Gain-of-function mutation in complement C2 protein identified in a patient with aHUS
Authors: Aleksandra Urban, Elena Volokhina, Anna Felberg, Grzegorz Stasiłojć, Anna M. Blom, Ilse Jongerius, Lambertus van den Heuvel, Marcel Thiel, Stanisław Ołdziej, Emilia Arjona, Santiago Rodriguez de Córdoba, Marcin Okrój
Journal: Journal of Allergy and Clinical Immunology (2020)

The complement system, a basic defense mechanism of innate immunity, protects us from invading pathogens and supports the maintenance of body homeostasis.1Ricklin D. Hajishengallis G. Yang K. Lambris J.D. Complement: a key system for immune surveillance and homeostasis.Nat Immunol. 2010; 11: 785-797Crossref PubMed Scopus (1945) Google Scholar As proper functioning of complement is based on the interplay between its activators and inhibitors, functional impairment of a particular component often leads to autoimmune or inflammatory diseases. The kidney is an organ that is especially susceptible to complement-mediated damage because of high blood flow combined with a delicate structure of the filtrating barrier. Additionally, glomerular vessels are vulnerable to thrombosis and damage by heme originating from local hemolysis of red blood cells. Complement attack on the kidney's structures can be mediated by abnormalities in the alternative complement pathway (AP).2De Vriese A.S. Sethi S. Van Praet J. Nath K.A. Fervenza F.C. Kidney disease caused by dysregulation of the complement alternative pathway: an etiologic approach.J Am Soc Nephrol. 2015; 26: 2917-2929Crossref PubMed Scopus (63) Google Scholar This route is constantly active at a low level and spontaneously deposits C3 activation fragments (C3b) on cell surfaces.1Ricklin D. Hajishengallis G. Yang K. Lambris J.D. Complement: a key system for immune surveillance and homeostasis.Nat Immunol. 2010; 11: 785-797Crossref PubMed Scopus (1945) Google Scholar Unlike pathogens, self-cells are equipped with a panel of complement inhibitors that block further pathway propagation. Therefore, the loss of control of the AP is a condition permissive for complement-mediated damage. The underlying mechanisms may involve aberrations that render AP convertases insensitive to regulation. Such aberrations include the presence of autoantibodies termed C3NeF, which stabilize AP convertases (key enzymatic complexes that amplify the complement cascade), autoantibodies binding to soluble complement inhibitors such as factor H, inheritance of rare variants of membrane complement inhibitors (eg, CD46) and factor H, or gain-of-function mutations in complement components such as C3 or factor B (FB).2De Vriese A.S. Sethi S. Van Praet J. Nath K.A. Fervenza F.C. Kidney disease caused by dysregulation of the complement alternative pathway: an etiologic approach.J Am Soc Nephrol. 2015; 26: 2917-2929Crossref PubMed Scopus (63) Google Scholar Classical complement-mediated renal diseases are C3 glomerulopathies (C3Gs) and atypical hemolytic uremic syndrome (aHUS). There are reports suggesting that next to dysregulation of the AP, abnormalities in the classical pathway/lectin pathway (CP/LP) may also play a role in pathogenesis. Previously, we screened 13 patients with C3G for alterations increasing or prolonging convertase activity and found 1 individual with no acknowledged risk factors but antibodies stabilizing the CP/LP convertases (C4NeF).3Blom A.M. Corvillo F. Magda M. Stasilojc G. Nozal P. Perez-Valdivia M.A. et al.Testing the is of C4NeF-mediated C3 glomerulonephritis.J Clin Immunol. 