retinal vein thrombosis ( rvt ) is the most common retinal vascular disease after diabetic retinopathy . it is a disease with multifactorial nature , which may affect small , medium , and large ocular vessels . it is generally accepted that a mildly elevated homocysteine ( hcy ) level is a risk factor for atherosclerosis . similarly , several studies have suggested that hyperhomocysteinemia is a risk factor for rvt , but not all studies have confirmed this . hyperhomocysteinemia may result from dietary deficiencies ( especially folate ) and/or inherited defects in the controlling enzymes of hcy metabolism . 5 , 10-methylenetetrahydrofolate reductase ( mthfr ) catalyses the conversion of 5 , 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate , which is the main circulating form of folate and the methyl donor for the vitamin b12 dependent remethylation of hcy to methionine . a common 677c->t transition in the mthfr gene results in a thermolabile variant with specific decreased enzymatic activity . the molecular basis of this thermolability is a missense mutation in exon 4 of the mthfr gene , a cytosine to thymine substitution at nucleotide 677 , which converts an alanine to a valine codon . homozygosity for the mthfr c677 t mutation has been associated with an increased plasma hcy level , particularly in the presence of suboptimal folate intake . studies investigating the role of hyperhomocysteinemia and mthfr c677 t mutation in patients with rvt have yielded conflicting results . some studies suggest that the thermolabile variant of mthfr c677 t polymorphism is a risk for rvt , whereas others do not support such an association . regarding this controversy , we performed a case control study to investigate the prevalence of elevated hcy level and mthfr c677 t genotypes among patients with rvt and compared the results with age and sex matched control group . our study population consisted of 78 consecutive patients with rvt referred to coagulation center of iranian blood transfusion organization for thrombophilia screening during 2008 and 2009 years in tehran . the clinical records and the objective documentation of rvt were reviewed by an ophthalmologist to confirm the diagnosis . five patients were excluded from the analysis because the diagnosis was uncertain and altogether 73 patients ( 50 - 78 years old ) were included in the study . the control subjects consisted of 73 individuals ( 51 - 80 years old ) were matched for age and sex with patients and were selected from iranian blood transfusion organization stuffs . in addition , some of the control subjects were selected from normal populations , which were resident around shahid bagheri dormitory in tehran . in order to exclude any history of thrombosis and/or use of vitamin b supplements and folate in control subjects , we completed questionnaires regarding many items including age , family history of eye diseases , history of thrombosis , renal failure , liver disease , hypertension , malignancy and use of drugs especially vitamin b supplements , and folate . all steps of our research were conducted according to ethical committee guidelines of iranian blood transfusion organization . overnight fasting ( 12 h ) blood samples were drawn from the rvt patients and control subjects . blood was collected into ethylenediaminetetraacetic acid ( edta ) -containing tubes and kept on ice . samples were centrifuged at 4c and plasma fraction was aspirated and transferred to plastic tube and stored at  20c until analysis for total hcy . total hcy was measured with a microplate enzyme immunoassay method using axis shield hcy enzyme immunoassay kit ( axis shield diagnostic , ltd , uk ) . dna was extracted from blood leukocyte according to the method described by lahiri et al . mthfr 677 c->t polymorphism was analyzed by polymerase chain reaction ( pcr)-restriction fragment length polymorphism method as previously described by frosst et al . with slight modification . pcr was carried out on 40 l reaction mixture containing 200 ng genomic dna in 1  pcr buffer , 2.2 mm mgcl2 , 200 m deoxyribonucleotide triphosphates ( dntps ) , 0.5 m primers , and 1.5 u amplitaq dna polymerase ( applied bio systems ) . the sequences of the primers were : 5-tga agg agg tgt ctg cgg ga-3 and 5-agg acg gtg cgg tga gag tg-3. the pcr conditions included an initial denaturation at 94c for 2 min , followed by 35 cycles of denaturation at 94c for 30 s , annealing at 62c for 30 s , extension at 72c for 30 s , and a final extension at 72c for 7 min . the amplified products were digested with 5 units of hinf i ( promega , madison , wi , usa ) at 37c for 12 h. the digested products were separated by electrophoresis on a 3% agarose gel and visualized using ethidium bromide . individuals with the cc ( wild - type homozygosity ) genotype showed a single band of 198 bp , those with the ct ( heterozygosity ) genotype showed bands of 198 bp and 175 bp , and those with the tt ( mutated homozygosity ) genotype showed a single band of 175 bp [ figure 1 ] . the polymerase chain reaction products were digested by restriction enzyme hinf i:1 ( ladder 100 bp ) ; 2 negative control ; 3 positive control for homozygote ( tt ) genotype ; 4 positive control for heterozygote ( ct ) genotype ; 5 , 6 , 8 , 9 , 10 cc genotype ( 198 bp ) ; 7 , 12 ct genotype ( 198 bp , 175 bp ) ; 11 tt genotype ( 175 bp ) allele frequencies were calculated by gene counting in rvt patients and control subjects . mean age and total hcy level differences between patients and controls were tested by student t - test . chi - square test was used to assess the association of mthfr c677 t mutation with rvt and odds ratio ( or ) along