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Johannes Roth - One of the best experts on this subject based on the ideXlab platform.
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PAPA Syndrome and the Spectrum of PSTPIP1-Associated Inflammatory Diseases
Auto-Inflammatory Syndromes, 2019Co-Authors: Dirk Holzinger, Johannes RothAbstract:Pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome was the first described autoinflammatory disease caused by mutations in the proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1, also known as CD2BP1) gene. However, in the last years, the spectrum of PSTPIP1-associated inflammatory diseases (PAID) has expanded encompassing PSTPIP1-associated myeloid-related proteinemia inflammatory (PAMI) syndrome and the anecdotal description of other clinical phenotypes.
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Alarming consequences - autoinflammatory disease spectrum due to mutations in proline-serine-threonine phosphatase-interacting protein 1.
Current opinion in rheumatology, 2016Co-Authors: Dirk Holzinger, Johannes RothAbstract:Purpose of reviewTo give an overview about the expanding spectrum of autoinflammatory diseases due to mutations in proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1) and new insights into their pathogenesis.Recent findingsIn addition to classical pyogenic sterile arthritis, pyoderm
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OP0193 Pyrin and PSTPIP1, Mutated in FMF, PAPA-, and PAMI Syndrome, are Involved in the Hypersecretion of Alarmins MRP8/14
Annals of the Rheumatic Diseases, 2015Co-Authors: Selina Kathleen Fassl, Marco Gattorno, A Omenetti, Dirk Holzinger, Judith Austermann, Thomas J. Vogl, Jaejin Chae, Ivona Aksentijevich, Johannes RothAbstract: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
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OP0128 The Regulation of MRP8/14 in PSTPIP1-Associated Myeloid-Related-Proteinaemia Inflammatory Syndrome (PAMI)
Annals of the Rheumatic Diseases, 2014Co-Authors: Selina Kathleen Fassl, Marco Gattorno, A Omenetti, Dirk Holzinger, Judith Austermann, Thomas J. Vogl, Jaejin Chae, Ivona Aksentijevich, Johannes RothAbstract: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 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
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op0128 the regulation of mrp8 14 in PSTPIP1 associated myeloid related proteinaemia inflammatory syndrome pami
Annals of the Rheumatic Diseases, 2014Co-Authors: Selina Kathleen Fassl, Marco Gattorno, A Omenetti, Dirk Holzinger, Judith Austermann, Thomas J. Vogl, Jaejin Chae, Ivona Aksentijevich, Johannes RothAbstract: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 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
Dirk Holzinger - One of the best experts on this subject based on the ideXlab platform.
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PAPA Syndrome and the Spectrum of PSTPIP1-Associated Inflammatory Diseases
Auto-Inflammatory Syndromes, 2019Co-Authors: Dirk Holzinger, Johannes RothAbstract:Pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome was the first described autoinflammatory disease caused by mutations in the proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1, also known as CD2BP1) gene. However, in the last years, the spectrum of PSTPIP1-associated inflammatory diseases (PAID) has expanded encompassing PSTPIP1-associated myeloid-related proteinemia inflammatory (PAMI) syndrome and the anecdotal description of other clinical phenotypes.
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Haematological involvement associated with a mild autoinflammatory phenotype, in two patients carrying the E250K mutation of PSTPIP1.
