Association of mutations in the NALP3/CIAS1/PYPAF1 gene with a broad phenotype including recurrent fever, cold sensitivity, sensorineural deafness, and AA amyloidosis




Familial cold urticaria (FCU) and Muckle-Wells syndrome (MWS) are dominantly inherited autoinflammatory disorders that cause rashes, fever, arthralgia, and in some subjects, AA amyloidosis, and have been mapped to chromosome 1q44. Sensorineural deafness in MWS, and provocation of symptoms by cold in FCU, are distinctive features. This study was undertaken to characterize the genetic basis of FCU, MWS, and an overlapping disorder in French Canadian, British, and Indian families, respectively.


Mutations in the candidate gene NALP3, which has also been named CIAS1 and PYPAF1, were sought in the study families, in a British/Spanish patient with apparent sporadic MWS, and in matched population controls. Identified variants were sought in 50 European subjects with uncharacterized, apparently sporadic periodic fever syndromes, 48 subjects with rheumatoid arthritis (RA), and 19 subjects with juvenile idiopathic arthritis (JIA).


Point mutations, encoding putative protein variants R262W and L307P, were present in all affected members of the Indian and French Canadian families, respectively, but not in controls. The R262W variant was also present in the subject with sporadic MWS. The V200M variant was present in all affected members of the British family with MWS, in 2 of the 50 subjects with uncharacterized periodic fevers, and in 1 of 130 Caucasian and 2 of 48 Indian healthy controls. No mutations were identified among the subjects with RA or JIA.


These findings confirm that mutations in the NALP3/CIAS1/PYPAF1 gene are associated with FCU and MWS, and that disease severity and clinical features may differ substantially within and between families. Analysis of this gene will improve classification of patients with inherited or apparently sporadic periodic fever syndromes.

The hereditary periodic fever syndromes comprise a group of multisystem disorders characterized by recurrent episodes of fever in association with inflammation that affects the skin, joints, and many other tissues (1). They include familial Mediterranean fever (FMF), tumor necrosis factor receptor (TNFR)–associated periodic syndrome (TRAPS), hyperimmunoglobulinemia D syndrome (HIDS), the Muckle-Wells syndrome (MWS), and familial cold urticaria (FCU). These syndromes are distinguished by their clinical presentation, although many features overlap, including the development of life-threatening systemic AA amyloidosis in some cases. The molecular defects underlying FMF, TRAPS, and HIDS have all been described in the last 4–5 years (1, 2), and these defects have been associated with newly identified and remarkably diverse aspects of inflammation in the different syndromes. Although inherited periodic fever syndromes are rare in the general population, they serve as valuable models for study of novel pathways in molecular mechanisms of inflammation (3).

MWS (MIM no. 191900) (4) and FCU (MIM no. 120100) (5) usually develop during childhood and are typically inherited as autosomal-dominant traits, but apparent sporadic cases also occur. Muckle and Wells described a triad of progressive sensorineural deafness, rashes, and systemic amyloidosis (4). Nonspecific limb pain and arthralgias are frequent, and the amyloidosis is of the AA type, susceptibility to which varies among individuals and families with various chronic inflammatory diseases. FCU is characterized by skin lesions, swollen and painful joints, conjunctivitis, chills, and fever after exposure to cold, usually with onset at a young age (5). Because skin biopsies in FCU do not generally demonstrate urticaria, it has been proposed that the condition be renamed familial cold autoinflammatory syndrome (FCAS) (6). Amyloidosis also occurs in some patients with FCU (6). The genetic defect in both MWS and FCU/FCAS in northern European Caucasian families has been located to chromosome 1q44 by linkage studies (7, 8). Refined mapping enabled the FCU/FCAS gene to be localized to the ∼10-cM region between D1S423 and D1S2682 (9), in 5 North American families.

We also mapped an overlap syndrome of MWS and FCU/FCAS in an Indian family to this locus (10), providing strong support for the notion that mutations in the same gene might be responsible for both conditions. In the present study, therefore, we specifically examined genes involved in apoptosis function from this interval as candidates for disease susceptibility in this Indian family, as well as in a large French Canadian family with FCU/FCAS (7), a previously unreported British family with MWS, and a British/Spanish man with sporadic MWS (i.e., without a family history). We had a particular interest in apoptosis-related genes because the FCU/FCAS and MWS phenotypes bear marked similarities to TRAPS (11). Ligand binding of TNF receptor I (TNFRSF1A; the protein that is mutated in TRAPS) is a key signal to the caspase activation cascade that mediates apoptosis.

