Optical coherence tomography-guided Brillouin microscopy highlights regional tissue stiffness differences during anterior neural tube closure in the Mthfd1l murine mutant.

Neurulation is a highly synchronized biomechanical process leading to the formation of the brain and spinal cord, and its failure leads to neural tube defects (NTDs). Although we are rapidly learning the genetic mechanisms underlying NTDs, the biomechanical aspects are largely unknown. To understand the correlation between NTDs and tissue stiffness during neural tube closure (NTC), we imaged an NTD murine model using optical coherence tomography (OCT), Brillouin microscopy and confocal fluorescence microscopy. Here, we associate structural information from OCT with local stiffness from the Brillouin signal of embryos undergoing neurulation. The stiffness of neuroepithelial tissues in Mthfd1l null embryos was significantly lower than that of wild-type embryos. Additionally, exogenous formate supplementation improved tissue stiffness and gross embryonic morphology in nullizygous and heterozygous embryos. Our results demonstrate the significance of proper tissue stiffness in normal NTC and pave the way for future studies on the mechanobiology of normal and abnormal embryonic development.

A non-coding insertional mutation of Grhl2 causes gene over-expression and multiple structural anomalies including cleft palate, spina bifida and encephalocele.

Orofacial clefts, including cleft lip and palate (CL/P) and neural tube defects (NTDs) are among the most common congenital anomalies, but knowledge of the genetic basis of these conditions remains incomplete. The extent to which genetic risk factors are shared between CL/P, NTDs and related anomalies is also unclear. While identification of causative genes has largely focused on coding and loss of function mutations, it is hypothesized that regulatory mutations account for a portion of the unidentified heritability. We found that excess expression of Grainyhead-like 2 (Grhl2) causes not only spinal NTDs in Axial defects (Axd) mice but also multiple additional defects affecting the cranial region. These include orofacial clefts comprising midline cleft lip and palate and abnormalities of the craniofacial bones and frontal and/or basal encephalocele, in which brain tissue herniates through the cranium or into the nasal cavity. To investigate the causative mutation in the Grhl2Axd strain, whole genome sequencing identified an approximately 4 kb LTR retrotransposon insertion that disrupts the non-coding regulatory region, lying approximately 300 base pairs upstream of the 5' UTR. This insertion also lies within a predicted long non-coding RNA, oriented on the reverse strand, which like Grhl2 is over-expressed in Axd (Grhl2Axd) homozygous mutant embryos. Initial analysis of the GRHL2 upstream region in individuals with NTDs or cleft palate revealed rare or novel variants in a small number of cases. We hypothesize that mutations affecting the regulation of GRHL2 may contribute to craniofacial anomalies and NTDs in humans.

Folate regulation of planar cell polarity pathway and F-actin through folate receptor alpha.

Folate deficiency contribute to neural tube defects (NTDs) which could be rescued by folate supplementation. However, the underlying mechanisms are still not fully understood. Besides, there is considerable controversy concerning the forms of folate used for supplementation. To address this controversy, we prepared culture medium with different forms of folate, folic acid (FA), and 5-methyltetrahydrofolate (5mTHF), at concentrations of 5 µM, 500 nM, 50 nM, and folate free, respectively. Mouse embryonic fibroblasts (MEFs) were treated with different folates continuously for three passages, and cell proliferation and F-actin were monitored. We determined that compared to 5mTHF, FA showed stronger effects on promoting cell proliferation and F-actin formation. We also found that FOLR1 protein level was positively regulated by folate concentration and the non-canonical Wnt/planar cell polarity (PCP) pathway signaling was significantly enriched among different folate conditions in RNA-sequencing analyses. We demonstrated for the first time that FOLR1 could promote the transcription of Vangl2, one of PCP core genes. The transcription of Vangl2 was down-regulated under folate-deficient condition, which resulted in a decrease in PCP activity and F-actin formation. In summary, we identified a distinct advantage of FA in cell proliferation and F-actin formation over 5mTHF, as well as demonstrating that FOLR1 could promote transcription of Vangl2 and provide a new mechanism by which folate deficiency can contribute to the etiology of NTDs.

