Biallelic hypomorphic mutations in a linear deubiquitinase define otulipenia, an early-onset autoinflammatory disease
Contributed by Daniel L. Kastner, August 2, 2016 (sent for review June 21, 2016; reviewed by Sudhir Gupta and Averil Ma)
Significance
We describe a human disease linked to mutations in the linear deubiquitinase (DUB) OTULIN, which functions as a Met1-specific DUB to remove linear polyubiquitin chains that are assembled by the linear ubiquitin assembly complex (LUBAC). OTULIN has a role in regulating Wnt and innate immune signaling complexes. Hydrolysis of Met1-linked ubiquitin chains attenuates inflammatory signals in the NF-κB and ASC-mediated pathways. OTULIN-deficient patients have excessive linear ubiquitination of target proteins, such as NEMO, RIPK1, TNFR1, and ASC, leading to severe inflammation. Cytokine inhibitors have been efficient in suppressing constitutive inflammation in these patients. This study, together with the identification of haploinsufficiency of A20 (HA20), suggests a category of human inflammatory diseases, diseases of dysregulated ubiquitination.
Abstract
Systemic autoinflammatory diseases are caused by mutations in genes that function in innate immunity. Here, we report an autoinflammatory disease caused by loss-of-function mutations in OTULIN (FAM105B), encoding a deubiquitinase with linear linkage specificity. We identified two missense and one frameshift mutations in one Pakistani and two Turkish families with four affected patients. Patients presented with neonatal-onset fever, neutrophilic dermatitis/panniculitis, and failure to thrive, but without obvious primary immunodeficiency. HEK293 cells transfected with mutated OTULIN had decreased enzyme activity relative to cells transfected with WT OTULIN, and showed a substantial defect in the linear deubiquitination of target molecules. Stimulated patients’ fibroblasts and peripheral blood mononuclear cells showed evidence for increased signaling in the canonical NF-κB pathway and accumulated linear ubiquitin aggregates. Levels of proinflammatory cytokines were significantly increased in the supernatants of stimulated primary cells and serum samples. This discovery adds to the emerging spectrum of human diseases caused by defects in the ubiquitin pathway and suggests a role for targeted cytokine therapies.
Sign up for PNAS alerts.
Get alerts for new articles, or get an alert when an article is cited.
Posttranslational modifications by ubiquitination are important for the regulation of many signaling complexes (1). Linear ubiquitin chains, also known as Met1-linked chains, are generated by the linear ubiquitin assembly complex (LUBAC) (2). LUBAC-mediated Met1 ubiquitination is critical for regulation of immune signaling and cell death (3). Absence of LUBAC attenuates NF-κB signaling and patients with loss-of-function mutations in LUBAC present with paradoxical features of susceptibility to infection and systemic inflammation, the latter due to increased responsiveness to IL-1β in monocytes (3–5). OTULIN and CYLD are deubiquitinases (DUBs) that cleave Met1-linked chains (6). Although OTULIN functions exclusively as a Met1 deubiquitinase (7, 8), CYLD may also hydrolyze Lys63-linked ubiquitin (9). OTULIN is an evolutionarily highly conserved protein, and in mice complete deficiency is embryonically lethal (8). Recently, we reported patients with heterozygous germline mutations in TNFAIP3/A20 (10), which has DUB activity for K63-linked polyubiquitin chains. Both OTULIN and A20 are important gatekeepers of innate immunity (7, 11).
Results
Identification of Loss-of-Function Mutations in OTULIN in Three Patients.
Using a combination of exome sequencing and candidate gene screening, we identified three homozygous mutations in the OTULIN/FAM105B gene in unrelated families of Pakistani and Turkish descent (Fig. 1, Fig. S1, Table 1, and Tables S1 and S2). Unaffected parents and siblings were carriers for the respective mutations. None of the mutations was reported in public databases or detected in 1,630 Turkish healthy controls. Two missense mutations, p.Leu272Pro and p.Tyr244Cys, are predicted to be deleterious by multiple algorithms (Table S3) and affect highly conserved amino acid residues (Fig. S2A). Similar to A20 disease-causing mutations (10), all three OTULIN mutations are located in the ovarian tumor (OTU) domain (Fig. S2B). The p.Gly174Aspfs*2 mutation introduces a premature stop codon, and mutant truncated protein was detectable by overexpression in HEK293 cells (Fig. 2A) but not in the patient’s fibroblasts (Fig. 2B, patient 3). The missense mutations likely affect the S1 site of the linear ubiquitin-binding domain (Fig. S2C). They reduce OTULIN protein stability (Fig. 2B) and may result in the instability of the LUBAC complex subunits, SHARPIN and HOIP (Fig. 2B).
Fig. 1.

Table 1.
Family | Ancestry | Nucleotide alteration† | cDNA alteration‡ | Amino acid alteration | Domain | ExAC | Turkish population | Software prediction§ | Conservation¶ |
---|---|---|---|---|---|---|---|---|---|
1 | Pakistani | chr5: 14690368T>C | c.815T>C | p.Leu272Pro | OTU | 0/122,972 | 0/3,260 | Damaging | Conserved |
2 | Turkish | chr5: 14690284A>G | c.731A>G | p.Tyr244Cys | OTU | 0/122,972 | 0/3,260 | Damaging | Conserved |
3 | Turkish | chr5: 14687678delC | c.517delC | p.Gly174Aspfs*2 | OTU | 0/122,972 | 0/3,260 | / | / |
†
Genome reference: GRCh37 (hg19).
‡
cDNA reference: NM_138348.4.
§
SIFT, PolyPhen2, LRT, Mutation Taster, and CADD.
¶
GERP, SiPhy 29 way, and CLUSTALW.
Fig. 2.

Fig. S1.

Table S1.
