Pyoderma gangrenosum (PG) is a prototypic autoinflammatory neutrophilic dermatosis, which is often associated with systemic disorders such as inflammatory bowel disease (IBD), rheumatoid arthritis (RA), seronegative arthritis, autoimmune hepatitis, and hematologic disorders (Alavi et al., 2017). The pathogenesis of PG is multifactorial, including abnormalities in the function of inflammatory cytokines, the immune system, and the neutrophils combined with specific genetic mutations (Braswell et al., 2015). Specific mutations in PSTPIP1, MTHFR, and JAK2 have been reported to be associated with the pathogenesis of PG (Defilippis et al., 2015).

The NF-κB signaling pathway is critically important for regulating both innate and adaptive immune responses (Boztug et al., 2016; Kaustio et al., 2017). The NF-κB transcription factor family consists of five members, NF-κB1 (p105/p50), NF-κB2 (p100/p52), RelA (p65), RelB, and c-Rel. NFKB1 encodes a 969-amino-acid precursor named p105, which is subsequently processed to the active subunit p50 (amino acids 1–433 of p105) by phosphorylation and poly-ubiquitination at the C-terminal portion of the protein (Fliegauf et al., 2015). In canonical NF-κB pathway, the p105 and RelA usually exist as heterodimers in the cytoplasm, sharing a Rel homology domain (RHD) at the N-terminal portions, to ensure their dimerization, DNA binding, and nuclear localization. Diseases related to the abnormal expression of NF-κB1 include autoimmunity, lymphoproliferation, non-infectious bowel disease, opportunistic infections, auto inflammation, and malignant tumors (Lorenzini et al., 2020). And genomic heterozygous loss-of-function mutations cause common variable immune deficiency (CVID) (Tuijnenburg et al., 2018). In this report, we described a PG patient with heterozygous mutation in intron 8 leading to the deletion of exon 8 in NFKB1 mRNA and a 53-amino-acid deletion in the RHD, which affects the stability of p105 and the generation of p50. Our report describes a novel mutation in NFKB1 that has not been previously described as a pathogenic variation.

The patient is a 66-year-old female with pain in both knee joints for more than 20 years. She was admitted to hospital with suppurative osteoarthritis and presented with a history of diabetes and hypertension. She developed lesions 5 days after the surgical of left knee replacement. 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. Antibiotic treatment was ineffective, and cultures for bacteria and fungi from pustules were negative. Routine blood tests showed that neutrophils, white blood cells, and high-sensitivity C-reactive protein (CRP) were significantly increased (Table 1). Histopathologic examination of skin biopsy revealed that a large number of neutrophils infiltrated in the dermis with granulomatous changes in the subcutaneous tissue. 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). After treatment, the patient was in remission, and blood neutrophils and CRP gradually decreased.

To identify the genetic cause of the disease, WES was performed for the patient. WES data were analyzed by GATK best practice as described before. A total of 105,754 high-quality variants were called. Variants were filtered step by step (Figure 1C). For dominant/de novo inheritance, variants were further filtered novel in gnomAD, Kaviar, dbSNP, and in-house database. Candidate variants were then manually interpreted and combined with clinical manifestations. A possible splicing site mutation in NFKB1 (c.730+5G>A) was considered as pathogenic mutation (Figure 1D) (dbscSNV_ADA_SCORE = 0.999, dbscSNV_RF_SCORE = 0.998) (Jian et al., 2014). Then we performed RNA sequencing in patient's PBMCs. The result showed that gene expression in the NF-κB signaling pathway was upregulated compared with unaffected healthy controls (Figure 1E), suggesting increased inflammation in the patient.

To verify whether the patient's mutation in intron 8 would affect the mRNA splicing of NFKB1 mRNA, we extracted RNA from PBMCs of the patient and healthy controls and amplified cDNA fragments spanning exons 7–10 (649 bp) by RT-PCR. The agarose electrophoresis results showed that an additional shorter band appeared below the normal band in the patient compared with that in the healthy control (Figure 1F). Sanger sequencing of the RT-PCR products confirmed that the patient had exon 7 and exon 9 spliced, resulting in the in-frame skipping of exon 8 (159 bp) (Figure 1G).

