title: The novel S59P mutation in the TNFRSF1A gene identified in an adult onset TNF receptor associated periodic syndrome (TRAPS) constitutively activates NF-κB pathway.
abstract: BACKGROUND
  Mutations in the TNFRSF1A gene, encoding tumor necrosis factor receptor 1 (TNF-R1), are associated with the autosomal dominant autoinflammatory disorder, called TNF receptor associated periodic syndrome (TRAPS). TRAPS is clinically characterized by recurrent episodes of long-lasting fever and systemic inflammation. A novel mutation (c.262 T > C; S59P) in the TNFRSF1A gene at residue 88 of the mature protein was recently identified in our laboratory in an adult TRAPS patient. The aim of this study was to functionally characterize this novel TNFRSF1A mutation evaluating its effects on the TNF-R1-associated signaling pathways, firstly NF-κB, under particular conditions and comparing the results with suitable control mutations.
  
  
  METHODS
  HEK-293 cell line was transfected with pCMV6-AC construct expressing wild-type (WT) or c.262 T > C (S59P), c.362G > A (R92Q), c.236C > T (T50M) TNFRSF1A mutants. Peripheral blood mononuclear cells (PBMCs) were instead isolated from two TRAPS patients carrying S59P and R92Q mutations and from five healthy subjects. Both transfected HEK-293 and PBMCs were stimulated with tumor necrosis factor (TNF) or interleukin 1β (IL-1β) to evaluate the expression of TNF-R1, the activation of TNF-R1-associated downstream pathways and the pro-inflammatory cytokines by means of immunofluorescent assay, array-based technique, immunoblotting and immunometric assay, respectively.
  
  
  RESULTS
  TNF induced cytoplasmic accumulation of TNF-R1 in all mutant cells. Furthermore, all mutants presented a particular set of active TNF-R1 downstream pathways. S59P constitutively activated IL-1β, MAPK and SRC/JAK/STAT3 pathways and inhibited apoptosis. Also, NF-κB pathway involvement was demonstrated in vitro by the enhancement of p-IκB-α and p65 nuclear subunit of NF-κB expression in all mutants in the presence of TNF or IL-1β stimulation. These in vitro results correlated with patients' data from PBMCs. Concerning the pro-inflammatory cytokines secretion, mainly IL-1β induced a significant and persistent enhancement of IL-6 and IL-8 in PBMCs carrying the S59P mutation.
  