Chr | Position | Reference | Variant | Gene | Consequence | Our 616 exomes | Clinseq | 1000 Genomes | ExAC | Polyphen | Patient 1 | Father | Mother | Healthy sibling |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
chr1 | 38397501 | C | T | INPP5B | c.439G>A:p.A147T | 0.0045 | 0.0016 | 0 | 3.22E-03 | Probably damaging | TT | CT | CT | CT |
chr1 | 54371889 | G | A | DIO1 | c.459G>A:p.M153I | 0.0041 | 0.0011 | 0 | 2.21E-03 | Probably damaging | AA | AG | AG | AG |
chr1 | 55166996 | G | A | MROH7 | c.3285+1G>A | 0.0043 | 0 | 0 | 4.33E-04 | / | AA | AG | AG | AG |
chr1 | 63329777 | G | T | ATG4C | c.1324G>T:p.D442Y | 0.0065 | 0.0032 | 0.0005 | 1.55E-03 | Possibly damaging | TT | GT | GT | GT |
chr3 | 39226171 | G | A | XIRP1 | c.4766C>T:p.T1589I | 0.0033 | 0.0011 | 0 | 5.55E-03 | Benign | AA | AG | AG | GG |
chr3 | 52537419 | A | G | STAB1 | c.754A>G:p.K252E | 0.0041 | 0.0005 | 0 | 2.26E-03 | Benign | GG | AG | AG | AG |
chr3 | 111842564 | C | T | GCSAM | c.281G>A:p.R94Q | 0.0033 | 0 | 0 | 1.63E-05 | Benign | TT | CT | CT | CC |
chr3 | 112648018 | A | G | CD200R1 | c.539T>C:p.L180S | 0.0033 | 0.0011 | 0 | 4.77E-03 | Benign | GG | AG | AG | AA |
chr3 | 119911827 | G | A | GPR156 | c.433C>T:p.L145F | 0.0033 | 0 | 0.0014 | 7.40E-04 | Probably damaging | AA | AG | AG | GG |
chr3 | 126255146 | C | T | CHST13 | c.130C>T:p.L44F | 0.0033 | 0 | 0 | 3.43E-03 | Benign | TT | CT | CT | CC |
chr3 | 127983506 | C | T | EEFSEC | c.668C>T:p.P223L | 0.0049 | 0.0021 | 0.0005 | 1.97E-03 | Probably damaging | TT | CT | CT | CC |
chr5 | 5463514 | C | T | KIAA0947 | c.4067C>T:p.T1356I | 0.0042 | 0 | 0 | 0 | Benign | TT | CT | CT | CT |
chr5 | 14690368 | T | C | FAM105B | c.815T>C:p.L272P | 0.0025 | 0 | 0 | 0 | Probably damaging | CC | CT | CT | CT |
chr6 | 131179287 | C | T | EPB41L2 | c.2548G>A:p.G850R | 0.0041 | 0.0016 | 0 | 2.02E-03 | Probably damaging | TT | CT | CT | CT |
chr12 | 10464155 | C | T | KLRD1 | c.256C>T:p.R86W | 0.0049 | 0 | 0 | 5.69E-05 | Probably damaging | TT | CT | CT | CT |
chr13 | 53603108 | T | A | OLFM4 | c.137T>A:p.F46Y | 0.0033 | 0 | 0 | 3.60E-03 | Benign | AA | AT | AT | TT |
chr15 | 34077949 | G | A | RYR3 | c.9355G>A:p.E3119K | 0.0058 | 0.001 | 0 | 2.47E-03 | Probably damaging | AA | AG | AG | GG |
chr15 | 43512935 | C | T | EPB42 | c.89G>A:p.S30N | 0.0041 | 0.0011 | 0.0027 | 1.35E-03 | Benign | TT | CT | CT | CT |
chr16 | 56709804 | C | A | MT1IP | c.134C>A:p.A45E | 0.0129 | 0 | 0.0046 | 2.25E-03 | NA | AA | AC | AC | AC |
chr16 | 57602000 | C | T | GPR114 | c.1054C>T:p.R352C | 0.0041 | 0 | 0.0005 | 9.03E-04 | Probably damaging | TT | CT | CT | CT |
chr19 | 1117467 | G | A | SBNO2 | c.1559C>T:p.A520V | 0.015 | 0 | 0 | 3.31E-05 | Probably damaging | AA | AG | AG | AG |
Boldface type indicates the only gene in common between family 1 and patient 2.
Table S2.
Chr | Position | Reference | Variant | Gene | Consequence | Our 616 exomes | Clinseq | 1000 Genomes | ExAC | Polyphen | Patient 2 |
---|---|---|---|---|---|---|---|---|---|---|---|
chr1 | 55068396 | A | G | ACOT11 | c.1084A>G:p.R362G | 0.0018 | 0 | 0 | 8.42E-06 | Probably damaging | GG |
chr3 | 169587465 | G | A | LRRC31 | c.131C>T:p.S44F | 0.0016 | 0.0027 | 0.0005 | 1.62E-03 | Benign | AA |
chr5 | 14690284 | A | G | FAM105B | c.731A>G:p.Y244C | 0.0016 | 0 | 0 | 0 | Probably damaging | GG |
chr5 | 24593481 | C | A | CDH10 | c.119G>T:p.R40L | 0.0016 | 0 | 0 | 8.13E-06 | Benign | AA |
chr5 | 39377259 | C | T | DAB2 | c.1630G>A:p.V544I | 0.0065 | 0.0053 | 0.0018 | 3.64E-03 | Benign | TT |
chr9 | 135202325 | A | C | SETX | c.4660T>G:p.C1554G | 0.0098 | 0.0011 | 0.0027 | 5.81E-03 | Probably damaging | CC |
chr9 | 136280025 | G | A | REXO4 | c.332C>T:p.S111L | 0.0082 | 0.0064 | 0.0023 | 5.68E-03 | Benign | AA |
chr19 | 40392655 | C | G | FCGBP | c.7849G>C:p.G2617R | 0.0023 | 0 | 0 | 7.59E-05 | Probably damaging | GG |
chr19 | 42583629 | C | T | ZNF574 | c.1141C>T:p.R381C | 0.0025 | 0.0016 | 0.0005 | 5.61E-04 | Probably damaging | TT |
chr19 | 43023175 | C | T | CEACAM1 | c.1171G>A:p.A391T | 0.0016 | 0 | 0 | 0 | Benign | TT |
chr19 | 45791007 | C | T | MARK4 | c.1177C>T:p.R393W | 0.0257 | 0 | 0.0027 | 6.54E-03 | Benign | TT |
chr19 | 49207114 | G | A | FUT2 | c.901G>A:p.G301R | 0.0024 | 0 | 0.0005 | 1.89E-03 | Probably damaging | AA |
Boldface type indicates the only gene in common between family 1 and patient 2.
