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

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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