2016; 36: 517-527Crossref PubMed Scopus (0) Google Scholar Another screening of a cohort of 168 patients with C3G revealed 3 patients who were positive only for C4NeF autoantibodies whereas genetic analysis showed no rare or novel variants of complement genes.4Zhang Y. Meyer N.C. Fervenza F.C. Lau W. Keenan A. Cara-Fuentes G. et al.C4 Nephritic factors in C3 glomerulopathy: a case series.Am J Kidney Dis. 2017; 70: 834-843Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar Because standard diagnostics of complement-mediated renal diseases has thus far been focused on abnormalities in the AP, the possible role of the CP/LP in pathogenesis may be underestimated. Up to now, no gain-of-function variants within CP/LP convertase components have been identified in patients. We decided to search for such naturally occurring mutations in complement C2 protein—a CP/LP analog and paralog of FB. These 2 proteins share a high degree of amino acid similarity, as well as almost identical length and structural organization.5Krishnan V. Xu Y. Macon K. Volanakis J.E. Narayana S.V. The structure of C2b, a fragment of complement component C2 produced during C3 convertase formation.Acta Crystallogr D Biol Crystallogr. 2009; 65: 266-274Crossref PubMed Scopus (18) Google Scholar Therefore, it is highly probable that they contain similar mutational hotspots, as already demonstrated by translation of gain-of-function mutations in FB to C2.6Kuttner-Kondo L.A. Dybvig M.P. Mitchell L.M. Muqim N. Atkinson J.P. Medof M.E. et al.A corresponding tyrosine residue in the C2/factor B type A domain is a hot spot in the decay acceleration of the complement C3 convertases.J Biol Chem. 2003; 278: 52386-52391Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar Above all, the fact that pathogenic, naturally occurring gain-of-function mutants of FB have already been characterized encouraged us to look for analogous pathogenic mutations in C2. DNA samples of 233 patients with aHUS and C3G who were included in the Spanish aHUS/C3G Registry (https://www.aHUSC3G.es) as of August 29, 2016, were analyzed for genetic variants by using a next-generation sequencing panel that included THBD, DGKE, and 42 complement genes. Missense mutations in C2 gene were identified in 8 patients (Table I). A complete list of analyzed genes, sequencing and data analysis procedures, diagnostic criteria, and all methods used for further characterization of mutated proteins are provided in this article's Online Repository (available in this article's Online Repository at www.jacionline.org).Table IDetailed characteristics of missense mutations in C2 identified in the cohort of 233 patientsPosition on chromosome 6IDNo. of patientscDNA substitutionAmino acid substitutionGenotypeDiagnosis of patients with mutationRisk polymorphismOther potential pathogenic complement genetic variantsAnti-FH antibodiesC3NefAutoantibodiesMCPFH31896638rs3679967211/233c.G386Ap.R129HHETaHUSHOMHOMNoneNo—ANA31901954rs3701210061/233c. C727Tp.R243CHETaHUSNOHETp.K591Sfs∗10: MASP1:HETNo—ANCA31901976rs1508272551/233c. C749Gp. S250CHETaHUSHOMNONone———31903804rs933273911/233∗Common polymorphism with a minor allele frequency greater than 0.01. The rest of the sequence variants display a minor allele frequency less than 0.001.c. G954Cp. E318DAll HETaHUS, C3GN——————31910761—1/233c. G1245Cp. K415NHETaHUSNOHETNoneNo—No31910765rs9078044611/233c. G1249Cp. D417HHETC3GNHETNONoneNoNoANA31911268rs1428021051/233c. G1531Ap. D511NHETC3GNHOMNONoneNoNoNo31911573—1/233c. T1720Cp. S574PHETC3GNHETHETNoneNoNoANAANA, Anti-nuclear antibody; ANCA, anti-neutrophil cytoplasm antibody; FH, factor H; HET, heterozygous; HOM, homozygous; ID, identifier; MCP, membrane cofactor protein/CD46.