Abstract

Neural tube defects (NTDs) are common, severe congenital malformations whose causation involves multiple genes and environmental factors. Although more than 200 genes are known to cause NTDs in mice, there has been rather limited progress in delineating the molecular basis underlying most human NTDs. Numerous genetic studies have been carried out to investigate candidate genes in cohorts of patients, with particular reference to those that participate in folate one-carbon metabolism. Although the homocysteine remethylation gene MTHFR has emerged as a risk factor in some human populations, few other consistent findings have resulted from this approach. Similarly, attention focused on the human homologues of mouse NTD genes has contributed only limited positive findings to date, although an emerging association between genes of the non-canonical Wnt (planar cell polarity) pathway and NTDs provides candidates for future studies. Priorities for the next phase of this research include: (i) larger studies that are sufficiently powered to detect significant associations with relatively minor risk factors; (ii) analysis of multiple candidate genes in groups of well-genotyped individuals to detect possible gene–gene interactions; (iii) use of high throughput genomic technology to evaluate the role of copy number variants and to detect ‘private’ and regulatory mutations, neither of which have been studied to date; (iv) detailed analysis of patient samples stratified by phenotype to enable, for example, hypothesis-driven testing of candidates genes in groups of NTDs with specific defects of folate metabolism, or in groups of fetuses with well-defined phenotypes such as craniorachischisis.

INTRODUCTION

Congenital malformations are the leading cause of infant mortality in developed countries and a major cause of health problems in surviving children. Neural tube defects (NTDs) are a common group of central nervous system anomalies affecting 0.5–2 per 1000 pregnancies worldwide. NTDs arise when the neural tube, the embryonic precursor of the brain and spinal cord, fails to close during neurulation. The cranial region (anencephaly) or the low spine (open spina bifida; myelomeningocele) are most commonly affected although, in the severe NTD craniorachischisis, almost the entire neural tube remains open, from midbrain to low spine.

Most individuals who survive with NTDs (particularly myelomeningocele) have a multiple system handicap and a limited life expectancy. However, despite the high prevalence and traumatic consequences for affected individuals and their families, the causes of NTD are poorly understood. Identification of causative factors is confounded by the fact that the majority of these malformations appear to result from a combination of genetic and environmental factors. A strong genetic component is indicated by the high recurrence risk for siblings of affected individuals (1,2). Syndromic cases of NTD also exist, often associated with chromosomal anomalies, but these represent <10% of all defects (1,3–5). The majority of NTDs are sporadic, with recurrence fitting a multifactorial polygenic or oligogenic pattern, rather than models on the basis of single gene dominant or recessives, with reduced penetrance (2).

GENETIC ANALYSIS OF HUMAN NTDS

Positional cloning strategies have been hampered by the paucity of large families with multiple affected members. Nevertheless, genome-wide studies using collections of smaller multiplex families have implicated chromosomes 2, 7 and 10 as harbouring candidate risk loci for spina bifida (6–8). Although the causative genes are yet to be identified, these studies may result in identification of candidate sequences that can be evaluated in larger populations. An alternative approach exploits the association of NTDs with chromosomal anomalies such as trisomies 13 and 18 (9), suggesting that gene-dosage can affect neural tube closure. Rearrangements involving deletions, duplications or balanced translocations are likely to be most informative, with fine mapping of chromosomal breakpoints enabling identification of specific loci (10).

In some studies, direct mutation screening of candidate genes has been carried out in cohorts of patients (11), but the vast majority involve statistical association analysis of sequence variants in or near candidate genes. Most work has involved case–control analysis, comparing the frequency of ‘risk’ alleles in affected individuals and/or mothers with a matched unaffected cohort. More sophisticated studies have used the transmission disequilibrium test (TDT) in family trios (mother, father and affected child), which is less dependent on population structure. In the remainder of this article, we review the main candidate gene studies which have arisen primarily from analysis of folate metabolic pathways and mouse models of NTDs. Boyles et al. (11) published a comprehensive review of this field up to 2004, and an updated candidate gene list is presented in Table 1.

Table 1.

