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Blood First Edition
Paper,
prepublished
20,
DOI
10.1182/blood-2003-07-2531
MOLECULAR BASIS OF THE SPECTRAL EXPRESSION OF CIAS1 MUTATIONS
ASSOCIATED WITH PHAGOCYTIC CELL-MEDIATED AUTO-INFLAMMATORY
DISORDERS (CINCA/NOMID, MWS, FCU)
Short title: CIAS1 genotype/phenotype analysis
Bénédicte Neven1,3, Isabelle Callebaut2, Anne-Marie Prieur3, Jérôme Feldmann1, Christine
Bodemer4, Loredana Lepore5, Beata Derfalvi6, Suata Benjaponpitak7, Richard Vesely8 , Marie
Jose Sauvain9, Stefan Oertle10, Roger Allen11, Gareth Morgan 12, Arndt Borkhardt13, Clare
Hill14, Janet Gardner-Medwin15, Alain Fischer1,3and Geneviève de Saint Basile1
1 Unité de Recherche sur le développement normal et pathologique du système immunitaire
INSERM U429 et 3Unité d'immuno-hématologie et rhumatologie pédiatriques, Hôpital NeckerEnfants Malades, 75743 Paris, France, 2Département de Biologie Structurale, LMCP, CNRS
UMR7590, Universités Paris 6 & Paris 7, case 115, 4 place Jussieu, 75252 Paris Cedex 05,
France,4Service de dermatologie, Hôpital Necker-Enfants Malades, 75743 Paris, France,
5Department of Pediatrics, IRCCS Burlo Garofolo Children's Hospital, Trieste, Italy, 6Department
of pediatrics, Semmelweis University of Medicine, 1089 Budapest, Hungary, 7Pediatric allergy
Ramathibodi hospice, Bangkok, Thailand, 8Faculty Hospital, Pediatric Rheumatology Unit , SK040 01 Kosice , Slovakia , 9Department of Pediatrics, University of Bern (Inselspital), 3010 Berne ,
Switzerland , 10Department of Rheumatology and Clinical Immunology/Allergology, University
Hospital (Inselspital),3010 Bern, Switzerland, 11Rheumatology and General Practice, Royal
Children's
Hospital,
3052
Melbourne,
Australia,
12Developmental
medicine
(Paediatrics/Immunology),University of Wales, Swansea SA2 8PP ,UK , 13Department of Pediatric
Hematology and Oncology, University of Giessen, 35 385 Giessen, Germany , 14Institute of
Medical Genetics, Institute of Medical genetics, University Hospital of Wales, CF4 4XW Cardiff,
UK , 15Department of Child Health, Glasgow University, UK.
This work was supported by grants from l’Institut National de la Santé et de la Recherche Médicale
(INSERM). B. N. holds a fellowship from the Fondation pour la Recherche Médicale (FRM).
1
Copyright (c) 2003 American Society of Hematology
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Corresponding author: Geneviève de Saint Basile, INSERM U429, Hôpital Necker-Enfants
malades, 149 rue de Sèvres, 75743 Paris, Cedex 15, France
Tel: 33 1 44 49 50 80
Fax: 33 1 42 73 06 40
email: [email protected]
Scientific section designation: phagocytes
Abstract Word Count: 210
Manuscript word count: 4108
2
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Abstract
NALPs proteins are recently identified members of the CATERPILLER family of proteins thought to
function in apoptotic and inflammatory signalling pathways. Mutations in the CIAS1 gene which
encodes a member of the NALP family, the cryopyrin/NALP3/PYPAF1 protein, expressed primarily
in phagocytic cells, were recently found to be associated with a spectrum of autoinflammatory
disorders: chronic infantile neurological cutaneous and articular (CINCA) syndrome (also known as
Neonatal-onset multisystem inflammatory disease (NOMID)), Muckle-Wells syndrome (MWS) and
familial cold urticaria (FCU). We describe herein seven new mutations in 13 unrelated patients with
CINCA syndrome and identify mutational hotspots in CIAS1 on the basis of all mutations described
to date. We also provide evidences of genotype/phenotype correlation. A three-dimensional model of
the nucleotide-binding domain (NBD) of cryopyrin suggested that this molecule is structurally and
functionally similar to members of the AAA+ protein family of ATPases. According to this model,
most of the mutations known to affect residues of the NBD are clustered on one side of this domain in
a region predicted to participate in intermolecular contacts. This suggests that this model is likely to
be biologically relevant and that defects in nucleotide binding, nucleotide hydrolysis or protein
oligomerization may lead to the functional dysregulation of cryopyrin in the MWS, FCU and
CINCA/NOMID disorders.
Introduction
CIAS1 gene encodes cryopyrin/NALP3/PYPAF1
1,2
, a member of the recently discovered
NALP/PYPAF subfamily of the CATERPILLER (CARD, transcription enhancer, R(purine)-binding,
pyrin, lots of LRR) protein family 3,4. Little is known about the structure and function of the proteins
of this subfamily. Each member of the NALP (NACHT
-,L RR- and PYD-containing
proteins)/PYPAF(PYRIN-containing Apaf-1like protein) family contains an amino-terminal pyrin
domain (PYD), a central NACHT domain including a nucleoside triphosphate (NTP)-binding site,
and carboxy terminal leucine-rich repeats (LRRs) (reviewed in 5). PYD contains six anti-parallel helices that form a compact bundle similar in structure to the CARD death, and death effector
domains 6. As a member of the death domain-fold superfamily, PYD probably mediates homotypic
interactions between PYD-containing proteins, resulting in the formation of a complex involved in
signal transduction. LRRs are 20 to 29 residue sequence motifs present in multiple proteins with
diverse functions. In various members of the CATERPILLER family as in the NOD (nucleotidebinding oligomerization domain) subfamily, LRRs may act as intracellular sensors of bacterial
invasion capable of initiating an inflammatory response. LRRs may thus play a role in detecting
3
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pathogen-derived molecules and possibly endogenous non foreign ‘alarm signal’ such as mammalian
DNA and heat shock proteins, ultimately leading to the induction of inflammatory responses
7,8
. The
NALP1 LRRs may exert their effects by means of inhibition as their removal in NALP1 makes the
protein constitutively active 9. The NACHT domain of NALP contains seven distinct motifs,
including an ATP/GTP-specific P-loop and a Mg2+-binding site typical of nucleoside triphosphatases
(NTPases) (Walker A and B motifs, respectively) 3. NACHT domains appear related to the NBD of
the AP-ATPases family10 or NB-ARC family
11
, sharing specific features with similarly positioned
motifs3. Some members of the AP-ATPases family, as the human APAF-1, are involved in
programmed cell death and inflammatory signaling pathways, a function which requires nucleotide
binding and protein oligomerization mediated through the NBD12. By analogy, the NACHT domain
of NALP may be involved in protein oligomerization.
