Molecular Basis of Familiar Mediterranean Fever
Molecular Basis of the Autoinflammation Developed in FMF and Related Disorders
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
8 Project completed
Senior Project Manager
Savinova N V
Center of Medical Genetics of NAS RA, Armenia, Yerevan
- Yerevan State Medical University, Armenia, Yerevan
- Institut National de la Santé et de la Recherche Médicale / Bases Moléculaires et Cellulaires des Maladies Génétiques, France, Creteil\nUniversita degli Studi di Genova / Cattedra di Pediatria, Italy, Genova
Project summaryFamilial Mediterranean Fever (FMF; MIM 294100) inherited as an autosomal-recessive trait, is a hereditary autoinflammatory disease most commonly affecting the ethnic groups originating from around the Mediterranean sea, including Armenians, Sephardic Jews, Greeks, Turks, and North African Arabs. This disease is characterized by self-limited recurrent episodes of fever and localized serosal inflammation, and is often complicated by amyloidosis, which is threatening for life. As many as 1 in 200 people in the above-mentioned populations have the disease, and as many as 1 in 5 to 1 in 7 carry a mutated FMF gene. The FMF gene, MEFV is localized on chromosome 16 (16p13.3) and more than 50 FMF-associated mutations have been identified in exons 2, 3, 5, and 10. As for the function of the protein encoded by MEFV and called pyrin, very few data are available: MEFV is expressed predominantly in innate immune cells. Pyrin is an intracellular protein, found either in the nucleus or the cytoplasm, depending on the cell type analyzed. It contains protein-protein interaction module known as the pyrin domain (PYD), which has now been found in several proteins that regulates apoptosis and inflammation. One of these is the adaptor protein ASC (apoptosis-associated speck-like protein with CARD), which can interact with pyrin through a PYD-PYD homotypic association. ASC is known to trigger autocatalysis of caspase-1, the enzyme, which cleaves the pro-inflammatory cytokine IL-1β, a cytokine central to the process of inflammation, into its active form. Targeted disruption of pyrin in the mouse shows an increase in IL-1β processing, and defective lipopolysacharide (LPS) and IL-4 induced apoptosis in peritoneal monocytes. Thus, it appears that pyrin is a suppressor of the activation of caspase-1. It is proposed that pyrin is a part of regulatory pathway of inflammation and apoptosis and that mutation in MEFV result in pyrin abnormalities that lead to uncontrolled inflammation. Physiological function of pyrin and its role in FMF have not yet been resolved; it is not conclusively known what exactly sets off the attacks.
We have provided the first demonstration of the diagnostic and prognostic value of MEFV analysis; our teams indeed described the first objective criterion for the diagnosis of FMF, and identified one particular MEFV genotype associated with a high risk of developing renal amyloidosis in Armenian patients. This latter result has been secondarily confirmed by numerous teams in different populations. We subsequently showed that susceptibility to renal amyloidosis is influenced by at least two MEFV-independent factors of genetic origin —SAA1 and sex— that act independently of each other. More recently, we showed that among patients from Karabakh and among all classically affected populations as well, the distribution of genotypes differed dramatically from Hardy-Weinberg equilibrium.
In order to further characterize the biological properties of pyrin, we identified a new isoform of pyrin (pyrin-d2) whose subcellular location (essentially nuclear) is dramatically different from that of the first identified (cytoplasm) isoform (pyrin-fl). Following a yeast two-hybrid screen, we recently identified 14.3.3 proteins as novel pyrin partners, and showed that this interaction occurs in an pyrin isoform-specific and phosphorylation-dependent manner that regulates the translocation of pyrin to the nucleus. We indeed showed that pyrin-fl but not pyrin-d2 is able to bind 14.3.3. and provide the first evidence that pyrin-fl is phosphorylated. In addition we subsequently studied the impact of the most frequent MEFV mutations on the formation of cytoplasm aggregates (called “specks”) in the presence of ASC and also identified the first transcription factors (NFκB and C/EBPβ) that play a crucial role in regulating the TNF-α-dependent transcription of MEFV.
