JaeJin Chae

Loss-of-function mutations in TNFAIP3 leading to A20 haploinsufficiency cause an early-onset autoinflammatory disease

Systemic autoinflammatory diseases are driven by abnormal activation of innate immunity. Herein we describe a new disease caused by high-penetrance heterozygous germline mutations in TNFAIP3, which encodes the NF-κB regulatory protein A20, in six unrelated families with early-onset systemic inflammation. The disorder resembles Behçets disease, which is typically considered a polygenic disorder with onset in early adulthood. A20 is a potent inhibitor of the NF-κB signaling pathway. Mutant, truncated A20 proteins are likely to act through haploinsufficiency because they do not exert a dominant-negative effect in overexpression experiments. Patient-derived cells show increased degradation of IκBα and nuclear translocation of the NF-κB p65 subunit together with increased expression of NF-κB–mediated proinflammatory cytokines. A20 restricts NF-κB signals via its deubiquitinase activity. In cells expressing mutant A20 protein, there is defective removal of Lys63-linked ubiquitin from TRAF6, NEMO and RIP1 after stimulation with tumor necrosis factor (TNF). NF-κB–dependent proinflammatory cytokines are potential therapeutic targets for the patients with this disease.

Recent advances in the molecular pathogenesis of hereditary recurrent fevers.

Purpose of review To discuss recent developments in the molecular basis of several hereditary recurrent fever syndromes, specifically the cryopyrin-associated periodic syndromes, familial Mediterranean fever and the tumor necrosis factor receptor associated periodic syndrome. Recent findings Mutations of CIAS1, the gene encoding cryopyrin/NALP3, lead to a spectrum of disease states termed the cryopyrinopathies. Recently, cryopyrin-deficient mice have been used to show that the protein is a key regulator of interleukin-1β production that functions by recognizing stimuli such as bacterial RNA and infectious agents. Tumor necrosis factor receptor-associated periodic syndrome was initially thought to be caused by deficient metalloprotease-induced tumor necrosis factor receptor shedding, however new findings suggest that mutations in this receptor may result in inappropriate protein folding, leading to a host of other functional abnormalities that may cause inflammatory disease. Finally, data are emerging that address the possible function of the C-terminal B30.2 domain of pyrin, the familial Mediterranean fever protein. This motif has recently been shown to interact with and inhibit caspase-1, and the modeled structure of this complex highlights how mutations may affect the binding interface. Summary Recent reports have advanced our understanding of the structural and functional biology underlying the hereditary recurrent fevers, and are beginning to suggest possible mechanisms by which specific mutations cause disease.

Human transthyretin intronic open reading frames are not independently expressed in vivo or part of functional transcripts.

The human transthyretin (TTR) gene encodes a protein composed of four identical subunits with an important role in the plasma transport of thyroid hormone T4 and retinol. TTR spans 7.6 kilobases and consists of four exons. Two independent open reading frames (ORFs) with putative regulatory sequences have been described in the first and third introns, but their function--if any--is unknown. We have screened human cDNA libraries to determine if these sequences are transcribed. Transcripts of both ORFs were found in liver, pancreas and brain. Hybridization of the two sequences with multiple-tissue Northern blots further confirmed these results and revealed transcript sizes of approximately 1.5 and approximately 2.2 kb for ORF 1, and approximately 5.2 and approximately 7.8 kb for ORF 2. Rapid Amplification of cDNA Ends (RACE) was performed to characterize the full-length cDNAs containing each sequence. All products containing the ORFs were continuous in the genomic sequence corresponding to unspliced or partially spliced TTR. No evidence was found for novel transcripts containing productively spliced products of either ORF, or for shorter transcripts using the promoter and polyadenylation signals associated with them. ORF 1 RACE products identified in liver, pancreas and brain correspond to TTR transcripts in which intron 1 had not been removed; the transcripts containing ORF 2 may represent TTR hnRNA. Neither ORF is productively expressed as part of a larger transcript, or as an independent polypeptide.