  
  CONCLUSIONS
  The novel S59P mutation leads to defective cellular trafficking and to constitutive activation of TNF-R1. This mutation also determines constitutive activation of the IL-1R pathway, inhibition of apoptosis and enhanced and persistent NF-κB activation and cytokine secretion in response to IL-1β stimulation.
authors.affiliation: University of Padova, Rheumatology Unit, Department of Medicine - DIMED, Via Giustiniani 2, 35128, Padova, Italy. eliana.greco@unipd.it.|University of Padova, Laboratory Medicine, Department of Medicine - DIMED, Via Giustiniani 2, 35128, Padova, Italy. eliana.greco@unipd.it.
authors.forename: Eliana
authors.initials: E
authors.lastname: Greco
authors.affiliation: University of Padova, Rheumatology Unit, Department of Medicine - DIMED, Via Giustiniani 2, 35128, Padova, Italy. ada.aita@studenti.unipd.it.|University of Padova, Laboratory Medicine, Department of Medicine - DIMED, Via Giustiniani 2, 35128, Padova, Italy. ada.aita@studenti.unipd.it.
authors.forename: Ada
authors.initials: A
authors.lastname: Aita
authors.affiliation: University of Padova, Rheumatology Unit, Department of Medicine - DIMED, Via Giustiniani 2, 35128, Padova, Italy. paola.galozzi@unipd.it.
authors.forename: Paola
authors.initials: P
authors.lastname: Galozzi
authors.affiliation: University of Padova, Rheumatology Unit, Department of Medicine - DIMED, Via Giustiniani 2, 35128, Padova, Italy. alessandra.gava@gmail.com.
authors.forename: Alessandra
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authors.lastname: Gava
authors.affiliation: University of Padova, Rheumatology Unit, Department of Medicine - DIMED, Via Giustiniani 2, 35128, Padova, Italy. paolo.sfriso@unipd.it.
authors.forename: Paolo
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authors.lastname: Sfriso
authors.affiliation: School of Life Sciences, The University of Nottingham, Queen's Medical Centre, Derby road, NG7 2UH, Nottingham, UK. ola.negm@nottingham.ac.uk.|Medical Microbiology and Immunology Department, Faculty of Medicine, Mansoura University, Elgomhouria Street, 35516, Mansoura City, Egypt. ola.negm@nottingham.ac.uk.
authors.forename: Ola H
authors.initials: OH
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authors.affiliation: School of Life Sciences, The University of Nottingham, Queen's Medical Centre, Derby road, NG7 2UH, Nottingham, UK. paddy.tighe@nottingham.ac.uk.
authors.forename: Patrick
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authors.lastname: Tighe
authors.affiliation: University of Padova, Rheumatology Unit, Department of Medicine - DIMED, Via Giustiniani 2, 35128, Padova, Italy. francescocaso@yahoo.it.
authors.forename: Francesco
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authors.lastname: Caso
authors.affiliation: University of Padova, Laboratory Medicine, Department of Medicine - DIMED, Via Giustiniani 2, 35128, Padova, Italy. filippo.navaglia@sanita.padova.it.
authors.forename: Filippo
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authors.lastname: Navaglia
authors.affiliation: Institute of Neuroscience of the National Research Council, Section of Padova, Corso Stati Uniti, 4, 3512, Padova, Italy. emanuela.dazzo@bio.unipd.it.
authors.forename: Emanuela
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authors.lastname: Dazzo
authors.affiliation: Department of Biology, University of Padova, Via U. Bassi, 58/B, 35121, Padova, Italy. marzia.debortoli@unipd.it.
authors.forename: Marzia
authors.initials: M
authors.lastname: De Bortoli
authors.affiliation: Department of Biology, University of Padova, Via U. Bassi, 58/B, 35121, Padova, Italy. alessandra.rampazzo@unipd.it.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Rampazzo
authors.affiliation: Amyloidosis Research and Treatment Center, Biotechnology Research laboratories, Fondazione IRCSS Policlinico San Matteo and University of Pavia, Viale Camillo Golgi 19, 27100, Pavia, Italy. l.obici@smatteo.pv.it.
authors.forename: Laura
authors.initials: L
authors.lastname: Obici
authors.affiliation: Amyloidosis Research and Treatment Center, Biotechnology Research laboratories, Fondazione IRCSS Policlinico San Matteo and University of Pavia, Viale Camillo Golgi 19, 27100, Pavia, Italy. simona_donadei@yahoo.it.
authors.forename: Simona
authors.initials: S
authors.lastname: Donadei
authors.affiliation: Amyloidosis Research and Treatment Center, Biotechnology Research laboratories, Fondazione IRCSS Policlinico San Matteo and University of Pavia, Viale Camillo Golgi 19, 27100, Pavia, Italy. gmerlini@unipv.it.
authors.forename: Giampaolo
authors.initials: G
authors.lastname: Merlini
authors.affiliation: University of Padova, Laboratory Medicine, Department of Medicine - DIMED, Via Giustiniani 2, 35128, Padova, Italy. mario.plebani@sanita.padova.it.
authors.forename: Mario
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authors.lastname: Plebani
authors.affiliation: School of Life Sciences, The University of Nottingham, Queen's Medical Centre, Derby road, NG7 2UH, Nottingham, UK. ian.todd@nottingham.ac.uk.
authors.forename: Ian
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authors.lastname: Todd
authors.affiliation: University of Padova, Laboratory Medicine, Department of Medicine - DIMED, Via Giustiniani 2, 35128, Padova, Italy. daniela.basso@sanita.padova.it.
authors.forename: Daniela
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authors.lastname: Basso
authors.affiliation: University of Padova, Rheumatology Unit, Department of Medicine - DIMED, Via Giustiniani 2, 35128, Padova, Italy. leonardo.punzi@unipd.it.
authors.forename: Leonardo
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mesh_terms: D000328:Adult; D000367:Age Factors; D000368:Aged; D017209:Apoptosis / Q000235:genetics; D016022:Case-Control Studies; D002478:Cells, Cultured; D010505:Familial Mediterranean Fever / Q000235:genetics* / Q000503:physiopathology; D020022:Genetic Predisposition to Disease; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D007558:Italy; D008297:Male; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics*; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000235:genetics*; D012016:Reference Values; D018570:Risk Assessment; D012494:Sampling Studies; D015398:Signal Transduction
chemical_list: D016328:NF-kappa B; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; C506235:TNFRSF1A protein, human