Table S3.
Amino acid alteration | SIFT | PolyPhen2 | LRT | Mutation taster | CADD | GERP | SiPhy 29way | CUPSAT | I-Mutant (protein stability) | DiANNA | |
---|---|---|---|---|---|---|---|---|---|---|---|
Overall stability | Torsion | ||||||||||
p.Leu272Pro | Deleterious | Probably damaging | Deleterious | Disease causing | 27.5 | 6.07 | 15.81 | Stabilizing | Unfavorable | Decrease | / |
p.Tyr244Cys | Deleterious | Probably damaging | Deleterious | Disease causing | 19.96 | 4.76 | 11.635 | Destabilizing | Unfavorable | Decrease | Create disulfide bond |
Fig. S2.

Clinical Manifestations of OTULIN Deficiency.
Patient 1, from a large consanguineous family, was born prematurely and soon after birth presented with fever and rash (Fig. 1 and Table S4). Two of his first cousins died from a similar disease in early childhood. Only one DNA sample was available for genotyping and was found to have the same homozygous mutation as patient 1. Other findings included failure to thrive, joint swelling, lipodystrophy, and diarrhea. Treatment with an IL-1β inhibitor (anakinra) was not steroid sparing; however, within 1 mo of starting a TNF inhibitor (infliximab) at the age of 3 y, his fevers and rash subsided. Eight years after initiation of the treatment, he is normal size for his age and fully functional (Fig. S3). Patient 2 presented at the age of 4.5 mo with prolonged fevers and pustular, scarring rashes. Skin biopsy revealed panniculitis and neutrophilic dermatosis. Initially, she responded to treatment with steroids, and subsequently symptoms improved on treatment with anakinra (Table S4). Patient 3 presented with neonatal-onset fever and prominent cutaneous lesions including an erythematous rash with painful skin nodules (Fig. 1B). Her skin biopsy showed a predominantly septal panniculitis with vasculitis of small and medium-sized blood vessels. Other manifestations included arthralgia, progressive lipodystrophy, and developmental delay. Her disease is partially controlled with a TNF inhibitor (etanercept), but she is still steroid dependent (Table S4). Patients did not have clear evidence for primary immunodeficiency and suffered from infections related to use of immunosuppressive therapies. Patients 1 and 3 had normal to high levels of T, B, and natural killer cells (Table S5), Ig levels were normal to high, and IgA was elevated in the two patients. T- and B-cell proliferative responses were normal (Fig. S4A). Patients had adequate specific antibody responses to vaccines or natural infections when tested.
Table S4.
Clinical features | Patient 1 | Patient 2 | Patient 3 |
---|---|---|---|
Ancestry | Pakistani | Turkish | Turkish |
Consanguinity | Yes | Yes | Yes |
Gender | Male | Female | Female |
Current age | 11 y | 4 y | 11 y |
Age of onset | 1 mo | 4.5 mo | 1 mo |
Mutation | Homozygous p.Leu272Pro | Homozygous p.Tyr244Cys | Homozygous p.Gly174Aspfs*2 |
Fevers | Yes | Yes (fever lasting for 20 d) | Yes (fever lasting 2–3 wk) |
Rash/nodular panniculitis | Erythematous with skin nodules first noted in the neonatal period | Pustular rash, multiple scars | Erythematous with painful skin nodules first noted in the neonatal period. Occasional pustular rash. |
Lipodystrophy | Yes | Yes | Yes |
Skin biopsy | Neutrophil-rich panniculitis with fat necrosis but no frank vasculitis | Panniculitis and neutrophilic dermatosis | Septal panniculitis, vasculitis of small and medium-sized arteries and capillaries |
Arthralgias/myalgias | Yes/yes | Yes/no | Yes/yes |
Lymphadenopathy | Yes | No, parotitis sialadenitis (accompanying 3-d attack) | Yes |
Abdominal pain/diarrhea | Yes/yes | No/no | No/no |
Therapies | Prednisone, partial response to a dose of 2 mg/kg of anakinra. Infliximab started at the age of 3 y. Methotrexate, 2.5 mg weekly. | Prednisone, anakinra treatment started at the age of 1 y, responsive to a dose of 6 mg/kg. Never been treated with anti-TNF therapy | Prednisone, she developed macrophage activation syndrome (MAS) on 1 dose of canakinumab. Anakinra was not effective at a dose of 1–3 mg/kg. Etanercept started at age of 3 y. Partial response, still requires prednisone for flares. |
History of infections | RSV pneumonia, two episodes of varicella and shingles, both well tolerated† | No | Frequent lymphadenitis typically during flares, varicella† |
Additional medical history | Born at 28 wk of gestation, failure to thrive | Born at 38 wk of gestation, failure to thrive | Born at 38 wk of gestation, failure to thrive, convulsions, hepatosplenomegaly, sterile encephalitis‡ |
Family history | Two affected cousins with similar disease | No family history | No family history |
Lab results at the time of NIH visit | ESR (8 mm/h), CRP (1 mg/L) | NA | ESR (60 mm/h), CRP (63 mg/L) |
WBC, 10K | WBC, 14K | ||
Lab results before coming to NIH | Before infliximab: CRP (147 mg/L), WBC, 31.6K | Before anakinra: ESR (59 mm/h); CRP, 91 mg/L; WBC, 27,4K | On etanercept: ESR (94 mm/h); CRP (>200 mg/L); WBC, 26,6K |
2 mo after infliximab: CRP, <1 mg/L | After anakinra: ESR (52 mm/h); CRP, 1.5 mg/L; WBC, 18,4K | ||
Autoantibodies | No | No | No |
CRP, C-reactive protein; ESR, erythrocytes sedimentation rate; NA, not available; WBC, white blood cell.
†
Intercurrent infections during immunosuppressive therapy.
‡
Possibly related to immunosuppressive therapy.
Fig. S3.

