Loss of Function Variants Contribute to Cerebral Folate Deficiency by Downregulating Expression

Cerebral folate deficiency (CFD) syndrome is characterised by a low concentration of 5-methyltetrahydrofolate in cerebrospinal fluid, while folate levels in plasma and red blood cells are in the low normal range. Mutations in several folate pathway genes, including , , and ) have been previously identified in patients with CFD. In an effort to identify causal mutations for CFD, we performed whole exome sequencing analysis on eight CFD trios and identified eight de novo mutations in seven trios. Notably, we found a de novo stop gain mutation in the (CIC) gene. Using 48 sporadic CFD samples as a validation cohort, we identified three additional rare variants in CIC that are putatively deleterious mutations. Functional analysis indicates that CIC binds to an octameric sequence in the promoter regions of folate transport genes: , and ). The nonsense variant (p.R353X) downregulated FOLR1 expression in HeLa cells as well as in the induced pluripotent stem cell (iPSCs) derived from the original CFD proband. Folate binding assay demonstrated that the p.R353X variant decreased cellular binding of folic acid in cells. This study indicates that loss of function variants can contribute to the genetic aetiology of CFD through regulating expression. Our study described the first mutations in a non-folate pathway gene that can contribute to the aetiology of CFD.