Clinical manifestations of the patients
Patient 1 (Fig 1A, P1) was a five-year-eight-month-old boy. At five years old, he had hospitalized for recurrent fever, vomit, dizziness, and bronchitis with elevated white blood cell (WBC) (8.92-28.34×10^9/L) and serum C-reactive protein (CRP) (normal range- 52mg/L). He was treated with antibiotics and antiemetics and the symptoms were gradually relieved. Since then, this boy has experienced repeated fevers, with each lasting about 3-5 days, mainly moderate and high fevers and with greater frequency than before. No significant pathogens were detected during the fever episodes, and it often relieved without antibiotics. The patient was admitted to our hospital six months later due to epigastralgia, oral ulcer, and fever. Physical examination of the boy revealed painful oral ulcers(Fig 1B, P1). Neither genital ulcer nor uveitis was confirmed, and gastrointestinal endoscopy showed normal mucosa. His mother (Fig 1A, P1M) had exhibited a long history of oral ulcers and hypothyroidism since she was 19-year-old . At that time, his elder sister and father had no clinical manifestation, but during the follow-up of this patient, his elder sister started to develop recurrent oral ulcers when she was 14 years and 6 months old (Fig 1A, P1S).
Patient 2 (Fig 1A, P2) was a seven-year-one-month-old boy. He had experienced recurrent and self-limited events of high fever (38.0℃–40.0℃) lasting for 1–3 days at a frequency of 1–2 months since six months of age. The child was prone to recurrent oral ulcers and abdominal pain, with no involvement of eye or skin, and showed no apparent evidence of inflammatory bowel disease. Gastrointestinal endoscopy also showed normal mucosa. Regarding inflammatory parameters, WBC, CRP, ESR, and plasma amyloid A were in the normal ranges. His mother (Fig 1A, P2M) had exhibited a long history of oral ulcers while his elder sister and father were healthy.
Laboratory investigations of these two patients showed no abnormalities in electrolytes, rheumatoid factor, antinuclear antibody, immunoglobulin level, complement components (C3, C4), or liver and renal functions. No abnormalities in the heart or lungs were found. After adding thalidomide, the symptoms of oral ulcer and fever were relieved in both patients. They are regularly followed up in the outpatient department of our hospital. At present, their condition is stable. Due to the quarantine policy of COVID-19, we were unable to obtain blood samples of P1S and P2M to further conduct the functional analysis.
Genetic Analysis revealed TNFAIP3 mutations in the patients
Based on the clinical symptoms and laboratory results, the primary autoinflammatory disease was considered for these two patients, and whole-exome sequencing was performed. WES analysis identified two missense heterozygous variants in these patients, including c.208 G>A, p.Asp70Asn (M1) in P1, P1S and P1M; c.770 T>C, p.Phe257Ser (M2) in P2. Both variants were predicted to be VUS according to the ACMG Standards and Guideline[20]. In Genome Aggregation Database (gnomAD), the mutation frequency of M1 was 0.000004. M2 was not found in other databases, including 1000 Genome, ExAC, HGMD, and gnomAD. These two variants were confirmed by Sanger sequencing (Fig 1C). TNFAIP3 p.Asp70 and p.Phe257 are located in the OTU domain and both are highly conserved across many species (Fig 1D, 1E). Pathogenicity prediction packages, including Polyphen2, Sorting Intolerant From Tolerant (SIFT), Mutation Taster, and PROVEAN, predicted that these two variants could be harmful.
Structure Analysis of the mutants TNFAIP3.
TNFAIP3 consists of an OTU domain and a ZnF domain (Fig.1D). Structural analysis showed that replacing acidic Asp70 with hydrophilic aspartic acid could promote the connection with Arg71, represented by the yellow dotted line (Fig.1F).Thus,this change suggested that this variant may cause a functional change to the protein. However, it also showed that replacing hydrophobic Phe257 with hydrophilic serine did not alter the protein structure.
Expression of A20 in the Patients
According to the current reports, frameshift and truncation mutations of TNFAIP3 gene reduced the expression of A20 protein, so we performed western blot analysis to determine whether the variants in our patients showed similar alteation. Compared with HCs, quantitative analysis of western blot showed that these two missense mutations in TNFAIP3 leaded to an insufficient expression of A20 in the patients' PBMCs as expected (Fig 2A).
Activation of NF-κB Pathway in the Patients-Derived Cells
A20 is well-known to inhibit NF-κB signaling[1], so we next examined whether the NF-κB pathway was activated in our patients with altered A20. Upon TNF-α stimulation, IkBα degradation and phosphorylation-p65 level were further evaluated in HA20 patients derived PBMCs(Fig 3A, 3B). Results showed that the IkBα degradation and phosphorylation-p65 level were increased in P1, P1M and P2, when compared with HCs.
The p.Asp70asn and p.phe257ser variants strongly increased NLRP3 inflammasome response and cytokine IL-1 β And IL-18 secretion
NLRP3 inflammasome triggers caspase-1-mediated proteolytic maturation and the secretion of proinflammatory cytokine IL-1β and IL-18[21]. NF- κB pathway and A20 play a regulatory role in activating NLRP3 inflammasome[22,23]. So we stimulated PBMCs of patients and HCs with LPS for 6 hours, then collected the supernatant and further examined the inflammatory factors. And compared with HCs, the secretion of IL-1β, IL-18, and TNF-α in patients was prominently elevated (Fig 4 ).