Functional characterization of DHFR and FPGS. (A) The luciferase reporter assay confirmed that enhancer activity of DHFR was negatively influenced by the nucleotide substitution from G (WT) to A (rs1382539) in 293T cells. Bars represent means from 3 triplicate experiments, and T bars indicate standard deviations. (B) WT or variant FPGS R466C protein (5 ng/reaction) was studied by the polyglutamation assay, using the Phosphate Sensor Kit with MTX_pg1-6 as a substrate. The error bar shows the mean ± standard deviation. *P < .05 (Student t test).