Functional consequences of a germline mutation in the leucine‐rich ...

ARTHRITIS & RHEUMATISM Vol. 62, No. 4, April 2010, pp 1176–1185 DOI 10.1002/art.27326 © 2010, American College of Rheumatology

Functional Consequences of a Germline Mutation in the Leucine-Rich Repeat Domain of NLRP3 Identified in an Atypical Autoinflammatory Disorder Isabelle Je´ru,1 Sandrine Marlin,2 Gae¨lle Le Borgne,3 Emmanuelle Cochet,4 Sylvain Normand,5 Philippe Duquesnoy,6 Florence Dastot-Le Moal,4 Laurence Cuisset,7 Ve´ronique Hentgen,8 Teresa Fernandes Alnemri,9 Jean-Claude Lecron,5 Robin Dhote,10 Gilles Grateau,11 Emad S. Alnemri,9 and Serge Amselem1 Objective. To gain insight into the pathophysiology of an atypical familial form of an autoinflammatory disorder, characterized by autosomal-dominant sensorineural hearing loss, systemic inflammation, increased secretion of interleukin-1␤ (IL-1␤), and the absence of any cutaneous manifestations, and to assess the functional consequences of a missense mutation identified in the leucine-rich repeat (LRR) domain of NLRP3.

Methods. Microsatellite markers were used to test the familial segregation of the NLRP3 locus with the disease phenotype. All NLRP3 exons were screened for mutations by sequencing. Functional assays were performed in HEK 293T cells to determine the effects of mutated (versus normal) NLRP3 proteins on NF-␬B activation, caspase 1 signaling, and speck formation. Results. A heterozygous NLRP3 missense mutation (p.Tyr859Cys) was identified in exon 6, which encodes the LRR domain of the protein. This mutation was found to segregate with the disease phenotype within the family, and had a moderate activating effect on speck formation and procaspase 1 processing and did not alter the inhibitory properties of NLRP3 on NF-␬B signaling. Conclusion. This report is the first to describe a familial form of a cryopyrinopathy associated with a mutation outside of exon 3 of NLRP3. This finding, together with the known efficacy of anti–IL-1 treatments in these disorders, underlines the importance of screening all exons of NLRP3 in patients who present with atypical manifestations. In addition, the gain of function associated with this mutation in terms of activation of caspase 1 signaling was consistent with the observed inflammatory phenotype. Therefore, this study of the functional consequences of an LRR mutation sheds new light on the clinical relevance of in vitro assays.

Supported by the Agence Nationale pour la Recherche (grant 06-MRAR-010-02), the Fondation pour la Recherche Me´dicale (grant SC 080910), the European Union Sixth Framework Programme (EURAMY project grant LSHM-CT-2006-037525), and the NIH (grants AGA-4357 and AR-055398). 1 Isabelle Je´ru, PharmD, PhD, Serge Amselem, MD, PhD: INSERM, U933, Universite´ Pierre et Marie Curie-Paris 6, UMR S933, and Assistance Publique Ho ˆ pitaux de Paris, Ho ˆ pital ArmandTrousseau, Paris, France; 2Sandrine Marlin, MD, PhD: INSERM, U587, and Assistance Publique Ho ˆpitaux de Paris, Ho ˆpital ArmandTrousseau, Paris, France; 3Gae¨lle Le Borgne, BS: INSERM, U933, and Universite´ Pierre et Marie Curie-Paris 6, UMR S933, Paris, France; 4Emmanuelle Cochet, BS, Florence Dastot-Le Moal, PhD: Assistance Publique Ho ˆpitaux de Paris, Ho ˆpital Armand-Trousseau, Paris, France; 5Sylvain Normand, PhD, Jean-Claude Lecron, PhD: Universite´ de Poitiers, EA 4331, and CHU de Poitiers, Poitiers, France; 6Philippe Duquesnoy, BS: INSERM, U933, Paris, France; 7 Laurence Cuisset, PhD: INSERM, U567, Assistance Publique Ho ˆpitaux de Paris, Ho ˆpital Cochin, and Universite´ Paris 5, Paris, France; 8 Ve´ronique Hentgen, MD: Centre Hospitalier de Versailles, Le Chesnay, France; 9Teresa Fernandes Alnemri, PhD, Emad S. Alnemri, PhD: Thomas Jefferson University, Philadelphia, Pennsylvania; 10 Robin Dhote, MD: Assistance Publique Ho ˆpitaux de Paris, Ho ˆpital Avicenne, and Universite´ Paris 13, Bobigny, France; 11Gilles Grateau, MD: Assistance Publique Ho ˆpitaux de Paris, Ho ˆpital Tenon, Paris, France. Address correspondence and reprint requests to Serge Amselem, MD, PhD, Institut National de la Sante´ et de la Recherche Me´dicale (INSERM), U933, Ho ˆpital Armand-Trousseau, 26 Avenue du Dr. Arnold-Netter, 75571 Paris Cedex 12, France. E-mail: [email protected]. Submitted for publication July 10, 2009; accepted in revised form December 28, 2009.