De novo heterozygous missense variants in CELSR1 as cause of fetal pleural effusions and progressive fetal hydrops.

Fetal hydrops as detected by prenatal ultrasound usually carries a poor prognosis depending on the underlying aetiology. We describe the prenatal and postnatal clinical course of two unrelated female probands in whom de novo heterozygous missense variants in the planar cell polarity gene CELSR1 were detected using exome sequencing. Using several in vitro assays, we show that the CELSR1 p.(Cys1318Tyr) variant disrupted the subcellular localisation, affected cell-cell junction, impaired planar cell polarity signalling and lowered proliferation rate. These observations suggest that deleterious rare CELSR1 variants could be a possible cause of fetal hydrops.

Dual functions of TET1 in germ layer lineage bifurcation distinguished by genomic context and dependence on 5-methylcytosine oxidation.

Gastrulation begins when the epiblast forms the primitive streak or becomes definitive ectoderm. During this lineage bifurcation, the DNA dioxygenase TET1 has bipartite functions in transcriptional activation and repression, but the mechanisms remain unclear. By converting mouse embryonic stem cells (ESCs) into neuroprogenitors, we defined how Tet1-/- cells switch from neuroectoderm fate to form mesoderm and endoderm. We identified the Wnt repressor Tcf7l1 as a TET1 target that suppresses Wnt/ß-catenin and Nodal signalling. ESCs expressing catalytic dead TET1 retain neural potential but activate Nodal and subsequently Wnt/ß-catenin pathways to generate also mesoderm and endoderm. At CpG-poor distal enhancers, TET1 maintains accessible chromatin at neuroectodermal loci independently of DNA demethylation. At CpG-rich promoters, DNA demethylation by TET1 affects the expression of bivalent genes. In ESCs, a non-catalytic TET1 cooperation with Polycomb represses primitive streak genes; post-lineage priming, the interaction becomes antagonistic at neuronal genes, when TET1's catalytic activity is further involved by repressing Wnt signalling. The convergence of repressive DNA and histone methylation does not inhibit neural induction in Tet1-deficient cells, but some DNA hypermethylated loci persist at genes with brain-specific functions. Our results reveal versatile switching of non-catalytic and catalytic TET1 activities based on genomic context, lineage and developmental stage.

Disruption of Fuz in mouse embryos generates hypoplastic hindbrain development and reduced cranial nerve ganglia.

BACKGROUND: The brain and spinal cord formation is initiated in the earliest stages of mammalian pregnancy in a highly organized process known as neurulation. Environmental or genetic interferences can impair neurulation, resulting in clinically significant birth defects known collectively as neural tube defects. The Fuz gene encodes a subunit of the CPLANE complex, a macromolecular planar polarity effector required for ciliogenesis. Ablation of Fuz in mouse embryos results in exencephaly and spina bifida, including dysmorphic craniofacial structures due to defective cilia formation and impaired Sonic Hedgehog signaling. RESULTS: We demonstrate that knocking Fuz out during embryonic mouse development results in a hypoplastic hindbrain phenotype, displaying abnormal rhombomeres with reduced length and width. This phenotype is associated with persistent reduction of ventral neuroepithelial stiffness in a notochord adjacent area at the level of the rhombomere 5. The formation of cranial and paravertebral ganglia is also impaired in these embryos. CONCLUSIONS: This study reveals that hypoplastic hindbrain development, identified by abnormal rhombomere morphology and persistent loss of ventral neuroepithelial stiffness, precedes exencephaly in Fuz ablated murine mutants, indicating that the gene Fuz has a critical function sustaining normal neural tube development and neuronal differentiation.

Systems biology analysis of human genomes points to key pathways conferring spina bifida risk.

