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1.  Autoantibodies to folate receptor during pregnancy and neural tube defect risk 
Periconceptional folic acid can reduce the occurrence of neural tube defects (NTDs) by up to 70%, and autoantibodies for folate receptors (FRs) have been observed in serum from women with a pregnancy complicated by an NTD. This population-based cohort study has examined serum from pregnant mothers for autoantibodies to FRs, antibodies to bovine folate binding protein (FBP), and inhibition of folic acid binding to FR and FBP in association with NTD risk. The mid-gestational maternal serum specimens used for this study were collected during the 15th–18th week of pregnancy. Samples were obtained from the California Birth Defects Monitoring Program; 29 mothers had a pregnancy complicated by spina bifida and 76 mothers had unaffected children. The presence of IgG and IgM antibodies to human FR, bovine FBP, and inhibition of folic acid binding to FR and FBP was determined. Higher activity of IgM to FBP in cases verses controls was observed (P=.04). Higher activity of IgM and IgG autoantibodies to FR was observed (P<0.001 and P=.04, respectively). Risk estimates at two standard deviations above average control antibody concentrations were OR=2.07 (CI=1.02, 4.06) for anti-FBPIgM, OR=2.15 (CI=1.02, 4.69) for anti-FRIgG and OR=3.19 (CI=1.47, 6.92) for anti-FR IgM. These data support the hypothesis that high titers of antibodies and blocking of folic acid binding to FRs by maternal serum should be regarded as risk factors for NTDs.
PMCID: PMC3998370  PMID: 18804286
Folate Receptor; Autoantibodies; Pregnancy; Neural Defects
Epidemiological studies have linked environmental arsenic (As) exposure to increased type 2 diabetes risk. Periconceptional hyperglycemia is a significant risk factor for neural tube defects (NTDs), the second most common structural birth defect. A suspected teratogen, arsenic (As) induces NTDs in laboratory animals.
We investigated whether maternal glucose homeostasis disruption was responsible for arsenate-induced NTDs in a well-established dosing regimen used in studies of arsenic’s teratogenicity in early neurodevelopment.
We evaluated maternal intraperitoneal (I.P.) exposure to As 9.6 mg/kg (as sodium arsenate) in LM/Bc/Fnn mice for teratogenicity and disruption of maternal plasma glucose and insulin levels. Selected compounds (insulin pellet, sodium selenate (SS), N-acetyl cysteine (NAC), L-methionine (L-Met), N-tert-Butyl-α-phenylnitrone (PBN)) were investigated for their potential to mitigate arsenate’s effects.
Arsenate caused significant glucose elevation during an I.P. glucose tolerance test (IPGTT). Insulin levels were not different between arsenate and control dams before (arsenate, 0.55 ng/dl; control, 0.48 ng/dl) or after glucose challenge (arsenate, 1.09 ng/dl; control, 0.81 ng/dl). HOMA-IR index was higher for arsenate (3.9) vs control (2.5) dams (p=0.0260). Arsenate caused NTDs (100%, p<0.0001). Insulin pellet and NAC were the most successful rescue agents, reducing NTD rates to 45% and 35%.
IPGTT, insulin assay, and HOMA-IR results suggest a modest failure of glucose stimulated insulin secretion and insulin resistance characteristic of glucose intolerance. Insulin’s success in preventing arsenate-induced NTDs provides evidence that these arsenate-induced NTDs are secondary to elevated maternal glucose. The NAC rescue, which did not restore maternal glucose or insulin levels, suggests oxidative disruption plays a role.
PMCID: PMC3998373  PMID: 19446573
arsenate; arsenic; glucose intolerance; insulin resistance; mouse; neural tube defect; oxidative stress; teratogen
3.  Mthfr gene ablation enhances susceptibility to arsenic prenatal toxicity 
In utero exposure to arsenic is known to adversely affect reproductive outcomes. Evidence of arsenic teratogenicity vary widely and depend on individual genotypic differences in sensitivity to As. In this study, we investigated the potential interaction between 5,10-methylenetetrahydrofolate reductase (Mthfr) genotype and arsenic embryotoxicity using the Mthfr knockout mouse model.
Pregnant dams were treated with sodium arsenate, and reproductive outcomes including: implantation, resorption, congenital malformation and fetal birth weight were recorded at E18.5.
When the dams in Mthfr+/− x Mthfr+/− matings were treated with 7.2mg/kg As, the resorption rate increased to 43.4%, from a background frequency of 7.2%. The As treatment also induced external malformations (40.9%) and significantly lowered the average fetal birth weight among fetuses, without any obvious toxic effect on the dam. When comparing the pregnancy outcomes resulting from different mating scenarios (Mthfr+/+ x Mthfr+/−, Mthfr+/− x Mthfr+/− and Mthfr−/− x Mthfr+/−) and arsenic exposure; the resorption rate showed a linear relationship with the number of null alleles (0, 1 or 2) in the Mthfr dams. Fetuses from nullizygous dams had the highest rate of external malformations (43%) and lowest average birth weight. When comparing the outcomes of reciprocal matings (nullizygote x wild-type versus wild-type x nullizygote) after As treatment, the null dams showed significantly higher rates of resorptions and malformations, along with lower fetal birth weights.
