There were two patients enrolled in this study. The proband was a 4-year-old boy and the other patient was his mother, both of them manifested with recurrent urticaria and arthralgia. Clinical data, family history and blood samples were collected when the proband first visited us in May 2019. Informed consent was obtained from all of the participants. This study was conducted in accordance with the tenets of the Declaration of Helsinki and was approved by the ethics committee of the Children's Hospital of Chongqing Medical University.
Peripheral blood was collected from members of this family after written informed consent obtained. Whole blood samples were sent to MyGenostics (Beijing, China) and subjected to whole-exome sequencing (WES). The pathogenicity of the mutations was assessed following the American College of Medical Genetics and Genomics guidelines (ACMG). Mutations in NLRC4 were confirmed by using Sanger sequencing. Briefly, genomic DNA was extracted from peripheral blood mononuclear cells (PBMCs) using the QIAamp DNA Mini Kit (Qiagen, Shanghai, China). Polymerase chain reaction (PCR) was performed to amplify the genomic NLRC4 gene. The products of the PCR reactions were sent for Sanger sequencing.
The crystal structure of NLRC4 (4KXF from the Protein Data Bank) was used as the template, which was determined by X-ray diffraction at a resolution of 3.20 A. Although the 4KXF is the crystal structure of mouse NLRC4, it does reveal the auto-inhibition mechanism. And previous studies also used it as the template to study the effect of NLRC4 mutation. Herein the structural impact of mutant Gly172Ser was analyzed by Swiss-PdbViewer. Residue 172 and certain nearby residues within 3 A are illustrated. For clear demonstration of the interresidue relationship, some residues were highlighted in the indicated colors with the computed hydrogen bonds being labeled.
Peripheral blood was collected in vacutainers containing sodium heparin. Centrifugation was performed at 2500 rpm for 5 minutes at room temperature to separate the plasma from the peripheral blood cells. The cells were diluted in PBS and added to the top of Ficoll-Paque PREMIUM (GE Healthcare, Sweden) and centrifuged at 800 g for 20 minutes at room temperature to separate the PBMCs fraction.
PBMCs were cultured in RPMI 1640 (Gibco, USA) at a density of 0.8x 106 cells/ml containing 10% FBS (Gibco, USA), and penicillin-streptomycin (100 U/mL each; Sigma-Aldrich, USA) was added. Lipopolysaccharide (Sigma Aldrich, USA) was added to cell culture at a final concentration of 100 ng/ml for stimulation. After 24 hours of culture, the cells were collected for the subsequent experiments.
After cell stimulation, the plate was centrifuged to pellet the cells. The presence of cytokines in the supernatants was determined by using Multi-Analyte Flow Assay Kit (Human Inflammation Panel(13-plex), Biolegend, San Diego, CA) according to the manufacturer's guidelines.
Caspase-Glo  1 inflammasome assay kit (Promega, USA) was used to measure the caspase-1 activation in the PBMCs culture supernatant. Caspase-1 and caspase-1 inhibitor reagents were prepared and added to the supernatant directly as described by the manufacturer. Fifty microliters of supernatant was transferred to a white 96-well plate. Caspase-Glo  1 Reagent or Caspase-Glo  1 YVAD-CHO Reagent was added to the wells (50 microl/well) and gently mixed on a plate shaker at 300 rpm for 30 s. The mixture was then incubated for 60 minutes at room temperature before measuring the luminescence on a Berthold Technologies luminometer.
Cells were immediately washed with PBS and lysed in RIPA buffer with complete protease inhibitor (Sigma-Aldrich) immediately. Protein lysates in protein loading buffer were separated in 10% SDS-PAGE gels, transferred to PVDF membranes (Millipore, Germany), blocked with 5% milk, and then probed overnight at 4 C with primary antibodies, including anti-NLRC4 (ab115537; Abcam, UK) and anti-GAPDH (Bioss antibodies, Woburn, MA). After incubation with an HRP-conjugated secondary antibody (Bioss, USA), the blots were washed with TBST and imaged using an enhanced chemiluminescence system (Thermo Scientific, USA).
A literature review of NLRC4 mutations was performed in PubMed using the terms "NLRC4" and ''mutation''. All types of publications (articles, reviews, editorials, letters, and correspondence), written in English, and published online between January 2014 and June 2020 were included. Only pathogenic and likely pathogenic mutations were considered.
All statistical analyses were conducted with GraphPad Prism 8 software (GraphPad Software, Inc., San Diego, CA). Data were analyzed using an unpaired two-tailed Student t test. P-values < 0.05 were considered to indicate a significant difference.