Mutations in NALP12 cause hereditary periodic fever syndromes

Abstract

NALP proteins, also known as NLRPs, belong to the CATERPILLER protein family involved, like Toll-like receptors, in the recognition of microbial molecules and the subsequent activation of inflammatory and immune responses. Current advances in the function of NALPs support the recently proposed model of a disease continuum bridging autoimmune and autoinflammatory disorders. Among these diseases, hereditary periodic fevers (HPFs) are Mendelian disorders associated with sequence variations in very few genes; these variations are mostly missense mutations whose deleterious effect, which is particularly difficult to assess, is often questionable. The growing number of identified sporadic cases of periodic fever syndrome, together with the lack of discriminatory clinical criteria, has greatly hampered the identification of new disease-causing genes, a step that is, however, essential for appropriate management of these disorders. Using a candidate gene approach, we identified nonambiguous mutations in NALP12 (i.e., nonsense and splice site) in two families with periodic fever syndromes. As shown by means of functional studies, these two NALP12 mutations have a deleterious effect on NF-kappaB signaling. Overall, these data identify a group of HPFs defined by molecular defects in NALP12, opening up new ways to manage these disorders. The identification of these first NALP12 mutations in patients with autoinflammatory disorder also clearly demonstrates the crucial role of NALP12 in inflammatory signaling pathways, thereby assigning a precise function to this particular member of an emerging family of proteins whose putative biological properties are currently inferred essentially through in vitro means.

Fig. 1.

Genealogical trees and mutational analysis of two families with periodic fever syndromes. Filled symbols represent patients with periodic fever syndromes, and open symbols indicate unaffected family members. Sequencing chromatograms are presented. Red circles indicate the positions of mutations. Upper and lowercases correspond to exonic and intronic sequences, respectively. Index cases are shown by arrows. (A) Family 1. (B) Family 2.

Fig. 2.

Impact of the c.2072+3insT mutation on NALP12 splicing. (A) (Upper) RT-PCR amplifications of NALP12 transcripts from HEK293T cells transfected with pNALP12g-WT and pNALP12g-c.2072+3insT mini-genes. β-actin was amplified on the same sample as control. L, 1-kb+ marker; EV, empty vector. (Lower) The diagram indicates the location of the primers used in RT-PCR experiments (arrows). Exons and introns are represented by gray boxes and thin lines, respectively. The c.2072+3insT insertion is indicated by an arrowhead. (B) Chromatograms obtained after sequencing of the RT-PCR products presented in A (middle gel) and corresponding to the exon 3–exon 4 junction. (C) Schematic representation of the effect of c.2072+3insT on NALP12 splicing. (Upper) Normal splicing of intron 3. (Lower) Splicing resulting from the c.2072+3insT mutation, indicated by a black arrowhead. Gray boxes correspond to NALP12 exons; upper and lowercases correspond to exonic and intronic sequences, respectively. The cryptic donor splice site is indicated in italics.

Fig. 3.

Functional consequences of the identified mutations on NALP12 function. (A) Schematic representation of human NALP12 and partial alignment of its amino acid sequence (GenBank accession no. NP_653288) with corresponding sequences in other species (chimpanzee, XP_524387.2; mouse, XP_978890.1; rat, XP_001066862.1; dog, Ensemble accession no. ENSCAFP00000003989). Alignment with human NALP3 (GenBank accession no. NP_004886.3) is also presented. The Arg-284 residue is indicated by an asterisk, and the Val-635 residue is indicated by an arrowhead. Black shading indicates identical residues, and gray shading indicates similar residues. PYD, pyrin domain; NBS, nucleotide-binding site; LRR; leucin-rich repeats. (B) Effect of NALP12-WT and NALP12-mutated proteins on NF-κB signaling. HEK293T cells were transfected with 100 ng of the pNF-κB-LUC luciferase reporter and with 800 ng of plasmids encoding NALP12-WT, NALP12-Arg284X, or NALP12-Val635ThrfsX12 as indicated. The NF-κB signaling pathway was induced by transfection of 100 ng of p65. Then, 24 h after transfection, luciferase activities were determined on cell lysates; results of one representative experiment done in triplicate are expressed as means ± SD. NALP12 proteins were quantitated by Western blot analysis on the same protein lysates. Membranes were reprobed with anti-α-tubulin antibodies as a loading control. EV, empty vector.