Candidate gene analysis in human NTDs

Human gene Type of candidate Reference Population studied Sample size Type of study Summary of results/conclusion 
AHCY (S-adenosylhomocysteine hydrolase) One carbon metabolism (56Dutch 180 SB patients, 190 controls Case–control study No association 
ALDH1L1 (Aldehyde dehydrogenase 1, member L1) One carbon metabolism (56Dutch 180 SB patients, 190 controls Case–control study Nominally significant association with Asp793Glu varianta 
ALDH1A2 (Retinaldehyde dehydrogenase Type 2, RALDH2) Retinol metabolism (104USA 318 SB families Family based association study One polymorphism associated with increased SB risk (tentative association for two others) 
AMD1 (Adenosyl methionine decarboxylase 1One carbon metabolism (56Dutch 180 SB patients, 190 controls Case–control study No association 
APE1 (apurinic endonuclease1) DNA repair (112Mixed USA 380 SB cases, 350 controls Case–control study Suggestion of reduced risk for Asp148Glu variant 
BHMT (betaine-homocysteine methyltransferase) One carbon metabolism (113Mixed USA 252 SB cases, 337 controls Case–control study No association for Arg239Gln polymorphism 
 (56Dutch 180 SB patients, 190 controls Case–control study No association 
  (103Mixed USA 259 SB cases, 359 controls Case–control study Modest increase in SB risk associated with 1 SNP (of eight tested) 
BHMT2 (betaine-homocysteine methyltransferase 2) One carbon metabolism (56Dutch 180 SB patients, 190 controls Association study No association detected 
  (103Mixed USA 259 SB cases, 359 controls Case–control study No association for 7 SNPs tested 
BRCA1 (breast cancer 1) NTDs in mouse mutant (114USA 268 SB patients and parents Family based association study (TDT) Association with SB for two microsatellite markers and A4956G SNP. Proposed polymorphisms affect level of lesion, not causative 
CAT (catalase) Oxidative stress (115Mixed USA 507 SB cases, 185 controls Case–control study No association 
CBS (cystathionine beta-synthase) Folate metabolism (116UK 207 NTD cases (200 mothers, 93 fathers). 601 controls, 542 control mothers Case–control study No association for 844ins68 
 (56Dutch 180 SB patients, 190 controls Case–control study No association 
 (103Mixed USA 259 SB cases, 359 controls Case–control (9 SNPs) Modest increase in SB risk associated with 2 SNPs 
CFL1 (n-cofilin) NTDs in mouse mutant (117Mixed USA 246 SB cases, 336 controls Case–control SNPs Mildly elevated risk of NTDs in non-Hispanic whites 
CHKA (choline kinase A) One carbon metabolism (118Mixed USA 103 SB cases, 338 controls Case–control study Possible association with reduced SB risk for 1 of 2 SNPs studied 
CITED2 NTDs in mouse mutant (119Mixed USA 64 SB cases, 72 controls Mutation screen and case–control No mutations. No association of three 5′-UTR SNPs with risk 
COQ3 (Coenzyme Q3 homolog, methyltransferase) Methylation (56Dutch 180 SB patients, 190 controls Case–control study No association 
CRABPI (cellular retinoic acid binding protein I) Retinol metabolism (104USA 230 SB cases, 318 SB families Mutation screen and family based association study No mutations. No association (3 SNPs tested) 
CRABPII (cellular retinoic acid binding protein II) Retinol metabolism (104USA 230 SB cases Mutation screen No mutations 
CTH (Cystathionase) One carbon metabolism (56Dutch 180 SB patients, 190 controls Case–control study No association 
CUBN (cubulin) Endocytosis (folate transport) (56Dutch 179 SB patients, 190 controls Case–control study GG genotype for rs1907362 significantly associated with reduced SB risk 
CYP26A1 (cytochrome P450) Retinol metabolism (104USA 230 SB cases Mutation screen No mutations 
CYP26B1 (cytochrome P450) Retinol metabolism (104USA 230 SB cases, 318 SB families Mutation screen and Family based association study No mutations. No association (5 SNPs tested) 
DHFR (Dihydrofolate reductase) Folate metabolism (120Mixed USA 61 SB cases and parents (multi-affected families) and 219 controls Case–control study of 19-bp intron-1 deletion The del/del genotype was more frequent in mothers of SB cases, compared with controls. No association in fathers or patients 
  (121Irish 283 cases (and 280 mothers, 279 fathers) and 256 controls. SB (95%) or encephalocele (5%) Case–control study. 19-bp deletion and two 3′-UTR variants. 19-bp Intron deletion shows protective effect. May increase mRNA levels 
  (122Dutch 109 patients, 121 mothers (SB). 234 paediatric controls, 292 control women Case–control study. 19-bp deletion and 9-bp repeat in 5′-UTR 19-bp Intron deletion not associated with NTDs. No effect on expression 
  (56Dutch 180 SB patients, 190 controls Case–control study No association 
  (103Mixed USA 259 SB cases, 359 controls Case–control study No association for 9 SNPs tested. Intron deletion not tested 
  (41Mixed UK 126 SB (open); 103 SB (closed); 49 anencephalic; 192 controls Case–control study No association 
FOLR1 (Folate receptor 1) Folate transport (56Dutch 180 SB patients, 190 controls Case–control study No association 
  (103Mixed USA 259 SB cases, 359 controls Case–control study No association for 3 SNPs tested 
FOLR2 (Folate receptor 2) Folate transport (56Dutch 180 SB patients, 190 controls Association study No association 
  (103Mixed USA 259 SB cases, 359 controls Case–control study No association for 3 SNPs tested 
FOLR3 (Folate receptor 3) Folate transport (56Dutch 180 SB patients, 190 controls Case–control study No association 
FPGS (Folylpolyglutamate synthase) Cellular folate retention (56Dutch 180 SB patients, 190 controls Case–control study No association 
FTCD (Formininotransferase cyclodeaminase One carbon metabolism (56Dutch 180 SB patients, 190 controls Case–control study No association 
GAMT (Guanidinoacetate N-methyl transferase) One carbon metabolism (56Dutch 180 SB patients, 190 controls Case–control study No association 
GAPD (glyceraldehyde 3 phosphate dehydrogenase) Glucose metabolism (115Mixed USA 507 SB cases, 185 controls Case–control study No association 
GART (Phosphoribosylglycinamide formyltransferase, phosphoribosyl glycinamide synthetase, phosphoribosyl aminoimidazole synthetase) Purine biosynthesis (one carbon metabolism) (56Dutch 180 SB patients, 190 controls Association study No association 
GCPII (glutamate carboxypeptidase), FOLH1 (Folate hydrolase) Folate metabolism (116UK 208 NTD cases (200 mothers, 92 fathers). 600 child, 531 mother controls Case–control study No association for 1561C>T 
 (56Dutch 180 SB patients, 190 controls Case–control study No association 
GGH (Gamma-glutamyl hydrolase) Folate metabolism (56Dutch 180 SB patients, 190 controls Case–control study No association 
GLUT1 (glucose transporter 1) Glucose metabolism (115Mixed USA 507 SB cases, 185 controls Case–control study Pro196 silent SNP associated with risk in TDT test 
GLUT4 (glucose transporter 4) Glucose metabolism (115Mixed USA 507 SB cases, 185 controls Case–control study No association 
HK1 (hexokinase 1) Glucose metabolism (115Mixed USA 507 SB cases, 185 controls Case–control study Lys481 SNP variant associated with risk in TDT test 
HK2 (hexokinase 2) Glucose metabolism (115Mixed USA 507 SB cases, 185 controls Case–control study No association 
ICMT (Isoprenylcysteine carboxyl methyltransferase) Protein methylation (56Dutch 180 SB patients, 190 controls Association study No association 
INS (insulin) Glucose metabolism (115Mixed USA 507 SB cases, 185 controls Case–control study No association 
INSR (insulin receptor) Glucose metabolism (115Mixed USA 507 SB cases, 185 controls Case–control study No association 
LEP (leptin) Glucose metabolism (115Mixed USA 507 SB cases, 185 controls Case–control study Arg109Lys variant associated with risk in TDT test 
LEPR (leptin receptor) Glucose metabolism (115Mixed USA 507 SB cases, 185 controls Case–control study Arg109Lys variant associated with risk in TDT test 
MAT1A (Methionine adenosyltransferase I, alpha) One carbon metabolism (56Dutch 180 SB patients, 190 controls Case–control study No association 
MAT2A (Methionine adenosyltransferase II, alpha) One carbon metabolism (56Dutch 180 SB patients, 190 controls Case–control study No association 
MGMT (O-6-Methylguanine DNA methyltransferase) One carbon metabolism DNA methylation (56Dutch 180 SB patients, 190 controls Case–control study No association 
MTHFD1 (methylenetetrahydrofolate dehydrogenase/methylenetetrahydrofolate-cyclohydrolase/formyltetrahydrofolate synthetase) Folate metabolism (50Irish 176 NTD cases (Mostly SB, few encephalocele). 245 mothers, 127 fathers (also includes parents of anencephalic cases). 770 controls Case–control to evaluate Arg653Gln (1958G>A; dbSNP rs 1801133) polymorphism Maternal AA genotype confers increased risk to offspring 
(51Italian 142 NTD cases (open and closed SB) (125 mothers, 108 fathers). 523 controls. Case–control study and family based association study (TDT) for 1958G>A Mildly increased risk for AA and GA genotypes in cases (and mothers). TDT shows excess transmission of A allele to cases 
(52Dutch 103 SB cases, 113 mothers, 98 fathers. 460 controls. Case–control and TDT for 1958G>A No association 
(54Irish 509 cases (with 485 mothers and 439 fathers)—mostly open SB. 966 controls. Case–control study for SNP rs1076991C>T Promoter variant not independent risk factor. Case and maternal risk factor when combined with Arg653Gln variant 
  (56Dutch 180 SB patients, 190 controls Case–control study No association 
  (103Mixed USA 259 SB cases, 359 controls Case–control (10 SNPs) Increased SB risk for one SNP, decreased risk for three others 
  (41Mixed UK 126 SB (open); 103 SB (closed); 49 anencephalic; 192 controls Case–control study No association 
MTHFD2 Folate metabolism (56Dutch 180 SB patients, 190 controls Case–control study No association 
  (103Mixed USA 259 SB cases, 359 controls Case–control study No association for 8 SNPs tested 
MTHFR (5,10-methylenetetrahydrofolate reductase) Folate metabolism (116UK 200 NTD cases (186 mothers, 92 fathers). 578 child, 512 mother controls Case–control study Mildly elevated risk for 677C>T. No association for 1298A>C 
  (123Polish 20 NTD cases, 262 controls. Case–control for C677T and A1298C No association 
  (124Irish 471 cases (451 SB, 20 encephalocele). Triads comprise >1300 samples. 922 controls. Mutation screen. Case–control and TDT for 116C>T (P39P) and 1793G>A (R594Q) Possible association with SB for variants tested. Unlikely to be independent risk factors (association likely due to linkage disequilibrium with 677C>T) 
  (125Mixed USA 350 cases, 328 mothers, 245 fathers, 167 siblings Family-based association study No association for 677C>T 
  (126Italian 15 cases (open SB), 18 fathers, 60 mothers. 43 control children and 100 control adults Case–control screen for C677T and A1298C T allele more frequent in cases than controls. A1298C not different between groups 
  (127Mexican 118 NTD case mothers (all anencephalic), 112 control mothers Case–control study for C677T and A1298C Maternal TT confers higher risk than CC for anencephaly. A1298C not associated with NTDs 
  (128Indian 83 mothers of NTD cases. 60 controls Case–control for C677T and A1298C 677T more frequent in mothers of cases than in controls (only for lower defects). No difference in A1298C frequency 
  (129Mexican (Yucatan) 108 cases (97 SB, 4 anencephalic, 7 encephalocele), with 147 parents. 120 controls Case–control, screen for A1298C No association 
  (130French 77 NTD mothers. 61 controls Case–control study No association for 677C>T. Reduced risk for 1298A>C allele 
  (131Chinese 38 mothers of NTD cases. 80 controls Case–control to evaluate 677C>T TT genotype less frequent in case mothers (but small numbers) 
  (56Dutch 180 SB patients, 190 controls Case–control study No association for 677C>T or 1298A>C 
  (103Mixed USA 259 SB cases, 359 controls Case–control (13 SNPs) Increased risk, OR2.0, of SB associated with 677C>T 
  (41Mixed UK 126 SB (open); 103 SB (closed); 49 anencephalic; 192 controls Case–control for C677T and A1298C Slight protective effect for open SB of 677TT genotype 
MTHFS (5,10-methylenetetrahydrofolate synthetase) Folate metabolism (56Dutch 180 SB patients, 190 controls Case–control study No association 
MTR (methionine synthase) One carbon metabolism (homocysteine remethylation) (105Hispanic USA 43 NTD cases, 122 mothers. 124 control infants and 127 mothers Case–control study for 2756A>G Not independent risk factor. May be associated with NTDs in combination with MTRR 66A>G 
 (130French 77 NTD mothers. 61 controls Case–control study No association for 66A>G 
 (56Dutch 180 SB patients, 190 controls Case–control study No association 
 (103Mixed USA 259 SB cases, 359 controls Case–control study No association with SB for 21 SNPs tested (including 2756A>G) 
  (41Mixed UK 126 SB (open); 103 SB (closed); 49 anencephalic; 192 controls Case–control study No association 
MTRR (methionine synthase reductase) One carbon metabolism (homocysteine remethylation) (105Hispanic USA 43 NTD cases, 122 mothers. 124 control infants and 127 mothers Case–control study for 66A>G (I22M) G allele associated with increased risk. Additional risk in combination with MTR 2756G allele 
 (116UK 201 NTD cases (203 mothers, 88 fathers). 601 child, 532 mother controls Case–control study Mildly reduced risk associated with 66A>G 
  (132Irish 575 NTD families 95% SB). 487 controls. Case–control and family-based analysis for three variants No association for 66A>G (I22M) with SB risk (except possible paternal effect). No association for S175L or K350R 
  (133Dutch 109 cases (open SB) and parents. 234 control children, 292 control women. Case–control screen for 66A>G In this study and meta-analysis (including previous studies) maternal GG is associated with increased risk in offspring, but GG in child not associated with NTDs 
  (130French 77 NTD mothers. 61 controls Case–control study Marginally increased risk associated with 66G allele 
  (56Dutch 180 SB patients, 190 controls Case–control study No association 
  (103Mixed USA 259 SB cases, 359 controls Case–control study Modest increase in SB risk for three linked SNPs, but not 66A>G 
  (41Mixed UK 126 SB (open); 103 SB (closed); 49 anencephalic; 192 controls Case–control study Marginal increased risk for combined NTDs 
MUT (methylmalonyl-CoA mutase) One carbon metabolism (134Irish 279 NTD triads (mostly SB), 256 controls Case–control and family-based study No association with NTDs for three variants tested 
NAT1 (N-acetyltransferase 1) Folate metabolism and acetylation reactions (135Mixed USA 354 NTD families Family based association study 1095C>A not associated with SB risk (except in combination with maternal smoking) 
 (136Mixed USA 374 NTD families Family based association study Composite genotype (6 SNPs) related to reduced function associated with lower SB risk for cases and mothers 
NAT2 (N-acetyltransferase 2)  (56Dutch 180 SB patients, 190 controls Case–control study No association 
NNMT (Nicotinamide N-methyltransferase) Methylation reactions (137Mixed USA 252 SB cases, 335 controls Case–control study No association between NNMT variants and SB risk 
  (56Dutch 180 SB patients, 190 controls Association study No association 
NCAM1 (neural cell adhesión molecule1) Embryonic cell adhesion (138USA 204 SB families Family based association study Risk of SB associated with intronic SNP (of 11 tested) in first 132 families, not in further 72 families 
NOS1 (nitric oxide synthase 1) Possible effect on one carbon metabolism (56Dutch 180 SB patients, 190 controls Case–control study No association for repeat polymorphisms 
NOS2 (nitric oxide synthase 2A)  (56Dutch 180 SB patients, 190 controls Case–control study No association for SNPs and repeat polymorphism 
NOS3 (nitric oxide synthase 3 endothelial)  (139Mixed US 301 families with SB Family based association study No association by TDT analysis. Maternal 894G>T associated with SB risk by log-linear modelling 
  (140Dutch 109 SB cases, 121 mothers, 103 fathers. 500 controls Case–control and TDT 894G>T not independent risk factor. Possible risk interaction with MTHFR 677TT 
  (56Dutch 180 SB patients, 190 controls Case–control study No association detected for SNPs and repeat polymorphism 
  (103Mixed USA 259 SB cases, 359 controls Case–control study No association 
hOGG1 (8-hydroxyguanine DNA-glycosylase1DNA repair (112Mixed USA 380 SB cases, 350 controls Case–control study No association 
PAX3 NTDs in mouse mutant (70Mixed USA 74 SB cases, 87 controls Mutation screen and case–control study rs16863657 and related haplotype associated with increased SB risk 
PCMT1 (l-isoaspartate O-methyltransferase) Methylation reactions (141Mixed USA 152 SB cases, 423 controls Case–control study of Ile120Val Val/Val genotype associated with possible reduction in SB risk 
  (56Dutch 180 SB patients, 190 controls Case–control study No association for Ile120Val 
PDGFRA (Platelet derived growth factor receptor alpha) NTDs in Patch mouse mutant (142US Hispanic 43 NTD cases, 122 NTD mothers. 124 control infants, 127 control mothers Case–control study for promoter haplotypes P1 promoter haplotypes with lower activity may be associated with maternal risk. Case numbers too small for conclusion 
 (143Mixed USA 407 parent-child triads (SB). 164 controls Case–control and family based association (TDT) No association for P1 promoter haplotypes 
  (108Dutch 88 SB cases, 56 SB mothers. 74 controls, 72 control mothers Evaluated H1 and H2 promoter haplotypes H1 promoter may be more frequent in cases than controls. Suggestion that BMI, glucose and inositol differentially interact with H1/H2 
PEMT (Phosphatidylethanolamine N-methyltransferase) One carbon and choline metabolism (144Mixed USA 360 SB cases, 595 controls Case–control study No association with SB for two non-synonymous SNPs 
PRKACA, PRKACB (cAMP-dependent protein kinase A catalytic subunits) NTDs in mouse mutants (145Mixed USA 207 SB cases, 209 controls Mutation screen and case–control study No mutation. No association 
PRMT1 (Protein arginine methyltransferase 1) Methylation reactions (56Dutch 180 SB patients, 190 controls Case–control study No association 
PRMT2 (Protein arginine methyltransferase 2) Methylation reactions (56Dutch 180 SB patients, 190 controls Case–control study No association 
PYCT1A (CTP:phosphocholine cytidyltransferase) One carbon metabolism (118Mixed USA 103 SB cases, 338 controls Case–control study Weak association with SB risk for 1 of 2 SNPs studied 
RNMT (RNA (guanine-7-) methyltransferase) Methylation reactions (56Dutch 180 SB patients, 190 controls Case–control study No association 
SARDH (Sarcosine dehydrogenase) One carbon metabolism (56Dutch 180 SB patients, 190 controls Case–control study Nominally significant association with two synonymous SNPsa 
SHMT (Serine hydroxyl methyltransferase I) One carbon metabolism (146UK 97 NTD mothers, 190 controls Case–control study 1420C>T associated with protective maternal effect 
  (56Dutch 180 SB patients, 190 controls Case–control study No association for Leu474Phe 
SLCA19A1, RFC-1 (reduced folate carrier), Folate transport (116UK 206 NTD cases (186 mothers, 92 fathers). 578 child, 512 mother controls Case–control study No association for 80G>A (H27R) 
  (125Mixed USA 350 cases, 328 mothers, 245 fathers, 167 siblings. Family-based association study No association for 80G>A 
  (147Irish 437 NTD families, 852 controls Case–control and family based association No association for 80G>A. 61-bp polymorphism under-represented in cases 
  (131Chinese 38 mothers of cases. 80 controls Case–control for 80G>A Association of GG genotype with risk of NTD offspring 
  (148Chinese 104 NTD families, 100 control families Case–control for 80G>A Elevated risk for GG genotype of cases or mother (if folic acid not used) 
  (56Dutch 180 SB patients, 190 controls Case–control study Nominally significant association for 80AA genotype and reduced riska 
  (103Mixed USA 259 SB cases, 359 controls Case–control study No association with risk of SB for 6 SNPs tested. 80G>A not tested 
  (41Mixed UK 126 SB (open); 103 SB (closed); 49 anencephalic; 192 controls Case–control study No association 
SOD2 (superoxide dismutase 2) Oxidative stress (115Mixed USA 507 SB cases, 185 controls Case–control study No association 
T (Brachyury) Axial development in mouse (149Mixed USA 316 SB families Family-based association study TIVS7 T/C allele more frequent in cases than expected 
TCNII (transcobalamin II) One carbon metabolism (150Irish ∼350 NTD families, ∼700 controls. Case–control and family based association study No association with SB risk for 6 SNPs 
  (130French 77 NTD mothers. 61 controls Case–control study No association for 776C>G 
  (56Dutch 180 SB patients, 190 controls Case–control study No association for Arg259Pro 
TXN2 (thioredoxin2) NTDs (SB) in mouse knockout (151Mixed USA 48 SB cases, 48 controls Case–control study A2 promoter allele associated with risk of NTDs but small sample size 
TP53 (p53) NTDs in mouse mutant (152Irish 549 NTD cases, 532 mothers, 481 fathers. 999 controls Case–control and family based association studies Two non-coding variants associated with case NTD risk and two variants with maternal risk (but no multiple testing correction) 
  (115Mixed USA 507 SB cases, 185 controls Case–control study No association 
TRDMT1 (tRNA aspartic acid methyltransferase 1) One carbon metabolism (56Dutch 180 SB patients, 190 controls Case–control study Nominally significant association for one SNPa 
TYMS (thymidylate synthase) Folate metabolism and pyrimidine biosynthesis (110Mixed USA 264 SB cases, 259 controls. Mutation screen and case–control for 28-bp repeat in 5′-UTR and 6-bp deletion in 3′-UTR 3′-UTR polymorphism associated with increased SB risk in non-Hispanic white population. Further increased risk with 5′-UTR polymorphism 
 (153UK 197 NTD cases, 194 mothers, 93 fathers. 179 control infants, 177 control mothers Case–control study of 28-bp repeat No association for TYMS 
  (56Dutch 180 SB patients, 190 controls Case–control study No association 
  (103Mixed USA 259 SB cases, 359 controls Case–control study Modest increase in SB risk for three linked SNPs (of 5 tested) 
UCP2 (uncoupling protein 2) Energy metabolism Previous study indicates association with NTDs (154Irish 169 cases, 163 mothers, 167 fathers Evaluated 866G/A, A55V, 3′-UTR 45-bp ins/del UCP2 not associated with NTD risk. Different frequency of risk allele in control population compared with previous study 
VANGL1 (van gogh-like 1) PCP gene homologue; Paralogue of VANGL2 (95Mixed UK and USA 66 (21 craniorachischisis, 24 SB, 21 anencephalic) and 200 controls Mutation screen No causative mutations. One missense variant, present in controls 
  (96Italian and French 144 (80 SB, 7 craniorachischisis, 22 closed spinal dysraphism, 35 caudal regression). 172 Italian and CEPH controls Mutation screen Three missense mutations (two in myelomeningocele, R274Q and M328T; 1 in caudal regression, V239I). V239I has functional effect on interaction with Dvl proteins 
  (97Italian and USA 673 cases (15 open cranial dysraphisms, 456 SB, 202 closed spinal dysraphisms) Mutation screen Ten missense variants in 13 individuals (absent in 1187–1462 controls). Five in highly conserved residues (two in myelomeningocele, three in closed spinal dysraphism/caudal regression syndrome) 
VANGL2 (van gogh-like 2) Mouse model: Craniorachischisis in loop-tail mice (95Mixed UK and USA 66 (21 craniorachischisis, 24 SB, 21 anencephalic) and 200 controls Mutation screen No causative mutations identified. 7 bp duplication in intron six in one craniorachischisis 
 (96Italian and French 144 (80 SB, 7 craniorachischisis, 22 closed spinal dysraphism, 35 caudal regression) and 172 Italian and CEPH controls Mutation screen No coding mutations 
XPD (DNA excision repair protein ERCC-2) DNA repair (112Mixed USA 380 SB cases, 350 controls Case–control study Mildly elevated risk associated with 751Gln 
XRCC1 (X-ray repair cross-complementing) DNA repair (112Mixed USA 380 SB cases, 350 controls Case–control study No association 
XRCC3 (X-ray repair cross-complementing) DNA repair (112Mixed USA 380 SB cases, 350 controls Case–control study No association 
ZIC1 Brain defects in mouse mutant (155Dutch 117 NTDs (SB, Anencephaly, Encephalocele), 364 controls Mutation screen No mutations 
ZIC2 NTDs in mouse mutant (155Dutch 117 NTDs (SB, Anencephaly, Encephalocele), 364 controls Mutation screen; case–control study Alanine deletion in one patient. Frequent polymorphism (1059C>T) has no association 
ZIC3 NTDs in mouse mutant (155Dutch 117 NTDs (SB, Anencephaly, Encephalocele), 364 controls Mutation screen One silent variant (858G>A) in one patient 
