Geryl Wood

Ancient missense mutations in a new member of the RoRet gene family are likely to cause Familial Mediterranean Fever

Familial Mediterranean fever (FMF) is a recessively inherited disorder characterized by dramatic episodes of fever and serosal inflammation. This report describes the cloning of the gene likely to cause FMF from a 115-kb candidate interval on chromosome 16p. Three different missense mutations were identified in affected individuals, but not in normals. Haplotype and mutational analyses disclosed ancestral relationships among carrier chromosomes in populations that have been separated for centuries. The novel gene encodes a 3.7-kb transcript that is almost exclusively expressed in granulocytes. The predicted protein, pyrin, is a member of a family of nuclear factors homologous to the Ro52 autoantigen. The cloning of the FMF gene promises to shed light on the regulation of acute inflammatory responses.Familial Mediterranean fever (FMF) is a recessively inherited disorder characterized by dramatic episodes of fever and serosal inflammation. This report describes the cloning of the gene likely to cause FMF from a 115-kb candidate interval on chromosome 16p. Three different missense mutations were identified in affected individuals, but not in normals. Haplotype and mutational analyses disclosed ancestral relationships among carrier chromosomes in populations that have been separated for centuries. The novel gene encodes a 3.7-kb transcript that is almost exclusively expressed in granulocytes. The predicted protein, pyrin, is a member of a family of nuclear factors homologous to the Ro52 autoantigen. The cloning of the FMF gene promises to shed light on the regulation of acute inflammatory responses.

Germline Mutations in the Extracellular Domains of the 55 kDa TNF Receptor, TNFR1, Define a Family of Dominantly Inherited Autoinflammatory Syndromes

Autosomal dominant periodic fever syndromes are characterized by unexplained episodes of fever and severe localized inflammation. In seven affected families, we found six different missense mutations of the 55 kDa tumor necrosis factor receptor (TNFR1), five of which disrupt conserved extracellular disulfide bonds. Soluble plasma TNFR1 levels in patients were approximately half normal. Leukocytes bearing a C52F mutation showed increased membrane TNFR1 and reduced receptor cleavage following stimulation. We propose that the autoinflammatory phenotype results from impaired downregulation of membrane TNFR1 and diminished shedding of potentially antagonistic soluble receptor. TNFR1-associated periodic syndromes (TRAPS) establish an important class of mutations in TNF receptors. Detailed analysis of one such mutation suggests impaired cytokine receptor clearance as a novel mechanism of disease.

Pyrin binds the PSTPIP1/CD2BP1 protein, defining familial Mediterranean fever and PAPA syndrome as disorders in the same pathway

Pyrin, the familial Mediterranean fever protein, is found in association with the cytoskeleton in myeloid/monocytic cells and modulates IL-1β processing, NF-κB activation, and apoptosis. These effects are mediated in part through cognate interactions with the adaptor protein ASC, which shares an N-terminal motif with pyrin. We sought additional upstream regulators of inflammation by using pyrin as the bait in yeast two-hybrid assays. We now show that proline serine threonine phosphatase-interacting protein [PSTPIP1, or CD2-binding protein 1 (CD2BP1)], a tyrosine-phosphorylated protein involved in cytoskeletal organization, also interacts with pyrin. Recently, PSTPIP1/CD2BP1 mutations were shown to cause the syndrome of pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA), a dominantly inherited autoinflammatory disorder mediated predominantly by granulocytes. Endogenous PSTPIP1/CD2BP1 and pyrin are coexpressed in monocytes and granulocytes and can be coimmunoprecipitated from THP-1 cells. The B box segment of pyrin was necessary and the B box/coiled-coil segment sufficient for this interaction, whereas the SH3 and coiled-coil domains of PSTPIP1/CD2BP1 were both necessary, but neither was sufficient, for pyrin binding. The Y344F PSTPIP1/CD2BP1 mutation, which blocks tyrosine phosphorylation, was associated with a marked reduction in pyrin binding in pervanadate-treated cells. PAPA-associated A230T and E250Q PSTPIP1/CD2BP1 mutations markedly increased pyrin binding as assayed by immunoprecipitation and, relative to WT, these mutants were hyperphosphorylated when coexpressed with c-Abl kinase. Consistent with the hypothesis that these mutations exert a dominant-negative effect on the previously reported activity of pyrin, we found increased IL-1β production by peripheral blood leukocytes from a clinically active PAPA patient with the A230T PSTPIP1/CD2BP1 mutation and in cell lines transfected with both PAPA-associated mutants.

