The clinical continuum of cryopyrinopathies: novel CIAS1 mutations in North American patients and a new cryopyrin model

Abstract

Objective: The cryopyrinopathies are a group of rare autoinflammatory disorders that are caused by mutations in CIAS1, encoding the cryopyrin protein. However, cryopyrin mutations are found only in 50% of patients with clinically diagnosed cryopyrinopathies. This study was undertaken to investigate the structural effect of disease-causing mutations on cryopyrin, in order to gain better understanding of the impact of disease-associated mutations on protein function.

Methods: We tested for CIAS1 mutations in 22 patients with neonatal-onset multisystem inflammatory disease/chronic infantile neurologic, cutaneous, articular syndrome, 12 with Muckle-Wells syndrome (MWS), 18 with familial cold-induced autoinflammatory syndrome (FCAS), and 3 probands with MWS/FCAS. In a subset of mutation-negative patients, we screened for mutations in proteins that are either homologous to cryopyrin or involved in the caspase 1/interleukin-1beta signaling pathway. CIAS1 and other candidate genes were sequenced, models of cryopyrin domains were constructed using structurally homologous proteins as templates, and disease-causing mutations were mapped.

Results: Forty patients were mutation positive, and 7 novel mutations, V262A, C259W, L264F, V351L, F443L, F523C, and Y563N, were found in 9 patients. No mutations in any candidate genes were identified. Most mutations mapped to an inner surface of the hexameric ring in the cryopyrin model, consistent with the hypothesis that the mutations disrupt a closed form of cryopyrin, thus potentiating inflammasome assembly. Disease-causing mutations correlated with disease severity only for a subset of known mutations.

Conclusion: Our modeling provides insight into potential molecular mechanisms by which cryopyrin mutations can inappropriately activate an inflammatory response. A significant number of patients who are clinically diagnosed as having cryopyrinopathies do not have identifiable disease-associated mutations.

Figures

Figure 1

Sequence alignment of cryopyrin with NALP1, NALP6, NALP10, and NALP12 for most of exon 3 of cryopyrin encoding the NACHT and NACHT-associated domains. Asterisks below the alignments show positions with perfect identity. AAA+ domain functional motifs that are identifiable in cryopyrin are shown in red. Disease-causing mutations are shown above the sequence; boxed mutations are those newly identified in this study. The consensus secondary structure prediction for all proteins from multiple prediction programs is depicted using bars for α-helices (α1–α22) and arrows for β-strands (β1–β6). Secondary structural elements shown in dark green correspond to the first subdomain of NACHT, those shown in light green correspond to the second (and possibly third) subdomain(s) of NACHT, and those shown in yellow-green correspond to the NACHT-associated domain.

Figure 2

A, The domain and exon arrangements of cryopyrin displayed with the positions of point mutations implicated in familial cold-induced autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), neonatal-onset multisystem inflammatory disease (NOMID), and other phenotypes. Domains are displayed as colored boxes; exons are numbered below and delineated by horizontal lines. Point mutations responsible for various clinical syndromes are displayed from the weakest phenotype (FCAS) on the light yellow portion of the gradient to the strongest phenotype (NOMID) on the red portion of the gradient. B, Three-dimensional models of the pyrin domain, subdomain 1 of NACHT, and the leucine-rich repeats (LRRs). Disease-causing mutations are displayed as spheres at the Cα positions, color-coded as yellow for FCAS, orange for MWS, red for NOMID, and gray for other. For amino acid positions identified in multiple syndromes, the most severe phenotype is indicated. The pyrin domain of cryopyrin was threaded onto the pyrin domain of human ASC (Protein Data Bank [PDB] code no. 1ucp [52]). The first domain of NACHT was generated using the structure of apoptotic protease–activating factor 1 (APAF1; PDB code no. 1z6t [53]). APAF1 and cryopyrin deviate between α5 and β4 and were modeled using consensus secondary structure predictions. This region includes the positions of the G326E and S331R mutations (red spheres that do not map to the common mutation surface). The LRR domains were modeled using the porcine ribonuclease inhibitor structure (PDB code no. 2bnh [54]). C, Superimposition of the model for NACHT subdomain 1 on the hexameric assembly of the AAA+ ATPase HslU (PDB code no. 1doo [48]). Most of the mutations (colored as in B) map to the inner, concave surface of the hexameric ring. In other AAA+ ATPases, this surface is where AAA+ subdomains 2 and 3 tend to contact subdomain 1 (46). As in other AAA+-ATPases, the hexameric assembly places nucleotide-binding sites at the interfaces between adjacent protomers. D, Location of the disease-causing mutations in cryopyrin, suggesting a model for inappropriate inflammasome assembly. In wild-type cryopyrin, opening of the protein to expose the pyrin and LRR domains is normally triggered due to an external stimulus. The open state of cryopyrin is presumably the active one that assembles a functional inflammasome. With mutations in the NACHT domain, the closed state is destabilized due to mutations at the interface of the hinge, tending to favor the activated and open state. The G755 mutations might similarly drive inflammasome assembly by producing a “kink” in the regular LRR structure that inappropriately exposes the LRR domains either to more readily open the structure or to become competent to directly assemble the inflammasome.