Clinical and experimental rheumatology, 2017Co-Authors: Elena Belelli, Dirk Holzinger, Chiara Passarelli, Manuela Pardeo, Fabrizio De Benedetti, Antonella InsalacoAbstract:Hyperzincaemia/hypercalprotectinemia (Hz/Hc) syndrome is a recently described condition caused by a specific de novo mutation (E250K) affecting PSTPIP1 gene. It has a phenotype distinct from classical pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome that includes severe systemic and cutaneous inflammation, hepatosplenomegaly, arthritis without sequelae, pancytopenia and failure to thrive. We describe an 8-year-old boy who presented recurrent right knee swelling mimicking septic arthritis and persistent bone marrow involvement, without cutaneous involvement. Molecular analysis of the PSTPIP1 gene revealed the presence of a heterozygous E250K mutation. No growth failure was detected nor in the patient neither in his mother, carrying the same variant. Blood zinc and calprotectin MRP8/14 concentrations of the patient were found to be markedly increased. Therapy with anakinra was started with rapid disappearance of clinical symptoms and normalization of CRP levels in 24 hours, but persistence of bone marrow involvement. The patient described has a milder phenotype, with no skin features, minor episodes of arthritis with no sequelae and normal growth. Compared to the patients with de novo mutations described in the literature, familial cases seem to have a milder phenotype. Our case further confirms the lack of efficacy of anakinra on bone marrow involvement.
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Haematological involvement associated with a mild autoinflammatory phenotype, in two patients carrying the E250K mutation of PSTPIP1
Clinical and Experimental Rheumatology, 2017Co-Authors: Elena Belelli, Dirk Holzinger, Chiara Passarelli, Manuela Pardeo, Fabrizio De Benedetti, Antonella InsalacoAbstract:Objectives Hyperzincaemia/hypercalprotectinemia (Hz/Hc) syndrome is a recently described condition caused by a specific de novo mutation (E250K) affecting PSTPIP1 gene. It has a phenotype distinct from classical pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome that includes severe systemic and cutaneous inflammation, hepatosplenomegaly, arthritis without sequelae, pancytopenia and failure to thrive. Methods We describe an 8-year-old boy who presented recurrent right knee swelling mimicking septic arthritis and persistent bone marrow involvement, without cutaneous involvement. Results Molecular analysis of the PSTPIP1 gene revealed the presence of a heterozygous E250K mutation. No growth failure was detected nor in the patient neither in his mother, carrying the same variant. Blood zinc and calprotectin MRP8/14 concentrations of the patient were found to be markedly increased. Therapy with anakinra was started with rapid disappearance of clinical symptoms and normalization of CRP levels in 24 hours, but persistence of bone marrow involvement. Conclusions The patient described has a milder phenotype, with no skin features, minor episodes of arthritis with no sequelae and normal growth. Compared to the patients with de novo mutations described in the literature, familial cases seem to have a milder phenotype. Our case further confirms the lack of efficacy of anakinra on bone marrow involvement.
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Alarming consequences - autoinflammatory disease spectrum due to mutations in proline-serine-threonine phosphatase-interacting protein 1.
Current opinion in rheumatology, 2016Co-Authors: Dirk Holzinger, Johannes RothAbstract:Purpose of reviewTo give an overview about the expanding spectrum of autoinflammatory diseases due to mutations in proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1) and new insights into their pathogenesis.Recent findingsIn addition to classical pyogenic sterile arthritis, pyoderm
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single amino acid charge switch defines clinically distinct proline serine threonine phosphatase interacting protein 1 PSTPIP1 associated inflammatory diseases
