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Factor H autoantibodies are associated with MPGN
Abstracts / Molecular Immunology 47 (2010) 2198–2294
90 Functional characterization of mutations in complement C3 that predispose to aHUS Elizabeth C. Miller a , Lubka Roumenina b , Veronique FremeauxBacchi b , John P. Atkinson a a
Department of Medicine, Division of Rheumatology, Washington University, St. Louis, MO 63110, United States b Centre de recherché des Cordeliers, Paris, France
Atypical haemolytic uraemic syndrome (aHUS) is a thromboangiopathy predominantly of the kidney microvasculature characterised by haemolytic anaemia, thrombocytopenia and acute renal failure. Mutations in the complement regulatory proteins, Factor H (FH), Membrane Cofactor Protein (MCP; CD46), and Factor I (FI) lead to a decreased ability to regulate the alternative pathway (AP). Mutations in Factor B (FB) and C3 also predispose to aHUS and are primary or secondary gain of function mutations. In all of these cases, a hyperactive AP is the functional hallmark. Previously, we analyzed C3 mutations in nine aHUS patients initially selected for analysis due to low serum C3 levels. In the majority, we identified defects in their interactions with negative regulators. We have now characterised five additional C3 mutations in aHUS patients presenting with normal C3. In these studies, we transiently express the mutant proteins and then perform functional analyses including MCP, FH, and CR1 binding ELISAs and fluid phase cofactor assays as well as FB and properdin binding assays. The five mutations are K43N, K133Q, R139W, I1073S and P1092L. All of these proteins were expressed normally and migrated on SDS-PAGE similarly to WT. K43N, in MG1, exhibited decreased binding to MCP but normal FH binding. K133Q, in MG2, had normal interactions with MCP and FH. Interestingly, P1092L, in the TED, bound to MCP comparably to WT C3 but had decreased binding to FH. R139W, in MG2, and two mutations in MG6 from our initial study, R570Q/W, demonstrated increased affinity for properdin. Of note, R570Q/W also had a defect in regulation by MCP and FH in our initial studies. Thus, relative to the AP, enhanced activity secondary to either decreased regulation by inhibitors (FH, MCP or FI) or increased activity of an enhancer (properdin) or a combination thereof leads to aHUS. These data also lend new insight as to where properdin may bind C3(H20)/C3b. doi:10.1016/j.molimm.2010.05.273 91 Factor H autoantibodies are associated with MPGN Isabel Y. Pappworth a , Mark Denton b , David Kavanagh a , Iain Moore a , Lisa Strain c , Paul N. Barlow d , Andrew P. Herbert d , Christoph Q. Schmidt c a
Institutes of Cellular Medicine and Human Genetics, Newcastle University, Newcastle-upon-Tyne, United Kingdom b Department of Renal Medicine, Derriford Hospital, Plymouth Hospitals NHS Trust, Plymouth, United Kingdom c Northern Molecular Genetics Service, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle-upon-Tyne, United Kingdom d School of Chemistry, University of Edinburgh, Edinburgh, United Kingdom Factor H autoantibodies are found in ∼10% of atypical haemolytic uraemic syndrome (aHUS) patients. The majority are associated with complete deficiency of factor H related proteins 1/3 and bind to the C terminal recognition domain. Membranoproliferative glomerulonephritis (MPGN), like aHUS, is characterised by complement activation. Therefore, we examined the hypothesis that factor H autoantibodies are associated with MPGN. We
2291
screened sera from 14 MPGN patients and 100 normal controls for factor H autoantibodies using enzyme-linked immunosorbent assay (ELISA). We detected strongly positive IgG factor H autoantibodies in two patients, which were confirmed by titration and western blotting. Further serum samples collected from both confirmed these findings and in one showed an increasing antibody titre with time. One patient (male aged 24 years) had type II (dense deposit disease; DDD) MPGN and one patient (female aged 26 years) had type I MPGN and both were C3 NeF negative. We identified the binding site of the autoantibodies using small SCR domain fragments in the ELISA and showed that the autoantibodies in both patients bound predominately to the N terminal complement regulatory domain of factor H. We measured CFHR1/3 copy number in paired DNA samples using multiplex ligation-dependent probe amplification (MLPA) and established both patients had two copies of CFHR1/3. Finally, functionality of the detected factor H autoantibodies was examined using purified patient IgG in haemolytic assays. We observed increased haemolysis when purified