1. Top of page
  2. Abstract
  6. Acknowledgements


To investigate the involvement of the CIAS1/PYPAF1/NALP3 gene in 7 unrelated Spanish families with recurrent autoinflammatory diseases characterized by early onset, recurrent fever, and a chronic urticarial rash, in whom a clinical diagnosis of cryopyrin-associated periodic syndromes (CAPS) is suspected.


Clinical symptoms, results of laboratory analyses, and data on previous treatments in members of the 7 families were recorded on a questionnaire specific for hereditary autoinflammatory diseases. All coding regions and intronic flanking boundaries of the CIAS1/PYPAF1/NALP3 gene were amplified by polymerase chain reaction and sequenced.


Five different missense mutations, including 2 de novo and 1 previously unreported mutation (R488K), were identified in exon 3 of the CIAS1/PYPAF1/NALP3 gene in 5 of the 7 affected families. Expanded genetic analysis among the healthy individuals identified incomplete penetrance in 2 families. No mutations were found in 2 of the 3 patients with chronic infantile neurologic, cutaneous, articular (CINCA) syndrome/neonatal-onset multisystem inflammatory disease (NOMID).


The clinical data suggested a diagnosis of familial cold-induced autoinflammatory syndrome in 3 families, CINCA/NOMID syndrome in 3 others, and a possible Muckle-Wells syndrome, whereas mutational analysis showed different CIAS1/PYPAF1/NALP3 missense mutations in 5 families. These data are consistent with a common molecular basis of these diseases and highlights the phenotypic heterogeneity among CIAS1/PYPAF1/NALP3 gene–associated syndromes. The previously unreported mutation and the incomplete penetrance found in 2 families expand the genetic basis underlying these autoinflammatory syndromes. These findings should alert clinicians to the possible genetic basis of these conditions, even in the absence of a family history, in their attempts to establish an accurate diagnosis and the optimal therapeutic approach.

The recently described group of systemic autoinflammatory diseases includes several syndromes characterized by recurrent episodes of inflammation in the absence of infectious and autoimmune causes. A genetic basis has been established in some of them, including the subgroup of hereditary periodic fever syndromes (HPFS) (1). Among the HPFS with an autosomal-dominant inheritance pattern, the familial cold-induced autoinflammatory syndrome (FCAS; MIM #120100) and the Muckle-Wells syndrome (MWS; MIM #191900) share several clinical features. Both syndromes have very early onset and present as fever, urticarial rash, and joint involvement. The inflammatory episodes of FCAS develop characteristically after generalized cold exposure and usually resolve within 24 hours, whereas the inflammatory episodes of MWS have a longer duration but no triggering factor. A progressive bilateral sensorineural hearing loss and/or secondary AA amyloidosis may also appear later in life in patients with MWS (1). Both conditions have been associated with missense mutations in the CIAS1/PYPAF1/NALP3 gene, which encodes the cryopyrin protein (2–4).

A third clinical entity, the chronic infantile neurologic, cutaneous, articular (CINCA) syndrome (MIM no. #670115), which is also known as neonatal-onset multisystem inflammatory disease (NOMID), has recently been associated with mutations in the same CIAS1/PYPAF1/NALP3 gene (5, 6). This condition is characterized by a neonatal onset, recurrent fever, urticarial rash, chronic arthropathy due to epiphyseal overgrowth, facial dysmorphia, and central nervous system involvement, which is manifested as chronic meningitis, bilateral papilledema, sensorineural deafness, mental retardation, and cerebral atrophy. Typically, no recognizable triggering factors for the episodes in these patients or family history of the condition are reported (7).

These 3 different clinical syndromes can now be considered as a single pathologic entity with different degrees of disease severity, FCAS being the mildest, CINCA/NOMID syndrome the most severe, and MWS an intermediate phenotype. Some cases have also been reported of overlap between FCAS and MWS and between MWS and CINCA/NOMID (4, 8). To underline their common molecular basis, the term cryopyrin-associated periodic syndromes (CAPS) has recently been proposed for all these syndromes (9).

The function of cryopyrin is not completely understood. However, its structural features suggest a role in the regulation of inflammatory and apoptotic pathways. It has been reported to interact with ASC through the pyrin domain, inducing NF-κB activation and increasing interleukin-1β (IL-1β) production (6, 10).