Human gene Type of candidate Reference Population studied Sample size Type of study Summary of results/conclusion 
AHCY (S-adenosylhomocysteine hydrolase) One carbon metabolism (56Dutch 180 SB patients, 190 controls Case–control study No association 
ALDH1L1 (Aldehyde dehydrogenase 1, member L1) One carbon metabolism (56Dutch 180 SB patients, 190 controls Case–control study Nominally significant association with Asp793Glu varianta 
ALDH1A2 (Retinaldehyde dehydrogenase Type 2, RALDH2) Retinol metabolism (104USA 318 SB families Family based association study One polymorphism associated with increased SB risk (tentative association for two others) 
AMD1 (Adenosyl methionine decarboxylase 1One carbon metabolism (56Dutch 180 SB patients, 190 controls Case–control study No association 
APE1 (apurinic endonuclease1) DNA repair (112Mixed USA 380 SB cases, 350 controls Case–control study Suggestion of reduced risk for Asp148Glu variant 
BHMT (betaine-homocysteine methyltransferase) One carbon metabolism (113Mixed USA 252 SB cases, 337 controls Case–control study No association for Arg239Gln polymorphism 
 (56Dutch 180 SB patients, 190 controls Case–control study No association 
  (103Mixed USA 259 SB cases, 359 controls Case–control study Modest increase in SB risk associated with 1 SNP (of eight tested) 
BHMT2 (betaine-homocysteine methyltransferase 2) One carbon metabolism (56Dutch 180 SB patients, 190 controls Association study No association detected 
  (103Mixed USA 259 SB cases, 359 controls Case–control study No association for 7 SNPs tested 
BRCA1 (breast cancer 1) NTDs in mouse mutant (114USA 268 SB patients and parents Family based association study (TDT) Association with SB for two microsatellite markers and A4956G SNP. Proposed polymorphisms affect level of lesion, not causative 
CAT (catalase) Oxidative stress (115Mixed USA 507 SB cases, 185 controls Case–control study No association 
CBS (cystathionine beta-synthase) Folate metabolism (116UK 207 NTD cases (200 mothers, 93 fathers). 601 controls, 542 control mothers Case–control study No association for 844ins68 
 (56Dutch 180 SB patients, 190 controls Case–control study No association 
 (103Mixed USA 259 SB cases, 359 controls Case–control (9 SNPs) Modest increase in SB risk associated with 2 SNPs 
CFL1 (n-cofilin) NTDs in mouse mutant (117Mixed USA 246 SB cases, 336 controls Case–control SNPs Mildly elevated risk of NTDs in non-Hispanic whites 
CHKA (choline kinase A) One carbon metabolism (118Mixed USA 103 SB cases, 338 controls Case–control study Possible association with reduced SB risk for 1 of 2 SNPs studied 
CITED2 NTDs in mouse mutant (119Mixed USA 64 SB cases, 72 controls Mutation screen and case–control No mutations. No association of three 5′-UTR SNPs with risk 
COQ3 (Coenzyme Q3 homolog, methyltransferase) Methylation (56Dutch 180 SB patients, 190 controls Case–control study No association 
CRABPI (cellular retinoic acid binding protein I) Retinol metabolism (104USA 230 SB cases, 318 SB families Mutation screen and family based association study No mutations. No association (3 SNPs tested) 
CRABPII (cellular retinoic acid binding protein II) Retinol metabolism (104USA 230 SB cases Mutation screen No mutations 
CTH (Cystathionase) One carbon metabolism (56Dutch 180 SB patients, 190 controls Case–control study No association 
CUBN (cubulin) Endocytosis (folate transport) (56Dutch 179 SB patients, 190 controls Case–control study GG genotype for rs1907362 significantly associated with reduced SB risk 
CYP26A1 (cytochrome P450) Retinol metabolism (104USA 230 SB cases Mutation screen No mutations 
CYP26B1 (cytochrome P450) Retinol metabolism (104USA 230 SB cases, 318 SB families Mutation screen and Family based association study No mutations. No association (5 SNPs tested) 
DHFR (Dihydrofolate reductase) Folate metabolism (120Mixed USA 61 SB cases and parents (multi-affected families) and 219 controls Case–control study of 19-bp intron-1 deletion The del/del genotype was more frequent in mothers of SB cases, compared with controls. No association in fathers or patients 
  (121Irish 283 cases (and 280 mothers, 279 fathers) and 256 controls. SB (95%) or encephalocele (5%) Case–control study. 19-bp deletion and two 3′-UTR variants. 19-bp Intron deletion shows protective effect. May increase mRNA levels 
  (122Dutch 109 patients, 121 mothers (SB). 234 paediatric controls, 292 control women Case–control study. 19-bp deletion and 9-bp repeat in 5′-UTR 19-bp Intron deletion not associated with NTDs. No effect on expression 
  (56Dutch 180 SB patients, 190 controls Case–control study No association 
  (103Mixed USA 259 SB cases, 359 controls Case–control study No association for 9 SNPs tested. Intron deletion not tested 
  (41Mixed UK 126 SB (open); 103 SB (closed); 49 anencephalic; 192 controls Case–control study No association 
FOLR1 (Folate receptor 1) Folate transport (56Dutch 180 SB patients, 190 controls Case–control study No association 
  (103Mixed USA 259 SB cases, 359 controls Case–control study No association for 3 SNPs tested 
FOLR2 (Folate receptor 2) Folate transport (56Dutch 180 SB patients, 190 controls Association study No association 
  (103Mixed USA 259 SB cases, 359 controls Case–control study No association for 3 SNPs tested 
FOLR3 (Folate receptor 3) Folate transport (56Dutch 180 SB patients, 190 controls Case–control study No association 
FPGS (Folylpolyglutamate synthase) Cellular folate retention (56Dutch 180 SB patients, 190 controls Case–control study No association 
FTCD (Formininotransferase cyclodeaminase One carbon metabolism (56Dutch 180 SB patients, 190 controls Case–control study No association 
GAMT (Guanidinoacetate N-methyl transferase) One carbon metabolism (56Dutch 180 SB patients, 190 controls Case–control study No association 
GAPD (glyceraldehyde 3 phosphate dehydrogenase) Glucose metabolism (115Mixed USA 507 SB cases, 185 controls Case–control study No association 
GART (Phosphoribosylglycinamide formyltransferase, phosphoribosyl glycinamide synthetase, phosphoribosyl aminoimidazole synthetase) Purine biosynthesis (one carbon metabolism) (56Dutch 180 SB patients, 190 controls Association study No association 
GCPII (glutamate carboxypeptidase), FOLH1 (Folate hydrolase) Folate metabolism (116UK 208 NTD cases (200 mothers, 92 fathers). 600 child, 531 mother controls Case–control study No association for 1561C>T 
 (56Dutch 180 SB patients, 190 controls Case–control study No association 
GGH (Gamma-glutamyl hydrolase) Folate metabolism (56Dutch 180 SB patients, 190 controls Case–control study No association 
GLUT1 (glucose transporter 1) Glucose metabolism (115Mixed USA 507 SB cases, 185 controls Case–control study Pro196 silent SNP associated with risk in TDT test 
GLUT4 (glucose transporter 4) Glucose metabolism (115Mixed USA 507 SB cases, 185 controls Case–control study No association 
HK1 (hexokinase 1) Glucose metabolism (115Mixed USA 507 SB cases, 185 controls Case–control study Lys481 SNP variant associated with risk in TDT test 
HK2 (hexokinase 2) Glucose metabolism (115Mixed USA 507 SB cases, 185 controls Case–control study No association 
ICMT (Isoprenylcysteine carboxyl methyltransferase) Protein methylation (56Dutch 180 SB patients, 190 controls Association study No association 
INS (insulin) Glucose metabolism (115Mixed USA 507 SB cases, 185 controls Case–control study No association 
INSR (insulin receptor) Glucose metabolism (115Mixed USA 507 SB cases, 185 controls Case–control study No association 
LEP (leptin) Glucose metabolism (115Mixed USA 507 SB cases, 185 controls Case–control study Arg109Lys variant associated with risk in TDT test 
LEPR (leptin receptor) Glucose metabolism (115Mixed USA 507 SB cases, 185 controls Case–control study Arg109Lys variant associated with risk in TDT test 
MAT1A (Methionine adenosyltransferase I, alpha) One carbon metabolism (56Dutch 180 SB patients, 190 controls Case–control study No association 
MAT2A (Methionine adenosyltransferase II, alpha) One carbon metabolism (56Dutch 180 SB patients, 190 controls Case–control study No association 
MGMT (O-6-Methylguanine DNA methyltransferase) One carbon metabolism DNA methylation (56Dutch 180 SB patients, 190 controls Case–control study No association 
MTHFD1 (methylenetetrahydrofolate dehydrogenase/methylenetetrahydrofolate-cyclohydrolase/formyltetrahydrofolate synthetase) Folate metabolism (50Irish 176 NTD cases (Mostly SB, few encephalocele). 245 mothers, 127 fathers (also includes parents of anencephalic cases). 770 controls Case–control to evaluate Arg653Gln (1958G>A; dbSNP rs 1801133) polymorphism Maternal AA genotype confers increased risk to offspring 
(51Italian 142 NTD cases (open and closed SB) (125 mothers, 108 fathers). 523 controls. Case–control study and family based association study (TDT) for 1958G>A Mildly increased risk for AA and GA genotypes in cases (and mothers). TDT shows excess transmission of A allele to cases 
(52Dutch 103 SB cases, 113 mothers, 98 fathers. 460 controls. Case–control and TDT for 1958G>A No association 
(54Irish 509 cases (with 485 mothers and 439 fathers)—mostly open SB. 966 controls. Case–control study for SNP rs1076991C>T Promoter variant not independent risk factor. Case and maternal risk factor when combined with Arg653Gln variant 
  (56Dutch 180 SB patients, 190 controls Case–control study No association 
  (103Mixed USA 259 SB cases, 359 controls Case–control (10 SNPs) Increased SB risk for one SNP, decreased risk for three others 
  (41Mixed UK 126 SB (open); 103 SB (closed); 49 anencephalic; 192 controls Case–control study No association 
MTHFD2 Folate metabolism (56Dutch 180 SB patients, 190 controls Case–control study No association 
  (103Mixed USA 259 SB cases, 359 controls Case–control study No association for 8 SNPs tested 
MTHFR (5,10-methylenetetrahydrofolate reductase) Folate metabolism (116UK 200 NTD cases (186 mothers, 92 fathers). 578 child, 512 mother controls Case–control study Mildly elevated risk for 677C>T. No association for 1298A>C 
  (123Polish 20 NTD cases, 262 controls. Case–control for C677T and A1298C No association 
  (124Irish 471 cases (451 SB, 20 encephalocele). Triads comprise >1300 samples. 922 controls. Mutation screen. Case–control and TDT for 116C>T (P39P) and 1793G>A (R594Q) Possible association with SB for variants tested. Unlikely to be independent risk factors (association likely due to linkage disequilibrium with 677C>T) 
  (125Mixed USA 350 cases, 328 mothers, 245 fathers, 167 siblings Family-based association study No association for 677C>T 
  (126Italian 15 cases (open SB), 18 fathers, 60 mothers. 43 control children and 100 control adults Case–control screen for C677T and A1298C T allele more frequent in cases than controls. A1298C not different between groups 
  (127Mexican 118 NTD case mothers (all anencephalic), 112 control mothers Case–control study for C677T and A1298C Maternal TT confers higher risk than CC for anencephaly. A1298C not associated with NTDs 
  (128Indian 83 mothers of NTD cases. 60 controls Case–control for C677T and A1298C 677T more frequent in mothers of cases than in controls (only for lower defects). No difference in A1298C frequency 
  (129Mexican (Yucatan) 108 cases (97 SB, 4 anencephalic, 7 encephalocele), with 147 parents. 120 controls Case–control, screen for A1298C No association 
  (130French 77 NTD mothers. 61 controls Case–control study No association for 677C>T. Reduced risk for 1298A>C allele 
  (131Chinese 38 mothers of NTD cases. 80 controls Case–control to evaluate 677C>T TT genotype less frequent in case mothers (but small numbers) 
  (56Dutch 180 SB patients, 190 controls Case–control study No association for 677C>T or 1298A>C 
  (103Mixed USA 259 SB cases, 359 controls Case–control (13 SNPs) Increased risk, OR2.0, of SB associated with 677C>T 
  (41Mixed UK 126 SB (open); 103 SB (closed); 49 anencephalic; 192 controls Case–control for C677T and A1298C Slight protective effect for open SB of 677TT genotype 
MTHFS (5,10-methylenetetrahydrofolate synthetase) Folate metabolism (56Dutch 180 SB patients, 190 controls Case–control study No association 
MTR (methionine synthase) One carbon metabolism (homocysteine remethylation) (105Hispanic USA 43 NTD cases, 122 mothers. 124 control infants and 127 mothers Case–control study for 2756A>G Not independent risk factor. May be associated with NTDs in combination with MTRR 66A>G 
 (130French 77 NTD mothers. 61 controls Case–control study No association for 66A>G 
 (56Dutch 180 SB patients, 190 controls Case–control study No association 
 (103Mixed USA 259 SB cases, 359 controls Case–control study No association with SB for 21 SNPs tested (including 2756A>G) 
  (41Mixed UK 126 SB (open); 103 SB (closed); 49 anencephalic; 192 controls Case–control study No association 
MTRR (methionine synthase reductase) One carbon metabolism (homocysteine remethylation) (105Hispanic USA 43 NTD cases, 122 mothers. 124 control infants and 127 mothers Case–control study for 66A>G (I22M) G allele associated with increased risk. Additional risk in combination with MTR 2756G allele 
 (116UK 201 NTD cases (203 mothers, 88 fathers). 601 child, 532 mother controls Case–control study Mildly reduced risk associated with 66A>G 
  (132Irish 575 NTD families 95% SB). 487 controls. Case–control and family-based analysis for three variants No association for 66A>G (I22M) with SB risk (except possible paternal effect). No association for S175L or K350R 
  (133Dutch 109 cases (open SB) and parents. 234 control children, 292 control women. Case–control screen for 66A>G In this study and meta-analysis (including previous studies) maternal GG is associated with increased risk in offspring, but GG in child not associated with NTDs 
  (130French 77 NTD mothers. 61 controls Case–control study Marginally increased risk associated with 66G allele 
  (56Dutch 180 SB patients, 190 controls Case–control study No association 
  (103Mixed USA 259 SB cases, 359 controls Case–control study Modest increase in SB risk for three linked SNPs, but not 66A>G 
  (41Mixed UK 126 SB (open); 103 SB (closed); 49 anencephalic; 192 controls Case–control study Marginal increased risk for combined NTDs 
MUT (methylmalonyl-CoA mutase) One carbon metabolism (134Irish 279 NTD triads (mostly SB), 256 controls Case–control and family-based study No association with NTDs for three variants tested 
NAT1 (N-acetyltransferase 1) Folate metabolism and acetylation reactions (135Mixed USA 354 NTD families Family based association study 1095C>A not associated with SB risk (except in combination with maternal smoking) 
 (136Mixed USA 374 NTD families Family based association study Composite genotype (6 SNPs) related to reduced function associated with lower SB risk for cases and mothers 
NAT2 (N-acetyltransferase 2)  (56Dutch 180 SB patients, 190 controls Case–control study No association 
NNMT (Nicotinamide N-methyltransferase) Methylation reactions (137Mixed USA 252 SB cases, 335 controls Case–control study No association between NNMT variants and SB risk 
  (56Dutch 180 SB patients, 190 controls Association study No association 
NCAM1 (neural cell adhesión molecule1) Embryonic cell adhesion (138USA 204 SB families Family based association study Risk of SB associated with intronic SNP (of 11 tested) in first 132 families, not in further 72 families 
NOS1 (nitric oxide synthase 1) Possible effect on one carbon metabolism (56Dutch 180 SB patients, 190 controls Case–control study No association for repeat polymorphisms 
NOS2 (nitric oxide synthase 2A)  (56Dutch 180 SB patients, 190 controls Case–control study No association for SNPs and repeat polymorphism 
NOS3 (nitric oxide synthase 3 endothelial)  (139Mixed US 301 families with SB Family based association study No association by TDT analysis. Maternal 894G>T associated with SB risk by log-linear modelling 
  (140Dutch 109 SB cases, 121 mothers, 103 fathers. 500 controls Case–control and TDT 894G>T not independent risk factor. Possible risk interaction with MTHFR 677TT 
  (56Dutch 180 SB patients, 190 controls Case–control study No association detected for SNPs and repeat polymorphism 
  (103Mixed USA 259 SB cases, 359 controls Case–control study No association 
hOGG1 (8-hydroxyguanine DNA-glycosylase1DNA repair (112Mixed USA 380 SB cases, 350 controls Case–control study No association 
PAX3 NTDs in mouse mutant (70Mixed USA 74 SB cases, 87 controls Mutation screen and case–control study rs16863657 and related haplotype associated with increased SB risk 
PCMT1 (l-isoaspartate O-methyltransferase) Methylation reactions (141Mixed USA 152 SB cases, 423 controls Case–control study of Ile120Val Val/Val genotype associated with possible reduction in SB risk 
  (56Dutch 180 SB patients, 190 controls Case–control study No association for Ile120Val 
PDGFRA (Platelet derived growth factor receptor alpha) NTDs in Patch mouse mutant (142US Hispanic 43 NTD cases, 122 NTD mothers. 124 control infants, 127 control mothers Case–control study for promoter haplotypes P1 promoter haplotypes with lower activity may be associated with maternal risk. Case numbers too small for conclusion 
 (143Mixed USA 407 parent-child triads (SB). 164 controls Case–control and family based association (TDT) No association for P1 promoter haplotypes 
  (108Dutch 88 SB cases, 56 SB mothers. 74 controls, 72 control mothers Evaluated H1 and H2 promoter haplotypes H1 promoter may be more frequent in cases than controls. Suggestion that BMI, glucose and inositol differentially interact with H1/H2 
PEMT (Phosphatidylethanolamine N-methyltransferase) One carbon and choline metabolism (144Mixed USA 360 SB cases, 595 controls Case–control study No association with SB for two non-synonymous SNPs 
PRKACA, PRKACB (cAMP-dependent protein kinase A catalytic subunits) NTDs in mouse mutants (145Mixed USA 207 SB cases, 209 controls Mutation screen and case–control study No mutation. No association 
PRMT1 (Protein arginine methyltransferase 1) Methylation reactions (56Dutch 180 SB patients, 190 controls Case–control study No association 
PRMT2 (Protein arginine methyltransferase 2) Methylation reactions (56Dutch 180 SB patients, 190 controls Case–control study No association 
PYCT1A (CTP:phosphocholine cytidyltransferase) One carbon metabolism (118Mixed USA 103 SB cases, 338 controls Case–control study Weak association with SB risk for 1 of 2 SNPs studied 
RNMT (RNA (guanine-7-) methyltransferase) Methylation reactions (56Dutch 180 SB patients, 190 controls Case–control study No association 
SARDH (Sarcosine dehydrogenase) One carbon metabolism (56Dutch 180 SB patients, 190 controls Case–control study Nominally significant association with two synonymous SNPsa 
SHMT (Serine hydroxyl methyltransferase I) One carbon metabolism (146UK 97 NTD mothers, 190 controls Case–control study 1420C>T associated with protective maternal effect 
  (56Dutch 180 SB patients, 190 controls Case–control study No association for Leu474Phe 
SLCA19A1, RFC-1 (reduced folate carrier), Folate transport (116UK 206 NTD cases (186 mothers, 92 fathers). 578 child, 512 mother controls Case–control study No association for 80G>A (H27R) 
  (125Mixed USA 350 cases, 328 mothers, 245 fathers, 167 siblings. Family-based association study No association for 80G>A 
  (147Irish 437 NTD families, 852 controls Case–control and family based association No association for 80G>A. 61-bp polymorphism under-represented in cases 
  (131Chinese 38 mothers of cases. 80 controls Case–control for 80G>A Association of GG genotype with risk of NTD offspring 
  (148Chinese 104 NTD families, 100 control families Case–control for 80G>A Elevated risk for GG genotype of cases or mother (if folic acid not used) 
  (56Dutch 180 SB patients, 190 controls Case–control study Nominally significant association for 80AA genotype and reduced riska 
  (103Mixed USA 259 SB cases, 359 controls Case–control study No association with risk of SB for 6 SNPs tested. 80G>A not tested 
  (41Mixed UK 126 SB (open); 103 SB (closed); 49 anencephalic; 192 controls Case–control study No association 
SOD2 (superoxide dismutase 2) Oxidative stress (115Mixed USA 507 SB cases, 185 controls Case–control study No association 
T (Brachyury) Axial development in mouse (149Mixed USA 316 SB families Family-based association study TIVS7 T/C allele more frequent in cases than expected 
TCNII (transcobalamin II) One carbon metabolism (150Irish ∼350 NTD families, ∼700 controls. Case–control and family based association study No association with SB risk for 6 SNPs 
  (130French 77 NTD mothers. 61 controls Case–control study No association for 776C>G 
  (56Dutch 180 SB patients, 190 controls Case–control study No association for Arg259Pro 
TXN2 (thioredoxin2) NTDs (SB) in mouse knockout (151Mixed USA 48 SB cases, 48 controls Case–control study A2 promoter allele associated with risk of NTDs but small sample size 
TP53 (p53) NTDs in mouse mutant (152Irish 549 NTD cases, 532 mothers, 481 fathers. 999 controls Case–control and family based association studies Two non-coding variants associated with case NTD risk and two variants with maternal risk (but no multiple testing correction) 
  (115Mixed USA 507 SB cases, 185 controls Case–control study No association 
TRDMT1 (tRNA aspartic acid methyltransferase 1) One carbon metabolism (56Dutch 180 SB patients, 190 controls Case–control study Nominally significant association for one SNPa 
TYMS (thymidylate synthase) Folate metabolism and pyrimidine biosynthesis (110Mixed USA 264 SB cases, 259 controls. Mutation screen and case–control for 28-bp repeat in 5′-UTR and 6-bp deletion in 3′-UTR 3′-UTR polymorphism associated with increased SB risk in non-Hispanic white population. Further increased risk with 5′-UTR polymorphism 
 (153UK 197 NTD cases, 194 mothers, 93 fathers. 179 control infants, 177 control mothers Case–control study of 28-bp repeat No association for TYMS 
  (56Dutch 180 SB patients, 190 controls Case–control study No association 
  (103Mixed USA 259 SB cases, 359 controls Case–control study Modest increase in SB risk for three linked SNPs (of 5 tested) 
UCP2 (uncoupling protein 2) Energy metabolism Previous study indicates association with NTDs (154Irish 169 cases, 163 mothers, 167 fathers Evaluated 866G/A, A55V, 3′-UTR 45-bp ins/del UCP2 not associated with NTD risk. Different frequency of risk allele in control population compared with previous study 
VANGL1 (van gogh-like 1) PCP gene homologue; Paralogue of VANGL2 (95Mixed UK and USA 66 (21 craniorachischisis, 24 SB, 21 anencephalic) and 200 controls Mutation screen No causative mutations. One missense variant, present in controls 
  (96Italian and French 144 (80 SB, 7 craniorachischisis, 22 closed spinal dysraphism, 35 caudal regression). 172 Italian and CEPH controls Mutation screen Three missense mutations (two in myelomeningocele, R274Q and M328T; 1 in caudal regression, V239I). V239I has functional effect on interaction with Dvl proteins 
  (97Italian and USA 673 cases (15 open cranial dysraphisms, 456 SB, 202 closed spinal dysraphisms) Mutation screen Ten missense variants in 13 individuals (absent in 1187–1462 controls). Five in highly conserved residues (two in myelomeningocele, three in closed spinal dysraphism/caudal regression syndrome) 
VANGL2 (van gogh-like 2) Mouse model: Craniorachischisis in loop-tail mice (95Mixed UK and USA 66 (21 craniorachischisis, 24 SB, 21 anencephalic) and 200 controls Mutation screen No causative mutations identified. 7 bp duplication in intron six in one craniorachischisis 
 (96Italian and French 144 (80 SB, 7 craniorachischisis, 22 closed spinal dysraphism, 35 caudal regression) and 172 Italian and CEPH controls Mutation screen No coding mutations 
XPD (DNA excision repair protein ERCC-2) DNA repair (112Mixed USA 380 SB cases, 350 controls Case–control study Mildly elevated risk associated with 751Gln 
XRCC1 (X-ray repair cross-complementing) DNA repair (112Mixed USA 380 SB cases, 350 controls Case–control study No association 
XRCC3 (X-ray repair cross-complementing) DNA repair (112Mixed USA 380 SB cases, 350 controls Case–control study No association 
ZIC1 Brain defects in mouse mutant (155Dutch 117 NTDs (SB, Anencephaly, Encephalocele), 364 controls Mutation screen No mutations 
ZIC2 NTDs in mouse mutant (155Dutch 117 NTDs (SB, Anencephaly, Encephalocele), 364 controls Mutation screen; case–control study Alanine deletion in one patient. Frequent polymorphism (1059C>T) has no association 
ZIC3 NTDs in mouse mutant (155Dutch 117 NTDs (SB, Anencephaly, Encephalocele), 364 controls Mutation screen One silent variant (858G>A) in one patient 