NLRP3 (also known as cryopyrin or NALP3) belongs to the NOD-like receptor (NLR) family of proteins. The NLRs are involved in the recognition of microbial molecules and trigger inflammatory as well as immune responses. NLRP3, which is primarily expressed in the myelomonocytic lineage (1), comprises an 1176

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N-terminal pyrin domain and a central nucleotidebinding site (NBS) domain, in addition to C-terminal leucine-rich repeats (LRRs). NLRP3 interacts via its pyrin domain with the pyrin domain of ASC, leading to the formation of intracellular aggregates, called specks (1). ASC is a protein involved in the activation of procaspase 1 (2–4), which in turn induces the proteolytic cleavage of pro–interleukin-1␤ (proIL-1␤) into the mature and active IL-1␤ (5). The NBS domain of NLRP3 is required for auto-oligomerization of the protein (6). As for the LRRs, they are composed of 20–29-residue sequence motifs involved in the recognition of different stimuli and subsequent activation of pathways of inflammation and cell death (7–9). However, the precise function of NLRP3 is still a controversial subject. Indeed, NLRP3 has been found to act either as an inducer (1,10–13) or as an inhibitor (6,14,15) of NF-␬B signaling. In addition, in vitro experiments have shown that, together with ASC and caspase 1, NLRP3 forms a multiprotein complex called the inflammasome (16), leading to activation of IL-1␤ secretion (6,17). Mutations in the NLRP3 gene (also designated CIAS1 or PYPAF1) have been found to underlie 3 autosomal-dominant autoinflammatory disorders defining the group known as the cryopyrinopathies (18,19), which belongs to the larger group of hereditary periodic fever syndromes (PFS). These disorders, initially considered to be 3 distinct clinical entities, include the familial cold-induced autoinflammatory syndrome (FCAS; MIM no. 120100), Muckle-Wells syndrome (MWS; MIM no. 191900), and neonatal-onset multisystem inflammatory disease, also known as chronic infantile neurologic, cutaneous, articular (CINCA) syndrome (MIM no. 607115). Patients with a cryopyrinopathy often present with recurrent episodes of fever, urticarial skin rash, arthralgia, and systemic inflammation. In patients with FCAS, attacks are often triggered by generalized exposure to the cold. In MWS, attacks are frequently complicated by progressive hearing loss and renal amyloidosis, whereas CINCA syndrome is characterized by the most severe phenotype, with severe arthropathy, central nervous system involvement, and frequent visual impairment and sensorineural hearing loss. However, many patients have symptoms that overlap among the different cryopyrinopathies (20–23), thereby revealing a continuum in the severity of the disease. In addition, it is getting increasingly more difficult to demonstrate the deleterious effect of new sequence variations identified in NLRP3, because most patients currently referred for a diagnosis of PFS have been considered to represent sporadic cases, thus pre-

