title: 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.
authors.affiliation: Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Mayu
authors.initials: M
authors.lastname: Kyotani
authors.forename: Kaoru
authors.initials: K
authors.lastname: Okumura
authors.forename: Akira
authors.initials: A
authors.lastname: Takagi
authors.forename: Takashi
authors.initials: T
authors.lastname: Murate
authors.forename: Koji
authors.initials: K
authors.lastname: Yamamoto
authors.forename: Tadashi
authors.initials: T
authors.lastname: Matsushita
authors.forename: Motoi
authors.initials: M
authors.lastname: Sugimura
authors.forename: Naohiro
authors.initials: N
authors.lastname: Kanayama
authors.forename: Takao
authors.initials: T
authors.lastname: Kobayashi
authors.forename: Hidehiko
authors.initials: H
authors.lastname: Saito
authors.forename: Tetsuhito
authors.initials: T
authors.lastname: Kojima
mesh_terms: D000328:Adult; D000991:Antithrombins / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism*; D044466:Asian People; D001778:Blood Coagulation Disorders / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D002460:Cell Line; D005260:Female; D006801:Humans; D009154:Mutation / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000991:Antithrombins; D011994:Recombinant Proteins