INTRODUCTION
Neural Tube Defects (NTDs) are severe congenital malformations in which the neural folds that ultimately give rise to the spine and brain failed to properly close during embryonic development. Spina bifida and anencephaly are the two most common subtypes of NTDs that affect the spinal cord and brain, respectively (Greene & Copp, 2014). Given their severity, NTDs are the leading cause of death in the first year of life. Standard of care for NTD varies greatly across nations. Including anencephaly, which is uniformly fatal, over 75% of NTD‐affected births resulted in death before 5 years of age (Blencowe, Kancherla, Moorthie, Darlison & Modell, 2018). In the industrialized nations, surgical and medical management has improved to the point that the majority of spina bifida affected infants will survive into adulthood (Oakeshott, Hunt, Poulton & Reid, 2010), but will suffer long-term associated disabilities and treatment costs remain major obstacles (Flores, Vellozzi, Valencia & Sniezek, 2014). The worldwide prevalence of NTDs ranges from 1 to 10 per 1000 births, varying widely among different countries and regions. The annual worldwide prevalence of NTD-affected birth was estimated to exceed 260,000 births (Blencowe et al. 2018; Au et al. 2010).
NTDs are complex malformations which have multifactorial etiologies, including genetic, environmental and dietary factors (CoppJ & Greene, 2013). There is both clinical and experimental evidence that maternal folate status is a nutritional modifier of NTD risks (Caffrey, McNulty, Irwin, Walsh & Pentieva, 2019). Folate molecules participate in one carbon metabolism to provide thymidine and purines for DNA synthesis and s-adenosyl methionine (SAM), which is the universal methyl group donor required for multiple methylation reactions (Froese Fowler & Baumgartner, 2019). Up to 70% of NTDs may be folate-sensitive and can be prevented by folic acid supplementation prior to conception and continued in early pregnancy (Shlobin, LoPresti, Du & Lam, 2020). Based on this, the United States Food and Drug Administration (FDA) has required mandatory folic acid fortification of grain products including flour, bread, pasta, rice, and cereal in 1998. The United States Centers for Disease Control and Prevention (CDC) has also urged every woman who may become pregnant to obtain at least 400ug of folate every day to prevent NTDs occurrence.
Genetic factors are also considered to be significant contributors to the occurrence of NTDs in both human and mice (Copp & Greene, 2010; Wallingford, Niswander, Shaw & Finnell, 2013; Wilde, Petersen & Niswander, 2014; Greene, Stanier & Copp, 2009). Several studies using Splotch mouse mutant have shown that NTDs can be related back to their compromised ability to maintain cellular proliferation and an undifferentiation state of their neuroepithelium which hampers normal neurulation and leads to the failure of neural tube closure. Folic acid supplementation restores proper proliferation in the cranial neuroepithelium and compensated for the loss of Pax3, which prevents cranial NTDs and thereby rescues the normal phenotype (Sudiwala et al. 2019). Mutations in the Vangl2 gene cause craniorachischisis through impaired Vangl2 interaction with Dvl1, Dvl2, and Dvl3 (Torban, Wang, Groulx & Gros, 2004). Furthermore, NTD caused by point mutation in the Lrp6Cd/Cd canonical WNT coreceptor are rescued both by folic acid and by inhibition of the WNT non-canonical pathway by RhoA inhibitors (Gray et al., 2013; Carter, Ulrich, Oofuji, Williams & Elizabeth Ross, 1999). To date, mutations in more than 300 genes have been reported to cause NTDs in mice (Wilde, Petersen & Niswander, 2014). Multiple signaling pathways, including the Wnt/planar cell polarity (PCP) pathway (Chen et al., 2018), sonic hedgehog (SHH) pathway (Murdoch & Copp, 2010) and mitochondrial folate metabolic pathway (Kim et al., 2018), have been reported to be involved in neural tube closure. However, only a few of these reported murine NTD genes have been positively associated with human NTDs, as the pattern of variants reported in human NTD patients supports a polygenic or oligogenic etiology (Copp & Greene, 2010).
CIC, homolog of the Drosophila capicua gene, was discovered in a screen for mutations affecting the anteroposterior pattern of Drosophila embryos (Bettegowda et al., 2011). It was found to function downstream of the receptor tyrosine kinase (RTK) pathways that includes the epidermal growth factor receptor, Torso, Ras, Raf, and mitogen-associated protein kinases (MAPKs), which are all related to embryonic pattern formation (Roch, Jiménez & Casanova, 2002). CIC can be transported into the nucleus through interactions with Karyopherin Subunit Alpha 3 (KPNA3) and serves as a transcription repressor. In the absence of EGFR-ERK signaling, CIC binds to target promoters and/or enhancers and represses downstream genes, while activation of the pathway leads to CIC degradation and activation of genes normally repressed by CIC (Jiménez, Shvartsman & Paroush, 2012; Astigarraga et al., 2007).
Recurrent mutations in CIC were initially identified in oligodendroglioma; subsequently, several CIC aberrations were found in multiple types of cancers including medulloblastoma, breast cancer, and small blue round cell tumors (Wong & Yip, 2020; Huang et al., 2016). In mammals, CIC forms a transcriptional repressor complex with ATXN1. Gain of function of the complex underlies the pathogenesis of spinocerebellar ataxia type 1 (Lam et al., 2006), and a spectrum of neurobehavior phenotypes which include hyperactivity, impaired learning and memory, and abnormal maturation and maintenance of upper-layer cortical neurons (Lu et al., 2017). Our previous study demonstrated that CIC acts as a transcription activator of FOLR1 and CIC loss of function contributes to the occurrence of cerebral folate deficiency through diminished FOLR1 expression (Cao et al., 2021). Although previous studies have identified mutations in the human CIC gene are associated with neurobehavioral phenotypes, there are no reports of CIC mutations in NTD patients.
To better understand the genetic etiology of human NTDs, we analyzed whole genome sequencing (WGS) data on 140 cases with isolated spina bifida from the U.S. and detected eight rare missense CIC variants. Functional analysis indicated that CIC missense variants identified in NTD cases downregulated the FOLR1 protein level and PCP signaling in Hela and NIH3T3 cell lines. Overall, our results support for the first time that CIC variants potentially contribute to the etiology of human NTDs.