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cluding analysis of a given sequence variant for possible segregation with the disease phenotype. Furthermore, the deleterious effects of NLRP3 variants are particularly difficult to assess in functional assays and often remain questionable. The majority of mutations in NLRP3 reported to date are missense mutations located in the third exon of the gene, which encodes the NBS domain. It has been shown that several of these mutations activate NF-␬B signaling in the presence of ASC (12,24), a finding that was, however, not confirmed by others (6). It has also been shown that mutations in the NBS domain affect NLRP3 self-oligomerization, activate procaspase 1 processing (6), and induce IL-1␤ secretion (6,24), in keeping with the increased levels of IL-1␤ secreted by mononuclear cells in patients with a cryopyrinopathy (for example, see ref. 25). As for the mutations located in the other NLRP3 domains, no functional studies have thus far been performed. In this study, we describe the first familial form of PFS associated with a missense mutation lying outside of the third exon of NLRP3. The perfect intrafamilial segregation of this mutation, located within the LRR domain of NLRP3, with the disease phenotype prompted us to study its functional consequences on the caspase 1 and NF-␬B signaling pathways. PATIENTS AND METHODS Patients. In this study, we investigated a French family in which family members had presented with an MWS–CINCA syndrome overlapping disease phenotype. Informed written consent for participation in the study was provided by all of the family members or their parents. Clinical features were recorded on a standardized form in a national reference center for autoinflammatory disorders. This study was approved by the local ethics committee (Comite´ de Protection des Personnes, Ile-de-France IV-SURDOM 2008/16NICB). Molecular analysis. Genomic DNA was extracted from the peripheral blood leukocytes of each subject, using standard procedures. Microsatellite markers (D1S2836 and D1S2682) were amplified by polymerase chain reaction (PCR) using primers labeled with 6-FAM. The PCR products were analyzed on an ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). The 9 coding exons of NLRP3 and their flanking intronic sequences were amplified by PCR and sequenced with an ABI PRISM Big Dye Terminator V3.1 Ready-Reaction Cycle-Sequencing kit (Applied Biosystems). Sequences were analyzed on an ABI 3100 Genetic Analyzer. The presence of the p.Tyr859Cys mutation was confirmed by forward and reverse sequencing, with the use of forward (5⬘TTGGCTGCAGATGGAATCTG-3⬘) and reverse (5⬘TTCTCCAAGTAGGAGGTCCTCTCC-3⬘) primers. Culture of peripheral blood mononuclear cells (PBMCs) and measurement of cytokines. Fresh heparinized blood was fractionated by density-gradient separation using

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Ficoll-Hypaque (Biochrom, Berlin, Germany). PBMCs (106 cells per ml) were cultured for 24 hours in 24-well plates in RPMI 1640 supplemented with Glutamax I (Invitrogen, Carlsbad, CA), 10% fetal calf serum (Sigma-Aldrich, St. Louis, MO), penicillin (100 IU/ml), and streptomycin (100 ␮g/ml). Each culture was performed in duplicate. Supernatants were collected, centrifuged, and frozen prior to cytokine measurements. The concentrations of IL-1␤ and tumor necrosis factor ␣ (TNF␣) were measured in the supernatants using a beadbased multiplex cytokine kit, flow-based protein detection, and the LabMAP multiplex system (Luminex) according to the manufacturer’s instructions (Millipore, Bedford, MA). Plasmid constructs. The FLAG-tagged NLRP3 expression vector has been described previously (24). Sitedirected mutagenesis (QuickChange; Stratagene, La Jolla, CA) was performed to generate the plasmid constructs pNLRP3-Arg260Trp-FLAG, pNLRP3-Asp303Asn-FLAG, and pNLRP3-Tyr859Cys-FLAG. In addition, an NF-␬B p65 expression plasmid (described previously [26]) was used. All constructs were checked with sequencing analysis. Culture of HEK 293T cells. HEK 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, penicillin (100 IU/ ml), and streptomycin (100 ␮g/ml). HEK 293T cells stably expressing FLAG-tagged procaspase 1 (procaspase 1–FLAG) and green fluorescent protein (GFP)–labeled ASC (ASCGFP) (27) were cultured in DMEM/Ham’s F12 medium (Invitrogen) supplemented with 10% fetal calf serum, penicillin (100 IU/ml), and streptomycin (100 ␮g/ml). NF-␬B luciferase assay. HEK 293T cells (5 ⫻ 105 cells) grown in 6-well plates were transfected using Lipofectamine, in accordance with the manufacturer’s protocol (Invitrogen), along with 100 ng of pNF-␬B-LUC luciferase reporter (Stratagene), 800 ng of each of the NLRP3 expression plasmids, and 100 ng of the expression vector encoding p65. Luciferase activities were determined in cell lysates of triplicate cultures using a Promega assay (Promega, Madison, WI), with results normalized to the protein concentration as determined on a Coomassie Plus protein assay (Pierce, Rockford, IL). Representative results from 1 of 3 experiments are presented, and values are expressed as the mean ⫾ SD of triplicate cultures. Forty micrograms of protein was then subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDSPAGE) through an 8% polyacrylamide gel, followed by transfer to nitrocellulose membranes. The membranes were successively incubated with horseradish peroxidise (HRP)– conjugated anti-FLAG antibodies (Sigma-Aldrich) for the detection of NLRP3 proteins, while anti–␣-tubulin antibodies (DM1A; Sigma-Aldrich) were used as a control for equal loading. Speck quantification assay. HEK 293T cells stably expressing procaspase 1–FLAG and ASC-GFP were grown in 6-well plates and transfected using Lipofectamine and Lipofectamine Plus reagents along with 500 ng of each of the NLRP3 expression plasmids. Twenty-seven hours after transfection, the percentage of cells containing ASC-GFP specks was calculated on a random selection of multiple fields (400 cells analyzed). Chi-square tests were then used to compare the percentages of speck-positive cells between cells expressing

