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Loss-of-function mutations in TNFAIP3 leading to A20 haploinsufficiency cause an early-onset autoinflammatory disease

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Systemic autoinflammatory diseases are driven by abnormal activation of innate immunity1. Herein we describe a new disease caused by high-penetrance heterozygous germline mutations in TNFAIP3, which encodes the NF-κB regulatory protein A20, in six unrelated families with early-onset systemic inflammation. The disorder resembles Behçet's disease, which is typically considered a polygenic disorder with onset in early adulthood2. A20 is a potent inhibitor of the NF-κB signaling pathway3. Mutant, truncated A20 proteins are likely to act through haploinsufficiency because they do not exert a dominant-negative effect in overexpression experiments. Patient-derived cells show increased degradation of IκBα and nuclear translocation of the NF-κB p65 subunit together with increased expression of NF-κB–mediated proinflammatory cytokines. A20 restricts NF-κB signals via its deubiquitinase activity. In cells expressing mutant A20 protein, there is defective removal of Lys63-linked ubiquitin from TRAF6, NEMO and RIP1 after stimulation with tumor necrosis factor (TNF). NF-κB–dependent proinflammatory cytokines are potential therapeutic targets for the patients with this disease.

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Figure 1: TNFAIP3 mutations cause a dominantly inherited systemic inflammatory disease.
Figure 2: Enhanced NF-κB signaling in transiently transfected 293T cells and patient-derived cells.
Figure 3: Impaired TNFR signaling and deubiquitinase function of mutant A20.
Figure 4: Patient-derived immune cells have a strong inflammatory signature.
Figure 5: Spontaneous NLRP3 inflammasome activity in PBMCs from patients with TNFAIP3 mutations.

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NCBI Reference Sequence

Change history

  • 16 December 2015

    In the version of this article initially published online, the name of author Jonathan J. Lyons was misspelled, and the p.Thr604Argfs*93 variant identified in family 4 was listed incorrectly in Figure 1a. These errors have been corrected for the print, PDF and HTML versions of this article


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We thank V. Dixit for helpful discussions. We also thank all the patients and their families, and the healthy children and adult controls, for their enthusiastic support during this research study. This research was supported by the Intramural Research Programs of the National Human Genome Research Institute, the National Institute of Arthritis and Musculoskeletal and Skin Diseases, the National Heart, Lung, and Blood Institute, the National Institute of Allergy and Infectious Diseases, and the US National Institutes of Health Clinical Center. S.O. received royalties for consulting and speaking from Novartis and SOBI. H.L.L. received royalties for consulting from Baxter. E.D. was recipient of the Research Fellowship Program for International Researchers, which is supported by the Scientific and Technological Research Council of Turkey (TUBITAK; B.14.2.TBT.0.06.01-219-84).

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Authors and Affiliations



Q.Z., H.W., D.L.K. and I.A. designed the study. Q.Z., H.W., M.S., Y.H.P., Y.Z., D.Y., M.T., W.L.T., J.J.L., X.Y., C.O., C.C., D.T.C., S.C.C., E.P.H. and Z.Y. performed experiments. Q.Z., H.W., K.Z., R.M.S., M.B., J.D.M., M.G. and J.C. analyzed and interpreted the data. D.M.S., E.D., A.K.O., D.L.S., P.H., S.A.H., A.J., B.K.B., H.L.L., A.v.R.-K., C.S., E.D.B., A.G., S.O., R.M.L. and D.L.K. enrolled the patients and collected and interpreted clinical information. K.Z., J.C.M. and I.E.W. provided technical support and comments. Q.Z., H.W., R.M.S., D.L.K. and I.A. wrote the manuscript. D.L.K. and I.A. directed and supervised the research. All authors contributed to the review and approval of the manuscript.

Corresponding author

Correspondence to Ivona Aksentijevich.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 TNFAIP3-associated haplotypes in family 1.

The p.Leu227* mutation (shown as a star) arose de novo in the first affected member of the family (P1). Red haplotypes carry the TNFAIP3 p.Leu227* mutation. Haplotypes of unavailable family members were inferred from reconstructed haplotypes and are shown in a purple font.

Supplementary Figure 2 Identification of TNFAIP3 mutations using exome sequencing, Sanger sequencing and targeted sequencing.

