Pyrin responds to pathogen signals and loss of cellular homeostasis by forming an inflammasome complex that drives the cleavage and secretion of IL-1β. We studied a family with dominantly inherited autoinflammatory disease characterised by childhood-onset recurrent episodes of neutrophilic dermatosis, fever, elevated acute-phase reactants, arthralgia, and myalgia/myositis. Disease was caused by a mutation in MEFV, the gene encoding pyrin (S242R). The clinical distinction from FMF, also caused by MEFV mutation, was due to loss of a 14-3-3 binding motif at phosphorylated S242. This interaction represents a guard regulating pyrin activation, which is downstream of bacterial effectors that trigger the pyrin inflammasome. S242R mutation recapitulated the effect of pathogen sensing, triggering inflammasome activation and IL-1β production. Successful therapy targeting IL-1β has been initiated in one patient, resolving Pyrin-Associated Autoinflammation with Neutrophilic Dermatosis (PAAND). This unique disease provides evidence that a guard mechanism, originally identified in plant innate immunity, also exists in humans.3 Main textAutoinflammatory diseases are characterized by recurrent episodes of fever with systemic and organ-specific inflammation, as well as uncontrolled activation of the innate immune response in the apparent absence of an infectious trigger(1). Familial Mediterranean fever (FMF, OMIM ID: 249100) is the most common of these monogenic diseases, characterized by short (24-72 h) episodes of fever associated with serositis, progressing to amyloidosis if untreated(2). FMF is an autosomal recessive disease caused by mutations in both alleles of the MEFV (MEditerranean FeVer) locus, which encodes the protein pyrin(3). Normally, pyrin functions as a link between intracellular pathogen sensing and activation of the inflammasome, allowing the production of inflammatory mediators during infection. As a potent checkpoint for the initiation of inflammation, the mechanisms of pyrin regulation are critical, and yet still poorly understood.We studied a three-generation Belgian family of 22 individuals, of whom 12 developed autoinflammatory disease (Figure 1a). The disease was characterized by neutrophilic dermatosis, childhood-onset recurrent episodes of fever lasting several weeks, increased levels of acute-phase reactants, arthralgia and myalgia/myositis (Figure 1b). The neutrophilic dermatosis comprised a spectrum of clinical manifestations including severe acne, sterile skin abscesses, pyoderma gangrenosum and neutrophilic small vessel vasculitis (Figure 1c,d).Pathological examination of affected skin showed a dense, predominantly neutrophilic, vascular, perivascular and interstitial infiltrate (Figure 1d). Serum cytokine analysis revealed elevated inflammatory mediators such as IL-1β, IL-6 and TNFα, and cytokines induced by inflammation such as IL-1Ra (Figure 1e, Figure S1a unlike some of the more typical FMF variants, that naturally occur in other species(7). Despite the association of MEFV mutations w...

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-B 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.genetics ͉ Mendelian disorder ͉ NLRP ͉ autoinflammatory disorder ͉ CATERPILLER

Although the number of NLRP3 test requests has doubled over the past 5 years, genetic screening has not contributed to enhanced detection of new index cases each year. There are two possible reasons for this: (i) no clinical prerequisite for genetic diagnosis and (ii) few new large families are now identified (unlike the initial study based on a selection by linkage). A set of initial clinical criteria have been drawn up which it is recommended should be fulfilled before a patient is tested: at least three recurrent bouts, age at disease onset < 20 years and elevated levels of C-reactive protein, especially in individuals with urticaria and moderate fever.

