spontaneous intracerebral hemorrhage ( ich ) is a devastating disease with an overall incidence of 24.6 per 100 000 person - years . the mortality of ich at 1 month was 3552% and only 20% regained functional independence by 6 months . hypertension , low cholesterol levels , heavy alcohol intake , advanced age , and male gender have been identified as important risk factors for ich . on the other hand , genome - wide association studies and large - scale genetic association studies have indicated an important role of genetic factors in ich pathogenesis [ 46 ] . elevated hcy has been associated with various vascular diseases , including coronary artery disease , venous thrombosis , and stroke . although not fully understood , endothelial dysfunction related to hyperhomocysteinemia has been suggested as one of the mechanisms . methylenetetrahydrofolate reductase ( mthfr ) , encoded by mtfhr gene , is a crucial enzyme regulating intracellular hcy and folate metabolism . mthfr catalyzes the transformation of 5,10-methylentetrahydrofolate to 5-methyltetrahydrofolate , which serves as the methyl group donor for converting homocysteine into methionine . the most investigated polymorphism in mthfr gene is c677 t , which can lead to the replacement at codon 222 for alanine to valine , causing increased thermolability and reduced activity of enzyme mthfr and , subsequently , an elevated plasma level of hcy . mthfr c677 t polymorphism has been speculated to be and extensively investigated as a risk factor for various vascular diseases , including ich . some reported a significant impact of c677 t polymorphism on ich risk [ 15 , 16 ] while some failed to replicate these findings . here , we conducted a case - control study to evaluate the influence of mthfr c677 t polymorphism on ich risk . furthermore , since ich incidence is heterogeneous among different chinese populations , this case - control study included subjects of chinese han ethnicity only . in addition , a meta - analysis was performed integrating data from the present samples and those available from previous studies focusing on chinese populations . consecutive adult ct - proved ich patients of chinese han ethnicity admitted to west china hospital of sichuan university were enrolled in this study from november 2011 to september 2012 . we excluded patients with secondary ich due to brain tumor , aneurysm , vascular malformation , head trauma , hemorrhagic transformation of cerebral infarction , coagulation disorder , or concurrent use of anticoagulation . age- , sex- , and ethnicity - matched control subjects without history of ich were recruited from those undergoing routine health examinations in our hospital . the following demographic and clinical data were collected from medical records of each participant : age , sex , hypertension , diabetes mellitus , hyperlipidemia , alcohol consumption , and smoking . hematoma location was classified as lobar or deep ( basal ganglia , thalamus , brain stem and cerebellum ) . this study was approved by the ethics committee of west china hospital of sichuan university . written informed consents were obtained from all participants or their legal surrogates . dna was isolated from peripheral whole blood using the dna blood kit ( bioteke corp . , the c677 t polymorphism was genotyped by the polymerase chain reaction- ligase detection reaction ( pcr - ldr ) sequencing method , as described previously . briefly , the pcr primers used were : 5-aggccagcctctcctgactgt-3 ( forward ) and 5-ccatgtcggtgcatgccttca-3 ( reverse ) . the pcr protocol was : an initial denaturation step of 5 min at 95c , 35 cycles of 94c for 15 s , 55c for 15 s , and 72c for 30 s , and a final extension step of 72c for 10 min . the following ldr was carried out in a total volume of 10l , containing 3 l pcr product , 1 l 10taq dna ligase buffer , 2 u of taq dna ligase ( neb ) , and 0.1 pmol of each probe . the ldr protocol was : 95c for 2 min , 35 cycles of 94c for 30 s , and 60c for 2 min . the fluorescent products of ldr were differentiated using abi 3730xl ( applied biosystems , usa ) . hardy - weinberg equilibrium ( hwe ) in the control subjects was tested by a goodness - of - fit chi - squared test . differences in baseline characteristics , genotype distribution , and allele frequency between ich patients and controls were evaluated using the t test or  test , as appropriate . multiple regression analysis was used to assess the independent impact of c677 t polymorphism after adjusting for age , sex , hypertension , diabetes , hyperlipidemia , smoking , and alcohol consumption . the alternative genetic models for the c677 t polymorphism included alleles ( t vs. c ) and genotypes for codominant , dominant , and recessive models . stratified analyses according to the hematoma location ( lobar or deep ) were also conducted . statistical analysis was performed with spss for windows , version 18.0 ( spss inc . , a meta - analysis was performed combining our data with previous published studies to further investigate the association of mtfhr c677 t polymorphism with ich risk in chinese populations . we searched pubmed , embase , and china national knowledge infrastructure ( cnki ) databases until july 2015 to screen for relevant genetic association articles . the following search terms were used : intracerebral hemorrhage , hemorrhagic stroke , mthfr , methylenetetrahydrofolate reductase , polymorphism , genotype , variant , and mutation . the inclusion criteria were : ( 1 ) a case - control design ; ( 2 ) conducted in a chinese population ; ( 3 ) provided information on genotype frequencies or sufficient data to calculate them ; ( 4 ) genotype distribution in control group was in accordance with hwe . for studies with overlapping subjects , for each included study , the following data were extracted : authors , year of publication , province , age , sex , sample size , source of control , matching criteria , and genotype distribution . the association of c677 t polymorphism with ich risk in chinese populations was evaluated by calculating pooled ors with 95% cis . we used cochran s q test and i statistic to assess between - study heterogeneity . when substantial heterogeneity was detected ( p<0.05 for q test or an i>50% ) , we used a random - effects model to assess the influence of genotype on ich risk . otherwise , a fixed - effects model was applied . to test the robustness of the findings , we performed sensitivity analysis by sequentially omitting each study . we assessed the potential publication bias visually by estimating the possible skewness in a funnel plot   and statistically with the methods described by egger . the latter is a weighted linear regression of standard normal deviates against the inverse of the standard error . the intercept of the regression is applied to measure the degree of funnel plot asymmetry . consecutive adult ct - proved ich patients of chinese han ethnicity admitted to west china hospital of sichuan university were enrolled in this study from november 2011 to september 2012 . we excluded patients with secondary ich due to brain tumor , aneurysm , vascular malformation , head trauma , hemorrhagic transformation of cerebral infarction , coagulation disorder , or concurrent use of anticoagulation . age- , sex- , and ethnicity - matched control subjects without history of ich were recruited from those undergoing routine health examinations in our hospital . the following demographic and clinical data were collected from medical records of each participant : age , sex , hypertension , diabetes mellitus , hyperlipidemia , alcohol consumption , and smoking . hematoma location was classified as lobar or deep ( basal ganglia , thalamus , brain stem and cerebellum ) . this study was approved by the ethics committee of west china hospital of sichuan university . written informed consents were obtained from all participants or their legal surrogates . dna was isolated from peripheral whole blood using the dna blood kit ( bioteke corp . , the c677 t polymorphism was genotyped by the polymerase chain reaction- ligase detection reaction ( pcr - ldr ) sequencing method , as described previously . briefly , the pcr primers used were : 5-aggccagcctctcctgactgt-3 ( forward ) and 5-ccatgtcggtgcatgccttca-3 ( reverse ) . the pcr protocol was : an initial denaturation step of 5 min at 95c , 35 cycles of 94c for 15 s , 55c for 15 s , and 72c for 30 s , and a final extension step of 72c for 10 min . the following ldr was carried out in a total volume of 10l , containing 3 l pcr product , 1 l 10taq dna ligase buffer , 2 u of taq dna ligase ( neb ) , and 0.1 pmol of each probe . the ldr protocol was : 95c for 2 min , 35 cycles of 94c for 30 s , and 60c for 2 min . the fluorescent products of ldr were differentiated using abi 3730xl ( applied biosystems , usa ) . hardy - weinberg equilibrium ( hwe ) in the control subjects was tested by a goodness - of - fit chi - squared test . differences in baseline characteristics , genotype distribution , and allele frequency between ich patients and controls were evaluated using the t test or  test , as appropriate . multiple regression analysis was used to assess the independent impact of c677 t polymorphism after adjusting for age , sex , hypertension , diabetes , hyperlipidemia , smoking , and alcohol consumption . the alternative genetic models for the c677 t polymorphism included alleles ( t vs. c ) and genotypes for codominant , dominant , and recessive models . stratified analyses according to the hematoma location ( lobar or deep ) were also conducted . statistical analysis was performed with spss for windows , version 18.0 ( spss inc . , a meta - analysis was performed combining our data with previous published studies to further investigate the association of mtfhr c677 t polymorphism with ich risk in chinese populations . we searched pubmed , embase , and china national knowledge infrastructure ( cnki ) databases until july 2015 to screen for relevant genetic association articles . the following search terms were used : intracerebral hemorrhage , hemorrhagic stroke , mthfr , methylenetetrahydrofolate reductase , polymorphism , genotype , variant , and mutation . the inclusion criteria were : ( 1 ) a case - control design ; ( 2 ) conducted in a chinese population ; ( 3 ) provided information on genotype frequencies or sufficient data to calculate them ; ( 4 ) genotype distribution in control group was in accordance with hwe . for studies with overlapping subjects , for each included study , the following data were extracted : authors , year of publication , province , age , sex , sample size , source of control , matching criteria , and genotype distribution . the association of c677 t polymorphism with ich risk in chinese populations was evaluated by calculating pooled ors with 95% cis . we used cochran s q test and i statistic to assess between - study heterogeneity . when substantial heterogeneity was detected ( p<0.05 for q test or an i>50% ) , we used a random - effects model to assess the influence