aNominally significant association which does not stand after correction for multiple testing. SB, defined as spina bifida (myelomeningocele) in study criteria. For studies labelled NTDs, populations were mixed (multiple types of NTDs) or undefined.

CANDIDATE GENES FROM FOLATE METABOLISM

Epidemiological studies provide an opportunity to identify risk factors for NTDs, such as dietary or teratogenic agents, to which susceptibility may be modified by genetic predisposition (12–14). Among environmental factors, folate status plays a key role in determining NTD risk (15,16). Maternal supplementation with folic acid during pregnancy reduces NTD frequency (17,18) whereas reduced serum folate and/or elevated homocysteine (an inverse indicator of folate status) are observed in some mothers of NTD fetuses, and are considered risk factors for NTDs (19–21). However, NTDs are not simply a condition of folate deficiency: maternal folate levels in most human NTD-affected pregnancies are in the ‘normal’ range (22), suggesting that low folate status may increase susceptibility but is not directly causative. Similarly, in mice dietary folate deficiency can cause significant embryonic growth retardation but does not cause NTDs (16,23,24). Hence, sub-optimal folate status may pre-dispose to NTDs in combination with additional factors, either environmental or genetic.

The intricate interplay and cross-regulation between elements of one-carbon (folate) metabolism (Fig. 1) complicates the teasing out of events that impinge on neural tube closure. In mice, key cellular functions in the developing embryo include methylation reactions and biosynthesis of nucleotides that support rapid cellular proliferation (2,25). Cranial NTDs arise when the methylation cycle is inhibited (26,27), and in null embryos for DNA methyltransferase 3B (28). In contrast, exogenous homocysteine does not cause NTDs (29–31), even in genetically predisposed splotch embryos (24) and may be an indicator of impaired folate or methylation cycle activity.

Figure 1.

Summary of folate one-carbon metabolism showing the main pathways and reactions that have been subject to analysis in the context of NTDs. Blue shading: proteins involved in processing of folates in the digestive tract, transport and cellular retention (by addition of glutamates). Yellow shading: the major part of the cycle involving transfer of 1C groups between folate molecules, as required for purine and pyrimidine biosynthesis. Pink shading: reactions of the methylation cycle. For clarity, mitochondrial reactions that include generation of formate and cleavage of glycine have been omitted. For explanation of abbreviations, see Table 1.

Figure 1.

Summary of folate one-carbon metabolism showing the main pathways and reactions that have been subject to analysis in the context of NTDs. Blue shading: proteins involved in processing of folates in the digestive tract, transport and cellular retention (by addition of glutamates). Yellow shading: the major part of the cycle involving transfer of 1C groups between folate molecules, as required for purine and pyrimidine biosynthesis. Pink shading: reactions of the methylation cycle. For clarity, mitochondrial reactions that include generation of formate and cleavage of glycine have been omitted. For explanation of abbreviations, see Table 1.

If folate status interacts with genetic factors in the causation of NTDs, this could involve either folate-related or folate-independent genes. To date, most emphasis has been placed on the evaluation of folate-related genes as NTD candidates (32,33) (Table 1). Further support comes from analysis of primary cell lines obtained from NTD fetuses, which indicates that a genetically-determined abnormality of folate metabolism is present, in at least a proportion of cases (34). However, identifying specific NTD-predisposing genetic lesions has proven far from straight forward. Although a number of variants have been widely studied, inconsistent results between different cohorts and populations (Table 1) indicate that very few, if any, have a major causative effect. Below, we sub-divide the candidate folate-related genes into three functional categories.

Methylation related genes

Among folate-related genes, 5,10-methylene tetrahydrofolate reductase (MTHFR) has been the principal focus of attention, following reports that the 677C>T (A222V; rs1801133) polymorphism is associated with increased risk of NTDs in Dutch and Irish populations (35–37). Other populations show no association (38,39) or even a protective effect (40,41) (Table 1). A meta-analysis, including genotype data from 27 studies up to 2004, suggests that the 677TT genotype confers an overall 1.9 times increase in NTD risk (Odds ratio: 1.9; 95% confidence interval: 1.6–2.2) (15). A more recent meta analysis (42) found a positive association only in non-latin groups, principally the Irish population.

The action of MTHFR generates 5-methylTHF for remethylation of homocysteine, at the expense of other folates required for purine and pyrimidine biosynthesis (Fig. 1). The A222V variant protein has reduced function and is associated with elevated plasma homocysteine (36). Nullizygosity for MTHFR in mice also results in elevated homocysteine and diminished DNA methylation (43), although NTDs are not observed under either normal or folate-deficient conditions. Moreover, MTHFR nullizygosity does not exacerbate the folate-responsive splotch mutation (43–45). These data suggest that in populations where MTHFR is a risk factor, additional interacting factors are likely to be present.

The link between reduced methylation/elevated homocysteine and NTDs has prompted analysis of variants in other genes that could influence the methylation cycle through remethylation (MTR, MTRR, BHMT and BHMT2) or transsulfuration (CBS) of homocysteine (11) (Table 1). In general, mildly elevated risks have been identified in some studies but rarely replicated. MTRR (methionine synthase reductase) functions to maintain activity of MTR (methionine synthase), and a variant form (I22M, encoded by 66A>G) was reported as an NTD case and maternal risk factor in some studies, but not others (Table 1). Mouse studies do not support a role for these genes in NTDs: targeted deletion of Mtr is embryonic lethal prior to neurulation stages and heterozygotes do not show NTDs (46). Similarly, reduced activity of Mtrr and loss of cbs function do not cause NTDs, although elevated plasma homocysteine is observed (47,48).

Folate cycle enzymes required for nucleotide biosynthesis

MTHFD1 encodes the cytoplasmic trifunctional C1THF synthase enzyme. A polymorphism (1958G>A; rs2236225) which results in an R653Q substitution in the 10-formylTHF synthetase domain was found to be both a maternal and NTD case risk factor, in the Irish and Italian populations (49–51), although not in the Dutch (51,52) or British (41). The R653Q polymorphism causes reduced C1THF synthase activity in cell lines, resulting in diminished purine biosynthesis (53). A promoter polymorphism (rs1076991C>T) in MTHFD1, that reduces transcriptional activity in vitro, was also associated with NTD case and maternal risk, in combination with R653Q (54).

Folate transport

Another attractive group of candidate genes are those encoding proteins required for transport, uptake and cellular retention of folates. This includes folate receptors FRα (Folr1 in mice), FRβ and FRγ, RFC1 (reduced folate carrier), GCPII (folyl-γ-glutamate carboxypeptidase) and FPGS (folylpolyglutamate synthetase) (32,33). Increased risks associated with variants in RFC1 and GCPII are not reproduced in all studies (Table 1), although the recently identified proton-coupled folate transporter PCFT (SLC46a1) is not required for embryonic survival or neural tube closure in mice (55), but has not yet been investigated in humans. A recent case–control study revealed a possible association with reduced risk of spina bifida for a polymorphism in CUBN (Cubulin), which encodes a membrane-associated multi-ligand endocytic receptor expressed in the neural folds and yolk sac (56). Together, cubulin and its partner protein megalin are involved in binding and endocytic uptake of a large number of different proteins, several of which could be important for neural tube closure, including the intrinsic factor-cobalamin complex (IF-B12) and folate binding protein (folate receptor) (57,58). Intriguingly, Cubn was one of the most up-regulated genes in a microarray analysis of Rfc1 null mouse embryos (59), which may reflect a compensatory mechanism to enhance endocytic folate uptake via Folr1. Hence, CUBN merits further attention as a potential risk factor, especially in conjunction with RFC1.

In view of the apparent resilience of mouse neurulation to specific genetic disturbance of the methylation cycle, analysis of compound mutants with other folate-related or NTD susceptibility alleles would be of considerable interest. In our analysis of NTD cell lines, impaired folate cycle activity did not correlate with known variants in MTHFR, MTHFD1, DHFR, GCPII, MTR, MTRR or RFC1 (34), encouraging the view that currently unknown genetic influences on folate metabolism remain to be identified in many NTD cases.

CANDIDATE GENES FROM THE MOUSE

The potential complexity of NTD genetics is illustrated by the fact that 200 or more different mouse genes result in NTD phenotypes either through naturally occurring, induced or targeted mutations (2,25). Many of the NTD-causing mouse mutations implicate specific signalling pathways such as non-canonical Wnt signalling (see below), maintenance of the cell cycle, regulation of the actin cytoskeleton, chromatin organization or epigenetic modifications including methylation and acetylation. Recently, NTDs were observed in mice null for Mib2 (60), Smurf1/2 (61) and Hectd1 (62), which all encode E3 ubiquitin ligases, suggesting a possible role in neurulation for protein ubiquitination and targeted degradation. The human homologues of some of these mouse NTD genes have been examined in case–control association studies or directly sequenced in mutation screens, although with very few significant findings to date (Table 1).

It is important to ask how appropriate are the mouse models as paradigms for human NTDs? At the embryonic level, the events of neurulation appear extremely similar between mice and humans. For example, the initial fusion event, Closure 1, occurs at a closely similar stage and body axial level in both species, as does initiation of closure in the forebrain (Closure 3) and completion of spinal closure at the posterior neuropore. One point of variation concerns de novo initiation of closure at the forebrain/midbrain boundary (Closure 2 in mice) which may be absent from human neurulation (63). Hence, brain closure could be a rather simpler process in humans than mice.

Another potential difference between mouse models and human NTDs is that many gene-specific homozygous null mouse embryos exhibit phenotypes additional to NTDs, such as prenatally lethal heart defects. Such syndromic examples do not appear particularly close models for human NTDs which are primarily non-syndromic (64). On the other hand, detailed analysis of a few of the mouse mutants suggests that isolated NTDs can also result from the effect of hypomorphic alleles, combinations of heterozygous mutations, genetic background effects and/or gene-environmental interactions. This partial loss of function or multifactorial aetiologies may more closely resemble human NTDs. For example, NTDs in splotch mice result from homozygosity for mutations in Pax3 (23,65) but can also occur, or be exacerbated, as a result of interaction with mutations in other genes including neurofibromin1 (66) and grainyhead-like-3 (67). Environmental factors including folate deficiency and arsenic can exacerbate NTDs in homozygous splotch mutants, or induce NTDs in the usually unaffected heterozygotes (24,68). Although association studies in humans have provided little evidence to implicate PAX3 mutations in human NTDs (69,70), the possible contribution of gene–gene and gene–environment interactions indicates that larger scale studies may be needed before a role for PAX3 in human NTDs can be completely ruled out.

The curly tail mouse also exhibits features typical of the multifactorial aetiology of human NTDs (71). Spinal NTDs are partially penetrant in homozygous ct/ct mutant embryos, with the frequency of defects strongly affected by genetic modifiers (72). The major ct gene is a hypomorphic allele of Grhl3, whose knockouts display completely penetrant spina bifida (73–75). The ct mutation appears to affect a regulatory region, emphasising the need for consideration of possible non-coding mutations in human NTDs. Moreover, there is a strong effect of environmental factors, including a protective effect of supplemental inositol (76). A key role for inositol in neural tube closure is supported by the finding that inositol deficiency in vitro causes NTDs (77), inositol may prevent diabetes-associated NTDs (78) and the recent finding of NTDs in embryos carrying a hypomorphic allele of inositol 1,3,4-trisphosphate 5/6-kinase (Itpk1), a key enzyme in inositol phosphate metabolism (79).

Planar cell polarity signalling and NTDs

A major advance in understanding the genetic basis of neurulation has been the finding that initiation of closure at the hindbrain-cervical boundary (Closure 1) requires non-canonical Wnt signalling: the so-called planar cell polarity (PCP) pathway (Fig. 2). PCP signalling was defined originally in Drosophila, as a genetic cascade involving the transmembrane receptor frizzled and the cytoplasmic protein dishevelled, but without a requirement for β-catenin (80–83). This pathway is required to specify planar polarity in epithelia including the wing and compound eye. In vertebrates, non-canonical Wnt signalling is highly conserved, underpinning tissue and cellular polarity during morphogenesis in systems as diverse as gastrulation and the coordinated orientation of stereociliary bundles in inner ear hair cells (84–90).

Figure 2.

Diagrammatic representation of non-canonical Wnt signalling in a mammalian cell. Black arrows indicate the signalling pathway necessary for establishment of planar cell polarity (PCP). Known biochemical interactions are indicated by blue arrows and genetic interactions are shown by red arrows. Ankdr6 is the mammalian homologue of Diego which in Drosophila interacts with Fz, Vang and Pk, but has not been studied in vertebrates.

Figure 2.