∗ Common polymorphism with a minor allele frequency greater than 0.01. The rest of the sequence variants display a minor allele frequency less than 0.001. Open table in a new tab ANA, Anti-nuclear antibody; ANCA, anti-neutrophil cytoplasm antibody; FH, factor H; HET, heterozygous; HOM, homozygous; ID, identifier; MCP, membrane cofactor protein/CD46. Wild-type (WT) C2 and all mutants were expressed in the eukaryotic system as C-terminal His-tagged proteins (see Fig E1, A and B in this article's Online Repository at www.jacionline.org). Such modification does not affect the function of C2, as evidenced by a comparison of the hemolytic activity of WT and plasma-purified C2 (see Fig E2, A and B in this article's Online Repository at www.jacionline.org). The variant Y347A, which was designed in silico and experimentally confirmed as resistant to CD55-, CD35- and C4BP-mediated convertase decay,6Kuttner-Kondo L.A. Dybvig M.P. Mitchell L.M. Muqim N. Atkinson J.P. Medof M.E. et al.A corresponding tyrosine residue in the C2/factor B type A domain is a hot spot in the decay acceleration of the complement C3 convertases.J Biol Chem. 2003; 278: 52386-52391Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar was included as a gain-of-function positive control. Assays performed on sensitized human erythrocytes in C2-depleted serum showed that the addition of each natural C2 mutant resulted in a lower hemolytic reaction (Fig 1, A) and lower CP/LP convertase activity (Fig 1, B) than did the addition of WT C2, except in the case of the variant S250C, which performed similarly to Y347A. These results were confirmed in the cytotoxic assay (Fig 1, C and D) and convertase assay (Fig 1, E and F) on sensitized human lymphoma cell lines. The gain-of-function S250C mutation did not affect C2 processing to C2a/C2b by C1s enzyme (see Fig E3 in this article's Online Repository at www.jacionline.org). Unlike in experiments on human cells, the activity of the S250C mutant was comparable to that of the WT when tested in the same serum on sensitized sheep erythrocytes (see Fig E4 in this article's Online Repository at www.jacionline.org). This led us to conclude that phenotype is dependent on membrane-bound convertase inhibitors. Further analysis of surface expression of these proteins (see Fig E5 in this article's Online Repository at www.jacionline.org) indicated CD55 as a candidate, as confirmed by complement deposition assay in the presence of CD55 ectodomain (see Fig E6 in this article's Online Repository at www.jacionline.org) and experiments with CD55-KO Raji cells in which the gain-of-function phenotype of the S250C mutant was lost (Fig 1, G and H). Serine 250 is highly conserved within sequences of C2 and FB (see Table E1 in this article's Online Repository at www.jacionline.org), and according to a model of C2a fragment (see Fig E7 in this article's Online Repository at www.jacionline.org), it is located only 22 Å from the Y347 residue, thus suggesting that these 2 residues together could form a binding site for a single molecule of complement inhibitor. Genetic screening of patients in whom aHUS and C3G has been diagnosed is usually limited to genes encoding AP elements such as CFB, CFH, C3, CFI, and CD46. Here, we present the first report of gain-of-function mutation in the CP/LP convertase component found in human disease. Moreover, S250C was the only rare variant in complement genes of a patient with aHUS who also carried the MCPggaac haplotype, which decreases transcriptional activity of CD46 promotor region and dramatically increases penetrance of gain-of-function mutation in FB.7Goicoechea de Jorge E. Harris C.L. Esparza-Gordillo J. Carreras L. Arranz E.A. Garrido C.A. et al.Gain-of-function mutations in complement factor B are associated with atypical hemolytic uremic syndrome.Proc Natl Acad Sci U S A. 2007; 104: 240-245Crossref PubMed Scopus (329) Google Scholar We propose that excessive activity of the CP/LP convertases together with predisposing haplotypes and environmental factors (eg, infections that