Family analysis: deficiency of CFHR1 and CFHR3 in aHUS patients and their family members. (A) A pedigree is shown for each family. Black boxes indicate patients; open symbols, family members with homozygous CFHR1 and CFHR3 deletion; and gray symbols, individuals with heterozygous CFHR1 and CFHR3 deficiency. (B) Plasma of the patients or their healthy family members were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a membrane, and analyzed by Western blotting using a mAB that identifies the conserved C-termini of CFH (150 kDa) and the 2 differently glycosylated forms CFHR1α and CFHR1β) (37 and 42 kDa). For detection of CFHR3, antiserum reacting with different glycosylated forms of CFHR3 (45 kDa, multiple bands) was used. Western blot analysis of plasma derived from individual family members demonstrated deficiency of CFHR1 in the aHUS patients (* lanes 2, 8, 13) and also in healthy relatives (lanes 3-5,9,14-17). CFHR1α and CFHR1β are detected in plasma of a healthy control (lanes 1,7,12). CFH is detected in all plasma samples. (C) Complete deficiency of CFHR3 is detected in the 3 aHUS patients (lanes 2,8,13) and several relatives (lanes 3-5,9,14-17), but CFHR3 is observed in the plasma of a healthy volunteer (lanes 1,7,12) and of heterozygous relatives (lanes 6,10,11). The band at 30 kDa in lane 2 is unspecific. (D) CFH autoantibody levels were detected by ELISA. CFH autoantibodies (black bars) are present in serum of the patients (AII₁, BII₁, and CII₁) but not of their relatives (dashed bars) and in plasma derived from controls (co, gray bars). The dotted line represents the background level (OD₄₅₀ − 0.35), that is, the highest absorbancy of plasma samples derived from 100 control individuals (Document S1).