Study of the potential role of CASPASE-10 mutations in the development of autoimmune lymphoproliferative syndrome

Abstract

Autoimmune lymphoproliferative syndrome (ALPS) is a primary disorder of lymphocyte homeostasis, leading to chronic lymphoproliferation, autoimmune cytopenia, and increased risk of lymphoma. The genetic landscape of ALPS includes mutations in FAS, FASLG, and FADD, all associated with apoptosis deficiency, while the role of CASP10 defect in the disease remains debated. In this study, we aimed to assess the impact of CASP10 variants on ALPS pathogenesis. We benefit from thousands of genetic analysis datasets performed in our Institute's genetic platform to identify individuals carrying CASP10 variants previously suspected to be involved in ALPS outcome: p.C401LfsX15, p.V410I and p.Y446C, both at heterozygous and homozygous state. Clinical and laboratory features of the six included subjects were variable but not consistent with ALPS. Two individuals were healthy. Comprehensive analyses of CASP10 protein expression and FAS-mediated apoptosis were conducted and compared to healthy controls and ALPS patients with FAS mutations. Missense CASP10 variants (p.V410I and p.Y446C), which are common in the general population, did not disrupt CASP10 expression, nor FAS-mediated apoptosis. In contrast, homozygous p.C401LfsX15 CASP10 variant lead to a complete abolished CASP10 expression but had no impact on FAS-mediated apoptosis function. At heterozygous state, this p.C401LfsX15 variant lead to a reduced CASP10 protein levels but remained associated with a normal FAS-mediated apoptosis function. These findings demonstrate that CASPASE 10 is dispensable for FAS-mediated apoptosis. In consequences, CASP10 defect unlikely contribute to ALPS pathogenesis, since they did not result in an impairment of FAS-mediated apoptosis nor in clinical features of ALPS in human. Moreover, the absence of FAS expression up-regulation in subjects with CASP10 variants rule out any compensatory mechanisms possibly involved in the normal apoptosis function observed. In conclusion, this study challenges the notion that CASP10 variants contribute to the development of ALPS.

Fig. 1. Reported CASP10 variants and family pedigrees of included subjects.

A Structure of (Pro)Caspase-10 and variants previously reported in literature. In bold are variants displayed by the enrolled subjects. CASc domain has two parts (p23/p17: residues 220–414 and p12: residues 415–522). C401 (central residue of the QACQG catalytic site) is highlighted in red. B Pedigrees of the six included individuals belonging to four different families. Subjects in gray have no clinical manifestations. L linker domains, DED1-2 Death Effector Domains, CASc Caspase proteolytic domain, S subject. This figure was created with Biorender.com and exported under a paid subscription.

Fig. 2. Western blot on SEE-stimulated T blasts.

A Western blot on SEE-stimulated T blasts generated from healthy controls, included subjects and ALPS-FAS patients with mutations in FAS extracellular (ECD) or intracellular (ICD) domain. Raji cell lines were used as negative controls due to their lack of Caspase-10 expression, while Ku70 (housekeeping protein) was used as loading control. The most expressed Caspase-10 isoforms (uncleaved Caspase-10A and D) are shown. B Quantitative determination of Caspase-10 protein levels normalized with respect to Ku70 levels. Western blot is representative of one experiment from n = 1 sample of SEE T blasts for each individual. HMZ homozygous, HTZ heterozygous, Ctrl control, kDa kiloDalton.

Fig. 3. FAS-mediated apoptosis assay on SEE and CD3/CD28 T blasts generated from included subjects.

Results of FAS-mediated apoptosis assay with optimal dosage (100 ng/ml) of the FAS agonist Apo1.3 was performed both on SEE-stimulated (A, C) and CD3/CD28-stimulated (B, D) T blasts. Results of one independent experiment for each condition performed in triplicate was depicted (A, B) and the global data obtained for each genetic conditions expressed as the percentage of apoptosis in healthy controls were used to calculate the median with interquartile range depicted in C and D. Patients affected by ALPS due to mutations in FAS extracellular (ECD) or intracellular domains (ICD) were used as positive controls of apoptosis defect. Plotted data in C and D are representative of two independent experiments on SEE T and CD3/CD28 T blasts. HMZ homozygous, HTZ heterozygous.

Fig. 4. Western blot for CASP10, CASP8 and FADD on B-LCL cell lines generated from S2 (C401Lfs homozygous).

A Western blot on B-LCL cell lines generated from healthy controls, CASP10 C401Lfs homozygous S2 and Raji cell lines (negative controls). The most expressed isoforms of CASP10 (uncleaved Caspase-10A and -D) and CASP8 (uncleaved Caspase-8A and -B) are shown. B–D Quantitative determination of CASP10, CASP8 and FADD protein levels normalized with respect of Ku70 levels. Western blot is representative of one experiment on n = 1 sample of B-LCL cell line for each individual. HMZ homozygous, FADD Fas-associated death domain, Ctrl control, kDa kiloDalton.