#3831 TRIMER FORMATION ASSAY SUCCESSFULLY EVALUATES THE EFFECTS OF COL4A VARIANTS IN FSGS PATIENTS

Abstract

Abstract Background and Aims Variants in COL4A3, COL4A4 and COL4A5 genes were the most common variants associated with focal segmental glomerulosclerosis (FSGS). Split-luciferase-based trimer formation assay of α345 collagen is a technique that test collagen structure formation by transfect COL4A3/4/5 plasmids into cells. This method was recently used to successfully determine the pathogenicity of variants in COL4A5 gene in Alport syndrome patients. We used the trimer formation assay to evaluate the variants found in our FSGS patients and correlate with genomic variants of COL4A gene. Method We transfected wild-type COL4A3-SmBiT, COL4A4-LgBiT and COL4A5 plasmids into human embryonic kidney cells. In wild-type plasmids, α345 collagen will be formed, then SmBiT and LgBiT will properly fuse, and luminescence will be measured (Figure 1A). We selected the pathogenic and likely pathogenic variants in three FSGS patients from our FSGS cohort, which are two missense variants in COL4A4 (c.1805G>A, p.Gly602Glu and c.2752G>A, p.Gly918Arg), and a deletion in COL4A4, leading to frameshift and premature stop codon (c.905delG, p.Gly302ValfsTer32). We also used COL4A4, c.2906C>G, p.Ser969Ter and COL4A4, c.1396G>A, p.Gly466Arg as nonsense and missense mutation negative controls, respectively. We did the mutagenesis in COL4A4-LgBiT plasmid and repeat the steps as in wild-type plasmids. If the variants caused abnormal protein structure, SmBiT and LgBiT will not properly fuse, and luminescence will be measured less than wild-type (Figure 1B). Results The luminescence of nonsense control and missense control were at 22.50% and 23.58% of relative light unit (RLU) from cells expressing wild-type, respectively. The luminescences of cells expressing mutated plasmid were at 33.68%, 30.94% and 19.74% of RLU from cells expressing wide type for c.905delG, c.1805G>A and c.2752G>A, respectively. (Figure 1C) The lower luminescence means cells with mutated plasmids produced less normal α345 collagen than cells with wild-type plasmids. The results confirmed that these three variants could really cause abnormal α345 collagen in cell model. Conclusion This is the first study to demonstrate that split-luciferase-based trimer formation assay of α345 collagen can be used as a functional study to evaluate the effect of COL4A3/4/5 variants in FSGS.