Research accomplishments of this project during the past year include: ? ? 1.) The identification of a heterozygous missense mutation in NRAS, one of the proto-oncogenes in the RAS family, in a patient with ALPS and a history of non-Hodgkin lymphoma, confirming a new molecular basis for this syndrome. Defective programmed cell death (apoptosis) was demonstrated in vitro employing IL-2 cytokine withdrawal assay. Results employing in vitro death receptor apoptosis assays, the standard apoptosis methodology in ALPS, were normal. The molecular basis of this disorder is a reduction in the apoptosis protein Bim caused by the de novo mutation in NRAS. ? We hypothesize that the NRAS mutation leads to ALPS and a predisposition to lymphoma. If confirmed, the implications will be similar to TNFRSF6 (Fas), if the presence of NRAS mutations defines a novel genetic basis for risk of lymphoma.? ? 2.) Designation of a seventh subtype of ALPS, ALPS Type IV, based on the identification of the new molecular defect described above. Provided is the classification scheme that we have devised for ALPS patients based on the particular molecular defect present: ? Type Ia: mutations in the TNFRSF6 (tumor necrosis factor receptor superfamily 6) gene, encodes the protein CD95 (Fas, Apo-1).? Type Ia: somatic mutant: TNFRSF6 gene defect in the double negative T (DNT) cell population. ? Type Ib: mutations in TNFRSF6 gene, encodes the protein CD95 ligand (Fas ligand).? Type IIa: mutations in CASP10 gene, encodes caspase-10.? Type IIb: mutations in the CASP8 gene, encodes caspase 8.? Type III: associated mutation unidentified to date.? Type IV: mutations in NRAS. ? ? 3.) The extended evaluation, management and duration of follow-up using Mycophenolate mofetil (MMF) for the treatment and prevention of ALPS-related complications. Our experience with MMF as an effective, well-tolerated steroid-sparing long-term maintenance immunomodulatory therapy in the subset of children with refractory ALPS-associated cytopenias associated with splenomegaly and/or autoimmunity is positive.? ? 4.) Extended in vitro and in vivo studies of potential therapeutic drugs in the MRL/lpr (ALPS) mice, testing their capacity to reduce lymphadenopathy and splenomegaly, and key biological markers in ALPS. Valproic acid, a histone deacetylase inhibitor, reduced splenomegaly, lymphadenopathy and CD3+CD4-CD8-alpha beta TCR+T cells (DNTs) in the spleen and blood of MRL/lpr mice. In vitro, using both normal and ALPS Type Ia peripheral blood mononuclear cells (PBMCs), valproic acid was able to induce cell death in a dose-dependent manner. Cell death appeared to be both caspase-dependent and -independent based on partial inhibition with the pan-caspase inhibitor, Z-VAD-FMK.? ? Mycophenolate mofetil resulted in reduced autoimmune kidney disease in the MRL/lpr (ALPS) mice, but had little effect on lymphadenopathy and splenomegaly, similar to that observed in ALPS patients.
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