cerebral ischemic stroke ( cis ) is a major cause of morbidity and mortality , and is expected to remain so until at least 2030 . high plasma levels of low - density lipoprotein cholesterol ( ldl - c ) have consistently been shown to be a risk factor for the development of atherosclerosis . plasma concentrations of ldl - c are determined primarily by the activity of the ldl receptor ( ldlr ) in the liver . proprotein convertase subtilisin - like kexin type 9 ( pcsk9 ) was recently discovered to be a major factor in cholesterol homeostasis through enhanced degradation of ldlr [ 36 ] and possibly in neural development . however , both rare mutations and common variants in the coding regions of pcsk9 can affect ldl cholesterol levels and stroke risk . recent studies identified several pcsk9 variants influencing circulating ldl - c levels . since the first identification mutation of pcsk9 was implicated in autosomal dominant hypercholesterolemia by abifadel , more than 53 missense variants have been identified . a common snp , e670 g ( rs505151 ) in exon 12 of pcsk9 , results in the substitution of glutamate for a glycine residue at position 670 in the protein   carriers of 670 gln in the general population presented increased plasma tc , ldl - c , and apob levels . another study suggested a key role played by the e670 g polymorphism in determining plasma ldl - c levels and the severity of coronary atherosclerosis in the united states . more recently , the presence of the 670 g allele was significantly associated with an increased risk of large - vessel atherosclerosis ( lva ) stroke   and intimal media thickness ( imt ) . however , these studies were inconsistent with previous studies [ 1416 ] , which were conducted in caucasian and african populations and failed to find this association . furthermore , the carriers of 670 g showed significantly increased ldl in men but not in women in a european population . in addition , the rs72555377 insertion polymorphism in exon 1 of pcsk9 is associated with lower ldl - c in caucasian populations , while the l11 allele , with insertion of 2 leucines , is associated with higher ldl - c , and rs562556 ( ile474val ) in exon9 of the pcsk9 gene is associated with approximately 7% lower ldl cholesterol levels in carriers in a japanese population . in our study , we used a single - base terminal extension ( snapshot ) method to detect the genotypes of the 20 single - nucleotide polymorphisms ( snp ) in the pcsk9 gene to assess the association between the human pcsk9 gene polymorphism and cis in members of the han and uygur populations of china . all participants explicitly provided permission for dna analyses as well as collection of relevant clinical data . this study was approved by the ethics committee of the first affiliated hospital of xinjiang medical university , urumqi , china ( no . 20120510 ) . subjects were from a han population and a uygur population who lived in the xinjiang uygur autonomous region of china . we recruited the cis group from the first affiliated hospital of xinjiang medical university neurology department between since october 2011 and may 2012 , and the control group came from the same hospital in the same period . in the cis group , there were 408 cis patients ( 158 uygur , 250 han ) , mean age 61.9711.80 years . inclusion criteria were : ( 1 ) diagnosed in accordance with the standards set at 10 international classifications of diseases ( icd10 ) ; ( 2 ) confirmed by mri . exclusion criteria were : ( 1 ) patients with chd ; ( 2 ) hemorrhagic cerebrovascular disease confirmed by ct or mri ; ( 3 ) refused to participate in trials . in the control group there were 348 of healthy controls ( 149 uygur , 199 han ) , mean age 61.8411.65 years . inclusion criteria were : ( 1 ) aged > 40 ; ( 2 ) no known family history of cerebrovascular disease ; ( 3 ) the cardiopulmonary physical examination and nervous system examination did not find abnormalities ; ( 4 ) mri negative except for cerebrovascular disease . all patients completed the standard test registration form , and disclosed the following data : ( 1 ) general information : age , sex , race . ( 2 ) personal history : smoking history ( daily average smoking , smoking an average of 1 day or more , time > 1 