Table S5.
Cell types | Patient 1 | Patient 3 | Normal range | ||
---|---|---|---|---|---|
CD3, no. | 3,789 | H | 6,072 | H | 1,900–3,700 |
CD4/CD3, % | 52.6 | 39.5 | 60–76% | ||
CD4/CD3, no. | 2,404 | H | 2,876 | H | 650–1,500/μL |
CD8/CD3, % | 22.3 | 31.7 | 11.2–34/8% | ||
CD8/CD3, no. | 1,019 | H | 2,308 | H | 370–1,100/μL |
CD19, % | 10 | L | 12.8 | L | 13–27% |
CD19, no. | 457 | 932 | H | 270–860/μL | |
NK cells, % | 7 | 3.7 | L | 4–17% | |
NK cells, no. | 320 | 269 | 100–480/μL |
Lymphocyte phenotyping in patients 1 and 3. Patient 2 was not available for immunophenotyping. NK, natural killer.
Fig. S4.

Mutations Do Not Disrupt OTULIN Interaction with LUBAC.
OTULIN is a 352-residue protein that consists of an N-terminal LUBAC-binding domain and a C-terminal ovarian tumor (OTU) domain (Fig. S2B). OTULIN interacts with the PUB domain of HOIP, and their interaction is required for the recruitment of OTULIN to the TNF receptor complex (12). The three mutations do not disrupt the OTULIN interaction with LUBAC, and mutant proteins maintain the intact N-terminal domain necessary for LUBAC interaction (Fig. 2A).
Increased NF-κB Signaling in OTULIN-Deficient Cells.
OTULIN restricts NF-κB signaling activity (7). We performed NF-κB luciferase assays to study the function of mutant OTULIN proteins in human embryonic kidney (HEK) 293 cells. Overexpressed mutant OTULIN plasmid p.Leu272Pro and p.Gly174Aspfs*2 failed to restrain NF-κB activity compared with WT OTULIN (Fig. 2C and Fig. S5A). Mutant p.Tyr244Cys plasmid suppressed the LUBAC-induced NF-κB activity similar to WT OTULIN. These results indicate that p. Leu272Pro and p.Gly174Aspfs*2 mutations affect the OTULIN enzyme activity, whereas the p.Tyr244Cys mutation retains sufficient residual OTULIN activity in the overexpression experiment, or may affect the protein function in a different manner. We then studied the activity of the NF-κB pathway in stimulated patients’ fibroblasts and peripheral blood mononuclear cells (PBMCs) (Fig. 2 D and E, and Fig. S5B). Sequential phosphorylation of IKKs and IκBα are essential steps in activation of the canonical NF-κB pathway (13). Patient-derived mononuclear leukocytes and fibroblasts sustained higher levels of phosphorylated IKKα/IKKβ and IκBα, and showed increased phosphorylation of P38 and JNK MAP kinases compared with healthy controls. These results demonstrate enhanced signaling of the NF-κB and MAPK pathways in OTULIN-deficient patients. Our data also suggest that NF-κB activation was not affected in patient’s lymphocytes in the context of T-cell receptor and B-cell receptor stimulation (Fig. S4 B and C).
Fig. S5.

Defect in the Deubiquitinase Function of Mutant OTULIN Proteins.
OTULIN cleaves Met1-linked linear polyubiquitin chains from target substrates, such as NEMO (IKKγ), RIPK1, ASC, and TNFR1 to restrict signaling activation and propagation (7, 14, 15). To investigate the effect of OTULIN mutations on its deubiquitinase function, we cotransfected WT and mutant OTULIN plasmids into HEK293 cells along with plasmids encoding the LUBAC subunits, mono specific-ubiquitin plasmid, and each of the OTULIN substrates NEMO (Fig. 3A), RIPK1 (Fig. 3B), ASC (Fig. 3C), and TNFR1 (Fig. 3D). Cells transfected with mutant p.Leu272Pro and p.Gly174Aspfs*2 proteins showed substantial defects in deubiquitination of the target substrate as indicated by accumulated high–molecular-weight linear-ubiquitin aggregates (Fig. 3 A–D). The defect in RIPK1 deubiquitination was more noticeable in cells transfected with WT monoubiquitin plasmid (Fig. 3B) than in cells transfected with the mutant monoubiquitin plasmid (Ub-KO), which can only form linear ubiquitin chains (Fig. S5C). RIPK1 and TNFR1 require the assembly of K63 and linear polyubiquitin chains for proper signaling activity, and they are subject to deubiquitination by A20 and OTULIN (14). The K63 ubiquitination of RIPK1 was not affected by the presence of mutant OTULIN proteins (Fig. 3B, second panel). Cells transfected with p.Tyr244Cys plasmid showed only mild, if any, defect compared with the other two mutant proteins (Fig. 3 A–D). The in vitro-observed defect in DUB activity of mutant proteins was rescued by cotransfection with WT OTULIN (Fig. 3 A–D).
Fig. 3.

Increased Linear Ubiquitination in Patients’ PBMCs and Fibroblasts.
Consistent with the data from overexpressed mutant proteins, TNF- or IL-1β–stimulated OTULIN-deficient primary patients’ cells showed accumulation of linear-ubiquitinated NEMO (Fig. 4 A and C), TNFR1 (Fig. 4B), RIPK1 (Fig. 4 B and C), and ASC (Fig. 4D), and accumulation of high-molecular linear Ub aggregates (Fig. 4 B and D) compared with healthy controls. In ex vivo experiments with cells from patient 2, who carries the p.Tyr244Cys mutation, we noted an increase in the linear ubiquitinated NEMO, TNFR1, RIPK1 (Fig. 4 A and B), and accumulation of high-molecular linear Ub aggregates (Fig. 4B). This ex vivo experiment supports the strong genetic data for pathogenicity of the p.Tyr244Cys mutation identified in patient 2. The combination of in vitro and ex vivo experiments provides compelling evidence that loss-of-function mutations in OTULIN result in increased linear ubiquitination of signaling molecules and lead to enhanced TNFR1, NF-κB, and ASC-dependent inflammation.
Fig. 4.

A Strong Inflammatory Signature in OTULIN-Deficient Patients’ Cells.