Since the description of the relationship between CAPS and the CIAS1/PYPAF1/NALP3 gene, more than 30 different missense mutations have been identified, all of them located on exon 3, which encodes the nucleotide binding site domain and boundary regions. Analyses of clinical phenotype and mutations in the CIAS1/PYPAF1/NALP3 gene identified in patients with different phenotypes of CAPS have not shown that a given clinical phenotype could be associated with a specific mutation or a specific gene region. Indeed, certain mutations have been associated with 2 different clinical phenotypes of CAPS (3, 5).

Herein, we present the clinical and laboratory data, treatment outcomes, and mutational analysis of the CIAS1/PYPAF1/NALP3 gene in 7 unrelated Spanish families with recurrent autoinflammatory disease in whom CAPS is clinically suspected.


  1. Top of page
  2. Abstract
  6. Acknowledgements


Twelve patients from 7 unrelated Spanish families who had recurrent autoinflammatory syndromes with early onset, periodic fever and urticarial rash were referred to our HPFS unit over the last 2 years to assess whether the different HPFS-associated genes might be the cause. Infectious and autoimmune causes had been ruled out. The clinical symptoms, findings of laboratory analyses, and data on previous treatments were recorded on a questionnaire specific for hereditary autoinflammatory diseases. Written informed consent was obtained from each patient or each patient's parents, and approval by the medical ethics committee of our hospital was obtained.

DNA extraction.

Genomic DNA obtained from whole blood samples was isolated using a QIAamp DNA Blood Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.

CIAS1/PYPAF1/NALP3 gene mutation analysis.

All coding regions and intronic flanking sequences of the CIAS1/PYPAF1/NALP3 gene were amplified by polymerase chain reaction (PCR), using specific intron primers and conditions as previously described (2). The PCR products were purified using a QIAquick PCR purification kit (Qiagen) according to the manufacturer's instructions. The products were sequenced using an ABI BigDye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA) and an ABI 3100 automatic sequencer.


  1. Top of page
  2. Abstract
  6. Acknowledgements

A family history of recurrent autoinflammatory syndrome was identified in only 1 family (family 3), which showed a clear autosomal-dominant pattern of inheritance, with 6 affected individuals in 4 generations (Figure 1A). In the other families, only 1 individual was affected. No consanguinity was reported for any of the families. The main clinical symptoms and the laboratory findings are summarized in Table 1.

thumbnail image

Figure 1. A, Pedigrees of the 7 Spanish families with recurrent autoinflammatory diseases. Solid circles and squares indicate affected females and males, respectively, who have the mutation. Shaded squares indicate asymptomatic males with the mutation. Open symbols indicate asymptomatic individuals. Numbers beneath the symbols represent the individuals in whom genetic analyses were performed. The CIAS1/PYPAF1/NALP3 genotype is shown below each index patient. B, DNA sequence electropherograms showing the heterozygous G-to-A transition at position 1463 (arrow) resulting in the new R488K mutation in the nucleotide binding site domain of cryopyrin, which was identified in the patient from family 2 (top). The sequence from a normal control subject (bottom) is shown for comparison. Note that only sense strands are shown. FCAS = familial cold-induced autoinflammatory syndrome; MWS = Muckle-Wells syndrome; CINCA = chronic infantile neurologic, cutaneous, articular (syndrome); wt = wild type.

Download figure to PowerPoint

Table 1. Clinical symptoms and laboratory data of patients with cryopyrin-associated periodic syndrome*
  • *

    Normal plasma IgD levels are <100 units/ml; levels >100 units/ml are considered to be increased. Features are defined as present (+), absent (–), unknown (?), and occasional (±). Two affected relatives in family 3 (unnumbered affected male and female subjects shown in Figure 1A) are not included in this table because neither a clinical examination nor a CIAS1/PYPAF1/NALP3 mutation analysis was performed. ESR = erythrocyte sedimentation rate; FCAS = familial cold-induced autoinflammatory syndrome; MWS = Muckle-Wells syndrome; CINCA = chronic infantile neurologic, cutaneous, articular (syndrome).

