ALBERT

Description: Abstract 3337 Introduction: Hemolytic uremic syndrome (HUS) is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and renal impairment. Approximately 10% of the cases are classified as atypical due to the absence of Shiga toxin-producing bacterial infection as a trigger. Compared to typical HUS, atypical HUS (aHUS) has a much poorer prognosis, with up to half of the patients progressing to end-stage renal disease, and a higher mortality. Uncontrolled activation of the complement system plays a role in the pathogenesis of aHUS. More than half of the patients with aHUS have the mutations of genes involved in the alternative pathway of the complement system. Mutations with loss-of-function of regulators (CFH, CFI, MCP and THBD) and gain-of-function of key of complement components (C3 and CFB) have been found to predispose to aHUS. In addition, genomic deletion of CFHR3 and CFHR1 has been linked to a risk of aHUS, sometimes together with the presence of CFH autoantibodies. Although many genetic studies on aHUS have been published in recent years, the Asian data are rare so far. It is important to perform genetic screening in patients with aHUS, because genotype-phenotype correlations have clinical significance in predicting renal recovery and transplant outcome. In this study, we analyzed 10 Japanese aHUS patients and the genotype-phenotype relationship was evaluated. Patients and Methods: Ten Japanese patients in Japan-Nara registry of thrombotic microangiopathy (TMA) with aHUS were investigated in this study; eight of them were sporadic and the other two were from one family. Diagnosis of aHUS was made by the simultaneous occurrence of microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure without association to Shiga toxin. The CFH antigen level was analyzed by a rocket-immunoelectrophoresis method, and hemolytic assay was performed using sheep red blood cells (Sanchez-Corral P, et al. Mol Immunol 2004). Genomic DNA was extracted from peripheral blood leukocytes of patients and their family members. The genetic mutations of 6 candidate genes and the gene deletion in complement factor H (CFH) and CFH-related genes were analyzed. Results: (1) Clinical features and laboratory data: Plasma ADAMTS13 activity was not severely decreased in all patients (29–119% of the normal). The first episode of aHUS occurred at childhood (≤10 yr) in 7 patients. Nine cases had probable triggering events. The plasma C3 level was low in 4 patients. Three patients showed apparent hemolytic activity against the sheep erythrocytes. Five patients had experienced relapses by the most recent follow-up. Five patients progressed to end-stage renal disease and could not be maintained without hemodialysis or peritoneal dialysis. (2) The result of gene analysis: We identified 7 causative or potentially causative mutations in 8 of these patients, and these mutations were heterozygous. Three of the mutations, p.R1215Q in CFH, p.I1157T in C3, and p.Y189D in MCP, were identified previously, indicating that these mutations are definitely causative for aHUS. The remaining 4 missense mutations, p.A359V in MCP, p.S562L and p.R425C in C3, and p.T500M in THBD, were novel. We considered them as potentially causative mutations based on the available information, including the PolyPhen-2 prediction, a search of the literature, and the position of the missense mutation in the three-dimensional structure. Two patients, who showed apparently an enhanced hemolytic activity, carried CFH p.R1215Q and 3 other patients carried C3 p.I1157T. One patient had 2 causative mutations in different genes. One patient was a compound heterozygote of the 2 MCP mutations. The patients carrying mutations in CFH or C3 had a high frequency of relapse and a worse prognosis. Conclusion: In this study, we identified the cause of aHUS in 8 out of 10 patients. Since the phenotype-genotype correlation of aHUS has clinical significance in predicting renal recovery and transplant outcome, a comprehensively accurate assessment of molecular variation would be necessary as part of the clinical management for aHUS patients. Such an assessment system would facilitate aHUS phenotype- genotype studies in Japan. Disclosures: Matsumoto: Alexion Pharma: Membership on an entity's Board of Directors or advisory committees. Fujimura:Baxter BioScience: Membership on an entity's Board of Directors or advisory committees; Alexion Pharma: Membership on an entity's Board of Directors or advisory committees.