The Journal of Allergy and Clinical Immunology, 2015Co-Authors: Dirk Holzinger, Marco Gattorno, A Omenetti, Selina Kathleen Fassl, Wilco De Jager, Peter Lohse, Ute F Rohrig, Sabrina Chiesa, Francesca Schena, Judith AustermannAbstract:Background Hyperzincemia and hypercalprotectinemia (Hz/Hc) is a distinct autoinflammatory entity involving extremely high serum concentrations of the proinflammatory alarmin myeloid-related protein (MRP) 8/14 (S100A8/S100A9 and calprotectin). Objective We sought to characterize the genetic cause and clinical spectrum of Hz/Hc. Methods Proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1) gene sequencing was performed in 14 patients with Hz/Hc, and their clinical phenotype was compared with that of 11 patients with pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome. PSTPIP1-pyrin interactions were analyzed by means of immunoprecipitation and Western blotting. A structural model of the PSTPIP1 dimer was generated. Cytokine profiles were analyzed by using the multiplex immunoassay, and MRP8/14 serum concentrations were analyzed by using an ELISA. Results Thirteen patients were heterozygous for a missense mutation in the PSTPIP1 gene, resulting in a p.E250K mutation, and 1 carried a mutation resulting in p.E257K. Both mutations substantially alter the electrostatic potential of the PSTPIP1 dimer model in a region critical for protein-protein interaction. Patients with Hz/Hc have extremely high MRP8/14 concentrations (2045 ± 1300 μg/mL) compared with those with PAPA syndrome (116 ± 74 μg/mL) and have a distinct clinical phenotype. A specific cytokine profile is associated with Hz/Hc. Hz/Hc mutations altered protein binding of PSTPIP1, increasing interaction with pyrin through phosphorylation of PSTPIP1. Conclusion Mutations resulting in charge reversal in the y-domain of PSTPIP1 (E→K) and increased interaction with pyrin cause a distinct autoinflammatory disorder defined by clinical and biochemical features not found in patients with PAPA syndrome, indicating a unique genotype-phenotype correlation for mutations in the PSTPIP1 gene. This is the first inborn autoinflammatory syndrome in which inflammation is driven by uncontrolled release of members of the alarmin family.
Selina Kathleen Fassl - One of the best experts on this subject based on the ideXlab platform.
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single amino acid charge switch defines clinically distinct proline serine threonine phosphatase interacting protein 1 PSTPIP1 associated inflammatory diseases
The Journal of Allergy and Clinical Immunology, 2015Co-Authors: Dirk Holzinger, Marco Gattorno, A Omenetti, Selina Kathleen Fassl, Wilco De Jager, Peter Lohse, Ute F Rohrig, Sabrina Chiesa, Francesca Schena, Judith AustermannAbstract:Background Hyperzincemia and hypercalprotectinemia (Hz/Hc) is a distinct autoinflammatory entity involving extremely high serum concentrations of the proinflammatory alarmin myeloid-related protein (MRP) 8/14 (S100A8/S100A9 and calprotectin). Objective We sought to characterize the genetic cause and clinical spectrum of Hz/Hc. Methods Proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1) gene sequencing was performed in 14 patients with Hz/Hc, and their clinical phenotype was compared with that of 11 patients with pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome. PSTPIP1-pyrin interactions were analyzed by means of immunoprecipitation and Western blotting. A structural model of the PSTPIP1 dimer was generated. Cytokine profiles were analyzed by using the multiplex immunoassay, and MRP8/14 serum concentrations were analyzed by using an ELISA. Results Thirteen patients were heterozygous for a missense mutation in the PSTPIP1 gene, resulting in a p.E250K mutation, and 1 carried a mutation resulting in p.E257K. Both mutations substantially alter the electrostatic potential of the PSTPIP1 dimer model in a region critical for protein-protein interaction. Patients with Hz/Hc have extremely high MRP8/14 concentrations (2045 ± 1300 μg/mL) compared with those with PAPA syndrome (116 ± 74 μg/mL) and have a distinct clinical phenotype. A specific cytokine profile is associated with Hz/Hc. Hz/Hc mutations altered protein binding of PSTPIP1, increasing interaction with pyrin through phosphorylation of PSTPIP1. Conclusion Mutations resulting in charge reversal in the y-domain of PSTPIP1 (E→K) and increased interaction with pyrin cause a distinct autoinflammatory disorder defined by clinical and biochemical features not found in patients with PAPA syndrome, indicating a unique genotype-phenotype correlation for mutations in the PSTPIP1 gene. This is the first inborn autoinflammatory syndrome in which inflammation is driven by uncontrolled release of members of the alarmin family.