The role of cryopyrin/NALP3/PYPAF1 is unclear. Its expression is restricted to immune cells and
chondrocytes
13
. Cryopyrin has been reported to interact with the protein ASC (apoptosis-associated
speckle-like protein containing a CARD), a PYD-CARD binding partner of procaspase-1, although
this interaction has been called into question2,14,15. The binding of procaspase-1 induces the
processing of pro-IL-1 to generate its active form, IL1, and the activation of NF- B. These findings
suggest that cryopyrin is involved in the regulation of apoptosis and/or inflammatory signalling
pathway. Clear evidence that cryopyrin plays a key role in inflammation was recently provided in
vivo, by the association of CIAS1 mutations with autoinflammatory diseases. FCU (MIM 120100),
MWS (MIM 191900) and CINCA/NOMID (MIM 607115) syndromes, are three autosomal dominant
disorders resulting from CIAS1 missense mutations
1,13
. All involve recurrent inflammatory episodes
generally associating fever, arthralgia, and urticaria. These features are brought on by exposure to
cold in FCU, the mildest of these conditions. In the MWS, transient arthritis, neurosensory deafness
and amyloidosis are frequently associated with these manifestations. Patients with CINCA syndrome
display the most severe phenotype with neonatal onset, chronic polymorphonuclear (PMN)
meningitis leading to progressive neurological impairment, and recurrent joint flare-ups of joint
inflammation. Joint involvement varies in severity from mild flare-ups to severe arthropathy with
radiological modifications. Progressive visual impairment and perceptive deafness may also be
observed with increasing age 16. This condition may be fatal. All of the mutations identified to date in
these three disorders are missense mutations within exon 3 of the CIAS1 gene. The mutations
associated with a particular condition 1,13,17-20 do not appear to be clustered. We analysed the clinical
and molecular features of 13 newly diagnosed patients with CINCA syndrome. We combined these
data with those previously obtained for patients with CINCA, MWS or FCU, which enabled us to
identify hotspots of mutation preferentially associated with particular disease expressions. We
4
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investigated the molecular consequences of these missense mutations in CIAS1, and the structurefunction relationships of the protein by generating a model of the three-dimensional structure of the
NBD of the cryopyrin NACHT domain.
Patients and Methods
Patients: twenty-two unrelated patients with suspected CINCA syndrome were analyzed in the
genetic study. We studied possible genotype/phenotype correlation in patients with CIAS1 gene
mutation within this cohort and in previously reported patients reported with autoinflammatory
disorders associated with CIAS1 mutations. For FCU, the diagnostic criteria were recurrent
intermittent episodes of fever, rash, conjunctivitis and articular manifestations primarily after general
exposure to cold, absence of deafness and amyloidosis. Patients with MWS were characterised by
similar recurrent episodes of inflammation without cold triggering, associated in some cases with
progressive deafness and amyloidosis. The diagnostic criteria for CINCA/NOMID syndrome were
presence of episodic fever, early-onset urticarial skin rash associated with chronic meningitis and in
some cases severe and deforming arthropathies. (Table1). Patients with NOMID/CINCA syndrome
differed considerably in terms of the severity of the condition. We therefore assigned patients to two
groups: one with transient joints flare-ups only and the other with permanent and deforming
arthropathies. Each patient was carefully examined by physicians experienced in the diagnosis of
CINCA/NOMID syndrome, and informed written consents for this study was obtained from the
patients or their parents.
Mutation detection: Genomic DNA was extracted from whole blood using standard procedures. We
searched for mutations in genomic DNA using exons with flanking intron sequences and bidirectional fluorescence sequencing as previously described13. A panel of control DNA samples was
tested for the presence of the CIAS1 mutations identified in each patient, by mutation sequencing
analysis (for CIAS1, GenBank accession number: AF427617).
Sequence analysis - modelling of three-dimensional structure
We used a battery of sequence analysis / structure prediction methods, including similarity searches
within the Protein Data Bank (PDB) using PSI-BLAST
21
with a protein specific score matrix
(PSSM) derived from the NACHT family of domains, and threading procedures (3D-PSSM
22
,
Fugue 23). The resulting alignments were manually checked for accuracy, refined and extended by
Hydrophobic Cluster Analysis (HCA)
24,25
, which makes it possible to consider the 1D sequence
5
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alignment in a structural context, as the hydrophobic clusters delineated with this approach are
generally consistent with the regular secondary structures
26,27
. The secondary structures predicted
using this approach are consistent with those predicted by the PSI-PRED
28
and PHD
29
programs
(the PSI-PRED prediction is reported above the CIAS1 sequence on Fig.2A). This careful analysis
was accompanied by visual inspection of the experimental three-dimensional structures. In
particular, the three-dimensional (3D) superimposition of the structures, together with the
associated multiple alignments, made it possible to distinguish core sequences from more variable
sequences (Fig 2A). It also led to the identification of position invariably occupied by hydrophobic
amino acids (buried positions) that are required for conservation of the typical fold of NACHT
family (Fig 2A). This method for modeling in conditions of low levels of sequence identity has
already been used successfully on many different protein targets (e.g. 30,31).
