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. During the 8 years leading up to the present reporting period, we have focused on several areas, including 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, with a 6 alpha helical structure similar to death domains, death effector domains, and caspase recruitment domains (CARDs), that is found in approximately 20 human proteins. We and others have demonstrated the cognate interaction of the PYRIN domain of pyrin to the homologous domain of the apoptosis-associated specklike protein with a CARD (ASC), placing 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? ? Direct interaction of the B30.2 domain of pyrin with caspase-1: During the last year we completed studies examining the possible interaction of the C-terminal B30.2 domain of pyrin with caspase-1. The B30.2 domain of pyrin is of particular importance because the preponderance of FMF-associated mutations occur in this domain, and carrier frequencies of several such mutations are extremely high in Middle Eastern populations. In lysates from cells not expressing ASC, reciprocal GST pull-downs demonstrated the interaction of pyrin with the p20 and p10 catalytic subunits of caspase-1. Coimmunoprecipitations of pyrin and caspase-1 from THP-1 human monocytic cells were consistent with the interaction of endogenous proteins. The C-terminal B30.2 domain of pyrin is necessary and sufficient for the interaction, and binding was reduced by FMF-associated B30.2 mutations. Full-length pyrin attenuated IL-1 beta production in cells transfected with a bicistronic caspase-1/IL-1 beta construct, an effect diminished by FMF-associated B30.2 mutations and in B30.2 deletion mutants. Modeling of the crystal structure of caspase-1 with the deduced structure of the pyrin B30.2 domain corroborated both the interaction and the importance of the M694V and M680I pyrin mutations. Consistent with a net inhibitory effect of pyrin on IL-1 beta activation, small interfering RNA (siRNA)-mediated pyrin knockdown in THP-1 cells augmented IL-1 beta production in response to bacterial LPS. Moreover, the IL-1 receptor antogonist anakinra suppressed acute-phase proteins in a patient with FMF and amyloidosis. Overall, these data support a direct, ASC-independent effect of pyrin on IL-1 beta activation and suggest heightened IL-1 responsiveness as one factor selecting for pyrin mutations.? ? Studies of murine models of FMF: During the last year we have continued studies of pyrin null mice begun in the previous reporting period. Gene expression profiling of resident peritoneal macrophages demonstrated marked differences in gene expression patterns between cells from wildtype and knockout mice, whether they were stimulated with lipopolysaccharide (LPS) or not. Several functional networks of up- and down-regulated genes were identified, and we are now in the process of testing functional hypotheses generated from these analyses. We have also generated knockin mice for the M680I, M694V, and V726A mutations. We have observed inflammatory phenotypes in all 3 knockin strains, but have been uncertain of the physiologic significance because of our previous inability to achieve germline transmission in the knockin of the wildtype human B30.2 domain. We have now achieved germline transmission of this important control, and will studying their phenotype. ? ? Studies of a canine model of FMF: The Chinese Shar-Pei dog has been proposed as a possible canine model for FMF. Some dogs of this breed suffer recurrent episodes of fever, arthritis, and skin swelling that may begin as early as two months of age, and systemic AA amyloidosis may also develop later in life. Pedigree analyses are consistent with a single-gene autosomal recessive mode of inheritance, prompting the term ?familial Shar-Pei fever? (FSF) for this disorder. Extending the similarity with human FMF, dogs with FSF often respond, at least partially, to colchicine. To test whether canine FSF might be caused by mutations in the canine MEFV, we cloned the CaMEFV from peripheral blood mononuclear cells from an unaffected Shar-Pei dog. CaMEFV is a 10-exon gene encoded on dog chromosome 6. The cDNA has an open reading frame of 2577 nucleotides, encoding a predicted protein of 858 amino acids. The deduced canine amino acid sequence was 57.1% identical to the human protein, with conserved PYRIN, B-box zinc-finger, nuclear localization signal, and B30.2 domains. CaMEFV is highly expressed in PBMCs, and to a lesser extent in brain, liver, and testis. We screened a panel of cDNAs from 7 affected Shar-Pei dogs, 3 unrelated and unaffected Shar-Pei dogs, and 10 unaffected dogs of other breeds. Although we identified a coding single nucleotide polymorphism (SNP), R130W, this did not associate with disease. To test the hypothesis that FSF might, instead, be a canine version of the TNF receptor-associated periodic syndrome, we also cloned the canine TNFRSF1A gene. This gene is encoded on the dog chromosome 27, encoding a predicted protein of 452 amino acids that is 73.4% identical to the human protein, with good conservation of the domain structure. Although we identified two SNPs, neither associated with disease. Given these data, we are about to conduct a chip-based genome-wide SNP analysis to identify the chromosomal location of the FSF susceptibility locus. ? ? Studies of patients: Among children with recurrent fever syndromes, over half have no identifiable mutations at any of the known periodic fever genes. We set out to determine the natural history of this set of patients. Among 65 patients seen in our clinic between January, 2001, and December, 2005, we were able to contact 51 for followup data. The average length of followup was 29.47 months, and the average age of patients was 9.1 years, with 26 males and 25 females. Twenty-five patients were still having fevers at the time of followup. Skin rash and pharyngitis appeared to be predictive of continued fevers in our cohort.? ? Conclusions and Significance? ? Biochemical and functional data obtained during the current reporting period corroboroate earlier findings implicating pyrin as an important regulator of inflammation. During the next year we plan to continue studies of pyrin biochemistry and function, continue investigations of murine models of FMF, attempt to map and possibly clone the canine FSF gene, and continue studies of patients with FMF and related phenotypes.
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