血栓症を発症した複合ヘテロ接合体性プロテインC欠乏症患者で認められた2種の遺伝子変異とそれぞれのプロテインC産生に及ぼす影響  [in Japanese] Hereditary protein C deficiency caused by the compound heterozygous mutations, Gly 282 Ser and Met 364 Ile ; Contribution of each protein C mutation on pathogenesis of thrombosis  [in Japanese]

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Author(s)

    • 油井 知雄 YUI Tomoo
    • 北海道医療大学歯学部口腔機能修復・再建学系クラウンブリッジ・インプラント補綴学分野 Division of Fixed Prosthodontics and Oral Implantrology, Department of Oral Rehabilitation
    • 越智 守生 OCHI Morio
    • 北海道医療大学歯学部口腔機能修復・再建学系クラウンブリッジ・インプラント補綴学分野 Division of Fixed Prosthodontics and Oral Implantrology, Department of Oral Rehabilitation
    • 高橋 伸彦 TAKAHASHI Nobuhiko
    • 北海道医療大学歯学部生体機能・病態学系内科学分野 Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido
    • 家子 正裕 IEKO Masahiro
    • 北海道医療大学歯学部生体機能・病態学系内科学分野 Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido
    • 林 辰弥 HAYASHI Tatsuya
    • 三重県立看護大学看護学部生化学 Department of Biochemistry, School of Nursing, Mie Prefectural College of Nursing
    • 鈴木 宏治 SUZUKI Koji
    • 三重大学大学院医学系研究科分子病態学 Department of Molecular Pathobiology, Mie University Graduate School of Medicine

Abstract

プロテインC（PC）はビタミンK依存性のセリンプロテアーゼ前駆体であり，先天性PC欠乏症は静脈系血栓症の発症に関与する．<br>本研究ではPC欠乏症と診断された家系の発端者PC遺伝子についてPCR-Direct sequence法によりPC遺伝子上の変異部位を同定するとともに，PCR-Restriction fragment length polymorphism（RFLP）法により，その家系の遺伝子型の精査を行った．PCR-Direct sequenceにより，発端者PC遺伝子の第9エクソン上にGly282→Ser変異（変異I）およびMet364→Ile（変異II）を認め，PCR-RFLP法による発端者家族の遺伝子型の解析により，2種の変異は異なるアレルに存在することが明らかになった．さらに，Site-directed mutagenesis法を用いて，変異Iあるいは変異IIを有するPCの発現ベクターを構築し，BHK-21細胞を用いて一過性の蛋白発現実験を行った．定量的PCRによるmRNAレベルの検討では野生型PCと両変異型PCに明らかなmRNAレベルの差異は認められなかったが，PC特異的ELISA法により測定した培養上清中のPC抗原量は，野生型PCを100％とした時，Gly282SerPCでは55.2％，Met364IlePCでは0.9％であった．<br>本研究の結果から2つの変異がPC欠乏症に関与し，特に本家系ではMet364Ile変異が血栓症の発症に関与したと考えられた．

Anticoagulant Protein C (PC) is a vitamin K-dependent serine protease zymogen, and is activated by thrombin complexed with thrombomodulin. Activated protein C inactivates blood coagulation cofactors, Factor Va and VIIIa. It is known that patients with inherited PC deficiency have increased risk of venous thrombosis. We investigated PC deficiency found in a family and analyzed its PC gene using PCR-direct sequence and PCR-RFLP. The result of PCR-direct sequence showed that the propositus is suffering from compound heterozygous protein C deficiency and that mutations, which is located in a different allele in exon IX, were Gly-282 (GGC) to Ser (AGC) [mutation I] and Met-364 (ATG) to Ile (ATA) [mutation II]. Transient expression assay of each mutant PC protein using BHK-21 cells followed by quantitative-PCR showed that there were no difference of mRNA levels between wild-type PC and each mutant PC. However, ELISA showed that concentrations of Gly282SerPC and Met364IlePC secreted into the culture medium were 55.2% and 0.9%, respectively, when concentration of wild type PC in culture medium is determined as 100 %. Our result suggests that each of the two mutations would be cause of type I PC deficiency, and the mutation II seems to be closely associated with thrombotic tendency observed in this family as compared with mutation I.

Journal

  • Japanese Journal of Thrombosis and Hemostasis

    Japanese Journal of Thrombosis and Hemostasis 22(3), 87-99, 2011-06-01

    The Japanese Society on Thrombosis and Hemostasis

References:  35

Codes

  • NII Article ID (NAID)
    10031162927
  • NII NACSIS-CAT ID (NCID)
    AN10353762
  • Text Lang
    JPN
  • Article Type
    ART
  • ISSN
    09157441
  • DOI
    10.2491/jjsth.22.87
  • Data Source
    CJP  J-STAGE 
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