We assessed alignments by calculating Z-scores (differences between the observed scores and the
mean scores of a distribution of scores calculated from the alignment of one sequence with 1000
randomised versions of the other). Z-score values are expressed in standard deviation units of the
random distribution. The mean Z-score values calculated for the alignment of CIAS1 with the four
sequences shown in Fig 2A were 6.0 (with a maximum of 7.1) and 7.5 (with a maximum of 8.4) for
identity and similarity (Blosum 62 matrix) scores, respectively, whereas the mean identity level is
11.6 %. These values are similar to those calculated from alignments of the NBDs of known threedimensional structures (e.g. identity and similarity Z-scores values for the alignment 1fnn/1hqc
(12.5 % identity) are 6.1 and 7.1, respectively).
We used Modeller-4
cdc6p (PDB 1fnn
33
32
for three-dimensional modeling with the three-dimensional structure of
), nsf (PDB 1d2n)
34,35
and p97 (PDB 1e32
36
) used as templates. Three-
dimensional structures were manipulated using Swiss-PdbViewer 37.
Results
Novel CIAS1 mutations identified in patients with CINCA syndrome.
We have previously reported seven different missense mutations in the CIAS1 gene associated with
CINCA syndrome in seven unrelated families 13. Since this first description, four additional CINCA
syndrome associated-mutations have been reported 20. CIAS1 mutations have also been reported in
additional MWS and FCU patients
1,17-19
. We studied 22 additional patients and identified CIAS1
gene mutations in thirteen, with CINCA/NOMID syndrome (Table1). Nine of these patients
displayed particularly severe disease with persistent arthropathy associated with radiologically
evident bone deformities (Table1). None of these patients had family history of the disease.
6
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Both strands of the CIAS1 coding sequence as all the exon/intron flanking sequences were screened
by direct sequencing of PCR fragments, as previously described 13. We also searched for mutations
in DNA of the parents' patients when available, and in a panel of control samples (Table1). All the
mutations identified in this cohort of patients, as in the previously reported ones, consist of
missense mutations located in exon 3 of CIAS1 (Table1 and Fig1). Seven of the 13 mutations
identified in CIAS1 are new. In three patients, the mutation affected a residue not previously
identified as involved in CIAS1 associated diseases: E354D in P21, T405P in P9, L632F in P15,
(Table1 and Fig1). In another four patients, the mutations affected residues previously reported to
be mutated in CIAS1 associated syndromes (CINCA syndrome, MWS or FCU), but with a different
substitution: R260L was identified in P11 and R260P in P12, whereas R260W was previously
reported in five families with either MWS or FCU (Fig1 and Table2), as independent events
occurring in each family. T436I was identified in P18 whereas T436N was previously observed in a
family with 3 members affected by CINCA syndrome
13
. D303G was found in P13, whereas a
different transition at the same residue, D303N, was previously observed in two members of a
family and one sporadic case with CINCA/MWS overlapping phenotype, and in one patient with a
CINCA syndrome
13,18,38
. Finally, six mutations identified in patients from this study had been
reported before: D303N (P8), T438M (P14) reported in 3 different families with MWS 18 and in one
sporadic case with CINCA syndrome 39, F309S (P17) was reported in one case of CINCA/NOMID
syndrome with a severe phenotype
13
and Y570C, observed in three patients (P10,16 and 19) was
previously reported in two others patients with a severe CINCA/NOMID syndrome
20,39
(Table1).
None of the mutations were found in controls as in patient 's parents when tested (Table1).
7
CIAS1
Mutation
Nucleotides Amino acid
Patient
number
Age
(year)
(1)
Neurological
involvement
(2)
Joint
involvement
(3)
Constant and
deforming
arthropathies
Frequency of CIAS1
mutations in controls
CIAS1 mutations in
the parents
(4)
11
3
NA
AG
No
G779T
R260L
0/74
-
14
45
+
TA
No
C1043T
T348M
0/98
ND
15
22
+
TA
No
G1896T
L632F
0/98
-
21
8
+++
TA
No
G1062T
E354D
0/98
-
12
5
NA
PA
Yes
G779C
R260P
0/74
-
8
13
++
PA
Yes
G907A
D303N
0/74
ND
13
13
+
TA/PA
Yes
A908G
D303G
0/112
-
17
8
++
TA/PA
Yes
T926C
F309S
0/112
-
9
22
+
PA
Yes
A1213C
T405P
0/90
ND
18
5
+
TA/ PA
Yes
C1307T
T436I
0/78
ND
10
3
++
PA
Yes
A1709G
Y570C
0/98
-
16
1
++
PA
Yes
A1709G
Y570C
0/98
-
19
22
+++
PA
Yes
A1709G
Y570C
0/98
ND
(1) : Current age
(2) : NA = not assessed ; + : chronic meningitis ; ++ : chronic meningitis, mental retardation ;+++ = chronic meningitis, mental retardation, epilepsia or cerebral atrophy
(3) : TA= transient arthritis ; AG : arthralgia ;PA : persistent arthritis
(4) : ND : not done
8
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Table 1: Clinical data, and CIAS1 mutations identified in the 14 tested patients with CINCA syndrome
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Overall, these findings show that irrespective of disease severity, all the CIAS1 mutations identified
to date are missense mutations. In addition, although they occur de novo, these mutations are
present in a number of patients, indicating the probable existence of mutation hotspots. We
therefore investigated distribution of these mutations according to disease severity, and whether
mutation hotspots designate critical functional residues on a predicted three-dimensional (3D)
structure of the cryopyrin NACHT domain.
Figure 1:
Locations of the mutations in CIAS1 encoding protein.
All the mutations identified to date in CIAS1 that cause FCU, MWS or CINCA/NOMID syndromes
are located in exon 3 which encodes the NACHT domain and its flanking regions. Mutations
previously reported are indicated above the protein structure whereas new mutations identified in
this study appear below the protein structure.