Spina bifida (SB) is a debilitating birth defect caused by multiple gene and environment interactions. Though SB shows non-Mendelian inheritance, genetic factors contribute to an estimated 70% of cases. Nevertheless, identifying human mutations conferring SB risk is challenging due to its relative rarity, genetic heterogeneity, incomplete penetrance, and environmental influences that hamper genome-wide association studies approaches to untargeted discovery. Thus, SB genetic studies may suffer from population substructure and/or selection bias introduced by typical candidate gene searches. We report a population based, ancestry-matched whole-genome sequence analysis of SB genetic predisposition using a systems biology strategy to interrogate 298 case-control subject genomes (149 pairs). Genes that were enriched in likely gene disrupting (LGD), rare protein-coding variants were subjected to machine learning analysis to identify genes in which LGD variants occur with a different frequency in cases versus controls and so discriminate between these groups. Those genes with high discriminatory potential for SB significantly enriched pathways pertaining to carbon metabolism, inflammation, innate immunity, cytoskeletal regulation, and essential transcriptional regulation consistent with their having impact on the pathogenesis of human SB. Additionally, an interrogation of conserved noncoding sequences identified robust variant enrichment in regulatory regions of several transcription factors critical to embryonic development. This genome-wide perspective offers an effective approach to the interrogation of coding and noncoding sequence variant contributions to rare complex genetic disorders.

Paternal genetic variants and risk of obstructive heart defects: A parent-of-origin approach.

Previous research on risk factors for obstructive heart defects (OHDs) focused on maternal and infant genetic variants, prenatal environmental exposures, and their potential interaction effects. Less is known about the role of paternal genetic variants or environmental exposures and risk of OHDs. We examined parent-of-origin effects in transmission of alleles in the folate, homocysteine, or transsulfuration pathway genes on OHD occurrence in offspring. We used data on 569 families of liveborn infants with OHDs born between October 1997 and August 2008 from the National Birth Defects Prevention Study to conduct a family-based case-only study. Maternal, paternal, and infant DNA were genotyped using an Illumina Golden Gate custom single nucleotide polymorphism (SNP) panel. Relative risks (RR), 95% confidence interval (CI), and likelihood ratio tests from log-linear models were used to estimate the parent-of-origin effect of 877 SNPs in 60 candidate genes in the folate, homocysteine, and transsulfuration pathways on the risk of OHDs. Bonferroni correction was applied for multiple testing. We identified 3 SNPs in the transsulfuration pathway and 1 SNP in the folate pathway that were statistically significant after Bonferroni correction. Among infants who inherited paternally-derived copies of the G allele for rs6812588 in the RFC1 gene, the G allele for rs1762430 in the MGMT gene, and the A allele for rs9296695 and rs4712023 in the GSTA3 gene, RRs for OHD were 0.11 (95% CI: 0.04, 0.29, P = 9.16x10-7), 0.30 (95% CI: 0.17, 0.53, P = 9.80x10-6), 0.34 (95% CI: 0.20, 0.57, P = 2.28x10-5), and 0.34 (95% CI: 0.20, 0.58, P = 3.77x10-5), respectively, compared to infants who inherited maternally-derived copies of the same alleles. We observed statistically significant decreased risk of OHDs among infants who inherited paternal gene variants involved in folate and transsulfuration pathways.

Exome-wide assessment of isolated biliary atresia: A report from the National Birth Defects Prevention Study using child-parent trios and a case-control design to identify novel rare variants.

The etiology of biliary atresia (BA) is unknown, but recent studies suggest a role for rare protein-altering variants (PAVs). Exome sequencing data from the National Birth Defects Prevention Study on 54 child-parent trios, one child-mother duo, and 1513 parents of children with other birth defects were analyzed. Most (91%) cases were isolated BA. We performed (1) a trio-based analysis to identify rare de novo, homozygous, and compound heterozygous PAVs and (2) a case-control analysis using a sequence kernel-based association test to identify genes enriched with rare PAVs. While we replicated previous findings on PKD1L1, our results do not suggest that recurrent de novo PAVs play important roles in BA susceptibility. In fact, our finding in NOTCH2, a disease gene associated with Alagille syndrome, highlights the difficulty in BA diagnosis. Notably, IFRD2 has been implicated in other gastrointestinal conditions and warrants additional study. Overall, our findings strengthen the hypothesis that the etiology of BA is complex.

CIC missense variants contribute to susceptibility for spina bifida.

