CIC missense variants downregulated PCP pathway protein Vangl2 and RhoA
The PCP signaling pathway has been shown to play an important role in neural tube closure in multiple previous studies (Copp, Greene & Murdoch, 2003; Wallingford, 2006; Wallingford, 2012; Murdoch et al., 2014).Genetic defects in the Vangl2 gene have been strongly associated with NTDs in mouse models, and there is growing evidence implicating Vangl genes (Vangl1 and Vangl2) in the genetic etiology of human NTDs (Kibar et al., 2007; Lei et al., 2010; Kibar et al., 2011; Tian et al., 2020). To investigate whether dysregulation of the PCP pathway also occurs in CIC variants associated spina bifida, we performed western blotting to examine the level of the PCP signaling pathway core protein Vangl2 and PCP downstream protein RhoA using overexpression wildtype CIC and CIC mutants in HeLa cells. As shown in Fig. 4, VANGL2 and RhoA protein levels in the wildtype CIC overexpression group were significantly increased compared to the pEGFP control (p<0.01), while VANGL2 and RhoA protein levels in those groups with overexpressed CIC mutants were less abundant compared to wildtype (p<0.05 and p<0.01, respectively). Protein levels of RhoA in each sample were consistent with those of Vangl2. We further investigated the effects of CIC LoF mutation on PCP pathway proteins Vangl2 and RhoA in NIH3H3 cells that were overexpressed with wildtype CIC, CIC-R353X and pEGFP control. As depicted in Fig. 5, both Vangl2 and RhoA showed increased protein levels in GFP-wildtype CIC transfection compared to EGCPC1 basic control vector transfection, and decreased protein level in CIC-R353X group compared to WT(p<0.01). These results support that CIC variants may also modify NTD risk through PCP signaling pathway.