An Ex Vivo Test of Complement Activation on Endothelium for Individualized Eculizumab Therapy in Hemolytic Uremic Syndrome.

RATIONALE & OBJECTIVE: Although primary atypical hemolytic uremic syndrome (aHUS) is associated with abnormalities in complement genes and antibodies to complement factor H, the role of complement in secondary aHUS remains debatable. We evaluated the usefulness of an ex vivo test to: (1) detect complement activation within the endothelium in primary and secondary aHUS, (2) differentiate active disease from remission, (3) monitor the effectiveness of eculizumab therapy, and (4) identify relapses during eculizumab dosage tapering and after discontinuation of treatment. STUDY DESIGN: Case series. SETTING & PARTICIPANTS: 121 patients with primary aHUS and 28 with secondary aHUS. Serum samples were collected during acute episodes, following remission, and during eculizumab treatment and were assessed using a serum-induced ex vivo C5b-9 endothelial deposition test. RESULTS: Serum-induced C5b-9 deposition on cultured microvascular endothelium was quantified by calculating the endothelial area covered by C5b-9 staining; values were expressed as percentage of C5b-9 deposits induced by a serum pool from healthy controls. Testing with adenosine diphosphate-activated endothelium demonstrated elevated C5b-9 deposits for all untreated patients with aHUS independent of disease activity, while testing with unstimulated endothelium demonstrated deposits only in active disease. Similar findings were observed in secondary aHUS. Serum-induced C5b-9 deposits on activated and unstimulated endothelium normalized during eculizumab treatment. 96% (22/23) of patients receiving eculizumab at extended 3- or 4-week dosing intervals demonstrated normal C5b-9 deposits on activated endothelium, despite most patients having CH50Eq (serum complement activity) > 20 UEq/mL, indicating that adequate complement control was achieved even with incomplete blockade of circulating C5. During eculizumab dosage tapering or after treatment discontinuation, all patients experiencing relapses versus only 6% (1/17) of those in stable remission had elevated C5b-9 deposits on unstimulated endothelium. LIMITATIONS: The C5b-9 endothelial deposition test can be performed in only specialized laboratories. Findings on eculizumab dosage tapering need to be confirmed with longitudinal monitoring of C5b-9 deposition. CONCLUSIONS: The C5b-9 endothelial deposition assay may represent an advance in our ability to monitor aHUS activity and individualize therapy.

Cluster Analysis Identifies Distinct Pathogenetic Patterns in C3 Glomerulopathies/Immune Complex-Mediated Membranoproliferative GN.

Membranoproliferative GN (MPGN) was recently reclassified as alternative pathway complement-mediated C3 glomerulopathy (C3G) and immune complex-mediated membranoproliferative GN (IC-MPGN). However, genetic and acquired alternative pathway abnormalities are also observed in IC-MPGN. Here, we explored the presence of distinct disease entities characterized by specific pathophysiologic mechanisms. We performed unsupervised hierarchical clustering, a data-driven statistical approach, on histologic, genetic, and clinical data and data regarding serum/plasma complement parameters from 173 patients with C3G/IC-MPGN. This approach divided patients into four clusters, indicating the existence of four different pathogenetic patterns. Specifically, this analysis separated patients with fluid-phase complement activation (clusters 1-3) who had low serum C3 levels and a high prevalence of genetic and acquired alternative pathway abnormalities from patients with solid-phase complement activation (cluster 4) who had normal or mildly altered serum C3, late disease onset, and poor renal survival. In patients with fluid-phase complement activation, those in clusters 1 and 2 had massive activation of the alternative pathway, including activation of the terminal pathway, and the highest prevalence of subendothelial deposits, but those in cluster 2 had additional activation of the classic pathway and the highest prevalence of nephrotic syndrome at disease onset. Patients in cluster 3 had prevalent activation of C3 convertase and highly electron-dense intramembranous deposits. In addition, we provide a simple algorithm to assign patients with C3G/IC-MPGN to specific clusters. These distinct clusters may facilitate clarification of disease etiology, improve risk assessment for ESRD, and pave the way for personalized treatment.

Mapping interactions between complement C3 and regulators using mutations in atypical hemolytic uremic syndrome.

