CIC missense variants affected FOLR1 protein level and cellular folate concentration
We have previously found that CIC is capable of binding to FOLR1 promoter region, thereby, regulating its transcription (Cao et al., 2021). FOLR1 is a critically important transport molecule involved in the uptake of folates into the cells and plays a crucial role in neural tube closure. In mice, Folr1 knock-out lead to 100% penetrant spina bifida. In humans, functional disruption of FOLR1 causes cerebral folate deficiency syndrome (CFD), while increased titers of FOLR1 autoantibody in maternal serum was reported to be associated with both NTDs (Rothenberg et al., 2004; Cabrera, et al., 2008) and CFD (Ramaekers et al., 2005). We examined the effect of variant containing CIC on FOLR1 protein levels in Hela cells. As presented in Figure 3A, wildtype GFP-CIC increased FOLR1 expression compared to the control pEGFP plasmid (p<0.001), while overexpression of all GFP-CIC mutants profoundly downregulated FOLR1 expression compared to wildtype(p<0.01).
We further investigated the effect of the CIC variants on folate absorption ability in HeLa cells. As expected, wildtype CIC overexpression increased intracellular folate concentrations compared to the pEGFP control, while overexpression of CIC mutants reduced cellular folate content compared to wildtype (p<0.05) (Fig. 3C), indicating that CIC mutants can reduce folate binding and/or absorption ability by downregulating FOLR1.