with 95% confidence interval ( ci ) was calculated . chi - square test also was used to analyze the distribution of mthfr c677 t genotypes between male and female in rvt patients and controls , and to test sex differences between patients and controls . statistical analysis was performed by statistical package for the social sciences 15 ( spss 15 ) software with a statistical significance level of p < 0.05 . our study population consisted of 78 consecutive patients with rvt referred to coagulation center of iranian blood transfusion organization for thrombophilia screening during 2008 and 2009 years in tehran . the clinical records and the objective documentation of rvt were reviewed by an ophthalmologist to confirm the diagnosis . five patients were excluded from the analysis because the diagnosis was uncertain and altogether 73 patients ( 50 - 78 years old ) were included in the study . the control subjects consisted of 73 individuals ( 51 - 80 years old ) were matched for age and sex with patients and were selected from iranian blood transfusion organization stuffs . in addition , some of the control subjects were selected from normal populations , which were resident around shahid bagheri dormitory in tehran . in order to exclude any history of thrombosis and/or use of vitamin b supplements and folate in control subjects , we completed questionnaires regarding many items including age , family history of eye diseases , history of thrombosis , renal failure , liver disease , hypertension , malignancy and use of drugs especially vitamin b supplements , and folate . all steps of our research were conducted according to ethical committee guidelines of iranian blood transfusion organization . overnight fasting ( 12 h ) blood samples were drawn from the rvt patients and control subjects . blood was collected into ethylenediaminetetraacetic acid ( edta ) -containing tubes and kept on ice . samples were centrifuged at 4c and plasma fraction was aspirated and transferred to plastic tube and stored at  20c until analysis for total hcy . total hcy was measured with a microplate enzyme immunoassay method using axis shield hcy enzyme immunoassay kit ( axis shield diagnostic , ltd , uk ) . dna was extracted from blood leukocyte according to the method described by lahiri et al . mthfr 677 c->t polymorphism was analyzed by polymerase chain reaction ( pcr)-restriction fragment length polymorphism method as previously described by frosst et al . with slight modification pcr was carried out on 40 l reaction mixture containing 200 ng genomic dna in 1  pcr buffer , 2.2 mm mgcl2 , 200 m deoxyribonucleotide triphosphates ( dntps ) , 0.5 m primers , and 1.5 u amplitaq dna polymerase ( applied bio systems ) . the sequences of the primers were : 5-tga agg agg tgt ctg cgg ga-3 and 5-agg acg gtg cgg tga gag tg-3. the pcr conditions included an initial denaturation at 94c for 2 min , followed by 35 cycles of denaturation at 94c for 30 s , annealing at 62c for 30 s , extension at 72c for 30 s , and a final extension at 72c for 7 min . the amplified products were digested with 5 units of hinf i ( promega , madison , wi , usa ) at 37c for 12 h. the digested products were separated by electrophoresis on a 3% agarose gel and visualized using ethidium bromide . individuals with the cc ( wild - type homozygosity ) genotype showed a single band of 198 bp , those with the ct ( heterozygosity ) genotype showed bands of 198 bp and 175 bp , and those with the tt ( mutated homozygosity ) genotype showed a single band of 175 bp [ figure 1 ] . the polymerase chain reaction products were digested by restriction enzyme hinf i:1 ( ladder 100 bp ) ; 2 negative control ; 3 positive control for homozygote ( tt ) genotype ; 4 positive control for heterozygote ( ct ) genotype ; 5 , 6 , 8 , 9 , 10 cc genotype ( 198 bp ) ; 7 , 12 ct genotype ( 198 bp , 175 bp ) ; 11 tt genotype ( 175 bp ) mean age and total hcy level differences between patients and controls were tested by student t - test . chi - square test was used to assess the association of mthfr c677 t mutation with rvt and odds ratio ( or ) along with 95% confidence interval ( ci ) was calculated . chi - square test also was used to analyze the distribution of mthfr c677 t genotypes between male and female in rvt patients and controls , and to test sex differences between patients and controls . statistical analysis was performed by statistical package for the social sciences 15 ( spss 15 ) software with a statistical significance level of p < 0.05 . our study population consisted of 73 patients with rvt and 73 apparently healthy subjects matched for age and sex as control . as shown in table 1 , between the rvt patients and control subjects , no significant differences were observed in mean age and gender . mean plasma hcy level was significantly higher in the rvt patients than in the control subjects ( p = 0.001 ) . homocysteine level and other characteristics in rvt patients and control subjects the distribution of genotypes and the relative allele frequency of the c677 t polymorphism of the mthfr gene in rvt patients and controls are given in table 2 . the frequency of the 677tt genotype in rvt patients was higher than control subjects , but the difference did not reach a significant value ( p = 0.07 ) . moreover , given to our null hypothesis , t allele variation may be involved in the disease process . therefore , in order to remove the effects of confounding factors such as age and sex , t allele frequency was adjusted for age and sex . the frequency of the mutant t allele in the rvt patients and control subjects after adjusting for age and sex was 26% and 21.2% , respectively