PMC8383449_01
A pyoderma gangrenosum (PG) patient with heterozygous splice-donor-site mutation in NFKB1. (A) After the left knee joint replacement surgery, purple erythema appeared on the left leg of the patient, and a large area of ulcers gradually formed. (B) The patient's ulcer gradually improved after treatment with intravenous immunoglobulin (IVIG) plus glucocorticoid. (C) Schematic of the whole-exome sequencing (WES) data-filtering approach under the assumption of dominant/de novo inheritance, leading to the identification of an NFKB1 variant. For details of variants in each assumed inheritance, see Supplementary Tables 1, 2. INDEL, frameshift, or non-frameshift insertions and deletions; SNP, single-nucleotide polymorphisms including missense, splice-site, and stop-codon variants. (D) The integrative genomics viewer revealed the exome sequencing reads covering a heterozygous mutation (c.730+5G>A) in intron 8 splice donor site of NFKB1. (E) RNA-sequencing analysis of NF-kappaB target genes in patient's peripheral blood mononuclear cells (PBMCs) compared with those of six unaffected controls (C1-C6). Analysis of each sample was performed in duplicate. For gene names, see Supplementary Figure 1. (F) The expression of NFKB1 mRNA in PBMCs of healthy controls and the patient were analyzed by RT-PCR. Primers located in exons 6 and 11 were used to amplify exons 7-10 (649 bp) of NFKB1. In addition to the expected band, a shorter product was observed in the patient, suggesting that the mutation caused the deletion of an exon (exon 7, exon 8, or exon 9). (G) Sequencing of RT-PCR products from healthy control showed normal splicing (top). The patient's heterozygous mutation (c.730+5G>A) caused the in-frame skipping of exon 8 and the fusion of exon 7 and exon 9 (105delEx8, p.Asp191_Lys244delinsGlu) (bottom). (H) PBMCs from the patient and healthy controls were stimulated with phorbol 12-myristate 13-acetate (PMA) plus ionomycin. The amounts of p105 and p50 and the phosphorylation of p105 (Ser933, P-p105) were analyzed by Western blotting. GAPDH was used as the loading control. (I) HEK293T cells were transiently transfected with cytomegalovirus promoter-driven ectopic expression vector; the amount of ectopic protein was analyzed based on the results of Western blotting. As shown in the figure, on the left are expressions of fusion proteins wild-type and mutant p105 and p50, and on the right are non-fusion proteins. The 150-kDa band is GFP-p105. The 75-kDa band is GFP-p50. The 50-kDa faint band in all lanes is endogenous p50. The molecular weight marker is shown on the right. Only a weak fluorescent signal was detected for the mutant protein. (J) Grayscale analysis of the western blot results in (I). (K) The immunofluorescence results showed that GFP-p50 was localized in the nucleus and GFP-p105 was in the cytoplasm. Green, green fluorescent protein (GFP) fusion; blue, nuclear. The scale bar represents 10 mum. *P < 0.05, **P < 0.001, ***P < 0.0001.
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F1
The patient's left lower leg initially presented as purplish erythema and then gradually developed into large area of ecchymosis and bullae, and the small pustules on the surface partly fused into a large ulceration (Figure 1A); along with these dermatology processes were high fever and temporary unconsciousness. Based on the clinical and histological features, the patient was diagnosed with PG and was treated with high-dose intravenous immunoglobulin (IVIG) plus corticosteroid, which induced a great improvement in her lesions (Figure 1B).