Neural tube defects (NTDs) are congenital malformations resulting from abnormal embryonic development of the brain, spine, or spinal column. The genetic etiology of human NTDs remains poorly understood despite intensive investigation. CIC, homolog of the Capicua transcription repressor, has been reported to interact with ataxin-1 (ATXN1) and participate in the pathogenesis of spinocerebellar ataxia type 1. Our previous study demonstrated that CIC loss of function (LoF) variants contributed to the cerebral folate deficiency syndrome by downregulating folate receptor 1 (FOLR1) expression. Given the importance of folate transport in neural tube formation, we hypothesized that CIC variants could contribute to increased risk for NTDs by depressing embryonic folate concentrations. In this study, we examined CIC variants from whole-genome sequencing (WGS) data of 140 isolated spina bifida cases and identified eight missense variants of CIC gene. We tested the pathogenicity of the observed variants through multiple in vitro experiments. We determined that CIC variants decreased the FOLR1 protein level and planar cell polarity (PCP) pathway signaling in a human cell line (HeLa). In a murine cell line (NIH3T3), CIC loss of function variants downregulated PCP signaling. Taken together, this study provides evidence supporting CIC as a risk gene for human NTD.

FKBP8 variants are risk factors for spina bifida.

Neural tube defects (NTDs) are a group of severe congenital malformations caused by a failure of neural tube closure during early embryonic development. Although extensively investigated, the genetic etiology of NTDs remains poorly understood. FKBP8 is critical for proper mammalian neural tube closure. Fkbp8-/- mouse embryos showed posterior NTDs consistent with a diagnosis of spina bifida (SB). To date, no publication has reported any association between FKBP8 and human NTDs. Using Sanger sequencing on genomic DNA samples from 472 SB and 565 control samples, we identified five rare (MAF ≤ 0.001) deleterious variants in SB patients, while no rare deleterious variant was identified in the controls (P = 0.0191). p.Glu140* affected FKBP8 localization to the mitochondria and created a truncated form of the FKBP8 protein, thus impairing its interaction with BCL2 and ultimately leading to an increase in cellular apoptosis. p.Ser3Leu, p.Lys315Asn and p.Ala292Ser variants decreased FKBP8 protein level. p.Lys315Asn further increased the cellular apoptosis. RNA sequencing on anterior and posterior tissues isolated from Fkbp8-/- and wildtype mice at E9.5 and E10.5 showed that Fkbp8-/- embryos have an abnormal expression profile within tissues harvested at posterior sites, thus leading to a posterior NTD. Moreover, we found that Fkbp8 knockout mouse embryos have abnormal expression of Wnt3a and Nkx2.9 during the early stage of neural tube development, perhaps also contributing to caudal specific NTDs. These findings provide evidence that functional variants of FKBP8 are risk factors for SB, which may involve a novel mechanism by which Fkbp8 mutations specifically cause SB in mice.

Multimodal imaging system combining optical coherence tomography and Brillouin microscopy for neural tube imaging.

To understand the dynamics of tissue stiffness during neural tube formation and closure in a murine model, we have developed a multimodal, coaligned imaging system combining optical coherence tomography (OCT) and Brillouin microscopy. Brillouin microscopy can map the longitudinal modulus of tissue but cannot provide structural images. Thus, it is limited for imaging dynamic processes such as neural tube formation and closure. To overcome this limitation, we have combined Brillouin microscopy and OCT in one coaligned instrument. OCT provided depth-resolved structural imaging with a micrometer-scale spatial resolution to guide stiffness mapping by Brillouin modality. 2D structural and Brillouin frequency shift maps were acquired of mouse embryos at gestational day (GD) 8.5, 9.5, and 10.5 with the multimodal system. The results demonstrate the capability of the system to obtain structural and stiffness information simultaneously.

Organoids as a new model system to study neural tube defects.

