Abstract

ALPS is a disorder of lymphocyte apoptosis associated with expansion of double negative T cells (DNT; TCRα/β+CD3+CD4−CD8−), an in vitro apoptosis defect and affected individuals typically present in early childhood with nonmalignant lymphadenopathy, splenomegaly, and multilineage cytopenias due to splenic sequestration and/or peripheral autoimmune destruction. ALPS classifications include Type Ia, Ib, and II associated with mutations in Fas, FasL, and Caspases, respectively, and Type III when the apoptosis pathway mutation is unidentified. Among our cohort of more than 200 patients, ALPS Type Ia with germline Fas mutations constitute the most common subtype (over 70%). These patients show an in vitro Fas mediated lymphocyte apoptosis defect and have a known increased risk of lymphoma. However, there are a number of patients that have an “ALPS-phenotype” manifesting typical historical and clinical features of ALPS and present with elevated circulating DNT cells, but have no demonstrable in vitro apoptosis defect. Based on the work of Holzelova et. al (

NEJM
,
2004
,
351
:
1409
) in which six such patients were reported with somatic Fas mutations in their DNT cell subsets, we sought to characterize our patient set without defined mutations. DNT cells from 20 patients, including 8 with ALPS-phenotype and 12 with ALPS Type III, were isolated using magnetic bead separation of frozen PBMCs. The sensitivity for PCR followed by sequencing required >20% abnormal cells using a mixture of normal and ALPS Type Ia PBMCs. However, the sensitivity proved to be lower (40%) using purified DNT cells from patients with potential somatic Fas mutations. Therefore, we chose an ungated DNT purity of 50% as a minimum requirement. Ungated DNT purities for the 20 evaluated patients ranged from 51% to 92%, with a mean of 66%. All nine exons of the Fas gene were PCR amplified prior to sequencing.

We found that 3/8 (37.5%) patients with ALPS-phenotype had somatic mutations in the Fas gene: 825G−>T, 846(−9)del11, and 846(−1)G−>T, with the first two mutations being new. Only 1/12 (8.3%) ALPS Type III patients had a somatic Fas muation: 851delAG, which also represents a new mutation. These four somatic mutations were considered significant as two were in a splice site and two were in coding regions. The ALPS Type III patient is atypical given a late age of presentation at age 17 and had milder cytopenias. The three ALPS-phenotype patients with somatic Fas mutations presented similar to cases of “classic” ALPS, with an early age of presentation (median, 1 yr; range, 4 months to 4 years), cytopenias and features of autoimmunity; two require chronic immunosuppression with Cellcept (mycophenolate mofitil) for chronic persistent cytopenias, one having previously undergone surgical splenectomy. From these data we conclude that patients with ALPS-phenotype or atypical ALPS Type III should be screened for somatic mutations in their peripheral blood DNT cell subset using ungated DNT purities of >50%, with priority given to evaluation by sequencing of exons 7–9 of the Fas gene. It is not yet evident if there is an increased risk of lymphoma in patients with somatic Fas mutations as is the case with intracellular germline Fas mutations. Somatic Fas mutations explain the molecular basis of a small subset of patients and are more readily observed in ALPS-phenotype patients.

Disclosure: No relevant conflicts of interest to declare.

Author notes

*

Corresponding author