Maternal genotype contributes to the sensitivity of As embryotoxicity in the Mthfr mouse model. The fetal genotype, however, does not appear to affect the reproductive outcome after in utero As exposure.
PMCID: PMC3943866  PMID: 24384392
Sodium arsenate; 5, 10-methylenetetrahydrofolate reductase (MTHFR); Mthfr knockout mice; embryotoxicity; gene-environment interaction; teratogenicity
4.  Excess S-adenosylmethionine reroutes phosphatidylethanolamine towards phosphatidylcholine and triglyceride synthesis 
Hepatology (Baltimore, Md.)  2013;58(4):1296-1305.
Methionine adenosyltransferase 1A (MAT1A) and glycine N-methyltransferase (GNMT) are the primary genes involved in hepatic S-adenosylmethionine (SAMe) synthesis and degradation, respectively. Mat1a ablation in mice induces a decrease in hepatic SAMe, activation of lipogenesis, inhibition of triglyceride (TG) release, and steatosis. Gnmt deficient mice, despite showing a large increase in hepatic SAMe, also develop steatosis. We hypothesized that as an adaptive response to hepatic SAMe accumulation, phosphatidylcholine (PC) synthesis via the phosphatidylethanolamine (PE) N-methyltransferase (PEMT) pathway is stimulated in Gnmt−/− mice. We also propose that the excess PC thus generated is catabolized leading to TG synthesis and steatosis via diglyceride (DG) generation. We observed that Gnmt−/− mice present with normal hepatic lipogenesis and increased TG release. We also observed that the flux from PE to PC is stimulated in the liver of Gnmt−/− mice and that this results in a reduction in PE content and a marked increase in DG and TG. Conversely, reduction of hepatic SAMe following the administration of a methionine deficient diet reverted the flux from PE to PC of Gnmt−/− mice to that of wild type animals and normalized DG and TG content preventing the development of steatosis. Gnmt−/− mice with an additional deletion of perilipin2, the predominant lipid droplet protein, maintain high SAMe levels, with a concurrent increased flux from PE to PC, but do not develop liver steatosis.
These findings indicate that excess SAMe reroutes PE towards PC and TG synthesis, and lipid sequestration.
PMCID: PMC3720726  PMID: 23505042
glycine N-methyltransferase; lipidomics; lipid droplets; steatosis; PLIN2
5.  Cannabinoid receptor 1 signaling in embryo neurodevelopment 
In utero exposure to THC, the psychoactive component of marijuana, is associated with an increased risk for neurodevelopmental defects in the offspring by interfering with the functioning of the endocannabinoid (eCB) system. At the present time it is not clearly known whether the eCB system is present prior to neurogenesis. Using an array of biochemical techniques we analyzed the levels of CB1 receptors, eCBs (AEA and 2-AG), and the enzymes (NAPE-PLD, DAGLα, DAGLβ MAGL and FAAH) involved in the metabolism of the eCBs in chick and mouse models during development. The findings demonstrate the presence of eCB system in early embryo, prior to neurogenesis. The eCB system might play a critical role in early embryogenesis and there might be adverse developmental consequences of in utero exposure to marijuana and other drugs of abuse during this period.
PMCID: PMC4175447  PMID: 22311661
Endocannabinoid; CB1; anandamide; 2-arachidonoylglycerol; G-protein; forebrain; chick; embryo
6.  Exon Sequencing of PAX3 and T (Brachyury) in Cases with Spina Bifida 
Based on studies in animals and humans, PAX3 and T (brachyury) are candidate genes for spina bifida. However, neither gene has been definitively identified as a risk factor for this condition.
Sanger sequencing was used to identify variants in all PAX3 and T exons and promoter regions in 114 spina bifida cases. For known variants, allele frequencies in cases were compared to those from public databases using unadjusted odds ratios. Novel variants were genotyped in parents and assessed for predicted functional impact.
We identified common variants in PAX3 (n=2) and T (n=3) for which the allele frequencies in cases were significantly different from those reported in at least one public database. We also identified novel variants in both PAX3 (n=11) and T (n=1) in spina bifida cases. Several of the novel PAX3 variants are predicted to be highly conserved and/or impact gene function or expression.
These studies provide some evidence that common variants of PAX3 and T are associated with spina bifida. Rare and novel variants in these genes were also identified in affected individuals. However, additional studies will be required to determine whether these variants influence the risk of spina bifida.
