The Molecular Diagnosis of Patients with Vein Thrombosis Due to Deficiency of Inherited Anticoagulant Proteins

Objective: To make genetic diagnosis and pedigree analysis for patients with recurrent venous thromboembolism due to inherited deficiency of protein C (PC), protein S (PS). Methods: The routine coagulation tests including activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) were performed. Chromogenic substrate assay was used to detect the activities of Protein C (PC:C), total Protein S (PS:C) and antithrombin (AT:C). All exons and their flanks of PC and PS gene were amplified by polymerase chain reaction (PCR). The PCR products were sequenced directly and blastered to normal sequence of corresponding anti-coagulant protein to find the gene mutations. Results: Totally nine probands with DVT or PE were enrolled, their peripheral blood and medical histories collected after informed consents. Proband 1, 2 and 3 were with combined deficiency of PC and PS, while proband 4 was with PC deficiency. Sequencing of PC gene showed there were polymorphism sites G4880A, C4867T and A5054T in promoter region for all four probands with PC deficiency. PC:C and PS:C for proband 1 was 48% and 26.3%, respectively. PC gene sequencing showed that there was a heterozygous mutation A6578T in exon2 region, resulting in Thr18Ser. Sequencing of PS gene showed there was G68395T heterozygous mutation in exon4 region, leading to Arg90Leu. PC:C and PS:C of proband 2 was 27% and 22.9%, respectively. Heterozygous mutations of G68428A and C68430T in exon4 region of PS gene were found, leading to Arg100His and Gln101Stop, respectively. Proband 3 was with PC deficiency and PS deficiency, PC:C and PS:C were 58% and 57.3%. Heterozygous AGA12702-12704 or AGA12705-12707 deletion mutation was found in Exon2 of PC gene resulting in Arg192del or Arg193del, and heterozygous missense mutation A15240G in Exon9 resulting in His370Arg. Heterozygous mutation G68395T and G825512C was found in Exon4 and Exon9 region of PS gene, respectively, resulting in Arg90Leu and Ser321Thr. Proband 4 was with PC deficiency, PC:C50%. There was no other mutation detected except for polymorphism sites in promoter region. Proband 5 was with PS deficiency, PS:C 38.8%. Heterozygous mutation G68395T in exon4 region was detected, leading to Arg90Leu. Homozygous mutation C102102T was found in Exon14 region,leading to Gla616Val. Proband 6 was with PS deficiency, PS:C 35.2%. Heterozygous mutations G68395T andC68430T in Exon4 were found, leading to Arg90Leu and Gln101Stop, respectively. Proband 7 was with PS deficiency, PS:C 43.7%.Homozygous mutation C102102T in exon14 region was detected, resulting in Gla616Val. Proband 8 was with PS deficiency, PS:C43.6%. Heterozygous mutation G68395T and C68430T in exon4 region was found, leading to Arg90Leu and Gln101Stop. Proband 9 was with PS deficiency, PS:C 7.7%. Two of his family members were also with PS deficiency (II2，III2） with heterozygous mutation G68395T in Exon4 region（II1，II2，III1，III2）, leading to Arg90Leu. Conclusions: Polymorphisms of G4880A, C4867T and A5054T in promoter region, missense mutation A6578T, A15240G, AGA12702-12704 or 12705-12707 deletion mutation in PC gene，missense mutation G68395T, G68428A, C86066T, G82512C, C102102T, and nonsense mutation C68430T in PS gene might be the cause of reduced activities of corresponding anticoagulant proteins. All these mutations, except for C86066T in PS gene, which had been reported in Hongkong, are de novo ones.