Background: An accurate estimation of the risk of life-threatening (LT) ventricular tachyarrhythmia (VTA) in patients with LMNA mutations is crucial to select candidates for implantable cardioverter defibrillator (ICD) implantation. Methods: We included 839 adult patients with LMNA mutations, including 660 from a French nationwide registry in the development sample, and 179 from other countries, referred to 5 tertiary centers for cardiomyopathies, in the validation sample. LTVTA was defined as a) sudden cardiac death or b) ICD-treated or hemodynamically unstable VTA. The prognostic model was derived using Fine-Gray's regression model. The net reclassification was compared with current clinical practice guidelines. The results are presented as means (standard deviation) or medians [interquartile range]. Results: We included 444 patients 40.6 (14.1) years of age in the derivation sample and 145 patients 38.2 (15.0) years in the validation sample, of whom 86 (19.3%) and 34 (23.4%) suffered LTVTA over 3.6 [1.0-7.2] and 5.1 [2.0-9.3] years of follow-up, respectively. Predictors of LTVTA in the derivation sample were: male sex, non-missense LMNA mutation, 1st degree and higher atrioventricular block, non-sustained ventricular tachycardia, and left ventricular ejection fraction. In the derivation sample, C-index (95% CI) of the model was 0.776 (0.711-0.842) and calibration slope 0.827. In the external validation sample, the C-index was 0.800 (0.642-0.959) and calibration slope 1.082 (95% CI, 0.643-1.522). A 5-year estimated risk threshold ≥7% predicted 96.2% of LTVTA and net reclassified 28.8% of patients with LTVTA compared with the guidelines-based approach. Conclusions: Compared to the current standard of care, this risk prediction model for LTVTA in laminopathies facilitated significantly the choice of ICD candidates. Clinical Trial Registration: URL: https://www.clinicaltrials.gov. Unique Identifier: NCT03058185.

Objective. To gain insight into the molecular bases of genetically unexplained periodic fever syndromes (PFS) by screening NLRP12, a gene in which only a nonsense and a splice site mutation have so far been identified, and to assess the functional consequences of the identified missense variation.Methods. NLRP12 was screened for mutations by direct sequencing. Functional assays were performed in HEK 293T cells stably expressing the proapoptotic protein ASC and procaspase 1, in order to determine the effects of normal and mutated NLRP12 proteins on speck formation, caspase 1 signaling, and NF-B activation.Results. A heterozygous NLRP12 missense mutation involving a CpG site (c.1054C>T; p.Arg352Cys) was identified in exon 3, which encodes the nucleotidebinding site (NBS) of the protein, in 2 patients from different countries and carrying different NLRP12 haplotypes. The mutation, which does not alter the inhibitory effect of NLRP12 on NF-B activation, increases speck formation and activates caspase 1 signaling. To define this new class of PFS, we propose the term NLRP12-associated disorders (NLRP12AD).Conclusion. Given the rarity of known NLRP12-associated disorders, the identification of this NLRP12 molecular defect contributes to the delineation of the clinical spectrum associated with mutations in this gene and highlights the importance of screening NLRP12 in patients presenting with unexplained PFS. This study also demonstrates, by means of functional assays, the deleterious effect of this recurrent missense mutation; the gain of function for speck formation and caspase 1 signaling associated with this NBS mutation is consistent with the inflammatory phenotype of PFS.Periodic fever syndromes (PFS) are Mendelian autoinflammatory disorders characterized by recurrent episodes of fever and systemic inflammation, sometimes complicated by amyloidosis. Recently, the first two recognized disease-causing mutations (a nonsense mutation and a splice site mutation) have been identified in the NLRP12 gene (also known as NALP12 or MONARCH-1) in patients with a clinical picture suggestive of a cryopyrin-associated periodic syndrome (CAPS) (1). CAPS represent a subgroup of PFS comprising the familial cold-induced autoinflammatory syndrome, the Muckle-Wells syndrome, and the chronic infantile neurologic, cutaneous, articular (CINCA) syndrome, also known as neonatal-onset multisystem inflammatory disease. These three disorders have been associated with mutations in NLRP3, which, like NLRP12, is a member of the NLRP family.Diagnosis of PFS remains difficult, for the following reasons: there is no pathognomonic sign of these disorders; many patients present with symptoms that do not fit any classification; the majority of patients referred today are sporadic cases, precluding intrafamilial segregation analyses; and the majority of mutations identified in PFS genes correspond to missense variations, whose deleterious effects are often questionable.