The pathogenesis of atypical hemolytic uremic syndrome (aHUS) is strongly linked to dysregulation of the alternative pathway of the complement system. Mutations in complement genes have been identified in about two-thirds of cases, with 5% to 15% being in C3. In this study, 23 aHUS-associated genetic changes in C3 were characterized relative to their interaction with the control proteins factor H (FH), membrane cofactor protein (MCP; CD46), and complement receptor 1 (CR1; CD35). In surface plasmon resonance experiments, 17 mutant recombinant proteins demonstrated a defect in binding to FH and/or MCP, whereas 2 demonstrated reduced binding to CR1. In the majority of cases, decreased binding affinity translated to a decrease in proteolytic inactivation (known as cofactor activity) of C3b via FH and MCP. These results were used to map the putative binding regions of C3b involved in the interaction with MCP and CR1 and interrogated relative to known FH binding sites. Seventy-six percent of patients with C3 mutations had low C3 levels that correlated with disease severity. This study expands our knowledge of the functional consequences of aHUS-associated C3 mutations relative to the interaction of C3 with complement regulatory proteins mediating cofactor activity.

Dynamics of complement activation in aHUS and how to monitor eculizumab therapy.

Atypical hemolytic-uremic syndrome (aHUS) is associated with genetic complement abnormalities/anti-complement factor H antibodies, which paved the way to treatment with eculizumab. We studied 44 aHUS patients and their relatives to (1) test new assays of complement activation, (2) verify whether such abnormality occurs also in unaffected mutation carriers, and (3) search for a tool for eculizumab titration. An abnormal circulating complement profile (low C3, high C5a, or SC5b-9) was found in 47% to 64% of patients, irrespective of disease phase. Acute aHUS serum, but not serum from remission, caused wider C3 and C5b-9 deposits than control serum on unstimulated human microvascular endothelial cells (HMEC-1). In adenosine 5'-diphosphate-activated HMEC-1, also sera from 84% and 100% of patients in remission, and from all unaffected mutation carriers, induced excessive C3 and C5b-9 deposits. At variance, in most patients with C3 glomerulopathies/immune complex-associated membranoproliferative glomerulonephritis, serum-induced endothelial C5b-9 deposits were normal. In 8 eculizumab-treated aHUS patients, C3/SC5b-9 circulating levels did not change posteculizumab, whereas serum-induced endothelial C5b-9 deposits normalized after treatment, paralleled or even preceded remission, and guided drug dosing and timing. These results point to efficient complement inhibition on endothelium for aHUS treatment. C5b-9 endothelial deposits might help monitor eculizumab effectiveness, avoid drug overexposure, and save money considering the extremely high cost of the drug.

A novel atypical hemolytic uremic syndrome-associated hybrid CFHR1/CFH gene encoding a fusion protein that antagonizes factor H-dependent complement regulation.

Genomic aberrations affecting the genes encoding factor H (FH) and the five FH-related proteins (FHRs) have been described in patients with atypical hemolytic uremic syndrome (aHUS), a rare condition characterized by microangiopathic hemolytic anemia, thrombocytopenia, and ARF. These genomic rearrangements occur through nonallelic homologous recombinations caused by the presence of repeated homologous sequences in CFH and CFHR1-R5 genes. In this study, we found heterozygous genomic rearrangements among CFH and CFHR genes in 4.5% of patients with aHUS. CFH/CFHR rearrangements were associated with poor clinical prognosis and high risk of post-transplant recurrence. Five patients carried known CFH/CFHR1 genes, but we found a duplication leading to a novel CFHR1/CFH hybrid gene in a family with two affected subjects. The resulting fusion protein contains the first four short consensus repeats of FHR1 and the terminal short consensus repeat 20 of FH. In an FH-dependent hemolysis assay, we showed that the hybrid protein causes sheep erythrocyte lysis. Functional analysis of the FHR1 fraction purified from serum of heterozygous carriers of the CFHR1/CFH hybrid gene indicated that the FHR1/FH hybrid protein acts as a competitive antagonist of FH. Furthermore, sera from carriers of the hybrid CFHR1/CFH gene induced more C5b-9 deposition on endothelial cells than control serum. These results suggest that this novel genomic hybrid mediates disease pathogenesis through dysregulation of complement at the endothelial cell surface. We recommend that genetic screening of aHUS includes analysis of CFH and CFHR rearrangements, particularly before a kidney transplant.

Combined complement gene mutations in atypical hemolytic uremic syndrome influence clinical phenotype.