increase the level of C-reactive protein) form multiple hits that together precipitate complement-related glomerulopathies. Therefore, routine diagnostics of such patients should not be focused only on the abnormalities in AP. We also underscore that commonly used methods such as hemolysis of sheep erythrocytes may be insufficient for functional analysis of gain-of-function variants of complement proteins, as is likewise evidenced in Felberg et al.8Felberg A. Urban A. Borowska A. Stasilojc G. Taszner M. Hellmann A. et al.Mutations resulting in the formation of hyperactive complement convertases support cytocidal effect of anti-CD20 immunotherapeutics.Cancer Immunol Immunother. 2019; 68: 587-598Crossref PubMed Scopus (7) Google Scholar The aHUS/C3G-causative nature of 7 C2 variants identified as loss-of-function mutants is less obvious than that of the S250C variant. Low C2 activity may result in higher susceptibility to bacterial infections, which often precede disease episodes.9Karpman D. Loos S. Tati R. Arvidsson I. Haemolytic uraemic syndrome.J Intern Med. 2017; 281: 123-148Crossref PubMed Scopus (50) Google Scholar However, the global minor allele frequency of the E318D mutation (a common polymorphism identified as a loss-of-function variant, which was the most frequent in our cohort) is 0.0297 (ClinVar identifier 12130). Because the analogous value in our cohort is lower (0.0236), we consider the representation of this loss-of-function mutation as not exceeding global distribution and probably not pivotal in the disease pathomechanism. For additional comments on the relevance and limitations of our finding, please see this article's Online Repository. We thank Dr Robbert Spaapen from the Sanquin Research and Landsteiner Laboratory, Amsterdam, The Netherlands, for his support and assistance with obtaining CD55-KO cells and Dr Paulina Czaplewska and Ms Aleksandra Lewandowska, MSc, from the Intercollegiate Faculty of Biotechnology the University of Gdańsk and Medical University of Gdańsk, Gdańsk, Poland, for mass spectrometry analysis. Patients in whom aHUS was diagnosed fulfilled the following criteria: platelet count less than 150 × 109/L or a decrease of more than 25% from baseline values, hemolytic anemia, and serum creatinine level greater than the upper limit of the normal range, together with a negative Coombs test result, normal activity of ADAMTS-13 and a negative Shiga toxin testing result. C3G was diagnosed by renal biopsy, which in many cases included analysis by electron microscopy. DNA from patients was analyzed for genetic variants by using an in-house next-generation sequencing panel including the following 44 genes: C1QA, C1QB, C1QC, C1R, C1S, C2, C3, C4A, C4BPA, C4BPB, C5, C7, C8A, C8B, C8G, C9, CD46, CD55, CD59, CFB, CFD, CFH, CFHR1, CFHR3, CFHR4, CFHR5, CFI, CFP, CLU, CR1, CR2, FCN1, FCN2, FCN3, ITGAX, ITGB2, MASP1, MASP2, MBL2, SERPING1, VSIG4, and VTN. Targeted sequences were captured by using the Nextera rapid capture custom Enrichment Kit (Illumina, San Diego, Calif) and sequencing data generated in a Miseq equipment using Miseq reagent kit v2 (300 cycles). Sequence data were analyzed by using Burrows-Wheeler Alignment and Picard software with additional filtering using customs tools. Variant calling was performed both with bcftools and VarScan, and the variant calling files generated were merged into a single file by using custom tools. Common variants with a minor allele frequency value greater than 1% in any population were excluded. To identified novel and/or pathogenic variants, we used different databases (the Exome Aggregation Consortium database, Genome Aggregation Database, 1000 Genomes, National Center for Biotechnology Information Single-Nucleotide Polymorphism Database, aHUS