The NLRP3 inflammasome is a multiprotein complex that mediates caspase-1 activation and the release of proinflammatory cytokines, including interleukin (IL)-1β and IL-18. Gain-of-function variants in the gene encoding NLRP3 (also called cryopyrin) lead to constitutive inflammasome activation and excessive IL-1β production in cryopyrin-associated periodic syndromes (CAPS). Here we present functional screening and automated analysis of 534 NLRP3 variants from the international INFEVERS registry and the ClinVar database. This resource captures the effect of NLRP3 variants on ASC speck formation spontaneously, at low temperature, after inflammasome stimulation and with the specific NLRP3 inhibitor MCC950. Most notably, our analysis facilitated the updated classification of NLRP3 variants in INFEVERS. Structural analysis suggested multiple mechanisms by which CAPS variants activate NLRP3, including enhanced ATP binding, stabilizing the active NLRP3 conformation, destabilizing the inactive NLRP3 complex and promoting oligomerization of the pyrin domain. Furthermore, we identified pathogenic variants that can hypersensitize the activation of NLRP3 in response to nigericin and cold temperature exposure. We also found that most CAPS-related NLRP3 variants can be inhibited by MCC950; however, NLRP3 variants with changes to proline affecting helices near the inhibitor binding site are resistant to MCC950, as are variants in the pyrin domain, which likely trigger activation directly with the pyrin domain of ASC. Our findings could help stratify the CAPS population for NLRP3 inhibitor clinical trials and our automated methodologies can be implemented for molecules with a different mechanism of activation and in laboratories worldwide that are interested in adding new functionally validated NLRP3 variants to the resource. Overall, our study provides improved diagnosis for patients with CAPS, mechanistic insight into the activation of NLRP3 and stratification of patients for the future application of targeted therapeutics.

NLRP3-associated autoinflammatory disease is a heterogenous group of monogenic conditions caused by NLRP3 gain-of-function mutations. The poor functional characterization of most NLRP3 variants hinders diagnosis despite efficient anti-IL-1 treatments. Additionally, while NLRP3 is controlled by priming and activation signals, gain-of-functions have only been investigated in response to priming. Here, we characterize 34 NLRP3 variants in vitro, evaluating their activity upon induction, priming, and/or activation signals, and their sensitivity to four inhibitors. We highlight the functional diversity of the gain-of-function mutants and describe four groups based on the signals governing their activation, correlating partly with the symptom severity. We identify a new group of NLRP3 mutants responding to the activation signal without priming, associated with frequent misdiagnoses. Our results identify key NLRP3 residues controlling inflammasome activity and sensitivity to inhibitors, and antagonistic mechanisms with broader efficacy for therapeutic strategies. They provide new insights into NLRP3 activation, an explanatory mechanism for NLRP3-AID heterogeneity, and original tools for NLRP3-AID diagnosis and drug development.

VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is a severe monogenic disorder caused by somatic mutations in ubiquitin-like modifier activating enzyme 1 (UBA1), characterized by inflammation, cytopenias, and frequent association with myelodysplastic neoplasms (MDS). Steroid dependence is common, and targeted therapies have demonstrated limited efficacy. Azacitidine (AZA), a hypomethylating agent used in MDS, has shown potential in VEXAS syndrome but data remain limited. This multicenter retrospective study assessed AZA efficacy and safety in 88 patients with genetically confirmed VEXAS syndrome from FRENVEX (French VEXAS study group), 80% meeting World Health Organization 2022 MDS criteria. Inflammatory response rates were 41% at 6 months and 54% at 12 months, regardless of MDS status. A total of 50 (61%) patients achieved inflammatory response, with 70% occurring at 6 months, suggesting a delayed median response. Among responders, relapse-free survival on AZA was 90% at 1 year and 85% at 5 years. Of the 12 responders who discontinued AZA, 9 relapsed after a median of 3.1 years (range, 0.4-5.6), with effective reexposure in 4 of 5 patients. Hematological responses included red blood cell transfusion independence in 65% and platelet improvement in 77% of patients. Molecular response, defined as a ≥25% reduction in UBA1 variant allele frequency (VAF), was observed in 65% of patients, all of whom achieved inflammatory and hematological responses; and VAF dropped to <2% in 43% of cases. Infections (34%) and cytopenias (36%) were common, particularly during the first 3 cycles. This study establishes AZA as an effective therapy for VEXAS syndrome, improving inflammation, cytopenias, and UBA1 clonal burden, warranting larger prospective trials.

The EU In-Vitro Diagnostic Device Regulation (IVDR) aims for transparent risk-and purpose-based validation of diagnostic devices, traceability of results to uniquely identified devices, and post-market surveillance. The IVDR regulates design, manufacture and putting into use of devices, but not medical services using these devices. In the absence of suitable commercial devices, the laboratory can resort to laboratory-developed tests (LDT) for in-house use. Documentary obligations (IVDR Art 5.5), the performance and safety specifications of ANNEX I, and development and manufacture under an ISO 15189-equivalent quality system apply. LDTs serve specific clinical needs, often for low volume niche applications, or correspond to the translational phase of new tests and treatments, often extremely relevant for patient care. As some commercial tests may disappear with the IVDR roll-out, many will require urgent LDT replacement. The workload will also depend on which modifications to commercial tests turns them into an LDT, and on how national legislators and competent authorities (CA) will handle new competences and responsibilities. We discuss appropriate interpretation of ISO 15189 to cover IVDR requirements. Selected cases illustrate LDT implementation covering medical needs with commensurate management of risk emanating from intended use and/or design of devices. Unintended collateral damage of the IVDR comprises loss of non-profitable niche applications, increases of costs and wasted resources, and migration of innovative research to more cost-efficient environments. Taking into account local specifics, the legislative framework should reduce the burden on and associated opportunity costs for the health care system, by making diligent use of existing frameworks.