PMCID: PMC3877942  PMID: 23913553
spina bifida; myelomeningocele; genetic epidemiology; sequencing; PAX3; T locus
7.  The Folate Pathway and Nonsyndromic Cleft Lip and Palate 
Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth malformation caused by genetic, environmental and gene-environment interactions. Periconceptional supplementation with folic acid, a key component in DNA synthesis and cell division, has reduced the birth prevalence of neural tube defects (NTDs) and may similarly reduce the birth prevalence of other complex birth defects including NSCLP. Past studies investigating the role of two common methylenetetrahydrofolate reductase (MTHFR) SNP polymorphisms, C677T (rs1801133) and A1298C (rs1801131), in NSCLP have produced conflicting results. Most studies of folate pathway genes have been limited in scope, as few genes/SNPs have been interrogated. In this study, we asked whether variations in a more comprehensive group of folate pathway genes were associated with NSCLP and, if so, were there detectable interactions between these genes and environmental exposures. In addition, we evaluated the data for a sex effect. Fourteen folate metabolism related genes were interrogated using eighty-nine SNPs in multiplex and simplex non-Hispanic White (NHW) (317) and Hispanic (128) NSCLP families. Evidence for a risk association between NSCLP and SNPs in nitrous oxide 3 (NOS3) and thymidylate synthetase (TYMS) was detected in the NHW group, whereas associations with methionine synthase (MTR), betaine-homocysteine methyltransferase (BHMT2), MTHFS and SLC19A1 were detected in the Hispanic group. Evidence for over-transmission of haplotypes and gene interactions in the methionine arm was detected. These results suggest that perturbations of the genes in the folate pathway may contribute to NSCLP. There was evidence for an interaction between several SNPs and maternal smoking, and for one SNP with sex of the offspring. These results provide support for other studies that suggest that high maternal homocysteine levels may contribute to NSCLP and should be further investigated.
PMCID: PMC4098909  PMID: 21254359
Nonsyndromic cleft lip and palate; NSCLP; folate metabolism; association; genetics; homocysteine; methionine
8.  Neural Tube Defects, Folate, and Immune Modulation 
Periconceptional supplementation with folic acid has led to a significant worldwide reduction in the incidence of neural tube defects (NTDs). However, despite increasing awareness of the benefits of folic acid supplementation and the implementation of food fortification programs in many countries, NTDs continue to be a leading cause of perinatal morbidity and mortality worldwide. Furthermore, there exists a significant subgroup of women who appear to be resistant to the protective effects of folic acid supplementation. The following review addresses emerging clinical and experimental evidence for a role of the immune system in the etiopathogenesis of NTDs, with the aim of developing novel preventative strategies to further reduce the incidence of NTD-affected pregnancies. In particular, recent studies demonstrating novel roles and interactions between innate immune factors such as the complement cascade, neurulation, and folate metabolism are explored.
PMCID: PMC4053177  PMID: 24078477
complement; C5a; neural tube defects; folate; neurulation
9.  Levels of PAH-DNA Adducts in Placental Tissue and the Risk of Fetal Neural Tube Defects in a Chinese Population 
We examined the relationship between PAH-DNA adduct levels in the placental tissue, measured by a highly sensitive 32P-postlabeling assay, and the risk of fetal neural tube defects (NTDs). We further explored the interaction between PAH-DNA adducts and placental PAHs with respect to NTD risk. Placental tissues from 80 NTD-affected pregnancies and 50 uncomplicated normal pregnancies were included in this case-control study. Levels of PAH-DNA adducts were lower in the NTD group (8.12 per 108 nucleotides) compared to controls (9.92 per 108 nucleotides). PAH-DNA adduct concentrations below the median was associated with a 3-fold increased NTD risk. Women with a low PAH-DNA adduct level in concert with a high placental PAH level resulted in a 10-fold elevated risk of having an NTD-complicated pregnancy. A low level of placental PAH-DNA adducts was associated with an increased risk of NTDs; this risk increased dramatically when a low adduct level was coupled with a high placental PAH concentration.
PMCID: PMC4040017  PMID: 23416326
Polycyclic aromatic hydrocarbons (PAHs); DNA adducts; Neural tube defects (NTDs); Placenta; Fetus
11.  Genes Encoding Catalytic Subunits of Protein Kinase A and Risk of Spina Bifida 
PRKACA and PRKACB are genes encoding the cAMP-dependent protein kinase A (PKA) catalytic subunits alpha and beta, respectively. PKA is known to be involved in embryonic development, as it downregulates the Hedgehog (Hh) signaling pathway, which is critical to normal pattern formation and morphogenesis. The PKA-deficient mouse model, which has only a single catalytic subunit, provided intriguing evidence demonstrating a relationship between decreased PKA activity and risk for posterior neural tube defects (NTDs) in the thoracic to sacral regions of gene-knockout mice. Unlike most other mutant mouse models of NTDs, the PKA-deficient mice develop spina bifida with 100% penetrance. We hypothesized that sequence variations in human genes encoding the catalytic subunits may alter the PKA activity and similarly increase the risk of spina bifida.
