This study aims to establish the role of novel single heterozygous missense variants in the RIPK1 gene in the development of autoinflammation and to evaluate whether RIPK1 can serve as a potential therapeutic target for monogenic autoinflammatory disorders associated with the TNF signaling pathway

Clinical phenotypes were collected from three Chinese Han male patients with adult-onset autoinflammatory syndrome. Genetic analysis via whole-exome sequencing identified three novel RIPK1 variants: V646E, A404S, and Y128*. Structural modeling was performed using PyMOL 2.0 based on AlphaFold database models. Functional assays were conducted on peripheral blood mononuclear cells (PBMCs) to assess inflammatory responses, including western blot for protein expression (p-RIPK1, p-RIPK3, NLRP3, caspase-1), ELISA for cytokine levels (TNFα, IL-1β, IL-6), and cell viability assays under necroptotic stimuli (TNFα, SM164, and z-VAD-fmk). Additionally, the modulatory effects of the RIPK1 inhibitor Nec-1S were investigated in PBMCs from patients with TNF receptor-associated periodic syndrome (TRAPS).

The three patients presented with periodic fever and lymphadenopathy, but lacked the typical early onset or severe immunodeficiency associated with known RIPK1 disorders like AIEFL or immunodeficiency 57. Conservation analysis indicated that the identified variants involve highly conserved residues. PBMCs from the patient carrying the V646E variant demonstrated increased sensitivity to necroptotic stimuli and significant elevation of TNFα and IL-1β levels upon stimulation. Notably, RIPK1 protein levels in this patient were lower than in healthy controls, leading to the hypothesis that the observed autoinflammation is driven by RIPK1 haploinsufficiency. Furthermore, while Nec-1S did not suppress the increased sensitivity in the V646E patient, it significantly reduced IL-6 and IL-1β production in treatment-naive TRAPS patients.

Our findings suggest that novel heterozygous non-324 variants in RIPK1 may lead to autoinflammatory diseases characterized by adult onset and periodic fever, potentially through a mechanism of RIPK1 haploinsufficiency. This defines a clinical phenotype distinct from previously reported RIPK1-associated conditions. Additionally, the inhibitory effect observed in TRAPS patients supports the broader application of RIPK1 targeting as a therapeutic strategy for TNF signaling pathway-associated disorders.