9
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Distribution of the CIAS1 mutations as a function of disease severity
We evaluated a possible genotype/phenotype correlation within the spectrum of CIAS1 mutations
found in this and other studies by classifying cases according to diagnosis and severity of disease
presentation. Patients with FCU and CIAS1 mutation were reported to have episodes of fever, rash
and articular manifestations primarily after natural and/or experimental generalized cold exposure,
without deafness or amyloidosis. In the other groups, patients presented similar recurrent episodes
of symptoms but without cold triggering, often associated with deafness and amyloidosis. Patients
with MWS were differentiated from patients with CINCA syndrome on the basis of chronic
meningitis that occurred in patients with CINCA syndrome but not in those with MWS. Finally,
within the group of patients with CINCA syndrome, the severity of disease expression was
estimated on the basis of the severity of neurological symptoms and development of arthropathy as
assessed by X rays (Table1 and Table.2). With these diagnostic criteria, several of the identified
mutations were found to be associated with the same phenotype. For instance, extremely severe
expression of CINCA syndrome was associated in five patients with a Y570C mutation (3 in our
group -P10, P16, P19- and 2 previously reported) 20 39. A detailed medical history was available for
four of these patients. They all presented severe arthropathy before one year of age resulting in
metaphyseal enlargement and severe contractures. Severe neurological symptoms with mental
retardation were observed in all, associated with epilepsy in one case (P19) and cerebral atrophia
and hydrocephalia in three cases (P16, P19) 39. Prematurity and dysmaturity were observed in three
cases (P10, P16, P19). They all failed to thrive (weight <-2DS), had growth failure (height <-2DS)
and dysmorphy. P19 died at 22 years of age. The F309S mutation was found in two patients with
severe articular and neurological diseases, one of whom died in early adulthood. The F523L
mutation was also found in two other patients presenting severe CINCA syndrome as reported by
Aksentijevich et al 20. Some mutations may be common to groups of patients contiguous in terms of
severity. For instance, R260W and V198M were found in several families with MWS or FCU
T348M occurred in three families with severe expression of MWS and in two patients
18,39
1,18
,
with
mild expression of CINCA syndrome involving acute episodes of arthritis and mild neurological
problems consisting of sporadic headache due to chronic meningitis as confirmed by CSF
examination. The D303N mutation was found in patients with severe or moderate expression of
CINCA syndrome. One patient with the D303N mutation was initially reported to have MWS
18
.
However, the clinical features of this patient were recently reported to be more consistent with
CINCA syndrome diagnosis
38
. This example highlights the limitations of such approach in
situations in which overlap exists in phenotype classification. However, this study clearly shows
10
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that none of the mutations identified to date in patients with the most severe expression of disease
(CINCA syndrome with chronic meningitis and arthropathy) were observed in patients with the
mildest phenotype (FCU). Although this analysis deals with a limited number of patients, these data
indicate a relative phenotype/genotype correlation, suggesting that distinct mutations differently
affect cryopyrin function and/or expression.
11
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FCU
MWS
CIAS1
CINCA
Chronic meningitis
transient joint flares
CINCA
Chronic meningitis
permanent and
deforming
arthropathies
‡V198M : family 12 (18) and 2 (1)
˚R260W : families 3 and 4 (18)
L305P (19)
L353P (17)
A439V : family 1 (1)
E627G : family 3 (1)
‡V198M (19)
˚R260W : families 1 and 2 (18) and (19)
A352V : family 4 (1)
*T348M : families 5, 6 and 7 (18)
A439T : family 11 (18)
G569R : family 10 (18)
#D303N : family 2(13), sporadic cases (18, 38)
Q306K : patient 2 (13)
*T348M : patient 14 and patient 2 (39)
E354D: patient 21
H358R : patient 4 (13)
T436N : patient 3a and 3b (13)
L632N : patient 15
M662T : patient 5 (13)
R260L : patient 12
R260P : patient 12
L264F : patient 997 (20)
#D303N : patient 922 (20) and patient 8
D303G : patient 13
F309S : patient 7 (13) and 11
A374N : patient 986 (20)
T405P : patient 9
T436I : patient 18
F523L : patients 975 and 996 (20)
Y570C : patients 10, 16, 19, patient 987(20) and patient 1 (39)
F573S : patient 1 (13)
Table 2:
Distribution of all the CIAS1 missense mutations identified to date, relative to the
spectrum of CIAS1 associated illnesses.
Independent mutations identified in several patients are indicated in bold typeface. Similar
mutations observed in patients from different groups are indicated with a specific sign ( , #, ‡, °).
References relating to previous descriptions of these mutations are indicated in italics.
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Mapping CIAS1 mutations on a three-dimensional model of the NACHT Nucleotide Binding
Domain
In the absence of experimental data, a three-dimensional model of the structure of the CIAS1
NACHT domain can be used to further evaluate the molecular impact of mutations and to
investigate the function of CIAS1 . The presence of Walker A (P-loop) and Walker B (Mg2+binding site) motifs clearly identifies NACHT domains as NTPases 3. However, apart from these
two signatures, NACHT domain sequences are fairly different from those of typical NBDs, for
which experimental 3D structures has been solved. NACHT domains are larger, with a predicted
fold (majority of -helices, data not shown) in the C-terminal region. We therefore used a battery of
sequence analysis/structure prediction methods, including the sensitive Hydrophobic Cluster
Analysis
24,25
to generate a relevant model for part of the CIAS1 NACHT domain. These methods
made it possible to detect significant relationship at low levels of sequence identity (< 20 %),
supported by relevant statistical scores, and to align the CIAS1 sequence accurately with sequences
of known three-dimensional structures constituting templates for homology modelling.
The CIAS1 NBD fold is predicted to consist of a typical five-stranded -sheet surrounded by helices, as observed in AAA+ ATPases, the structures of which have been used as templates for
modelling (Fig.2). Walker A , Walker B, as well and a “sensor” motif are found at the end of the
parallel strands, forming the nucleotide-binding site, which in AAA+ ATPases also involves
residues from a second -helical domain following the NBD. NACHT domains may have a similar
structure, but the lack of accurate alignment for the -helical domain precluded its modelling on
AAA+ ATPase templates.