Targeted Disruption of Pyrin, the FMF Protein, Causes Heightened Sensitivity to Endotoxin and a Defect in Macrophage Apoptosis

Familial Mediterranean fever (FMF) is an inherited disorder characterized by recurrent episodes of fever and inflammation. Most patients with FMF carry missense mutations in the C-terminal half of the pyrin protein. To study the physiologic role of pyrin, we generated mice expressing a truncated pyrin molecule that, similar to FMF patients, retains the full PYRIN domain. Bacterial lipopolysaccharide (LPS) induces accentuated body temperatures and increased lethality in homozygous mutant mice. When stimulated, macrophages from these mice produce increased amounts of activated caspase-1 and, consequently, elevated levels of mature IL-1beta. Full-length pyrin competes in vitro with caspase-1 for binding to ASC, a known caspase-1 activator. Apoptosis is impaired in macrophages from pyrin-truncation mice through an IL-1-independent pathway. These data support a critical role for pyrin in the innate immune response, possibly by acting on ASC, and suggest a biologic basis for the selection of hypomorphic pyrin variants in man.

Mutation and Haplotype Studies of Familial Mediterranean Fever Reveal New Ancestral Relationships and Evidence for a High Carrier Frequency with Reduced Penetrance in the Ashkenazi Jewish Population

Familial Mediterranean fever (FMF) is a recessive disorder characterized by episodes of fever with serositis or synovitis. The FMF gene (MEFV) was cloned recently, and four missense mutations were identified. Here we present data from non-Ashkenazi Jewish and Arab patients in whom we had not originally found mutations and from a new, more ethnically diverse panel. Among 90 symptomatic mutation-positive individuals, 11 mutations accounted for 79% of carrier chromosomes. Of the two mutations that are novel, one alters the same residue (680) as a previously known mutation, and the other (P369S) is located in exon 3. Consistent with another recent report, the E148Q mutation was observed in patients of several ethnicities and on multiple microsatellite haplotypes, but haplotype data indicate an ancestral relationships between non-Jewish Italian and Ashkenazi Jewish patients with FMF and other affected populations. Among approximately 200 anonymous Ashkenazi Jewish DNA samples, the MEFV carrier frequency was 21%, with E148Q the most common mutation. Several lines of evidence indicate reduced penetrance among Ashkenazi Jews, especially for E148Q, P369S, and K695R. Nevertheless, E148Q helps account for recessive inheritance in an Ashkenazi family previously reported as an unusual case of dominantly inherited FMF. The presence of three frequent MEFV mutations in multiple Mediterranean populations strongly suggests a heterozygote advantage in this geographic region.

Genome-wide association analysis identifies new susceptibility loci for Behcet's disease and epistasis between HLA-B*51 and ERAP1.

Individuals with Behçets disease suffer from episodic inflammation often affecting the orogenital mucosa, skin and eyes. To discover new susceptibility loci for Behçets disease, we performed a genome-wide association study (GWAS) of 779,465 SNPs with imputed genotypes in 1,209 Turkish individuals with Behçets disease and 1,278 controls. We identified new associations at CCR1, STAT4 and KLRC4. Additionally, two SNPs in ERAP1, encoding ERAP1 p.Asp575Asn and p.Arg725Gln alterations, recessively conferred disease risk. These findings were replicated in 1,468 independent Turkish and/or 1,352 Japanese samples (combined meta-analysis P < 2 × 10−9). We also found evidence for interaction between HLA-B*51 and ERAP1 (P = 9 × 10−4). The CCR1 and STAT4 variants were associated with gene expression differences. Three risk loci shared with ankylosing spondylitis and psoriasis (the MHC class I region, ERAP1 and IL23R and the MHC class I–ERAP1 interaction), as well as two loci shared with inflammatory bowel disease (IL23R and IL10) implicate shared pathogenic pathways in the spondyloarthritides and Behçets disease.

Early-onset stroke and vasculopathy associated with mutations in ADA2

BACKGROUND We observed a syndrome of intermittent fevers, early-onset lacunar strokes and other neurovascular manifestations, livedoid rash, hepatosplenomegaly, and systemic vasculopathy in three unrelated patients. We suspected a genetic cause because the disorder presented in early childhood. METHODS We performed whole-exome sequencing in the initial three patients and their unaffected parents and candidate-gene sequencing in three patients with a similar phenotype, as well as two young siblings with polyarteritis nodosa and one patient with small-vessel vasculitis. Enzyme assays, immunoblotting, immunohistochemical testing, flow cytometry, and cytokine profiling were performed on samples from the patients. To study protein function, we used morpholino-mediated knockdowns in zebrafish and short hairpin RNA knockdowns in U937 cells cultured with human dermal endothelial cells. RESULTS All nine patients carried recessively inherited mutations in CECR1 (cat eye syndrome chromosome region, candidate 1), encoding adenosine deaminase 2 (ADA2), that were predicted to be deleterious; these mutations were rare or absent in healthy controls. Six patients were compound heterozygous for eight CECR1 mutations, whereas the three patients with polyarteritis nodosa or small-vessel vasculitis were homozygous for the p.Gly47Arg mutation. Patients had a marked reduction in the levels of ADA2 and ADA2-specific enzyme activity in the blood. Skin, liver, and brain biopsies revealed vasculopathic changes characterized by compromised endothelial integrity, endothelial cellular activation, and inflammation. Knockdown of a zebrafish ADA2 homologue caused intracranial hemorrhages and neutropenia - phenotypes that were prevented by coinjection with nonmutated (but not with mutated) human CECR1. Monocytes from patients induced damage in cocultured endothelial-cell layers. CONCLUSIONS Loss-of-function mutations in CECR1 were associated with a spectrum of vascular and inflammatory phenotypes, ranging from early-onset recurrent stroke to systemic vasculopathy or vasculitis. (Funded by the National Institutes of Health Intramural Research Programs and others.).