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Figure 1. Levels of interleukin-1␤ (IL-1␤) and tumor necrosis factor ␣ (TNF␣) in supernatants of cultures of peripheral blood mononuclear cells (PBMCs). PBMCs from the proband (patient III.1) and from 5 healthy controls were cultured for 24 hours, and levels of IL-1␤ and TNF␣ were measured in the culture supernatants using multiplex immunoanalytic technology. Bars show the mean and SD results of duplicate assays.

NLRP3–wild-type (WT) and cells transfected with each protein mutant. Procaspase 1 processing assay. HEK 293T cells (5 ⫻ 105 cells) stably expressing procaspase 1–FLAG and ASC-GFP were plated in 6-well plates and transfected using Lipofectamine (Invitrogen) along with 500 ng of each of the NLRP3 expression plasmids. Cells were harvested 27 hours after transfection and then lysed in a buffer containing 0.1% CHAPS, 25 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EGTA, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 0.5 mM Na3VO4. Forty micrograms of protein was then subjected to SDS-PAGE through a 12.5% polyacrylamide gel, followed by transfer to nitrocellulose membranes. The membranes were successively incubated with HRP-conjugated anti-FLAG antibodies (Sigma-Aldrich) for the detection of caspase 1 and NLRP3 proteins, while antiASC (Alexis Biochemicals, Florence, Italy) and HRPconjugated anti-rabbit antibodies (Sigma-Aldrich) were used for the detection of ASC as a control for equal loading. Detection was performed with chemiluminescence reagents (Pierce). Representative results from 1 of 3 experiments are presented.

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Figure 2. Genealogy tree and mutation analysis of the family with an atypical autoinflammatory disorder. Top, In the genealogy tree, solid symbols represent family members who presented with a cryopyrinopathy, while open symbols indicate healthy relatives. The proband (patient III.1) is indicated by an arrow. Values in color below the symbols indicate the allele sizes (in basepairs) for each microsatellite marker (blue font, marker D1S2836; green font, marker D1S2682). The haplotype associated with the disease is boxed. Bottom, Sequencing chromatograms show the mutation identified in family members III.1, III.2, II.1, and II.2 (left) compared with the sequence in healthy relatives (family members IV.1 and II.3) as controls (right). The heterozygous transition generating the missense mutation is circled.

RESULTS Familial form of an atypical periodic fever syndrome. The proband (patient III.1 in Figures 1 and 2 and Table 1) was a 37-year-old woman who presented with bilateral sensorineural hearing loss, permanent bilateral tinnitus, and a history of vertigo since childhood, leading to the use of hearing aids at the age of 14 years. Symptoms of a PFS appeared in her teenage years. She experienced more than 1 episode per month. The attacks, which were more frequent in the winter and lasted no more than 1 day, were characterized by fever (38.5– 39°C), headaches, arthralgias, myalgias, and adenopathy, but she never experienced any cutaneous manifestations. In addition, the patient was found to have uveitis and papillitis, and presented with clinodactyly of the 2 fifth

digits. During the attacks, the C-reactive protein (CRP) levels and erythrocyte sedimentation rate (ESR) in this patient were elevated, to 30–45 mg/liter and 80–107 mm/hour, respectively. The secretion of IL-1␤ and TNF␣ in PBMCs from patient III.1, as compared with that in PBMCs from 5 healthy control subjects, was measured in the supernatants of cells after 24 hours of culture. Spontaneous secretion of IL-1␤ was found to be dramatically higher in the proband, with an increase of ⬃80-fold, as compared with that in the healthy controls, whereas TNF␣ levels were only moderately elevated in the proband (⬃5-fold higher compared with that in controls) (Figure 1). The patient’s brother and mother as well as 1 of