We sequenced the coding regions and the exon/intron boundaries of PRKACA and PRKACB. We also examined 3 common single-nucleotide polymorphisms (SNPs) of these 2 genes by allele discrimination.
Five sequence variants in coding region and 2 intronic sequence variants proximal to exons were detected. None of the 3 SNPs examined in the association study appeared to be associated with substantially increased risk for spina bifida.
Our results did not reveal a strong association between these PKA SNPs and spina bifida risk. Nonetheless, it is important to examine the possible gene-gene interactions between PRKACA and PRKACB when evaluating the risk for NTDs, as well as genes encoding regulatory subunits of PKA. In addition, interactions with other genes such as Sonic Hedgehog (SHH) should also be considered for future investigations.
PMCID: PMC2970525  PMID: 16080189
PRKACA; PRKACB; case-control study; spina bifida
12.  The planar cell polarity effector Fuz is essential for targeted membrane trafficking, ciliogenesis, and mouse embryonic development 
Nature cell biology  2009;11(10):1225-1232.
The planar cell polarity (PCP) signaling pathway is essential for embryonic development because it governs diverse cellular behaviors, and the “core PCP” proteins, such as Dishevelled and Frizzled, have been extensively characterized1–4. By contrast, the “PCP effector” proteins, such as Intu and Fuz, remain largely unstudied5, 6. These proteins are essential for PCP signaling, but they have never been investigated in a mammal and their cell biological activities remain entirely unknown. We report here that Fuz mutant mice display neural tube defects, skeletal dysmorphologies, and Hedgehog signaling defects stemming from disrupted ciliogenesis. Using bioinformatics and imaging of an in vivo mucociliary epithelium, we establish a central role for Fuz in membrane trafficking, showing that Fuz is essential for trafficking of cargo to basal bodies and to the apical tips of cilia. Fuz is also essential for exocytosis in secretory cells. Finally, we identify a novel, Rab-related small GTPase as a Fuz interaction partner that is also essential for ciliogenesis and secretion. These results are significant because they provide novel insights into the mechanisms by which developmental regulatory systems like PCP signaling interface with fundamental cellular systems such as the vesicle trafficking machinery.
PMCID: PMC2755648  PMID: 19767740
13.  Identification of Novel CELSR1 Mutations in Spina Bifida 
PLoS ONE  2014;9(3):e92207.
Spina bifida is one of the most common neural tube defects (NTDs) with a complex etiology. Variants in planar cell polarity (PCP) genes have been associated with NTDs including spina bifida in both animal models and human cohorts. In this study, we sequenced all exons of CELSR1 in 192 spina bifida patients from a California population to determine the contribution of CELSR1 mutations in the studied population. Novel and rare variants identified in these patients were subsequently genotyped in 190 ethnically matched control individuals. Six missense mutations not found in controls were predicted to be deleterious by both SIFT and PolyPhen. Two TG dinucleotide repeat variants were individually detected in 2 spina bifida patients but not detected in controls. In vitro functional analysis showed that the two TG dinucleotide repeat variants not only changed subcellular localization of the CELSR1 protein, but also impaired the physical association between CELSR1 and VANGL2, and thus diminished the ability to recruit VANGL2 for cell-cell contact. In total, 3% of our spina bifida patients carry deleterious or predicted to be deleterious CELSR1 mutations. Our findings suggest that CELSR1 mutations contribute to the risk of spina bifida in a cohort of spina bifida patients from California.
PMCID: PMC3954890  PMID: 24632739
14.  Folate Transport Gene Inactivation in Mice Increases Sensitivity to Colon Carcinogenesis 
Cancer research  2005;65(3):887-897.