Several abnormalities in complement genes reportedly contribute to atypical hemolytic uremic syndrome (aHUS), but incomplete penetrance suggests that additional factors are necessary for the disease to manifest. Here, we sought to describe genotype-phenotype correlations among patients with combined mutations, defined as mutations in more than one complement gene. We screened 795 patients with aHUS and identified single mutations in 41% and combined mutations in 3%. Only 8%-10% of patients with mutations in CFH, C3, or CFB had combined mutations, whereas approximately 25% of patients with mutations in MCP or CFI had combined mutations. The concomitant presence of CFH and MCP risk haplotypes significantly increased disease penetrance in combined mutated carriers, with 73% penetrance among carriers with two risk haplotypes compared with 36% penetrance among carriers with zero or one risk haplotype. Among patients with CFH or CFI mutations, the presence of mutations in other genes did not modify prognosis; in contrast, 50% of patients with combined MCP mutation developed end stage renal failure within 3 years from onset compared with 19% of patients with an isolated MCP mutation. Patients with combined mutations achieved remission with plasma treatment similar to patients with single mutations. Kidney transplant outcomes were worse, however, for patients with combined MCP mutation compared with an isolated MCP mutation. In summary, these data suggest that genotyping for the risk haplotypes in CFH and MCP may help predict the risk of developing aHUS in unaffected carriers of mutations. Furthermore, screening patients with aHUS for all known disease-associated genes may inform decisions about kidney transplantation.

Atypical haemolytic uraemic syndrome with underlying glomerulopathies. A case series and a review of the literature.

BACKGROUND: Primary or secondary glomerulonephritis has been anecdotally reported in association with atypical haemolytic uraemic syndrome (aHUS). We here report a series of six patients who developed aHUS and glomerulopathy, and review the literature on aHUS and glomerulonephritis. METHODS: Out of all patients diagnosed at our unit with biopsy-proven glomerular diseases between March 2007 and October 2011, selected cases developing aHUS during the follow-up are presented. The following tests were performed in all six patients: serum C3 and C4 levels, ADAMTS13 activity, CFH levels and anti-CFH autoantibodies and genetic screening for CFH, MCP, CFI, C3 and CFHR1-3 mutations and risk haplotypes associated with aHUS. RESULTS: Two hundred and forty-eight patients received a biopsy-proven diagnosis of glomerulopathy and were followed for a median of 31 months (range 2-58). Of these, six developed aHUS, within a median of 15 months (range 1-36) of their initial diagnosis of glomerulopathy. One of these patients had focal segmental glomerulosclerosis (FSGS), two membranoproliferative glomerulonephritis (MPGN) type I, one C3 glomerulonephritis and two systemic small vessel vasculitis [one granulomatosis with polyangiitis (Wegener's), one Henoch-Schoenlein purpura]. Five patients (one of them heterozygous for a CFH mutation) carried, in homo- or heterozygosity, the risk haplotype CFH-H3 (CFH tgtgt), previously described to be associated with aHUS, while another one patient was homozygous for the MCPggaac risk haplotype predisposing to aHUS when present on both alleles. CONCLUSIONS: Different types of glomerulopathies can be complicated by aHUS. Several mechanisms can contribute to this association, such as nephrotic-range proteinuria, mutations or aHUS-risk haplotypes involving genes encoding alternative complement regulatory proteins in some patients and inflammatory triggers associated with systemic immune-mediated diseases.

A GWAS in the pandemic epicenter highlights the severe COVID-19 risk locus introgressed by Neanderthals.

Large GWAS indicated that genetic factors influence the response to SARS-CoV-2. However, sex, age, concomitant diseases, differences in ancestry, and uneven exposure to the virus impacted the interpretation of data. We aimed to perform a GWAS of COVID-19 outcome in a homogeneous population who experienced a high exposure to the virus and with a known infection status. We recruited inhabitants of Bergamo province-that in spring 2020 was the epicenter of the SARS-Cov-2 pandemic in Europe-via an online questionnaire followed by personal interviews. Cases and controls were matched by age, sex and risk factors. We genotyped 1195 individuals and replicated the association at the 3p21.31 locus with severity, but with a stronger effect size that further increased in gravely ill patients. Transcriptome-wide association study highlighted eQTLs for LZTFL1 and CCR9. We also identified 17 loci not previously reported, suggestive for an association with either COVID-19 severity or susceptibility.