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Table 1. Clinical presentation of the family members carrying the p.Tyr859Cys mutation, as compared with a previously described sporadic case* Present study Patient III.1† Age at onset of febrile episodes ⬃20 years Duration of episodes ⬍1 day Frequency of episodes ⬎1/month (more frequent in winter) Fever Yes (38.5–39°C) Abdominal manifestations No Bilateral sensorineural hearing Moderate (associated loss with tinnitus and vertigo) Visual impairment Uveitis, papillitis Other neurologic signs Headache

Patient III.2

Patient II.1 ⬃20 years No episode No episode

Childhood 2–3 days 1/month Yes No Moderate

No No Profound (associated with tinnitus and vertigo) Papillitis Uveitis, keratitis Headache, elevation of Headache, lymphocytic cerebrospinal fluid meningitis pressure during infancy, lymphocytic meningitis No No

Cutaneous lesions

No

Musculoskeletal signs

Myalgia arthralgia, bilateral clinodactyly

Myalgia, arthralgia

No

Lymphatic signs Proteinuria Other signs

Adenopathy No Parathyroid cysts, facial palsy 30–45

Adenopathy No Ulcerative colitis

No No Parathyroid cysts

53–97

50–100

CRP during febrile episodes, mg/liter

Patient II.2

Patient described by Frenkel et al

No episode No episode No episode

2 years ND ⬎1/month

No No Moderate

No Vomiting Yes

Uveitis No

Papilledema Headache, elevation of cerebrospinal fluid pressure

No

One episode of erythematous eruption No Recurrent episodes of pain (ankles, knees, and back), digital clubbing, growth retardation, prominent forehead No No No No Cold feeling Delayed pubertal development 78 60–140

* The sporadic case was described in a report by Frenkel et al (28). ND ⫽ not determined; CRP ⫽ C-reactive protein. †Proband.

her uncles and her grandmother on the maternal side also presented with bilateral sensorineural hearing loss and permanent systemic inflammation, an observation consistent with an autosomal-dominant mode of inheritance of the disease (Figure 2). In her brother (patient III.2 in Table 1 and Figure 2), the disease began early, during infancy, and was associated with papillitis, aseptic chronic meningitis, elevation of the cerebrospinal fluid pressure, and episodes of fever. Sensorineural hearing loss appeared in his teenage years; at that time, his symptoms were similar to those of his sister, although temperature variations (cold or seasonal temperature changes) had no triggering effect. The mother of these 2 patients (patient II.1 in Table 1 and Figure 2) also had manifestations of the autoinflammatory disorder, which started around the age of 18 years and was characterized by aseptic lymphocytic meningitis, uveitis, keratitis, and prelingual sensorineural hearing loss, leading to cochlear implantation at the age of 45 years. She had never experienced any bout of fever. The proband’s uncle

(patient II.2 in Table 1 and Figure 2) had a milder phenotype, characterized by a frequent cold feeling and bilateral sensorineural hearing loss. After 20–50 years of disease evolution, none of the patients in this family developed amyloidosis. No precise clinical information is available for the grandmother. Detailed clinical manifestations in each patient, compared with that described by Frenkel et al in a sporadic case (28), are presented in Table 1. Segregation of a missense mutation in exon 6 of NLRP3 with the disease phenotype. Although no urticarial skin rash was observed in any of the affected family members, the clinical and biologic manifestations as well as the inheritance mode were consistent with a diagnosis of a cryopyrinopathy. Since the majority of mutations in NLRP3 identified to date are located in exon 3 (29), we first analyzed this gene region. No mutation was identified, but, as shown in Figure 2, microsatellite markers flanking NLRP3 segregated perfectly with the disease phenotype. This result prompted

