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Biological analysis of Fas- or Fas ligand gene mutations and basic analysis of gene therapy

Fas或Fas配体基因突变的生物学分析和基因治疗的基础分析

基本信息

批准号：
10670710
负责人：
KASAHARA Yoshihito
金额：
$ 2.05万
依托单位：
Kanazawa 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-10670710/
关键词：
CD95(Fas)Apoptosis Lymphoproliferation lpr mouse gld mouse Fas ligand double negative T cell Apoptosis アポトーシス自己免疫性リンパ球増殖症候群 lprマウス gldマウス CD95リガンド

项目摘要

Fas-mediated apoptosis and mutation of Fas and Fas ligand (FasL) gene were evaluated in four cases from three families with autoimmune lymphoproliferative syndrome. They showed clinical manifestations of hepatosplenomegaly, Iymphoadenopathy, autoimmune pancytopenia, hypergamma-globulinemia,. Apoptosis induced by anti-Fas antibody was determined PHA-activated T cells, EBV-transformed B cells and freshly isolated granulocytes from patients. Fas and Fas-ligand mRNA from patients' cells was amplified by RT-PCR with primers specific for Fas and Fas-ligand gene and mutations of Fas gene was detected from PCR products. All cells from four cases showed resistance to Fas-induced apoptosis and the surface expression of Fas-receptor was completely diminished on cells from case 3. CD3-indeced activation cell death was also decreased in patients' T cells. Three a different Fas gene mutations were detected. The heterozygous point mutations of intron 7 were detected in case 1 and 2 who are familial cases, resulting in skipping of exon 7 and premature termination. These mutation resulted in production of truncated Fas protein which lacks death domain. Case 3 showed homozygous mutation of intron 3 with lack of exon 4, In case 4, point mutation with exon 9 encoding death domain was detected. All four cases showed an increase of TCRαβ+CD4-CD8- T cells in circulation, which is a specific characteristic in mouse with Fas and FasL mutations.. Familial analysis showed that mutation of Fas gene were inherited from mother in Case 1 and 2 and from both parents in case 3. This is first report of Fas gene mutations in Japan. These results suggest that Fas-mediated apoptosis play pivotal role in maintenance of immune function

对3个自身免疫性淋巴组织增生综合征家系中的4例患者进行了Fas介导的细胞凋亡及Fas和Fas配体（FasL）基因突变检测。临床表现为肝脾肿大、淋巴结肿大、自身免疫性全血细胞减少、高丙种球蛋白血症等。用PHA活化的T细胞、EBV转化的B细胞和新鲜分离的粒细胞检测抗Fas抗体诱导的细胞凋亡。用特异性引物RT-PCR扩增患者细胞Fas和Fas配体mRNA，检测Fas基因突变。所有的细胞从4例显示出抵抗Fas诱导的细胞凋亡和Fas受体的表面表达完全减少的细胞从案件3。在患者的T细胞中，CD 3诱导的活化细胞死亡也减少。检测到三种不同的Fas基因突变。例1和例2均为家族性病例，检测到内含子7的杂合子点突变，导致外显子7跳读和提前终止。这些突变导致产生缺失死亡结构域的截短Fas蛋白。例3为内含子3纯合突变，缺失外显子4;例4为外显子9点突变，缺失外显子9编码死亡结构域。所有4例病例均显示循环中TCRαβ+ CD 4-CD 8- T细胞增加，这是Fas和FasL突变小鼠的特异性特征。家系分析表明，例1和例2的Fas基因突变遗传自母亲，例3的突变遗传自双亲。这是日本首次报道Fas基因突变。这些结果表明Fas介导的细胞凋亡在维持免疫功能中起着关键作用