Co-inherited mutations of Fas and caspase-10 in development of the autoimmune lymphoproliferative syndrome

Abstract

Background: Autoimmune lymphoproliferative syndrome (ALPS) is a rare inherited disorder characterized by defective function of Fas, autoimmune manifestations that predominantly involve blood cells, polyclonal accumulation of lymphocytes in the spleen and lymph nodes with lymphoadenomegaly and/or splenomegaly, and expansion of TCRalphabeta+ CD4/CD8 double-negative (DN) T cells in the peripheral blood. Most frequently, it is due to Fas gene mutations, causing ALPS type Ia (ALPS-Ia). However, other mutations, namely of the FasL gene (ALPS-Ib) and the caspase-10 gene (ALPS-II) are occasionally detected, whereas some patients do not present any known mutations (ALPS-III). Recently, mutations of the NRAS gene have been suggested to cause ALPS-IV.

Results: This work reports two patients that are combined heterozygous for single nucleotide substitutions in the Fas and caspase-10 genes. The first patient carried a splice site defect suppressing allele expression in the Fas gene and the P501L substitution in caspase-10. The second had a mutation causing a premature stop codon (Q47X) in the Fas gene and the Y446C substitution in caspase-10. Fas expression was reduced and caspase-10 activity was decreased in both patients. In both patients, the mutations were inherited from distinct healthy parents.

Conclusion: These data strongly suggest that co-transmission of these mutation was responsible for ALPS.

Figure 1

Pedigrees of Family 1 and Family 2. Inheritance of the CASP10 and TNFRSF6 mutations and electropherograms of the sequences performed on the genomic DNA of Pt.1 and Pt.2. Circles represent females; squares, males; subjects carrying a CASP10 mutation are marked in black, those with a TNFRSF6 are marked with striped lines. Numbers indicate the cell survival upon Fas triggering by mAb in T cell lines generated form each subject; Fas function was defective in Pt.1 and borderline in the other subjects (normal values of cell survival: median 60%, 95^th percentile 82%)

Figure 2

Fas expression and caspase-10 activity in subjects carrying the TNFRSF6 and CASP10 mutation. a) Fas expression was evaluated in T cell lines obtained by activating PBMC with PHA (1 μg/ml) and cultured for 6 days in RPMI 1640 +10% FCS+rIL-2 (2 U/mL). Before activation (day 0), and at day 3 and day 6 of culture, cells were stained with a FITC-conjugated anti-Fas mAb and analyzed with a cytofluorimeter. The upper panel shows the cytofluorimetric staining of cells from Pt.1, Pt.2, and a control donor after 6 days of culture. The lower panel shows the MFI ratio calculated for each subject at different times of culture. Control data are the medians ± interquartile ranges (25–75% range) from 5 control donors; their 5^th percentile value at day 6 was MFI-R = 6.48. b) Caspase-10 activity was evaluated in PHA-activated T cells cultured for 12 days (see Methods) in RPMI 1640 +10% FCS+rIL-2 (10 U/mL) and then treated or not with an anti-Fas mAb for 3 hours. Results are expressed as relative caspase activity % calculated as follows: (result displayed by each subject/mean of the results displayed by the 2 controls run in the same experiment) × 100; 100% indicates the mean of the results obtained with the 2 control donors run in parallel with the patient samples in each experiment; the dotted horizontal lines indicate the 5^th percentile of the activity displayed by all normal controls. The color code is the same in all panels.

Figure 3

Caspase-10 activity in 293T cells transfected with P501L-caspase-10. Analysis of 293T cells transiently transfected with the mock, WT^FLAG, P501L^HA, or P501L^HA+WT^FLAG plasmids, as indicated in the panel. a) Western blot analysis of cell transfectant lysates performed with anti-caspase-10 antibody; expression of the transfected molecules was confirmed using anti-HA and -FLAG antibodies (data not shown). The white arrow shows the pro-caspase-10; black arrows indicate the cleaved forms. b) Fluorimetric enzyme assay of caspase-10 activity evaluated in the cell transfectant lysates 24 h after transfection; data are relative to those displayed by mock-transfected cells (indicated as 100%) and are the means ± SE of data from 6 independent experiments. The asterisks mark the data significantly different from those obtained with P501L^HA-transfected cells (p < 0.01, Mann Whitney test).