We found mutations in the NALP3 (for NACHT-, LRR- and PYD-containing proteins) gene associated with FCU/FCAS (affected members of French Canadian family), MWS (British family and sporadic case), as well as in those with the overlap syndrome (Indian family). During the course of our study, mutations of the same gene (given the novel name CIAS1, for cold-induced autoinflammatory syndrome 1 gene; the protein was named cryopyrin) were reported in 4 of the North American families described above (12). This protein has also been given yet another name, PYPAF1 (for pyrin-containing Apaf-1–like protein) (13). We have retained the NALP terminology, rather than adopting the cryopyrin (12) or PYPAF1 (13) terminology, because of the homology of the NALP3 protein to other NALP proteins that have been identified (AF310105, AF310106, AF442488). The NALP3 sequence has been submitted to GenBank (accession no. AF418985 for the short isoform, AY092033 for the intermediate isoform, AF468522 for the long isoform). In order to avoid confusion among investigators specializing in the genetics of these diseases we have used a compromised version, NALP3/CIAS1/PYPAF1, for the gene throughout this text.


Subjects were from 3 multiplex families, 1 of French Canadian ancestry, 1 of British ancestry, and 1 of Indian ancestry, whose members had been diagnosed, respectively, as having FCU/FCAS (7), MWS, and an MWS-FCU/FCAS overlap syndrome (10), on the basis of their clinical features. A British/Spanish man, who had apparent sporadic MWS that had developed during infancy with rash, arthralgia, and progressive sensorineural deafness and in whom AA amyloidosis was diagnosed at age 22, was also studied. The phenotype in the Indian family with MWS-FCU/FCAS overlap syndrome (family A) included rashes, periorbital edema, arthralgia, provocation of symptoms by cold, and variable development of AA amyloidosis. This complete kindred includes at least 25 living first-degree family members. In the French Canadian family (family B), 126 family members in 7 generations were identified, of whom 48 had symptoms consistent with FCU/FCAS that usually developed before 6 months of age. The main clinical features were cold-induced pruritic skin lesions accompanied by malaise, fatigue, generalized weakness, myalgia, joint pains and swelling, and chills; 1 member of this family died of amyloidosis. No family member had deafness. The susceptibility gene in this family had also been mapped to chromosome 1q44 (7). The British family with MWS (family C) comprised a mother and her son and daughter, all of whom had fever, rashes, and arthropathy that began during the first few weeks of life. They had subsequently developed deafness during childhood, but not amyloidosis to date. This pedigree had not undergone microsatellite analysis, because it was not sufficiently large to permit conclusive linkage studies.

To examine the hypothesis that NALP3/CIAS1/PYPAF1 mutations might be associated with less characteristic inflammatory conditions, we studied 50 subjects of European ancestry who had experienced uncharacterized, apparently sporadic, periodic fever syndromes, to investigate for the 3 variants identified in this study. All of these subjects were referred on clinical suspicion of having TRAPS and not MWS or FCU/FCAS; however, we had excluded mutations in exons 2–5 of the TNFRSF1A gene in all subjects. Each of these subjects had experienced recurrent fevers for at least 5 years, with objective evidence of systemic inflammation lasting from days to weeks; those with a diagnosis of FMF or HIDS were specifically excluded. In addition, to investigate the possibility that NALP3/CIAS1/PYPAF1 mutations might contribute to the expression of common chronic inflammatory diseases, we studied 48 patients with rheumatoid arthritis (RA) as defined by the American College of Rheumatology (formerly, the American Rheumatism Association) criteria (14) and 19 patients with juvenile idiopathic arthritis (JIA). The NALP3/CIAS1/PYPAF1 gene was also screened in 90 healthy Indian controls and 68 Caucasian (white European) controls for the R262W variant, in 68 Caucasian controls for L307P, and in 48 Indian and 130 Caucasian controls for V200M.

The study was approved by the East London and City Health Authority Research Ethics Committee, and all the participants gave informed consent. A blood sample was obtained from each individual, and genomic DNA was extracted using the Puregene kit (Gentra Systems, Minneapolis, MN).

Database screening and identification of candidate genes.