Strikingly, most of the CIAS1 mutations in the NBD (arrows on Fig. 2A) are located on one side of
this domain (Fig. 2B) along the nucleotide-binding cleft or in prolongation of the cleft. They are
found in loops next to the parallel -strands (loop after -strand S2: R260, L264; loop after -strand
S3, near the Walker B motif: D303, L305, Q306, F309; loop after
-strand S4: T348 (which
corresponds to the “sensing” residue of the sensor motif for the detection of nucleotide binding and
hydrolysis, it is located close to the ATP -phosphate in AAA+ ATPases
19,40
). Mutations are also
found in -helix H5 (A352, L353, E354, H358) and in the N-terminus of helix H1B (V198). Most of
these regions are also involved in oligomeric interactions in AAA+ ATPases. Surimposition of our
three-dimensional model of CIAS1 NBD on the NBD subunit of the AAA+ nsf ring-forming
hexamer (data not shown) suggests that CIAS1 may be involved in similar oligomeric organization.
Thus, mutations within the NBD located outside the structure core, are most likely to affect
13
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nucleotide binding and/or hydrolysis or to disturb conformational changes affecting the quaternary
structure 30.
Other CIAS1 mutations are located in the C-terminal part of the NACHT domain, after the NBD
(residues A374, T405, T436, A439, F523, G569, Y570, F573, E627, L632 and M662). Although no
accurate alignment and model can be built for this region, the predicted secondary structures are
consistent with a mainly -helical domain following the NBD, which should run from residue 374
to residue 451.
Fig. 2 A) Alignment of the CIAS1 sequence with four nucleotide-binding domain sequences of
known three-dimensional structures corresponding to proteins of the AAA+
ATPases
superfamily. p97: d1 AAA domain of membrane fusion ATPase p97 (PDB identifier: 1e32
(chainA) 36; nsf: D2 hexamerization domain of N-ethylmaleimide sensitive factor (PDB 1d2n (chain
A) 34,35; Cdc6p (PDB 1fnn (chain A) 33 and RuvB (PDB 1hqc (chain A) 40.
The alignment was generated by threading and PSI-BLAST procedures and was carefully refined
and extended using the sensitive Hydrophobic Cluster Analysis (HCA) (see Methods). The
14
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positions of CIAS1 mutations are indicated by vertical arrows. The amino acids of nsf involved in
inter-subunit contacts 35 are boxed in red.
Identitical amino acids are shaded in black, whereas similar amino acids are shaded in gray with
white and black letters for hydrophobic (or amino acids that can substitute for them) and nonhydrophobic amino acids, respectively. The positions most frequently occupied by hydrophobic
amino acids in the NACHT family of domains are indicated by green circles. These residues mainly
correspond to amino acid which, in the aligned NBDs, are buried within the considered structures
and which can serve as anchors for the alignment procedure. Moreover, positions of the observed
regular secondary structures (underlined and labeled under the sequence (S= -strand, H= helix)) in
most cases match those of the predicted secondary structures of CIAS1, with respect to its sequence
(E=extended ( -strand), H=helix, C=coil). No accurate structural alignment could be obtained for
helix H5 (indicated in brackets). However, the N-termini of the corresponding sequences could be
aligned, highlighting the conservation of two hydrophobic amino acids.
The main original features of the CIAS1 NBD fold with respect to the NBD core structures shown
here are: i) the presence of another helix (H3C) after helix H3B (nsf labeling). In this respect, the
predicted structure of CIAS1 is suspected to be similar that of the Cdc6p 33, in which a longer helix
H3C is also present between helix H3B and strand S3 (yellow); ii) a large loop linking strand S3 to
helix H4. This loop is seven amino acids longer than the corresponding loop in nsf
34,35
but also
contains an IGP sequence, which in the nsf structure forms a tight turn involved in hexamer
interactions 34,35.
B) Mapping of mutations on the three-dimensional model of CIAS1 NBD.
Two orthogonal views are shown in ribbon representation. The model was constructed based of the
alignment shown in panel A, Strands and helices are labeled and colored according to panel A.
Positions of mutations are shown and labeled, as are shown the positions of the Walker A T231
(blue on helix H2) and Walker B D300 (orange in strand S3) motifs, and the positions of ATP and
of the magnesium ion, as in the nsf structure
35
. Note that the conformation of the loop linking
strand S3 to helix H4B is hypothetical. A C -trace of the
AAA+ ATPases (nsf D2
35
-helical domain following NBD in
) is shown on right to illustrate its position with respect to the NDB.
According to HCA (data not shown), T305 can be tentatively located in the C-terminal end of an
extended structure, following two -helices, that lies near the ATP-binding site (gray ball).
15
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Discussion
FCU, MWS and CINCA syndrome, three conditions associated with CIAS1 mutations, are inherited
as dominant traits. Almost 50 independent mutations, including those described in this study, have
now been characterized. All these mutations are missense mutations affecting exon 3 of CIAS1
causing a wide spectrum of disease expression. These findings strongly suggest that the mutated
protein exerts a dominant negative or a gain of function over the wild-type product and that the null
mutation of one allele would probably have no effect or lead to a different phenotypic expression
due to haplo-insufficiency. Although we cannot rule out an effect of unknown modifier genes in
phenotypic expression, specific CIAS1 mutations seem likely to affect disease expression, as shown
by some degree of genotype/phenotype correlation observed within this spectrum of phenotypic
expression. This correlation is particularly clear if we consider the extreme groups defined by the
magnitude of phenotypic expression, i.e. FCU and the severe CINCA syndrome. Patients from
different groups do not share mutations whereas, within each group, several unrelated patients carry
the same mutation, occurring as an independent event in each case. In contrast, a few patients from
contiguous groups, such as FCU/MWS or MWS and milder forms of CINCA syndrome, may share
mutations. In such cases, the moderate expression of symptoms, as for chronic meningitis, may
have been missed, or patients may not yet have developed the features used to discriminate between
the various groups. Fine analysis of a larger number of patients with each condition is required to
confirm and strengthen these observations. If confirmed, these data may be of outmost importance
for prognostic assessment and for adjustment of treatment for patients with CIAS1 associated
diseases. Our analysis also confirms the previously suggested genetic heterogeneity of these
disorders 13,20, because mutations in CIAS1 were identified in only 60% of the patients analysed.
