Research On Relativity Between C46T Polymorphism Of The Coagulation Factor Ⅻ Gene And Thrombosis In The Northeast Area

BackgroundIt has been important in many countries in the world because of the highmorbidity and mortality of thrombus disease. In the aged, the cerebralthrombosis has threatened their health because the sickness rate is high, theprognosis is badly and the mutilation rate is high, too. However, the diseaseincidence of vein thrombosis is 1‰ in the developed country and the veinthrombosis often exists in the deep vein of the lower limb. The principalcomplication is post-thrombus symptom complex and fatal pulmonaryembolism, meanwhile its disease incidence separately is 20% and 1%~2%.Since the pathogenesy of the thrombus disease is complicated, the risk factormainly includes two categories: one is acquired character risk factor eg: theaged, obesity and so on;the other is heritage risk factor, it can be consideredas biological parameter to predict thrombosis or hypercoagulabale state. Thearticle intent to research the distributing frequency of the polymorphism(C46T) in the exon-1 region of the coagulation factorⅫ(FⅫ) gene andexplore the relation of the polymorphism (C46T) in the exon1 region of FⅫgene with the formation of thrombosis in the northeast of China. We candeepen the recognition of thrombopoiesis mechanism and can hope to offerreference for the thrombus disease clinical sieve, prophylaxis and healing.The coagulation factorⅫ(FⅫ: Hageman factor) is an 80-kDa serineprotease circulating at an average concentration of 30ug/ml. In vitro, FXIIplays a central role in the initiation of coagulation and fibrinolysis, but itsphysiological role is still under discussing. The polymorphism (C46T) in theexon1 region of FⅫgene exists in the upstream of translation initiation. AfterT displaces C, the polymorphism destroys FⅫ gene expression and result inthe lower translation efficiency and a decrease in FⅫ plasma level. In theforeign, there are some researches on the polymorphism (C46T) of FⅫ gene,but the role of the polymorphism for the development and progression of thethrombosis is still under discussion. Thus, we apply PCR-RFLP method toinvestigate the distributing frequency of the polymorphism (C46T) in theexon-1 region of the coagulation factorⅫ(FⅫ) gene and explore the relationof the polymorphism (C46T) in the exon1 region of FⅫgene with theformation of thrombosis in the northeast of China.Material and Methods:1. Subject:Case group: 92 cerebral thrombosis persons from the Nerve MedicalDepartment of Ji-Lin province electric power hospital (51 male, 41 female),aged extent: 42-85 years old, mean age is 65.57 years old. All of them arediagnosed as cerebral thrombosis persons by CT or MRI;87 vein thrombosispersons from the vascular surgery Department of JiLin University first hospital(41 male, 46 female), aged extent: 19-92 years old, mean age is 53.14 years old.All of them are diagnosed as vein thrombosis persons by Color DopplerUltrasonography.Control group: 129 health persons from basic medicine college of JiLinUniversity, (53 male, 76 female), aged extent: 26-77 years old, mean age is40.19 years old. All of them are diagnosed without Heard-Cerebral vesselsdisease, liver and Kidney disease, or thrombus disease case history2. Methods:2.1 Blood sample collection: 1ml venous blood was mixed with 50ul 1.5mg/mlEDTA,was centrifugalized 10 min, 3000rmp/min,was subpackaged upperstratum blood plasma and was stored at -80℃.2.2 Isolation of DNA: DNA in the peripheral blood was pending extraction bythe guanidinium thiocyanate, dissolved in the TE and stored at -20 ℃.2.3 PCR: PCR amplifies the exon1 region of FXII gene (162-530), fragmentsare 369bp. Sense primer:5`-ccagtcccactatctagaaaa g-3`, Antisense primer:5`-atggctcatggctgtgatag-3`.The action system of PCR is 25ul, including DNAtemplate 80ng, primer 2.25pmol, MgCl2 1.5mmol/L, KCl 50mmol/L,Tris-HCl(pH8.3) 10mmol/L, dNTPs 0.2mmol/L,1.25μl DMSO 和1.5UTaqpclymerase. PCR reaction condition: 94℃/4min , force-degeneration;94℃/1min,57℃/1min,72℃/ 1min,2circles;94℃/30s,57℃/ 30s,72℃/30s,30 circles;72℃ progradation 10 min。