Typer 4

Peripheral blood samples were collected from each subject. DNA was isolated from venous blood sample by the GoldMag DNA purification kit (GoldMag Co. Ltd, Xi′an, China) in accordance with the user’s protocol, then quantified by NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA). The SNPs in the MAD1L1, TSNARE1 genes were chosen based on the minor allele frequency (MAF) > 0.05 in Han Chinese from the 1000 Genome Projects. Three SNPs (rs10275045, rs12666575, rs1107592) in MAD1L1 and two SNPs (rs4976976, rs67756423) in TSNARE1 were selected in the present study.
Primers of the five SNPs are listed in Additional file 1: Table S1. PCR reactions were performed in a buffer containing 1 μl DNA, 0.5 μl PCR Buffer, 0.4 μl MgCl₂, 0.1 μl dNTP Mix, 1.0 μl primer mix, and 0.2 μl Taq ligase in a final reaction volume of 5 μl. The reaction mixture was heated to 94 °C for 15 min for denaturation. Then, the sample was subjected to 45 cycles of 94 °C 20 s, annealing at 56 °C 30 s and extension at 72 °C 60 s, followed by a final extension step at 72 °C for 3 min. The PCR product was used to genotype using the Agena MassArray platform (Agena Bioscience, San Diego, CA, USA). The raw data was analyzed and managed using Agena Typer 4.0 software (Agena Bioscience, San Diego, CA, USA).

We selected rs3806265 (chr1: 247423034(GRCh38.p12); T>A or T>C; Intron variant) and rs4612666 (chr1: 247435768 (GRCh38.p12); T>C; Intron variant) of the NLRP3 gene for analysis. These two SNPs were nominated using a widely used nomenclature system. We selected these two SNPs because they were previously demonstrated to be correlated with many autoimmune and inflammatory diseases, and rs4612666 was even shown to serve as a functional variant that influenced the process of inflammation. In addition, we prepared SNPs with minor allele frequencies (MAFs) >0.05 in Asian people. Those selected SNPs were located in different linkage regions.
MassARRAY was used for genotyping. ADS2.0 from Agena Bioscience was used to design the sequences of the primers for the two SNPs, which were then synthesized by BGI. Typer 4.0 software (Agena Bioscience Inc.) was used to collect genotyping data after clustering. A specificity of 0.90 and a sensitivity of 0.95 were set to cluster all loci. All genotyping assays were performed when the clinical information of the research subjects was unknown in the laboratory of BGI Genomics, BGI-Shenzhen, Shenzhen 518083, China.

We selected three single nucleotide polymorphisms (SNPs) (rs1940475 and rs3765620 in MMP8, and rs17860949 in MMP10) from the 1000 Genomes Project database with minor allele frequency (MAF) large than 0.05. Genomic DNA in peripheral venous blood samples was extracted by DNA extraction kit. PCR primers for SNPs genotyping were designed by Agena Bioscience Assay Design software and listed in Table 1. The genotyping of MMP8/MMP10 genetic polymorphisms was identified by the Agena MassARRAY iPLEX platform. Additionally, analysis of the genotyping data was conducted by the Agena Bioscience TYPER 4.0 software.