PMC

Scatter plot of the observed frequency variation in both strands. The colours depict known SNPs (green), heterozygous and mosaic mutations (orange) and errors (grey).

The amplicon analysis for NLRP3 exons and its error rate. (A) Exon–intron structure of the NLPR3 gene. Thick and thin rectangles depict exons and introns, respectively. Blue thick rectangles indicate the CDS region. The 14 designed amplicons (red) for nine exons are shown under the exon–intron structure. (B) Amplicon design schema. (C) Error rate for each error category in the region of entire amplicon (pale blue), that without designed primer regions (light blue), and the target regions (CDS + flanking intron; dark blue), respectively. (D) Strand-wise error rate for each amplicon. (E) Error rates along the amplicon sequence of exon 1 in each strand for insertions and deletions in the upper panel and mismatches and ambiguous base calls in the lower panel. The orange and blue lines depict the primer and target regions, respectively. The yellow shaded area depicts the homonucleotide (n > 3) region. The colour representation for the bars is the same as (D). (F) Co-occurrence error rate in both strands. The fraction of positions where a certain error occurred with the error rate for insertions, deletions, and mismatches. The colour representation is the same as in (D) and (E).

In vitro functional analysis of the identified NLRP3 mosaic mutations. (A) Rapid cell death in transfected THP-1 cells. A GFP-fused wild-type or mutant NLRP3 was transfected into THP-1 cells and incubated with PMA (10 ng/ml) for 4 h. The percentage of dead cells (7-amino-Actinomycin D [7-AAD]-positive) among the GFP-positive cells is shown. Data represent the means ± SD of triplicate experiments and are representative of two independent experiments. The data for previously reported mutations as well as the mutations found in this study are shown. (B) ACS-dependent NF-κB activation in transfected HEK293FT cells. HEK293FT cells were co-transfected with wild-type or mutant NLRP3 in the presence or absence of ASC. NF-κB induction is shown as the fold-change compared with cells that were transfected with a control vector without ASC (set equal to one). Values are the means ± SD of triplicate experiments, and the data are representative of three independent experiments. The data for previously reported mutations (p.Arg260Trp and p.Tyr570Cys) and the mutations found in this study are shown. For each mutation, the data obtained in the presence and absence of ASC are shown. These findings identified p.Phe302Leu as a novel disease-causing mutation.