Stimulated patient whole-blood samples showed an increased production of IL-1β, IL-6, IL-12, IL-18, and IFN-γ in response to LPS, and increased levels of multiple cytokines and chemokines in response to staphylococcal enterotoxin B stimulation (Fig. 5A and Fig. S6A). Purified patients’ monocytes had significantly higher secretion of IL-1β, IL-6, IL-16, IL-18, and TNF in response to LPS, TNF, or IL-1β stimulation relative to cells from healthy controls (Fig. 5B). Intracellular staining for TNF and IL-6 from patient 1 and 3 was higher at basal levels in monocytes (Fig. 5C and Fig. S6B), T cells (Fig. S6C), and dendritic cells (Fig. S6D) than in controls. Fig. 5D represents the average of TNF responses assayed on the three patients individually. Cytokine profiling in serum samples was consistent with disease activity. Patient 2, who had the most active disease at the time of sampling, had the highest levels of proinflammatory cytokines. Patients 1 and 3 had less active disease phenotypes at the time of sampling and substantially lower cytokine levels (Table S4 and Fig. 5E). Transcriptome profiling of patient whole-blood samples and stimulated fibroblasts showed similar results. Patients 2 and 3 displayed strong inflammatory signatures enriched for NF-κB, Jak-STAT, and TNF signaling (Fig. S7 A and B). In contrast, patient 1, whose disease was both clinically and biochemically inactive at the time of visit, had a transcriptome profile similar to controls. These data provide evidence that a malfunction in linear deubiquitination leads to an up-regulation in cytokine production and that the disease is amenable to targeted anticytokine treatment.
Fig. 5.

Fig. S6.

Fig. S7.

Discussion
We describe a recessively inherited autoinflammatory disease caused by excessive linear ubiquitination in innate immune signaling pathways, which we denote as “otulipenia.” We show that OTULIN deficiency leads to increased linear ubiquitination of target proteins, which is associated with enhanced NF-κB activity, increased TNFR1 signaling, and NLRP3 inflammasome activity. The phenotype is very severe and potentially lethal if left untreated.
This is a report of a human disease caused by excessive linear ubiquitination. Conversely, patients with LUBAC deficiency have impaired linear ubiquitination of the same target molecules, which leads to immunodeficiency due to decreased NF-κB activity in fibroblasts, and a concomitant inflammatory phenotype due to hyperresponsiveness to IL-1β in monocytes (4). These latter studies demonstrate cell type-specific functions of the LUBAC subunits HOIP and HOIL-1. No human disease has yet been linked to SHARPIN deficiency. In contrast, OTULIN-deficient patients have a broader constitutive inflammatory phenotype in fibroblasts and monocytes and no overt primary immunodeficiency. Heterozygote carriers of these mutations are asymptomatic, which suggests that OTULIN expression levels do not appear to be critical for immune homeostasis. The importance of the linear ubiquitin pathway in the regulation of innate immune responses has been demonstrated in murine models. Mice deficient in LUBAC subunits have variable degrees of inflammation, from a mild phenotype in HOIL-1–deficient mice (16) to more severe inflammation and dermatitis in SHARPIN KO (2, 17) to defective vascularization and embryonic lethality in HOIP KO (18). Consistent with the essential function of OTULIN in regulation of multiple signaling pathways, OTULIN-deficient mice (gumby/gumby) are embryonic lethal due to vascular and neuronal defects caused by dysregulation in canonical Wnt signaling (8).
This is the second report of human germline mutations in a deubiquitinase protein leading to an inflammatory phenotype, the first being mutations in DUB A20 (10). In contrast, deficiency of another deubiquitinase CYLD, which hydrolyzes both Met1 and K63 ubiquitin chains, leads to cylindromatosis (19). Although A20 and OTULIN have roles in attenuating common signaling pathways, patients with otulipenia have a more severe inflammatory phenotype than patients with A20 haploinsufficiency (HA20) likely for two reasons: (i) OTULIN has a unique and nonredundant function in regulation of the linear ubiquitin pathway, and (ii) patients with otulipenia have a more profound protein deficiency than patients with HA20, who still retain 50% of nonmutant A20 protein. The discoveries of otulipenia, HA20, and LUBAC deficiencies demonstrate a complex interplay between LUBAC and deubiquitinases in controlling immune signaling complexes.
Materials and Methods
Patients.
We studied three patients in this report. All subjects and their family members were enrolled in an Institutional Review Board-approved protocol and provided written informed consent. Samples from patient 1 were available for all experiments, whereas samples from patients 2 and 3 were limited. More detailed information is reported in SI Materials and Methods.
Genetic and Functional Analysis.
We performed whole-exome sequencing in patients 1 and 2 and their family members, candidate-gene sequencing in patient 3 and her parents, and mutation-specific genotyping in 1,630 DNA samples from the Turkish population. To study protein function, we used short hairpin RNA (shRNA) knockdowns in 293 cells and NF-κB luciferase assay, and Met1-linked linear polyubiquitin deubiquitination assay in 293 cells. Immunoprecipitation and immunoblotting, flow cytometry, Nanostring, intracellular cytokine staining, and cytokine profiling were performed on samples from the patients and healthy controls. SI Materials and Methods describes the methods used for all these procedures.
SI Materials and Methods
Human Subjects.
Patients 1 and 3 were evaluated at the NIH Clinical Center, patients 2 and 3 were evaluated at the Hacettepe University Faculty of Medicine Department of Pediatric Nephrology and Rheumatology in Turkey and Familial Mediterranean Fever Arthritis Vasculitis and Orphan Disease Research Center, Gulhane Military Medical Academy, in Turkey. All of the three patients enrolled in this study were evaluated under protocols approved by the respective institutional review boards and all patients and family members provided written informed consent including consent to publish (the National Institute of Arthritis and Musculoskeletal and Skin Diseases/National Institute of Diabetes and Digestive and Kidney Diseases combined institutional review board at the NIH, and the Hacettepe University Institutional Review Board in Turkey), and all patients and family members provided written informed consent including consent to publish.
Whole-Exome Sequencing.
