Novel heterozygous C243Y A20/TNFAIP3 gene mutation is responsible for chronic inflammation in autosomal-dominant Behçet's disease

Abstract

Objective: Although Behçet's disease (BD) is a chronic inflammatory disorder of uncertain aetiology, the existence of familial BD with autosomal-dominant traits suggests that a responsibility gene (or genes) exists. We investigated a Japanese family with a history of BD to search for pathogenic mutations underlying the biological mechanisms of BD.

Methods: 6 patients over 4 generations who had suffered from frequent oral ulcers, genital ulcers and erythaema nodosum-like lesions in the skin were assessed. Whole-exome sequencing was performed on genomic DNA, and cytokine production was determined from stimulated mononuclear cells. Inflammatory cytokine secretion and Nod2-mediated NF-κB activation were analysed using the transfected cells.

Results: By whole-exome sequencing, we identified a common heterozygous missense mutation in A20/TNFAIP3, a gene known to regulate NF-κB signalling, for which all affected family members carried a heterozygous C243Y mutation in the ovarian tumour domain. Mononuclear cells obtained from the proband and his mother produced large amounts of interleukin 1β, IL-6 and tumour necrosis factor α (TNF-a) on stimulation as compared with those from normal controls. Although inflammatory cytokine secretion was suppressed by wild-type transfected cells, it was suppressed to a much lesser extent by mutated C243Y A20/TNFAIP3-transfected cells. In addition, impaired suppression of Nod2-mediated NF-κB activation by C243Y A20/TNFAIP3 was observed.

Conclusions: A C243Y mutation in A20/TNFAIP3 was likely responsible for increased production of human inflammatory cytokines by reduced suppression of NF-κB activation, and may have accounted for the autosomal-dominant Mendelian mode of BD transmission in this family.

Keywords: Behcet's disease; Cytokines; Fever Syndromes; Inflammation.

Figure 1

Family tree and A20/TNFAIP3 mutations. Sequence analysis of the A20/TNFAIP3 gene among the proband and family members revealed a heterozygous mutation of C243Y in exon 5, which was absent in the healthy younger brother. Sequence analysis was not possible on patient 6. Sequence analysis results are shown using reverse primer.

Figure 2

Cytokine synthesis from mononuclear cells. (A) Mononuclear cells obtained from patient 1 (●) and healthy control (○) were cultured with the indicated concentrations of LPS for 24 h, after which supernatant cytokine concentrations were measured using CBA kits. The results of triplicate experiments are shown as mean±SD. One representative result of three independent experiments is shown. (B) Production of IL-1β, IL-6, TNF-α and IL-8 by mononuclear cells obtained from patient 2 (▪) and the healthy younger brother of the proband (□). Mononuclear cells were cultured with medium alone, LPS (100 pg/mL), poly (I:C) (25 μg/mL), MDP (10 ng/mL), or CpG DNA (1 μg/mL) for 24 h. Cytokine concentrations in culture supernatants were measured using CBA kits. The results of triplicate experiments were expressed as mean±SD. The data shown are representative of two independent experiments with different healthy controls, and one representative result is shown. CBA, cytometric bead array; IL-1β, interleukin 1β; LPS, lipopolysaccharide; MDP, muramyl dipeptide; TNF-α, tumour necrosis factor α.

Figure 3

Inflammatory cytokine secretion from THP-1 cells, luciferase reporter assay of Nod2-mediated NF-κB activation and NLRP3 expression. (A) THP-1 cells were transfected with vector control (□), pcDNA3.1-C243Y A20 (), or pcDNA3.1-WT A20 (▪) expression plasmids together with pGL4.74 [hRluc/TK]. Eight hours after LPS stimulation, the concentrations of IL-1β, IL-6, IL-8 and TNF-α in the culture supernatants were measured using ELISA. One representative result of two independent experiments is shown. (B) HEK293T cells were cotransfected with vector control (□), pcDNA3.1-C243Y A20 (▪), or pcDNA3.1-WT A20 (▪) expression plasmids together with pcDNA3-Nod2-Flag, pcDNA3-RICK-Myc, NF-κB-dependent pBxVI-luc reporter and pGL4.74[hRluc/TK]. NF-κB luciferase reporter activity was measured 24 h post-transfection. One representative result of two independent experiments is shown. Transfection efficiency of THP-1 cells (A) or HEK293T (B) were normalised using Renilla luciferase activity generated by cotransfection of pGL4.74[hRluc/TK]. (C) Total RNA was extracted from patient 1, normal individuals and THP-1 cells transfected by C243Y or WT A20 with or without LPS stimulation, after 8 h incubation. The mRNA expression of NLRP3 was determined using RT-PCR analysis with β2-MG as a control. The same results were obtained with 14 h incubation. The hold induction based on β2-MG (NLRP3/β2-MG) is shown on each upper band. β2-MG, β2-microglobulin; HEK293T, human embryonic kidney 293T; IL-1β, interleukin 1β; LPS, lipopolysaccharide; RIP2, receptor-interacting protein 2; TNF-α, tumour necrosis factor α; WT, wild-type.

Figure 4

(A) Domain structure of A20. The N-terminal ovarian tumour (OTU) domain is essential for deubiquitinase activity and C-terminal zinc finger (ZF1-7) domains mediate E3 ubiquitin-ligase activity. The location of the present mutation is represented with a bold up arrow and recently published mutations are indicated with down arrows.