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OP0193 Pyrin and PSTPIP1, Mutated in FMF, PAPA-, and PAMI Syndrome, are Involved in the Hypersecretion of Alarmins MRP8/14
Annals of the Rheumatic Diseases, 2015Co-Authors: Selina Kathleen Fassl, Marco Gattorno, A Omenetti, Dirk Holzinger, Judith Austermann, Thomas J. Vogl, Jaejin Chae, Ivona Aksentijevich, Johannes RothAbstract: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
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OP0128 The Regulation of MRP8/14 in PSTPIP1-Associated Myeloid-Related-Proteinaemia Inflammatory Syndrome (PAMI)
Annals of the Rheumatic Diseases, 2014Co-Authors: Selina Kathleen Fassl, Marco Gattorno, A Omenetti, Dirk Holzinger, Judith Austermann, Thomas J. Vogl, Jaejin Chae, Ivona Aksentijevich, Johannes RothAbstract: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 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
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op0128 the regulation of mrp8 14 in PSTPIP1 associated myeloid related proteinaemia inflammatory syndrome pami
Annals of the Rheumatic Diseases, 2014Co-Authors: Selina Kathleen Fassl, Marco Gattorno, A Omenetti, Dirk Holzinger, Judith Austermann, Thomas J. Vogl, Jaejin Chae, Ivona Aksentijevich, Johannes RothAbstract: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 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
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op0024 the function of PSTPIP1 and s100a8 s100a9 in hyperzincaemia hypercalprotectinaemia and the papa syndrom
Annals of the Rheumatic Diseases, 2013Co-Authors: Selina Kathleen Fassl, Dirk Holzinger, Judith Austermann, Thomas J. Vogl, Johannes RothAbstract:Background Hypercalprotectinaemia and hyperzincaemia is a very rare autoinflammatory syndrome that is characterized by excessively high S100A8/S100A9 complex (calprotectin) serum concentrations (0.9-12.0 g/l, normal range Objectives The molecular link between PSTPIP1 mutations and elevated S100A8/S100A9 concentrations is currently unknown. The present project focuses therefore on the identification of molecular links between PSTPIP1 and S100A8/S100A9 and the potential function of PSTPIP1 for the release of S100A8/S100A9. Methods The intracellular distribution of E250K and wildtype PSTPIP1 was determined in transfected cell lines by immunofluorescence analysis. The interactions between PSTPIP1, S100A8/S100A9 and microtubules were characterized via immunoprecipitations. These interaction studies were confirmed and further characterized by in vitro interaction studies using a modified S100A8/S100A9-ELISA and microtubule binding assays. Results The immunofluorescence and immunoprecipitation results point towards an interaction between PSTPIP1 and S100A8/S100A9 in vivo . Moreover we could specify this interaction in vitro as direct and strictly calcium-dependent. Using several deletion constructs of PSTPIP1 we have some first evidences indicating that the S100A8/S100A9 binding motif is located within the same region described to be mutated in hyperzincaemia and hypercalprotectinaemia patients. Microtubule co-sedimentation experiments indicate a mutual interference of PSTPIP1 and S100A8/S100A9 on their interaction with microtubules which is altered by using the E250K mutated PSTPIP1. Conclusions PSTPIP1 and S100A8/S100A9 directly interact in a calcium-dependent manner and the E250K mutation is apparently located within the S100A8/S100A9 binding region. Therefore this mutation might have an influence on the S100A8/S100A9-PSTPIP1 interaction. In addition PSTPIP1 and S100A8/S100A9 seem to have a regulatory function on their tubulin binding capability. This in turn might be of important relevance for the tubulin-dependent secretion of the S100A8/S100A9 proteins and putatively the cause for the high serum concentrations of S100A8/S100A9 in hyperzincaemia and hypercalprotectinaemia patients. References Vogl et al. (2007) Nat. Med. 13, 1042-1049; Rammes et al. (1997) J. Biol. Chem. 272, 9496-9502; Wise et al. (2002) Hum. Mol. Gen. 11(8), 961-969. Disclosure of Interest None Declared
Katherine A. Siminovitch - One of the best experts on this subject based on the ideXlab platform.