Ig from both patients (but not from control) was added to normal human sera prior to incubation with rabbit red blood cells. Thus, in a small cohort of MPGN patients, we have found a high titre of functionally significant factor H autoantibodies in two patients. Antibody depleting therapy may have a role in such patients and we suggest that screening for factor H autoantibodies should be undertaken in all patients with MPGN (particularly DDD). doi:10.1016/j.molimm.2010.05.274 92 Factor I autoantibodies are associated with atypical haemolytic uraemic syndrome David Kavanagh a , Isabel Y. Pappworth a , Pietro Roversi d , John S. Tapson b , Iain Moore a , Lisa Strain c , Susan Lea d , Timothy H.J. Goodship a , Kevin J. Marchbank a a
Institutes of Cellular Medicine and Human Genetics, Newcastle University, Newcastle-upon-Tyne, UK b Renal Services Centre, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle-upon-Tyne, UK c Northern Molecular Genetics Service, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle-upon-Tyne, UK d Sir William Dunn School of Pathology, University of Oxford, South Parks Road, UK Background: Atypical haemolytic uraemic syndrome (aHUS) is a disease of complement over activation. Mutations in the genes encoding both regulators (factor H, membrane cofactor protein and factor I) and activators (factor B and C3) are associated with aHUS. Factor H autoantibodies have also been demonstrated in around ∼10% of aHUS patients. These autoantibodies bind to the C-terminal recognition domain of factor H and impair complement regulation at the cell surface. In this study we have examined the hypothesis that factor I (fI) autoantibodies are associated with aHUS. Methods: We screened sera from 142 aHUS patients and 100 controls for fI autoantibodies using ELISA. We confirmed positive results by titration and western blotting. We screened CFH, CFI, MCP, CFB and C3 for mutations using direct fluorescent sequencing. We screened for genomic disorders using muliplex ligation-dependent probe amplification and assessed the functional significance of co-existing mutations in co-factor assays using recombinant proteins. Results: We detected IgG fI autoantibodies in the sera of three aHUS patients. One was strongly positive and two were moderately positive. We confirmed these results with western blotting, which also showed that the autoantibodies bind predominantly to
90 Functional characterization of mutations in complement C3 that predispose to aHUS Elizabeth C. Miller a , Lubka Roumenina b , Veronique FremeauxBacchi b , John P. Atkinson a a
Department of Medicine, Division of Rheumatology, Washington University, St. Louis, MO 63110, United States b Centre de recherché des Cordeliers, Paris, France
Atypical haemolytic uraemic syndrome (aHUS) is a thromboangiopathy predominantly of the kidney microvasculature characterised by haemolytic anaemia, thrombocytopenia and acute renal failure. Mutations in the complement regulatory proteins, Factor H (FH), Membrane Cofactor Protein (MCP; CD46), and Factor I (FI) lead to a decreased ability to regulate the alternative pathway (AP). Mutations in Factor B (FB) and C3 also predispose to aHUS and are primary or secondary gain of function mutations. In all of these cases, a hyperactive AP is the functional hallmark. Previously, we analyzed C3 mutations in nine aHUS patients initially selected for analysis due to low serum C3 levels. In the majority, we identified defects in their interactions with negative regulators. We have now characterised five additional C3 mutations in aHUS patients presenting with normal C3. In these studies, we transiently express the mutant proteins and then perform functional analyses including MCP, FH, and CR1 binding ELISAs and fluid phase cofactor assays as well as FB and properdin binding assays. The five mutations are K43N, K133Q, R139W, I1073S and P1092L. All of these proteins were expressed normally and migrated on SDS-PAGE similarly to WT. K43N, in MG1, exhibited decreased binding to MCP but normal FH binding. K133Q, in MG2, had normal interactions with MCP and FH. Interestingly, P1092L, in the TED, bound to MCP comparably to WT C3 but had decreased binding to FH. R139W, in MG2, and two mutations in MG6 from our initial study, R570Q/W, demonstrated increased affinity for properdin. Of note, R570Q/W also had a defect in regulation by MCP and FH in our initial studies. Thus, relative to the AP, enhanced activity secondary to either decreased regulation by inhibitors (FH, MCP or FI) or increased activity of an enhancer (properdin) or a combination thereof leads to aHUS. These data also lend new insight as to where properdin may bind C3(H20)/C3b. doi:10.1016/j.molimm.2010.05.273 91 Factor H autoantibodies are associated with MPGN Isabel Y. Pappworth a , Mark Denton b , David Kavanagh a , Iain Moore a , Lisa Strain c , Paul N. Barlow d , Andrew P. Herbert d , Christoph Q. Schmidt c a
