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

研究了3个家族4例自身免疫性淋巴细胞增生性综合征Fas介导的细胞凋亡及Fas和Fas配体（FasL）基因突变。临床表现为肝脾肿大、淋巴腺病、自身免疫性全血细胞减少症、高γ -球蛋白血症。检测患者体内pha活化的T细胞、ebv转化的B细胞和新鲜分离的粒细胞诱导的fas抗体诱导的细胞凋亡。用Fas和Fas-配体基因特异性引物RT-PCR扩增患者细胞Fas和Fas-配体mRNA， PCR产物检测Fas基因突变。4例细胞均表现出抗fas诱导的凋亡，且病例3细胞表面fas受体表达完全减少。患者T细胞中cd3诱导的活化细胞死亡也有所减少。检测到三种不同的Fas基因突变。病例1和病例2均为家族性病例，发现7号内含子杂合点突变，导致7号外显子跳变，提前终止。这些突变导致缺乏死亡结构域的Fas蛋白被截断。病例3为内含子3纯合突变，缺失外显子4；病例4为编码死亡结构域的外显子9点突变。所有4例病例均显示循环中TCRαβ+CD4-CD8- T细胞增加，这是Fas和FasL突变小鼠的特异性特征。家族性分析表明，病例1和2的Fas基因突变遗传自母亲，病例3的Fas基因突变遗传自父母双方。这是日本首次报道Fas基因突变。这些结果提示fas介导的细胞凋亡在维持免疫功能中起关键作用

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