To localize mutated residues at the three-dimensional level and to investigate further the function of
CIAS1, we constructed a model of the cryopyrin NACHT domain by homology modeling based on
known structures of NBD domains. The NACHT domain of cryopyrin can be accurately aligned
with nucleotide-binding domains from members of the AAA+ class of proteins, which generally
have a
helical domain following the NBD. The proposed model for the NACHT domain of
cryopyrin is highly consistent with the NB-ARC domain model of CED4 protein proposed by
Jaroszewski and colleagues 41. These two nucleotide binding related domains share specific features
including a sensor 1 region within the motif IV and a highly conserved proline in motif V that
distinguishes these proteins from the rest of the ATPases
42
. These two families of domains differ,
however, in their C-terminal regions, which cannot be aligned. As the aa sequence of CIAS1 differs
16
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considerably from those of the templates used for modelling and despite the adapted methodology
and cautions applied to this analysis, we cannot totally exclude the possibility that in, some places,
the alignment, and thus the structure prediction remains imperfect. However, as most of the
mutations in the NBD are located in conserved regions close to invariant regular secondary
structures, the model may be considered reliable. Experimental structure determination would be
necessary to refine the alignment in some regions, to obtain atomic details and to determine the
fold(s) adopted by region following NBD, in which several other mutations are located. Although
members of the AAA+ ATPases, have very different cellular activities
regulatory subunits of macromolecular protein complexes
43,45
43,44
, they all function as
. Their function depends on the
binding and hydrolysis of a nucleotide resulting in conformational changes that promote assembly,
disassembly, or functional operation of another part of the protein complex. In addition, many of
these proteins form hexamers, in a process demonstrated to be ATP-dependent in the case of nsf-D2
35
. Based on the similarity of the NACHT cryopyrin sequence to the sequence of these proteins, it is
tempting to speculate that nucleotide binding to cryopyrin induces conformational change in this
protein or promote its oligomerization. Although the similarity to AAA+ ATPases suggests that the
CIAS1 NACHT domain forms a hexamer structure, we cannot exclude the possibility that CIAS1
oligomers, if indeed they exist, display different stochiometries or arrangements.
Twelve of the 23 different substitutions identified affect residues of the NACHT NBD domain.
Remarkably, all are clustered on one side of the protein, near the nucleotide-binding cleft, within a
region possibly involved in oligomeric interactions, based on the known oligomeric structures of
AAA+ ATPases. Based on sequence similarity, the mutations appear to affect residues directly
involved in “sensing” of the nucleotide-binding state, in predicted subunits interactions or residues
that are located very close to the Walker A and B motifs. None of these mutations target highly
conserved positions intimately involved in the binding of the metal ion or of the nucleotide.
Although we cannot rule out the possibility that nucleotide binding is impaired, this observation
may suggest that defective hydrolysis and conformational change and oligomerization of the protein
in particular, may be the main mechanism by which the mutated protein exerts its dominant effect.
As half of the cryopyrin monomers expressed in patients’ cells are translated from the wild type
allele, the mutated monomer should exert a transdominant effect over the normal protein function.
This may occurs through the formation of oligomers containing mixtures of active and inactive
monomers that fail to support protein activity. Further attempts to correlate the location of the
mutations with disease severity were uninformative. This may be explained by the fact that residues
clustered in the same loop, like L305 and Q306, predicted to be either buried (L305) or exposed at
17
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the surface (Q306), will differentially affect the stability of the loop as well as its ability to interact
with potential partners. In addition, the nature of the substitution at a given position, may determine
the extent to which it impairs the protein function or oligomerization. Several substitutions beyond
A374N have been identified in CIAS1 sequence, in a region for which no accurate structural
information is available. These mutations are also associated with a relative genotype/phenotype
correlation. Most of the mutations in this region (such as F523L or Y570C), leads to the most
severe expression of the disease. Based on sequence similarities with AAA ATPases, this region
may regulate the oligomerization process.
One of the main functions of AAA+ proteins is to form and to regulate transient macromolecular
complexes. The pyrin domain and LRR repeats of cryopyrin are expected to mediate intermolecular
interactions. The pyrin domain can interact with the ASC adaptor, which in turn recruits effector
protein via its CARD domain to generate an heterocomplex. 2. The CARD domain ASC has been
shown to bind to that of procaspase-1, inducing the processing and activation of caspase-1 and the
activation of NF- B. Furthermore, the LRRs of cryopyrin may contain a ligand-binding domain that
may constitute a molecular on/off “switch” as reported for NOD proteins. By analogy to the
NALP1/ASC/caspase-1 and caspase-5 protein complex which assembles to form the inflammasome
9
, the NACHT domain oligomerization of cryopyrin may be essential for the formation of a
macromolecular heterocomplex, bringing into close proximity several effectors, thereby inducing
their activation. However, such a mechanism remains to be demonstrated. Given the phenotypic
expression of cryopyrin-associated disorders and the in vitro studies performed with this protein, the
function of this complex is likely to be connected with critical processes such as apoptosis
regulation, NF- B activation, caspase-1 activation and cytokine secretion, all elements of
inflammatory responses.
Acknowledgments
We thank the patients and their families for their cooperation. We also thank Cécile Dumont,
Stéphanie Certain and Nathalie Lambert for excellent technical assistance and Jean-Paul Mornon
for helpful discussion and critical reading of this manuscript.
Bibliography
1. Hoffman HM, Mueller JL, Broide DH, Wanderer AA, Kolodner RD. Mutation of a new gene
encoding a putative pyrin-like protein causes familial cold autoinflammatory syndrome and Muckle-Wells
syndrome. Nat Genet. 2001;29:301-305.