PCR production wasidentified by 1.5%agarose gel electrophoresis and ultraviolet transilluminator.2.4 Genotypic identification in the exon1 region of FXII gene (46C/T): AfterPCR products were purified by the purification kit, they were digested by therestriction enzyme Hin1Ⅰ. The action system is 10ul: PCR product 8.25ul,Hin1Ⅰ7.5U, 37℃ incubation overnight. The result was identified by2.5%agarose gel electrophoresis and ultraviolet transilluminator and wrotegenotypes.3. Statistical analysis:Using the SPSS13.0 for window statistical package, count data wasperformed by the t-test and measure data was performed by chi-squared test.Genotype and allele frequencies between groups were analyzed by Logisticun-conditional regression.Results1. Comparison of age and sex among the control group, cerebral thrombosisgroup and vein thrombosis group:The significant difference of mean age exits among the control group,cerebral thrombosis group and vein thrombosis group(P<0.05). On the sex,significant difference is between the control group and cerebral thrombosisgroup(P<0.05). On the contrary, un-difference is between the control groupand vein thrombosis group(P>0.05).2. Electrophoresis result of the polymorphism (C46T) of FⅫgene and analyzingthe genotype:C, T allele and genotype of CC, CT, TT genotype can be founded in thecontrol group, the cerebral thrombosis group and the vein thrombosis group.The fragment length of CC genotype separately was 130bp and 239bp, thefragment length of CT genotype was 130bp, 239bp and 369bp and thefragment length of TT genotype was 369bp.3. Distributing frequency of the polymorphism (C46T) of FⅫgene:The genotype of CC, CT, TT was found in the 308 persons in the northeastof China. The distributing frequency of the genotype separately is 5.52%,42.21% and 52.27%. The distributing frequency of C, T allele separately is26.6% and 73.4%.4. Polymorphism (C46T) of FⅫgene effect on cerebral thrombosis and veinthrombosis:By the comparison between the control group and cerebral thrombosisgroup, C allele frequency separately is 26.3% and 22.1%. There is no significantdifference by the statistical analysis, P=0.080(X2=3.06). By referring to TTgenotype, the risk odd of CC genotype is higher 2.11 times than of TT genotype.OR=2.11, no significant difference (P=0.222).By the comparison between the control group and vein thrombosis group, Callele frequency separately is 30.1% and 22.1%. There is no significantdifference by the statistical analysis, P=0.084(X2=2.994). By referring to TTgenotype, the risk odd of CC genotype is higher 1.86 times than of TT genotype.OR=1.86, no significant difference (P=0.309).5. Age and sex effect on cerebral thrombosis and vein thrombosis:By the statistical analysis, age factor is risk factor for cerebral thrombosis andvein thrombosis, the analysis result is separately P=0.000(X2=62.228)andP=0.000(X2=34.586).On the sex, there is significant difference, P=0.036(X2=4.405)OR=0.561, So, the risk of female in the cerebral thrombosisdevelopment is higher 0.561 times than of male;There is no significantdifference in the vein thrombosis group, P=0.380 (X2=0.770).6. According to multiplicity, polymorphism (C46T) of FⅫgene effect oncerebral thrombosis and vein thrombosis:By Logistic un-conditional regression analysis, including age, sex, genotype,the result indicated the risk of CC genotype is higher 14.533 times than TTgenotype in the cerebral thrombosis group. There is significant differenceP=0.023(X2=5.201,OR=14.533,95%CI:1.457-144.988);There is nosignificant difference in the vein thrombosis group P=0.074(X2=3.203,OR=2.063,95%CI:0.933-4.559).ConclusionsThere is FⅫC46T polymorphism in the northeast of China. C, T allelefrequencies are separately 26.6% and 73.4%. By Logistic un-conditionalregression analysis, the result is that CC genotype is risk factor in the cerebralthrombosis, but CC genotype is not relativity with the vein thrombosis. It can beseen that the relationship between polymorphism and thrombus disease is stilldiscussing. We are going on deepening to research by enlarging sample size.