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.