NLRP3 LRR GERMLINE MUTATION AND AUTOINFLAMMATION

us to sequence all exons and flanking intronic sequences of NLRP3 in the proband. A missense mutation, c.2576A⬎G (p.Tyr859Cys), was identified in the heterozygous state in exon 6 (Figure 2); this mutation is identical to the mutation that was previously found in a patient with atypical manifestations of the CINCA syndrome (28). The other 3 affected members of the family for whom DNA was available (patients II.1, II.2, and III.2 in Figure 2) also carried the mutation in the heterozygous state, whereas the DNA of 2 healthy relatives (control subjects II.3 and IV.1 in Figure 2) displayed a normal sequence. These results are consistent with a perfect segregation of the mutation with the disease phenotype within this family. Response of the patients to anakinra treatment. These findings prompted us to treat 3 of the affected family members (patients III.1, III.2, and II.1) with anakinra, a recombinant human IL-1 receptor antagonist, at a dosage of 2 mg/kg/day. This anti–IL-1 treatment led to an immediate and marked improvement of their clinical symptoms and normalization of the CRP levels, in keeping with the known efficacy of anti–IL-1 in patients with cryopyrinopathies. In all cases, clinical and biologic remission persisted after 6 months of treatment; however, no hearing improvement was observed. Functional consequences of the p.Tyr859Cys mutation. NF-␬B signaling. To assess the functional consequences of the p.Tyr859Cys mutation on NF-␬B signaling, we compared the biologic properties of NLRP3-Tyr859Cys with those of the NLRP3-WT protein as well as those of 2 NLRP3–NBS domain mutants (p.Arg260Trp and p.Asp303Asn), whose pathogenicity has already been assessed in in vitro experiments. In an NF-␬B reporter assay, we observed that NLRP3-WT, when transiently expressed in HEK 293T cells, strongly inhibited the NF-␬B activation induced by p65 (Figures 3A and B), which is consistent with the findings in previous reports (14,15). A similar inhibition was observed in the presence of the p.Tyr859Cys LRR mutation, as well as in the presence of the p.Arg260Trp and p.Asp303Asn NBS domain mutations (Figure 3). In addition, when ASC was coexpressed, no synergistic activation of NF-␬B was observed (results not shown). Taken together, these data strongly suggest that NLRP3 is a protein with antiinflammatory properties in the regulation of NF-␬B signaling, and that the p.Tyr859Cys LRR missense mutation, identified in this familial form of cryopyrinopathy, has no direct effect on this pathway. Speck formation. A previous study revealed that, compared with the NLRP3-WT form of the protein, the

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Figure 3. Effect of the p.Tyr859Cys mutation on NF-␬B signaling. HEK 293T cells were transfected with the pNF-␬B-LUC luciferase reporter (100 ng) together with each of the NLRP3 expression plasmids (800 ng), and the NF-␬B signaling pathway was induced by transfection of the cells with an expression vector encoding p65 (100 ng). A, Luciferase activities were determined on the cell lysates. Bars show the mean and SD results from triplicate cultures, with results representative of 1 of 3 experiments. B, FLAG-tagged NLRP3 proteins were analyzed by Western blotting. The same blot was reprobed with anti–␣-tubulin antibodies as a loading control. EV ⫽ empty vector; WT ⫽ wild-type.

NLRP3-Arg260Trp and NLRP3-Asp303Asn mutations display higher self-oligomerization properties and increased abilities to induce speck formation in the presence of ASC (6). To test the effect of the p.Tyr859Cys mutation on NLRP3–ASC interactions and speck formation, we used HEK 293T cells stably expressing ASC-GFP and procaspase 1–FLAG. These cells were transiently transfected with plasmids encoding normal NLRP3 or mutant NLRP3 (p.Tyr859Cys, p.Arg260Trp, and p.Asp303Asn). Seventeen percent of the cells transfected with p.NLRP3-WT displayed specks (Figures 4A and B). In comparison, the 3 missense mutations induced significantly more speck formation than that observed with the wild-type vector, with 24% of NLRP3Tyr859Cys–expressing cells (P ⫽ 0.02), 26% of NLRP3Arg260Trp–expressing cells (P ⫽ 3 ⫻ 10⫺3), and 35% of NLRP3-Asp303Asn–expressing cells (P ⫽ 1.5 ⫻ 10⫺8) showing speck formation (Figure 4). Of note, the Tyr859Cys mutant exhibited the lowest ability to aggregate with ASC.

