| Part I Association of antithrombin variant with venous thrombosisOBJECTIVES:Despite the key anticoagulant role of antithrombin and the high risk of thrombosis associated to its deficient state, the prevalence of antithrombin deficiency among patients with venous thromboembolism (VTE) is very low. Increasing evidences are suggesting that antithrombin deficiency might be underestimated. The objective of the study is to identity possible variants in SERPINC1 that could increase the risk of thrombosis and explore the potential mechanisms.METHODS AND RESULTS:The analysis of SERPINC1, the gene encoding antithrombin, in randomly selected 60 non-acute VTE patients (no more than 65years old) without protein C and protein S deficiency found a novel variant c.-11G>A (rs483352843) in the 5'-untranslated region (5'UTR) of SERPINC1 from two VTE patients. Genotyping c.-11G>A in 1,760 consecutive VTE patients and 2,200 healthy controls revealed that the mutation severely increases the risk of venous thrombosis, with an odds ratio of 6.91 (95% CI:1.53-31.22; P=0.0035). The antithrombin levels and anticoagulant activity of carriers are 78.4%(P<0.0001) and 88.9%(P=0.0004) of controls, respectively. The recombinant pGL4 luciferase reporter vectors were transfected into HEK-293T and COS-7 cells, the expression levels of the reporter gene containing the-11A variant are 62.9%(P=0.006) and 53.1%(P=0.004) of the-11G wild type. Real-time PCR showed that mRNA levels in blood cells, HEK-293T, and COS-7 cells containing the variant are 35.8%(P=0.026),57.1% (P=0.010), and 52.8%(P=0.004) of controls,respectively. Electrophoretic mobility shift assay (EMSA) indicated that the c.-11G>A significantly enhanced the protein binding affinity of the sequence surrounding the variant. Conclusions:The mutation c.-11G>A in 5'UTR of SERPINC1 enhanced the protein binding affinity, which significantly inhibit the transcription of SERPINC1, leading to plasma anthrombin levels of carriers reduced remarkablely. As a result, the mutation c.-11G>A significantly increases the risk of VTE.Part ? Variation in TFPI and ADTRP and the Risk of Venous ThrombosisOBJECTIVES:Tissue factor (TF) pathway inhibitor (TFPI) is an important inhibitor of TF-factor ?a (TF-FVIIa)-dependent FXa generation, which impedes early stage of the blood coagulation cascade. Low plasma TFPI levels increase the risk of thrombosis. Androgen-dependent TFPI-regulating protein (ADTRP) regulates TFPI expression, intracellular localization, and activity. The objective of the study is to identity possible variants in TFPI and ADTRP that could increase the risk of thrombosis.METHODS AND RESULTS:By using enzyme linked immunosorbent assay (ELISA), plasma TFPI antigen levels were assayed in randomly selected 176 non-acute VTE patients who were under the age of 65 years and were free of protein C, protein S, and antithrombin deficiency. Plasma from 100 healthy donors was pooled to generate the reference sample used to generate the standard curve for the assays. TFPI and ADTRP gene in 48 patients with plasma TFPI levels<50% of control were resequneced. As a result,10 variants in TFPI and 11 variants in ADTRP were found, ADTRP c.154-18delT variant was previously unreported. Genotyping the variant TFPI c.662A>G (rs7586970) and ADTRPc.154-18delT in 901 consecutive VTE patients and 799 healthy controls unveiled that both of the variants didn't increase the risk of VTE. The odds ratio for c.662A>G and c.154-18delT were 0.85 (95% CI; 0.71-1.03; P= 0.106) and 1.07 (95% CI; 0.998-1.14; P=0.058), respectively.Conclusions:There were no variants in TFPI gene associate with VTE in Chinese. The ADTRP variant c.154-18delT may be a potential risk factor of VTE, but further study is warranted.Part III Rare THBD mutations in the pathogenesis of thrombotic diseasesOBJECTIVES:Thrombomodulin (TM), encoded by the THBD gene, is an endothelial transmembrane glycoprotein that acts as a cofactor for thrombin in the activation of protein C (PC). It has been clearly demonstrated that the anticoagulant and profibrinolytic functions of the thrombin-TM-PC system play critical roles in the prevention of thromboembolic diseases.METHODS AND RESULTS:Using direct sequencing, seven novel mutations were identified in THBD gene of eight patients with either arterial or venous thrombosis: c.376G>T (p.Asp126Tyr), c.569C>G (p.Serl90Trp), c.659T>G (p.Leu220X), c.1208G>A (p.Arg403Lys), c.1288G>A (p.Gly430Ser), c.*6_*23del and c.*23_*40del. Of the mutations, c.376G>T and c.*6_*23del were detected in a 10-year old child with venous thrombosis (VTE), who died one year later. The c.376G>T point mutation predicting p.Asp126Tyr in thrombomodulin protein was also detected in a 38-year old VTE patient. The nonsense mutation c.659T>G recurred in a 55-year old patient with coronary artery disease (CAD) and a 46-year old VTE patient, who died from repeated venous thrombosis two years later. ELISA showed that plasma TM antigen levels in carriers were lower than that of controls. The wild type and the mutant thrombomodulin were expressed in HEK-293t cells. Two mutations were found to have reduced TM protein expression compared to wild-type (100%), Ser190Trp (71.2%±12.8%, P< 0.05), p.Leu220X (20.1%± 10.4%, P< 0.01). Comparing to wild type, four point mutations (c.569C>G, c.659T>G, c.1208G>A, c.1288G>A) diminished the cofactor activity of TM according to the reduced level of activation of PC in cultured cells. As for the two 18-bp deletions, a psiCHECKTM-2 vector containing wild type and mutant 3'UTR of TM was constructed. Compared with the wild-type, the deletions significantly reduced the reporter gene-expression level. The antigen level and activity in cultured cells with c.376G>T have no significant difference with the wild type, indicating that the mutant may cause a thrombophilic state through other unknown pathological mechanisms.Conclusions:The results suggest that gene mutation of thrombomodulin may be important in the pathogenesis of thrombotic diseases. Further study on genetics of thrombosis should focus on resequencing of THBD gene in different populations. |
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