Molecular analyses of C4 alleles.

Similar to Sekar et al.¹⁶, we measured the CNs of each individual C4 structural allele (C4A, C4B, C4L, and C4S) using droplet digital PCR, on genomic DNA isolated from blood samples from a total of 130 controls and 54 SZ patients from the MGH Neuropsychiatric Biobank. In summary, 50 ng of genomic DNA was fragmented by digestion with the restriction enzyme AluI¹⁶. The CNs of C4A, C4B, C4L, and C4S were then quantified from genomic DNA using droplet digital PCR on a QX200 Droplet Digital PCR System (Bio-Rad Laboratories) using specific and reference primers and probes as described by Wu et al.⁵⁷. Data were analyzed with the QuantaSoft software (v.1.4.0.99; Bio-Rad Laboratories), which estimated the absolute copies of C4 allele-specific DNA templates by comparison to the reference RPP30 template present as two copies per genome. C4AL, C4AS, C4BL, and C4BS CNs were determined based on the assumption of C4A CN + C4B CN = C4L CN + C4S CN, and given the low prevalence of C4S CN (around 1% in the normal population)¹⁶ C4L = C4AL CN if C4L CN ≥ C4A CN. In total, according to this imputation, we observed two patients with C4AS in the sample used for the C3 deposition assay; no patients displayed C4AS in the sample used contributing to our engulfment assays.