This project is based on our discovery that genetic mutations in molecules that control the programmed death, or apoptosis, of lymphocytes are responsible for the Autoimmune Lymphoproliferative Syndrome (ALPS). ALPS is a disease affecting children that leads to loss of normal lymphocyte homeostasis leading to swollen lymph glands and organs. Because lymphocytes are the primary cell mediating immune reactions, this excess of lymphocytes leads to a pathological autoimmune attack on the patients own tissues. We have identified mutations in a death-inducing cell surface receptor termed Fas (also known as APO-1 or CD95) and in other molecules that regulate apoptosis. The FAS receptor is homotrimeric and activated by the cognate FAS ligand (FASL), another homotrimeric protein complex homologous to tumor necrosis factor (TNF). Following stimulation, the intracellular death domain (DD) portion of FAS nucleates an extended helical complex of the FADD adaptor protein and caspases-8 and -10. These caspases undergo proteolytic autoprocessing and cleave, in a signaling cascade, downstream effector caspases and other targets leading to the cellular death process of apoptosis. The FAS gene contains 9 exons spanning 26 Kb on chromosome 10q24.118. The first 5 exons encode the extracellular portion containing three cysteine-rich domains that control receptor trimerization and FASL binding. Exon 6 represents the transmembrane domain (TM), and the intracellular portion is encoded by exons 7 through 9. The FAS DD encoded by exon 9 is 85-amino acids long and critical for apoptosis signaling. ALPS-FAS is most frequently caused by heterozygous mutations that generate mutant FAS proteins, often with defective DDs. The defective FAS chains associate with wild-type chains via the pre-ligand assembly domain (PLAD) resulting in functionally defective receptor trimers, a phenomenon termed dominant interference. Less frequently, heterozygous mutations cause decreased FAS protein and haploinsufficiency. Mutations in genes encoding FASL, FADD, and CASP10, also cause ALPS termed ALPS-FASL, ALPS-FADD, and ALPS-CASP10,respectively. Germline mutations in CASP8 or somatic mutations in NRAS and KRAS cause ALPS-related syndromes with distinct clinical phenotypes. Importantly, FAS mutations are associated with in vitro lymphocyte apoptosis defects but may show variable clinical penetrance which has not been fully defined. in a study completed this year, we determined the full clinical, molecular and laboratory assessments of 150 ALPS-FAS patients and 63 healthy mutations positive relatives (HMPRs) to broaden the current understanding of the diagnosis and management of ALPS-FAS. We have found that ALPS presents in childhood with nonmalignant lymphadenopathy and splenomegaly associated with a characteristic expansion of mature CD4 and CD8 negative or double negative TCRalpha/beta+ T cells. Patients often present with chronic multilineage cytopenias due to autoimmune peripheral destruction and/or splenic sequestration of blood elements and have an increased risk of B cell lymphoma. Deleterious heterozygous mutations in the FAS gene are the most common cause of this condition, termed ALPS-FAS. We have determined the natural history and pathophysiology of 150 ALPS-FAS patients and 63 healthy mutation-positive relatives evaluated at the NIH clinical center over the last two decades. Our principal findings are that FAS mutations have a clinical penetrance of less than 60%, elevated serum vitamin B12 is a reliable and accurate biomarker of ALPS-FAS, and the major causes of morbidity and mortality in these patients are the complications of surgical splenectomy and the development of lymphoma. The fact that 41% (27/66) of the splenectomized patients in our cohort had at least one episode of post splenectomy sepsis and 6 of them have died profoundly reinforces the recommendation of avoiding splenectomy and managing chronic cytopenias pharmaceutically. With the long follow up of our study, we observed a significantly greater relative risk of lymphoma than previously reported. We uncovered 18 B cell lymphomas in ALPS-FAS individuals and HMPRs and one non-lymphoid hematopoietic malignancy. The age at diagnosis ranged from 5-60 years (median 17 years). The male to female ratio was 14:5. This included 10 cases of Hodgkins lymphoma in 150 ALPS-FAS patients compared to 0.067 expected giving an observed to expected ratio (O/E) of 149 (95%, confidence interval (CI) = 71 274) and 6 cases of Non-Hodgkin lymphoma compared to 0.099 expected (O/E = 61; 95% CI = 22 132) in ALPS-FAS. The O/E ratios were both highly significant. Standardized incidence ratio (SIR) values are 149 and 61 for Hodgkin and Non-Hodgkin lymphoma, respectively, in the current report versus 51 and 14, respectively, in a previous report we published in 2001. This difference may be related to the cumulative increase of lymphoma with age and accrual of additional events and person-years. Surveillance for lymphoma and avoiding splenectomy while controlling hypersplenism using corticosteroid-sparing treatments such as mycophenolate or sirolimus improves the outcome in ALPS-FAS patients. We are presently studying a class of these patients called ALPS Type III which do not display mutations in the Fas receptor, its ligand (Fas ligand), or caspase-10. We are using a variety of molecular analyses to determine the gene mutation that underlies disease in ALPS Type III. These experiments have been successful in uncovering the molecular basis of a new class of this disease, ALPS type IV. Patients with this disorder have typical clinical features of autoimmunity and abnormal lymphocyte homeostasis that are detected in ALPS, type I and II. however, these patients differ in that they have a strikingly decreased death in response to cytokine withdrawal rather than a defect in death receptor apoptosis. The molecular basis of this disorder is a reduction in the apoptosis protein Bim due to an inherited germline mutation in the N-Ras oncogene. We plan to continue to examine unusual Alps Type III cases to understand their molecular basis. Our guiding principle is that patient specimens from poorly understood diseases can yield valuable insights into disease mechanisms and normal physiology if investigated properly at the molecular level. We have found several new mutations in these unusual patient disorders and are currently characterizing their role in lymphocyte homeostasis and apoptosis.