(a) Whole-exome sequencing identified a common gene mutated in two families. Schematic representation of the exome data-filtering approach used to select for novel and dominantly inherited variants segregating with disease in family 1 and family 2. TNFAIP3 is the only gene in common for these two families. SNV, single-nucleotide variants, including missense variants, splice-site variants and stop codon variants; INDEL, frameshift and non-frameshift insertions and deletions. (b) Electropherograms for the five TNFAIP3 mutations identified in five families. M1, M2, M3, M4 and M5 indicate TNFAIP3 mutant alleles. (c) A p.Pro268Leufs*19 mutation was identified by targeted gene sequencing. Sanger sequencing validated the p.Pro268Leufs*19 mutation in the proband and confirmed its presence in the other two affected family members. Age of onset of the proband was 29 years; her older daughter was 15 years old, and her younger daughter was 13 years old. The early symptoms included oral and genital ulcers, mild fever and skin rash.

Supplementary Figure 3 NF-κB reporter assay in a human T cell lymphoblast-like cell line (Jurkat cells).

NF-κB activity was assayed in cells transiently transfected with a mock control or with wild-type (WT) or mutant TNFAIP3 plasmid. NF-κB activity was determined on the basis of gated GFP+ cells (bottom). Mutant plasmids were less efficient in suppressing NF-κB activity than the wild-type plasmid. The mean fluorescence intensity (MFI) of Thy1 is the indicator of NF-κB activity.

Supplementary Figure 4 TNFAIP3 haploinsufficiency causes upregulation of the NF-κB signaling pathway.

(a) Patient 2 (P2) showed increased IκBα degradation and increased phosphorylation of p38 and JNK following stimulation with TNF. (b) Increased ratio of phosphorylated IκBα to total IκBα in PBMCs and fibroblasts in patients (P2 and P6) by ImageJ analysis.

Supplementary Figure 5 TNFAIP3 haploinsufficiency causes upregulation of the NF-κB signaling pathway.

(a) Patient 6 (P6) showed evidence for increased nuclear translocation of the p65 NF-κB subunit in TNF-stimulated PBMCs. (b) Immunofluorescence staining of NF-κB p65 in control (top) and patient-derived (bottom) fibroblasts under no stimulation (left) and with stimulation by 0.2 ng/ml TNF for 30 min (right). p65 translocation is indicated by red arrowheads. (c) The accompanying frequency plot for the different levels of activation under no stimulation (left) and stimulation by 0.2 ng/ml TNF for 30 min (right). Patient-derived fibroblasts are significantly more activated than control fibroblasts (Mann-Whitney test, P < 0.0001) under basal resting conditions and after TNF stimulation, as shown by the shifted distribution of nuclear p65 intensities.

Supplementary Figure 6 Impaired deubiquitinase function of mutant A20 in PBMCs and fibroblasts.

(a) In PBMCs, A20-deficient patients accumulated high-molecular-weight ubiquitin aggregates and K63-ubiquitinated RIP1. Top, ImageJ analysis of high-molecular-weight K63-linked ubiquitin aggregates for Figure 3e, top. Middle, increased K63-ubiquitinated RIP1 in patients. Bottom, RIP1 expression in lysate as control. (b) In fibroblasts, A20-deficient patients accumulated high-molecular-weight ubiquitin aggregates and K63-ubiquitinated RIP1. First panel, ImageJ analysis of high-molecular-weight K63-linked ubiquitin aggregates for Figure 3f, top. Second panel, increased K63-ubiquitinated RIP1 in patients. Third panel, increased high-molecular-weight ubiquitin aggregates of TNFR1 in patients. Fourth and fifth panels, RIP1 and TNFR1 expression in lysate as control, respectively.

Supplementary Figure 7 Gene expression of proinflammatory cytokines in differentiated M1 macrophages.

Human monocytes were isolated and differentiated into M1 macrophages using human GM-CSF (20 ng/ml) as described in the Online Methods. Cells were stimulated with or without LPS (100 ng/ml) for 6 h. Relative mRNA expression of IL-1β and TNF was analyzed in four patients and four healthy controls.

Supplementary Figure 8 Intracellular staining of TNF in T cells.

Increased staining for TNF in CD3+ T cells from patients 1, 4 and 6 is observed in SEB (staphylococcal enterotoxin B)-stimulated cells compared to three non-carrier controls.

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Supplementary Figures 1–8 and Supplementary Tables 1 and 2. (PDF 1350 kb)

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Zhou, Q., Wang, H., Schwartz, D. et al. Loss-of-function mutations in TNFAIP3 leading to A20 haploinsufficiency cause an early-onset autoinflammatory disease. Nat Genet 48, 67–73 (2016).

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