year , defined as smoking ) , ( drinking alcohol an average of 3 times per week , more than 50 g each time > 1 year , defined as drinking ) , hypertension , diabetes , hyperlipidemia , transient ischemic attack ( tia ) , atrial fibrillation ( af ) , heart valve disease , heart valve replacement , peripheral vascular disease . hypertension : the seventh world health organization /international society of hypertension league conference defined the new standard for the diagnosis of hypertension ; in our study , the diagnosis of hypertension was established if patients were treated with antihypertensive medication or if the mean of 3 measurements of systolic blood pressure ( sbp ) > 140 mm hg or diastolic blood pressure ( dbp ) > 90 mm hg , respectively . mmol / l or after fasting glucose 7.0mmol / l or glucose in line 2 h 11.1mmol / l or with a history of diabetes or treatment with insulin were considered diabetic . ( 3 ) medical history prior to admission : treatment with antihypertensive drugs , antiplatelet drugs and anticoagulants , diabetes , lipid drug , anti - seizure medication , birth control pills , hormones . ( 4 ) family history : whether grandparents , parents , siblings , and children had hypertension , diabetes , cerebral hemorrhage , cerebral infarction , myocardial infarction , coronary heart disease , or arrhythmia incidence . ( 5 ) physical examination : height , weight , blood pressure , pulse , temperature . ( 6 ) special tests : electrocardiogram , chest x - ray , heart neck ultrasound , blood routine , blood glucose , blood lipids . serum concentrations of total cholesterol ( tc ) , triglyceride ( tg ) , glucose ( glu ) , high - density lipoprotein cholesterol ( hdl - c ) , and low - density lipoprotein cholesterol ( ldl - c ) , apolipo protein a1 ( apoa1 ) , apolipo protein b ( apob ) , and apolipo lipoprotein a ( apolpa ) were measured using standard methods in the central laboratory of first affiliated hospital of xinjiang medical university . fasting blood samples ( 5 ml ) drawn by venipuncture were taken from all participants early in the morning . the blood samples were drawn into a 5-ml ethylene diamine tetraacetic acid ( edta ) tube and centrifuged at 4000g for 5 min to separate the plasma content . the dna samples were stored at 80c until use , then diluted to 50 ng/l concentration . we selected the genotypes of the 20 snps in the pcsk9 gene using the haploview 4.2 software and the hapmap phrase ii database by using minor allele frequency ( maf ) 0.1 and linkage disequilibrium patterns with r2 0.5 as a cut - off . the position of the 20 snps was by order of increasing distance from the gene pcsk9 5 end ( table 1 ) . snapshot reactions were performed as described by the manufacturer ( applied biosystems , warrington , uk ) . briefly , 4.0-l of pcr product was incubated at 37c for 60 min with 2-u shrimp alkaline phosphatase ( sap ) and 2-u exonuclease i ( exoi ) . following a 15-min incubation to inactivate the enzymes , 1 ul of digested pcr product was mixed with 5 ul of ready reaction premix , 1 ul of 1.0- um primer ( table 1 ) , and 3 ul of dh2o . this mixture was placed in the thermal cycler and underwent 25 cycles of 96c for 10 s , 50c for 5 s , and 60c for 30 s. when completed , 0.5-u sap was added and the reaction mixture was incubated for 60 min . prior to loading onto the prism 310 , 10 ul of formamide was added to 1 ul of reaction mixture and samples were heated to 95c for 5 min . all continuous variables ( e.g. , age , bmi , pulse , and cholesterol levels ) are presented as means  standard deviation ( s.d . ) . the difference between the cis and control groups was analyzed using an independent - sample t - test . the differences in the frequencies of sex , hypertension , diabetes mellitus , smoking , drinking , and genotypes were analyzed using chi - square test or fisher s exact test , as appropriate . logistic regression analyses with effect ratios ( odds ratio [ or ] and 95% ci ) were used to assess the contribution of the major risk factors . all statistical analyses were performed using spss 17.0 for windows ( spss institute , chicago , usa ) . all participants explicitly provided permission for dna analyses as well as collection of relevant clinical data . this study was approved by the ethics committee of the first affiliated hospital of xinjiang medical university , urumqi , china ( no . 