Diagrammatic representation of non-canonical Wnt signalling in a mammalian cell. Black arrows indicate the signalling pathway necessary for establishment of planar cell polarity (PCP). Known biochemical interactions are indicated by blue arrows and genetic interactions are shown by red arrows. Ankdr6 is the mammalian homologue of Diego which in Drosophila interacts with Fz, Vang and Pk, but has not been studied in vertebrates.

A potential role for PCP in NTDs first came to light following positional cloning of Vangl2 (the homologue of Drosophila strabismus/Van gogh) in the loop-tail mouse mutant which exhibits the severe NTD, craniorachischisis (91,92). Subsequently, the same NTD phenotype was found in other mouse mutants and targeted gene knockouts (Table 2) almost all of which have been implicated biochemically in the PCP pathway (e.g. Celsr1, Dvl) or which interact genetically with recognized PCP genes (e.g. Scrb, Ptk7) (93,94). Interestingly, the double knockout for Smurf1 and Smurf2 was recently found to display craniorachischisis and other characteristic PCP defects. These genes encode ubiquitin ligases whose targets include Prickle1 (Fig. 2), supporting the crucial nature of PCP signalling for initiation of neural tube closure (61).

Table 2.

Genes of the planar cell polarity pathway—involvement in mouse NTDs

Gene Mutant/genotype Protein NTD phenotype References 
Single gene effects 
Vangl2 Loop-tail (Lp, Lpm1Jus); Lp/Lp Transmembrane protein Craniorachischisis. Occasional spina bifida in Lp/+ (91,92,156,157
Scrb1 Circle-tail; Crc/Crc Cytoplasmic polarity protein Craniorachischisis (93
Celsr1 Crash; Crsh/Crsh Seven-pass transmembrane protein Craniorachischisis (158
Spin-cycle; Scy/Scy  Craniorachischisis 
Ptk7 (CCK-4Ptk7mu/mu (truncating gene-trap allele) Transmembrane receptor tyrosine kinase-like protein Craniorachischisis (94
Paralogous gene interactions 
Dv11/2 Dvl1−/−; Dvl2−/− Cytoplasmic signalling proteins Craniorachischisis (159
Dvl2/3 Dv12−/−; Dvl3+/− (Dv12−/−; 3−/− die before E8.5) Craniorachischisis (101
Fz3/6 Fz3−/−; Fz6−/− Transmembrane receptor protein Craniorachischisis (160
Smurf1/2 Smurf1−/−; Smurf2−/− Ubiquitin ligase No closure at E8.5 (61
Smurf1−/−; 2+/− or 1+/−;2−/− Spina bifida and/or exencephaly 
Interactions between different genes 
Vangl2/Scrb1 Lp/+; Crc/+ See above Craniorachischisis (102
Vangl2/Dvl3 Lp/+;Dvl3−/− See above Craniorachischisis (101
Lp/+;Dvl3+/− Craniorachishisis or exencephaly 
Vangl2/Vangl1 Lp/+; Vang1gt/+ Transmembrane protein Craniorachischisis (98
Vangl2/Ptk7 Lp/+; Ptk7+/mu See above Spina bifida (94
Vangl2/Wnt5a Lp/+; Wnt5a−/− Secreted signalling protein Craniorachischisis (161
Vangl2/Sfrp1,2,5 Lp/+; Sfrp1−/−; Sfrp2−/−; Sfrp5+/− Secreted Wnt antagonist Craniorachischsis (162
Lp/+; Sfrp1−/−; Sfrp2+/−; Sfrp5−/− Spina bifida 
Vangl2/Cthrc1 Lp/+; Cthrc1LacZ/LacZ Secreted glycoprotein, Wnt co-factor Exencephaly (100
Vangl2/Grhl3 Lp/+; Grhl3ct/ct Transcription factor Severe spina bifida (163
Vangl2/cobl Lp/+; coblC101/C101 Actin nucleator Exencephaly (99
Gene Mutant/genotype Protein NTD phenotype References 
Single gene effects 
Vangl2 Loop-tail (Lp, Lpm1Jus); Lp/Lp Transmembrane protein Craniorachischisis. Occasional spina bifida in Lp/+ (91,92,156,157
Scrb1 Circle-tail; Crc/Crc Cytoplasmic polarity protein Craniorachischisis (93
Celsr1 Crash; Crsh/Crsh Seven-pass transmembrane protein Craniorachischisis (158
Spin-cycle; Scy/Scy  Craniorachischisis 
Ptk7 (CCK-4Ptk7mu/mu (truncating gene-trap allele) Transmembrane receptor tyrosine kinase-like protein Craniorachischisis (94
Paralogous gene interactions 
Dv11/2 Dvl1−/−; Dvl2−/− Cytoplasmic signalling proteins Craniorachischisis (159
Dvl2/3 Dv12−/−; Dvl3+/− (Dv12−/−; 3−/− die before E8.5) Craniorachischisis (101
Fz3/6 Fz3−/−; Fz6−/− Transmembrane receptor protein Craniorachischisis (160
Smurf1/2 Smurf1−/−; Smurf2−/− Ubiquitin ligase No closure at E8.5 (61
Smurf1−/−; 2+/− or 1+/−;2−/− Spina bifida and/or exencephaly 
Interactions between different genes 
Vangl2/Scrb1 Lp/+; Crc/+ See above Craniorachischisis (102
Vangl2/Dvl3 Lp/+;Dvl3−/− See above Craniorachischisis (101
Lp/+;Dvl3+/− Craniorachishisis or exencephaly 
Vangl2/Vangl1 Lp/+; Vang1gt/+ Transmembrane protein Craniorachischisis (98
Vangl2/Ptk7 Lp/+; Ptk7+/mu See above Spina bifida (94
Vangl2/Wnt5a Lp/+; Wnt5a−/− Secreted signalling protein Craniorachischisis (161
Vangl2/Sfrp1,2,5 Lp/+; Sfrp1−/−; Sfrp2−/−; Sfrp5+/− Secreted Wnt antagonist Craniorachischsis (162
Lp/+; Sfrp1−/−; Sfrp2+/−; Sfrp5−/− Spina bifida 
Vangl2/Cthrc1 Lp/+; Cthrc1LacZ/LacZ Secreted glycoprotein, Wnt co-factor Exencephaly (100
Vangl2/Grhl3 Lp/+; Grhl3ct/ct Transcription factor Severe spina bifida (163
Vangl2/cobl Lp/+; coblC101/C101 Actin nucleator Exencephaly (99

In view of these findings in mice, PCP genes emerge as excellent candidates for causation of craniorachischisis in humans. Nevertheless, sequence analysis has so far failed to identify mutations in human VANGL2 or its paralogue VANGL1 in a group of patients with craniorachischisis (95,96). Reports of other PCP gene analysis in similar patients are awaited. Although craniorachischisis is the obvious NTD phenotype for study, the VANGL genes have also been analysed in patients with anencephaly and open and closed spina bifida. No mutations were reported in VANGL2 (95,96) but several highly conserved and unique, heterozygous missense variants were identified in VANGL1 in patients with either myelomeningocele or closed spina bifida, as well as caudal regression syndrome (96,97). To date a functional effect has been demonstrated for one of these putative mutations, where V239I (identified in caudal regression syndrome) results in loss of interaction between VANGL1 and DVL proteins (96,).

Interestingly, loss of Vangl1 function is insufficient to cause NTDs in mice, although compound heterozygotes with Vangl2 (loop-tail) develop craniorachischisis (98). Nevertheless, there is increasing evidence that PCP genes can contribute to NTDs other than craniorachischisis (Table 2). For example, double heterozygotes carrying both Vangl2 and Ptk7 develop spina bifida (94) although Vangl2 double mutants with cordon-bleuC101 or Cthrc1 develop exencephaly (99,100). In contrast, Vangl2:Scrb and Vangl2:Dvl3 double heterozygotes develop craniorachischisis (101,102). It remains to be determined why Vangl2 displays this variable phenotypic behaviour when combined with different PCP and other mutants. Hence, although non-canonical Wnt signalling has been firmly linked with Closure 1 in mice, it is possible that genes in this pathway play more diverse roles in human neural tube closure.

CONCLUSIONS

The identification of genetic risk factors for human NTDs is complicated by the multiplicity of genes participating in neurulation, and the importance of gene–environment interactions. Sequence analysis of candidate genes implicated from their role in mouse models has revealed putative mutations in a few genes, but each in only a small number of patients. Association studies of common polymorphic variants, particularly related to folate one-carbon metabolism, indicate risk factors such as MTHFR. However, no specific folate-related gene has yet been implicated as a major determinant of risk for NTDs. Large-scale studies will be required to provide sufficient statistical power to convincingly test whether such variants are truly NTD susceptibility factors (56,103). It will also be essential, to evaluate multiple genes (folate-related and others) in the same individuals in order to detect possible compounding effects of combinations of risk alleles that, individually, might not be statistically significant (11,39). To date, very few studies have been sufficiently large to overcome issues of multiple testing bias in screening for gene–gene interactions (39,56,104). Examination of specific hypotheses may be fruitful where fewer NTD cases are available, particularly if combined with stratified sample sets in which cases are sub-divided on the basis of phenotype. For example, NTDs with abnormal folate metabolism have enabled a combined analysis of MTR and MTRR (105), and fetuses with craniorachischisis provide a focus for determining the role of PCP genes. Gene–environment interactions appear likely to contribute to NTD predisposition, with examples including interactions of MTHFR with multivitamin use (106), MTRR with vitamin B12 (107) and PDGFRA with inositol and zinc (108).

One limitation of the association studies of multiple folate-related candidate genes in NTDs is the predominant focus on known polymorphisms. In future, it will be necessary also to consider the possible existence of ‘private’ disease-causing mutations. Moreover, the potential for deleterious gene expression changes resulting from promoter mutations or copy number variation has been addressed in relatively few studies (10,108–110). Emerging technologies for high throughput sequencing and analysis of genomic deletions and copy number variations (111) offer the prospect, in the coming years, of progress in identification of candidate genes and screening for novel mutations in human NTDs.

FUNDING

The authors’ NTD research is funded by the Medical Research Council, the Wellcome Trust and SPARKS. Funding to pay the Open Access publication charges for this article was provided by UCL using funds provided by Wellcome Trust.

ACKNOWLEDGEMENTS

The authors thank the NTD research community for many helpful discussions, and particularly Muriel Harris and Diana Juriloff for their insightful contributions.

Conflict of Interest statement. None declared.