and did not differ significantly between the two groups ( p = 0.33 ) . in addition , the or for rvt related to the 677 t allele was 1.3 , 95% ci ( 0.75 - 2.24 ) , confirming the absence of an association . furthermore , there was a significant increase in mean serum hcy level of subjects carrying tt genotype compared to subjects carrying cc and ct genotype in both patients ( p = 0.001 ) and controls ( p = 0.03 ) as shown in table 3 . according to table 4 , no significant difference in the distribution of genotypes of the mthfr c677 t mutation was observed between males and females in rvt patients ( p = 0.79 ) and also control subjects ( p = 0.96 ) . mthfr c677 t genotypes and allelic frequencies of rvt patients and control subjects effects of mthfr c677 t genotypes on total hcy level in patients and controls the prevalence rate of mthfr c677 t polymorphism according to gender in rvt patients and control subjects rvt is a disease that is relatively common in elderly people . according to blue mountains eye study , the frequency of retinal vein occlusion is 0.7% in individuals younger than 60 years , 1.2% in individuals aged 60 - 69 years , and 4.6% in individuals older than 80 years . the identification of risk factors may have a significant role in the prevention of this potentially warning disorder . our study demonstrated that elevated plasma hcy level is a risk factor for rvt ; thus , confirming the results of other investigators reported a positive correlation between hyperhomocysteinemia and rvt . our result also is inconsistent with some studies reporting no association of hyperhomocysteinemia with rvt . increased concentration of plasma hcy level may result from acquired factors ( e.g. , deficiencies of vitamins such as folic acid , pyridoxine , and cobalamin ) , and genetic factors ( e.g. , mutation in mthfr gene ) . the c677 t mutation , the best characterized mutation of mthfr gene has been reported to result in a less active enzymatic variant that putatively leads to increased total hcy level . our study also demonstrates that 677tt genotype is associated with elevated hcy level , so verifying the results of other studies . despite the relationship of 677tt genotype with elevated hcy level , some studies have indicated mthfr 677tt genotype as a risk factor for rvt while other studies did not prove such an association . although , in our study , a higher prevalence of 677tt genotype was seen in patients than controls , but the difference was not significant . so , our study was consistent with those studies showed no relationship of mthfr 677 tt genotype with rvt . the reasons for non - replication of association studies are numerous and many factors such as population heterogeneity , ethnic stratification , and sample size may contribute to variable association results . population - specific linkage disequilibrium between markers and causal variants , variation in study design , confounding sampling bias , misclassification of phenotypes , and gene - gene and gene - environment interactions are other factors that influence genetic association results . the results of the current study show a relationship between hyperhomocysteinemia and rvt , but do not confirm a causal relationship . whether the elevated plasma hcy level is a cause or merely a consequence of rvt interventional trials will be needed to evaluate its potential causal role and the effectiveness of hcy lowering treatment for diminishing the risk of rvt development . possible mechanisms by which elevated plasma total hcy level may contribute to rvt include activation of factor v , inhibition of protein c activation , promoting endothelial tissue factor expression and suppressing endothelial cell surface heparin sulfate . since hyperhomocysteinemia can be safely and easily corrected by vitamin supplementation , hcy should be routinely measured in rvt patients , and even slightly elevated plasma hcy level should be treated to decrease the risks associated with rvt . based on this study , we suggest that hyperhomocysteinemia , but not mthfr c677 t polymorphism is a significant risk factor for rvt development .
background : elevated plasma homocysteine ( hcy ) level has been established as a significant risk factor for venous thrombosis and cardiovascular disease . 
 homozygosity for the methylenetetrahydrofolate reductase ( mthfr ) c677 t mutation has been associated with elevated plasma hcy concentration and may contribute to retinal vein thrombosis ( rvt ) development . 
 the aim of the present study was to investigate whether the hyperhomocysteinemia and/or homozygosity for the mthfr c677 t mutation are associated with an increased risk for rvt.materials and methods : our study population consisted of 73 consecutive patients ( 50 - 78 years old ) with rvt and 73 control subjects ( 51 - 80 years old ) , matched for age and sex . 
 genotyping for the mthfr c677 t mutation was performed by polymerase chain reaction - restriction fragment length polymorphism technique and hcy level was determined by an enzyme immunoassay kit.results:the prevalence of 677tt genotype was higher in patients than control subjects , but the difference in frequency did n't reach a significant value ( p = 0.07 ) . 
 the frequency of the 677 t allele was 26% and 21.2% in patients and controls , respectively and did not differ significantly between the two groups ( odds ratio = 1.3 , 95% confidence interval ( 0.75 - 2.24 ) , p = 0.33 ) . 
 fasting plasma total hcy level was significantly higher in patients than controls ( p = 0.001).conclusion : our study demonstrated that hyperhomocysteinemia , but not the mthfr c677 t mutation , is associated with rvt .