The neural tube is the first critically important structure that develops in the embryo. It serves as the primordium of the central nervous system; therefore, the proper formation of the neural tube is essential to the developing organism. Neural tube defects (NTDs) are severe congenital defects caused by failed neural tube closure during early embryogenesis. The pathogenesis of NTDs is complicated and still not fully understood even after decades of research. While it is an ethically impossible proposition to investigate the in vivo formation process of the neural tube in human embryos, a newly developed technology involving the creation of neural tube organoids serves as an excellent model system with which to study human neural tube formation and the occurrence of NTDs. Herein we reviewed the recent literature on the process of neural tube formation, the progress of NTDs investigations, and particularly the exciting potential to use neural tube organoids to model the cellular and molecular mechanisms underlying the etiology of NTDs.

Exome sequencing identifies genetic variants in anophthalmia and microphthalmia.

Anophthalmia and microphthalmia (A/M) are rare birth defects affecting up to 2 per 10,000 live births. These conditions are manifested by the absence of an eye or reduced eye volumes within the orbit leading to vision loss. Although clinical case series suggest a strong genetic component in A/M, few systematic investigations have been conducted on potential genetic contributions owing to low population prevalence. To overcome this challenge, we utilized DNA samples and data collected as part of the National Birth Defects Prevention Study (NBDPS). The NBDPS employed multi-center ascertainment of infants affected by A/M. We performed exome sequencing on 67 family trios and identified numerous genes affected by rare deleterious nonsense and missense variants in this cohort, including de novo variants. We identified 9 nonsense changes and 86 missense variants that are absent from the reference human population (Genome Aggregation Database), and we suggest that these are high priority candidate genes for A/M. We also performed literature curation, single cell transcriptome comparisons, and molecular pathway analysis on the candidate genes and performed protein structure modeling to determine the potential pathogenic variant consequences on PAX6 in this disease.

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.

Upregulation of reduced folate carrier by vitamin D enhances brain folate uptake in mice lacking folate receptor alpha.

Folates are critical for central nervous system function. Folate transport is mediated by 3 major pathways, reduced folate carrier (RFC), proton-coupled folate transporter (PCFT), and folate receptor alpha (FRα/Folr1), known to be regulated by ligand-activated nuclear receptors. Cerebral folate delivery primarily occurs at the choroid plexus through FRα and PCFT; inactivation of these transport systems can result in very low folate levels in the cerebrospinal fluid causing childhood neurodegenerative disorders. These disorders have devastating effects in young children, and current therapeutic approaches are not sufficiently effective. Our group has previously reported in vitro that functional expression of RFC at the blood-brain barrier (BBB) and its upregulation by the vitamin D nuclear receptor (VDR) could provide an alternative route for brain folate uptake. In this study, we further demonstrated in vivo, using Folr1 knockout (KO) mice, that loss of FRα led to a substantial decrease of folate delivery to the brain and that pretreatment of Folr1 KO mice with the VDR activating ligand, calcitriol (1,25-dihydroxyvitamin D3), resulted in over a 6-fold increase in [13C5]-5-formyltetrahydrofolate ([13C5]-5-formylTHF) concentration in brain tissues, with levels comparable to wild-type animals. Brain-to-plasma concentration ratio of [13C5]-5-formylTHF was also significantly higher in calcitriol-treated Folr1 KO mice (15-fold), indicating a remarkable enhancement in brain folate delivery. These findings demonstrate that augmenting RFC functional expression at the BBB could effectively compensate for the loss of Folr1-mediated folate uptake at the choroid plexus, providing a therapeutic approach for neurometabolic disorders caused by defective brain folate transport.

Unraveling the complex genetics of neural tube defects: From biological models to human genomics and back.

Neural tube defects (NTDs) are a classic example of preventable birth defects for which there is a proven-effective intervention, folic acid (FA); however, further methods of prevention remain unrealized. In the decades following implementation of FA nutritional fortification programs throughout at least 87 nations, it has become apparent that not all NTDs can be prevented by FA. In the United States, FA fortification only reduced NTD rates by 28-35% (Williams et al., 2015). As such, it is imperative that further work is performed to understand the risk factors associated with NTDs and their underlying mechanisms so that alternative prevention strategies can be developed. However, this is complicated by the sheer number of genes associated with neural tube development, the heterogeneity of observable phenotypes in human cases, the rareness of the disease, and the myriad of environmental factors associated with NTD risk. Given the complex genetic architecture underlying NTD pathology and the way in which that architecture interacts dynamically with environmental factors, further prevention initiatives will undoubtedly require precision medicine strategies that utilize the power of human genomics and modern tools for assessing genetic risk factors. Herein, we review recent advances in genomic strategies for discovering genetic variants associated with these defects, and new ways in which biological models, such as mice and cell culture-derived organoids, are leveraged to assess mechanistic functionality, the way these variants interact with other genetic or environmental factors, and their ultimate contribution to human NTD risk.