Sex/age, yearsM/13M/7F/7F/4M/32M/62M/3F/7F/14F/8
Age at onset10 years6 yearsBirthBirthNeonateBirth9 monthsNeonateNeonateNeonate
Episode durationHoursHours10–12 hours10–12 hours10–12 hours10–12 hours5–8 days3–7 days?2–3 days
Periodicity??1–2 days2–3 days2–3 days2–3 days4–6 weeksIrregular2–3 monthsIrregular
Cold-triggering factor++++++
Recurrent fever++++++
Urticarial skin rash++++++++++
Patellar overgrowth+
Chronic arthropathy+++
Sensorineural deafness+
Chronic meningitis++++
Mental retardation+
Growth retardation++
Abdominal pain+±+
AA amyloidosis
Elevated ESR++++++++++
Plasma IgD levelNormalNormalNormalNormalNormalNormalIncreasedIncreasedIncreasedIncreased
CIAS1/PYPAF1/NALP3 mutationV198MR488KL305PL305PL305PL305PT348MD303N

A diagnosis of FCAS was made in families 1–3 based on short-duration episodes of inflammation triggered after a generalized exposure to cold. The main clinical symptom identified was the skin rash, although small differences in age at onset and extracutaneous symptoms (periodic fever, arthralgias) were also identified. These patients had usually been treated with different drugs (nonsteroidal antiinflammatory drugs [NSAIDs], antihistamines) and experienced a partial response, probably due to the short duration of the self-limited episodes.

Mutational analysis of the CIAS1/PYPAF1/NALP3 gene identified 3 different heterozygous missense mutations in all affected individuals in these 3 families. There was a V198M mutation in family 1, a G-to-A transition at position 1463 of the complementary DNA sequence (GenBank accession no. NM_004895), which resulted in the identification of a new R488K missense mutation in family 2 (Figure 1B), and an L305P mutation in family 3. Mutational analysis performed in all available unaffected individuals belonging to the 3 families with FCAS identified 2 healthy individuals who carried the same mutations identified in their offspring (V198M in family 1 and R488K in family 2), suggesting incomplete penetrance of these mutations. In order to exclude the possibility that these mutations (V198M and R488K) were common polymorphisms, we investigated their presence in 150 healthy unrelated Caucasian blood bank donors; no chromosomes harboring these variants were detected.

The affected child in family 4 experienced long episodes of inflammation. Mild, chronic meningitis and occipital headache had recently been reported. However, the absence of cold exposure as the triggering factor and the absence of severe neurologic or articular involvement suggested the diagnosis of MWS, although the most typical signs of MWS (sensorineural hearing loss and AA amyloidosis) were not present in this patient. The patient had been treated with NSAIDs alone, and the response was always partial, decreasing the clinical symptoms during the episode but not preventing the recurrence of further episodes. Mutational analysis of the CIAS1/PYPAF1/NALP3 gene in this family identified a heterozygous T348M missense mutation in the affected child but not in his parents. Paternity was confirmed by HLA haplotype segregation (data not shown), suggesting that the T348M mutation was a de novo mutation in this affected child.

In the affected individuals in families 5–7, a clinical diagnosis of CINCA/NOMID syndrome was suspected because of the neonatal onset, the chronic arthropathy with functional impairment, and the neurologic involvement (chronic meningitis, papilledema, or sensorineural hearing loss) that was present in all subjects. Antiinflammatory treatment with corticosteroids in the patient in family 6 resulted in a partial clinical response, with improvement of the skin rash and periodic fever, but the effects on the articular and neurologic involvement were limited. The affected girl in family 5 developed severe intracranial hypertension and bilateral papilledema, and after several medical regimens were tried, a ventriculoperitoneal shunt was required to prevent a fatal compromise of her vision. Analysis of the CIAS1/PYPAF1/NALP3 gene in these patients identified only a heterozygous missense mutation (D303N) in the affected girl in family 5. No mutations were found in any coding regions of this gene in the affected individuals in families 6 and 7. Paternity was confirmed in all affected individuals by HLA haplotype segregation (data not shown), suggesting that the D303N mutation identified in the patient in family 5 was a de novo mutation.

The laboratory findings during episodes of inflammation showed leukocytosis with neutrophilia and a large increase in the levels of all acute-phase response proteins, including plasma C-reactive protein and serum amyloid A1 protein. Interestingly, increased plasma levels of IgD (>100 units/ml) were detected only in patients with MWS or CINCA/NOMID syndrome, both during episodes of inflammation as well as during asymptomatic intervals. This finding suggests a stronger underlying inflammatory response in these patients than in those with FCAS.