Periodic fever, aphthous stomatitis, pharyngitis, and adenitis (PFAPA) is a disorder of innate immunity and Th1 activation responsive to IL-1 blockade

The syndrome of periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) is the most common periodic fever disease in children. However, the pathogenesis is unknown. Using a systems biology approach we analyzed blood samples from PFAPA patients whose genetic testing excluded hereditary periodic fevers (HPFs), and from healthy children and pediatric HPF patients. Gene expression profiling could clearly distinguish PFAPA flares from asymptomatic intervals, HPF flares, and healthy controls. During PFAPA attacks, complement (C1QB, C2, SERPING1), IL-1–related (IL-1B, IL-1RN, CASP1, IL18RAP), and IFN-induced (AIM2, IP-10/CXCL10) genes were significantly overexpressed, but T cell-associated transcripts (CD3, CD8B) were down-regulated. On the protein level, PFAPA flares were accompanied by significantly increased serum levels of chemokines for activated T lymphocytes (IP-10/CXCL10, MIG/CXCL9), G-CSF, and proinflammatory cytokines (IL-18, IL-6). PFAPA flares also manifested a relative lymphopenia. Activated CD4+/CD25+ T-lymphocyte counts correlated negatively with serum concentrations of IP-10/CXCL10, whereas CD4+/HLA-DR+ T lymphocyte counts correlated positively with serum concentrations of the counterregulatory IL-1 receptor antagonist. Based on the evidence for IL-1β activation in PFAPA flares, we treated five PFAPA patients with a recombinant IL-1 receptor antagonist. All patients showed a prompt clinical and IP-10/CXCL10 response. Our data suggest an environmentally triggered activation of complement and IL-1β/-18 during PFAPA flares, with induction of Th1-chemokines and subsequent retention of activated T cells in peripheral tissues. IL-1 inhibition may thus be beneficial for treatment of PFAPA attacks, with IP-10/CXCL10 serving as a potential biomarker.

The familial Mediterranean fever protein, pyrin, is cleaved by caspase-1 and activates NF-κB through its N-terminal fragment

Familial Mediterranean fever (FMF) is an autoinflammatory disease caused by mutations in MEFV, which encodes a 781-amino acid protein denoted pyrin. We have previously shown that pyrin regulates caspase-1 activation and IL-1beta production through interaction of its N-terminal PYD motif with the ASC adapter protein, and also modulates IL-1beta production by interaction of its C-terminal B30.2 domain with the catalytic domains of caspase-1. We now asked whether pyrin might itself be a caspase-1 substrate, and found that pyrin is cleaved by caspase-1 at Asp330, a site remote from the B30.2 domain. Pyrin variants harboring FMF-associated B30.2 mutations were cleaved more efficiently than wild-type pyrin. The N-terminal cleaved fragment interacted with the p65 subunit of NF-kappaB and with IkappaB-alpha through its 15-aa bZIP basic domain and adjacent sequences, respectively, and translocated to the nucleus. The interaction of the N-terminal fragment with p65 enhanced entrance of p65 into the nucleus. The interaction of N-terminal pyrin with IkappaB-alpha induced calpain-mediated degradation of IkappaB-alpha, thus potentiating NF-kappaB activation. Absolute and relative quantities of cleaved pyrin and IkappaB-alpha degradation products were substantially increased in leukocytes from FMF patients compared with healthy controls. Our data support a new pyrin/caspase-1 pathway for NF-kappaB activation.

Pyrin inflammasome activation and RhoA signaling in the autoinflammatory diseases FMF and HIDS

Mutations in the genes encoding pyrin and mevalonate kinase (MVK) cause distinct interleukin-1β (IL-1β)-mediated autoinflammatory diseases: familial Mediterranean fever (FMF) and hyperimmunoglobulinemia D syndrome (HIDS). Pyrin forms an inflammasome when mutant or in response to bacterial modification of the GTPase RhoA. We found that RhoA activated the serine-threonine kinases PKN1 and PKN2 that bind and phosphorylate pyrin. Phosphorylated pyrin bound to 14-3-3 proteins, regulatory proteins that in turn blocked the pyrin inflammasome. The binding of 14-3-3 and PKN proteins to FMF-associated mutant pyrin was substantially decreased, and the constitutive IL-1β release from peripheral blood mononuclear cells of patients with FMF or HIDS was attenuated by activation of PKN1 and PKN2. Defects in prenylation, seen in HIDS, led to RhoA inactivation and consequent pyrin inflammasome activation. These data suggest a previously unsuspected fundamental molecular connection between two seemingly distinct autoinflammatory disorders.