Genomic DNA samples from patients and family members were isolated from peripheral blood. We performed whole-exome sequencing on patient 1 and his three family members as well as patient 2 from the second family (Fig. S1A). Whole-exome sequencing and data analysis were performed as previously described (10). Exonic variants including missense, splice site, and stop codon variants as well as small INDELs (frameshift and nonframeshift insertions and deletions) were filtered by ExAC, 1000 Genomes, dbSNP, NHLBI GO Exome Sequencing Project, ClinSeq database, and our internal database with over 600 exomes, based on a minor allele frequency below 1%. The variants were further filtered by homozygous inheritance due to the consanguinity of the patients.
Sanger Sequencing and Single-Base Extension Genotyping.
We performed Sanger sequencing to confirm the OTULIN mutations identified by exome sequencing and the candidate gene screening in additional patients with similar clinical presentations including patient 3. We used the BigDye Terminator, Version 1.1, Cycle Sequencing kit (Applied Biosystems) for sequencing the OTULIN exons. Sequencing was performed on a 3130xl Genetic Analyzer (Applied Biosystems), and Sequencher (Gene Codes) was used to analyze the sequencing data.
We performed single-base extension genotyping for the three OTULIN mutations (p.Leu272Pro, p.Tyr244Cys, p.Gly174Aspfs*2) in a total of 1,630 Turkish healthy controls using the Sequenom iPLEX gold method (Sequenom). Genotypes were determined with Typer 4.0 software (Sequenom).
Antibodies and Expression Plasmids.
We used the following antibodies for immunoprecipitation and immunoblotting. Antibodies specific for NEMO (sc-8330), ASC (N-15, sc-22514-R), and HOIL1 (sc-365523) were from Santa Cruz; phospho-IKKα/β (#2697), IKKα (#11930), IκBα (#4814, #9242), phospho-IκBα (#2859), TNFR1 (#3736), RIP (#3493), phospho-p38 MAPK (#4511), phospho-SAPK/JNK (#4668), SAPK/JNK (#9252), p38 MAPK (#8690), SHARPIN (#12541), OTULIN (#14127), β-actin (#4970), and HSP90 (#4874) were from Cell Signaling; HOIP (TA329873) was from Origene; polyubiquitin (sc-271289) was from Santa Cruz; linear polyubiquitin (AB130) was from Lifesensors; and linear polyubiquitin (1F11/3F5/Y102L) and K63 polyubiquitin (Apu3.A8) were from Genentech.
The antibodies used for intracellular cytokine staining were anti-TNF (554514; BD Biosciences), anti-IL6 (56-7069-42; eBioscience), and anti-CD14 (555398; BD Biosciences).
The WT OTULIN plasmid (RC224840) was from Origene, and the three mutant OTULIN plasmids (Y244C, L272P, and G174Dfs*2) were constructed by site-directed mutagenesis. Plasmids for RIPK1 (HW1506051), NEMO (SC117828), TNFRSF1A (RC204008), and ASC (RC215592) were from Origene; plasmids for SHARPIN (#50014), HOIP (#50015), and HOIL-1 (#50016) were from Addgene; GFP-Ub (#11928) and GFP-Ub KO (#11934) were also from Addgene, and Ub-WT and Ub-KO were constructed by site-directed mutagenesis of removing the GFP tag from GFP-Ub and GFP-Ub KO, respectively.
Cell Cultures, Peripheral Blood Mononuclear Cell Preparation, and Monocyte Isolation and Stimulation.
Human embryonic kidney 293 cells (HEK293 cells) (negative for mycoplasma, originally obtained from the American Type Culture Collection) and skin fibroblast cells derived from OTULIN-deficient patients or normal donors were grown in DMEM (Life Technologies) plus 10% (vol/vol) fetal bovine serum (FBS) and 1× antibiotics (Life Technologies). Heparinated peripheral blood mononuclear cells (PBMCs) were separated by Ficoll (Ficoll-Paque PLUS; GE Healthcare) gradient centrifugation. Monocytes were purified from PBMCs by negative selection (Monocyte Isolation Kit II; Miltenyi Biotec). Monocytes were then suspended in monocyte attachment medium (PromoCell) and seeded at a density of 150,000 per cm2 for 2 h. After washing out suspension cells, cells were cultured in RPMI 1640 (Gibco) supplemented with 10% FBS, 2 mM l-glutamine, penicillin (100 U/mL), and streptomycin (100 μg/mL) for 1 h.
Recombinant human TNF (PeproTech; 20 ng/mL) and human recombinant IL-1β (PeproTech; 10 ng/mL) were used to stimulate PBMCs and fibroblasts for certain time periods indicated in the figures.
OTULIN Gene Silencing by Short Hairpin RNA Transduction.
Human short hairpin RNA (shRNA) constructs for OTULIN (TL304698) and scrambled controls were purchased from Origene and prepared according to manufacturer’s protocol. HEK293 cells were transduced by incubating with shRNA virus. Puromycin (2 μg/mL) was added to the medium [DMEM (Life Technologies) plus 10% FBS and 1× antibiotics (Life Technologies)] to generate stable knockdown cell lines.
NF-κB Reporter Assay.
NF-κB pathway reporter plasmids (Promega) (pGL4.32[luc2P/NF-κB-RE/Hygro] luciferase reporter and the pRLCMV-Renilla vector) were used to cotransfect in HEK293 cells with shRNA knocked-down OTULIN, together with expression plasmids for OTULIN WT or mutants, LUBAC (SHARPIN, HOIL-1, and HOIP), and Ub-KO with or without NEMO using Lipofectamine 2000 reagents (Invitrogen). Luciferase activity was measured 36 h later by the DualGlo Luciferase Assay System (Promega). Results for NF-κB activity are expressed as fold induction by normalizing firefly luciferase activity to Renilla luciferase activity.
Immunoprecipitation and Immunoblotting.
