Nature Genetics: doi: /ng Supplementary Figure 1. TNFAIP3-associated haplotypes in family 1.

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1 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.

2 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.

3 Supplementary Figure 3 NF- B reporter assay in a human T cell lymphoblast-like cell line (Jurkat cells). NF- B activity was assayed in cells transiently transfected with a mock control or with wild-type (WT) or mutant TNFAIP3 plasmid. NF- B activity was determined on the basis of gated GFP + cells (bottom). Mutant plasmids were less efficient in suppressing NF- B activity than the wild-type plasmid. The mean fluorescence intensity (MFI) of Thy1 is the indicator of NF- B activity.

4 Supplementary Figure 4 TNFAIP3 haploinsufficiency causes upregulation of the NF- B signaling pathway. (a) Patient 2 (P2) showed increased I B degradation and increased phosphorylation of p38 and JNK following stimulation with TNF. (b) Increased ratio of phosphorylated I B to total I B in PBMCs and fibroblasts in patients (P2 and P6) by ImageJ analysis.

5 Supplementary Figure 5 TNFAIP3 haploinsufficiency causes upregulation of the NF- B signaling pathway. (a) Patient 6 (P6) showed evidence for increased nuclear translocation of the p65 NF- B subunit in TNF-stimulated PBMCs. (b) Immunofluorescence staining of NF- B 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 < ) under basal resting conditions and after TNF stimulation, as shown by the shifted distribution of nuclear p65 intensities.

6 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.

7 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-1 and TNF was analyzed in four patients and four healthy controls.

8 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.

9 Supplementary Table 1 Clinical manifestations of individuals with mutations in TNFAIP3 Family Family 1 Family 2 Family 3 Family 4 Family 5 Patient P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 Ancestry Canadian Canadian Canadian American American American Turkish Turkish American Dutch Dutch Gender Female Female Female Female Female Female Male Male Female Female Female Current age 51y 22y 20y 52y 57y 25y 45y 7y 13y 46y 14y Age of onset 11y 7m 2y 5y 6y 10y 13y 7y 2y 16y 5y Oral ulcers Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Genital ulcers Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Skin rash Erythematous papules Folliculitis Folliculitis Malar rash Yes Ophtho Uveitis Bilateral anterior uveitis Retinal vasculitis, anterior uveitis CNS Headache GI Evidence of inflammation on biopsy CNS vasculitis, chorea, migraine Diffuse ulcers in oropharynx and colon Colitis and typical ulcer in the terminal ileum N/V, anorexia, weight loss Mild undifferentiated colitis Pathergy NA Yes Yes Yes NA Autoantibodies ANA Lupus anticoagulant, anti dsdna Anticardiolipin, lupus anticoagulant RNP, ANA, Lupus anticoagulant NA ANA 1/100 (ANA weakly positive)

10 Arthritis Polyarthritis (non-deforming, large and small joint) ne Asymmetrical polyarthritis (nondeforming, large and small joint) Polyarthritis (nondeforming, large and small joint but predominantly small joint) Polyarthritis (nondeforming, large and small joint but predominantly small joint) Polyarthritis (non-deforming, large and small joint but predominantly small joint) Arthralgia HLA typing NA NA NA NA NA NA HLA-B51 HLA-B51 HLA-B15 NA HLA-B39/B44 Other phenotypes NA Tendonitis NA Periodic fever, hemolytic anemia, asthma Periodic fevers Idiopathic thrombocytopenic purpura (ITP) NA Pericarditis in infancy NA IgG2 and 4 subclass deficiency, low antipolysaccharide antibodies lymphopenia IgG2 subclass deficiency, low antipolysaccharide antibodies lymphopenia Treatment Responded to treatment with infliximab and experienced significant decrease in daily joint stiffness and pain treatment On infliximab therapy for over 10 years treatment treatment Responded to treatment with anakinra. In the past, anti-tnfs worked too, but lost efficacy Treatment has been successful with colchicine Treatment has been successful with colchicine and oral prednisone Responded to treatment with anti- TNF agents, but still has flares Treatment has been successful with infliximab, patient is free from oral ulcers, and genital ulcers are much less frequent and less severe Responded to treatment with infliximab. The patient occasionally has oral aphthous lesions, no perianal lesions

11 Supplementary Table 2 List of 11 candidate variants identified by whole exome sequencing in Family 1 and Sanger sequencing validation in the three patients and three unaffected family members Chr Position Nucleotide change Gene cdna alteration Protein change ExAC a P1 b P2 b P3 b H1 c H2 c H3 c chr8 42,608,329 T>C CHRNA6 c.1478a>g p.k493r 0 CT CT CT CT TT TT chr3 7,456,749 A>C GRM7 c.1073a>c p.n358t 0 AC AC AC AC AC AA chr5 137,056,158 G>A KLHL3 c.130c>t p.r44w 0 AG AG AG AG GG GG chr11 65,365,802 A>C MAP3K11 c.2504t>g p.m835r 0 AC AC AC AC AA AA chr16 88,801,170 A>T PIEZO1 c.1885t>a p.f629i 0 AT AT AT AT AT AA chr3 38,753,851 A>C SCN10A c.3890t>g p.i1297s 0 AC AC AC AA AC AA chr4 107,151,563 G>T TBCK c.1731c>a p.y577x 0 GT GT GT GT GT GG chr6 138,197,178 T>A TNFAIP3 c.680t>a p.l227x 0 AT AT AT TT TT TT chr1 7,998,365 A>C TNFRSF9 c.234t>g p.c78w 0 AC AC AC AA AA AA chr11 88,924,586 G>A TYR c.1036g>a p.g346r 0 AG AG AG AG AG GG chr5 71,756,680 C>T ZNF366 c.644g>a p.r215q 0 CT CT CT CC CT CC a ExAC. The Exome Aggregation Consortium includes 61,486 exomes ( b Exome sequencing identification and Sanger sequencing validation of variants for patients P1, P2, and P3. c Sanger sequencing generated genotypes in the three unaffected family members. H1, grandmother; H2, maternal uncle; H3, paternal aunt.