  1. Top of page
  2. Abstract
  6. Acknowledgements

The term cryopyrin-associated periodic syndromes has recently been proposed to refer to a group of heterogeneous clinical conditions that share as a common molecular basis mutations in the CIAS1/PYPAF1/NALP3 gene (9). All of these conditions are characterized clinically by episodes of inflammation, urticarial rash, periodic fever, and articular involvement that begin early in life, usually during the neonatal period.

In the present study, we evaluated 7 unrelated Spanish families with a clinical diagnosis of CAPS and the phenotype differences described above (3 with FCAS, 3 with CINCA/NOMID syndrome, and 1 with probable sporadic MWS). Mutational analysis of the CIAS1/PYPAF1/NALP3 gene was performed to confirm the hypothesis that all affected subjects have altered cryopyrin protein. This analysis identified 5 different missense mutations of the gene, all of which were located on exon 3. Surprisingly, a dominant family history was present in only 1 of the 7 families, suggesting that clinicians should be aware of a possible dominant hereditary disease even in the absence of a family history. Our analyses of the patients' clinical features and the CIAS1/PYPAF1/NALP3 gene mutations identified revealed the phenotypic heterogeneity that has been described by other investigators, and our findings add evidence suggesting the involvement of the CIAS1/PYPAF1/NALP3 gene in these clinical disorders.

The L305P mutation identified in a Spanish family with FCAS was previously reported in a French Canadian family in association with the same condition (4). The different ethnic origins of these families suggest that the mutational event occurred independently, probably due to the presence of a “hot spot” on the CIAS1/PYPAF1/NALP3 gene. Haplotype analysis must be performed in order to answer this question.

The V198M mutation with incomplete penetrance identified in another Spanish family with FCAS has been previously reported both in FCAS and in MWS families, showing heterogeneity of the phenotype (2, 4). Interestingly, this variant has also been identified in healthy Indian and Caucasian control subjects (4). Taken together, these data suggest that this CIAS1/PYPAF1/NALP3 variant may be a mild low-penetrance mutation rather than a benign polymorphism.

In FCAS family 2, genetic analysis showed the previously unreported missense R488K mutation affecting an amino acid residue located in the nucleotide binding site domain of the cryopyrin protein. This variant was identified both in the affected child and in his unaffected father, suggesting incomplete penetrance. This genetic phenomenon has been identified in both the family with FCAS and V198M (family 1) in the present study and in some CAPS families previously described by other investigators (3).

With regard to the de novo mutations identified in the present study, the T348M mutation identified in the family with sporadic MWS has been previously reported in patients with MWS and with CINCA/NOMID syndrome (3, 11, 12). The D303N mutation identified in the patient with CINCA/NOMID syndrome has been previously described both in MWS patients and in CINCA/NOMID syndrome patients (3, 5, 6). Together, these data emphasize the phenotypic heterogeneity observed in these diseases that have a common genetic basis.

The occurrence of more than 1 mutation at a given position of the cryopyrin protein associated with CAPS has been reported to occur elsewhere (amino acid residues 260, 303, 436, and 439) (2, 3, 12). Furthermore, in their analysis of the CIAS1/PYPAF1/NALP3 gene haplotype, Dode at al (3) showed the absence of an ancestral haplotype for the T348M mutation. These data, together with the detection of the D303N and T348M de novo mutations among our Spanish CAPS patients, suggest the presence of a mutational hot spot in these codons.

The absence of mutations in the CIAS1/PYPAF1/NALP3 gene in the patients with CINCA/NOMID syndrome from families 6 and 7 suggests the possibility that another gene(s) may be the basis of this phenotype or that there may be acquired, nongenetic forms of this severe disease, a possibility that has previously been proposed by other investigators (5).

The results obtained in this study reflect the genetic complexity of these autoinflammatory syndromes. The question of which genetic or environmental modifying factors could explain the phenotypic differences (cold sensitivity, sensorineural hearing loss, severe neurologic involvement) remains to be answered.

With regard to treatment, the different antiinflammatory drugs administered to these CAPS patients have only produced a partial resolution of the symptoms. This effect is probably due to the self-limited nature of the episodes rather than an effective response to the drugs. An improvement in the articular symptoms in CINCA/NOMID syndrome patients has recently been reported with the tumor necrosis factor–blocking agent etanercept (13). In addition, anakinra, a human recombinant IL-1 receptor antagonist, has recently been proposed as a new therapeutic approach to MWS patients. Anakinra treatment in patients with MWS has shown rapid resolution of all symptoms, normalization of levels of acute-phase reactants, and no recurrence of the episodes (14, 15). Although these therapeutic results should be tested in FCAS and CINCA/NOMID syndrome patients, the common molecular basis underlying CAPS suggests that blockade of the IL-1β signaling pathway could represent a successful therapeutic approach for the most severe phenotypes of CAPS.