Low dietary folate intake is associated with an increased risk for colon cancer; however, relevant genetic animal models are lacking. We therefore investigated the effect of targeted ablation of two folate transport genes, folate binding protein 1 (Folbp1) and reduced folate carrier 1 (RFC1), on folate homeostasis to elucidate the molecular mechanisms of folate action on colonocyte cell proliferation, gene expression, and colon carcinogenesis. Targeted deletion of Folbp1 (Folbp1+/− and Folbp1−/−) significantly reduced (P < 0.05) colonic Folbp1 mRNA, colonic mucosa, and plasma folate concentration. In contrast, subtle changes in folate homeostasis resulted from targeted deletion of RFC1 (RFC1+/−). These animals had reduced (P < 0.05) colonic RFC1 mRNA and exhibited a 2-fold reduction in the plasma S-adenosylmethionine/S-adenosylhomocysteine. Folbp1+/− and Folbp1−/− mice had larger crypts expressed as greater (P < 0.05) numbers of cells per crypt column relative to Folbp1+/+ mice. Colonic cell proliferation was increased in RFC1+/− mice relative to RFC1+/+ mice. Microarray analysis of colonic mucosa showed distinct changes in gene expression specific to Folbp1 or RFC1 ablation. The effect of folate transporter gene ablation on colon carcinogenesis was evaluated 8 and 38 weeks post-azoxymethane injection in wild-type and heterozygous mice. Relative to RFC1+/+ mice, RFC1+/− mice developed increased (P < 0.05) numbers of aberrant crypt foci at 8 weeks. At 38 weeks, RFC1+/− mice developed local inflammatory lesions with or without epithelial dysplasia as well as adenocarcinomas, which were larger relative to RFC1+/+ mice. In contrast, Folbp1+/− mice developed 4-fold (P < 0.05) more lesions relative to Folbp1+/+ mice. In conclusion, Folbp1 and RFC1 genetically modified mice exhibit distinct changes in colonocyte phenotype and therefore have utility as models to examine the role of folate homeostasis in colon cancer development.
PMCID: PMC3938162  PMID: 15705887
15.  Folate modulates Hox gene controlled skeletal phenotypes 
Genesis (New York, N.Y. : 2000)  2004;39(3):155-166.
Hox genes are well known regulators of pattern formation (Capecchi, 1996; Krumlauf, 1994) and cell differentiation (Goff and Tabin, 1997; Papenbrock et al., 2000; Yueh et al., 1998) in the developing vertebrate skeleton. Although skeletal variations are not uncommon in humans (Hald et al., 1995), few mutations in human HOX genes have been described (Goodman and Scambler, 2001). If such mutations are compatible with life, there may be physiological modifiers for the manifestation of Hox gene-controlled phenotypes, masking underlying mutations. We here present evidence that the essential nutrient folate modulates genetically induced skeletal defects in Hoxd4 transgenic mice. We also show that chondrocytes require folate for growth and differentiation and that they express folate transport genes, providing evidence for a direct effect of folate on skeletal cells. To our knowledge, this is the first report of nutritional influence on Hox gene controlled phenotypes, and implicates gene-environment interactions as important modifiers of Hox gene function. Taken together, our results demonstrate a beneficial effect of folate on skeletal development that may also be relevant to disorders and variations of the human skeleton.
PMCID: PMC3938166  PMID: 15282741
16.  Developmental consequences of in utero sodium arsenate exposure in mice with folate transport deficiencies 
Previous studies have demonstrated that mice lacking a functional folate binding protein 2 gene (Folbp2−/−) were significantly more sensitive to in utero arsenic exposure than were the wild-type mice similarly exposed. When these mice were fed a folate-deficient diet, the embryotoxic effect of arsenate was further exacerbated. Contrary to expectations, studies on 24-h urinary speciation of sodium arsenate did not demonstrate any significant difference in arsenic biotransformation between Folbp2−/− and Folbp2+/+ mice. To better understand the influence of folate pathway genes on arsenic embryotoxicity, the present investigation utilized transgenic mice with disrupted folate binding protein 1 (Folbp1) and reduced folate carrier (RFC) genes. Because complete inactivation of Folbp1 and RFC genes results in embryonic lethality, we used heterozygous animals. Overall, no RFC genotype-related differences in embryonic susceptibility to arsenic exposure were observed. Embryonic lethality and neural tube defect (NTD) frequency in Folbp1 mice was dose-dependent and differed from the RFC mice; however, no genotype-related differences were observed. The RFC heterozygotes tended to have higher plasma levels of S-adenosylhomocysteine (SAH) than did the wild-type controls, although this effect was not robust. It is concluded that genetic modifications at the Folbp1 and RFC loci confers no particular sensitivity to arsenic toxicity compared to wild-type controls, thus disproving the working hypothesis that decreased methylating capacity of the genetically modified mice would put them at increased risk for arsenic-induced reproductive toxicity.
PMCID: PMC3938173  PMID: 15694460
Arsenic; Teratogenicity; Biotransformation; Detoxification; Folbp1; RFC; Neural tube defects
17.  Diabetes and Obesity-Related Genes and the Risk of Neural Tube Defects in the National Birth Defects Prevention Study 
American Journal of Epidemiology  2012;176(12):1101-1109.