Whole-cell lysates were prepared using ice-cold cell lysis buffer containing 20 mM Tris⋅HCl, pH 7.5, 140 mM NaCl, and 1% Triton X-100, and supplemented with protease inhibitor mixture (Roche) and 1% phosphatase inhibitor (Thermo Fisher). For immunoprecipitations, antibodies (5 μg) were added to 1 mg of total protein extract and incubated overnight at 4 °C. For linear-polyubiquitin immunoprecipitation, cells were lysed in a cell lysis buffer containing 8 M urea (Qiagen) and incubated with an antibody in cell lysis buffer containing 7 M urea at room temperature. Protein G-agarose beads (Thermo Fisher) were added to the samples and incubated for 2 h at 4 °C. Then beads were washed three times with lysis buffer and resuspended in 1× NuPAGE LDS sample buffer (Thermo Fisher) with 1% 2-mercaptoethanol (Sigma-Aldrich). Proteins were separated by SDS/PAGE and transferred to PVDF membranes. Immunoreactive proteins were visualized using ECL Plus Western blotting substrate (Thermo Fisher).
T- and B-Cell Stimulation.
For B-cell activation, human B cells were purified by negative selection using the StemSep Human B-Cell Enrichment kit according to the manufacturer’s instructions. B cells were stimulated with F(ab′)2 anti-IgM (10 μg/mL; Jackson ImmunoResearch Laboratory) for 30 min. For T-cell receptor (TCR) stimulation, total PBMCs (1 × 106) were stimulated with CD3/CD28 (Dynabeads T-cell activator from Thermo Fisher Scientific) for 30 min. Cell lysates [50 mM Tris-Cl, pH 7.4, 0.5% Nonidet P-40, 0.5% Triton X-100, 0.15 M NaCl, 2 mM EDTA, protease inhibitor (Thermo Fisher Scientific)] were prepared and resolved on NuPAGE Novex 4-12% Bis–Tris gels (polyacrylamide concentration: 4–12% gradient) (Thermo Fisher Scientific). Proteins were transferred on a nitrocellulose membrane and probed using indicated antibodies.
Met1-Linked Linear Polyubiquitin Deubiquitination Assay.
For deubiquitination assay, HEK293 cells were transfected with plasmids expressing target protein (NEMO, ASC, RIP1, or TNFR1), together with Ub-KO or Ub-WT, SHARPIN, HOIP, HOIL-1, OTULIN WT or mutants, or WT together with one mutant. Cells were harvested 36 h after transfection. Cell lysates were prepared and immunoprecipitated as previously described.
Immune Cells Cytokine Production and Serum Cytokine Detection.
Whole blood was stimulated with bacterial lipopolysaccharide (LPS) (Sigma) and staphylococcal enterotoxin B (SEB) (Sigma) for 24 h. Purified monocytes (1 × 106 cells per mL) were stimulated with LPS (Sigma; 10 ng/mL), human recombinant TNF (PeproTech; 20 ng/mL), or human recombinant IL-1β (PeproTech; 10 ng/mL) for 48 h. The concentrations of cytokines in the supernatants of stimulated and nonstimulated whole blood, monocytes, and serum were determined using Bio-Plex Pro Human Cytokine 27-plex and 21-plex immunoassay kits (Bio-Rad Hercules). The Bio-Plex pro human cytokine standard group I and group II were used as standards for the assays. The 48 cytokines and growth factors assayed are as follows: IL-1α, IL-2Rα, IL-3, IL-12 (p40), IL-16, IL-18, CTACK, GRO-α, HGF, IFN-α2, LIF, MCP-3, M-CSF, MIF, MIG, β-NGF, SCF, SCGF-β, SDF-1α, TNF-β, TRAIL, IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, basic FGF, eotaxin, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1 (MCAF), MIP-1α, MIP-1β, PDGF-BB, RANTES, TNF-α, and VEGF.
PBMCs (2 × 106 cells per mL) were stimulated with human recombinant IL-1β (PeproTech; 10 ng/mL), LPS (Sigma) for 24 h. The concentrations of cytokines in the supernatants of stimulated and nonstimulated PBMCs were determined using ProcartaPlex 13plex (Affymetrix). The 13 cytokines are IL-1α, IL-1β, IL-1ra, IL17A, IL-17F, IL-2, IL-6, IL-8, IP-10, MCP-1, MIP-1β, RANTES, and TNF. The differences in the cytokine concentrations were statistically analyzed using unpaired t test, and plotted with GraphPad Prism software package.
Intracellular Cytokine Staining.
Intracellular cytokine staining for TNF and IL-6 following LPS stimulation in PBMCs gated on CD14+ CD11+ for monocytes, gated on CD14+CD11+CD123− for dendritic cells, and gated on CD3+CD45RO+ for T cells were performed as previously described. Briefly, cells were washed twice with PBS, stained with the Live/Dead Fixable Aqua Dead Cell Stain Kit (Invitrogen) for 15 min at 4 °C, immediately fixed with 4% paraformaldehyde for 10 min at room temperature and permeabilized with cold methanol at −20 °C overnight. Cells were stained as indicated with the following antibodies: CD3-Qdot605 (Life Technologies), CD11c-PB (BD Biosciences), CD14-PE (BD Biosciences), CD123-PEcy5 (BD Biosciences), CD45RO-Texas-Red (Beckman Coulter), and TNF-FITC and IL6-AF700 (BD Biosciences). All events were collected on an LSRFortessa (BD Biosciences) and analyzed with FlowJo (Tree Star).
NanoString Assay.
RNA was extracted by PAXgene Blood RNA Kit (Qiagen), and gene expression analysis was conducted using the nCounter Analysis System (NanoString Technologies) with a codeset designed to target 594 immunologically related genes. A total of 100 ng of total RNA was mixed with the codeset and reporter probes and hybridized for 24 h. The complexes were then bound to the imaging surface using the nCounter Prep Station and then were quantified on the nCounter digital analyzer. nSolver software was used for data analysis. The expression data were normalized by using a geometric mean of 15 housekeeping genes (EEF1G, TUBB, TBP, POLR2A, GUSB, HPRT1, GAPDH, SDHA, OAZ1, PPIA, G6PD, RPL19, POLR1B, ABCF1, and ALAS1).
Accession Codes: Referenced Accessions.
NCBI reference sequence: NM_138348.4.
URLs.
