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Molecular pathogenesis of the patients with congenital disorder of thrombosis and hemostasis

先天性血栓止血障碍患者的分子发病机制

基本信息

批准号：
10670933
负责人：
HAYASHI Tomihiro
金额：
$ 1.09万
依托单位：
YAMAGATA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10670933/
关键词：
hereditary qualitative platelet disorder congenital deficiencies of coagulation factors gene analysis Bernard-Soulier syndrome gene expression mutation splicing abnormality 凝固第X因子欠乏症

项目摘要

I. Analysis of Bernard-Soulier syndrome : PheィイD155ィエD1 (TTT) to Ser (TCT) replacement in the leucine rich motif (LRM) of the GPIX polypeptide does cause BSS phenotype. We have certified this by means of in vitro transfection studies with plasmid for mutant GPIX and other plasmids for GPIb/IX complex. Mutant GPIX could not increase the surface expression of GPIb-α nor surface expression of GPIX itself.II. Analysis of coagulation factor X deficiency : We have identified the molecular defect underlying congenital factor X (FX) deficiency. A novel 3-base-pair deletion was observed within intron D of the FX gene. This mutation was not detected in 53 unrelated Japanese (106 alleles) by an allele-specific restriction analysis, indicating a likely cause of the FX deficiency. The deletion resides within a polypyrimidine tract of the acceptor splicing site where U2 snRNP binds to form spliceosomes. This defect could alter the formation of splicesomes, then result in incorrect splicing and decre … More ased FX production.III. Analysis of coagulation factor XI deficiency : We have identified a novel single-base point mutation, a GT->AT transition of the factor XI (FXI) gene at the donor splicing site of its intron J.A reverse transcription-polymerase chain reaction with ectopic RNA revealed that propositus cDNA showed 20-base truncation of the 3'-end of the FXI exon 10, resulting in the frame-shift and generation of premature stop codon at 56-base downstream of the correct exon 10/11 junction. A phenotypic consequence of this mutation was then investigated through mammalian cell expression system. Recombinant plasmids harboring wild type FXI gene did direct the production of the FXI antigen into the medium, but the plasmids lacking 20 bases of the FXI exon 10 failed to produce the antigen while the mRNA expression thereof was unchanged. The mutation described here is novel, and ectopic RNA from peripheral mononuclear cells was first proved to be useful for analyzing the resultant FXI mRNA sequence. Less

I. Bernard-Soulier综合征的分析：GPIX多肽的富亮氨酸基序（LRM）中Phe D155丝氨酸D1（TTT）替换为Ser（TCT）确实导致BSS表型。我们已经通过用突变体GPIX的质粒和GPIb/IX复合物的其他质粒进行体外转染研究来证明这一点。GPIX突变体既不能增加GPIb-α的表面表达，也不能增加GPIX本身的表面表达。凝血因子X缺乏症的分析：我们已经确定了先天性凝血因子X（FX）缺乏症的分子缺陷。在FX基因的内含子D内观察到一种新的3个碱基对缺失。通过等位基因特异性限制性分析，在53个不相关的日本人（106个等位基因）中未检测到这种突变，这表明FX缺陷的可能原因。该缺失位于U2 snRNP结合形成剪接体的受体剪接位点的聚嘧啶段内。这种缺陷会改变剪接体的形成，从而导致错误的剪接， ...更多信息基础外汇生产。凝血因子XI缺乏症分析：我们发现了一个新的单碱基点突变，即因子XI（FXI）基因内含子J的供体剪接位点的GT->AT转换。用异位RNA进行逆转录-聚合酶链反应显示，先证者cDNA显示FXI外显子10的3 '端有20个碱基的截短，导致移码和在正确的外显子10/11接合处下游56个碱基处产生提前终止密码子。然后通过哺乳动物细胞表达系统研究该突变的表型结果。携带野生型FXI基因的重组质粒确实将FXI抗原的产生引导到培养基中，但是缺少FXI外显子10的20个碱基的质粒不能产生抗原，而其mRNA表达没有改变。这里描述的突变是新的，并且首次证明来自外周单核细胞的异位RNA可用于分析所得的FXI mRNA序列。少