18
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
2. Manji GA, Wang L, Geddes BJ, Brown M, Merriam S, Al-Garawi A, Mak S, Lora JM, Briskin M,
Jurman M, Cao J, DiStefano PS, Bertin J. PYPAF1: A PYRIN-containing Apaf1-like protein that assembles with
ASC and regulates activation of NF-kB. J Biol Chem. 2002;10:10
3. Koonin EV, Aravind L. The NACHT family- a new group of predicted NTPases implicated in
apoptosis and MHC transcription activation. Trends Biochem. Sci. 2000;25:223-224
4. Harton JA, Linhoff MW, Zhang J, Ting JP. Cutting edge: CATERPILLER: a large family of
mammalian genes containing CARD, pyrin, nucleotide-binding, and leucine-rich repeat domains. J Immunol.
2002;169:4088-4093.
5. Tschopp J, Martinon F, Burns K. NALPs: a novel protein family involved in inflammation. Nat Rev
Mol Cell Biol. 2003;4:95-104.
6. Martinon F, Hofmann K, Tschopp J. The pyrin domain: a possible member of the death domain-fold
family implicated in apoptosis and inflammation. Curr Biol. 2001;11:R118-120.
7. Inohara N, Koseki T, Lin J, del Peso L, Lucas PC, Chen FF, Ogura Y, Nunez G. An induced
proximity model for NF-kappa B activation in the Nod1/RICK and RIP signaling pathways. J Biol Chem.
2000;275:27823-27831.
8. Girardin SE, Sansonetti PJ, Philpott DJ. Intracellular vs extracellular recognition of pathogens-common concepts in mammals and flies. Trends Microbiol. 2002;10:193-199.
9. Martinon F, Burns K, Tschopp J. The inflammasome: a molecular platform triggering activation of
inflammatory caspases and processing of proIL-beta. Mol Cell. 2002;10:417-426.
10. Aravind L, Dixit VM, Koonin EV. The domains of death: evolution of the apoptosis machinery.
Trends Biochem Sci. 1999;24:47-53.
11. van der Biezen EA, Jones JD. The NB-ARC domain: a novel signalling motif shared by plant
resistance gene products and regulators of cell death in animals. Curr Biol. 1998;8:R226-227.
12. Acehan D, Jiang X, Morgan DG, Heuser JE, Wang X, Akey CW. Three-dimensional structure of the
apoptosome: implications for assembly, procaspase-9 binding, and activation. Mol Cell. 2002;9:423-432.
13. Feldmann J, Prieur AM, Quartier P, Berquin P, Certain S, Cortis E, Teillac-Hamel D, Fischer A, de
Saint Basile G. Chronic infantile neurological cutaneous and articular syndrome is caused by mutations in
CIAS1, a gene highly expressed in polymorphonuclear cells and chondrocytes. Am J Hum Genet. 2002;71:198203.
14. Gumucio DL, Diaz A, Schaner P, Richards N, Babcock C, Schaller M, Cesena T. Fire and ICE: the
role of pyrin domain-containing proteins in inflammation and apoptosis. Clin Exp Rheumatol. 2002;20:S45-53
15. Srinivasula SM, Poyet JL, Razmara M, Datta P, Zhang Z, Alnemri ES. The PYRIN-CARD protein
ASC is an activating adaptor for caspase-1. J Biol Chem. 2002;277:21119-21122.
16. Prieur AM, Griscelli C, Lampert F, Truckenbrodt H, Guggenheim MA, Lovell DJ, Pelkonnen P,
Chevrant-Breton J, Ansell BM. A chronic, infantile, neurological, cutaneous and articular (CINCA) syndrome. A
specific entity analysed in 30 patients. Scand J Rheumatol Suppl. 1987;66:57-68.
17. Hoffman HM, Gregory SG, Mueller JL, Tresierras M, Broide DH, Wanderer AA, Kolodner RD.
Fine structure mapping of CIAS1: identification of an ancestral haplotype and a common FCAS mutation,
L353P. Hum Genet. 2003;112:209-216.
18. Dode C, Le Du N, Cuisset L, Letourneur F, Berthelot JM, Vaudour G, Meyrier A, Watts RA, Scott
DG, Nicholls A, Granel B, Frances C, Garcier F, Edery P, Boulinguez S, Domergues JP, Delpech M, Grateau G.
New mutations of CIAS1 that are responsible for Muckle-Wells syndrome and familial cold urticaria: a novel
mutation underlies both syndromes. Am J Hum Genet. 2002;70:1498-1506.
19. Aganna E, Martinon F, Hawkins PN, Ross JB, Swan DC, Booth DR, Lachmann HJ, Bybee A,
Gaudet R, Woo P, Feighery C, Cotter FE, Thome M, Hitman GA, Tschopp J, McDermott MF. Association of
mutations in the NALP3/CIAS1/PYPAF1 gene with a broad phenotype including recurrent fever, cold
sensitivity, sensorineural deafness, and AA amyloidosis. Arthritis Rheum. 2002;46:2445-2452.
20. Aksentijevich I, Nowak M, Mallah M, Chae JJ, Watford WT, Hofmann SR, Stein L, Russo R,
Goldsmith D, Dent P, Rosenberg HF, Austin F, Remmers EF, Balow JE, Jr., Rosenzweig S, Komarow H,
Shoham NG, Wood G, Jones J, Mangra N, Carrero H, Adams BS, Moore TL, Schikler K, Hoffman H, Lovell DJ,
Lipnick R, Barron K, O'Shea JJ, Kastner DL, Goldbach-Mansky R. De novo CIAS1 mutations, cytokine
activation, and evidence for genetic heterogeneity in patients with neonatal-onset multisystem inflammatory
disease (NOMID): a new member of the expanding family of pyrin-associated autoinflammatory diseases.
Arthritis Rheum. 2002;46:3340-3348.