Aberrant tRNA processing causes an autoinflammatory syndrome responsive to TNF inhibitors

Objectives To characterise the clinical features, immune manifestations and molecular mechanisms in a recently described autoinflammatory disease caused by mutations in TRNT1, a tRNA processing enzyme, and to explore the use of cytokine inhibitors in suppressing the inflammatory phenotype. Methods We studied nine patients with biallelic mutations in TRNT1 and the syndrome of congenital sideroblastic anaemia with immunodeficiency, fevers and developmental delay (SIFD). Genetic studies included whole exome sequencing (WES) and candidate gene screening. Patients’ primary cells were used for deep RNA and tRNA sequencing, cytokine profiling, immunophenotyping, immunoblotting and electron microscopy (EM). Results We identified eight mutations in these nine patients, three of which have not been previously associated with SIFD. Three patients died in early childhood. Inflammatory cytokines, mainly interleukin (IL)-6, interferon gamma (IFN-γ) and IFN-induced cytokines were elevated in the serum, whereas tumour necrosis factor (TNF) and IL-1β were present in tissue biopsies of patients with active inflammatory disease. Deep tRNA sequencing of patients’ fibroblasts showed significant deficiency of mature cytosolic tRNAs. EM of bone marrow and skin biopsy samples revealed striking abnormalities across all cell types and a mix of necrotic and normal-appearing cells. By immunoprecipitation, we found evidence for dysregulation in protein clearance pathways. In 4/4 patients, treatment with a TNF inhibitor suppressed inflammation, reduced the need for blood transfusions and improved growth. Conclusions Mutations of TRNT1 lead to a severe and often fatal syndrome, linking protein homeostasis and autoinflammation. Molecular diagnosis in early life will be crucial for initiating anti-TNF therapy, which might prevent some of the severe disease consequences.

Brief Report: Deficiency of Complement 1r Subcomponent in Early-Onset Systemic Lupus Erythematosus: The Role of Disease-Modifying Alleles in a Monogenic Disease: ROLE OF C1r DEFICIENCY IN EARLY-ONSET SLE

To identify a genetic cause of early‐onset systemic lupus erythematosus (SLE) in a large consanguineous family from Turkey and to study the mechanisms of the disease.

Deficiency of Complement 1r Subcomponent in Early‐Onset Systemic Lupus Erythematosus: The Role of Disease‐Modifying Alleles in a Monogenic Disease

To identify a genetic cause of early‐onset systemic lupus erythematosus (SLE) in a large consanguineous family from Turkey and to study the mechanisms of the disease.

Deficiency of Complement 1r subcomponent in early-onset SLE: Role for disease-modifying alleles in a monogenic disease

To identify a genetic cause of early‐onset systemic lupus erythematosus (SLE) in a large consanguineous family from Turkey and to study the mechanisms of the disease.

OP0193 Pyrin and PSTPIP1, Mutated in FMF, PAPA-, and PAMI Syndrome, are Involved in the Hypersecretion of Alarmins MRP8/14

