Whole-exome-sequencing-based discovery of human FADD deficiency

Abstract

Germline mutations in FASL and FAS impair Fas-dependent apoptosis and cause recessively or dominantly inherited autoimmune lymphoproliferative syndrome (ALPS). Patients with ALPS typically present with no other clinical phenotype. We investigated a large, consanguineous, multiplex kindred in which biological features of ALPS were found in the context of severe bacterial and viral disease, recurrent hepatopathy and encephalopathy, and cardiac malformations. By a combination of genome-wide linkage and whole-exome sequencing, we identified a homozygous missense mutation in FADD, encoding the Fas-associated death domain protein (FADD), in the patients. This FADD mutation decreases steady-state protein levels and impairs Fas-dependent apoptosis in vitro, accounting for biological ALPS phenotypes in vivo. It also impairs Fas-independent signaling pathways. The observed bacterial infections result partly from functional hyposplenism, and viral infections result from impaired interferon immunity. We describe here a complex clinical disorder, its genetic basis, and some of the key mechanisms underlying its pathogenesis. Our findings highlight the key role of FADD in Fas-dependent and Fas-independent signaling pathways in humans.

Figure 1

Characterization of an Inherited FADD Mutation (A) Pedigree of the Pakistani family. Black symbols indicate patients. Gray symbols indicate siblings in the parents' generation who died early in childhood with clinical symptoms related to the disorder observed in P1–P4. Clinical information was incomplete for these members of the parents' generation. Haplotypes of FADD are indicated: M stands for c.315T>G, and WT for the wild-type allele. (B) Schematic diagram of FADD protein showing the death effector domain (DED) and the death domain (DD). The location corresponding to the mutation is indicated by an arrow, and the predicted amino acid substitution is shown. (C) Evolutionary conservation of the FADD region containing amino acid residue C105 (indicated by the arrow). (D) FADD immunoblot in primary fibroblasts and EBV-B cells from patients and controls. Experiments were carried out with two different antibodies: a mouse monoclonal antibody against the C terminus of FADD (#610399, BD Biosciences) and a rabbit polyclonal antibody against the residues surrounding Ser194 in human FADD (#2782, Cell Signaling). Similar results were obtained with both antibodies. GAPDH was used as a loading control. A representative blot with the monoclonal antibody is shown (n = 6). (E and F) FADD protein levels in primary fibroblasts (E) and EBV-B cells (F) determined on the basis of the intensity of the signal on immunoblots and normalized with respect to GAPDH levels. A mean of six experiments is shown. Error bars indicate the SEM. (G and H) Effect of the C105W mutation on the stability of FADD DD folding and the Fas-FADD complex. (G) Differential scanning calorimetry (DSC) analysis of WT and C105W His₆-FADD DD proteins. (H) Fas-FADD complex stability assay. The retention of FADD DD (WT or C105W) with immobilized His₆-Fas DD was assessed with various concentrations of NaCl, as previously described (E: imidazole elution of remaining complex after the final NaCl concentration).

Figure 2

Characterization of the ALPS Phenotype (A) PHA blasts from normal volunteers (Ctrls), patient P4, and a heterozygous Fas-deficient patient were stimulated with recombinant FasL (r-FasL), and cell viability was analyzed. The proportion of live cells in the presence of r-FasL is displayed as a percentage of live cells in the presence of enhancer alone, for each sample. Error bars indicate the SEM. (B) EBV-B cells from P3, IV.1, and a Fas-deficient patient were treated with an antibody against Fas (Apo-1.3), in the presence of rabbit anti-mouse Ig (Ramig), for 6 hr and analyzed for early apoptosis. The percentage of cells that were apoptotic was calculated as the percentage of cells positive for AnnexinV and negative for propidium iodide staining. NS indicates that the cells were incubated with Ramig only. A mean of three independent experiments is shown. Error bars indicate the SEM. (C) FasL levels in the serum of P4 and eight healthy controls (Ctrls). Error bars indicate the SEM. (D) IL-10 levels in the serum of P4 and eight healthy controls. IL-10 values were normalized with respect to the mean of the control samples. Error bars indicate the SEM. (E) Proliferation assay. Proliferative responses to various stimuli (PHA, 5 μg/ml; IL-2, 500 U/ml; concanavalin A, 5 μg/ml; anti-CD3 [OKT3], 0.1 μg/ml; PMA, 50 ng/ml; ionomycin, 0.1 μg/ml). P3 and P4 responded similarly in comparison to the two controls, C1 and C2.

