A novel FAS mutation with variable expressivity in a family with unicentric and idiopathic multicentric Castleman disease

Abstract

1. FAS can be mutated in individuals diagnosed with unicentric and idiopathic multicentric Castleman disease.

2. Defective lymphocyte apoptosis may be a pathological mechanism shared between Castleman disease and autoimmune lymphoproliferative syndrome.

Graphical abstract

Figure 1.

A missense Fas mutation in a family with CD. (A) The pedigree with FAS genotype. Blue shading indicates iMCD diagnosis, and gray shading indicates UCD diagnosis. (B) Hematoxylin and eosin (H&E) staining of P1’s lymph node. Prominent vascularity and follicular hyperplasia with occasional involuted follicles rich in dendritic cells (left panel), germinal center with a hyalinized vessel (middle panel), and paracortical expansion rich in vessels and plasma cells with occasional plasmablasts, close to an atrophic follicle (lower left corner) (right panel). (C) H&E staining of P2’s lymph node. Hyperplastic follicles and paracortical expansion (left panel), 2 germinal centers sharing the same mantle layer (“twinning”) (middle panel), and an involuted germinal center from which a hyalinized vessel (“lollipop”) arises (right panel). (D) Schematic representation of the Fas protein domains. Numbers shown below the boxes indicate the amino acid residue number. (E) Multiple sequence alignment of human Fas and its orthologs. R68, mutated in the family shown in panel A, is in red. (F) PHA-activated T cells were exposed to recombinant human FasL for 18 hours. Cell viability was determined by quantifying adenosine triphosphate contents. The percentages of viable cells relative to the untreated control (healthy donor [HD]) for each sample are plotted. (G) FasL binding to PHA-activated T cells was measured by flow cytometry. Median fluorescence intensity (MFI) normalized to that of an HD is shown. (H) Fas expression on the surface of PHA-activated T cells was measured with 2 monoclonal anti-human Fas antibodies: DX2 and SM1/23. MFI was normalized to a single HD mean for each separate experiment. All experiments were performed 3 times. Means and standard errors of the mean are shown. **P ≤ .01, ***P ≤ .001, ****P ≤ .0001 vs HD average; 1-way analysis of variance. CRD, cysteine-rich domain; DD, death domain; n.s., not significant (P > .05); SP, signal peptide; TM, transmembrane domain.

Figure 2.

R68G impairs surface Fas expression and binding to FasL. The BW5147.3 (BW) cell line was transduced with lentiviruses encoding WT or mutated Fas, along with GFP as a marker for transduction. GFP⁺ cells were sorted and used for experiments. (A) Cell surface Fas expression was analyzed with flow cytometry using an anti-Fas antibody, SM1/23. Similar results were obtained with DX2 (data not shown). (B) FasL binding was analyzed with flow cytometry using recombinant human FasL. (C) Intracellular trafficking of Fas was analyzed with immunoblotting. Where indicated, samples were treated with endoglycosidase H (EndoH). PLC-γ1 was used as a loading control. (D) Apoptosis was analyzed by incubating BW cells with recombinant human FasL or cycloheximide for 6 hours and staining with annexin V. (E) Dominant negativity was analyzed by transducing WT-transduced BW cells with empty vector, WT or mutated Fas. Fas-mediated apoptosis was measured as in panel D. All experiments were performed 3 times. Means and standard errors of the mean are shown. **P ≤ .01, ****P ≤ .0001, 1-way analysis of variance. n.s., P > .05.