3.1 The patients presented with a severe phenotype of ALPS
Herein, we present a case of patient with clinical and genetic characteristics of ALPS and TCRαβ + CD4/CD8 double-negative T cells (DNT) elevation. From Sep, 2016, he showed unexplained large-scale splenomegaly and lymphadenopathy, along with severe thrombocytopenia. The patient was unresponsive to steroids, immunoglobulins, platelet transfusions, and recombinant human platelet growth factors. The elevated proportion of DNTs/CD3+ (5.8%) made us consider the dignosis of ALPS. however, he was positive for anti-platelet antibodies, anti-Epstein-Barr viruses, rubella viruses, cytomegalovirus, herpes simplex virus immunoglobulin G, and antinuclear antibodies. In addition, the analysis of markers for measles virus, parvovirus B19, schistosomiasis, and Leishmania donovani infection was negative. T lymphocytes (CD3+CD19-) and T helper cells (CD3 + CD4 +) in peripheral blood samples were slightly increased. However, Treg/CD4+ ratio was significantly decreased (2.5%). Moreover, the patient displayed amplified proportions of cytokine IFN-γ, IL-10, and IL-6.
Histological analysis of the bone marrow and spleen did not show signs of hemophagocytosis with obviously splenomegaly. Because of the severe clinical manifestation, we tried rapamycin mTOR signal pathway inhibitor before the NGS genetic results, after which platelet count was restored, and spleen retracted quickly.
3.2 Immunophenotyping after sirolimus
Routine blood results indicated thrombocytopenia (0-5×109/L). Four days after the application of rapamycin, the platelet count returned to 150×109/L. The patient displayed persisting T-cell lymphocytosis with an increased proportion of DNT, while NK and Treg cells (CD3+CD4+CD25+Foxp3+) were reduced. After 4 weeks rapamycin treatment, DNTs/CD3+ (3.5%) was decreased; however, it was still not within normal levels, unlike Treg and the serum levels of TNF, IL-6, and IL-10, which returned to a normal level.
3.3 ALPS was associated with a novel type of FAS mutation
After receiving informed consent from the parents, we sequenced the ALPS-related genes (FAS, FASLG, CASP8, CASP10) and genes known to cause common variable immunodeficiency. We identified novel spontaneous somatic heterozygous missense mutations on the FAS gene (c.857G>A, p.G286E) in exon 9, causing an amino acid exchange. Sanger sequencing verification of peripheral blood and nail confirmed the somatic mutation. The identified variation, which has been previously observed, is the first disease-causing mutation detected in the extracellular domain of the Fas (neither in the 1000 Genomes Project, the HapMap Project, Exome Variant Server data sets nor in dbSNP database or ExAC-Asian database). No mutations were detected by Sanger sequencing of the FASLG，CASP8, CASP10 gene.
3.4 The abnormal ability of the mutant to inhibit cell proliferation
According to the results of CCK8, after 96 h, higher inhibition of Jurkat cell proliferation was observed in the overexpressing wild-type FAS (LW1866) and FAS-G286E (LW1579) groups compared with the NC group. However, the G286E had less ability to inhibit compared to wild-type.
3.5. Cell cycle results: mutant vs. wild-type
According to the flow cytometry results, compared with the NC group, the proportion of overexpressed wild-type FAS (LW1866) and FAS-G286E (LW1579) G2 cells increased, indicating that overexpressed wild-type FAS (LW1866) and FAS-G286E (LW1579) could block Jurkat cells in G2 phase. Although the trend of the G286E was more obvious, there was no significant difference between them.
3.6. Mutant and wild-type apoptosis results
Compared with the NC group, the over-expressing wild-type FAS (LW1866) and FAS-G286E (LW1579) groups could increase Jurkat cells' early number. The proportion of apoptotic cells in the G286E was lower than that in the wild type. Compared to the overall cells, the degree of speculation of these three groups was not obvious.
Medical history, clinical presentation, and laboratory tests results led to the diagnosis of the autoimmune lymphoproliferative syndrome (ALPS). Consequently, the treatment with sirolimus (1mg/d, blood concentration 5-15ng/L) was initiated. Subsequently, the patient’s platelet count and clinical condition improved rapidly.