REFERENCES

1
Detrait
E.R.
George
T.M.
Etchevers
H.C.
Gilbert
J.R.
Vekemans
M.
Speer
M.C.
Human neural tube defects: developmental biology, epidemiology, and genetics
Neurotoxicol. Teratol.
 , 
2005
, vol. 
27
 (pg. 
515
-
524
)
2
Harris
M.J.
Juriloff
D.M.
Mouse mutants with neural tube closure defects and their role in understanding human neural tube defects
Birth Defects Res. A Clin Mol. Teratol.
 , 
2007
, vol. 
79
 (pg. 
187
-
210
)
3
Kennedy
D.
Chitayat
D.
Winsor
E.J.T.
Silver
M.
Toi
A.
Prenatally diagnosed neural tube defects: ultrasound, chromosome, and autopsy or postnatal findings in 212 cases
Am. J. Med. Genet.
 , 
1998
, vol. 
77
 (pg. 
317
-
321
)
4
Chen
C.P.
Chromosomal abnormalities associated with neural tube defects (I): full aneuploidy
Taiwan J Obstet. Gynecol.
 , 
2007
, vol. 
46
 (pg. 
325
-
335
)
5
Chen
C.P.
Chromosomal abnormalities associated with neural tube defects (II): partial aneuploidy
Taiwan J Obstet. Gynecol.
 , 
2007
, vol. 
46
 (pg. 
336
-
351
)
6
Rampersaud
E.
Bassuk
A.G.
Enterline
D.S.
George
T.M.
Siegel
D.G.
Melvin
E.C.
Aben
J.
Allen
J.
Aylsworth
A.
Brei
T.
, et al.  . 
Whole genomewide linkage screen for neural tube defects reveals regions of interest on chromosomes 7 and 10
J. Med. Genet.
 , 
2005
, vol. 
42
 (pg. 
940
-
946
)
7
Stamm
D.S.
Rampersaud
E.
Slifer
S.H.
Mehltretter
L.
Siegel
D.G.
Xie
J.
Hu-Lince
D.
Craig
D.W.
Stephan
D.A.
George
T.M.
, et al.  . 
High-density single nucleotide polymorphism screen in a large multiplex neural tube defect family refines linkage to loci at 7p21.1-pter and 2q33.1–q35
Birth Defects Res. A Clin. Mol. Teratol.
 , 
2006
, vol. 
76
 (pg. 
499
-
505
)
8
Stamm
D.S.
Siegel
D.G.
Mehltretter
L.
Connelly
J.J.
Trott
A.
Ellis
N.
Zismann
V.
Stephan
D.A.
George
T.M.
Vekemans
M.
, et al.  . 
Refinement of 2q and 7p loci in a large multiplex NTD family
Birth Defects Res. A Clin. Mol. Teratol.
 , 
2008
, vol. 
82
 (pg. 
441
-
452
)
9
Goetzinger
K.R.
Stamilio
D.M.
Dicke
J.M.
Macones
G.A.
Odibo
A.O.
Evaluating the incidence and likelihood ratios for chromosomal abnormalities in fetuses with common central nervous system malformations
Am. J. Obstet. Gynecol.
 , 
2008
, vol. 
199
 (pg. 
285
-
286
)
10
Gustavsson
P.
Schoumans
J.
Staaf
J.
Borg
A.
Nordenskjold
M.
Anneren
G.
Duplication 16q12.1–q22.1 characterized by array CGH in a girl with spina bifida
Eur. J. Med. Genet.
 , 
2007
, vol. 
50
 (pg. 
237
-
241
)
11
Boyles
A.L.
Hammock
P.
Speer
M.C.
Candidate gene analysis in human neural tube defects
Am. J. Med. Genet. C Semin. Med. Genet.
 , 
2005
, vol. 
135
 (pg. 
9
-
23
)
12
Frey
L.
Hauser
W.A.
Epidemiology of neural tube defects
Epilepsia
 , 
2003
, vol. 
44
 (pg. 
4
-
13
)
13
Mitchell
L.E.
Epidemiology of neural tube defects
Am. J. Med. Genet.
 , 
2005
, vol. 
135C
 (pg. 
88
-
94
)
14
Carmichael
S.L.
Witte
J.S.
Shaw
G.M.
Nutrient pathways and neural tube defects: a semi-Bayesian hierarchical analysis
Epidemiology
 , 
2009
, vol. 
20
 (pg. 
67
-
73
)
15
Blom
H.J.
Shaw
G.M.
Den Heijer
M.
Finnell
R.H.
Neural tube defects and folate: case far from closed
Nat. Rev. Neurosci.
 , 
2006
, vol. 
7
 (pg. 
724
-
731
)
16
Beaudin
A.E.
Stover
P.J.
Folate-mediated one-carbon metabolism and neural tube defects: balancing genome synthesis and gene expression
Birth Defects Res. C Embryo. Today
 , 
2007
, vol. 
81
 (pg. 
183
-
203
)
17
Smithells
R.W.
Sheppard
S.
Schorah
C.J.
Seller
M.J.
Nevin
N.C.
Harris
R.
Read
A.P.
Fielding
D.W.
Possible prevention of neural-tube defects by periconceptional vitamin supplementation
Lancet
 , 
1980
, vol. 
1
 (pg. 
339
-
340
)
18
Wald
N.
Sneddon
J.
Densem
J.
Frost
C.
Stone
R.
MRC Vitamin Study Res Group
Prevention of neural tube defects: results of the medical research council vitamin study
Lancet
 , 
1991
, vol. 
338
 (pg. 
131
-
137
)
19
Kirke
P.N.
Molloy
A.M.
Daly
L.E.
Burke
H.
Weir
D.G.
Scott
J.M.
Maternal plasma folate and vitamin B12 are independent risk factors for neural tube defects
Q. J. Med.
 , 
1993
, vol. 
86
 (pg. 
703
-
708
)
20
Steegers-Theunissen
R.P.M.
Boers
G.H.J.
Trijbels
F.J.M.
Finkelstein
J.D.
Blom
H.J.
Thomas
C.M.G.
Borm
G.F.
Wouters
M.G.A.J.
Eskes
T.K.A.B.
Maternal hyperhomocysteinemia: a risk factor for neural tube defects
Metabolism
 , 
1994
, vol. 
43
 (pg. 
1475
-
1480
)
21
Mills
J.L.
McPartlin
J.M.
Kirke
P.N.
Lee
Y.J.
Conley
M.R.
Weir
D.G.
Scott
J.M.
Homocysteine metabolism in pregnancies complicated by neural- tube defects
Lancet
 , 
1995
, vol. 
345
 (pg. 
149
-
151
)
22
Scott
J.M.
Folate and vitamin B12
Proc. Nutr. Soc.
 , 
1999
, vol. 
58
 (pg. 
441
-
448
)
23
Greene
N.D.
Massa
V.
Copp
A.J.
Understanding the causes and prevention of neural tube defects: insights from the splotch mouse model
Birth Defects Res. A Clin. Mol. Teratol.
 , 
2009
, vol. 
85
 (pg. 
322
-
330
)
24
Burren
K.A.
Savery
D.
Massa
V.
Kok
R.M.
Scott
J.M.
Blom
H.J.
Copp
A.J.
Greene
N.D.E.
Gene–environment interactions in the causation of neural tube defects: folate deficiency increases susceptibility conferred by loss of Pax3 function
Hum. Mol. Genet.
 , 
2008
, vol. 
17
 (pg. 
3675
-
3685
)
25
Copp
A.J.
Greene
N.D.E.
Murdoch
J.N.
The genetic basis of mammalian neurulation
Nat. Rev. Genet.
 , 
2003
, vol. 
4
 (pg. 
784
-
793
)
26
Dunlevy
L.P.E.
Burren
K.A.
Chitty
L.S.
Copp
A.J.
Greene
N.D.E.
Excess methionine suppresses the methylation cycle and inhibits neural tube closure in mouse embryos
FEBS Lett.
 , 
2006
, vol. 
580
 (pg. 
2803
-
2807
)
27
Dunlevy
L.P.E.
Burren
K.A.
Mills
K.
Chitty
L.S.
Copp
A.J.
Greene
N.D.E.
Integrity of the methylation cycle is essential for mammalian neural tube closure
Birth Defects Res. (Part A)
 , 
2006
, vol. 
76
 (pg. 
544
-
552
)
28
Okano
M.
Bell
D.W.
Haber
D.A.
Li
E.
DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development
Cell
 , 
1999
, vol. 
99
 (pg. 
247
-
257
)
29
VanAerts
L.A.G.J.M.
Blom
H.J.
Deabreu
R.A.
Trijbels
F.J.M.
Eskes
T.K.A.B.
Peereboom-Stegeman
J.H.J.C.
Noordhoek
J.
Prevention of neural tube defects by and toxicity of L-homocysteine in cultured postimplantation rat embryos
Teratology
 , 
1994
, vol. 
50
 (pg. 
348
-
360
)
30
Greene
N.D.E.
Dunlevy
L.E.
Copp
A.J.
Homocysteine is embryotoxic but does not cause neural tube defects in mouse embryos
Anat. Embryol.
 , 
2003
, vol. 
206
 (pg. 
185
-
191
)
31
Bennett
G.D.
Vanwaes
J.
Moser
K.
Chaudoin
T.
Starr
L.
Rosenquist
T.H.
Failure of homocysteine to induce neural tube defects in a mouse model
Birth Defects Res. B Dev. Reprod. Toxicol.
 , 
2006
, vol. 
77
 (pg. 
89
-
94
)
32
Molloy
A.M.
Brody
L.C.
Mills
J.L.
Scott
J.M.
Kirke
P.N.
The search for genetic polymorphisms in the homocysteine/folate pathway that contribute to the etiology of human neural tube defects
Birth Defects Res. A Clin. Mol. Teratol.
 , 
2009
, vol. 
85
 (pg. 
285
-
294
)
33
Beaudin
A.E.
Stover
P.J.
Insights into metabolic mechanisms underlying folate-responsive neural tube defects: a minireview
Birth Defects Res. A Clin Mol. Teratol
 , 
2009
, vol. 
85
 (pg. 
274
-
284
)
34
Dunlevy
L.P.E.
Chitty
L.S.
Doudney
K.
Burren
K.A.
Stojilkovic-Mikic
T.
Stanier
P.
Scott
R.
Copp
A.J.
Greene
N.D.E.
Abnormal folate metabolism in foetuses affected by neural tube defects
Brain
 , 
2007
, vol. 
130
 (pg. 
1043
-
1049
)
35
Frosst
P.
Blom
H.J.
Milos
R.
Goyette
P.
Sheppard
C.A.
Matthews
R.G.
Boers
G.J.H.
Den Heijer
M.
Kluijtmans
L.A.J.
Van den Heuvel
L.P.
, et al.  . 
A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase
Nat. Genet.
 , 
1995
, vol. 
10
 (pg. 
111
-
113
)
36
Van der Put
N.M.J.
Steegers-Theunissen
R.P.M.
Frosst
P.
Trijbels
F.J.M.
Eskes
T.K.A.B.
Van den Heuvel
L.P.
Mariman
E.C.M.
Den Heyer
M.
Rozen
R.
Blom
H.J.
Mutated methylenetetrahydrofolate reductase as a risk factor for spina bifida
Lancet
 , 
1995
, vol. 
346
 (pg. 
1070
-
1071
)
37
Shields
D.C.
Kirke
P.N.
Mills
J.L.
Ramsbottom
D.
Molloy
A.M.
Burke
H.
Weir
D.G.
Scott
J.M.
Whitehead
A.S.
The ‘thermolabile’ variant of methylenetetrahydrofolate reductase and neural tube defects: an evaluation of genetic risk and the relative importance of the genotypes of the embryo and the mother
Am. J. Hum. Genet.
 , 
1999
, vol. 
64
 (pg. 
1045
-
1055
)
38
Stegmann
K.
Ziegler
A.
Ngo
E.T.K.M.
Kohlschmidt
N.
Schröter
B.
Ermert
A.
Koch
M.C.
Linkage disequilibrium of MTHFR genotypes 677C/T-1298A/C in the german population and association studies in probands with neural tube defects(NTD)
Am. J. Med. Genet.
 , 
1999
, vol. 
87
 (pg. 
23
-
29
)
39
Boyles
A.L.
Billups
A.V.
Deak
K.L.
Siegel
D.G.
Mehltretter
L.
Slifer
S.H.
Bassuk
A.G.
Kessler
J.A.
Reed
M.C.
Nijhout
H.F.
, et al.  . 
Neural tube defects and folate pathway genes: family-based association tests of gene–gene and gene–environment interactions
Environ. Health Perspect.
 , 
2006
, vol. 
114
 (pg. 
1547
-
1552
)
40
De Marco
P.
Calevo
M.G.
Moroni
A.
Arata
L.
Merello
E.
Finnell
R.H.
Zhu
H.
Andreussi
L.
Cama
A.
Capra
V.
Study of MTHFR and MS polymorphisms as risk factors for NTD in the Italian population
J. Hum. Genet.
 , 
2002
, vol. 
47
 (pg. 
319
-
324
)
41
Doudney
K.
Grinham
J.
Whittaker
J.
Lynch
S.A.
Thompson
D.
Moore
G.E.
Copp
A.J.
Greene
N.D.E.
Stanier
P.
Evaluation of folate metabolism gene polymorphisms as risk factors for open and closed neural tube defects
Am. J. Med. Genet.
 , 
2009
, vol. 
149A
 (pg. 
1585
-
1589
)
42
Amorim
M.R.
Lima
M.A.
Castilla
E.E.
Orioli
I.M.
Non-Latin European descent could be a requirement for association of NTDs and MTHFR variant 677C>T: a meta-analysis
Am. J. Med. Genet. A
 , 
2007
, vol. 
143A
 (pg. 
1726
-
1732
)
43
Chen
Z.T.
Karaplis
A.C.
Ackerman
S.L.
Pogribny
I.P.
Melnyk
S.
Lussier-Cacan
S.
Chen
M.F.
Pai
A.
John
S.W.M.
Smith
R.S.
, et al.  . 
Mice deficient in methylenetetrahydrofolate reductase exhibit hyperhomocysteinemia and decreased methylation capacity, with neuropathology and aortic lipid deposition
Hum. Mol. Genet.
 , 
2001
, vol. 
10
 (pg. 
433
-
443
)
44
Li
D.
Pickell
L.
Liu
Y.
Wu
Q.
Cohn
J.S.
Rozen
R.
Maternal methylenetetrahydrofolate reductase deficiency and low dietary folate lead to adverse reproductive outcomes and congenital heart defects in mice
Am. J. Clin. Nutr.
 , 
2005
, vol. 
82
 (pg. 
188
-
195
)
45
Li
D.
Pickell
L.
Liu
Y.
Rozen
R.
Impact of methylenetetrahydrofolate reductase deficiency and low dietary folate on the development of neural tube defects in splotch mice
Birth Defects Res. A Clin. Mol. Teratol.
 , 
2006
, vol. 
76
 (pg. 
55
-
59
)
46
Swanson
D.A.
Liu
M.L.
Baker
P.J.
Garrett
L.
Stitzel
M.
Wu
J.M.
Harris
M.
Banerjee
R.
Shane
B.
Brody
L.C.
Targeted disruption of the methionine synthase gene in mice
Mol. Cell. Biol.
 , 
2001
, vol. 
21
 (pg. 
1058
-
1065
)
47
Elmore
C.L.
Wu
X.
Leclerc
D.
Watson
E.D.
Bottiglieri
T.
Krupenko
N.I.
Krupenko
S.A.
Cross
J.C.
Rozen
R.
Gravel
R.A.
, et al.  . 
Metabolic derangement of methionine and folate metabolism in mice deficient in methionine synthase reductase
Mol. Genet. Metab.
 , 
2007
, vol. 
91
 (pg. 
85
-
97
)
48
Watanabe
M.
Osada
J.
Aratani
Y.
Kluckman
K.
Reddick
R.
Malinow
M.R.
Maeda
N.
Mice deficient in cystathionine β-synthase: Animal models for mild and severe homocyst(e)inemia
Proc. Natl. Acad. Sci. U.S.A.
 , 
1995
, vol. 
92
 (pg. 
1585
-
1589
)
49
Brody
L.C.
Conley
M.
Cox
C.
Kirke
P.N.
McKeever
M.P.
Mills
J.L.
Molloy
A.M.
O’Leary
V.B.
Parle-McDermott
A.
Scott
J.M.
, et al.  . 
A polymorphism, R653Q, in the trifunctional enzyme methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase/formyltetrahydrofolate synthetase is a maternal genetic risk factor for neural tube defects: report of the Birth Defects Research Group
Am. J. Hum. Genet.
 , 
2002
, vol. 
71
 (pg. 
1207
-
1215
)
50
Parle-McDermott
A.
Kirke
P.N.
Mills
J.L.
Molloy
A.M.
Cox
C.
O’Leary
V.B.
Pangilinan
F.
Conley
M.
Cleary
L.
Brody
L.C.
, et al.  . 
Confirmation of the R653Q polymorphism of the trifunctional C1-synthase enzyme as a maternal risk for neural tube defects in the Irish population
Eur. J. Hum. Genet.
 , 
2006
, vol. 
14
 (pg. 
768
-
772
)
51
De Marco
P.
Merello
E.
Calevo
M.G.
Mascelli
S.
Raso
A.
Cama
A.
Capra
V.
Evaluation of a methylenetetrahydrofolate-dehydrogenase 1958G>A polymorphism for neural tube defect risk
J. Hum. Genet.
 , 
2006
, vol. 
51
 (pg. 
98
-
103
)
52
Van der Linden
I.
Heil
S.G.
Kouwenberg
I.C.
den
H.M.
Blom
H.J.
The methylenetetrahydrofolate dehydrogenase (MTHFD1) 1958G>A variant is not associated with spina bifida risk in the Dutch population
Clin. Genet.
 , 
2007
, vol. 
72
 (pg. 
599
-
600
)
53
Christensen
K.E.
Rohlicek
C.V.
Andelfinger
G.U.
Michaud
J.
Bigras
J.L.
Richter
A.
Mackenzie
R.E.
Rozen
R.
The MTHFD1 p.Arg653Gln variant alters enzyme function and increases risk for congenital heart defects
Hum. Mutat.
 , 
2009
, vol. 
30
 (pg. 
212
-
220
)
54
Carroll
N.
Pangilinan
F.
Molloy
A.M.
Troendle
J.
Mills
J.L.
Kirke
P.N.
Brody
L.C.
Scott
J.M.
Parle-McDermott
A.
Analysis of the MTHFD1 promoter and risk of neural tube defects
Hum. Genet.
 , 
2009
, vol. 
125
 (pg. 
247
-
256
)
55
Jakubowski
H.
Perla-Kajan
J.
Finnell
R.H.
Cabrera
R.M.
Wang
H.
Gupta
S.
Kruger
W.D.
Kraus
J.P.
Shih
D.M.
Genetic or nutritional disorders in homocysteine or folate metabolism increase protein N-homocysteinylation in mice
FASEB J.
 , 
2009
, vol. 
23
 (pg. 
1721
-
1727
)
56
Franke
B.
Vermeulen
S.H.
Steegers-Theunissen
R.P.
Coenen
M.J.
Schijvenaars
M.M.
Scheffer
H.
den
H.M.
Blom
H.J.
An association study of 45 folate-related genes in spina bifida: Involvement of cubilin (CUBN) and tRNA aspartic acid methyltransferase 1 (TRDMT1)
Birth Defects Res. A Clin. Mol. Teratol.
 , 
2009
, vol. 
85
 (pg. 
216
-
226
)
57
Birn
H.
Zhai
X.
Holm
J.
Hansen
S.I.
Jacobsen
C.
Christensen
E.I.
Moestrup
S.K.
Megalin binds and mediates cellular internalization of folate binding protein
FEBS J.
 , 
2005
, vol. 
272
 (pg. 
4423
-
4430
)
58
Christensen
E.I.
Birn
H.
Megalin and cubilin: multifunctional endocytic receptors
Nat. Rev. Mol. Cell Biol.
 , 
2002
, vol. 
3
 (pg. 
256
-
266
)
59
Gelineau-van Waes
J.
Maddox
J.R.
Smith
L.M.
van
W.M.
Wilberding
J.
Eudy
J.D.
Bauer
L.K.
Finnell
R.H.
Microarray analysis of E9.5 reduced folate carrier (RFC1; Slc19a1) knockout embryos reveals altered expression of genes in the cubilin–megalin multiligand endocytic receptor complex
BMC Genomics
 , 
2008
, vol. 
9
 pg. 
156
 
