Mutations in Proteins of the Alternative Pathway of Complement and the Pathogenesis of Atypical Hemolytic Uremic Syndrome

Time course of therapeutic interventions and hematologic parameters in the patient. Asterisks denote red blood cell transfusions, and arrows point to the 4 hemolytic uremic syndrome (HUS) episodes. Periods of plasma therapy (plasma exchange [PE] or infusion [PI]) and supportive treatment to restore renal function (hemodialysis [HD] or peritoneal dialysis [PD]) also are indicated. To convert serum hemoglobin in g/dL to g/L, multiply by 10; creatinine in mg/dL to μmol/L, multiply by 88.4.

Anomalous hemolytic activity in the patient′s serum. (A) Hemolytic activity observed after incubation of sheep erythrocytes with a control human serum, or (B) serum sample from the patient drawn before the beginning of plasma therapy. The percentage of sheep erythrocytes lysed is shown in the Y axis. Inset: hemolysis observed with 15 μL of the patient′s serum in the presence or absence of increasing amounts of factor H purified from control sera. A schematic representation of sheep erythrocytes, C3b, and factor H is included in the right part of the figure to illustrate the molecular basis for the results observed in A and B. White circles indicate polyanionic molecules on the erythrocyte surface.

Genetic analysis of factor H in the patient and her parents. The patient has a missense mutation inherited from her mother in exon 22 of the CFH gene (c.3425G→A) that substitutes a cysteine residue in short consensus repeat (SCR) 19 for a tyrosine. The figure also illustrates inheritance of the CFH haplotypes H1 and H3 from the mother and father, respectively.

Detection of the mutant factor H polypeptide (containing cysteine at amino acid 1142) in the patient′s plasma. (A) Electrophoretic separation of the proteins eluted from the Sepharose anti–human factor H immunosorbent incubated with plasma from the patient. Proteins were visualized by using Coomassie staining, and several spots from the band corresponding to factor H were picked, digested with trypsin, and analyzed further by using mass spectrometry. Positions of molecular-weight markers are shown at the left. (B) Matrix-assisted laser desorption-ionization (MALDI) time-of-flight (TOF) spectrum of the tryptic digest of factor H isolated from the patient. Two peptides potentially containing histidine (m/z 2031.8890) or tyrosine (m/z 2057.8933) at position 402 were fragmented further and analyzed to determine their amino acid sequence. Peptides from both the paternal and maternal alleles were detected in factor H isolated from the patient′s plasma. (C) Amino acid differences between factor H polypeptides of paternal and maternal origin, deduced from genetic analyses. The paternal polypeptide has tyrosine (Y) at position 402, whereas the maternal polypeptide has histidine (H) and also contains the mutated cysteine (C) residue at position 1142. Identification of factor H tryptic peptides was performed in the Proteomics Unit of the UCM-PCM (Universidad Complutense, Madrid, Spain) by using MALDI-TOF/TOF spectrometry (4700 Proteomics Analyzer; PerSeptives Biosystems, Framingham, MA) and ESI-IT (Electrospray Ionization-Ionic Trap spectrometer; Finnigan-LTQ, Thermo Electron Corp, Waltham, MA). Abbreviation: SCR, short consensus repeat.

Time course of complement components and hemolytic activity. Levels of C3, C4, and factor H and degree of hemolytic activity observed in serum samples from the patient drawn at different dates. Lysis denotes anomalous complement regulation. Plasma exchange (PE) and plasma infusion (PI) periods are indicated by boxes. To convert C3, C4, and factor H in mg/dL to g/L, multiply by 0.01. Abbreviation: HUS, hemolytic uremic syndrome.