of genotype on ich risk . otherwise , a fixed - effects model was applied . to test the robustness of the findings , we performed sensitivity analysis by sequentially omitting each study . we assessed the potential publication bias visually by estimating the possible skewness in a funnel plot   and statistically with the methods described by egger . the latter is a weighted linear regression of standard normal deviates against the inverse of the standard error . the intercept of the regression is applied to measure the degree of funnel plot asymmetry . no significant difference was observed regarding the genotype distribution ( p=0.748 ) or t allele frequency ( p=0.492 ) between the ich patients and healthy controls ( table 2 ) . when stratified by hematoma location , no significant difference in genotype frequencies between ich patients and controls was detected in lobar ich ( p=0.193 ) or in deep ich ( p=0.682 ) . in the multiple logistic regression model adjusted for age , sex , hypertension , diabetes , hyperlipidemia , smoking , and alcohol consumption , no significant association of c677 t polymorphism with ich risk was detected in allelic , codominant , dominant , or recessive models ( table 3 ) . after a comprehensive literature search and records screening , full texts of 16 studies were included for further evaluation . eight studies were then excluded : five for departure from hwe [ 2630 ] , one for insufficient data , one   for duplication with another study , and one for mongolian population . one more eligible study was detected through screening reference lists of achieved studies and reviews . finally , nine previous studies together with the current study , forming a total of 1030 ich patients and 1474 controls , were enrolled in this meta - analysis [ 15,16,3440 ] ( table 3 ) . overall , a significant association between c677 t polymorphism and ich risk in chinese populations were observed ( dominant model : or=1.57 , 95%ci=1.291.91 , table 4 , figure 1 ) . when focusing on the chinese han population , a significant association was detected in the recessive model ( dominant model : or=1.36 , 95%ci=1.051.75 ) . when combining studies with population - based controls , c677 t polymorphism was found to be significantly associated with an increased ich risk ( or=1.39 , 95%ci=1.091.77 ) . when stratified by match status , we also detected a significant role of c677 t polymorphism in ich susceptibility ( or=1.29 , 95%ci=1.051.59 ) . in sensitivity analysis , we repeated the meta - analysis by omitting each study sequentially and found that the results did not change substantially under the genetic models except for the heterozygous codominant model . the funnel plots ( figure 2 ) and egger s test ( data not shown ) suggested that there was no publication bias in the present meta - analysis . no significant difference was observed regarding the genotype distribution ( p=0.748 ) or t allele frequency ( p=0.492 ) between the ich patients and healthy controls ( table 2 ) . when stratified by hematoma location , no significant difference in genotype frequencies between ich patients and controls was detected in lobar ich ( p=0.193 ) or in deep ich ( p=0.682 ) . in the multiple logistic regression model adjusted for age , sex , hypertension , diabetes , hyperlipidemia , smoking , and alcohol consumption , no significant association of c677 t polymorphism with ich risk was detected in allelic , codominant , dominant , or recessive models ( table 3 ) . after a comprehensive literature search and records screening , full texts of 16 studies were included for further evaluation . eight studies were then excluded : five for departure from hwe [ 2630 ] , one for insufficient data , one   for duplication with another study , and one for mongolian population . one more eligible study was detected through screening reference lists of achieved studies and reviews . finally , nine previous studies together with the current study , forming a total of 1030 ich patients and 1474 controls , were enrolled in this meta - analysis [ 15,16,3440 ] ( table 3 ) . overall , a significant association between c677 t polymorphism and ich risk in chinese populations were observed ( dominant model : or=1.57 , 95%ci=1.291.91 , table 4 , figure 1 ) . when focusing on the chinese han population , a significant association was detected in the recessive model ( dominant model : or=1.36 , 95%ci=1.051.75 ) . when combining studies with population - based controls , c677 t polymorphism was found to be significantly associated with an increased ich risk ( or=1.39 , 95%ci=1.091.77 ) . when stratified by match status , we also detected a significant role of c677 t polymorphism in ich susceptibility ( or=1.29 , 95%ci=1.051.59 ) . in sensitivity analysis , we repeated the meta - analysis by omitting each study sequentially and found that the results did not change substantially under the genetic models except for the heterozygous codominant model . the funnel plots ( figure 2 ) and egger s test ( data not shown ) suggested that there was no publication bias in the present meta - analysis . despite advances in neurocritical care in recent decades , ich remains a devastating disease causing substantial death and chronic disability . this status highlights the critical role of primary and secondary preventive strategies , which is now most promising for reducing the burden imposed by ich . however , the current limited understanding of ich pathogenesis remains a formidable barrier to the development