Clinical description, genetic analysis and characterization of two NLRP12 heterozygous VUS variants

Background: Systemic autoinflammatory diseases (SAIDs) are a rare, heterogeneous group of disorders caused by dysregulated innate immune activation. Objective: variant of uncertain significance (VUS)-one previously reported and one newly identified in Europeans-and to evaluate their functional and genetic relevance. Methods: variants not associated with Familial Cold Autoinflammatory Syndrome type 2 (FCAS2). Functional assays using peripheral blood mononuclear cells (PBMCs) were performed to assess the impact of these variants on cytokine production. Clinical features were compared among affected and unaffected family members and correlated with molecular findings. Results: gene. The first, c.2854G>A (p.Asp952Asn), previously reported but still classified as a VUS, substitutes a negatively charged aspartic acid with an uncharged asparagine at residue 952. Structural modeling suggested disruption of local interactions between the pyrin domain (PYD) and leucine-rich repeat (LRR) domains, potentially impairing protein activation. The second variant, c.616C>G (p.Arg206Gly), represents a novel finding in Europeans, replacing a large positively charged arginine with a small, flexible glycine at residue 206. Modeling predicted destabilization of a loop near the nucleotide oligomerization core domain (NACHT) and loss of key interactions. Despite their classification as VUS according to the American College of Medical Genetics and Genomics (ACMG) criteria, both variants demonstrated functional impact. Carriers of p.Asp952Asn (index case, mother, and sibling) showed a marked, generalized increase in proinflammatory cytokines, particularly IL-1β, TNF-α, and IL-17A. The index case exhibited exceptionally high basal IL-1β levels, consistent with an activated inflammatory state, and clinical data confirmed elevated C-reactive protein (CRP) levels. In contrast, the individual carrying p.Arg206Gly displayed a selective inflammatory response characterized by increases in IL-1β and TNF-α only after lipopolysaccharide (LPS) stimulation, not after muramyl dipeptide (MDP), with no elevation in IL-10 or IL-17A. Conclusion: VUS in the context of Human Inborn Errors of Immunity and primary immunodeficiencies.