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Fyn and PTP-PEST–mediated Regulation of Wiskott-Aldrich Syndrome Protein (WASp) Tyrosine Phosphorylation Is Required for Coupling T Cell Antigen Receptor Engagement to WASp Effector Function and T Cell Activation
The Journal of experimental medicine, 2004Co-Authors: Karen Badour, Jinyi Zhang, Fabio Shi, Yan Leng, Michael P. Collins, Katherine A. SiminovitchAbstract:Involvement of the Wiskott-Aldrich syndrome protein (WASp) in promoting cell activation requires its release from autoinhibitory structural constraints and has been attributed to WASp association with activated cdc42. Here, however, we show that T cell development and T cell receptor (TCR)-induced proliferation and actin polymerization proceed normally in WASp−/− mice expressing a WASp transgene lacking the cdc42 binding domain. By contrast, mutation of tyrosine residue Y291, identified here as the major site of TCR-induced WASp tyrosine phosphorylation, abrogated induction of WASp tyrosine phosphorylation and its effector activities, including nuclear factor of activated T cell transcriptional activity, actin polymerization, and immunological synapse formation. TCR-induced WASp tyrosine phosphorylation was also disrupted in T cells lacking Fyn, a kinase shown here to bind, colocalize with, and phosphorylate WASp. By contrast, WASp was tyrosine dephosphorylated by protein tyrosine phosphatase (PTP)-PEST, a tyrosine phosphatase shown here to interact with WASp via proline, serine, threonine phosphatase interacting protein (PSTPIP)1 binding. Although Fyn enhanced WASp-mediated Arp2/3 activation and was required for synapse formation, PTP-PEST combined with PSTPIP1 inhibited WASp-driven actin polymerization and synapse formation. These observations identify key roles for Fyn and PTP-PEST in regulating WASp and imply that inducible WASp tyrosine phosphorylation can occur independently of cdc42 binding, but unlike the cdc42 interaction, is absolutely required for WASp contributions to T cell activation.
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the wiskott aldrich syndrome protein acts downstream of cd2 and the cd2ap and PSTPIP1 adaptors to promote formation of the immunological synapse
Immunity, 2003Co-Authors: Karen Badour, Mary K H Mcgavin, Vik Rampersad, Lynne A. Hardy, Deborah J. Field, Jinyi Zhang, Katherine A. SiminovitchAbstract:Abstract The Wiskott-Aldrich syndrome protein (WASp) couples actin cytoskeletal rearrangement to T cell activation, but the mechanisms involved are unknown. Here, we show that antigen-induced formation of T cell:APC conjugates and synapses is abrogated in WASp-deficient T cells and that CD2 engagement evokes interactions between the proline-rich region required for WASp translocation to the synapse and the PSTPIP1 adaptor SH3 domain and between the PSTPIP1 coiled-coil domain and both CD2 and another CD2-binding adaptor, CD2AP. The induced colocalization of these proteins at the synapse is disrupted by expression of coiled-coil domain-deleted PSTPIP1. These data, together with the impairment in CD2-induced actin polymerization observed in WASp-deficient cells, suggest that PSTPIP1 acts downstream of CD2/CD2AP to link CD2 engagement to the WASp-evoked actin polymerization required for synapse formation and T cell activation.
Judith Austermann - One of the best experts on this subject based on the ideXlab platform.
