Somatic Fas Mutations Account for Nearly One Third of Autoimmune Lymphoproliferative Syndrome (ALPS) Cases with Previously Unknown Genetic Mutations.

Autoimmune lymphoproliferative syndrome (ALPS) is characterized by childhood onset of lymphadenopathy, hepatosplenomegaly, autoimmune cytopenias, elevated (>1%) double negative T (DNT; CD3⁺, TCRalpha-beta⁺, CD4⁻, CD8⁻, B220⁺) lymphocytes in peripheral blood and an increased risk of lymphoma; primarily due to impaired lymphocyte apoptosis. Most cases (65%; 173 individuals in our cohort), known as ALPS Type Ia, are associated with dominant heterozygous germline mutations in the gene TNFRSF6 encoding the protein for CD95 (Fas, Apo-1). Another 5% of patients have mutations in FasL (Type Ib), caspases (Type II) or NRAS (Type IV). However, approximately 30% of ALPS patients in our cohort have no mutation found upon genomic sequencing of DNA from peripheral blood. We designated these patients as ALPS Type III (16%) if they met all the criteria of ALPS, i.e., elevated DNT cell numbers, nonmalignant chronic lymphadenopathy/splenomegaly and defective lymphocyte apoptosis by in vitro assay; we called them ALPS phenotype (14%) if they had all the features as described above but did not demonstrate an in vitro apoptosis defect. Holzelova et. al. (NEJM 2004; 351:1409) previously identified somatic Fas mutations in DNT cell population in 6 patients with ALPS phenotype. However, there was no information as to the proportion of ALPS patients expressing a somatic Fas mutation or any differences in their clinical phenotype.

Over the last 5 years we sought to determine the proportion of ALPS Type III and ALPS phenotype patients with somatic Fas mutations in their DNT cell population and to further clinically characterize these patients. DNT cells were purified to >50% by magnetic bead separation and DNA was sequenced for Fas. We found somatic Fas mutations in the DNT cells of 11/31 (35.5%) patients; 5/15 with ALPS Type III (5 males; age range = 1-17 years; median = 12 years), and 6/16 with ALPS phenotype (3 males and 3 females; age range = 3 mo–48 years; median age = 1 year). All the mutations clustered to an approximately 150 base region of the intracellular domain of Fas (exons 7, 8 and 9), except for one mutation in exon 3. All mutations resulted in functional loss of normal Fas signaling based on the mutation type or having been previously observed in ALPS Typa Ia. The somatic ALPS patients showed a similar clinical phenotype to that of ALPS Type Ia with increased DNT cell numbers (Median 6%; Range 4-19%), and increased levels of biomarkers like serum vitamin B12, IL-10 and sFasL. All 4 patients with onset of symptoms in infancy have required long-term treatment of their refractory autoimmune cytopenias with mycophenolate mofetil for the last 2-8years, while one of them underwent splenectomy. Inability to demonstrate defective lymphocyte apoptosis by in vitro testing of unseparated lymphocytes in ALPS phenotype patients with somatic Fas mutations is likely due to the relatively small number of affected cells (<20%) in their peripheral blood. Additionally, we hypothesize that the late onset of clinical disease in ALPS Type III patients with somatic Fas mutations may reflect the time required to accumulate a threshold number of cells that are able to confer clinical manifestations.

Thus, the majority of somatic ALPS mutations can be detected by sequencing exons 7-9 of isolated DNT cell DNA. Patients with somatic Fas mutations now comprise the second largest group of known genetic mutations in ALPS, followed by patients with caspase 10, NRAS, caspase 8 and FasL germline mutations, respectively. We recommend testing for somatic Fas mutations in ALPS Type III patients (particularly those with late onset) and all ALPS phenotype patients, using ungated DNT purities of >50%. These data also highlight the role of somatic mutations in the pathogenesis of nonmalignant hematological conditions in adults and children, as well as in clonal malignancies.

No relevant conflicts of interest to declare.