20120510 ) . subjects were from a han population and a uygur population who lived in the xinjiang uygur autonomous region of china . we recruited the cis group from the first affiliated hospital of xinjiang medical university neurology department between since october 2011 and may 2012 , and the control group came from the same hospital in the same period . in the cis group , there were 408 cis patients ( 158 uygur , 250 han ) , mean age 61.9711.80 years . inclusion criteria were : ( 1 ) diagnosed in accordance with the standards set at 10 international classifications of diseases ( icd10 ) ; ( 2 ) confirmed by mri . exclusion criteria were : ( 1 ) patients with chd ; ( 2 ) hemorrhagic cerebrovascular disease confirmed by ct or mri ; ( 3 ) refused to participate in trials . in the control group there were 348 of healthy controls ( 149 uygur , 199 han ) , mean age 61.8411.65 years . inclusion criteria were : ( 1 ) aged > 40 ; ( 2 ) no known family history of cerebrovascular disease ; ( 3 ) the cardiopulmonary physical examination and nervous system examination did not find abnormalities ; ( 4 ) mri negative except for cerebrovascular disease . all patients completed the standard test registration form , and disclosed the following data : ( 1 ) general information : age , sex , race . ( 2 ) personal history : smoking history ( daily average smoking , smoking an average of 1 day or more , time > 1 year , defined as smoking ) , ( drinking alcohol an average of 3 times per week , more than 50 g each time > 1 year , defined as drinking ) , hypertension , diabetes , hyperlipidemia , transient ischemic attack ( tia ) , atrial fibrillation ( af ) , heart valve disease , heart valve replacement , peripheral vascular disease . hypertension : the seventh world health organization /international society of hypertension league conference defined the new standard for the diagnosis of hypertension ; in our study , the diagnosis of hypertension was established if patients were treated with antihypertensive medication or if the mean of 3 measurements of systolic blood pressure ( sbp ) > 140 mm hg or diastolic blood pressure ( dbp ) > 90 mm hg , respectively . mmol / l or after fasting glucose 7.0mmol / l or glucose in line 2 h 11.1mmol / l or with a history of diabetes or treatment with insulin were considered diabetic . ( 3 ) medical history prior to admission : treatment with antihypertensive drugs , antiplatelet drugs and anticoagulants , diabetes , lipid drug , anti - seizure medication , birth control pills , hormones . ( 4 ) family history : whether grandparents , parents , siblings , and children had hypertension , diabetes , cerebral hemorrhage , cerebral infarction , myocardial infarction , coronary heart disease , or arrhythmia incidence . ( 5 ) physical examination : height , weight , blood pressure , pulse , temperature . ( 6 ) special tests : electrocardiogram , chest x - ray , heart neck ultrasound , blood routine , blood glucose , blood lipids . serum concentrations of total cholesterol ( tc ) , triglyceride ( tg ) , glucose ( glu ) , high - density lipoprotein cholesterol ( hdl - c ) , and low - density lipoprotein cholesterol ( ldl - c ) , apolipo protein a1 ( apoa1 ) , apolipo protein b ( apob ) , and apolipo lipoprotein a ( apolpa ) were measured using standard methods in the central laboratory of first affiliated hospital of xinjiang medical university . fasting blood samples ( 5 ml ) drawn by venipuncture were taken from all participants early in the morning . the blood samples were drawn into a 5-ml ethylene diamine tetraacetic acid ( edta ) tube and centrifuged at 4000g for 5 min to separate the plasma content . the dna samples were stored at 80c until use , then diluted to 50 ng/l concentration . we selected the genotypes of the 20 snps in the pcsk9 gene using the haploview 4.2 software and the hapmap phrase ii database by using minor allele frequency ( maf ) 0.1 and linkage disequilibrium patterns with r2 0.5 as a cut - off . the position of the 20 snps was by order of increasing distance from the gene pcsk9 5 end ( table 1 ) . snapshot reactions were performed as described by the