Institutes of Cellular Medicine and Human Genetics, Newcastle University, Newcastle-upon-Tyne, United Kingdom b Department of Renal Medicine, Derriford Hospital, Plymouth Hospitals NHS Trust, Plymouth, United Kingdom c Northern Molecular Genetics Service, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle-upon-Tyne, United Kingdom d School of Chemistry, University of Edinburgh, Edinburgh, United Kingdom Factor H autoantibodies are found in ∼10% of atypical haemolytic uraemic syndrome (aHUS) patients. The majority are associated with complete deficiency of factor H related proteins 1/3 and bind to the C terminal recognition domain. Membranoproliferative glomerulonephritis (MPGN), like aHUS, is characterised by complement activation. Therefore, we examined the hypothesis that factor H autoantibodies are associated with MPGN. We
2291
screened sera from 14 MPGN patients and 100 normal controls for factor H autoantibodies using enzyme-linked immunosorbent assay (ELISA). We detected strongly positive IgG factor H autoantibodies in two patients, which were confirmed by titration and western blotting. Further serum samples collected from both confirmed these findings and in one showed an increasing antibody titre with time. One patient (male aged 24 years) had type II (dense deposit disease; DDD) MPGN and one patient (female aged 26 years) had type I MPGN and both were C3 NeF negative. We identified the binding site of the autoantibodies using small SCR domain fragments in the ELISA and showed that the autoantibodies in both patients bound predominately to the N terminal complement regulatory domain of factor H. We measured CFHR1/3 copy number in paired DNA samples using multiplex ligation-dependent probe amplification (MLPA) and established both patients had two copies of CFHR1/3. Finally, functionality of the detected factor H autoantibodies was examined using purified patient IgG in haemolytic assays. We observed increased haemolysis when purified Ig from both patients (but not from control) was added to normal human sera prior to incubation with rabbit red blood cells. Thus, in a small cohort of MPGN patients, we have found a high titre of functionally significant factor H autoantibodies in two patients. Antibody depleting therapy may have a role in such patients and we suggest that screening for factor H autoantibodies should be undertaken in all patients with MPGN (particularly DDD). doi:10.1016/j.molimm.2010.05.274 92 Factor I autoantibodies are associated with atypical haemolytic uraemic syndrome David Kavanagh a , Isabel Y. Pappworth a , Pietro Roversi d , John S. Tapson b , Iain Moore a , Lisa Strain c , Susan Lea d , Timothy H.J. Goodship a , Kevin J. Marchbank a a
Institutes of Cellular Medicine and Human Genetics, Newcastle University, Newcastle-upon-Tyne, UK b Renal Services Centre, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle-upon-Tyne, UK c Northern Molecular Genetics Service, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle-upon-Tyne, UK d Sir William Dunn School of Pathology, University of Oxford, South Parks Road, UK Background: Atypical haemolytic uraemic syndrome (aHUS) is a disease of complement over activation. Mutations in the genes encoding both regulators (factor H, membrane cofactor protein and factor I) and activators (factor B and C3) are associated with aHUS. Factor H autoantibodies have also been demonstrated in around ∼10% of aHUS patients. These autoantibodies bind to the C-terminal recognition domain of factor H and impair complement regulation at the cell surface. In this study we have examined the hypothesis that factor I (fI) autoantibodies are associated with aHUS. Methods: We screened sera from 142 aHUS patients and 100 controls for fI autoantibodies using ELISA. We confirmed positive results by titration and western blotting. We screened CFH, CFI, MCP, CFB and C3 for mutations using direct fluorescent sequencing. We screened for genomic disorders using muliplex ligation-dependent probe amplification and assessed the functional significance of co-existing mutations in co-factor assays using recombinant proteins. Results: We detected IgG fI autoantibodies in the sera of three aHUS patients. One was strongly positive and two were moderately positive. We confirmed these results with western blotting, which also showed that the autoantibodies bind predominantly to