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.


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.

Author notes


Asterisk with author names denotes non-ASH members.