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.