manufacturer ( applied biosystems , warrington , uk ) . briefly , 4.0-l of pcr product was incubated at 37c for 60 min with 2-u shrimp alkaline phosphatase ( sap ) and 2-u exonuclease i ( exoi ) . following a 15-min incubation to inactivate the enzymes , 1 ul of digested pcr product was mixed with 5 ul of ready reaction premix , 1 ul of 1.0- um primer ( table 1 ) , and 3 ul of dh2o . this mixture was placed in the thermal cycler and underwent 25 cycles of 96c for 10 s , 50c for 5 s , and 60c for 30 s. when completed , 0.5-u sap was added and the reaction mixture was incubated for 60 min . prior to loading onto the prism 310 , 10 ul of formamide was added to 1 ul of reaction mixture and samples were heated to 95c for 5 min . all continuous variables ( e.g. , age , bmi , pulse , and cholesterol levels ) are presented as means  standard deviation ( s.d . ) . the difference between the cis and control groups the differences in the frequencies of sex , hypertension , diabetes mellitus , smoking , drinking , and genotypes were analyzed using chi - square test or fisher s exact test , as appropriate . logistic regression analyses with effect ratios ( odds ratio [ or ] and 95% ci ) were used to assess the contribution of the major risk factors . all statistical analyses were performed using spss 17.0 for windows ( spss institute , chicago , usa ) . table 2 showed the clinical characteristics of the cis patients ( n=408 ) and control participants ( n=348 ) . for all han and uygur subjects , there were no significant differences in age and sex between cis patients and control subjects , indicating the study was an age- and sex - matched case - control study . as expected , several common risk factors for cis were significantly different between the 2 subgroups : glu , low hdl - c , high ldl - c , eh , and dm . other cis risk factors , such as high tc , tg levels , and cigarette smoking and drinking , were not significantly different . table 1 shows the basic information and the distribution of genotypes and alleles of the 20 snps for the pcsk9 gene . the position of the 20 snps was by order of increasing distance from the gene pcsk9 5 end . we observed that the distribution of genotypes and alleles of 3 snps ( snp1 , snp2 , and snp8 ) were significantly different between cis group and control participants . the 3 snps among the 3 groups ( total , han , and uyghur ) were examined by hardy - weinberg equilibrium test and no significant differences were found in these 3 groups ( data not shown ) . in the study , we confirmed the distribution of genotypes and alleles of the 3 snps ( snp1 , snp2 , and snp8 ) for the pcsk9 gene . for snp1 ( rs17111503 ) , the distribution of alleles showed a significant difference between cis and control participants ( p=0.028 ) in the han group , but not in the total group and uygur group . for snp2 ( rs2479408 ) , the distribution of alleles , the dominant model ( cc vs. cg + gg ) , and the additive model ( cg vs. cc + gg ) showed a significant difference between cis and control participants in total and han groups , but not in the uygur group . c allele of rs2479408 was significantly higher in cis patients than in control participants ( total : 96.94% vs. 94.97% ; han : 99.80% vs. 98.24% ) . for snp3 ( rs529787 ) , the distribution of alleles , the dominant model ( cc vs. cg + gg ) and the additive model ( cg vs. cc + gg ) showed a significant difference between cis and control participants in the total and han groups , but not in the uygur group . c allele of rs529787 was significantly higher in cis patients than in control participants ( total : 96.81% vs. 94.68% ; han : 99.80% vs. 97.99% ) ( data no shown ) . table 3 and figure 1 show patterns of linkage disequilibrium in the pcsk9 gene , with their |d| and r values . |d| values from 0.25 to 0.7 indicate moderate ld and |d| values of 00.25 indicate low ld . in the study , 3 strong ld patterns were observed between snp1 and snp2 ( |d|=0.999 ) , snp2 and snp8 ( |d|=0.983 ) , and snp1 and snp8 ( |d|=0.999 ) . the r value of snp2snp8 > 0.5 means the snp2 and snp8 can replace each other   and they can not construct haplotypes simultaneously . therefore , given that the position of snp1 and snp2 are both in 2 kb upstream of pcsk9 gene and the