21. Altschul S, Madden T, Schaffer A, Zhang J, Zhang Z, Miller W, Lipman D. Gapped-BLAST and
PSI-BLAST : a new generation of protein database search programs. Nucl. Acids Res. 1997;25:33893402
22. Kelley LA, MacCallum RM, Sternberg MJ. Enhanced genome annotation using structural profiles
in the program 3D-PSSM. J Mol. Biol. 2000;299:499-520
19
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
23. Shi J, Blundell TL, Mizugushi K. FUGUE: sequence-structure homology recognition using
environment-specific substitution tables and structure-dependent gap penalties. J. Mol. Biol. 2001;310: 243-257
24. Gaboriaud C, Bissery V, Benchetrit T, Mornon J-P. Hydrophobic cluster analysis: An efficient new
way to compare and analyse amino-acid sequences. FEBS Lett. 1987;224:149–155
25. Callebaut I, Labesse G, Durand P, Poupon A, Canard L, Chomilier J, Henrissat B, Mornon J-P.
Deciphering protein sequence information through hydrophobic cluster analysis (HCA): current status and
perspectives. Cell Mol Life Sci. 1997;53:621-645
26. Woodcock S, Mornon JP, Henrissat B. Detection of secondary structure elements in proteins by
Hydrophobic Cluster Analysis. Prot Eng. 1992;5:629-635
27. Hennetin J, LeTuan K, Canard L, Colloc'h N, Mornon J-P, Callebaut I. Non intertwined binary
patterns of hydrophobic/non hydrophobic amino acids are considerably better markers of regular secondary
structures than non constrained binary patterns. Proteins. 2003;51:236-244
28. Jones D. Protein secondary structure prediction based on position-specific scoring matrices. J Mol
Biol. 1999;292:195-202
29. Rost B, Sander C. Combining evolutionary information and neural networks to predict protein
secondary structure. Proteins. 1994;19:55-72
30. Paoletti P, Perin-Dureau F, Fayyazuddin A, Le Goff A, Callebaut I, Neyton J. Molecular
organization of a zinc binding N-terminal modulatory domain in a NMDA receptor subunit. Neuron.
2000;28:911-925
31. Callebaut I, Curcio-Morelli C, Mornon JP, Gereben B, Buettner C, Huang S, Castro B, Fonseca TL,
Harney JW, Larsen PR, Bianco AC. The iodothyronine selenodeiodinases are thioredoxin-fold family proteins
containing a glycoside hydrolase clan GH-A-like structure. J Biol Chem. 2003;278:36887-36896.
32. Sali A, Potterton L, Yuan F, van Vlijmen H, Karplus M. Evaluation of comparative protein
modelling by MODELLER. Proteins. 1995;23:318-326
33. Liu J, Smith C, DeRyckere D, DeAngelis K, Martin G, Berger J. Structure and function Of
Cdc6/Cdc18: implications for orgin recognition and checkpoint control. Mol Cell. 2000;6:637-648
34. Lenzen CU, Steinmann D, Whiteheart SW, Weis WI. Crystal structure of the hexamerization
domain of N-ethylmaleimide-sensitive fusion protein. Cell. 1998;94:525-536.
35. Yu R, Hanson P, Jahn R, Brünger A. Structure of the ATP-dependent oligomerization domain of Nethylmaleimide sensitive factor complexed with ATP. Nat Struct Biol. 1998;5:803-811
36. Zhang X, Shaw A, Bates P, Newman R, Gowen B, Orlova E, Gorman M, Kondo H, Dokurno P,
Lally J, Leonard G, Meyer H, van Heel M, Freemont P. Structure of the AAA ATPase p97. Mol Cell.
2000;6:1473-1484
37. Guex N, Peitsch MC. SWISS-MODEL and the Swiss-PdbViewer: An environment for comparative
protein modeling. Electrophoresis. 1997;18:2714-2723
38. Granel B, Philip N, Serratrice J, Ene N, Grateau G, Dode C, Cuisset L, Disdier P, Berbis P, Delpech
M, Weiller PJ. CIAS1 Mutation in a Patient with Overlap between Muckle-Wells and Chronic Infantile
Neurological Cutaneous and Articular Syndromes. Dermatology. 2003;206:257-259.
39. Rosen-Wolff A, Quietzsch J, Schroder H, Lehmann R, Gahr M, Roesler J. Two German CINCA
(NOMID) patients with different clinical severity and response to anti-inflammatory treatment. Eur J Haematol.
2003;71:215-219.
40. Yamada K, Kunishima N, Mayanagi K, Ohnishi T, Nishino T, Iwasaki H, Shinagawa H, Morikawa
K. Crystal structure of the Holliday junction migration motor protein RuvB from Thermus thermophilus HB8.
Proc Natl Acad Sci USA. 2001;98:1442-1447
41. Jaroszewski L, Rychlewski L, Reed J, Godzik A. ATP-activated oligomerization as a mechanism for
apoptosis regulation: fold and mechanism prediction for CED-4. Proteins. 2000;39:197-203
42. Aravind L, Wolf YI, Koonin EV. The ATP-cone: an evolutionarily mobile, ATP-binding regulatory
domain. J Mol Microbiol Biotechnol. 2000;2:191-194.
43. Neuwald A, Aravind L, Spouge J, Koonin E. AAA+: a class of chaperone-like ATPases associated
with the assembly, operation and dissambly of protein complexes. Genome Res. 1999;9:27-43
44. Patel S, Latterich M. The AAA team: related ATPases with diverse functons. Trends Cell Biol.
1998;8:65-71
45. Vale RD. AAA proteins. Lords of the ring. J Cell Biol. 2000;150:F13-19.
20
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
Prepublished online November 20, 2003;
doi:10.1182/blood-2003-07-2531
Molecular basis of the spectral expression of CIAS1 mutations
associated with phagocytic cell-mediated auto-inflammatory disorders
(CINCA/NOMID, MWS, FCU)
Benedicte Neven, Isabelle Callebaut, Anne-Marie Prieur, Jerome Feldmann, Christine Bodemer,
Loredana Lepore, Beata Derfalvi, Suata Benjaponpitak, Richard Vesely, Marie Jose Sauvain, Stefan
Oertle, Roger Allen, Gareth Morgan, Arndt Borkhardt, Clare Hill, Janet Gardner-Medwin, Alain Fischer
and Genevieve de Saint Basile
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