The finding that FMF-associated mutations induce heightened sensitivity to endotoxin has important implications for understanding the episodic nature of FMF. It remains unknown how heightened sensitivity to endotoxin during FMF could be a result of innate immune cell activation switching and the impaired resolution of inflammation. In FMF patients, during the period of remission, we observed up-regulation of neutrophil and monocyte phagocyte activity and chemotaxis- and phagocytosis-dependent oxidative burst followed by down-modulation in FMF patients during an attack. We demonstrated that LPS-induced oxidative burst and CD11a/CD18 integrin surface expression is higher in FMF patients during an attack compared to patients in remission. The induction of homologous tolerance of monocytes to the repeated action of LPS is observed in FMF patients during an attack, whereas monocytes from patients in remission failed to induce LPS homologous tolerance and exhibit heightened sensitivity to bacterial endotoxin. Moreover, we found that colchicine is able to restore impaired LPS-homologous tolerance induction in FMF patients in the period of remission upon the increase of IL-4 synthesis in FMF patient monocytes. Thus, chronic inflammation during FMF is characterized by periodic changes in monocyte activation and heightened sensitivity to endotoxin which is associated with the episodic nature of FMF. Although these data contribute to a better understanding of the structure/function relationships of the pyrin protein, the precise mechanism by which the normal and mutated pyrin isoforms act on inflammatory pathways is, however, still to be elucidated.
These results, combined with other population genetics-based data, reveal the existence of an FMF-like condition unrelated to MEFV and that is today indiscernible from MEFV-related FMF. Most importantly, as demonstrated in our studies, this condition explains nearly all the cases of FMF in whom no MEFV mutation has been identified (i.e. 85 to 99% of those patients, depending on their ethnic origin). Subsequent study of recurrent fevers in western European Caucasian patients, which showed that MEFV analysis is of particular weak diagnostic value in those patients, led to the similar conclusion of the existence of a newly recognized entity still called FMF, but unrelated to MEFV.
These last data raise two important questions:
- what are the gene(s) involved in this disease condition unrelated to MEFV? (we will call this disorder “FMF-like condition”, in order avoid any confusion with the FMF disease caused by mutations in MEFV).
- is it possible to identify new biological phenotypic markers that could discriminate between these two clinically similar entities?
The aim of this project is to better describe and identify the molecular basis of the FMF condition related to MEFV and the FMF-like condition unrelated to MEFV. The specific objectives are the following:
- To discover by positional cloning the genes involved in the families with a FMF-like phenotype unrelated to MEFV and in which no genetic explanation has been found.
- To search for phenotypic markers that could discriminate between the two following entities: the FMF condition due to mutations in MEFV and the FMF-like condition unrelated to MEFV, through the accurate determination of the cytokine profiling associated with each of these conditions.
- To decipher the mechanisms underlying the auto-inflammation observed in FMF related to MEFV.
We aim to focus our efforts on the Karabakhian population of patients, a clinically and genetically homogeneous population in which FMF-like phenotypes are particularly frequent. In order to characterize loci containing genes responsible for the FMF-like condition and to determine intervals as narrow as possible, we will perform, a genome scan using Affymetrix Gene chips which allow the genotyping of at least 250,000 SNP of an inpidual with one chip. It will then be possible for us to characterize haplotypes of these blocks, and thus to detect ancestral recombination events making it possible to reduce the intervals. We will also use a candidate-gene approach to search for genetic factors involved in the FMF-like disease including the analysis of the recently recognized gene family encoding proteins with domains of striking similarity with those found in pyrin or in cryopyrin. All the candidate genes will be sequenced (sequencing limited to exons and introns/exons boundaries). Any sequence variations (compared to the databases) will be searched in all members of the larger families and a co-segregation between the variant and the disease will be searched in order to distinguish between polymorphisms and mutations. The validated genes will be analyzed in all the corresponding families, and in the FMF-like families with only one mutation detected in the MEFV gene (assuming a digenic model). To search for phenotypic markers that could discriminate between the FMF condition due to mutations in MEFV and the FMF-like condition unrelated to MEFV we plan to determine the cytokine profile associated with each of these conditions, endotoxin homologous- and cross-tolerance induction in patients with FMF and FMF-like condition; and influence of colchicine, TLR4, TLR2, TLR9, NALP3 and NOD2 agonists on monocyte endotoxin tolerance induction. We will also use PCR array profiling the expression of key genes related to NFκB- and TLR-mediated signal transduction, and expression of genes involved in the inflammatory response including cytokines and their receptors. To decipher the mechanisms underlying the auto-inflammation observed in FMF related to MEFV we plan to study the activation of the NFB pathway in the presence or absence of ASC; production of various cytokines in response to the silencing of the endogenously expressed MEFV and the role of wild-type and various mutant pyrin proteins on the ASC-dependent cleavage of procaspase 1 into caspase 1.
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