position of snp8 is in intron3 , we constructed the haplotypes using snp1 and snp2 . the overall distribution of the haplotypes were significantly different between the cis patients and the control subjects ( all p<0.05 ) . the most frequent haplotype in this study was a - c haplotype . for han , the frequency of a - c was significantly higher in the cis patients than in the control subjects ( p=0.0011 ) . in addition , the frequency of the g - c haplotype was lower in the cis patients than in the control subjects ( p=0.0011 ) . table 5 showed that multiple logistic regression analyses were performed with age , sex , bmi , hdl - c , ldl - c , tc , tg , apoa1 apob , apolpa , eh , dm , and smoking and drinking , because these variables were the major confounding factors for cis . the significant difference of the dominant model ( cc vs. cg + gg ) of rs2479408 was retained after adjustment for covariates in the han , but not in the uygur group ( or : 75.262 , 95% confidence interval [ ci ] : 7.232783.278 , p<0.001 ) . pcsk9 , also known as neural apoptosis - regulated convertase 1 ( narc1 ) , is the ninth member of the proprotein convertase ( pc ) family . the human pcsk9 gene is located on chromosome 1p32.3 ; it encompasses 12 exons and encodes a 692 amino acid glycoprotein . pcsk9 is synthesized as an inactive zymogen , pro - pcsk9 ( 73 kda ) and contains a signal peptide , a prodomain ( residues 31152 ) and a catalytic domain ( residues 153451 ) followed by a c - terminal domain ( residues 452692 ) . pcsk9 acts as a serine protease and molecular chaperone that reduces both hepatic and extrahepatic low - density lipoprotein receptor levels through an endosomal / lysosomal pathway and increases plasma ldl cholesterol . recent advances revealed a large number of genetic variants of pcsk9 that may modulate plasma cholesterol levels either positively or negatively . gain of function  missense mutations in pcsk9 were associated with autosomal - dominant hypercholesterolemia ( adh ) , a rare form of familial hypercholesterolemia ( fh ) in which neither the ldlr nor the ligand binding domain of apolipoprotein ( apo ) b100 are mutated . loss of function  nonsense mutations in pcsk9 were associated with low plasma ldl - c levels and a reduced incidence of cardiovascular disease . later , many in vitro and in vivo overexpression and knockout / knockdown studies confirmed that pcsk9 targets the ldlr for degradation [ 3234 ] . studies have confirmed that both rare mutations and common variants in the coding regions of pcsk9 affect ldl cholesterol levels and stroke risk . in this study , we selected 20 snps of pcsk9 and used case - control analyses to assess the association between the human pcsk9 gene polymorphism and cis in the han and uygur populations . our findings showed the distribution of snp8 ( rs529787 ) genotypes were significantly different between cis and control participants ( p=0.049 ) . however , when analyzing han and uygur groups separately , we found that only in the han population was the distribution of snp1 ( rs1711503 ) , snp2 ( rs2479408 ) , and snp8 ( rs529787 ) alleles significantly different between cis and control participants ( p=0.028 , p=0.013 , p=0.006 , respectively ) . for snp1 ( rs17111503 ) , the frequency of a allele was higher in cis than in control participants ( p=0.028 , 39.80% vs. 32.66% ) in the han group , indicating that the risk of cis was increased with the a allele of rs17111503 . for snp2 ( rs2479408 ) , the distribution of alleles , the dominant model ( cc vs. cg + gg ) , and the additive model ( cg vs. cc + gg ) showed a significant difference between cis and control participants in total and han groups , but not in the uygur group . c allele of rs2479408 was significantly higher in cis patients than in control participants ( total : 96.94% vs. 94.97% ; han : 99.80% vs. 98.24% ) . moreover , the significant difference of the dominant model ( cc vs. cg + gg ) of rs2479408 was retained after adjustment for covariates : age , sex , bmi , hdl - c , ldl - c , tc , tg , apoa1 apob , apolpa , eh , dm , and smoking and drinking in the han group ( or : 75.262 , 95% confidence interval [ ci ] : 7.232783.278 , p<0.001 ) , indicating that the risk of cis was increased with the c allele of