Thrombomodulin Gene Mutations Associated with Thromboembolic Diseases

Background

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, six 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.Ser190Trp), 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. 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 (69.1% ±5%, P < .05), p.Leu220X (19.1% ± 7.6%, P < .001). Comparing to wild type, four point mutations 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 psiCHECK^TM-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.

Conclusion

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.