Description: Backgrounds and Aims Atypical hemolytic uremic syndrome (aHUS) is a life-threatening generalized disease, featured by a clinical triad of microangiopathic hemolytic anemia, thrombocytopenia, and renal failure. Now it is well established that most of aHUS is caused by uncontrolled complement activation due to gene mutations involved in the alternative pathway, which includes C3, factor H (CFH), factor I (CFI), membrane cofactor protein (MCP), thrombomodulin (THBD) and factor B (CFB). Gene mutations in complement factor H- related proteins 1-5 (CFHR1-5) are also included in a category of aHUS. On the other hand, HUS and thrombotic thrombocytopenic purpura (TTP) are both categorized with a common pathological diagnosis, thrombotic microangiopathy (TMA). TTP, however, is now clearly defined by a deficient activity of ADAMTS13 Our laboratory of Nara Medical University has been functioning as a TMA referral center in Japan through analyzing ADAMTS13 since 1998. Through this study we identified 51 patients with hereditary deficiency of ADAMTS13 activity and 63 patients with aHUS with almost normal ADAMTS13 activity. To characterize these aHUS patients, as a first step we prepared 6 murine monoclonal antibodies (mAbs) against CFH, purified from normal plasma. One of anti-CFH mAbs, termed O-72 (IgG1-k), totally inhibited CFH function in the hemolytic assay described below. Epitope analysis of the mAb O-72 using yeast constructs clearly indicated that it resides on short consensus repeat 18 of CFH molecule. Patients and Methods (1) Patients: Of 63 patients with aHUS, 35 patients whose blood specimen were obtained within 3 months were extensively analyzed in this study. (2) Hemolytic assay: Using the mAb O-72, sheep red blood cells, and citrated plasmas, we were able to establish a quantitative hemolytic assay, according to the method of Sanchez-Corral et al (Mol Immunol 2004). The hemolysis obtained in the presence of the mAb O-72 (200 µg IgG/ml, final) was defined as a 100% hemolysis as the control. In this study, we consistently used citrated plasmas as test specimen, which were either freshly prepared or deep-frozen at -80oC within 3 months, and did not use sera. This is because our preliminary experiments clearly indicated that hemolytic activity using freshly prepared plasmas gives a consistent result, but that using sera was not. (3) Anti-CFH autoantibody: This was performed by western blot (WB) analysis using purified plasma derived CFH, (4) The comprehensive gene analyses on complement and complement regulatory factors, such as C3, CFH, CFI, MCP, THBD and CFB: These were performed as previously described [Fan et al. Molecular Immunology 2013], (5) Semi-quantitative WB analysis on antigens of CFHR1 and CFHR3, and (6) MLPA analyses for the exons of CFHR1 and CFHR3. Results and Discussion In the hemolytic assay, 3 unrelated patients with CFH-R1215Q mutation had a strong hemolysis (100% of the control). Interestingly, 4 individuals belonged to these 3 families, but without clinical signs of HUS, also showed an enhanced hemolysis as did the patients. Thus, it is important to note that our hemolytic assay can detect asymptomatic carriers of CFH-R1215Q mutation. To strengthen this observation, 3 patients with positive anti-CFH autoantibody but without CFH gene mutations also had an enhanced hemolysis (50-70% of the control), and one patient indeed had the homozygous deletion of CFHR1 gene. Fifteen patients carried C3 gene mutations, of which 13 patients had the same mutation of C3-I1157T (13/35, 37%). None of these patients, however, showed an enhanced hemolysis in our hemolytic assay. But, the C3-I1157T mutation can be easily detected with PCR followed by restriction fragments length polymorphism (RFLP) assay. Thus, using these combined assays of hemolysis, anti-CFH autoantibody detection, and RFLP for C3-I1157T, we can make a solid diagnosis on approximately 60% of Japanese aHUS patients within 5 days. But, the remaining 40% of the aHUS patients required the comprehensive gene analysis. A summary of the gene mutations in our 35 patients with aHUS is shown in Table 1. As consequence, here we have shown a major cause of aHUS (approximately 37% of the total) in Japan is a missense mutation of C3-I1157T, and a missense mutation of CFH (-R1215Q) is likely to be less (approximately 9% of the total) than those in Western countries-US. Disclosures: Matsumoto: Alexion Pharma: Membership on an entity’s Board of Directors or advisory committees. Fujimura:Baxter BioScience: Membership on an entity’s Board of Directors or advisory committees; Alexion Pharma: Membership on an entity’s Board of Directors or advisory committees.