Mutation Database [www.fh-hus.org], or our in-house database). Pathogenicity was established by using multiple functional prediction methods (SIFT, PolyPhen2, etc) included in the ANNOVAR server and the functional data available in our laboratory and in the literature. Variants were categorized as pathogenic, variants of uncertain significance, and benign. The analysis of copy number variations within the CFH-CFHR gene region was performed by multiplex ligation-dependent probe amplification with the P236 A1 ARMD mix 1 (MRC-Holland, Amsterdam, The Netherlands). The human lymphoma cell lines Raji and Ramos (both obtained from the American Type Culture Collection, Manassas, Va) were cultured in RPMI 1640 medium with l-glutamine (Mediatech, New York, NY) supplemented with 10% FBS (PANBiotech, Aidenbach, Germany). Cells were cultivated at 37°C and humidified in a 5% CO2 atmosphere. Raji cells with CD55 knockout were produced by clustered regularly interspaced short palindromic repeats/Cas9 technology as described in Thielen et al.E1Thielen A.J.F. van Baarsen I.M. Jongsma M.L. Zeerleder S. Spaapen R.M. Wouters D. CRISPR/Cas9 generated human CD46, CD55 and CD59 knockout cell lines as a tool for complement research.J Immunol Methods. 2018; 456: 15-22Crossref PubMed Scopus (16) Google Scholar Wild-type C2 cDNA sequence (accession number NM_000063) additionally containing 6 histidine codons at the 3′ terminus, as well as sequences for R129H, R243C, S250C, E318D, K415N, D417H, D511N, S574P, and Y347A variants were codon-optimized, synthesized, and cloned into a pCEP4 vector in the framework of GeneArt Gene Synthesis service by Thermo Fisher Scientific (Waltham, Mass). Proteins were expressed in a eukaryotic system and purified as described in Urban et al.E2Urban A. Borowska A. Felberg A. van den Heuvel L. Stasilojc G. Volokhina E. et al.Gain of function mutant of complement factor B K323E mimics pathogenic C3NeF autoantibodies in convertase assays.Autoimmunity. 2018; 51: 18-24Crossref PubMed Scopus (11) Google Scholar Western blotting was developed with anti-C2 antibody (Complement Technology, Tyler, Tex) diluted 1:1,000, followed by horseradish peroxidase (HRP)-conjugated donkey anti-goat antibody diluted 1:10,000 (Jackson ImmunoResearch, Cambridgeshire, United Kingdom). Hemolytic assays evaluating the activity of recombinant, his-tagged C2 mutants were performed as described previously,E3Blom A.M. Volokhina E.B. Fransson V. Stromberg P. Berghard L. Viktorelius M. et al.A novel method for direct measurement of complement convertases activity in human serum.Clin Exp Immunol. 2014; 178: 142-153Crossref PubMed Scopus (36) Google Scholar with some modifications. Sheep erythrocytes (Biomaxima, Gdańsk, Poland) were sensitized with amboceptor (Behring Bern, Switzerland) diluted 1:1000 in 1 mL of dextrose-gelatin-Veronal buffer (DGVB+++) for 20 minutes at 37°C.E3Blom A.M. Volokhina E.B. Fransson V. Stromberg P. Berghard L. Viktorelius M. et al.A novel method for direct measurement of complement convertases activity in human serum.Clin Exp Immunol. 2014; 178: 142-153Crossref PubMed Scopus (36) Google Scholar Human erythrocytes were sensitized with anti-human red blood cells antibodies (Rockland, Limerick, Pa) diluted 1:75. Afterward, cells were washed 3 times, pelleted, and resuspended in 1 mL of DGVB++. Ten microliters of these suspensions were overlaid with serial dilutions of C2-depleted serum (ΔC2) (Complement Technology) in DGVB++ supplemented with the particular C2 mutant and incubated for 30 minutes at 37oC. Erythrocytes were centrifuged, and the hemoglobin released to the supernatant was measured at 405 nm in a Synergy H1 microplate reader (Biotek, Winooski, Vt). For the CP/LP convertase activity assay, sensitized erythrocytes were mixed with 2.5 μg/mL of the C5 inhibitor OmCI.E3Blom A.M. Volokhina E.B. Fransson V. Stromberg P. Berghard L. Viktorelius M. et al.A novel method for direct measurement of complement convertases activity in human serum.Clin Exp Immunol. 