Mycoplasma pneumoniae Infection Associated with Anti-Factor H Autoantibodies in Atypical Hemolytic Uremic Syndrome.

Hemolytic uremic syndrome (HUS) is a rare disease characterized by hemolytic anemia, thrombocytopenia, and renal impairment mostly triggered by strains of Shiga-like toxin-producing Escherichia coli (STEC-HUS). A rarer form of HUS, defined as atypical HUS (aHUS), is associated with genetic or acquired dysregulation of the alternative pathway of the complement system and presents a poorer prognosis than STEC-HUS. Factor H autoantibodies (anti-FHs) have been reported in aHUS in 5-11% of cases and are strongly associated with the homozygous deletion of CFHR3-CFHR1 genes. In the large majority of patients, anti-FH-associated aHUS is commonly preceded by gastrointestinal or respiratory tract infections. Here, we described the clinical case of a 3-year-old boy who was hospitalized for aHUS preceded by Mycoplasma pneumoniae (MP) infection. He resulted positive for anti-FHs and carried the homozygous deletion of CFHR3-CFHR1. Of relevance, he also showed a variant of unknown significance in the C5 gene. The patient was successfully treated with eculizumab and achieved hematological and renal remission. The anti-FH titer decreased, became negative after 6 months of mycophenolate mofetil (MMF) treatment, and remained negative for 21-month follow-up indicating that immunosuppression was effective and could prevent the reappearance of anti-FHs. We hypothesized that MP, likely through an evasion strategy of immunosurveillance based on binding of pathogen to FH, triggers anti-FH antibody generation and aHUS in a subject genetically predisposed. In conclusion, to the best of our knowledge, here, we reported the first case of anti-FH-mediated aHUS after an MP infection who benefited from eculizumab and immunosuppressive therapy based on MMF. Hence, monitoring of anti-FHs in patients with post-MP infection glomerulonephritis could be recommended, especially in those with low C3 plasma levels.

CFH and CFHR structural variants in atypical Hemolytic Uremic Syndrome: Prevalence, genomic characterization and impact on outcome.

Introduction: Atypical hemolytic uremic syndrome (aHUS) is a rare disease that manifests with microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure, and is associated with dysregulation of the alternative complement pathway. The chromosomal region including CFH and CFHR1-5 is rich in repeated sequences, favoring genomic rearrangements that have been reported in several patients with aHUS. However, there are limited data on the prevalence of uncommon CFH-CFHR genomic rearrangements in aHUS and their impact on disease onset and outcomes. Methods: In this study, we report the results of CFH-CFHR Copy Number Variation (CNV) analysis and the characterization of resulting structural variants (SVs) in a large cohort of patients, including 258 patients with primary aHUS and 92 with secondary forms. Results: We found uncommon SVs in 8% of patients with primary aHUS: 70% carried rearrangements involving CFH alone or CFH and CFHR (group A; n=14), while 30% exhibited rearrangements including only CFHRs (group B; n=6). In group A, 6 patients presented CFH::CFHR1 hybrid genes, 7 patients carried duplications in the CFH-CFHR region that resulted either in the substitution of the last CFHR1 exon(s) with those of CFH (CFHR1::CFH reverse hybrid gene) or in an internal CFH duplication. In group A, the large majority of aHUS acute episodes not treated with eculizumab (12/13) resulted in chronic ESRD; in contrast, anti-complement therapy induced remission in 4/4 acute episodes. aHUS relapse occurred in 6/7 grafts without eculizumab prophylaxis and in 0/3 grafts with eculizumab prophylaxis. In group B, 5 subjects had the CFHR31-5::CFHR410 hybrid gene and one had 4 copies of CFHR1 and CFHR4. Compared with group A, patients in group B exhibited a higher prevalence of additional complement abnormalities and earlier disease onset. However, 4/6 patients in this group underwent complete remission without eculizumab treatment. In secondary forms we identified uncommon SVs in 2 out of 92 patients: the CFHR31-5::CFHR410 hybrid and a new internal duplication of CFH. Discussion: In conclusion, these data highlight that uncommon CFH-CFHR SVs are frequent in primary aHUS and quite rare in secondary forms. Notably, genomic rearrangements involving the CFH are associated with a poor prognosis but carriers respond to anti-complement therapy.

