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.

Interaction between Multimeric von Willebrand Factor and Complement: A Fresh Look to the Pathophysiology of Microvascular Thrombosis.

von Willebrand factor (VWF), a multimeric protein with a central role in hemostasis, has been shown to interact with complement components. However, results are contrasting and inconclusive. By studying 20 patients with congenital thrombotic thrombocytopenic purpura (cTTP) who cannot cleave VWF multimers because of genetic ADAMTS13 deficiency, we investigated the mechanism through which VWF modulates complement and its pathophysiological implications for human diseases. Using assays of ex vivo serum-induced C3 and C5b-9 deposits on endothelial cells, we documented that in cTTP, complement is activated via the alternative pathway (AP) on the cell surface. This abnormality was corrected by restoring ADAMTS13 activity in cTTP serum, which prevented VWF multimer accumulation on endothelial cells, or by an anti-VWF Ab. In mechanistic studies we found that VWF interacts with C3b through its three type A domains and initiates AP activation, although assembly of active C5 convertase and formation of the terminal complement products C5a and C5b-9 occur only on the VWF-A2 domain. Finally, we documented that in the condition of ADAMTS13 deficiency, VWF-mediated formation of terminal complement products, particularly C5a, alters the endothelial antithrombogenic properties and induces microvascular thrombosis in a perfusion system. Altogether, the results demonstrated that VWF provides a platform for the activation of the AP of complement, which profoundly alters the phenotype of microvascular endothelial cells. These findings link hemostasis-thrombosis with the AP of complement and open new therapeutic perspectives in cTTP and in general in thrombotic and inflammatory disorders associated with endothelium perturbation, VWF release, and complement activation.

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.

Withdrawals/Retractions: Insights into the effects of complement factor H on the assembly and decay of the alternative pathway C3 proconvertase and C3 convertase.

VOLUME 291 (2016) PAGES 8214­8230 This article has been withdrawn by the authors. Lanes 1 and 7 of Fig. 4B were duplicated.

Insights into the Effects of Complement Factor H on the Assembly and Decay of the Alternative Pathway C3 Proconvertase and C3 Convertase.

The activated fragment of C3 (C3b) and factor B form the C3 proconvertase (C3bB), which is cleaved by factor D to C3 convertase (C3bBb). Older studies (Conrad, D. H., Carlo, J. R., and Ruddy, S. (1978)J. Exp. Med.147, 1792-1805; Pangburn, M. K., and Müller-Eberhard, H. J. (1978)Proc. Natl. Acad. Sci. U.S.A.75, 2416-2420; Kazatchkine, M. D., Fearon, D. T., and Austen, K. F. (1979)J. Immunol.122, 75-81) indicated that the complement alternative pathway regulator factor H (FH) competes with factor B for C3b binding; however, the capability of FH to prevent C3bB assembly has not been formally investigated. Moreover, in the few published studies FH did not favor C3bB dissociation. Whether FH may affect C3bBb formation from C3bB is unknown. We set up user-friendly assays based on combined microplate/Western blotting techniques that specifically detect either C3bB or C3bBb, with the aim of investigating the effect of FH on C3bB assembly and decay and C3bBb formation and decay. We document that FH does not affect C3bB assembly, indicating that FH does not efficiently compete with factor B for C3b binding. We also found that FH does not dissociate C3bB. FH showed a strong C3bBb decay-accelerating activity, as reported previously, and also exerted an apparent inhibitory effect on C3bBb formation. The latter effect was not fully attributable to a rapid FH-mediated dissociation of C3bBb complexes, because blocking decay with properdin and C3 nephritic factor did not restore C3bBb formation. FH almost completely prevented release of the smaller cleavage subunit of FB (Ba), without modifying the amount of C3bB complexes, suggesting that FH inhibits the conversion of C3bB to C3bBb. Thus, the inhibitory effect of FH on C3bBb formation is likely the sum of inhibition of C3bB conversion to C3bBb and of C3bBb decay acceleration. Further studies are required to confirm these findings in physiological cell-based settings.

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.

A case of familial glomerulopathy with fibronectin deposits caused by the Y973C mutation in fibronectin.

Glomerulopathy with fibronectin deposits is a rare hereditary kidney disease characterized by the extensive deposition of fibronectin in glomeruli, particularly in mesangial regions and subendothelial zones. Prognostically, the disease is known as slowly progressive, leading to kidney failure in most cases. We recently diagnosed glomerulopathy with fibronectin deposits in a 24-year-old man in whom proteinuria was detected incidentally. Genetic analysis of the fibronectin 1 (FN1) gene showed heterozygosity for the Y973C mutation. The same mutation was found in his elder brother, who similarly experienced proteinuria. Both patients had normal kidney function but persistent proteinuria after 30 months and 11 years of follow-up, respectively.