60
Wu
J.I.
Rajendra
R.
Barsi
J.C.
Durfee
L.
Benito
E.
Gao
G.
Kuruvilla
M.
Hrdlickova
R.
Liss
A.S.
Artzt
K.
Targeted disruption of Mib2 causes exencephaly with a variable penetrance
Genesis
 , 
2007
, vol. 
45
 (pg. 
722
-
727
)
61
Narimatsu
M.
Bose
R.
Pye
M.
Zhang
L.
Miller
B.
Ching
P.
Sakuma
R.
Luga
V.
Roncari
L.
Attisano
L.
, et al.  . 
Regulation of planar cell polarity by Smurf ubiquitin ligases
Cell
 , 
2009
, vol. 
137
 (pg. 
295
-
307
)
62
Zohn
I.E.
Anderson
K.V.
Niswander
L.
The Hectd1 ubiquitin ligase is required for development of the head mesenchyme and neural tube closure
Dev. Biol.
 , 
2007
, vol. 
306
 (pg. 
208
-
221
)
63
Greene
N.D.
Copp
A.J.
Development of the vertebrate central nervous system: formation of the neural tube
Prenat. Diagn.
 , 
2009
, vol. 
29
 (pg. 
303
-
311
)
64
Harris
M.J.
Why are the genes that cause risk of human neural tube defects so hard to find?
Teratology
 , 
2001
, vol. 
63
 (pg. 
165
-
166
)
65
Epstein
D.J.
Vekemans
M.
Gros
P.
Splotch (Sp2H), a mutation affecting development of the mouse neural tube, shows a deletion within the paired homeodomain of Pax-3
Cell
 , 
1991
, vol. 
67
 (pg. 
767
-
774
)
66
Lakkis
M.M.
Golden
J.A.
O'Shea
K.S.
Epstein
J.A.
Neurofibromin deficiency in mice causes exencephaly and is a modifier for Splotch neural tube defects
Dev. Biol.
 , 
1999
, vol. 
212
 (pg. 
80
-
92
)
67
Estibeiro
J.P.
Brook
F.A.
Copp
A.J.
Interaction between splotch (Sp) and curly tail (ct) mouse mutants in the embryonic development of neural tube defects
Development
 , 
1993
, vol. 
119
 (pg. 
113
-
121
)
68
Martin
L.J.
Machado
A.F.
Loza
M.A.
Mao
G.E.
Lee
G.S.
Hovland
D.N.
Jr
Cantor
R.M.
Collins
M.D.
Effect of arsenite, maternal age, and embryonic sex on spina bifida, exencephaly, and resorption rates in the splotch mouse
Birth Defects Res. (Part A)
 , 
2003
, vol. 
67
 (pg. 
231
-
239
)
69
Trembath
D.
Sherbondy
A.L.
Vandyke
D.C.
Shaw
G.M.
Todoroff
K.
Lammer
E.J.
Finnell
R.H.
Marker
S.
Lerner
G.
Murray
J.C.
Analysis of select folate pathway genes, PAX3, and human T in a Midwestern neural tube defect population
Teratology
 , 
1999
, vol. 
59
 (pg. 
331
-
341
)
70
Lu
W.
Zhu
H.
Wen
S.
Laurent
C.
Shaw
G.M.
Lammer
E.J.
Finnell
R.H.
Screening for novel PAX3 polymorphisms and risks of spina bifida
Birth Defects Res. A Clin. Mol. Teratol.
 , 
2007
, vol. 
79
 (pg. 
45
-
49
)
71
Van Straaten
H.W.M.
Copp
A.J.
Curly tail: a 50-year history of the mouse spina bifida model
Anat. Embryol.
 , 
2001
, vol. 
203
 (pg. 
225
-
237
)
72
Neumann
P.E.
Frankel
W.N.
Letts
V.A.
Coffin
J.M.
Copp
A.J.
Bernfield
M.
Multifactorial inheritance of neural tube defects: localization of the major gene and recognition of modifiers in ct mutant mice
Nat. Genet.
 , 
1994
, vol. 
6
 (pg. 
357
-
362
)
73
Ting
S.B.
Wilanowski
T.
Auden
A.
Hall
M.
Voss
A.K.
Thomas
T.
Parekh
V.
Cunningham
J.M.
Jane
S.M.
Inositol- and folate-resistant neural tube defects in mice lacking the epithelial-specific factor Grhl-3
Nat. Med.
 , 
2003
, vol. 
9
 (pg. 
1513
-
1519
)
74
Gustavsson
P.
Greene
N.D.
Lad
D.
Pauws
E.
de Castro
S.C.
Stanier
P.
Copp
A.J.
Increased expression of Grainyhead-like-3 rescues spina bifida in a folate-resistant mouse model
Hum. Mol. Genet.
 , 
2007
, vol. 
16
 (pg. 
2640
-
2646
)
75
Gustavsson
P.
Copp
A.J.
Greene
N.D.
Grainyhead genes and mammalian neural tube closure
Birth Defects Res. A Clin Mol. Teratol.
 , 
2008
, vol. 
82
 (pg. 
728
-
735
)
76
Greene
N.D.E.
Copp
A.J.
Inositol prevents folate-resistant neural tube defects in the mouse
Nat. Med.
 , 
1997
, vol. 
3
 (pg. 
60
-
66
)
77
Cockroft
D.L.
Brook
F.A.
Copp
A.J.
Inositol deficiency increases the susceptibility to neural tube defects of genetically predisposed (curly tail) mouse embryos in vitro
Teratology
 , 
1992
, vol. 
45
 (pg. 
223
-
232
)
78
Khandelwal
M.
Reece
E.A.
Wu
Y.K.
Borenstein
M.
Dietary myo-inositol therapy in hyperglycemia-induced embryopathy
Teratology
 , 
1998
, vol. 
57
 (pg. 
79
-
84
)
79
Wilson
M.P.
Hugge
C.
Bielinska
M.
Nicholas
P.
Majerus
P.W.
Wilson
D.B.
Neural tube defects in mice with reduced levels of inositol 1,3,4-trisphosphate 5/6-kinase
Proc. Natl. Acad. Sci. U. S. A
 , 
2009
, vol. 
106
 (pg. 
9831
-
9835
)
80
Krasnow
R.E.
Wong
L.L.
Adler
P.N.
Dishevelled is a component of the frizzled signaling pathway in Drosophila
Development
 , 
1995
, vol. 
121
 (pg. 
4095
-
4102
)
81
Strutt
D.I.
Weber
U.
Mlodzik
M.
The role of RhoA in tissue polarity and Frizzled signalling
Nature
 , 
1997
, vol. 
387
 (pg. 
292
-
295
)
82
McEwen
D.G.
Peifer
M.
Wnt signaling: Moving in a new direction
Curr. Biol.
 , 
2000
, vol. 
10
 (pg. 
R562
-
R564
)
83
Strutt
D.
The planar polarity pathway
Curr. Biol.
 , 
2008
, vol. 
18
 (pg. 
R898
-
R902
)
84
Wallingford
J.B.
Rowning
B.A.
Vogeli
K.M.
Rothbächer
U.
Fraser
S.E.
Harland
R.M.
Dishevelled controls cell polarity during Xenopus gastrulation
Nature
 , 
2000
, vol. 
405
 (pg. 
81
-
85
)
85
Park
M.
Moon
R.T.
The planar cell-polarity gene stbm regulates cell behaviour and cell fate in vertebrate embryos
Nat. Cell Biol.
 , 
2002
, vol. 
4
 (pg. 
20
-
25
)
86
Jessen
J.R.
Topczewski
J.
Bingham
S.
Sepich
D.S.
Marlow
F.
Chandrasekhar
A.
Solnica-Krezel
L.
Zebrafish trilobite identifies new roles for Strabismus in gastrulation and neuronal movements
Nat. Cell Biol.
 , 
2002
, vol. 
4
 (pg. 
610
-
615
)
87
Montcouquiol
M.
Rachel
R.A.
Lanford
P.J.
Copeland
N.G.
Jenkins
N.A.
Kelley
M.W.
Identification of Vangl2 and Scrb1 as planar polarity genes in mammals
Nature
 , 
2003
, vol. 
423
 (pg. 
173
-
177
)
88
Henderson
D.J.
Conway
S.J.
Greene
N.D.E.
Gerrelli
D.
Murdoch
J.N.
Anderson
R.H.
Copp
A.J.
Cardiovascular defects associated with abnormalities in midline development in the loop-tail mouse mutant
Circ. Res.
 , 
2001
, vol. 
89
 (pg. 
6
-
12
)
89
Phillips
H.M.
Rhee
H.J.
Murdoch
J.N.
Hildreth
V.
Peat
J.D.
Anderson
R.H.
Copp
A.J.
Chaudhry
B.
Henderson
D.J.
Disruption of planar cell polarity signaling results in congenital heart defects and cardiomyopathy attributable to early cardiomyocyte disorganization
Circ. Res.
 , 
2007
, vol. 
101
 (pg. 
137
-
145
)
90
Vandenberg
A.L.
Sassoon
D.A.
Non-canonical Wnt signaling regulates cell polarity in female reproductive tract development via van gogh-like 2
Development
 , 
2009
, vol. 
136
 (pg. 
1559
-
1570
)
91
Kibar
Z.
Vogan
K.J.
Groulx
N.
Justice
M.J.
Underhill
D.A.
Gros
P.
Ltap, a mammalian homolog of Drosophila Strabismus/Van Gogh, is altered in the mouse neural tube mutant Loop-tail
Nat. Genet.
 , 
2001
, vol. 
28
 (pg. 
251
-
255
)
92
Murdoch
J.N.
Doudney
K.
Paternotte
C.
Copp
A.J.
Stanier
P.
Severe neural tube defects in the loop-tail mouse result from mutation of Lpp1, a novel gene involved in floor plate specification
Hum. Mol. Genet.
 , 
2001
, vol. 
10
 (pg. 
2593
-
2601
)
93
Murdoch
J.N.
Henderson
D.J.
Doudney
K.
Gaston-Massuet
C.
Phillips
H.M.
Paternotte
C.
Arkell
R.
Stanier
P.
Copp
A.J.
Disruption of scribble (Scrb1) causes severe neural tube defects in the circletail mouse
Hum. Mol. Genet.
 , 
2003
, vol. 
12
 (pg. 
87
-
98
)
94
Lu
X.
Borchers
A.G.
Jolicoeur
C.
Rayburn
H.
Baker
J.C.
Tessier-Lavigne
M.
PTK7/CCK-4 is a novel regulator of planar cell polarity in vertebrates
Nature
 , 
2004
, vol. 
430
 (pg. 
93
-
98
)
95
Doudney
K.
Ybot-Gonzalez
P.
Paternotte
C.
Stevenson
R.E.
Greene
N.D.
Moore
G.E.
Copp
A.J.
Stanier
P.
Analysis of the planar cell polarity gene Vangl2 and its co-expressed paralogue Vangl1 in neural tube defect patients
Am. J. Med. Genet. A
 , 
2005
, vol. 
136A
 (pg. 
90
-
92
)
96
Kibar
Z.
Torban
E.
McDearmid
J.R.
Reynolds
A.
Berghout
J.
Mathieu
M.
Kirillova
I.
De Marco
P.
Merello
E.
Hayes
J.M.
, et al.  . 
Mutations in VANGL1 associated with neural-tube defects
N. Engl. J Med.
 , 
2007
, vol. 
356
 (pg. 
1432
-
1437
)
97
Kibar
Z.
Bosoi
C.M.
Kooistra
M.
Salem
S.
Finnell
R.H.
De
M.P.
Merello
E.
Bassuk
A.G.
Capra
V.
Gros
P.
Novel mutations in VANGL1 in neural tube defects
Hum. Mutat.
 , 
2009
, vol. 
30
 (pg. 
E706
-
E715
)
98
Torban
E.
Patenaude
A.M.
Leclerc
S.
Rakowiecki
S.
Gauthier
S.
Andelfinger
G.
Epstein
D.J.
Gros
P.
Genetic interaction between members of the Vangl family causes neural tube defects in mice
Proc. Natl. Acad. Sci. U. S. A.
 , 
2008
, vol. 
105
 (pg. 
3449
-
3454
)
99
Carroll
E.
Gerrelli
D.
Gasca
S.
Berg
E.
Beier
D.
Copp
A.
Klingensmith
J.
Cordon-bleu is a conserved gene involved in neural tube formation
Dev. Biol.
 , 
2003
, vol. 
262
 (pg. 
16
-
31
)
100
Yamamoto
S.
Nishimura
O.
Misaki
K.
Nishita
M.
Minami
Y.
Yonemura
S.
Tarui
H.
Sasaki
H.
Cthrc1 selectively activates the planar cell polarity pathway of Wnt signaling by stabilizing the Wnt-receptor complex
Dev. Cell
 , 
2008
, vol. 
15
 (pg. 
23
-
36
)
101
Etheridge
S.L.
Ray
S.
Li
S.
Hamblet
N.S.
Lijam
N.
Tsang
M.
Greer
J.
Kardos
N.
Wang
J.
Sussman
D.J.
, et al.  . 
Murine dishevelled 3 functions in redundant pathways with dishevelled 1 and 2 in normal cardiac outflow tract, cochlea, and neural tube development
PLoS Genet.
 , 
2008
, vol. 
4
 pg. 
e1000259
 
102
Murdoch
J.N.
Rachel
R.A.
Shah
S.
Beermann
F.
Stanier
P.
Mason
C.A.
Copp
A.J.
Circletail, a new mouse mutant with severe neural tube defects: Chromosomal localisation and interaction with the loop-tail mutation
Genomics
 , 
2001
, vol. 
78
 (pg. 
55
-
63
)
103
Shaw
G.M.
Lu
W.
Zhu
H.
Yang
W.
Briggs
F.B.
Carmichael
S.L.
Barcellos
L.F.
Lammer
E.J.
Finnell
R.H.
118 SNPs of folate-related genes and risks of spina bifida and conotruncal heart defects
BMC Med. Genet.
 , 
2009
, vol. 
10
 pg. 
49
 
104
Deak
K.L.
Dickerson
M.E.
Linney
E.
Enterline
D.S.
George
T.M.
Melvin
E.C.
Graham
F.L.
Siegel
D.G.
Hammock
P.
Mehltretter
L.
, et al.  . 
Analysis of ALDH1A2, CYP26A1, CYP26B1, CRABP1, and CRABP2 in human neural tube defects suggests a possible association with alleles in ALDH1A2.
Birth Defects Res. A Clin. Mol. Teratol.
 , 
2005
, vol. 
73
 (pg. 
868
-
875
)
105
Zhu
H.
Wicker
N.J.
Shaw
G.M.
Lammer
E.J.
Hendricks
K.
Suarez
L.
Canfield
M.
Finnell
R.H.
Homocysteine remethylation enzyme polymorphisms and increased risks for neural tube defects
Mol. Genet. Metab.
 , 
2003
, vol. 
78
 (pg. 
216
-
221
)
106
Volcik
K.A.
Shaw
G.M.
Lammer
E.J.
Zhu
H.P.
Finnell
R.H.
Evaluation of infant methylenetetrahydrofolate reductase genotype, maternal vitamin use, and risk of high versus low level spina bifida defects
Birth Defects Res. (Part A)
 , 
2003
, vol. 
67
 (pg. 
154
-
157
)
107
Wilson
A.
Platt
R.
Wu
Q.
Leclerc
D.
Christensen
B.
Yang
H.
Gravel
R.A.
Rozen
R.
A common variant in methionine synthase reductase combined with low cobalamin (vitamin B12) increases risk for spina bifida
Mol. Genet. Metab.
 , 
1999
, vol. 
67
 (pg. 
317
-
323
)
108
Toepoel
M.
Steegers-Theunissen
R.P.
Ouborg
N.J.
Franke
B.
Ladd
A.M.
Joosten
P.H.
van Zoelen
E.J.
Interaction of PDGFRA promoter haplotypes and maternal environmental exposures in the risk of spina bifida
Birth Defects Res. A Clin. Mol. Teratol.
 , 
2009
, vol. 
85
 (pg. 
629
-
636
)
109
Joosten
P.H.
Toepoel
M.
Mariman
E.C.
Van Zoelen
E.J.
Promoter haplotype combinations of the platelet-derived growth factor alpha-receptor gene predispose to human neural tube defects
Nat. Genet.
 , 
2001
, vol. 
27
 (pg. 
215
-
217
)
110
Volcik
K.A.
Shaw
G.M.
Zhu
H.P.
Lammer
E.J.
Laurent
C.
Finnell
R.H.
Associations between polymorphisms within the thymidylate synthase gene and spina bifida
Birth Defects Res. Part A Clin Mol. Teratol.
 , 
2003
, vol. 
67
 (pg. 
924
-
928
)
111
Henrichsen
C.N.
Chaignat
E.
Reymond
A.
Copy number variants, diseases and gene expression
Hum. Mol. Genet.
 , 
2009
, vol. 
18
 (pg. 
R1
-
R8
)
112
Olshan
A.F.
Shaw
G.M.
Millikan
R.C.
Laurent
C.
Finnell
R.H.
Polymorphisms in DNA repair genes as risk factors for spina bifida and orofacial clefts
Am. J. Med. Genet.
 , 
2005
, vol. 
135A
 (pg. 
268
-
273
)
113
Zhu
H.
Curry
S.
Wen
S.
Wicker
N.J.
Shaw
G.M.
Lammer
E.J.
Yang
W.
Jafarov
T.
Finnell
R.H.
Are the betaine-homocysteine methyltransferase (BHMT and BHMT2) genes risk factors for spina bifida and orofacial clefts?
Am. J. Med. Genet. A
 , 
2005
, vol. 
135
 (pg. 
274
-
277
)
114
King
T.M.
Au
K.S.
Kirkpatrick
T.J.
Davidson
C.
Fletcher
J.M.
Townsend
I.
Tyerman
G.H.
Shimmin
L.C.
Northrup
H.
The impact of BRCA1 on spina bifida meningomyelocele lesions
Ann. Hum. Genet.
 , 
2007
, vol. 
71
 (pg. 
719
-
728
)
115
Davidson
C.M.
Northrup
H.
King
T.M.
Fletcher
J.M.
Townsend
I.
Tyerman
G.H.
Au
K.S.
Genes in glucose metabolism and association with spina bifida
Reprod. Sci.
 , 
2008
, vol. 
15
 (pg. 
51
-
58
)
116
Relton
C.L.
Wilding
C.S.
Pearce
M.S.
Laffling
A.J.
Jonas
P.A.
Lynch
S.A.
Tawn
E.J.
Burn
J.
Gene-gene interaction in folate-related genes and risk of neural tube defects in a UK population
J. Med. Genet.
 , 
2004
, vol. 
41
 (pg. 
256
-
260
)
117
Zhu
H.
Enaw
J.O.
Ma
C.
Shaw
G.M.
Lammer
E.J.
Finnell
R.H.
Association between CFL1 gene polymorphisms and spina bifida risk in a California population
BMC. Med. Genet.
 , 
2007
, vol. 
8
 pg. 
12
 
118
Enaw
J.O.
Zhu
H.
Yang
W.
Lu
W.
Shaw
G.M.
Lammer
E.J.
Finnell
R.H.
CHKA and PCYT1A gene polymorphisms, choline intake and spina bifida risk in a California population
BMC Med.
 , 
2006
, vol. 
4
 pg. 
36
 
