Mutation of a new gene encoding a putative pyrin-like protein causes familial cold autoinflammatory syndrome and Muckle-Wells syndrome

Abstract

Familial cold autoinflammatory syndrome (FCAS, MIM 120100), commonly known as familial cold urticaria (FCU), is an autosomal-dominant systemic inflammatory disease characterized by intermittent episodes of rash, arthralgia, fever and conjunctivitis after generalized exposure to cold. FCAS was previously mapped to a 10-cM region on chromosome 1q44 (refs. 5,6). Muckle-Wells syndrome (MWS; MIM 191900), which also maps to chromosome 1q44, is an autosomal-dominant periodic fever syndrome with a similar phenotype except that symptoms are not precipitated by cold exposure and that sensorineural hearing loss is frequently also present. To identify the genes for FCAS and MWS, we screened exons in the 1q44 region for mutations by direct sequencing of genomic DNA from affected individuals and controls. This resulted in the identification of four distinct mutations in a gene that segregated with the disorder in three families with FCAS and one family with MWS. This gene, called CIAS1, is expressed in peripheral blood leukocytes and encodes a protein with a pyrin domain, a nucleotide-binding site (NBS, NACHT subfamily) domain and a leucine-rich repeat (LRR) motif region, suggesting a role in the regulation of inflammation and apoptosis.

Fig. 1

Pedigree, linkage and mutational analysis of a family with FCAS with a de novo mutation. Illustrated is the pedigree of family 1, with filled squares (male) and circles (female) indicating individuals with FCAS and open squares and circles indicating unaffected individuals; the subject number is indicated below each symbol. Listed to the left of subject 1 are the microsatellite markers used to genotype the family. Below each subject are indicated the allele numbers for each microsatellite marker observed for the two chromosome 1q44 haplotypes inherited by each individual; the disease haplotype is boxed. Indicated below each pair of haplotypes is the genotype for each subject, C being the wild type allele (C1317) and T the mutant allele (T1317). The upper sequencing chromatogram is a representative chromatogram from a mutation carrier (the subject being indicated by the arrow) showing the heterozygous genotype (C/T), and the lower sequencing chromatogram is a representative chromatogram showing the same region from an unaffected individual.

Fig. 2

Pedigree and mutational analysis of two families with FCAS and one with MWS. The key to the pedigrees and sequence analysis is as described in Fig. 1. a,b, Families with FCAS possessing the G592A and A1880G mutations, respectively. c, A family with MWS possessing the C1055T mutation. All living family members shown were studied. All of the affected individuals shown were found to contain the indicated mutation; arrows indicate the individual whose DNA was sequenced to generate the mutation-containing chromatogram presented below each pedigree.

Fig. 3

Structural features of CIAS1. a, Illustration of the intron–exon structure of CIAS1. The coding exons are illustrated by the boxes, the number of coding nucleotides in each exon (including the stop codon) is indicated below the box and the relative positions of each mutation are identified above the exon 3 box. The exact and estimated sizes of the introns are indicated above each intervening segment. Exons 4–8 each encode two LRRs and exon 9 encodes one LRR. b, Schematic diagram of the structural features of the CIAS1 protein (cryopyrin), which are indicated by the boxes. The pyrin domain comprises aa 13–83, the NBS domain aa 217–533 and the LRR domain aa 697–920 (of the initial isoform). The positions of the amino-acid substitutions caused by the mutations identified are indicated above the illustration of the protein. c, Alignment of the amino-acid sequence of the pyrin domain of cryopyrin with the pyrin domain of human, mouse and rat pyrin and zebrafish caspase (GenBank accession numbers AAB70557, AAF03766, AAF03767 and AAF66964). d, Alignment of the amino-acid sequence of the seven exon initial isoform (exons 1–3, 5 and 7–9) with the amino-acid sequence of NALP2 (GenBank accession number AAG30289). The observed identity and similarity were 28% and 42%, respectively, the identities being indicated by gray shading. The positions within cryopyrin containing the pyrin, NBS and LRR domains are indicated by the boxes; these domains show significant homology between cryopyrin and NALP2. The large gaps in the alignment all lie in the LRR domain and result simply from the fact that NALP2 contains six more LRRs than this isoform of cryopyrin.

Fig. 4

Northern-blot analysis of FCAS expression in different tissues. Northernblot analysis was carried out with an exon 1–specific probe. The upper panel illustrates hybridization with the exon 1 probe and the lower panel hybridization with the β-actin control probe. The positions of the molecular-mass markers are indicated to the left and right of the upper panels.