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.

Post-transplant recurrence of atypical hemolytic uremic syndrome in a patient with thrombomodulin mutation.

HUS is characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. While "typical" HUS is usually associated with Shiga toxin-producing Escherichia coli infections and recovers in the majority of cases, aHUS is caused by mutations of complement components or antibodies against CFH leading to uncontrolled activation of alternative complement pathway and often to ESRD. Recently, THBD gene mutations have been reported in aHUS. Theoretically, the risk of disease recurrence after renal transplantation should be low because THBD is primarily a membrane-bound protein expressed by endothelial cells; however, a small proportion of THBD is present as a soluble form in plasma. We report the case of a 19-yr-old man with aHUS secondary to a THBD mutation that relapsed twice after two renal transplantations performed 12 yr apart. Despite successful control of HUS with plasma exchange and eculizumab after the second transplantation, the graft was ultimately lost due to severe steroid-resistant cellular rejection. The present report suggests that THBD mutations may favor-relapse of aHUS after renal transplantation.

Successful long-term outcome after renal transplantation in a patient with atypical haemolytic uremic syndrome with combined membrane cofactor protein CD46 and complement factor I mutations.

BACKGROUND: Atypical haemolytic uremic syndrome (aHUS) is often associated with a high risk of disease recurrence and subsequent graft loss after isolated renal transplantation. Evidence-based recommendations for a mutation-based management after renal transplantation in aHUS caused by a combined mutation with complement factor I (CFI) and membrane cofactor protein CD46 (MCP) are limited. CASE-DIAGNOSIS/TREATMENT: We describe a 9-year-old boy with a first manifestation of aHUS at the age of 9 months carrying combined heterozygous mutations in the CFI and MCP genes. At the age of 5 years, he underwent isolated cadaveric renal transplantation. Fresh frozen plasma was administered during and after transplantation, tapered and finally stopped after 3 years. CONCLUSIONS: During the 5-year follow-up after transplantation there have been no signs of aHUS recurrence and graft function has remained good. The combination of heterozygous MCP and CFI mutations with aHUS might have a positive impact on the post-transplant course, possibly predicting a lower risk of aHUS recurrence after an isolated cadaveric renal transplantation.

Mutation Analysis of PKD1 and PKD2 Genes in a Large Italian Cohort Reveals Novel Pathogenic Variants Including A Novel Complex Rearrangement.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited disease of the kidney. It occurs in adulthood but is also rarely diagnosed in early childhood. The majority of the disease-causing variants observed in ADPKD patients are in two genes: PKD1 and PKD2. METHODS: 237 patients from 198 families with a clinical diagnosis of ADPKD were screened for PKD1 and PKD2 genetic variants using Sanger sequencing and Multiple Ligation-dependent Probe Amplification (MLPA) analysis. RESULTS: Disease-causing (diagnostic) variants were identified in 173 families (211 patients), 156 on PKD1 and 17 on PKD2. Variants of unknown significance (VUS) were detected in 6 additional families, while no mutations were found in the remaining 19 families. Among the diagnostic variants detected, 51 were novel. In ten families, seven large rearrangements were found and the molecular breakpoints of 3 rearrangements were identified. Renal survival was significantly worse for PKD1 mutated patients, particularly those carrying truncating mutations. In patients with PKD1 truncating ( PKD1-T) mutations, disease onset was significantly earlier than in patients with PKD1 non-truncating (PKD1-NT) variants or PKD2 mutated patients. CONCLUSIONS: Comprehensive genetic testing confirms its utility in diagnosing patients with ADPKD and contributes to explaining the clinical heterogeneity observed in this disease. Moreover, the genotype-phenotype correlation can allow a more accurate disease prognosis.

An ex vivo test to investigate genetic factors conferring susceptibility to atypical haemolytic uremic syndrome.