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Caspase 1 signaling. We subsequently investigated the effect of the pTyr859Cys mutation on caspase 1 signaling in HEK 293T cells stably expressing ASC-GFP and procaspase 1–FLAG. Consistent with the findings in a previous report (6), we observed that NLRP3-WT activated procaspase 1 processing, and that NLRP3Arg260Trp and NLRP3-Asp303Asn had enhanced abilities to induce caspase 1 activation (Figure 5). Interestingly, the caspase 1 processing associated with NLRP3Tyr859Cys was also found to be higher than that with NLRP3-WT, but was weaker than that observed in cells transfected with NLRP3-Arg260Trp or NLRP3Asp303Asn (Figure 5). Therefore, in this cell system, our results support the hypothesis that NLRP3 acts as a proinflammatory protein, and that these 3 diseaseassociated variants, located in the LRR and NBS domains, represent gain-of-function mutations.

Figure 5. Effect of the p.Tyr859Cys mutation on caspase 1 processing. HEK 293T cells stably expressing ASC–green fluorescent protein and procaspase 1–FLAG were transfected with 500 ng of empty vector (EV), wild-type (WT), or each of the NLRP3 expression plamsids. Twenty-four hours after transfection, cell lysates were collected and procaspase 1 processing was detected by Western blot analysis.

DISCUSSION

Figure 4. Effect of the p.Tyr859Cys mutation on speck formation. HEK 293T cells stably expressing ASC–green fluorescent protein (GFP) and procaspase 1–FLAG were transfected with 500 ng of empty vector (EV), wild-type (WT), or each of the NLRP3 expression plasmids. Immunofluorescence studies were performed 24 hours after transfection. A, The percentage of cells containing ASC-GFP specks was calculated, with results expressed as the mean ⫾ SD from 3 independent experiments. B, Examples of representative fields obtained by immunofluorescence microscopy are shown.

This report is the first to describe a familial form of a cryopyrinopathy associated with a mutation located in the LRR domain of NLRP3. The atypical clinical presentation, characterized by late-onset symptoms that overlapped between MWS and the CINCA syndrome and the absence of urticarial skin rash, was greatly improved by anakinra treatment. In vitro assays, performed to determine the functional consequences of this LRR variant, revealed the subtle effects of the mutation, characterized by a moderate increase in caspase 1 signaling and speck formation. The 3 non–exon 3 mutations in NLRP3 identified to date have been found in sporadic cases of the CINCA syndrome (29–31). In the present study, we identified a missense mutation (p.Tyr859Cys) in exon 6 of NLRP3 in a familial form of a cryopyrinopathy. The involvement of this sequence variation in the pathophysiology of the disease is attested to by the fact that 1) it perfectly segregated with the disease phenotype within this large family, 2) it affected an amino acid highly conserved throughout evolution, and 3) this mutation was previously identified as a neomutation in a sporadic case described by Frenkel et al (28). Interestingly, the patient in that previous study also had atypical symptoms, with only 1 episode of erythematous rash and no fever (28). Although the number of mutations identified outside of exon 3 is not sufficient to establish genotype– phenotype correlations, our data underline the fact that non–exon 3 mutations can be associated with atypical presentations of the disease. Indeed, although urticarial rash is usually considered to be a hallmark of the