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single amino acid charge switch defines clinically distinct proline serine threonine phosphatase interacting protein 1 PSTPIP1 associated inflammatory diseases
The Journal of Allergy and Clinical Immunology, 2015Co-Authors: Dirk Holzinger, Marco Gattorno, A Omenetti, Selina Kathleen Fassl, Wilco De Jager, Peter Lohse, Ute F Rohrig, Sabrina Chiesa, Francesca Schena, Judith AustermannAbstract:Background Hyperzincemia and hypercalprotectinemia (Hz/Hc) is a distinct autoinflammatory entity involving extremely high serum concentrations of the proinflammatory alarmin myeloid-related protein (MRP) 8/14 (S100A8/S100A9 and calprotectin). Objective We sought to characterize the genetic cause and clinical spectrum of Hz/Hc. Methods Proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1) gene sequencing was performed in 14 patients with Hz/Hc, and their clinical phenotype was compared with that of 11 patients with pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome. PSTPIP1-pyrin interactions were analyzed by means of immunoprecipitation and Western blotting. A structural model of the PSTPIP1 dimer was generated. Cytokine profiles were analyzed by using the multiplex immunoassay, and MRP8/14 serum concentrations were analyzed by using an ELISA. Results Thirteen patients were heterozygous for a missense mutation in the PSTPIP1 gene, resulting in a p.E250K mutation, and 1 carried a mutation resulting in p.E257K. Both mutations substantially alter the electrostatic potential of the PSTPIP1 dimer model in a region critical for protein-protein interaction. Patients with Hz/Hc have extremely high MRP8/14 concentrations (2045 ± 1300 μg/mL) compared with those with PAPA syndrome (116 ± 74 μg/mL) and have a distinct clinical phenotype. A specific cytokine profile is associated with Hz/Hc. Hz/Hc mutations altered protein binding of PSTPIP1, increasing interaction with pyrin through phosphorylation of PSTPIP1. Conclusion Mutations resulting in charge reversal in the y-domain of PSTPIP1 (E→K) and increased interaction with pyrin cause a distinct autoinflammatory disorder defined by clinical and biochemical features not found in patients with PAPA syndrome, indicating a unique genotype-phenotype correlation for mutations in the PSTPIP1 gene. This is the first inborn autoinflammatory syndrome in which inflammation is driven by uncontrolled release of members of the alarmin family.
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OP0193 Pyrin and PSTPIP1, Mutated in FMF, PAPA-, and PAMI Syndrome, are Involved in the Hypersecretion of Alarmins MRP8/14
Annals of the Rheumatic Diseases, 2015Co-Authors: Selina Kathleen Fassl, Marco Gattorno, A Omenetti, Dirk Holzinger, Judith Austermann, Thomas J. Vogl, Jaejin Chae, Ivona Aksentijevich, Johannes RothAbstract: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
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OP0128 The Regulation of MRP8/14 in PSTPIP1-Associated Myeloid-Related-Proteinaemia Inflammatory Syndrome (PAMI)
Annals of the Rheumatic Diseases, 2014Co-Authors: Selina Kathleen Fassl, Marco Gattorno, A Omenetti, Dirk Holzinger, Judith Austermann, Thomas J. Vogl, Jaejin Chae, Ivona Aksentijevich, Johannes RothAbstract: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 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
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op0128 the regulation of mrp8 14 in PSTPIP1 associated myeloid related proteinaemia inflammatory syndrome pami
Annals of the Rheumatic Diseases, 2014Co-Authors: Selina Kathleen Fassl, Marco Gattorno, A Omenetti, Dirk Holzinger, Judith Austermann, Thomas J. Vogl, Jaejin Chae, Ivona Aksentijevich, Johannes RothAbstract: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 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
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PW02-018 - Impact of PSTPIP1 mutaions on clinical phenotype.
Pediatric Rheumatology, 2013Co-Authors: Dirk Holzinger, Marco Gattorno, Judith Austermann, Peter Lohse, Steven M Holland, S. Faßl, Thomas Vogl, W. De Jager, Carlos Rodríguez-gallego, Juan I. ArósteguiAbstract:Hyperzincaemia and hypercalprotectinaemia (Hz/Hc), a rare condition within the spectrum of autoinflammatory diseases, is associated with hepatosplenomegaly, arthritis, anemia, cutaneous inflammation, and failure to thrive. So far, no genetic cause has been identified. While the clinical appearance is heterogeneous, all affected individuals present with extremely elevated MRP8/MRP14 (calprotectin) serum concentrations (0.9-12.0 g/l (normal range < 0.001 g/l)).