In conclusion, we identified 5 different missense mutations in affected members of 7 unrelated Spanish families with CAPS, including 1 previously unreported mutation and 2 mutations for which penetrance was incomplete. Our findings confirm the involvement of the CIAS1/PYPAF1/NALP3 gene in these autoinflammatory syndromes. The fact that only 1 family had a dominant medical history suggests that clinicians should be alert to the possibility of a genetic basis even in the absence of a family history, and that genetic testing should be performed to diagnose these patients. No mutations were found in 2 CINCA/NOMID syndrome patients, suggesting the involvement of other genes or the existence of acquired forms of this clinical phenotype. The recent reports of the successful use of anakinra therapy in patients with MWS open up new therapeutic possibilities for the most severe forms of these conditions.


  1. Top of page
  2. Abstract
  6. Acknowledgements

We thank the patients and their families for their participation in the study.


  1. Top of page
  2. Abstract
  6. Acknowledgements
  • 1
    Grateau G. Clinical and genetic aspects of the hereditary periodic fever syndromes. Rheumatology (Oxford) 2004; 43: 4105.
  • 2
    Hoffman HM, Mueller JL, Broide DH, Wanderer AA, Koloder RD. Mutations of a new gene encoding a putative pyrin-like protein causes familial cold autoinflammatory syndrome and Muckle-Wells syndrome. Nature Genet 2001; 29: 3015.
  • 3
    Dode C, Le Du N, Cuisset L, Letourneur F, Berthelot JM, Vaudour G, et al. 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: 1498506.
  • 4
    Aganna E, Martinon F, Hawkins PN, Ross JB, Swan DC, Booth DR, et al. 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: 244552.
  • 5
    Feldmann J, Prieur AM, Quartier P, Berquin P, Certain S, Cortis E, et al. 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.
  • 6
    Aksentijevich I, Nowak M, Mallah M, Chae JJ, Watford WT, Hofmann SR, et al. 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: 33408.
  • 7
    Prieur AM, Griscelli C, Lampert F, Truckenbrodt H, Guggenheim MA, Lovell DJ, et al. A chronic, infantile, neurological, cutaneous and articular (CINCA) syndrome: a specific entity analysed in 30 patients. Scand J Rheumatol 1987; 66: 5768.
  • 8
    Granel B, Philip N, Serratrice J, Ene N, Grateau G, Dode C, et al. CIAS1 mutation in a patient with overlap between Muckle-Wells and chronic infantile neurological cutaneous and articular syndromes. Dermatology 2003; 206: 2579.
  • 9
    Houten SM, Frenkel J, Waterham HR. Isoprenoid biosynthesis in hereditary periodic fever syndromes and inflammation. Cell Mol Life Sci 2003; 60: 111834.
  • 10
    Manji GA, Wang L, Geddes BJ, Brown M, Merriam S, Al-Garawi A, et al. PYPAF1, a PYRIN-containing Apaf1-like protein that assembles with ASC and regulates activation of NF-κB. J Biol Chem 2002; 277: 115705.
  • 11
    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: 2159.
  • 12
    Neven B, Callebaut I, Prieur AM, Feldmann J, Bodemer C, Lepore L, et al. Molecular basis of the spectral expression of CIAS1 mutations associated with phagocytic cell-mediated auto-inflammatory disorders CINCA/NOMID, MWS, and FCU. Blood 2004; 103: 280915.
  • 13
    Federico G, Rigante D, Pugliese AL, Ranno O, Catania S, Stabile A. Etanercept induces improvement of arthropathy in chronic infantile neurological cutaneous articular (CINCA) syndrome. Scand J Rheumatol 2003; 32: 3124.
  • 14
    Hawkins PN, Lachmann HJ, McDermott MF. Interleukin-1-receptor antagonist in the Muckle-Wells syndrome. N Engl J Med 2003; 348: 25834.
  • 15
    Hawkins PN, Lachmann HJ, Aganna E, McDermott MF. Spectrum of clinical features in Muckle-Wells syndrome and response to anakinra. Arthritis Rheum 2004; 50: 60712.