Molecular basis of antithrombin deficiency in four Japanese patients with antithrombin gene abnormalities including two novel mutations

Abstract

We analyzed the antithrombin (AT) gene in four unrelated Japanese patients with an AT deficiency, and individually identified four distinct mutations in the heterozygous state. There were two novel mutations, 2417delT leading to a frameshift with a premature termination at amino acid -3 (FS-3Stop) and C2640T resulting in a missense mutation (Ala59Val). Previously reported mutations, T5342C (Ser116Pro) and T72C (Met-32Thr), were also found in the other two patients. To understand the molecular basis responsible for the AT deficiency in these patients, in vitro expression experiments were performed using HEK293 cells transfected with either wild type or respective mutant AT expression vector. We found that -3Stop-AT and -32Thr-AT were not secreted into the culture media, whereas 116Pro-AT and 59Val-AT were secreted normally. We further studied the heparin cofactor activity and the binding to heparin of each recombinant AT molecule. Ser116Pro mutation significantly impaired the binding affinity to heparin resulting in a reduced heparin cofactor activity. In contrast, we found that Ala59Val mutant AT unexpectedly showed a normal affinity to heparin, but severely impaired the heparin cofactor activity. Our findings suggested that FS-3Stop and Met-32Thr mutations are responsible for type I AT deficiency, whereas Ser116Pro and Ala59Val mutations contribute to type II AT deficiency, confirming that there were diverse molecular mechanisms of AT deficiency depend upon discrete AT gene abnormalities as reported previously. Am. J. Hematol. 82:702-705, 2007. V

Molecular Defects Associated with Antithrombin Deficiency and Dilated Cardiomyopathy in a Japanese Patient

“…3C). We observed an unexpected normal heparin affinity in the recombinant 439Thr-AT, whereas the recombinant 148Pro-AT (AT Nagasaki) showed an impaired affinity to heparin as reported previously (11,17). (Fig.…”

“…All exons including splice junctions of the SERPINC1 gene were amplified by polymerase chain reaction (PCR) and analyzed by direct sequencing (11). Similarly, TNNT2 and LMNA genes were also analyzed by direct sequencing, after PCR amplification of all exons including splice junctions using the primer sets listed in Table 2.…”

“…We prepared a mutant human AT expression vector (pcDNA/439Thr-AT: the initial Met residue is denoted amino acid +1) using the recombinant PCR method (11 …”

“…We established stable transformants of HEK293 cells highly expressing the recombinant AT molecules and measured their antigen levels in the culture media by enzymelinked immunosorbent assay (11). Heparin cofactor activity of the AT was measured using Ntest AT III-S kit (Nittobo; Tokyo, Japan).…”

“…We also performed affinity chromatography of each re- combinant AT molecule on a heparin-sepharose column as described previously (11 …”