Figure 3

Impaired Antiviral Immunity (A) EBV-B cells from P3, IV.1, and a STAT1-deficient patient were infected with VSV at a multiplicity of infection (MOI) of 1, with or without prior treatment with 100 IU/ml of IFN-α for 24 hr. Cell mortality was assessed by measuring the amount of LDH released into the medium. For STAT1-deficient cells, results are shown only until 36 hr after infection because of high levels of spontaneous LDH release at later time points. A mean of three independent experiments is shown. Error bars indicate the SEM. (B) EBV-B cells from P3, IV.1, and a STAT1-deficient patient were either left untreated (NT, bars in white) or treated with 100 IU/ml IFN-α for 24 hr (IFN-α, bars in black), prior to infection with VSV at a MOI of 0.1, and were harvested 48 hr after infection. VSV titers (TCID50, ml) were determined on Vero cells. A mean of three independent experiments is shown. Error bars indicate the SEM. (C) EBV-B cells from P3 or IV.1 were infected with VSV at a MOI of 10. Cells were harvested at the indicated time points, and total RNA was extracted. IFN-β and IRF7 mRNA levels were determined by quantitative RT-PCR. Threshold cycles normalized with respect to those of GUS (ΔC_T) are plotted as 2^−ΔCT. A mean of three independent experiments is shown. Error bars indicate the SEM.

Figure 4

Histopathology of P2 and P4 (A–K) postmortem histopathology of P2 at 14 months. (A) Normal thymic architecture; increased cortical tingible body macrophages consistent with a stress response (P2, hematoxylin and eosin [H&E] stain, original magnification × 100). (B) Normal-appearing reactive lymph node with follicular hyperplasia (P2, H&E stain, original magnification × 40). (C) Low-power view of the spleen showing expanded, congested red pulp with relatively reduced white pulp (P2, H&E stain, original magnification × 20). (D) White pulp nodule containing a follicle with a reactive germinal centre, but no marginal zone hyperplasia, arising from a periarteriolar lymphoid sheath (P2, H&E stain, original magnification × 200). (E) Immunohistochemistry for CD3 shows a normal distribution of CD3-positive T cells in a white pulp nodule (P2, Ventana Benchmark XT immunostainer, monoclonal antibody LN10, original magnification × 100). (F) Immunohistochemistry for CD20 shows a normal distribution of CD20-positive B cells in a white pulp nodule (P2, Ventana Benchmark XT immunostainer, monoclonal antibody L26, original magnification × 100). (G) Congested, disorganized red pulp containing increased neutrophils consistent with sepsis (P2, H&E stain, original magnification × 400). (H) Disorganized reticulin staining pattern of splenic red pulp. Inset: “barrel hoop” arrangement of sinusoidal reticulin fibers in a normal control spleen (P2, Gordon and Sweet's reticulin stain, original magnification × 400). (I) Immunohistochemistry for CD68 shows a haphazard arrangement of macrophages in the splenic red pulp. Inset: orderly distribution of cordal macrophages in a normal control spleen (P2, Ventana Benchmark XT immunostainer, monoclonal antibody PG-M1, original magnification × 400). (J) Liver showing portal-portal linkage and expansion of portal tracts by a moderate chronic inflammatory cell infiltrate without interface activity (P2, H&E stain, original magnification × 100). (K) Sirius red staining of liver shows collagen deposition (red) within portal-portal bridges. Elastin could also be demonstrated (not shown), consistent with established portal-portal fibrosis (P2, Sirius red and Fast green stain, original magnification × 100). (L) Histopathology of P4 at 22 months. The liver shows mild expansion of portal tracts by a chronic inflammatory cell infiltrate but no bridging fibrosis (H&E stain, original magnification × 200).