Introduction: Comprehensive genetic analysis is essential to clinical care of patients with atypical haemolytic uremic syndrome (aHUS) to reinforce diagnosis, and to guide treatment. However, the characterization of complement gene variants remains challenging owing to the complexity of functional studies with mutant proteins. This study was designed: 1) To identify a tool for rapid functional determination of complement gene variants; 2) To uncover inherited complement dysregulation in aHUS patients who do not carry identified gene variants. Methods: To address the above goals, we employed an ex-vivo assay of serum-induced C5b-9 formation on ADP-activated endothelial cells in 223 subjects from 60 aHUS pedigrees (66 patients and 157 unaffected relatives). Results: Sera taken from all aHUS patients in remission induced more C5b-9 deposition than control sera, independently from the presence of complement gene abnormalities. To avoid the possible confounding effects of chronic complement dysregulation related to aHUS status, and considering the incomplete penetrance for all aHUS-associated genes, we used serum from unaffected relatives. In control studies, 92.7% of unaffected relatives with known pathogenic variants exhibited positive serum-induced C5b-9 formation test, documenting a high sensitivity of the assay to identify functional variants. The test was also specific, indeed it was negative in all non-carrier relatives and in relatives with variants non-segregating with aHUS. All but one variants in aHUS-associated genes predicted in-silico as likely pathogenic or of uncertain significance (VUS) or likely benign resulted as pathogenic in the C5b-9 assay. At variance, variants in putative candidate genes did not exhibit a functional effect, with the exception of a CFHR5 variant. The C5b-9 assay in relatives was helpful in defining the relative functional effect of rare variants in 6 pedigrees in which the proband carried more than one genetic abnormality. Finally, for 12 patients without identified rare variants, the C5b-9 test in parents unmasked a genetic liability inherited from an unaffected parent. Discussion: In conclusion, the serum-induced C5b-9 formation test in unaffected relatives of aHUS patients may be a tool for rapid functional evaluation of rare complement gene variants. When combined with exome sequencing the assay might be of help in variant selection, to identify new aHUS-associated genetic factors.

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.

Congenital thrombotic thrombocytopenic purpura (cTTP) with two novel mutations.

Congenital thrombotic thrombocytopenic purpura (cTTP) is a rare disorder of childhood that has clinical and laboratory similarities to other, more common conditions. Prompt recognition is required as delays in therapy are associated with significant morbidity and failure to treat may lead to death. While the principles of treatment have not changed, enormous progress in the genetic and molecular understanding has taken place. Emerging treatment options may offer some hope of improved quality of life in future. We describe a Chinese patient with cTTP which resulted from two previously undescribed mutations in the ADAMTS13 gene.

Membrano-proliferative glomerulonephritis, atypical hemolytic uremic syndrome, and a new complement factor H mutation: report of a case.

BACKGROUND: Complement protein factor H (CFH) is a regulatory protein of the alternative complement pathway (AP); CFH mutations lead to a spectrum of different phenotypical manifestations of renal disease. CASE-DIAGNOSIS/TREATMENT: We report the case of a boy with a novel CFH gene mutation who presented with a membranoproliferative (MPGN) pattern of glomerular injury and developed 2 years later atypical hemolytic uremic syndrome (aHUS); this description shows that CFH alteration leads to two different renal diseases in the same patient. CONCLUSIONS: Our case suggests the possibility that complement dysregulation could determine different renal conditions, which may be part of the same disease spectrum. Early recognition of an evolution of glomerulopathies into aHUS may allow appropriate management and prevention of life-threatening consequences.

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.

IgA nephropathy and atypical hemolytic uremic syndrome: a case series and a literature review.

BACKGROUND: IgA nephropathy (IgAN) has been anecdotally reported in association with atypical hemolytic uremic syndrome (aHUS). The association likely portends poor renal outcome, and the possible relationship with complement overactivation has yet to be elucidated. We evaluated a series of IgAN patients with aHUS and reviewed the available literature. METHODS: Adult patients who received a diagnosis of IgAN and developed aHUS between January 2009 and December 2019 were included in this retrospective review. RESULTS: We identified six IgAN-aHUS patients, all of whom developed end-stage kidney disease. At aHUS presentation all patients had decreased serum C3 levels. Predisposing pathogenetic variants and risk haplotypes for aHUS in CFH gene heterozygosity were documented in four out of six patients. Anti-CFH antibodies were found to be negative in the five tested patients. In the literature we identified 21 case reports involving aHUS-IgAN and six retrospective studies evaluating the presence of TMA at the time of renal biopsy. Hypertension, severe proteinuria, reduced sC3 and a worse renal prognosis were the common features of most cases. CONCLUSION: Our case series and literature review show that the onset of either aHUS or renal TMA in the course of IgAN are associated with very poor renal outcome. Activation of the alternative pathway revealed by consumption of serum C3 seems to play a major role. Our hypothesis is that the presence of a predisposing factor (e.g. dysregualtion of complement alternative pathway and/or other intrarenal precipitating factors) might be at the heart of aHUS-IgAN pathophysiology.