Few studies have evaluated genetic susceptibility related to diabetes and obesity as a risk factor for neural tube defects (NTDs). The authors investigated 23 single nucleotide polymorphisms among 9 genes (ADRB3, ENPP1, FTO, LEP, PPARG, PPARGC1A, SLC2A2, TCF7L2, and UCP2) associated with type 2 diabetes or obesity. Samples were obtained from 737 NTD case-parent triads included in the National Birth Defects Prevention Study during 1999–2007. Log-linear models were used to evaluate maternal and offspring genetic effects. After application of the false discovery rate, there were 5 significant maternal genetic effects. The less common alleles at the 4 FTO single nucleotide polymorphisms showed a reduction of NTD risk (for rs1421085, relative risk (RR) = 0.73 (95% confidence interval (CI): 0.62, 0.87); for rs8050136, RR = 0.79 (95% CI: 0.67, 0.93); for rs9939609, RR = 0.79 (95% CI: 0.67, 0.94); and for rs17187449, RR = 0.80 (95% CI: 0.68, 0.95)). Additionally, maternal LEP rs2071045 (RR = 1.31, 95% CI: 1.08, 1.60) and offspring UCP2 rs660339 (RR = 1.32, 95% CI: 1.06, 1.64) were associated with NTD risk. Furthermore, the maternal genotype for TCF7L2 rs3814573 suggested an increased NTD risk among obese women. These findings indicate that maternal genetic variants associated with glucose homeostasis may modify the risk of having an NTD-affected pregnancy.
PMCID: PMC3571234  PMID: 23132673
case-parent triads; diabetes; genetics; neural tube defects; obesity
18.  A GCH1 Haplotype and Risk of Neural Tube Defects in the National Birth Defects Prevention Study 
Molecular genetics and metabolism  2012;107(3):592-595.
Tetrahydrobiopterin (BH4) is an essential cofactor and an important cellular antioxidant. BH4 deficiency has been associated with diseases whose etiologies stem from excessive oxidative stress. GTP cyclohydrolase I (GCH1) catalyzes the first and rate-limiting step of de novo BH4 synthesis. A 3-SNP haplotype in GCH1 (rs8007267, rs3783641, and rs10483639) is known to modulate GCH1 gene expression levels and has been suggested as a major determinant of plasma BH4 bioavailability. As plasma BH4 bioavailability has been suggested as a mechanism of neural tube defect (NTD) teratogenesis, we evaluated the association between this GCH1 haplotype and the risk of NTDs. Samples were obtained from 760 NTD case-parent triads included in the National Birth Defects Prevention Study (NBDPS). The three SNPs were genotyped using TaqMan® SNP assays. An extension of the log-linear model was used to assess the association between NTDs and both offspring and maternal haplotypes. Offspring carrying two copies of haplotype C-T-C had a significantly increased NTD risk (risk ratio [RR] = 3.40, 95% confidence interval [CI]: 1.02–11.50), after adjusting for the effect of the maternal haplotype. Additionally, mothers carrying two copies of haplotype C-T-C had a significantly increased risk of having an NTD-affected offspring (RR = 3.46, 95% CI: 1.05–11.00), after adjusting for the effect of the offspring haplotype. These results suggest offspring and maternal variation in the GCH1 gene and altered BH4 biosynthesis may contribute to NTD risk.
PMCID: PMC3704723  PMID: 23059057
GCH1 gene; GTP cyclohydrolase I; haplotype; neural tube defects; tetrahydrobiopterin (BH4)
19.  Maternal and Infant Gene-Folate Interactions and the Risk of Neural Tube Defects 
Neural tube defects (NTDs) are common, serious malformations with a complex etiology that suggests involvement of both genetic and environmental factors. The authors evaluated maternal or offspring folate-related gene variants and interactions between the gene variants and maternal intake of folates on the risk of NTDs in their offspring. A case-control study was conducted on mothers and/or their fetuses and infants who were born in California from 1999–2003 with an NTD (cases n = 222, including 24 mother-infant pairs) or without a major malformation (controls n = 454, including 186 mother-infant pairs). Maternal intake of folates was assessed by food frequency questionnaire and genotyping was performed on samples from mothers and infants. For mothers in the lowest folate-intake group, risk of NTDs in offspring was significantly decreased for maternal MTHFR SNPs rs1476413, rs1801131 and rs1801133 (odds ratio (OR) = 0.55, 80% confidence interval (CI): 0.20, 1.48; OR = 0.58, 80% CI: 0.24, 1.43; OR = 0.69, 80% CI: 0.41, 1.17, respectively), and TYMS SNPs rs502396 and rs699517 (OR= 0.91, 80% CI: 0.53, 1.56; OR = 0.70, 80% CI: 0.38, 1.29). A gene-only effect was observed for maternal SHMT1 SNP rs669340 (OR = 0.69, 95% CI: 0.49, 0.96). When there was low maternal folate intake, risk of NTDs was significantly increased for infant MTHFD1 SNPs rs2236224, rs2236225 and rs11627387 (OR = 1.58, 80% CI: 0.99, 2.51; OR = 1.53, 80% CI: 0.95, 2.47; OR = 4.25, 80% CI: 2.33, 7.75, respectively) and SHMT1 SNP rs12939757 (OR = 2.01, 80% CI: 1.20, 3.37), but decreased for TYMS SNP rs2847153 (OR = 0.73, 80% CI: 0.37, 1.45). Although power to detect interaction effects was low for this birth defects association study, the gene-folate interactions observed in this study represent preliminary findings that will be useful for informing future studies on the complex etiology of NTDs.
