Antithrombin deficiency in three Japanese families: one novel and two reported point mutations in the antithrombin gene.
INTRODUCTION
Inherited antithrombin (AT) deficiency is associated with a predisposition to familial venous thromboembolic disease. We analyzed the AT gene in three unrelated patients with an AT deficiency who developed thrombosis.


MATERIALS AND METHODS
We analyzed the SERPINC1 gene in three patients. Additionally, we expressed the three mutants in the COS-1 cells and compared their secretion rates and levels of AT activity with those of the wild-type (WT).


RESULTS
We identified three distinct heterozygous mutations of c.2534C>T: p.56Arginine → Cysteine (R56C), c.13398C>A: p.459Alanine → Aspartic acid (A459D) and c.2703C>G: p.112 Proline → Arginine (P112R). In the in vitro expression experiments, the AT antigen levels in the conditioned media (CM) of the R56C mutant were nearly equal to those of WT. In contrast, the AT antigen levels in the CM of the A459D and P112R mutants were significantly decreased. The AT activity of R56C was decreased in association with a shorter incubation time in a FXa inhibition assay and a thrombin inhibition-based activity test. However, the AT activity of R56C was comparable to that of WT when the incubation time was increased.


CONCLUSIONS
We concluded that the R56C mutant is responsible for type II HBS deficiency. We considered that the A459D and P112R mutants can be classified as belonging to the type I AT deficiency.
['Maruyama|Keiko|K|', 'Morishita|Eriko|E|', 'Karato|Megumi|M|', 'Kadono|Tadaaki|T|', 'Sekiya|Akiko|A|', 'Goto|Yukie|Y|', 'Sato|Tomomi|T|', 'Nomoto|Haruka|H|', 'Omi|Wataru|W|', 'Tsuzura|Sachie|S|', 'Imai|Hidenori|H|', 'Asakura|Hidesaku|H|', 'Ohtake|Shigeki|S|', 'Nakao|Shinji|S|']
D000328:Adult D000368:Aged D000818:Animals D000990:Antithrombin III D020152:Antithrombin III Deficiency D001780:Blood Coagulation Tests D019556:COS Cells D002522:Chlorocebus aethiops D005260:Female D006801:Humans D007564:Japan D017354:Point Mutation D055815:Young Adult
Antithrombin Antithrombin deficiency AT Venous thromboembolism COS-1 cells Wild-type WT Cysteine Aspartic acid Proline Arginine Conditioned media CM FXa type II HBS Ala Alanine Arg Asp CL Cell lysates Complementary DNA Cys DVT Deep vein thrombosis ELISA Enzyme-linked immunosorbent assay Factor Xa Green monkey kidney cells Lys Lysine PE PMSF Phenylmethyl sulfonylfluoride Pro Pulmonary thromboembolism RCL RFLP Reactive center loop Restriction fragment length polymorphism analysis Serine protease inhibitor Type II heparin binding site defects Type II reactive site defects Type II with pleiotropic defects VTE cDNA factor Xa inhibition activity assay serpin thrombin inhibition activity assay type II PE type II RS