Candidate genes within the D1S423 and D1S2682 minimal interval were sought, using data provided by the International Human Genome Sequencing Consortium (15, 16). At least 7 genes and 39 expressed sequence tags (ESTs) were mapped to this critical interval. First, LOC50129 (accession no. XM_051928), which encodes a protein (CGI-146) homologous to an apoptosis-related protein (PNAS-4), was screened and excluded as a candidate gene. We excluded the remaining 6 genes, including 3 zinc-finger genes, on the basis of their not being functionally relevant to the pathogenesis of inflammation. While assigning functionality to the 39 ESTs, we identified a transcript annotated as AK027194, whose product contains a pyrin domain, implicated in apoptosis and inflammation, a feature of the NALP family (16). This gene, subsequently referred to as NALP3/CIAS1/PYPAF1 (Figure 1), was selected for investigation because it contains both pyrin and NACHT (for NAIP, CIITA, HET-E, and TP1) domains, and is therefore a member of the death domain–fold family (17, 18). Variant forms of pyrin are the cause of FMF, which is the most common inherited periodic fever syndrome (19, 20). Human sequencing was done by assembling the nucleotide sequences AC104335 for the first 6 exons and NT004536.7 for exons 7–9.

Figure 1.

Schematic representation of the NALP3/CIAS1/PYPAF1 gene, cDNA, and protein. A, Diagram of the genomic structure with the variable-length transcripts. Exons 1–3 code for the short isoform (GenBank accession no. AF418985), while the intermediate isoform is produced from a 7-exon transcript (exons 1–3, 5, and 7–9) that is extensively spliced (GenBank accession no. AY092033). B, The full-length cDNA of 3.1 kb (GenBank accession no. AF468522) encodes a protein of 1,036 amino acids (aa), identical to PYPAF1 (GenBank accession no. AF420469). The first ATG Met start codon and the stop codon TGA correspond to nucleotides (nt) 133 and 3243 of the PYPAF1 sequence. The locations of the pyrin domain (PYD), the NACHT domain, and the 3 mutations (V200M, R262W, L307P) are indicated. The locations of the overlapping primer pairs used for polymerase chain reaction (PCR) are also shown. An is the poly(A) tail of the NALP3 gene. LRR = leucine-rich repeat.

DNA and RNA extraction and microsatellite genotyping.

Genomic DNA was extracted from the blood of each subject, using Qiagen (Crawley, UK) kits (96-well format). Total RNA was extracted from a total of 6 selected individuals from the 3 families (all of whom were unrelated), as well as from the individual with MWS and an unaffected member of the French Canadian family, using PureScript RNA isolation kits (Gentra Systems, Minneapolis, MN). Aliquots were quantified and stored at −80°C. Complementary DNA (cDNA) was synthesized from total RNA by priming with random hexamers. Reverse transcription was performed in a reaction mixture containing 5 μg total RNA, 50 ng/μl random hexamers, 100 mM dithiothreitol, 10 mM dNTP mix (Gibco BRL, Gaithersburg, MD), and 200 units of SuperScript reverse transcriptase in 1× reverse transcription buffer (50 mM Tris HCl [pH 8.3], 75 mM KCl, 3 mM MgCl2) according to the instructions of the manufacturer (Gibco BRL). A negative control sample lacking RNA was run concurrently.

Specific amplification of NALP3/CIAS1/PYPAF1 cDNA was performed by polymerase chain reaction (PCR) using the 9 primer pairs listed in Figure 2 (designated A–I in Figure 1). This combination of primers allows amplification of the entire translated NALP3/CIAS1/PYPAF1 sequence. To ensure coverage of the 5′ and 3′ untranslated regions, CIAS1 exon 1 and 9 primers (12) were used on genomic DNA. The PCR cycling conditions for the primer sets were as follows: initial denaturing at 94°C for 15 minutes, 30–45 cycles of denaturing at 94°C for 45 seconds, annealing at 60°C for 45 seconds, extension at 72°C for 1 minute, and final extension at 72°C for 10 minutes. PCR products were purified and sequenced using an ABI (Foster City, CA) BigDye Terminators v2.0 Cycle Sequencing Kit and run on an ABI 3100 sequencer. Sequence data were analyzed with Sequence Navigator (ABI).

Figure 2.

Primer pairs used for NALP3 cDNA amplification.

Restriction fragment length polymorphism assays.

The sequences of the primers used for detection of these mutations in restriction fragment length polymorphism assays were as follows: forward 5′-CCTGTGCACACTGTGGTGTT-3′, reverse 5′-TGTGTCACAAGGCTCACCTCT-3′ for R262W, primer pair C for V200M, and primer pair D for L307P. The underlined nucleotide in the reverse primer for R262W was modified by changing nucleotide G at position 948 of the reference sequence to an A; because the reverse primer is antisense, it thus becomes a T. The R262W mutation abolishes a Taq I site, the V200M mutation creates an Nla III site, and the L307P mutation abolishes an Alu I site.