Background Familial Mediterranean fever (FMF) and pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome are inherited autoinflammatory diseases. FMF patients have mutations in pyrin, a protein that has been suggested to play a role in the regulation of the inflammasome, PAPA patients have mutations in PSTPIP1, which interacts with pyrin. In both diseases, extraordinarily elevated serum levels of MRP8/14 were detected. Recently, we have identified two novel autosomal dominant mutations in the PSTPIP1 gene, p.E250K or p.E257K substitution, as genetic cause for a novel, more severe autoinflammatory entity: PAMI syndrome (PSTPIP1-associated myeloid-related-proteinaemia inflammatory syndrome). PAMI patients present with even higher levels of MRP8/14. MRP8 and MRP14 belong to the family of proinflammatory Damage Associated Molecular Pattern (DAMP) proteins, are mostly expressed in phagocytes and activate innate immune cells via TLR4 (Fassl et al. J. Immunol. 2015) after released via a so-called alternative pathway. However, the mechanism of secretion of MRP8/14 is still unknown. Objectives We investigated if pyrin and PSTPIP1 play a role in the secretion of MRP8/14. Methods MRP8/14 serum concentrations of patients were determined by ELISA. PSTPIP1 gene sequencing was performed in 14 patients with PAMI syndrome. Monocytes from patients were isolated and MRP8/14 levels were measured in culture supernatants prior and after activation. PSTPIP1-Pyrin-MRP8/14 interactions were investigated by immunoprecipitations and structural analysis. Results Monocytes from patients with PSTPIP1 mutations release significantly higher amounts of MRP8/14 than control cells. Immunoprecipitation studies demonstrated that p.E250K-PSTPIP1 is hyperphsphorylated and binds stronger to pyrin compared to wildtype protein. Furthermore, we could prove a direct interaction of both PSTPIP1 and pyrin with MRP8/14. By using deletion constructs of PSTPIP1 we could demonstrate that the MRP8/14 binding motif is mutated in PAMI syndrome. Conclusions Phagocytes seem to be the responsible cell type for the high serum concentration of MRP8/14 in PAMI syndrome. The DAMP proteins MRP8 and MRP14 interact directly with PSTPIP1 and mutations found in all patients are apparently located inside the PSTPIP1-MRP8/14 binding region. Moreover, the PSTPIP1 mutations influence the interaction with pyrin. Overall, our data indicate that hypersecretion of MRP8/14 is a relevant pathomechanism in PSTPIP1- and pyrin-associated diseases. Disclosure of Interest None declared

OP0128 The Regulation of MRP8/14 in Pstpip1-Associated Myeloid-Related-Proteinaemia Inflammatory Syndrome (PAMI)

Background MRP8 and MRP14 are phagocyte-derived Damage Associated Molecular Pattern (DAMP) proteins and can be used as biomarkers in inflammatory diseases e.g. juvenile idiopathic arthritis. We found that the MRP8/14 complex (calprotectin) is highly elevated in the serum of patients with pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome and is even significantly higher in hypercalprotectinaemia and hyperzincaemia (Hz/Hc) [1]. For PAPA, mutations in the proline serine threonine phosphatase-interacting protein 1 (PSTPIP1) gene are described [2]. We have recently identified novel autosomal dominant mutations in PSTPIP1 Hz/Hc patients. All are heterozygous carrier of an E250K or E257K substitution encoded by exon 11 of the PSTPIP1 gene. These patients show an excessively high serum concentration (0.9-12.0 g/l, normal range <0.001 g/l) of MRP8/14 not present in any other inflammatory condition. The mechanism of secretion of MRP8/14 is not clear but depends on a tubulin-mediated alternative secretory pathway. MRP8/14 induces pro-inflammatory effects through interaction with Toll-like receptor 4 dependent signalling pathways [3]. Objectives The molecular link between PSTPIP1 mutations and elevated MRP8/14 concentrations is currently unknown. Therefore we investigated the role of PSTPIP1 during release of MRP8/14. Methods MRP8/14 serum concentrations of patients were determined by ELISA. Monocytes from patients were isolated and MRP8/14 levels were measured in culture supernatants prior and after activation. Intracellular distribution of E250K and wildtype PSTPIP1 was analysed in transfected cells by immunofluorescence and interactions between PSTPIP1, MRP8/14 and microtubules were characterized via immunoprecipitations or microtubule binding assays. Protein interactions were further quantified in vitro by using a modified MRP8/14-ELISA with different constructs of PSTPIP1. Results Monocytes from patients with PSTPIP1 mutations release significantly higher amounts of MRP8/14 than control cells. A co-localization of PSTPIP1 and MRP8/14 could be shown in monocytes and in vitro studies confirm this interaction as a calcium dependent and direct binding. By using deletion constructs of PSTPIP1 we could demonstrate that the MRP8/14 binding motif is mutated in PAPA and Hz/Hc. A mutual interference of PSTPIP1 and MRP8/14 on their interaction with microtubules could be shown which is altered by using the E250K mutated PSTPIP1. Conclusions Phagocytes seem to be the responsible cell type for the high serum concentration of MRP8/14 in Hz/Hc and PAPA. The alarmin MRP8/14 interacts directly with PSTPIP1 in a calcium-dependent manner and mutations found in all patients are apparently located inside the PSTPIP1-MRP8/14 binding region. Interaction of these proteins seem to have a regulatory function on their tubulin binding capability and could be of important relevance for the tubulin-dependent secretion of MRP8/14 which may be a pathogenetic mechanism of MRP-driven inflammation in PAMI. References Sampson et al. (2002) Lancet 360(9347), 1742-1745. Wise et al. (2002) Hum. Mol. Gen. 11(8), 961-969. Rammes et al. (1997) J. Biol. Chem. 272, 9496-9502. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2822