PMCID: PMC3448841  PMID: 22903727
Congenital Abnormalities; Folic Acid; Genetic Association Studies; Molecular Epidemiology; Neural Tube Defects; Maternal Nutritional Physiological Phenomena; Nervous System Malformations; Nutrigenomics
20.  Transcriptional Analyses of Two Mouse Models of Spina Bifida 
Spina bifida is one of the most common of all human structural birth defects. Despite considerable effort over several decades, the causes and mechanisms underlying this malformation remain poorly characterized. In order to better understand the pathogenesis of this abnormality, we conducted a microarray study using Mouse Whole Genome Bioarrays which have ~36,000 gene targets, to compare gene expression profiles between two mouse models; CXL-Splotch and Fkbp8Gt(neo)which express a similar spina bifida phenotype. We anticipated that there would be a collection of overlapping genes or shared genetic pathways at the molecular level indicative of a common mechanism underlying the pathogenesis of spina bifida during embryonic development.
A total of 54 genes were determined to be differentially expressed (25 down regulated, 29 upregulated) in the Fkbp8Gt(neo) mouse embryos; while 73 genes were differentially expressed (56 down regulated, 17 upregulated) in the CXL-Splotch mouse relative to their wildtype controls. Remarkably, the only two genes that showed decreased expression in both mutants were v-ski sarcoma viral oncogene homolog (Ski), and Zic1, a transcription factor member of the zinc finger family. Confirmation analysis using real time qRT-PCR indicated that only Zic1 was significantly decreased in both mutants. Gene Ontology analysis revealed striking enrichment of genes associated with mesoderm and central nervous system development in the CXL-Splotch mutant embryos, whereas in the Fkbp8Gt(neo) mutants, the genes involved in dorsal/ventral pattern formation, cell fate specification, and positive regulation of cell differentiation were most likely to be enriched. These results indicate that there are multiple pathways and gene networks perturbed in mouse embryos with shared phenotypes.
PMCID: PMC3505988  PMID: 23024056
spina bifida; gene expression; microarray; mouse mutants
21.  C5a Receptor Signaling Prevents Folate Deficiency-Induced Neural Tube Defects in Mice 
The complement system is involved in a range of diverse developmental processes including cell survival, growth, differentiation, and regeneration. However, little is known about the role of complement in embryogenesis. Herein we demonstrate a novel role for the canonical complement 5a receptor (C5aR) in the development of the mammalian neural tube under conditions of maternal dietary folic acid deficiency. Specifically, we found C5aR and C5 to be expressed throughout the period of neurulation in wildtype mice and localized the expression to the cephalic regions of the developing neural tube. C5aR was also found to be expressed in the neuroepithelium of early human embryos. Ablation of the C5ar1 gene or the administration of a specific C5aR peptide antagonist to folic acid-deficient pregnant mice resulted in a high prevalence of severe anterior neural tube defect-associated congenital malformations. These findings provide a new and compelling insight into the role of the complement system during mammalian embryonic development.
PMCID: PMC3608813  PMID: 23420882
22.  Thymidylate Synthase (TYMS) Polymorphisms and Risk of Conotruncal Heart Defects 
In this study, we investigated whether the two TYMS functional variants (28bp VNTR and 1494del6) (275 cases and 653 controls) and six selected SNPs (265 case infants, 535 control infants; 169 case mothers and 276 control mothers) were associated with risks of conotruncal heart defects. Further, we evaluated interaction effects between these gene variants and maternal folate intake for risk of CTD. Cases with diagnosis of single gene disorders or chromosomal aneusomies were excluded. Controls were randomly selected from area hospitals in proportion to their contribution to the total population of live-born infants. DNA samples were collected using buccal brushes or drawn from the repository of newborn screening blood specimens when available. Genetic variants were treated as categorical variables (homozygous referent, heterozygote, homozygous variant). Odds ratios and 95% confidence intervals (CI) were computed to estimate risks among all subjects, Hispanic and non-Hispanic whites, respectively using logistic regression. Gene-folate interactions were assessed for these variants by adding an interaction term to the logistic model. A dichotomized composite variable, “combined folate intake”, was created by combining maternal peri-conceptional use of folic acid-containing vitamin supplements with daily dietary intake of folate. In general, the results do not show strong gene-only effects on risk of CTD. We did, however, observe a 3.6-fold increase in CTD risk (95%CI:1.1 – 11.9) among infants who were homozygotes for the 6bp deletion in the 3’-untranslated region (UTR) (1694del6) and whose mothers had low folate intake during the peri-conceptional period.
