517 ) .
these 560 case and 153 control samples gave a power of 60% for detection of small effect size and 100% for detection of medium effect size .
analysis with the cocaphase program showed that rs3918181 was associated with ischemic stroke in males (  = 4.04 ,
p = 0.044 , or = 1.43 , 95%ci 1.012.02 ) and frequency of allele a of rs3918181 was significantly higher in male patients than male control subjects ( table 2 ) .
however , female patients did not show such an association ( table 2 ) . as shown in table 3 ,
the  test revealed genotypic association between rs3918181 and ischemic stroke in males ( x = 4.26 , df = 1 , p = 0.039 , or = 1.61 , 95% ci 1.022.53 ) but not the genotypic association was not observed in female subjects .
the gene coding for enos is mapped to the long arm of chromosome 7 ( 7q36 ) and spans 21 kb of dna , although it contains 26 exons
. it should be sufficient to capture a linkage disequilibrium ( ld ) signal by genotyping a highly polymorphic dna marker present in the middle of the gene .
snp rs3918181 is located in intron 14 of the gene and has heterozygosity of 0.43 .
this finding gives further evidence in support of the enos association with ischemic stroke in the chinese population , although female patients did not show such an association in our samples ( tables 2 and 3 ) .
the sample power test indicates that the study is not sufficiently powered at present for detection of small effect - size gene .
however , we will continue to collect controls matched for age , sex and ethnicity in order to confirm the results and 80% of power will be needed for combined samples in our future study .
 we recruited 560 unrelated patients with ischemic stroke and 153 unrelated controls to undertake a genetic analysis for association between the nos3 gene and ischemic stroke . 
 all the subjects were chinese of han descent . because the nos3 gene spans about 21 kb of dna and contains 26 exons , we selected a single nucleotide polymorphism ( snp ) rs3918181 , an a to g base change located in intron 14 of the gene , as a dna marker . 
 pcr - based restriction fragment length polymorphism analysis was applied to genotype rs3918181 ( rsai site ) . 
 the chi - square ( 2 ) goodness - of - fit test showed that the genotypic distributions of the marker were not deviated from hardy - weinberg equilibrium in both the patient group ( 2 = 0.166 , p = 0.684 ) and the control group ( 2 = 0.421 , p = 0.517 ) . 
 the cocaphase analysis showed allelic association of rs3918181 with ischemic stroke in males ( 2 = 4.04 , p = 0.044 , or = 1.43 , 95% ci 1.012.02 ) and frequency of allele a was significantly higher in male patients than male control subjects . 
 the 2 test revealed genotypic association between rs3918181 and ischemic stroke in males ( 2 = 4.26 , df = 1 , p = 0.039 , or = 1.61 , 95% ci 1.022.53 ) but not in females . 
 the present work suggests that rs3918181 is associated with ischemic stroke in male patients . 
 this finding gives further evidence in support of the enos association with ischemic stroke in the chinese population . 
Introduction
Materials and methods
Results
Discussion