The deletion of exon 8 would result in the absence of 53 amino acids from the N-terminal RHD (p.Asp191_Lys244delinsGlu, p105delEx8). The stability of RHD is critical for generation of p50–p105 heterodimers and is required for effective p50 production (Lin et al., 2000; Lin and Kobayashi, 2003). Therefore, this deletion would affect the normal function of NF-κB1 proteins. To investigate whether the deletion of the specific fragment at the mRNA level would lead to the formation of truncated NF-κB1 proteins (with a molecular weight reduced by ~5.8 kDa) in the patient (Fliegauf et al., 2015), we prepared protein extracts from PBMCs of the patient and healthy controls to perform Western blotting analysis. The results showed that, under the PMA plus ionomycin stimulation, the expression levels of p105 and p50 proteins were lower in the patient than in the control groups, and truncated p105delEx8 bands appeared in the patient's lanes (Figure 1H). The phosphorylation of p105 at Ser933 was also reduced in the patient's mutant allele (Figure 1H). Accordingly, only wild-type p105 was further processed into p50, and no p50delEx8 (~44 kDa) processed from truncated p105delEx8 was detected (Figure 1H). These observations suggest that the splice-donor-site mutation in NFKB1 (c.730+5G>A) leads to the degradation of truncated p105delEx8, which further influences the formation of p50delEx8.

To verify the effects of mutation on p105/p50 stability, we transiently transfected the p50, p50delEx8, p105, p105delEx8, and N-terminal GFP-fusion constructs (GFP-p50, GFP-p50delEx8, GFP-p105, and GFP-p105delEx8) into HEK293T cells, using a cytomegalovirus promoter-driven ectopic expression vector. Western blotting results showed that the protein level of truncated p105delEx8 was significantly lower than the non-mutant protein p105, regardless of whether it was fused with GFP or not (Figures 1I,J). In addition, the amounts of p50delEx8 and GFP-p50delEx8 processed from p105delEx8 variants were also decreased compared with those of p50 and GFP-p50 processed from p105 (Figure 1I). Consistently, the immunofluorescence results confirmed this result; we observed strong fluorescent signals in the GFP-p50 and GFP-p105 transfected cells in the nucleus and cytoplasm, respectively (Figure 1K), while only weak fluorescence signals were detected in GFP-p50delEx8 and GFP-p105delEx8 transfected cells (Figure 1K).

Our functional study identified a heterozygous NFKB1 (c.730+5G>A) mutation causing the in-frame skipping of exon 8, which led to P50 haploinsufficiency. Many previous studies have shown that NF-κB1 haploinsufficiency can lead to CVID (Bryant and Tangye, 2016; Schipp et al., 2016; Dieli-Crimi et al., 2018; Duan and Feanny, 2019; Schroder et al., 2019); typical symptoms are repeated infections and IgA/IgM deficiency caused by hypogammaglobulinemia. Thompson et al. reported that heterozygous mutations in NFKB1 were associated with PG and CVID (Thompson et al., 2018). In contrast to previous reports, this patient did not present hypogammaglobulinemia, and the levels of IgA and IgM were normal. The patient showed recurrent fevers, PG, and increased neutrophil and white blood cell counts. In the studies of liver and lung inflammatory diseases, the loss or abnormal expression of NF-κB1 often leads to the accumulation of neutrophils, which leads to inflammation (Fiona Oakley et al., 2005; Wilson et al., 2015; Mcminn et al., 2019), so we speculate that the number of neutrophils may be part of the reason for the high inflammation of PG due to neutrophil hyperactivation. However, mechanisms explaining the PG and autoinflammation caused by NFKB1 heterozygous mutation still need to be further studied.

The datasets for this article are not publicly available due to concerns regarding participant/patient anonymity. Requests to access the datasets should be directed to the corresponding author.

The studies involving human participants were reviewed and approved by The Ethic Committee of Sir Run Run Shaw Hospital of Zhejiang University School of Medicine, China. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

RF and JW contributed equally. QZ and HC designed the study, directed and supervised the research, and critically revised the manuscript. RF performed the experiments and wrote the manuscript. JW performed genetic bioinformatics analyses and corrected the manuscript. X-yJ enrolled the patient and collected and interpreted the clinical information. S-hW performed the experiments and assisted in manuscript editing. All authors contributed to the approval of the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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