CFH and CFHR Copy Number Variations in C3 Glomerulopathy and Immune Complex-Mediated Membranoproliferative Glomerulonephritis.

C3 Glomerulopathy (C3G) and Immune Complex-Mediated Membranoproliferative glomerulonephritis (IC-MPGN) are rare diseases characterized by glomerular deposition of C3 caused by dysregulation of the alternative pathway (AP) of complement. In approximately 20% of affected patients, dysregulation is driven by pathogenic variants in the two components of the AP C3 convertase, complement C3 (C3) and Factor B (CFB), or in complement Factor H (CFH) and Factor I (CFI), two genes that encode complement regulators. Copy number variations (CNVs) involving the CFH-related genes (CFHRs) that give rise to hybrid FHR proteins also have been described in a few C3G patients but not in IC-MPGN patients. In this study, we used multiplex ligation-dependent probe amplification (MLPA) to study the genomic architecture of the CFH-CFHR region and characterize CNVs in a large cohort of patients with C3G (n = 103) and IC-MPGN (n = 96) compared to healthy controls (n = 100). We identified new/rare CNVs resulting in structural variants (SVs) in 5 C3G and 2 IC-MPGN patients. Using long-read single molecule real-time sequencing (SMRT), we detected the breakpoints of three SVs. The identified SVs included: 1) a deletion of the entire CFH in one patient with IC-MPGN; 2) an increased number of CFHR4 copies in one IC-MPGN and three C3G patients; 3) a deletion from CFHR3-intron 3 to CFHR3-3'UTR (CFHR34 - 6 Δ) that results in a FHR3-FHR1 hybrid protein in a C3G patient; and 4) a CFHR31 - 5-CFHR410 hybrid gene in a C3G patient. This work highlights the contribution of CFH-CFHR CNVs to the pathogenesis of both C3G and IC-MPGN.

Molecular Studies and an ex vivo Complement Assay on Endothelium Highlight the Genetic Complexity of Atypical Hemolytic Uremic Syndrome: The Case of a Pedigree With a Null CD46 Variant.

Atypical hemolytic uremic syndrome (aHUS) is an ultra-rare disease characterized by microangiopathic hemolysis, thrombocytopenia, and renal impairment and is associated with dysregulation of the alternative complement pathway on the microvascular endothelium. Outcomes have improved greatly with pharmacologic complement C5 blockade. Abnormalities in complement genes (CFH, CD46, CFI, CFB, C3, and THBD), CFH-CFHR genomic rearrangements, and anti-FH antibodies have been reported in 40-60% of cases. The penetrance of aHUS is incomplete in carriers of complement gene abnormalities; and multiple hits, including the CFH-H3 and CD46 GGAAC risk haplotypes and the CFHR1 * B risk allele, as well as environmental factors, contribute to disease development. Here, we investigated the determinants of penetrance of aHUS associated with CD46 genetic abnormalities. We studied 485 aHUS patients and found CD46 rare variants (RVs) in about 10%. The c.286+2T>G RV was the most prevalent (13/485) and was associated with <30% penetrance. We conducted an in-depth study of a large pedigree including a proband who is heterozygous for the c.286+2T>G RV who experienced a severe form of aHUS and developed end-stage renal failure. The father and paternal uncle with the same variant in homozygosity and six heterozygous relatives are unaffected. Flow cytometry analysis showed about 50% reduction of CD46 expression on blood mononuclear cells from the heterozygous proband and over 90% reduction in cells from the proband's unaffected homozygous father and aunt. Further genetic studies did not reveal RVs in known aHUS-associated genes or common genetic modifiers that segregated with the disease. Importantly, a specific ex vivo test showed excessive complement deposition on endothelial cells exposed to sera from the proband, and also from his mother and maternal uncle, who do not carry the c.286+2T>G RV, indicating that they share a circulating defect that results in complement dysregulation on the endothelium. These results highlight the complexity of the genetics of aHUS and indicate that CD46 deficiency may not be enough to induce aHUS. We hypothesize that the proband inherited from his mother a genetic abnormality in a complement circulating factor that has not been identified yet, which synergized with the CD46 RV in predisposing him to the aHUS phenotype.

Hemolytic Uremic Syndrome in an Infant with Primary Hyperoxaluria Type II: An Unreported Clinical Association.