Candidate genes in the 3 families with MWS and FCU/FCAS and in the British/Spanish patient with apparent sporadic MWS were sequenced within a 10-cM critical interval. After excluding LOC50129 (CGI-146), an apoptosis-related gene, for disease susceptibility in these subjects, we focused on a gene predicted to encode a protein (NALP3) with sequence homology to the pro-apoptotic Apaf-1 protein (Figure 1). There are 3 apparent isoforms of NALP3, consisting of the short, intermediate, and long forms (Figure 1). The short isoform is encoded by exons 1, 2, and a long exon 3 including a stop codon and a poly(A) site. The coding region of the intermediate form consists of exons 1–3, 5, and 7–9 (exclusion of exons 4 and 6). The long form is likely to be the most important and is very similar to the predicted mouse protein sequence (not shown). We propose that exon 1 begins at the first ATG and that the coding region finishes in exon 9 at the stop codon. There are 2 potential start codons in exon 1, and we decided to follow the standard practice of using the first ATG as the initiation codon, despite the fact that the second ATG codon has a better Kozak consensus, which refers to the nucleotides preceding the AUG initiator codon. This genomic organization supports previously reported findings (12) except for the position of the initiation codon, which results in a 2–amino acid discrepancy between the numbering systems, i.e., V198M in the report by Hoffman et al (12) and V200M in our study refer to the same amino acid variants.

Three point mutations were identified in the NALP3/CIAS1/PYPAF1 gene of affected individuals (Table 1), producing the single–amino acid substitutions V200M, R262W, and L307P in its putative protein product. The mutations segregated completely within families in which subjects had inflammatory disease, and were identified in all available DNA samples from affected members of each of the 3 families (Figures 3 and 4). Screening of DNA samples from the parents of the British/Spanish patient with sporadic MWS did not reveal the presence of the R262W variant seen in the patient, probably indicating a de novo mutation. The R262W variant was also present in affected members of family A from India, with MWS and FCU/FCAS overlap syndrome. The L307P variant segregated with disease in family B with FCU/FCAS (Figure 3). Neither the R262W nor the L307P changes had been found in any of the families described previously by Hoffman et al (12), or among a panel of 158 unaffected, ethnically matched Indian and Caucasian European controls (90 Indian and 68 Caucasian European); therefore, they appear to be 2 novel private mutations of NALP3/CIAS1/PYPAF1.

Table 1. Patients/families studied and point mutations identified
Ethnic groupNucleic acid changeAmino acid changeDistinctive clinical features
French CanadianT920CL307PCold sensitivity
IndianC784TR262WCold sensitivity, amyloidosis
British/Spanish (sporadic case)C784TR262WDeafness, amyloidosis
BritishG598AV200MDeafness, no amyloidosis
Figure 3.

Pedigrees of family A (Indian) and family B (French Canadian), showing the results of Taq I and Alu I restriction fragment length polymorphism assays for the R262W and L307P mutations, respectively. Samples were loaded on 16% polyacrylamide gels (Novex, San Diego, CA) and stained with ethidium bromide. In the gel image, M corresponds to the molecular size marker while U refers to the undigested polymerase chain reaction product; lane numbers correspond to the numbers for the family members in the pedigrees. Positive and negative controls were included in each run.

Figure 4.

Pedigree of family C (British). The V200M variant was detected using an Nla III restriction fragment length polymorphism assay. Fragments were resolved on a 3% ethidium bromide–stained agarose gel. V200M did not segregate with disease in the unaffected grandfather. See Figure 3 for details.

In contrast, V200M, which was present in all subjects with MWS in family C but also in the unaffected grandfather (Figure 4), has also been associated with FCU susceptibility (12). The V200M variant was present in 1 of 130 Caucasian controls and 2 of 48 Indian controls and was also identified in 2 cases among a group of 50 subjects with apparently sporadic periodic inflammatory disorders. Review of these 2 subjects, both of whom had periodic fever, revealed that the clinical features bore some resemblance to those of MWS and FCU/FCAS, although the resemblance was not sufficient to make a diagnosis by clinical criteria. The first patient had limb pains with atypical rash, and the other had urticarial/edematous-appearing skin lesions with histologic findings showing a predominantly polymorphonuclear leukocytic infiltrate consistent with FCU/FCAS, but no sensitivity to cold. The V200M, R262W, and L307P variants were not found in any of the 48 subjects with RA or 19 subjects with JIA.


We have demonstrated that mutations in the NALP3/CIAS1/PYPAF1 gene underlie susceptibility to both FCU/FCAS and MWS, which confirms that the NALP protein family has an important role in inflammation. We found a novel mutation shared by an Indian family and a British/Spanish patient, associated with an MWS-FCU/FCAS overlap syndrome and classic MWS respectively, and another novel mutation in a French Canadian family with a classic FCU/FCAS syndrome. Although the V200M variant (annotated as V198M) was reported by Hoffman et al (12) to be associated with FCU, it was associated in the present study with classic infantile-onset MWS with severe deafness in all 3 affected members, as well as 1 unaffected member, of a British family, and with chronic inflammatory disorders in 2 other subjects with sporadic disease that did not meet clinical diagnostic criteria for either MWS or FCU/FCAS.

