CIC de novo loss of function variants contribute to cerebral folate deficiency by downregulating FOLR1 expression.

Background 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 FOLR1 (folate receptor alpha, FRα), DHFR (dihydrofolate reductase) and PCFT (proton coupled folate transporter) have been previously identified in patients with CFD. Methods 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. Results Notably, we found a de novo stop gain mutation in the capicua (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: FOLR1, PCFT and reduced folate carrier (Slc19A1; RFC1). The CIC 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. Conclusion This study indicates that CIC loss of function variants can contribute to the genetic aetiology of CFD through regulating FOLR1 expression. Our study described the first mutations in a non-folate pathway gene that can contribute to the aetiology of CFD.

背景 脑叶酸缺乏（CFD）综合征的特征是脑脊液中5 - 甲基四氢叶酸浓度较低，而血浆和红细胞中的叶酸水平处于正常范围下限。此前在CFD患者中已发现包括FOLR1（叶酸受体α，FRα）、DHFR（二氢叶酸还原酶）和PCFT（质子偶联叶酸转运体）在内的几个叶酸途径基因的突变。 方法 为了确定CFD的致病突变，我们对8个CFD三联体进行了全外显子组测序分析，并在7个三联体中鉴定出8个新生突变。 结果 值得注意的是，我们在capicua（CIC）基因中发现了一个新生的终止获得突变。我们使用48个散发的CFD样本作为验证队列，在CIC中又鉴定出3个可能有害的罕见变异。功能分析表明，CIC与叶酸转运基因（FOLR1、PCFT和还原叶酸载体（Slc19A1；RFC1））启动子区域的八聚体序列结合。CIC无义变异体（p.R353X）在HeLa细胞以及源自原始CFD先证者的诱导多能干细胞（iPSCs）中下调了FOLR1的表达。叶酸结合试验表明，p.R353X变异体降低了细胞对叶酸的细胞结合能力。 结论 这项研究表明，CIC功能缺失变异可通过调节FOLR1的表达导致CFD的遗传病因。我们的研究描述了首个非叶酸途径基因中的突变可导致CFD的病因。