Background Familial Mediterranean fever (FMF) is a recessively inherited disorder characterized by self-limited attacks of fever with serosal, synovial, or cutaneous inflammation, sometimes complicated by systemic amyloidosis. In 1992 our laboratory mapped the FMF locus to chromosome 16p13.3, and in 1997 we isolated the underlying gene, MEFV, and demonstrated that it is highly expressed in granulocytes. We have subsequently studied FMF population genetics, the regulation of FMF gene expression in leukocyte subpopulations, the biochemistry and cell biology of pyrin (the FMF protein), and the development of animal models of FMF. The N-terminal 90 amino acids of pyrin comprise a motif, commonly called the PYRIN domain, found in approximately 20 human proteins. The cognate interaction of the PYRIN domain of pyrin with the homologous domain of the apoptosis-associated specklike protein with a CARD (ASC) places pyrin upstream in the regulation caspase-1-mediated interleukin-1 (IL-1) beta activation. In studies of peritoneal macrophages from a mouse strain expressing a truncated form of pyrin, we found increased caspase-1 activation and IL-1 beta processing, and impaired apoptosis through a caspase-8-dependent, IL-1 beta-independent pathway. By yeast two-hybrid studies, we demonstrated that pyrin interacts with the cytoskeletal protein PSTPIP1, and that PSTPIP1 mutations associated with the syndrome of pyogenic arthritis with pyoderma gangrenosum and acne (PAPA) lead to markedly increased pyrin-binding and IL-1 beta activation. Results of the Last Year Mutational studies in FMF patients: We completed studies of a cohort of 46 patients with clinical FMF but only one demonstrable high-penetrance MEFV mutation, and published the findings in Arthritis and Rheumatism. Using standard capillary electrophoresis sequencing, 46 FMF patients with 1 MEFV mutation were screened for a second disease-associated mutation in all 10 exons of MEFV, including the exon-flanking regions. No new mutation was identified in any of the patients analyzed. A subset of 10 DNA samples was selected for additional sequencing of the entire 15-kb MEFV genomic region using hybridization-based chip technology. A novel heterozygous mutation was identified in the putative regulatory region 1 kb upstream of the start codon at position c.-888G->A in a North American patient, but population screening demonstrated that this variant is a polymorphism in the Caucasian population. Haplotype analysis did not identify a common haplotype that might be associated with the transmission of a second FMF allele. Western blots did not demonstrate a significant difference in pyrin levels between patients with a single mutation and those with a double mutation. However, FMF patients of both types showed higher protein expression as compared with controls and with non-FMF patients with active inflammation. Screening of genes encoding pyrin-interacting proteins identified rare mutations in a small number of patients, suggesting the possibility of digenic inheritance. Overall our data indicate that a single mutation in MEFV may, in some circumstances, be sufficient for clinical FMF. Studies of knockin (KI) models of FMF: To study the role of FMF-associated B30.2 domain mutations in the molecular pathogenesis of disease, we generated KI mouse models by inserting the B30.2 domain of wild-type (WT) or FMF-associated M680I, M694V, and V726A mutant pyrin into mouse pyrin, which ordinarily does not include a B30.2 domain. While the WT human B30.2 was embryonic lethal, the mice homozygous for human FMF mutations exhibited a phenotype similar to the human disease, but that was chronic rather than episodic, and generally more severe. KI mice exhibited growth retardation, spontaneous dermatitis and arthritis, and increased CD11b+ cells (especially Ly-6G+ neutrophils) in the blood. Bone marrow (BM) cells of KI mice transferred the KI phenotype into WT mice, and WT BM cells rescued the diseased KI mice, suggesting that BM-derived cells are necessary and sufficient for the disease. Lymphocytes are not required for inflammation since Rag-1 deficient KI mice showed phenotypes similar to Rag-1 sufficient KI mice. In CD45 congenic mixed bone marrow chimeras we found evidence that KI hematopoietic cells could induce WT CD11b expansion. Proinflammatory cytokines, such as IL-1beta, are significantly increased in KI mouse sera, and CD11b+ cells secrete active IL-1beta when stimulated with LPS alone without ATP in vitro. Moreover, the inflammatory phenotype did not appear when KI mice were crossed with IL-1 receptor KO or ASC KO mice. On the other hand, NLRP3-deficient KI mice exhibited the autoinflammatory phenotype. These data suggest that in FMF KI mice there is activation of an ASC-dependent NLRP3-independent inflammasome. We are currently preparing a manuscript describing these findings. Discovery of a novel recessively-inherited autoinflammatory syndrome: During the last year we completed collaborative studies with Dr. Raphaela Goldbach-Mansky establishing the existence of a novel disorder we term the deficiency in IL-1 receptor antagonist (DIRA), and published the initial description of this illness in the New England Journal of Medicine. The clinical findings of this disease include neonatal-onset sterile multifocal osteomyelits, periostitis, and pustulosis. We identified homozygous mutations in IL1RN in a total of nine affected children. One child from a family from Newfoundland, Canada, harbored a two base-pair deletion that caused a frame-shift mutation, N52KfsX25, followed by the incorporation of 24 amino aberrant amino acids and a termination codon. Five children from three unrelated families from the Netherlands were homozygous for a nonsense mutation (E77X). Two children from a consanguineous Lebanese family were homozygous for a second nonsense mutation (Q54X). A ninth patient, from Puerto Rico, was homozygous for a deletion of approximately 175 kb on chromosome 2q that includes six genes IL1RN and five interleukin-1 related genes. The Canadian, Dutch, and Lebanese mutations result in a truncated protein that is not secreted, while the Puerto Rican mutation results in no IL-1 receptor antagonist protein at all. In all cases, this renders patient leukocytes hyperresponsive to IL-1beta stimulation. Patients treated with anakinra responded rapidly. Systems biology analysis of the syndrome of periodic fever with aphthous stomatitis, pharyngitis, and/or cervical adenitis (PFAPA): PFAPA is perhaps the most common recurrent fever syndrome of childhood. The precise pathogenesis is unknown. We therefore undertook a systematic analysis in which we compared whole blood gene expression, serum inflammatory measures, and multicolor lymphocyte immunophenotyping in PFAPA patients during and between flares, healthy controls, and patients with hereditary periodic fever (HPF) syndromes. Principal components analysis revealed the overall mRNA expression profile during PFAPA flares to be remarkably distinct from that in asymptomatic intervals and HPF flares. PFAPA attacks were characterized by a significant overexpression of complement, interleukin-1-related, and interferon-induced genes, whereas T cell-associated transcripts were downregulated. PFAPA flares were most strongly associated with increased serum levels of chemokines for activated T lymphocytes, as well as granulocyte colony-stimulating factor. Activated T lymphocytes correlated inversely with serum concentrations of IP-10/CXCL10. All of five PFAPA patients subsequently treated with anakinra during febrile attacks showed a prompt clinical response. We are currently preparing a manuscript describing our findings.
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