Objective. Pyrin, the familial Mediterranean fever gene product, exists in several isoforms of unknown functions. The recombinant full-length isoform (pyrin.fl) is cytoplasmic, whereas an alternatively spliced isoform lacking exon 2 (pyrin.⌬Ex2) concentrates in the nucleus. Native pyrin, mainly consisting of pyrin.fl, is also cytoplasmic in monocytes but is predominantly nuclear in other cell types. To understand pyrin-dependent biologic pathways and to decipher the mechanisms accounting for such different patterns of subcellular compartmentalization, binding partners and posttranslational modifications of pyrin were assessed. Conclusion. These results disclose the role played by 14.3.3 in the regulation of the subcellular compartmentalization of pyrin in a phosphorylation-and isoform-dependent manner. They also reconcile the observations made in vitro with those made in vivo, while providing a direct link between 14.3.3-dependent pathways and pyrin.Pyrin, also called marenostrin, is the product of the MEFV gene involved in familial Mediterranean fever (FMF) (MIM no. 249100) (1,2). FMF is an autosomal recessive condition that primarily affects populations of Mediterranean extraction and is characterized by recurrent episodes of fever and serosal inflammation, manifested by sterile peritonitis, arthritis, and/or pleurisy. MEFV, which is composed of 10 coding exons and spans 15 kb on the 16p13.3 region, is expressed mainly in granulocytes and monocytes (3,4). Although the physiologic role of the pyrin/marenostrin protein is yet to be elucidated, the identification of a domain (called pyrin, or PYD) in its N-terminal region has revealed the existence of an expanding family of related PYDcontaining proteins involved in both inflammatory and

Methods. Patients' disease manifestations and cytokine measurements were recorded before anakinra treatment was started, during 14 months of therapy, and after discontinuation of anakinra treatment.Results. Spontaneous secretion of IL-1␤ by patients' PBMCs was found to be dramatically increased (80-175 fold) compared to healthy controls. Consistent with these findings, anakinra initially led to a marked clinical improvement and to a rapid near-normalization of IL-1␤ secretion. However, a progressive clinical relapse occurred secondarily, associated with an increase in TNF␣ secretion, persistent elevated levels of IL-1Ra and IL-6, and a reactivation of IL-1␤ secretion. Anakinra was discontinued after 14 months of therapy.Conclusion. Our findings provide in vivo evidence of the crucial role of IL-1␤ in the pathophysiology of NLRP12AD. This is the first time anakinra has been used to treat this disorder. This study provides new insights into the mechanisms underlying resistance to anti-IL-1 therapy observed in a few patients with autoinflammatory syndromes. Our data also point to the potential of ex vivo cytokine measurements as predictors of response to treatment.Recurrent fever syndromes are autoinflammatory disorders characterized by recurrent episodes of fever, systemic inflammation, sterile peritonitis, arthritis, and/or cutaneous manifestations. Among them, cryopyrin-associated periodic syndromes have been associated with mutations in NLRP3, a gene of myelomonocytic expression. Peripheral blood mononuclear cells (PBMCs) from patients carrying NLRP3 mutations secrete excessive amounts of interleukin-1␤ (IL-1␤) (e.g., refs. 1 and 2), a crucial cytokine in systemic inflammation. IL-1␤ acts on multiple organs and amplifies inflammation by inducing the expression of proinflammatory cytokines (including IL-1␤ itself) and numerous genes involved in inflammatory processes. In patients with cryopyrin-associated periodic syndromes, there is constitutive activation of a multiprotein complex, called the NLRP3 inflammasome, which triggers the cleavage of procaspase 1 into caspase 1 and leads to the conversion of proIL-1␤ to mature IL-1␤. Once

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