The significance of the V200M variant may differ from that of the 2 other mutations in that it was identified in 1 of 130 healthy Caucasian controls and 2 of 48 Indian controls, as well as in 2 of 50 subjects with disabling periodic inflammatory disorders, who had no family history of similar illness. It appears that V200M can occur in apparently healthy people, or it can be associated with MWS, as shown in family C. This variant may thus represent a low-penetrance mutation rather than a benign polymorphism, which may contribute to inflammatory disease processes other than those directly within the MWS-FCU/FCAS spectrum. Evidence against the notion that any of the 3 variants we found might contribute frequently to the more common chronic autoimmune diseases, analogous to the role of the R92Q mutation of the TNFRSF1 gene in inflammatory arthritis (21), was provided by their absence among 48 subjects with RA and 19 with JIA. However, these numbers are relatively small, and a larger study is in progress.

It is notable that several affected members of the Indian family with the same R262W mutation had relatively mild symptoms, which in several female family members had substantially decreased with advancing age. This same R262W mutation had arisen de novo in the British/Spanish patient, and in this regard it is significant that 2 of the 4 mutations identified in North American families with FCU/FCAS were de novo in origin (12).

The N-terminal pyrin domain, which is integral to both NALP3 and pyrin/marenostrin proteins (mutated in FMF) (19, 20), is another member of the death domain–fold superfamily (18), and has been shown to interact with an apoptosis-associated speck-like recruitment domain, also involved in caspase recruitment and regulation of nuclear factor κB transcription factor (18). Therefore, mutations involving either TNFRSF1A, pyrin/marenostrin, or NALP3/CIAS1/PYPAF1 molecules might all be postulated to result in defective apoptosis and transcriptional regulation, albeit through different intracellular signaling pathways. Such defects may be central to the overexuberant inflammatory response seen with some periodic fever conditions.

Some potential mechanisms by which the R262W, L307P, and V200M variants might influence disease susceptibility could include impaired binding of a putative inhibitor of NALP3 or, alternatively, increased activity of NALP3 in intracellular signaling mechanisms. The R262W and L307P mutations are found in the conserved NACHT domain (Figure 1), which is predicted to exert NTPase activity (22). The arginine residue of the R262W mutation is highly conserved in all NALP proteins, and interestingly, the corresponding amino acid is also mutated in the NACHT domain of NOD2 in subjects with the Blau syndrome (23), another inherited autoinflammatory disease. Of wider interest, mutations in other parts of the CARD15/NOD2 gene (the leucine-rich repeat domain) are associated with susceptibility to Crohn's disease (24, 25).

One of the major findings in this study was that the same NALP3/CIAS1/PYPAF1 genetic defect can cause vastly different phenotypes, e.g., the presence of periorbital edema has been considered a distinguishing clinical feature of TRAPS, but was also found in some affected members of the Indian family. Two features of the MWS-FCU/FCAS disease spectrum are especially curious, and are not recognized in other inherited periodic fever syndromes. The cold sensitivity in FCU/FCAS is unlike that in other cold-related disorders such as cryoglobulinemia, in that it is not induced by a cool absolute ambient temperature, but by a rapid decrease in temperature. Air-conditioning may be very problematic for patients with FCU/FCAS who are in hot climates, and provides a clear example of an environmental influence on a genetic disease, in that the phenotypic expression of FCU/FCAS can presumably be modulated by meteorologic conditions. Skin histologic findings are too nonspecific and variable to be of any major help regarding diagnosis of FCU/FCAS, which should now be assisted by DNA analysis.

Characterization of mutations in the NALP3/CIAS1/PYPAF1 gene provides physicians with a new diagnostic tool and may in time suggest new therapeutic approaches to these particular disorders, and possibly inflammatory diseases in general. DNA analysis has had a major impact on clinical diagnosis of TRAPS, formerly known as familial Hibernian fever, and has shown that, far from there being relatively few affected families in the world, variants of the TNFRSF1A gene that sometimes cause TRAPS (R92Q and P46L) may occur in at least 1% of the general Caucasian population (21, 26). Our preliminary data from NALP3/CIAS1/PYPAF1 screening support the inclusion of DNA analysis of this gene also among subjects with longstanding but apparently sporadic “periodic” inflammatory disease.


We are grateful to the patients and family members for their participation in the study