A 6-month-old boy presented with acute renal failure, thrombocytopenia, and severe non-immune hemolytic anemia. Infection by Shiga-like toxin-producing Escherichia coli and other causes of microangiopathic hemolysis were ruled out, leading to a diagnosis of atypical hemolytic uremic syndrome (aHUS). Neither pathogenic variants in HUS-associated genes nor anti-factor H antibodies were identified. Copy number variation analysis uncovered 4 copies of complement factor H related genes, CFHR1-CFHR4, conceivably leading to higher than normal levels of the corresponding proteins. However, this abnormality was also found in the healthy relatives, neither explaining the disease nor the excessive complement deposition on endothelial cells detected by an ex-vivo test. Whole-exome sequencing revealed a pathogenic homozygous variant in GRHPR encoding the glyoxylate and hydroxypyruvate reductase. Recessive GRHPR mutations cause primary hyperoxaluria type 2 (PH2). The presence of renal calculi in the patient and elevated oxalate levels in the urine were consistent with the genetic diagnosis of PH2. We hypothesize that, in this patient, hyperoxaluria caused by the GRHPR genetic defect triggered endothelial perturbation and complement activation, which was amplified by impaired factor H regulatory activity due to the increased -CFHR1-CFHR4 copy numbers, resulting in aHUS.

Rare Functional Variants in Complement Genes and Anti-FH Autoantibodies-Associated aHUS.

Atypical hemolytic uremic syndrome (aHUS) is a rare disease characterized by microangiopathic hemolytic anemia, thrombocytopenia and renal failure. It is caused by genetic or acquired defects of the complement alternative pathway. Factor H autoantibodies (anti-FHs) have been reported in 10% of aHUS patients and are associated with the deficiency of factor H-related 1 (FHR1). However, FHR1 deficiency is not enough to cause aHUS, since it is also present in about 5% of Caucasian healthy subjects. In this study we evaluated the prevalence of genetic variants in CFH, CD46, CFI, CFB, C3, and THBD in aHUS patients with anti-FHs, using healthy subjects with FHR1 deficiency, here defined "supercontrols," as a reference group. "Supercontrols" are more informative than general population because they share at least one risk factor (FHR1 deficiency) with aHUS patients. We analyzed anti-FHs in 305 patients and 30 were positive. The large majority were children (median age: 7.7 [IQR, 6.6-9.9] years) and 83% lacked FHR1 (n = 25, cases) due to the homozygous CFHR3-CFHR1 deletion (n = 20), or the compound heterozygous CFHR3-CFHR1 and CFHR1-CFHR4 deletions (n = 4), or the heterozygous CFHR3-CFHR1 deletion combined with a frameshift mutation in CFHR1 that generates a premature stop codon (n = 1). Of the 960 healthy adult subjects 48 had the FHR1 deficiency ("supercontrols"). Rare likely pathogenetic variants in CFH, THBD, and C3 were found in 24% of cases (n = 6) compared to 2.1% of the "supercontrols" (P-value = 0.005). We also found that the CFH H3 and the CD46GGAAC haplotypes are not associated with anti-FHs aHUS, whereas these haplotypes are enriched in aHUS patients without anti-FHs, which highlights the differences in the genetic basis of the two forms of the disease. Finally, we confirm that common infections are environmental factors that contribute to the development of anti-FHs aHUS in genetically predisposed individuals, which fits with the sharp peak of incidence during scholar-age. Further studies are needed to fully elucidate the complex genetic and environmental factors underlying anti-FHs aHUS and to establish whether the combination of anti-FHs with likely pathogenetic variants or other risk factors influences disease outcome and response to therapies.

Impact of a Complement Factor H Gene Variant on Renal Dysfunction, Cardiovascular Events, and Response to ACE Inhibitor Therapy in Type 2 Diabetes.