2014; 178: 142-153Crossref PubMed Scopus (36) Google Scholar The plate was incubated at 37oC, and 2% of the C2-depleted serum mixed with a particular C2 mutant diluted in DGVB++ was added at the indicated time points. Then, the erythrocytes were washed with EDTA–gelatin-Veronal buffer (GVB),E3Blom A.M. Volokhina E.B. Fransson V. Stromberg P. Berghard L. Viktorelius M. et al.A novel method for direct measurement of complement convertases activity in human serum.Clin Exp Immunol. 2014; 178: 142-153Crossref PubMed Scopus (36) Google Scholar pelleted, overlaid with EDTA-GVB containing 1:40 dilution of guinea pig serum (Harlan Laboratories, Itingen, Switzerland), and incubated for 30 minutes at 37oC. Lysis was determined by measuring the absorbance at 405 nm. The readout of the sample of erythrocytes mixed with water was considered as 100% (full) lysis. Complement-dependent cytotoxicity (CDC) was measured by calcein release assay as described in Stasilojc et al,E4Stasilojc G. Felberg A. Urban A. Kowalska D. Ma S. Blom A.M. et al.Calcein release assay as a method for monitoring serum complement activity during monoclonal antibody therapy in patients with B-cell malignancies.J Immunol Methods. 2020; 476: 112675Crossref PubMed Scopus (4) Google Scholar but ΔC2 serum was used instead of normal human serum. Cell lysis was calculated in reference to the fluorescence readout (490/520 nm) obtained for the supernatant of cells treated with 2% NP40 (full lysis). Ofatumumab (100 μg/mL) and 30% of the C5- or C3-depleted serum (Complement Technology) supplemented with 7.5 μg/mL of the analyzed C2 variant were added to calcein-loaded cells at the indicated time points. Complement activation in these conditions was arrested at the stage of C3 CP/LP convertase or C5 CP/LP convertase, respectively. Then, cells were washed with EDTA-GVB buffer, pelleted, and overlaid with EDTA-GVB containing a 1:20 dilution of guinea pig serum followed by a 30-minute incubation (37oC, 600 g). Fluorescence readout was performed as already described in the section on CDC assay. Each C2 mutant was incubated for 2 hours with 4 nM C1s enzyme (Complement Technology) diluted in 5 mM veronal buffer supplemented with 1 mM of Ca2+ and Mg2+. Cleavage into C2a and C2b was analyzed by Western blot. C2 polyclonal goat anti-human antibody (No. A212, Complement Technology) diluted 1:1,000 and secondary donkey anti-goat antibody conjugated with HRP (Jackson ImmunoResearch) diluted 1:10,000 were used for detection, followed by 3,3'-diaminobenzidine (Vector Laboratories, Burlingame, Calif) applied for visualization. ELISA plates were coated with 50 mg/mL of human immunoglobulin solution (Pentaglobin, Biotest) for 1 hour at 37oC and then blocked for 30 minutes with 3% fish gelatin (Sigma). Afterward, the plates were incubated with 0.5% ΔC2 serum and the particular C2 variant was diluted in GVB with or without the addition of soluble ectodomain of CD55 inhibitor (produced as described in Okroj et alE5Okroj M. Mark L. Stokowska A. Wong S.W. Rose N. Blackbourn D.J. et al.Characterization of the complement inhibitory function of rhesus rhadinovirus complement control protein (RCP).J Biol Chem. 2009; 284: 505-514Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) for 1 hour at 37oC. C3b detection was performed with polyclonal goat-anti human C3 antibody (Complement Technology), followed by rabbit anti-goat antibody conjugated with HRP (Dako, Glostrup, Denmark) diluted 1:10,000 and 1:5,000 in PBS, respectively. The assay was developed by using o-phenylenediamine dihydrochloride (Sigma Aldrich, Darmstadt, Germany) according to manufacturer's instructions. Expression of CD35, CD46, and CD55 in CD20+ cells and human erythrocytes was assessed by flow cytometry as described in Okroj et al.E6Okroj M. Eriksson I. Osterborg A. Blom A.M. Killing of CLL and NHL cells by rituximab and ofatumumab under limited availability of complement.Med Oncol. 