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cryopyrinopathies, none of the patients investigated herein ever reported having any cutaneous manifestations. The identification of this genetically unambiguous mutation underscores the importance of screening for mutations not only in exon 3, which is usually screened on a routine basis, but also in other exons of the NLRP3 gene in patients with an atypical presentation of a cryopyrinopathy. This is all the more important because patients with cryopyrinopathies usually display a rapid clinical response to anti–IL-1 treatment. Consistent with this notion, the 3 affected members of this family who were treated with anakinra displayed marked improvement in clinical and biologic responses to anakinra treatment. In our functional assays, NLRP3 acted both as a proinflammatory protein with regard to caspase 1 signaling and speck formation and as a protein with antiinflammatory properties with regard to NF-␬B activation. These findings therefore reveal that it would be simplistic to deduce the physiologic role of NLRP3 only from in vitro data, especially because it is well established that NF-␬B and IL-1␤ are part of interconnected networks (32,33) and that NF-␬B cannot only act on the induction of inflammation but also contribute to the resolution of inflammation (34). The conflicting data on NLRP3 function that have been reported, especially the observations of its effects on NF-␬B signaling (1,6,10– 15), may also reflect differences in the experimental conditions used or could be an indication of the classic limitations inherent to in vitro assays (e.g., overexpression of the protein or use of recombinant proteins fused to tags). The p.Tyr859Cys mutation is located in the C-terminal LRR domain. Although the results of several studies have suggested the importance of LRRs in the proper functioning of NLRP3 (1,14,24), the functional consequences of the LRR variants identified in patients has never before been investigated. The present findings demonstrated that, similar to NBS domain mutations, the p.Tyr859Cys mutation lying in the LRRs increases speck formation and procaspase 1 processing, suggesting that the dominant mode of inheritance of the disease results from a gain of function. Consistent with this hypothesis, cultured PBMCs from the proband spontaneously secreted abnormally elevated amounts of IL-1␤, although TNF␣ levels remained slightly above those obtained in healthy controls. These results are similar to those obtained in PBMCs from patients carrying mutations in the NBS domain (35). The present findings demonstrate that the p.Tyr859Cys NLRP3 protein mutant is freed from the requirement of any stimulus

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when contributing to caspase 1 processing and inflammasome activation, and confirm that IL-1␤ signaling is a key element in the pathophysiology of the cryopyrinopathies, in keeping with its pivotal role in autoinflammatory disorders. Our results also show that the 3 mutations investigated have no direct effect on the major inhibitory properties of NLRP3 on NF-␬B signaling. Using the same experimental system, we previously showed that a nonsense mutation (p.Arg554X) located between the NBS domain and LRRs of NLRP3 partly inhibits the antiinflammatory effects of NLRP3 on NF-␬B signaling, in keeping, in that case, with a partial loss of function (15). The molecular and cellular mechanisms underlying the cryopyrinopathies may therefore vary according to both the nature of the mutation identified in NLRP3 and the signaling pathway investigated. Interestingly, the mild effects of the p.Tyr859Cys mutation observed on caspase 1 signaling and speck formation are consistent with the hypothesis that this mutation would have only a minor impact on the structure of the LRR domain (36). Although these observations show the difficulty in setting up relevant in vitro assays, they underline the importance of such functional experiments in demonstrating the pathogenicity of newly identified NLRP3 sequence variants, especially in sporadic cases. The present study, which relies on an unusual clinical observation and on functional studies, addresses the clinical relevance of in vitro functional assays and provides both in vivo and in vitro evidence to support the notion that missense mutations in LRRs are critical for the activity of NLRP3. The data presented herein should also encourage complete screening of NLRP3 in patients with atypical symptoms. The results are indeed of particular importance for determining the best approach to management of the disease in terms of genetic counseling and initiation of effective treatment. ACKNOWLEDGMENTS We are grateful to all of the members of this family who agreed to participate in the study. AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Amselem had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Je´ru, Amselem. Acquisition of data. Je´ru, Marlin, Le Borgne, Cochet, Normand,

´ RU ET AL JE

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Duquesnoy, Dastot-Le Moal, Cuisset, Hentgen, T. F. Alnemri, Lecron, Dhote, Grateau, E. S. Alnemri. Analysis and interpretation of data. Je´ru, Normand, Lecron, Amselem.

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DOI 10.1002/art.27339

Clinical Images: Visualization of overcompensated rebound temperature against ice-water cold challenge

Digital thermography is a noninvasive, operator-dependent test based on changes in surface temperature after ice-cold challenge. Decreased temperature rebound is regarded as indicating vascular dysfunction. We used this technique to evaluate a patient who reported that her hands were sensitive to cold. Temperatures of both of the patient’s hands were similar or only slightly different, with the right hand temperature being somewhat lower than the left. After the cold challenge, vivid rebound was observed in the right hand, with 1.54 times overcompensation. We do not know the exact phenomenon that would explain such a finding, but it is interesting to observe. A, Hands with initial equivalent temperatures. B–D, Immediately after (B), 5 minutes after (C), and 10 minutes after (D) the challenge with ice-cold water. Ji-Hyeon Ju, MD Sung-Hwan Park, MD Seoul St. Mary’s Hospital and Catholic University of Korea School of Medicine Seoul, South Korea