Complement activation has been increasingly implicated in the pathogenesis of type 2 diabetes and its chronic complications. It is unknown whether complement factor H (CFH) genetic variants, which have been previously associated with complement-mediated organ damage likely due to inefficient complement modulation, influence the risk of renal and cardiovascular events and response to therapy with angiotensin-converting enzyme inhibitors (ACEi) in type 2 diabetic patients. Here, we have analyzed the c.2808G>T, (p.Glu936Asp) CFH polymorphism, which tags the H3 CFH haplotype associated to low plasma factor H levels and predisposing to atypical hemolytic uremic syndrome, in 1,158 type 2 diabetics prospectively followed in the Bergamo nephrologic complications of type 2 diabetes randomized, controlled clinical trial (BENEDICT) that evaluated the effect of the ACEi trandolapril on new onset microalbuminuria. At multivariable Cox analysis, the p.Glu936Asp polymorphism (Asp/Asp homozygotes, recessive model) was associated with increased risk of microalbuminuria [adjusted hazard ratio (HR) 3.25 (95% CI 1.46-7.24), P = 0.0038] and cardiovascular events [adjusted HR 2.68 (95% CI 1.23-5.87), P = 0.013]. The p.Glu936Asp genotype significantly interacted with ACEi therapy in predicting microalbuminuria. ACEi therapy was not nephroprotective in Asp/Asp homozygotes [adjusted HR 1.54 (0.18-13.07), P = 0.691 vs. non-ACEi-treated Asp/Asp patients], whereas it significantly reduced microalbuminuria events in Glu/Asp or Glu/Glu patients [adjusted HR 0.38 (0.24-0.60), P < 0.0001 vs. non-ACEi-treated Glu/Asp or Glu/Glu patients]. Among ACEi-treated patients, the risk of developing cardiovascular events was higher in Asp/Asp homozygotes than in Glu/Asp or Glu/Glu patients [adjusted HR 3.26 (1.29-8.28), P = 0.013]. Our results indicate that type 2 diabetic patients Asp/Asp homozygotes in the p.Glu936Asp CFH polymorphism are at increased risk of microalbuminuria and cardiovascular complications and may be less likely to benefit from ACEi therapy. Further studies are required to confirm our findings.

Unraveling the Molecular Mechanisms Underlying Complement Dysregulation by Nephritic Factors in C3G and IC-MPGN.

Membranoproliferative glomerulonephritis (MPGN) was recently classified as C3 glomerulopathies (C3G), and immune-complex (IC) mediated MPGN. Dysregulation of the complement alternative pathway, driven by acquired and/or genetic defects, plays a pathogenetic role in C3G. However, alternative pathway abnormalities were also found in IC-MPGN. The most common acquired drivers are the C3 nephritic factors (C3NeFs), heterogeneous autoantibodies that stabilize the C3 convertase, C3bBb. C3NeFs are traditionally detected by hemolytic assays based on sheep erythrocyte lysis, which however do not provide a direct molecular estimation of C3bBb formation and decay. We set up a microplate/western blot assay that specifically detects and quantifies C3bBb, and its precursor, the C3 proconvertase C3bB, to investigate the complex mechanistic effects of C3NeFs from patients with primary IC-MPGN (n = 13) and C3G (n = 13). In the absence of properdin, 9/26 patients had C3NeF IgGs stabilizing C3bBb against spontaneous and FH-accelerated decay. In the presence of properdin the IgGs of all but one patient had C3bBb-stabilizing activity. Properdin-independent C3NeFs were identified mostly in DDD patients, while properdin-dependent C3NeFs associated with either C3GN or IC-MPGN and with higher incidence of nephrotic syndrome. When we grouped patients based on our recent cluster analysis, patients in cluster 3, with highly electron-dense intramembranous deposits, low C3, and mostly normal sC5b-9 levels, had a higher prevalence of properdin-independent C3NeFs than patients in clusters 1 and 2. Conversely, about 70% of cluster 1 and 2 patients, with subendothelial, subepithelial, and mesangial deposits, low C3 levels and high sC5b-9 levels, had properdin-dependent C3NeFs. The flexibility of the assay allowed us to get deep insights into C3NeF mechanisms of action, showing that: (1) most C3NeFs bind strongly and irreversibly to C3 convertase; (2) C3NeFs and FH recognize different epitopes in C3 convertase; (3) C3NeFs bind rapidly to C3 convertase and antagonize the decay accelerating activity of FH on newly formed complexes; (4) C3NeFs do not affect formation and stability of the C3 proconvertase. Thus, our study provides a molecular approach to detecting and characterizing C3NeFs. The results highlight different mechanisms of complement dysregulation resulting in different complement profiles and patterns of glomerular injury, and this may have therapeutic implications.

Complement gene variants determine the risk of immunoglobulin-associated MPGN and C3 glomerulopathy and predict long-term renal outcome.

