Revisiting autoimmune lymphoproliferative syndrome caused by Fas ligand mutations.

BACKGROUND: Fas ligand (FasL) is expressed by activated T cells and induces death in target cells upon binding to Fas. Loss-of-function FAS or FASLG mutations cause autoimmune-lymphoproliferative syndrome (ALPS) characterized by expanded double-negative T cells (DNT) and elevated serum biomarkers. While most ALPS patients carry heterozygous FAS mutations, FASLG mutations are rare and usually biallelic. Only 2 heterozygous variants were reported, associated with an atypical clinical phenotype. OBJECTIVE: We revisited the significance of heterozygous FASLG mutations as a cause of ALPS. METHODS: Clinical features and biomarkers were analyzed in 24 individuals with homozygous or heterozygous FASLG variants predicted to be deleterious. Cytotoxicity assays were performed with patient T cells and biochemical assays with recombinant FasL. RESULTS: Homozygous FASLG variants abrogated cytotoxicity and resulted in early-onset severe ALPS with elevated DNT, raised vitamin B12, and usually no soluble FasL. In contrast, heterozygous variants affected FasL function by reducing expression, impairing trimerization, or preventing Fas binding. However, they were not associated with elevated DNT and vitamin B12, and they did not affect FasL-mediated cytotoxicity. The dominant-negative effects of previously published variants could not be confirmed. Even Y166C, causing loss of Fas binding with a dominant-negative effect in biochemical assays, did not impair cellular cytotoxicity or cause vitamin B12 and DNT elevation. CONCLUSION: Heterozygous loss-of-function mutations are better tolerated for FASLG than for FAS, which may explain the low frequency of ALPS-FASLG.

Cleavage of the Ewing tumour-specific EWSR1-FLI1 mRNA by hammerhead ribozymes.

BACKGROUND: Ewing family tumours (EFT) are the second most common bone tumours in children and adolescents. In the majority of EFT, EWSR1-FLI1 (Ewing sarcoma breakpoint region 1-Friend leukaemia virus integration 1) fusion proteins can be detected and EWSR1-FLI1 substantially contributes to the malignant phenotype of EFT. Therefore, inactivation of EWSR1-FLI1 is an interesting strategy for EFT therapy. MATERIALS AND METHODS: A ribozyme with specificity for EWSR1-FLI1 was developed and the activity in vitro was investigated. Synthetic RNAs corresponding to EWSR1-FLI1 were used as substrates. In addition, the total RNA from EFT cells was used as substrate and the rapid amplification of cDNA ends method for the detection of the cleavage products was used. RESULTS: The ribozyme cleaved the synthetic RNA in a sequence specific manner with high efficiency in vitro. Furthermore, the expected cleavage products were detected after digestion of the total cellular RNA with this ribozyme. A point mutation in the catalytic centre of the ribozyme abolished enzymatic activity. CONCLUSION: The RNA corresponding to EWSR1-FLI1 is accessible for ribozyme mediated inactivation and ribozymes are able to cleave EWSR1-FLI1 specific RNA in the presence of a high background of normal cellular RNAs.