Dominant negative GPR161 rare variants are risk factors of human spina bifida.

Spina bifida (SB) is a complex disorder of failed neural tube closure during the first month of human gestation, with a suspected etiology involving multiple gene and environmental interactions. GPR161 is a ciliary G-protein coupled receptor that regulates Sonic Hedgehog (Shh) signaling. Gpr161 null and hypomorphic mutations cause neural tube defects (NTDs) in mouse models. Herein we show that several genes involved in Shh and Wnt signaling were differentially expressed in the Gpr161 null embryos using RNA-seq analysis. To determine whether there exists an association between GPR161 and SB in humans, we performed direct Sanger sequencing on the GPR161 gene in a cohort of 384 SB patients and 190 healthy controls. We identified six rare variants of GPR161 in six SB cases, of which two of the variants were novel and did not exist in any databases. Both of these variants were predicted to be damaging by SIFT and/or PolyPhen analysis. The novel GPR161 rare variants mislocalized to the primary cilia, dysregulated Shh and Wnt signaling and inhibited cell proliferation in vitro. Our results demonstrate that GPR161 mutations cause NTDs via dysregulation of Shh and Wnt signaling in mice, and novel rare variants of GPR161 can be risk factors for SB in humans.

The TFAP2A-IRF6-GRHL3 genetic pathway is conserved in neurulation.

Mutations in IRF6, TFAP2A and GRHL3 cause orofacial clefting syndromes in humans. However, Tfap2a and Grhl3 are also required for neurulation in mice. Here, we found that homeostasis of Irf6 is also required for development of the neural tube and associated structures. Over-expression of Irf6 caused exencephaly, a rostral neural tube defect, through suppression of Tfap2a and Grhl3 expression. Conversely, loss of Irf6 function caused a curly tail and coincided with a reduction of Tfap2a and Grhl3 expression in tail tissues. To test whether Irf6 function in neurulation was conserved, we sequenced samples obtained from human cases of spina bifida and anencephaly. We found two likely disease-causing variants in two samples from patients with spina bifida. Overall, these data suggest that the Tfap2a-Irf6-Grhl3 genetic pathway is shared by two embryologically distinct morphogenetic events that previously were considered independent during mammalian development. In addition, these data suggest new candidates to delineate the genetic architecture of neural tube defects and new therapeutic targets to prevent this common birth defect.

Genome-wide investigation identifies a rare copy-number variant burden associated with human spina bifida.

PURPOSE: Next-generation sequencing has implicated some risk variants for human spina bifida (SB), but the genome-wide contribution of structural variation to this complex genetic disorder remains largely unknown. We examined copy-number variant (CNV) participation in the genetic architecture underlying SB risk. METHODS: A high-confidence ensemble approach to genome sequences (GS) was benchmarked and employed for systematic detection of common and rare CNVs in two separate ancestry-matched SB case-control cohorts. RESULTS: SB cases were enriched with exon disruptive rare CNVs, 44% of which were under 10 kb, in both ancestral populations (P = 6.75 × 10-7; P = 7.59 × 10-4). Genes containing these disruptive CNVs fall into molecular pathways, supporting a role for these genes in SB. Our results expand the catalog of variants and genes with potential contribution to genetic and gene-environment interactions that interfere with neurulation, useful for further functional characterization. CONCLUSION: This study underscores the need for genome-wide investigation and extends our previous threshold model of exonic, single-nucleotide variation toward human SB risk to include structural variation. Since GS data afford detection of CNVs with greater resolution than microarray methods, our results have important implications toward a more comprehensive understanding of the genetic risk and mechanisms underlying neural tube defect pathogenesis.