BACKGROUND: Membranoproliferative glomerulonephritis (MPGN) is an uncommon cause of chronic nephropathy recently reclassified into immunoglobulin-associated MPGN (Ig-MPGN) and C3 glomerulopathy (C3G). In this study we aimed: (1) to evaluate the complement genetic and biochemical profile in patients with Ig-MPGN/C3G; (2) to investigate whether genetic variants and different patterns of complement activation (i.e., fluid versus solid phase) correlate with disease manifestations and outcomes. METHODS: In 140 patients with idiopathic Ig-MPGN or C3G we performed complement biochemical and genetic screening and correlated genetic, biochemical and histology data with clinical features. RESULTS: Mutations in genes encoding alternative pathway complement proteins were found in both Ig-MPGN and C3G, and mutations in the two components of the C3 convertase are the most prevalent. We also report a mutation in THBD encoding thrombomodulin in a C3G patient. The presence of mutations alone does not significantly increase the risk of Ig-MPGN or C3G, but it does so when combined with common susceptibility variants (CD46 c.-366A in Ig-MPGN; CFH V62 and THBD A473 in C3G). Finally, patients without complement gene mutations or C3NeFs--autoantibodies that stabilize the alternative pathway C3 convertase--have a higher risk of progressing to end-stage renal disease than patients with identified mutations and/or C3NeFs, suggesting the existence of different pathogenetic mechanisms that lead to renal disease. CONCLUSIONS: We provide new insights into the pathogenesis of Ig-MPGN/C3G that underscore the complex nature of these diseases and suggest that the current C3G classification may miss many cases associated with abnormalities of the complement alternative pathway.

Characterization of a New DGKE Intronic Mutation in Genetically Unsolved Cases of Familial Atypical Hemolytic Uremic Syndrome.

BACKGROUND AND OBJECTIVES: Genetic and acquired abnormalities causing dysregulation of the complement alternative pathway contribute to atypical hemolytic uremic syndrome (aHUS), a rare disorder characterized by thrombocytopenia, nonimmune microangiopathic hemolytic anemia, and acute kidney failure. However, in a substantial proportion of patients the disease-associated alterations are still unknown. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Whole-exome and whole-genome sequencing were performed in two unrelated families with infantile recessive aHUS. Sequencing of cDNA from affected individuals was used to test for the presence of aberrant mRNA species. Expression of mutant diacylglycerol kinase epsilon (DGKE) protein was evaluated with western blotting. RESULTS: Whole-exome sequencing analysis with conventional variant filtering parameters did not reveal any obvious candidate mutation in the first family. The report of aHUS-associated mutations in DGKE, encoding DGKE, led to re-examination of the noncoding DGKE variants obtained from next-generation sequencing, allowing identification of a novel intronic DGKE mutation (c.888+40A>G) that segregated with disease. Sequencing of cDNA from affected individuals revealed aberrant forms of DGKE mRNA predicted to cause profound abnormalities in the protein catalytic site. By whole-genome sequencing, the same mutation was found in compound heterozygosity with a second nonsense DGKE mutation in all affected siblings of another unrelated family. Homozygous and compound heterozygous patients presented similar clinical features, including aHUS presentation in the first year of life, multiple relapsing episodes, and proteinuria, which are prototypical of DGKE-associated aHUS. CONCLUSIONS: This is the first report of a mutation located beyond the exon-intron boundaries in aHUS. Intronic mutations such as these are underreported because conventional filtering parameters used to process next-generation sequencing data routinely exclude these regions from downstream analyses in both research and clinical settings. The results suggest that analysis of noncoding regions of aHUS-associated genes coupled with mRNA sequencing might provide a tool to explain genetically unsolved aHUS cases.

Posttransplant recurrence of atypical hemolytic uremic syndrome.

Hemolytic uremic syndrome (HUS) is a rare disease characterized by microangiopathic hemolytic anemia, thrombocytopenia and acute renal failure. It is usually secondary to infections by strains of Escherichia coli (STEC) that produce Shiga-like toxin. In about 10% of patients, no STEC infections are reported. In these cases of atypical HUS (aHUS), mutations in genes encoding proteins of the complement system have been described. Atypical HUS is characterized by poor prognosis and by high risk of posttransplant recurrence which greatly depends on the specific gene mutation involved in the disease. Plasma therapy, eculizumab treatment and, in some cases, combined liver-kidney transplant have been used to prevent and/or treat posttransplant aHUS recurrences.