ABSTRACT
Familial Mediterranean fever (FMF) is a hereditary, autoinflammatory disease that causes recurrent fever, arthritis, and serositis. The diagnosis of FMF is based on the presentation of typical clinical symptoms and the Mediterranean fever gene (MEFV) test. However, the challenge lies in diagnosing atypical cases. In this report, we have described a pediatric patient with complex FMF whose diagnosis required trio-whole exome sequencing (WES) and functional validation of a rare MEFV variant. A 3-year-old boy presented with recurrent episodes of elevated liver enzymes and arthralgia. He was diagnosed with autoimmune hepatitis (AIH), and his liver enzymes improved rapidly with steroid treatment. However, he exhibited recurrent arthralgia and severe abdominal attacks. Trio-WES identified compound heterozygous mutations in MEFV (V726A and I692del). Ex vivo functional assays of the patient's monocytes and macrophages, which had been pre-treated with Clostridium difficile toxin A (TcdA) and colchicine, were comparable to those of typical FMF patients, thereby confirming the diagnosis of FMF. Although he was intolerant to colchicine because of liver toxicity, subsequent administration of canakinumab successfully ameliorated his abdominal attacks. However, it was ineffective against liver injury, which recurred after steroid tapering. Therefore, in this case, the pathogenesis of AIH was probably interleukin-1ß (IL-1ß)-independent. In fact, AIH might have been a concurrent disease with FMF, rather than being one of its complications. Nevertheless, further studies are necessary to determine whether FMF-induced inflammasome activation contributes to AIH development. Moreover, we must consider the possibility of mixed phenotypes in such atypical patients who present distinct pathologies simultaneously.
Subject(s)
Familial Mediterranean Fever , Hepatitis, Autoimmune , Arthralgia , Child , Colchicine/therapeutic use , Familial Mediterranean Fever/complications , Familial Mediterranean Fever/diagnosis , Familial Mediterranean Fever/drug therapy , Hepatitis, Autoimmune/complications , Hepatitis, Autoimmune/diagnosis , Hepatitis, Autoimmune/drug therapy , Humans , Male , Mutation , Pyrin/geneticsABSTRACT
PURPOSE: Pathogenic MEFV variants cause pyrin-associated autoinflammatory diseases (PAADs), which include familial Mediterranean fever (FMF), FMF-like disease, and pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND). The diagnosis of PAADs is established by clinical phenotypic and genetic analyses. However, the pathogenicity of most MEFV variants remains controversial, as they have not been functionally evaluated. This study aimed to establish and validate a new functional assay to evaluate the pathogenicity of MEFV variants. METHODS: We transfected THP-1 monocytes with 32 MEFV variants and analyzed their effects on cell death with or without stimulation with Clostridium difficile toxin A (TcdA) or UCN-01. These variants were classified using hierarchical cluster analysis. Macrophages were obtained from three healthy controls and two patients with a novel homozygous MEFVP257L variant, for comparison of IL-1ß secretion using a cell-based assay and a novel THP-1-based assay. RESULTS: Disease-associated MEFV variants induced variable degrees of spontaneous or TcdA/UCN-01-induced cell death in THP-1. Cell death was caspase-1 dependent and was accompanied by ASC speck formation and IL-1ß secretion, indicating that pathogenic MEFV variants induced abnormal pyrin inflammasome activation and subsequent pyroptotic cell deaths in this assay. The MEFV variants (n = 32) exhibiting distinct response signatures were classified into 6 clusters, which showed a good correlation with the clinical phenotypes. Regarding the pathogenicity of MEFVP257L variants, the results were consistent between the cell-based assay and the THP-1-based assay. CONCLUSION: Our assay facilitates a rapid and comprehensive assessment of the pathogenicity of MEFV variants and contributes to a refined definition of PAAD subtypes.
Subject(s)
Flow Cytometry/methods , Genetic Variation/genetics , Pyrin/genetics , Cell Death/genetics , Cell Line , Female , Genetic Predisposition to Disease/genetics , Humans , Inflammasomes/genetics , Male , Middle Aged , Monocytes/metabolism , Phenotype , THP-1 CellsABSTRACT
The aim of this present study was to establish a new in vitro assay, double artificial membrane permeation assay (DAMPA), to evaluate the human intestinal permeability of drugs. A double artificial membrane with an intracellular compartment was constructed in side-by-side chambers by sandwiching a filter containing buffer solution with impregnated lipophilic filters with dodecane containing 2w/v% phosphatidylcholine. Permeation data of ionic compounds clearly indicated that not only the pH value of the apical solution but also that of the intracellular compartment affected the permeability across the double artificial membrane. DAMPA was performed with 20 compounds at physiological pH (apical; 6.5, intracellular and basal; 7.4). Paracellular and transcellular permeabilities of compounds in human epithelium were estimated based on the characteristics of the paracellular pathway using physicochemical properties of compounds with the Renkin function and the area factor i.e. the difference in the effective surface area between human epithelium and the double artificial membrane, respectively. The human intestinal permeability of each compound was predicted by the sum of estimated transcellular and paracellular permeabilities. Predicted human intestinal permeability was significantly correlated with the fraction of absorbed dose in humans, indicating that DAMPA has the potential to predict oral absorption of drugs in humans.