of these strategies . data on familial aggregation suggests the role of genetics in ich pathogenesis . in our case - control study , we detected a similar genotype distribution regarding mthfr c677 t polymorphism between ich patients and healthy controls in the chinese han population . however , when combined with previously published data , we found a significant association between this polymorphism and an increased ich risk in chinese populations . the association remained significant when confined to han ethnicity , studies using matched controls , and studies including population - based controls . the impact of mthfr c677 t polymorphism on ich risk has also been evaluated in other populations , with conflicting results . hultdin et al . , investigating swedish cohorts , reported that mthfr tt carriers had a 3.62-fold higher risk of hemorrhagic stroke when compared with cc carriers after adjusting for other confounding factors . did not detect any significant role of c677 t polymorphism in hemorrhagic stroke risk in a turkish population . in vivo and in vitro studies suggested that subjects homozygous for the t allele have significantly elevated hcy levels . interestingly , some studies , although still controversial , observed significant associations of mthfr c677 t polymorphism with increased risk of ischemic stroke . the explanation underlying this phenomenon was suggested to be that elevated hcy can cause either ischemic stroke through its coagulative effect or ich through promoting plaque rupture . one plausible application of genetic information on ich susceptibility is its potential to improve risk assessment and thus to provide immediate clinical impact , such as decision - making regarding coagulation . however , the further application of mthfr polymorphism , at least for coagulation adjustment , seems to be hampered by its dual effect on both ischemic stroke and ich . notably , ich is a polygenic disease , with many genes thought to be involved , while a specific gene might make only a small contribution to ich risk . we could not exclude the possibility that the ability to predict ich would overwhelm ischemic stroke when incorporating c677 t polymorphism into other specific genetic or environmental risk factors . therefore , mthfr c677 t polymorphism still has the potential to contribute to the construction of future risk assessment algorithms . firstly , some studies , including one multicenter case - control study , were excluded due to deviation from hwe . secondly , although it was mentioned in all studies that participants were chinese , it was not clearly if they were of han ethnicity . thus , only four studies were combined in the subgroup analysis focusing on the chinese han population . thirdly , adjusted pooled ors could not be calculated due to the limited information available in the included studies . fourthly , although it is suggested that cerebral amyloid angiopathy might contribute to lobar ich , while deep ich is related to hypertensive vasculopathy , it remains unknown whether genes play different roles in lobar vs. deep ich . therefore , we could not calculate pooled ors estimating the potential role of mthfr c677 t polymorphism in lobar ich and deep ich , respectively . firstly , this is the first meta - analysis evaluating the role of mthfr c677 t polymorphism in ich risk in chinese populations . in the present case - control study , we did not observe that mthfr c677 t polymorphism is associated with ich risk in the chinese han population . however , when combined with previously published studies , a significant association of c677 t polymorphism with an increased risk of ich was detected in chinese populations , and also in the subgroup analysis focusing on han ethnicity .
backgroundmethylenetetrahydrofolate reductase ( mthfr ) c677 t polymorphism has been speculated to be and extensively investigated as a risk factor for various vascular diseases , including intracerebral hemorrhage ( ich ) . 
 however , results from published studies regarding the role of c677 t polymorphism in ich risk in chinese populations were contradictory rather than conclusive.material/methodsin this study , a total of 180 ich patients and 180 matched controls of chinese han ethnicity were enrolled . 
 the mthfr c677 t polymorphism was genotyped by polymerase chain reaction - ligation detection reaction ( pcr - ldr ) . 
 a meta - analysis was conducted by combining our data with previous relevant studies in chinese populations.resultsin our case - control study , similar allele frequency ( p=0.492 ) and genotype distribution ( p=0.748 ) of mthfr c677 t polymorphism were detected between ich patients and controls . further analysis based on hematoma location 
 did not show a significant association . 
 when combined with previous studies , however , c677 t polymorphism was found to be significantly associated with an increased risk for ich in chinese populations ( recessive model : or=1.57 , 95%ci=1.291.91 ) . 
 when focusing on the han ethnicity , carriers of the tt genotype had an increased risk of ich ( recessive model : or=1.36 , 95%ci=1.051.75).conclusionsin this case - control study we did not observe that the mthfr c677 t polymorphism was associated with ich risk in people of chinese han ethnicity . 
 however , when combined with previous published studies , a significant association of c677 t polymorphism with an increased risk of ich was detected in chinese populations , and also in the subgroup analysis focusing on han ethnicity .