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Functional consequences of a germline mutation in the leucine-rich repeat domain of NLRP3 identified in an atypical autoinflammatory disorder.

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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 Jéru,1 Sandrine Marlin,2 Gaëlle Le Borgne,3 Emmanuelle Cochet,4 Sylvain Normand,5
Philippe Duquesnoy,6 Florence Dastot-Le Moal,4 Laurence Cuisset,7 Véronique 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 Médicale
(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).
Isabelle Jéru, PharmD, PhD, Serge Amselem, MD, PhD:
INSERM, U933, Université Pierre et Marie Curie-Paris 6, UMR S933,
and Assistance Publique Hôpitaux de Paris, Hôpital ArmandTrousseau, Paris, France; 2Sandrine Marlin, MD, PhD: INSERM,
U587, and Assistance Publique Hôpitaux de Paris, Hôpital ArmandTrousseau, Paris, France; 3Gaëlle Le Borgne, BS: INSERM, U933,
and Université Pierre et Marie Curie-Paris 6, UMR S933, Paris,
France; 4Emmanuelle Cochet, BS, Florence Dastot-Le Moal, PhD:
Assistance Publique Hôpitaux de Paris, Hôpital Armand-Trousseau,
Paris, France; 5Sylvain Normand, PhD, Jean-Claude Lecron, PhD:
Université de Poitiers, EA 4331, and CHU de Poitiers, Poitiers,
France; 6Philippe Duquesnoy, BS: INSERM, U933, Paris, France;
Laurence Cuisset, PhD: INSERM, U567, Assistance Publique Hôpitaux de Paris, Hôpital Cochin, and Université Paris 5, Paris, France;
Véronique Hentgen, MD: Centre Hospitalier de Versailles, Le
Chesnay, France; 9Teresa Fernandes Alnemri, PhD, Emad S. Alnemri,
PhD: Thomas Jefferson University, Philadelphia, Pennsylvania;
Robin Dhote, MD: Assistance Publique Hôpitaux de Paris, Hôpital
Avicenne, and Université Paris 13, Bobigny, France; 11Gilles Grateau,
MD: Assistance Publique Hôpitaux de Paris, Hôpital Tenon, Paris,
Address correspondence and reprint requests to Serge Amselem, MD, PhD, Institut National de la Santé et de la Recherche
Médicale (INSERM), U933, Hôpital Armand-Trousseau, 26 Avenue
du Dr. Arnold-Netter, 75571 Paris Cedex 12, France. E-mail:
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
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-
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. 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 (Comité 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
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
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
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
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.
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
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)
Yes (38.5–39°C)
Abdominal manifestations
Bilateral sensorineural hearing Moderate (associated
with tinnitus and
Visual impairment
Uveitis, papillitis
Other neurologic signs
Patient III.2
Patient II.1
⬃20 years
No episode
No episode
2–3 days
Profound (associated
with tinnitus and
Uveitis, keratitis
Headache, elevation of Headache, lymphocytic
cerebrospinal fluid
pressure during
infancy, lymphocytic
Cutaneous lesions
Musculoskeletal signs
Myalgia arthralgia,
Myalgia, arthralgia
Lymphatic signs
Other signs
Parathyroid cysts,
facial palsy
Ulcerative colitis
Parathyroid cysts
CRP during febrile episodes,
Patient II.2
Patient described by
Frenkel et al
No episode
No episode
No episode
2 years
Headache, elevation of
cerebrospinal fluid
One episode of
Recurrent episodes of
pain (ankles, knees,
and back), digital
clubbing, growth
prominent forehead
Cold feeling Delayed pubertal
* The sporadic case was described in a report by Frenkel et al (28). ND ⫽ not determined; CRP ⫽ C-reactive protein.
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
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
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.
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.
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
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
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
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.
We are grateful to all of the members of this family
who agreed to participate in the study.
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. Jéru, Amselem.
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Duquesnoy, Dastot-Le Moal, Cuisset, Hentgen, T. F. Alnemri, Lecron,
Dhote, Grateau, E. S. Alnemri.
Analysis and interpretation of data. Jéru, Normand, Lecron,
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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
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domain, disorder, atypical, germline, repeat, identifier, leucine, mutation, autoinflammatory, nlrp3, function, consequences, rich
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