rs2479408 . for snp3 ( rs529787 ) , the distribution of alleles , the dominant model ( cc vs. cg + gg ) , and the additive model ( cg vs. cc + gg ) showed a significant difference between cis and control participants in total and han groups , but not in uygurs . c allele of rs529787 was significantly higher in cis patients than in control participants ( total : 96.81% vs. 94.68% ; han : 99.80% vs. 97.99% ) . when we constructed the haplotypes using snp1 and snp2 , we found that the most frequent haplotype in this study was a - c haplotype . for han , the frequency of a - c was significantly higher in the cis patients than in the control subjects ( p=0.0011 ) , but the frequency of the g - c haplotype was lower in the cis patients than in the control subjects ( p=0.0011 ) . this fully showed that a allele of rs17111503 and c allele of rs2479408 may be the risk factor of cis , and g allele of rs17111503 and g allele of rs2479408 may be the protective factor of cis . snp20 ( rs505151 ) was observed in the exon12 of the pcsk9 gene and the polymorphisms caused the substitution of glutamate for a glycine residue at position 670 in the protein . the studies about the association between rs505151 of pcsk9 gene polymorphisms ( e670 g ) and the cardiovascular risk have provided inconsistent results , as the introduction of description . our study was consistent with previous studies [ 1416 ] showing no significant association between the polymorphism of pcsk9 ( rs505151 ) and cis . by comparison , we found the age of our control subjects was higher than the other studies   and the study by afef slimani . in our study , there were no significant difference in age between cis patients ( age : 63.5611.37 ) and control subjects ( age : 62.3511.79 ) ( p=0.269 ) , but in the study by afef slimani , there were significant difference in age between cis patients ( age : 66/54.576.50 ) and control subjects ( age : 49/4555 ) ( p<0.0001 ) . age is a risk factor for stroke , and this may be why our conclusions were not consistent with their conclusions . in addition , there may be differences in populations and geographical factors that explain some differences . we found that both rs1711503 and 2479408 of pcsk9 were associated with cis in the han population of china . a - c haplotype may be a risk genetic marker of cis in han in china . a allele of rs17111503 and c allele of rs2479408 may be the risk marker of cis . studies with statistically significant numbers of clinical samples are needed for further research in china .
backgroundcerebral ischemic stroke ( cis ) is a major cause of morbidity and mortality . 
 its main pathological basis is atherosclerosis ( as ) ; in turn , the main risk factor in as is dyslipidemia . 
 human proprotein convertase subtilisin / kexin9 ( pcsk9 ) plays a key role in regulating plasma low - density lipoprotein ( ldl ) cholesterol levels . 
 we sought to assess the association between pcsk9 and cis in chinese han and uygur populations.material/methodswe selected 408 cis patients and 348 control subjects and used a single - base terminal extension ( snapshot ) method to detect the genotypes of the 20 single - nucleotide polymorphisms ( snps ) in pcsk9.resultsdistribution of snp8 ( rs529787 ) genotypes showed a significant difference between cis and control participants ( p=0.049 ) . 
 however , when analyzing han and uygur populations separately , we found that only han subjects showed distribution of snp1 ( rs1711503 ) , snp2 ( rs2479408 ) , and snp8 ( rs529787 ) alleles that was significantly different between cis and control participants ( p=0.028 , p=0.013 , p=0.006 , respectively ) , and distribution of snp2 ( rs2479408 ) in the dominant model ( cc vs. cg + gg ) was significantly different between cis and control participants ( p=0.013 ) , even after adjustment for covariates ( or : 75.262 , 95% confidence interval [ ci ] : 7.232783.278 , p<0.001 ) . 
 distribution of the 2 haplotypes ( a - c and g - c ) ( rs1711503 and rs2479408 ) was significantly different between cis and control participants ( both , p=0.011).conclusionsboth rs1711503 and rs2479408 of pcsk9 genes were associated with cis in the han population of china . 
 a - c haplotype may be a genetic marker of cis risk in this population .