Fig 1.
Production of six anti-complement factor H (CFH) murine monoclonal antibodies (mAbs), and their effects on lysis of sheep red blood cells (RBCs).
(A) Western blot analysis. All six anti-CFH mAbs reacted with purified human CFH on Western blot under both non-reducing (NR) and reducing (R) conditions. (B) Effects of six anti-CFH mAbs on lysis of sheep RBCs. Twenty microliters of normal plasmas spiked with anti-CFH mAbs were incubated with sheep RBCs at a final concentration of 50 μg/ml to 400 μg/ml. Two plasma samples, treated with anti-CFH mAb O52 and O72, induced strong hemolysis in a dose-dependent manner. Three mAbs (R27, R35, and O37) resulted in slightly enhanced hemolysis. As for mAb Q34, appreciable hemolysis was not detected, similar to control IgG. (C) Epitope analysis of mAb O72 using recombinant CFH expressed by yeast. The mAb O72 reacted with full length CFH (left) and short consensus repeat (SCR) 18–20 (middle). Moreover, the right panel shows that mAb O72 reacted with peptide of SCR18−19, and SCR18 and 20, but not SCR19−20, indicating that the epitope of mAb O72 resided in SCR18.
Fig 2.
Optimization of the hemolytic assay: sheep red blood cells (RBCs) concentration, anti-complement factor H (CFH) monoclonal antibody (mAb) O72, and blood specimen type.
(A) Evaluation of the optimal sheep RBC count. Various counts of sheep RBCs were incubated with 20 μl of normal plasma spiked with anti-CFH mAb O72 (200 μg IgG/ml, NP + O72) or control IgG. Maximum hemolysis was observed at the sheep RBC count of 2.5×106/μl; thus, all subsequent experiments were performed using this sheep RBC count. (B) Comparison of hemolysis induced by mAb O72 and CFH mutant protein. The degree of hemolysis caused by normal plasma spiked with mAb O72 or plasma from a patient with a CFH-p.R1215Q mutation was quite comparable up to 40 μl. (C) Screening of optimal blood specimen type. Three different specimens (serum, EDTA plasma, and citrated plasma) from one healthy donor were analyzed using the hemolytic assay. Citrated normal plasma spiked with mAb O72 was used as a positive control. In serum and EDTA plasma, mild hemolysis of sheep RBCs was detected at 40μl. No appreciable hemolysis was observed at any volume of citrated plasma. (D) Evaluation of the influence of the coagulation time interval on hemolytic reaction. Serum obtained 2 and 4 hours after blood collection showed mildly enhanced hemolysis at 40 μl. This mild hemolysis was lower in serum obtained 24 and 48 hours prior. No appreciable hemolysis was detected in serum obtained 7 days prior.
Fig 3.
Relationship between hemolytic activity and complement abnormalities in 45 patients with atypical hemolytic uremic syndrome (aHUS).
The results of the hemolytic assay for 45 aHUS patients are shown according to the predisposing (bold type) or potentially predisposing mutations and/or the acquired abnormalities. The patients carrying two mutations were classified according to the predisposing mutation, and the other mutations were described alongside the predisposing mutations or black bars. Two patients (2V1 and H2) were categorized into the C3 group, although they had two predisposing mutations (C3-p.I1157T and THBD-p.D486Y). In two patients (2P1 and 3G1), the hemolysis induced by serum was represented by gray bars. The normal range of hemolysis (5.4±1.8%: mean ± standard deviation) obtained from 20 healthy individuals (10 males and 10 females) is shown at the bottom of Fig 3. The results of the restriction fragment length polymorphism (RFLP) analysis of C3-p.I1157T are summarized to the right of the hemolytic assay results. Regarding the RFLP analysis of C3-p.I1157T (c.3470T>C), the heterozygous mutation (T/C) showed two bands (278 bp, 314 bp) and the homozygous mutation (C/C) showed one band (314 bp). † H2 had a homozygous mutation in C3-p.I1157T.
Fig 4.
Family 3O with a C3-p.K1105Q mutation.
(A) Patient 3O1 (female) developed atypical hemolytic uremic syndrome (aHUS) at the age of five months. (B) In the hemolytic assay, plasma samples from three family members (patient: P, father: F, and brother: B) induced strong hemolysis of sheep red blood cells (RBCs), but her mother (M) had no appreciable hemolysis, with results similar to normal plasma (NP). (C) Lysis of sheep RBCs detected in the three family members was corrected by the addition of complement factor H (CFH) in a dose-dependent manner. Genetic analysis showed that the patient and her father and brother, whose plasma clearly showed hemolysis, carried a potentially predisposing mutation p.K1105Q in C3, not in CFH.
Fig 5.
Family 2I with a CFH-p.R1215Q mutation.
(A) Patient 2I1 (male) had episodes of atypical hemolytic uremic syndrome (aHUS) at the age of 28 and 29. Gray squares or circles indicate individuals who were not analyzed in this study. (B) The hemolytic assay showed that plasma from the patient (P), his mother (M), and one brother (B1) induced severe hemolysis, whereas samples from his father (F) and the other brother (B2) showed no appreciable hemolysis. (C) The hemolysis detected in the three family members was corrected by the addition of purified complement factor H (CFH). Genetic analysis showed that the patient, his mother, and brother 1 carried a CFH-p.R1215Q mutation, but his mother and brother 1 have never had the episodes of aHUS.
Fig 6.
Family 2K with no predisposing or potentially predisposing mutations.
(A) Patient 2K1 (female) developed atypical hemolytic uremic syndrome (aHUS) at the age of 35. Her husband (gray square) was not analyzed in this study. (B) The hemolytic assay showed that plasma from the patient (P), her father (F), and two daughters (D1 and D2) induced severe hemolysis, which was not observed in her mother (M). (C) The enhanced hemolysis detected in these four family members was corrected by the addition of purified complement factor H (CFH). However, no predisposing or potentially predisposing mutations were detected in Patient 2K1.
Fig 7.
Family 2A with a C3-p.I1157T mutation.
(A) Patient 2A1 (male) had six bouts of atypical hemolytic uremic syndrome (aHUS), at the age of 9, 15, 18, 22 and 29 (twice). Gray squares or circle indicate individuals who were not analyzed in this study. (B) The hemolytic assay showed that neither patient (P) nor his parents (father: F and mother: M) had appreciable hemolysis. However, Patient 2A1 and his father carried the same predisposing mutation p.I1157T in C3, confirmed by restriction fragment length polymorphism (RFLP) analysis (C) and direct DNA sequencing.
Table 1.
Comparison of genetic or acquired abnormalities among Western countries and Japan.