2013; 30: 759Crossref PubMed Scopus (21) Google Scholar Primary antibodies (clone UJ11 for CD35, clone MEM-258 for CD46, and clone HI-55a for CD55) and isotype controls were purchased from Immunotools (Friesoythe, Germany). Amino acids sequences of C2 and FB proteins were retrieved from the UniProtE7Boutet E. Lieberherr D. Tognolli M. Schneider M. Bansal P. Bridge A.J. et al.UniProtKB/Swiss-Prot, the manually annotated section of the UniProt KnowledgeBase: how to use the entry view.Methods Mol Biol. 2016; 1374: 23-54Crossref PubMed Scopus (432) Google Scholar amino acids sequence database searched for the term complement C2, and the results were further manually filtered out to obtain only sequences of C2 and FB proteins. For further analysis, 151 sequences were selected. All selected sequences were subjected to multiple sequence alignment by using a multiple alignment using fast Fourier transform algorithm.E8Katoh K. Standley D.M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability.Mol Biol Evol. 2013; 30: 772-780Crossref PubMed Scopus (21622) Google Scholar Statistical calculations were performed with GraphPad Prism 7 software (GraphPad Software Inc, San Diego, Calif). The Kruskal-Wallis test was applied for column analysis (see Fig E3). The Dunnett multiple comparison test for nonrepeated measures was applied for grouped analysis (see Figs E1, E2, and Fig E4, Fig E5, Fig E6, Fig E7). The domain structure of FB and complement C2 share a high degree of similarity.E10Krishnan V. Xu Y. Macon K. Volanakis J.E. Narayana S.V. The structure of C2b, a fragment of complement component C2 produced during C3 convertase formation.Acta Crystallogr D Biol Crystallogr. 2009; 65: 266-274Crossref PubMed Scopus (24) Google Scholar The von Willenbrand type A domain of FB contains several mutational hot spots, alterations of which may provoke a potentially pathogenic gain-of-function phenotype.E11Goicoechea de Jorge E. Harris C.L. Esparza-Gordillo J. Carreras L. Arranz E.A. Garrido C.A. et al.Gain-of-function mutations in complement factor B are associated with atypical hemolytic uremic syndrome.Proc Natl Acad Sci U S A. 2007; 104: 240-245Crossref PubMed Scopus (367) Google Scholar, E12Marinozzi M.C. Vergoz L. Rybkine T. Ngo S. Bettoni S. Pashov A. et al.Complement factor B mutations in atypical hemolytic uremic syndrome-disease-relevant or benign?.J Am Soc Nephrol. 2014; 25: 2053-2065Crossref PubMed Scopus (75) Google Scholar We identified 1 rare variant of C2 with mutation at conserved position 250 adjacent to α-helixes forming the von Willenbrand type A domain, which leads to the formation of hyperactive CP/LP convertases. No previous studies have shown the importance of this position for the biologic activity of C2 or FB. Moreover, no prior studies have presented patients with gain-of-function mutations in complement genes coding for CP/LP convertase components. Thus, to the best of our knowledge, this is the first such report confirmed by functional studies. Genetic screening in patients in whom aHUS and C3G have been diagnosed is usually limited to genes encoding AP elements such as FB, CFH, C3, CFI, and CD46,E13Loirat C. Fremeaux-Bacchi V. Atypical hemolytic uremic syndrome.Orphanet J Rare Dis. 2011; 6: 60Crossref PubMed Scopus (461) Google Scholar and on the basis of this scheme, abnormalities in complement are found in 70% of patients with aHUS whereas 30% remain unexplained.E14Joseph C. Gattineni J. Complement disorders and hemolytic uremic syndrome.Curr Opin Pediatr. 