Background/Purpose: Systemic juvenile idiopathic arthritis (sJIA) is an inflammatory condition characterized by recurring fevers, arthritis and hyperinflammation, but its exact etiology is unknown.  Tumor necrosis factor receptor 1 (TNFR1), the prototypical TNF superfamily receptor, is constitutively expressed on most cell types and activated by TNF.  Signaling through TNFR1 is mediated by its death domain (DD), which acts as the scaffold upon which signaling complexes are formed.  These signaling complexes activate pro-inflammatory pathways in an NFkB dependent manner, while also promoting cell death via either caspase-dependent apoptosis or necroptosis.  Using a combination of genetic studies and in vitro investigations, we have discovered a novel class of TNFRSF1A variations in children with sJIA.

Methods: Trio exome sequencing was performed in a child with sJIA.  Targeted resequencing of TNFRSF1A was performed in 525 INCHARGE sJIA cases.  Protein modeling was used to predict variant effects.  Flow cytometric Förster resonance energy transfer (FRET) was used to measure the strength of homotypic and heterotypic interactions of TNFR1.  Variants were studied in vitro in HEK-293, HT1080 and Jurkat cell overexpression systems.  TNFR1-nucleated signaling complexes were examined by immunoprecipitation (IP).  TNFR1 function was evaluated with NFkB reporter assays and flow cytometric cell death assays.  The locaization of TNFR1 in transfected cells was visualized by confocal microscopy.  Soluble TNFR1 (sTNFR1) in the serum of sJIA patients and controls was measured by ELISA.

Results: We identified a novel, de novo truncating variant of TNFRSF1A (K203X) that completely eliminated the transmembrane and DD in a child with sJIA.  Targeted resequencing identified 2 children with rare variants in the DD of TNFRSF1A (P367S, E398K).  Modeling predicted that E398K would reduce TNFR1-TNFR1 interactions while P367S would reduce the interaction of TNFR1 with its adaptor molecule, TRADD (TNFR1 associated death domain protein).  The predictions were affirmed experimentally by FRET: E398K reduced homotypic interactions, P367S reduced heterotypic interactions and K203X abolished nearly all interactions.  Similarly, IP studies demonstrated dramatic reductions in proximal signaling complex formation in HT1080 cells overexpressing each of the variants, relative to WT.  Functionally, each of these variants led to significantly less cell death and NFkB activation at steady state in the HT1080 system relative to WT.  In the Jurkat cell system, each of the variants abrogated responses to TNF stimulation.  Although serum sTNFR1 was higher in sJIA patients than in controls, the K203X carrier had the lowest level of sTNFR1 in the study.  Consistent with this, imaging of HT1080 cells expressing K203X showed intracellular retention of TNFR1, alluding to possible sequestration.

Conclusion: We have discovered a new class of TNFRSF1A variants in sJIA patients that reduces DD-dependent signaling complex formation, leading to decreased inflammatory signaling and cell death.  Investigations of primary patient cells are underway.  Future studies will investigate how these loss-of-function mutations paradoxically lead to an inflammatory phenotype.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/novel-loss-of-function-variants-in-the-death-domain-of-tumor-necrosis-factor-superfamily-receptor-1a-tnfrsf1a-in-children-with-systemic-juvenile-idiopathic-arthritis-sjia/