119
Volcik
K.A.
Zhu
H.
Finnell
R.H.
Shaw
G.M.
Canfield
M.
Lammer
E.J.
Evaluation of the Cited2 gene and risk for spina bifida and congenital heart defects
Am. J Med. Genet.
 , 
2004
, vol. 
126A
 (pg. 
324
-
325
)
120
Johnson
W.G.
Stenroos
E.S.
Spychala
J.R.
Chatkupt
S.
Ming
S.X.
Buyske
S.
New 19 bp deletion polymorphism in intron-1 of dihydrofolate reductase (DHFR): a risk factor for spina bifida acting in mothers during pregnancy?
Am. J. Med. Genet.
 , 
2004
, vol. 
124A
 (pg. 
339
-
345
)
121
Parle-McDermott
A.
Pangilinan
F.
Mills
J.L.
Kirke
P.N.
Gibney
E.R.
Troendle
J.
O’Leary
V.B.
Molloy
A.M.
Conley
M.
Scott
J.M.
, et al.  . 
The 19-bp deletion polymorphism in intron-1 of dihydrofolate reductase (DHFR) may decrease rather than increase risk for spina bifida in the Irish population
Am. J. Med. Genet. A
 , 
2007
, vol. 
143A
 (pg. 
1174
-
1180
)
122
Van der Linden
I.
Nguyen
U.
Heil
S.G.
Franke
B.
Vloet
S.
Gellekink
H.
den
H.M.
Blom
H.J.
Variation and expression of dihydrofolate reductase (DHFR) in relation to spina bifida
Mol. Genet. Metab.
 , 
2007
, vol. 
91
 (pg. 
98
-
103
)
123
Gos
M.
Sliwerska
E.
Szpecht-Potocka
A.
Mutation incidence in folate metabolism genes and regulatory genes in Polish families with neural tube defects
J. Appl. Genet.
 , 
2004
, vol. 
45
 (pg. 
363
-
368
)
124
O’Leary
V.B.
Mills
J.L.
Parle-McDermott
A.
Pangilinan
F.
Molloy
A.M.
Cox
C.
Weiler
A.
Conley
M.
Kirke
P.N.
Scott
J.M.
, et al.  . 
Screening for new MTHFR polymorphisms and NTD risk
Am. J. Med. Genet. A
 , 
2005
, vol. 
138A
 (pg. 
99
-
106
)
125
Vieira
A.R.
Murray
J.C.
Trembath
D.
Orioli
I.M.
Castilla
E.E.
Cooper
M.E.
Marazita
M.L.
Lennon-Graham
F.
Speer
M.
Studies of reduced folate carrier 1 (RFC1) A80G and 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T polymorphisms with neural tube and orofacial cleft defects
Am. J. Med. Genet. A
 , 
2005
, vol. 
135
 (pg. 
220
-
223
)
126
Grandone
E.
Corrao
A.M.
Colaizzo
D.
Vecchione
G.
Di
G.C.
Paladini
D.
Sardella
L.
Pellegrino
M.
Zelante
L.
Martinelli
P.
, et al.  . 
Homocysteine metabolism in families from southern Italy with neural tube defects: role of genetic and nutritional determinants
Prenat. Diagn.
 , 
2006
, vol. 
26
 (pg. 
1
-
5
)
127
Munoz
J.B.
Lacasana
M.
Cavazos
R.G.
Borja-Aburto
V.H.
Galaviz-Hernandez
C.
Garduno
C.A.
Methylenetetrahydrofolate reductase gene polymorphisms and the risk of anencephaly in Mexico
Mol. Hum. Reprod.
 , 
2007
, vol. 
13
 (pg. 
419
-
424
)
128
Dalal
A.
Pradhan
M.
Tiwari
D.
Behari
S.
Singh
U.
Mallik
G.K.
Das
V.
Agarwal
S.
MTHFR 677C–>T and 1298A–>C polymorphisms: evaluation of maternal genotypic risk and association with level of neural tube defect
Gynecol. Obstet. Invest.
 , 
2007
, vol. 
63
 (pg. 
146
-
150
)
129
Gonzalez-Herrera
L.
Castillo-Zapata
I.
Garcia-Escalante
G.
Pinto-Escalante
D.
A1298C polymorphism of the MTHFR gene and neural tube defects in the state of Yucatan, Mexico
Birth Defects Res. A Clin. Mol. Teratol.
 , 
2007
, vol. 
79
 (pg. 
622
-
626
)
130
Candito
M.
Rivet
R.
Herbeth
B.
Boisson
C.
Rudigoz
R.C.
Luton
D.
Journel
H.
Oury
J.F.
Roux
F.
Saura
R.
, et al.  . 
Nutritional and genetic determinants of vitamin B and homocysteine metabolisms in neural tube defects: a multicenter Case–control study
Am. J Med. Genet. A
 , 
2008
, vol. 
146A
 (pg. 
1128
-
1133
)
131
Shang
Y.
Zhao
H.
Niu
B.
Li
W.I.
Zhou
R.
Zhang
T.
Xie
J.
Correlation of polymorphism of MTHFRs and RFC-1 genes with neural tube defects in China
Birth Defects Res. A Clin. Mol. Teratol.
 , 
2008
, vol. 
82
 (pg. 
3
-
7
)
132
O’Leary
V.B.
Mills
J.L.
Pangilinan
F.
Kirke
P.N.
Cox
C.
Conley
M.
Weiler
A.
Peng
K.
Shane
B.
Scott
J.M.
, et al.  . 
Analysis of methionine synthase reductase polymorphisms for neural tube defects risk association
Mol. Genet. Metab.
 , 
2005
, vol. 
85
 (pg. 
220
-
227
)
133
Van der Linden
I.
Den Heijer
M.
Afman
L.A.
Gellekink
H.
Vermeulen
S.H.
Kluijtmans
L.A.
Blom
H.J.
The methionine synthase reductase 66A>G polymorphism is a maternal risk factor for spina bifida
J. Mol. Med.
 , 
2006
, vol. 
84
 (pg. 
1047
-
1054
)
134
Parle-McDermott
A.
McManus
E.J.
Mills
J.L.
O’Leary
V.B.
Pangilinan
F.
Cox
C.
Weiler
A.
Molloy
A.M.
Conley
M.
Watson
D.
, et al.  . 
Polymorphisms within the vitamin B12 dependent methylmalonyl-coA mutase are not risk factors for neural tube defects
Mol. Genet. Metab.
 , 
2003
, vol. 
80
 (pg. 
463
-
468
)
135
Jensen
L.E.
Hoess
K.
Whitehead
A.S.
Mitchell
L.E.
The NAT1 C1095A polymorphism, maternal multivitamin use and smoking, and the risk of spina bifida
Birth Defects Res. A Clin. Mol. Teratol.
 , 
2005
, vol. 
73
 (pg. 
512
-
516
)
136
Jensen
L.E.
Hoess
K.
Mitchell
L.E.
Whitehead
A.S.
Loss of function polymorphisms in NAT1 protect against spina bifida
Hum. Genet.
 , 
2006
, vol. 
120
 (pg. 
52
-
57
)
137
Lu
W.
Zhu
H.
Wen
S.
Yang
W.
Shaw
G.M.
Lammer
E.J.
Finnell
R.H.
Nicotinamide N-methyl transferase (NNMT) gene polymorphisms and risk for spina bifida
Birth Defects Res. A Clin. Mol. Teratol.
 , 
2008
, vol. 
82
 (pg. 
670
-
675
)
138
Deak
K.L.
Boyles
A.L.
Etchevers
H.C.
Melvin
E.C.
Siegel
D.G.
Graham
F.L.
Slifer
S.H.
Enterline
D.S.
George
T.M.
Vekemans
M.
, et al.  . 
SNPs in the neural cell adhesion molecule 1 gene (NCAM1) may be associated with human neural tube defects
Hum. Genet.
 , 
2005
, vol. 
117
 (pg. 
133
-
142
)
139
Brown
K.S.
Cook
M.
Hoess
K.
Whitehead
A.S.
Mitchell
L.E.
Evidence that the risk of spina bifida is influenced by genetic variation at the NOS3 locus
Birth Defects Res. Part A Clin. Mol. Teratol.
 , 
2004
, vol. 
70
 (pg. 
101
-
106
)
140
Van der Linden
I.J.
Heil
S.G.
den
H.M.
Blom
H.J.
The 894G>T variant in the endothelial nitric oxide synthase gene and spina bifida risk
J. Hum. Genet.
 , 
2007
, vol. 
52
 (pg. 
516
-
520
)
141
Zhu
H.
Yang
W.
Lu
W.
Zhang
J.
Shaw
G.M.
Lammer
E.J.
Finnell
R.H.
A known functional polymorphism (Ile120Val) of the human PCMT1 gene and risk of spina bifida
Mol. Genet. Metab.
 , 
2006
, vol. 
87
 (pg. 
66
-
70
)
142
Zhu
H.
Wicker
N.J.
Volcik
K.
Zhang
J.
Shaw
G.M.
Lammer
E.J.
Suarez
L.
Canfield
M.
Finnell
R.H.
Promoter haplotype combinations for the human PDGFRA gene are associated with risk of neural tube defects
Mol. Genet. Metab.
 , 
2004
, vol. 
81
 (pg. 
127
-
132
)
143
Au
K.S.
Northrup
H.
Kirkpatrick
T.J.
Volcik
K.A.
Fletcher
J.M.
Townsend
I.T.
Blanton
S.H.
Tyerman
G.H.
Villarreal
G.
King
T.M.
Promotor genotype of the platelet-derived growth factor receptor-alpha gene shows population stratification but not association with spina bifida meningomyelocele
Am. J. Med. Genet. A
 , 
2005
, vol. 
139
 (pg. 
194
-
198
)
144
Zhang
J.
Zhu
H.
Yang
W.
Shaw
G.M.
Lammer
E.J.
Finnell
R.H.
Phosphatidylethanolamine N-methyltransferase (PEMT) gene polymorphisms and risk of spina bifida
Am. J. Med. Genet. A
 , 
2006
, vol. 
140
 (pg. 
785
-
789
)
145
Zhu
H.P.
Lu
W.
Laurent
C.
Shaw
G.M.
Lammer
E.J.
Finnell
R.H.
Genes encoding catalytic subunits of protein kinase A and risk of spina bifida
Birth Defects Res. A Clin. Mol. Teratol.
 , 
2005
, vol. 
73
 (pg. 
591
-
596
)
146
Relton
C.L.
Wilding
C.S.
Laffling
A.J.
Jonas
P.A.
Burgess
T.
Binks
K.
Tawn
E.J.
Burn
J.
Low erythrocyte folate status and polymorphic variation in folate-related genes are associated with risk of neural tube defect pregnancy
Mol. Genet. Metab.
 , 
2004
, vol. 
81
 (pg. 
273
-
281
)
147
O’Leary
V.B.
Pangilinan
F.
Cox
C.
Parle-McDermott
A.
Conley
M.
Molloy
A.M.
Kirke
P.N.
Mills
J.L.
Brody
L.C.
Scott
J.M.
Reduced folate carrier polymorphisms and neural tube defect risk
Mol. Genet. Metab.
 , 
2006
, vol. 
87
 (pg. 
364
-
369
)
148
Pei
L.
Liu
J.
Zhang
Y.
Zhu
H.
Ren
A.
Association of reduced folate carrier gene polymorphism and maternal folic acid use with neural tube defects
Am. J Med. Genet. B Neuropsychiatr. Genet.
 , 
2008
 
Epub
149
Jensen
L.E.
Barbaux
S.
Hoess
K.
Fraterman
S.
Whitehead
A.S.
Mitchell
L.E.
The human T locus and spina bifida risk
Hum. Genet.
 , 
2004
, vol. 
115
 (pg. 
475
-
482
)
150
Swanson
D.A.
Pangilinan
F.
Mills
J.L.
Kirke
P.N.
Conley
M.
Weiler
A.
Frey
T.
Parle-McDermott
A.
O’Leary
V.B.
Seltzer
R.R.
, et al.  . 
Evaluation of transcobalamin II polymorphisms as neural tube defect risk factors in an Irish population
Birth Defects Res. A Clin. Mol. Teratol.
 , 
2005
, vol. 
73
 (pg. 
239
-
244
)
151
Wen
S.
Lu
W.
Zhu
H.
Yang
W.
Shaw
G.M.
Lammer
E.J.
Islam
A.
Finnell
R.H.
Genetic polymorphisms in the thioredoxin 2 (TXN2) gene and risk for spina bifida
Am. J. Med. Genet. A
 , 
2009
, vol. 
149A
 (pg. 
155
-
160
)
152
Pangilinan
F.
Geiler
K.
Dolle
J.
Troendle
J.
Swanson
D.A.
Molloy
A.M.
Sutton
M.
Conley
M.
Kirke
P.N.
Scott
J.M.
, et al.  . 
Construction of a high resolution linkage disequilibrium map to evaluate common genetic variation in TP53 and neural tube defect risk in an Irish population
Am. J Med. Genet. A
 , 
2008
, vol. 
146A
 (pg. 
2617
-
2625
)
153
Wilding
C.S.
Relton
C.L.
Sutton
M.J.
Jonas
P.A.
Lynch
S.A.
Tawn
E.J.
Burn
J.
Thymidylate synthase repeat polymorphisms and risk of neural tube defects in a population from the northern United Kingdom
Birth Defects Res. Part A Clin. Mol. Teratol.
 , 
2004
, vol. 
70
 (pg. 
483
-
485
)
154
Mitchell
A.
Pangilinan
F.
Van der
M.J.
Molloy
A.M.
Troendle
J.
Conley
M.
Kirke
P.N.
Scott
J.M.
Brody
L.C.
Mills
J.L.
Uncoupling protein 2 polymorphisms as risk factors for NTDs
Birth Defects Res. A Clin. Mol. Teratol.
 , 
2009
, vol. 
85
 (pg. 
156
-
160
)
155
Klootwijk
R.
Groenen
P.
Schijvenaars
M.
Hol
F.
Hamel
B.
Straatman
H.
Steegers-Theunissen
R.
Mariman
E.
Franke
B.
Genetic variants in ZIC1, ZIC2 and ZIC3 are not major risk factors for neural tube defects in humans
Am. J. Med. Genet.
 , 
2004
, vol. 
124A
 (pg. 
40
-
47
)
156
Copp
A.J.
Checiu
I.
Henson
J.N.
Developmental basis of severe neural tube defects in the loop-tail (Lp) mutant mouse: use of microsatellite DNA markers to identify embryonic genotype
Dev. Biol.
 , 
1994
, vol. 
165
 (pg. 
20
-
29
)
157
Kibar
Z.
Underhill
D.A.
Canonne-Hergaux
F.
Gauthier
S.
Justice
M.J.
Gros
P.
Identification of a new chemically induced allele (Lp(m1Jus)) at the loop-tail locus: morphology, histology, and genetic mapping
Genomics
 , 
2001
, vol. 
72
 (pg. 
331
-
337
)
158
Curtin
J.A.
Quint
E.
Tsipouri
V.
Arkell
R.M.
Cattanach
B.
Copp
A.J.
Fisher
E.M.
Nolan
P.M.
Steel
K.P.
Brown
S.D.M.
, et al.  . 
Mutation of Celsr1 disrupts planar polarity of inner ear hair cells and causes severe neural tube defects in the mouse
Curr. Biol.
 , 
2003
, vol. 
13
 (pg. 
1
-
20
)
159
Wang
J.
Hamblet
N.S.
Mark
S.
Dickinson
M.E.
Brinkman
B.C.
Segil
N.
Fraser
S.E.
Chen
P.
Wallingford
J.B.
Wynshaw-Boris
A.
Dishevelled genes mediate a conserved mammalian PCP pathway to regulate convergent extension during neurulation
Development
 , 
2006
, vol. 
133
 (pg. 
1767
-
1778
)
160
Wang
Y.
Guo
N.
Nathans
J.
The role of Frizzled3 and Frizzled6 in neural tube closure and in the planar polarity of inner-ear sensory hair cells
J. Neurosci.
 , 
2006
, vol. 
26
 (pg. 
2147
-
2156
)
161
Qian
D.
Jones
C.
Rzadzinska
A.
Mark
S.
Zhang
X.
Steel
K.P.
Dai
X.
Chen
P.
Wnt5a functions in planar cell polarity regulation in mice
Dev. Biol.
 , 
2007
, vol. 
306
 (pg. 
121
-
133
)
162
Satoh
W.
Matsuyama
M.
Takemura
H.
Aizawa
S.
Shimono
A.
Sfrp1, Sfrp2 and Sfrp5 regulate the Wnt/beta-catenin and the planar cell polarity pathways during early trunk formation in mouse
Genesis
 , 
2008
, vol. 
46
  
spcone
163
Stiefel
D.
Shibata
T.
Meuli
M.
Duffy
P.
Copp
A.J.
Tethering of the spinal cord in mouse fetuses and neonates with spina bifida
J. Neurosurg. (Spine)
 , 
2003
, vol. 
99
 (pg. 
206
-
213
)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.