OP0260 The pathophysiology of S100A8/A9 in familial mediterranean fever (FMF)

Background Familial Mediterranean Fever (FMF) is an auto-inflammatory syndrome caused by mutations within the MEFV gene encoding pyrin protein. The FMF syndromeis associated with activation of phagocytic cells and secretion of IL-1β. The production and secretion of IL-1β is regulated by interactions of pyrin and the NALP3-inflammasome, but exact pathogenic mechanisms are still elusive. Like IL-1β, the pro-inflammatory Damage Associated Molecular Pattern (DAMP) molecules S100A8/A9 are released by a Golgi-independent but tubulin-dependent, so called alternative secretory pathway (1, 2). S100A8/A9 have been recently identified as endogenous activators of TLR4 and several studies on inflammatory disorders demonstrated their potential as biomarkers of inflammation (3). Objectives Our goal was i) to correlate S100A8/A9 serum concentrations in FMF-patients to disease activity and to evaluate the potential of S100A8/A9 as biomarker, ii) to study S100A8/A9 -pyrin interaction, iii) to analyze S100A8/A9 serum-concentrations in FMF-mice. Methods 52 genetically proven FMF-patients were studied longitudinally over 18 months. Serum-levels of S100A8/A9 (ELISA), ESR, CRP and SAA were analysed before and during colchicine treatment and compared to other autoinflammatory syndromes. S100A8/A9 serum-levels of homozygous Pyrin V726A knock-in mice (“FMF-mice”) were determined for secretion analysis. Interaction and co-localization of S100A8/A9 and pyrin was analysed by immunofluorescence-, co-immunoprecipitation- and affinity-chromatography experiments. Results The mean serum-levels of S100A8/A9 during inflammatory episodes of FMF (Mean ± SEM 343,210±202,210 ng/ml) were significantly higher compared to chronic infantile neurological, cutaneous and articular (CINCA) syndrome (2,830±580 ng/ml; p<0.001) or Muckle-Wells syndrome (MWS) (3,205±585 ng/ml; p<0.001), and correlated to disease activity. Similar, sera of homozygous FMF-mice showed increased S100A8/A9 -levels (1,260±540 ng/ml) compared to WT- (150±40 ng/ml), or heterozygous FMF-mice (340±180 ng/ml). Immunofluorescence stainings of human monocytes showed a colocalisation of pyrin, S100A8/A9 and tubulin. Moreover a direct molecular interaction of pyrin with S100A8/A9 could be demonstrated in immunoprecipitation and affinity-chromatographyexperiments. Conclusions S100A8/A9 are strongly secreted in FMF-patients compared to other IL-1 driven diseases. Measurement of S100A8/A9 -levels in FMF might be a valuable tool to reflect disease activity and response to anti-inflammatory therapy. Pyrin interacts directly with S100A8/A9 pointing towards an important role in the alternative secretion of those proteins in FMF. References Vogl et al. (2007) Nat. Med. 13, 1042-1049. Rammes et al. (1997) J. Biol. Chem. 272, 9496-9502. Frosch et al. (2000) Arthritis Rheum. 43, 628-637. Disclosure of Interest None Declared