2013; 25: 209-215Crossref PubMed Scopus (28) Google Scholar Typically, patients with aHUS are heterozygous in complement mutations, and the same mutation can be carried by healthy relatives. Often, the difference between healthy relatives carrying pathogenic mutations and patients is the presence of predisposing polymorphisms in genes coding complement regulators.E15Noris M. Caprioli J. Bresin E. Mossali C. Pianetti G. Gamba S. et al.Relative role of genetic complement abnormalities in sporadic and familial aHUS and their impact on clinical phenotype.Clin J Am Soc Nephrol. 2010; 5: 1844-1859Crossref PubMed Scopus (698) Google Scholar, E16Esparza-Gordillo J. Goicoechea de Jorge E. Buil A. Carreras Berges L. Lopez-Trascasa M. Sanchez-Corral P. et al.Predisposition to atypical hemolytic uremic syndrome involves the concurrence of different susceptibility alleles in the regulators of complement activation gene cluster in 1q32.Hum Mol Genet. 2005; 14: 703-712Crossref PubMed Scopus (240) Google Scholar, E17Fremeaux-Bacchi V. Kemp E.J. Goodship J.A. Dragon-Durey M.A. Strain L. Loirat C. et al.The development of atypical haemolytic-uraemic syndrome is influenced by susceptibility factors in factor H and membrane cofactor protein: evidence from two independent cohorts.J Med Genet. 2005; 42: 852-856Crossref PubMed Scopus (148) Google Scholar Moreover, aHUS can be preceded by bacterial and viral infections, surgical procedures, use of certain medications, and pregnancy in women.E18Nester C.M. Barbour T. de Cordoba S.R. Dragon-Durey M.A. Fremeaux-Bacchi V. Goodship T.H. et al.Atypical aHUS: state of the art.Mol Immunol. 2015; 67: 31-42Crossref PubMed Scopus (192) Google Scholar, E19Jokiranta T.S. HUS and atypical HUS.Blood. 2017; 129: 2847-2856Crossref PubMed Scopus (154) Google Scholar One of the limitations of our study is the lack of a medical record for the patient with aHUS who was carrying the S250C allele, which was not available. Such detailed clinical data, as well as the family history and a genotype/phenotype analysis of family members, would help to evaluate whether this case is similar to or different from cases of aHUS connected to gain-of-function mutations in FB or C3 proteins. On the other hand, it is highly plausible that overactive CP/LP convertases are responsible for the percentage of aHUS cases previously classified as being of unknown etiology. Unlike AP, CP and LP require specific stimuli (eg, antibodies, certain sugar moieties, or C-reactive protein.E20Ricklin D. Hajishengallis G. Yang K. Lambris J.D. Complement: a key system for immune surveillance and homeostasis.Nat Immunol. 2010; 11: 785-797Crossref PubMed Scopus (2458) Google Scholar Therefore, 2 conditions permissive for CP/LP-mediated autoimmune disease are necessary, namely, loss of proper pathway regulation (eg, formation of decay-resistant CP/LP convertase) and presence of stimuli driving the pathway. Without a detailed clinical report, we can only speculate on a hypothetical explanation of disease development in the patient with the 250C mutation. Nonetheless, infections and presence of autoantibodies (or appearance of antibodies cross-reactive to patient cells and/or tissues) emerge as possible drivers of pathomechanism. In line with reports on C4NeF as the only complement abnormality found in patients with C3G,E21Blom A.M. Corvillo F. Magda M. Stasilojc G. Nozal P. Perez-Valdivia M.A. et al.Testing the activity of complement convertases in serum/plasma for diagnosis of C4NeF-mediated C3 glomerulonephritis.J Clin Immunol. 2016; 36: 517-527Crossref PubMed Scopus (20)