As C3 p.W1034R was considered the most probable candidate variant for a causative mutation in this family with aHUS (Zhao et al., 2020 (link); Table S1), a functional analysis was conducted using a recombinant glutathione S‐transferase (GST)‐tagged C3 protein in which the amino acid at the 1034 position was substituted with arginine to demonstrate the functional effects of the variant (Supplementary Methods). Briefly, the PCR‐amplified fragments of C3 from 3004 to 3909 (Figure 2a), corresponding to C3d, were inserted into the pGEX6P‐1 vector (Cytiva, Tokyo, Japan), and the recombinant GST‐tagged C3d protein was expressed and purified using a glutathione Sepharose 4B column (Cytiva, Tokyo, Japan). Subsequently, the GST tag was removed by overnight incubation with PreScission Protease (Cytiva, Tokyo, Japan) at 4°C. A 3100T>C mutation in C3d introduced into the pGEX6P‐1 vector was induced using a mutagenesis kit (Toyobo, Osaka, Japan). As controls that had been reported to have deleterious effects on the binding between C3b protein and factor H (Schramm et al., 2015 (link)), the vectors harboring the 3187 A > C mutation, which leads to an amino‐acid substitution of serine to arginine (S1063R), and the 3281 C > A mutation, which leads to an amino‐acid substitution of alanine to aspartate (A1094D), were also constructed.