URLs are as follows: Exome Aggregation Consortium (ExAC) database, exac.broadinstitute.org/; 1000 Genomes Project, www.1000genomes.org/; dbSNP, www.ncbi.nlm.nih.gov/projects/SNP/; Picard, broadinstitute.github.io/picard/; Genome Analysis Toolkit (GATK), https://software.broadinstitute.org/gatk/; ANNOVAR, www.openbioinformatics.org/annovar; CLUSTALW, www.genome.jp/tools/clustalw/.
Acknowledgments
We thank all the patients and their families, and the healthy controls, for their enthusiastic support during this research study. We thank Drs. Alejandra Negro and Xiaodong Fu from National Heart, Lung, and Blood Institute (NHLBI) for technical support, and Dr. Eric P. Hanson from National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) for helpful suggestions. This research was supported by the Intramural Research Programs of the National Human Genome Research Institute, NIAMS, NHLBI, National Institute of Allergy and Infectious Diseases, and the NIH Clinical Center. E.D. received grant support from Tübitak 1003, Primary Subjects R&D Funding Program (Project 315S122), which is supported by the Scientific and Technological Research Council of Turkey.
Supporting Information
Supporting Information (PDF)
Supporting Information
- Download
- 1.76 MB
References
1
B Gerlach, et al., Linear ubiquitination prevents inflammation and regulates immune signalling. Nature 471, 591–596 (2011).
2
F Tokunaga, et al., SHARPIN is a component of the NF-κB-activating linear ubiquitin chain assembly complex. Nature 471, 633–636 (2011).
3
B Boisson, et al., Immunodeficiency, autoinflammation and amylopectinosis in humans with inherited HOIL-1 and LUBAC deficiency. Nat Immunol 13, 1178–1186 (2012).
4
B Boisson, et al., Human HOIP and LUBAC deficiency underlies autoinflammation, immunodeficiency, amylopectinosis, and lymphangiectasia. J Exp Med 212, 939–951 (2015).
5
MJ Ombrello, DL Kastner, JD Milner, HOIL and water: The two faces of HOIL-1 deficiency. Nat Immunol 13, 1133–1135 (2012).
6
P Draber, et al., LUBAC-recruited CYLD and A20 regulate gene activation and cell death by exerting opposing effects on linear ubiquitin in signaling complexes. Cell Rep 13, 2258–2272 (2015).
7
K Keusekotten, et al., OTULIN antagonizes LUBAC signaling by specifically hydrolyzing Met1-linked polyubiquitin. Cell 153, 1312–1326 (2013).
8
E Rivkin, et al., The linear ubiquitin-specific deubiquitinase gumby regulates angiogenesis. Nature 498, 318–324 (2013).
9
M Hrdinka, et al., CYLD limits Lys63- and Met1-linked ubiquitin at receptor complexes to regulate innate immune signaling. Cell Rep 14, 2846–2858 (2016).
10
Q Zhou, et al., Loss-of-function mutations in TNFAIP3 leading to A20 haploinsufficiency cause an early-onset autoinflammatory disease. Nat Genet 48, 67–73 (2016).
11
A Ma, BA Malynn, A20: Linking a complex regulator of ubiquitylation to immunity and human disease. Nat Rev Immunol 12, 774–785 (2012).
12
V Schaeffer, et al., Binding of OTULIN to the PUB domain of HOIP controls NF-κB signaling. Mol Cell 54, 349–361 (2014).
13
A Israël, The IKK complex, a central regulator of NF-kappaB activation. Cold Spring Harb Perspect Biol 2, a000158 (2010).
14
IE Wertz, et al., Phosphorylation and linear ubiquitin direct A20 inhibition of inflammation. Nature 528, 370–375 (2015).
15
MA Rodgers, et al., The linear ubiquitin assembly complex (LUBAC) is essential for NLRP3 inflammasome activation. J Exp Med 211, 1333–1347 (2014).
16
DA MacDuff, et al., Phenotypic complementation of genetic immunodeficiency by chronic herpesvirus infection. eLife 4, 4 (2015).
17
F Ikeda, et al., SHARPIN forms a linear ubiquitin ligase complex regulating NF-κB activity and apoptosis. Nature 471, 637–641 (2011).
18
N Peltzer, et al., HOIP deficiency causes embryonic lethality by aberrant TNFR1-mediated endothelial cell death. Cell Reports 9, 153–165 (2014).
19
GR Bignell, et al., Identification of the familial cylindromatosis tumour-suppressor gene. Nat Genet 25, 160–165 (2000).
Information & Authors
Information
Published in
Classifications
Copyright
Freely available online through the PNAS open access option.
Submission history
Published online: August 24, 2016
Published in issue: September 6, 2016
Keywords
Acknowledgments
We thank all the patients and their families, and the healthy controls, for their enthusiastic support during this research study. We thank Drs. Alejandra Negro and Xiaodong Fu from National Heart, Lung, and Blood Institute (NHLBI) for technical support, and Dr. Eric P. Hanson from National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) for helpful suggestions. This research was supported by the Intramural Research Programs of the National Human Genome Research Institute, NIAMS, NHLBI, National Institute of Allergy and Infectious Diseases, and the NIH Clinical Center. E.D. received grant support from Tübitak 1003, Primary Subjects R&D Funding Program (Project 315S122), which is supported by the Scientific and Technological Research Council of Turkey.
Authors
Competing Interests
Conflict of interest statement: S.Ö. received royalties for consulting and speaking from Novartis and SOBI. All other authors declare no conflict of interest.
Metrics & Citations
Metrics
Citation statements
Altmetrics
Citations
Cite this article
Biallelic hypomorphic mutations in a linear deubiquitinase define otulipenia, an early-onset autoinflammatory disease, Proc. Natl. Acad. Sci. U.S.A.
113 (36) 10127-10132,
https://doi.org/10.1073/pnas.1612594113
(2016).
Copied!
Copying failed.
Export the article citation data by selecting a format from the list below and clicking Export.
Cited by
Loading...
View Options
View options
PDF format
Download this article as a PDF file
DOWNLOAD PDFLogin options
Check if you have access through your login credentials or your institution to get full access on this article.
Personal login Institutional LoginRecommend to a librarian
Recommend PNAS to a LibrarianPurchase options
Purchase this article to access the full text.