PMCID: PMC3429723  PMID: 22887475
thymidylate synthase; TYMS; congenital heart defects; conotruncal defects; folate
23.  Antiepileptic Drugs and Pregnancy Outcomes 
The treatment of epilepsy in women of reproductive age remains a clinical challenge. While most women with epilepsy require anticonvulsant drugs for adequate control of their seizures, the teratogenicity associated with some antiepileptic drugs is a risk that needs to be carefully addressed. Antiepileptic medications are also used to treat an ever broadening range of medical conditions such as bipolar disorder, migraine prophylaxis, cancer and neuropathic pain. Despite the fact that the majority of pregnancies of women with epilepsy who are receiving pharmacological treatment are normal, studies have demonstrated that the risk of having a pregnancy complicated by a major congenital malformation is doubled when comparing the risk of untreated pregnancies. Furthermore, when antiepileptic drugs (AEDs) are used in polytherapy regimens, the risk is tripled, especially when valproic acid (VPA) is included. However, it should be noted that the risks are specific for each anticonvulsant drug. Some investigations have suggested that the risk of teratogenicity is increased in a dose-dependent manner. More recent studies have reported that in utero exposure to AEDs can have detrimental effects on the cognitive functions and language skills in later stages of life. In fact, the FDA just issued a safety announcement on the impact of VPA on cognition (Safety Announcement 6-30-2011). The purpose of this document is to review the most commonly used compounds in the treatment of women with epilepsy, and to provide information on the latest experimental and human epidemiological studies of the effects of antiepileptic drugs in the exposed embryos.
PMCID: PMC3402584  PMID: 22711424
antiepileptic drugs; teratogenicity; pregnancy; birth defects; epilepsy
24.  Mutations in Planar Cell Polarity Gene SCRIB Are Associated with Spina Bifida 
PLoS ONE  2013;8(7):e69262.
Neural tube defects (NTDs) (OMIM #182940) including anencephaly, spina bifida and craniorachischisis, are severe congenital malformations that affect 0.5–1 in 1,000 live births in the United States, with varying prevalence around the world. Mutations in planar cell polarity (PCP) genes are believed to cause a variety of NTDs in both mice and humans. SCRIB is a PCP-associated gene. Mice that are homozygous for the Scrib p.I285K and circletail (Crc) mutations, present with the most severe form of NTDs, namely craniorachischisis. A recent study reported that mutations in SCRIB were associated with craniorachischisis in humans, but whether SCRIB mutations contribute to increased spina bifida risk is still unknown. We sequenced the SCRIB gene in 192 infants with spina bifida and 190 healthy controls. Among the spina bifida patients, we identified five novel missense mutations that were predicted-to-be-deleterious by the PolyPhen software. Of these five mutations, three of them (p.P1043L, p.P1332L, p.L1520R) significantly affected the subcellular localization of SCRIB. In addition, we demonstrated that the craniorachischisis mouse line-90 mutation I285K, also affected SCRIB subcellular localization. In contrast, only one novel missense mutation (p.A1257T) was detected in control samples, and it was predicted to be benign. This study demonstrated that rare deleterious mutations of SCRIB may contribute to the multifactorial risk for human spina bifida.
PMCID: PMC3724847  PMID: 23922697
25.  Fuz Mutant Mice Reveal Shared Mechanisms between Ciliopathies and FGF-Related Syndromes 
Developmental Cell  2013;25(6):623-635.
Ciliopathies are a broad class of human disorders with craniofacial dysmorphology as a common feature. Among these is high arched palate, a condition that affects speech and quality of life. Using the ciliopathic Fuz mutant mouse, we find that high arched palate does not, as commonly suggested, arise from midface hypoplasia. Rather, increased neural crest expands the maxillary primordia. In Fuz mutants, this phenotype stems from dysregulated Gli processing, which in turn results in excessive craniofacial Fgf8 gene expression. Accordingly, genetic reduction of Fgf8 ameliorates the maxillary phenotypes. Similar phenotypes result from mutation of oral-facial-digital syndrome 1 (Ofd1), suggesting that aberrant transcription of Fgf8 is a common feature of ciliopathies. High arched palate is also a prevalent feature of fibroblast growth factor (FGF) hyperactivation syndromes. Thus, our findings elucidate the etiology for a common craniofacial anomaly and identify links between two classes of human disease: FGF-hyperactivation syndromes and ciliopathies.
•A genetic model for high arched palate, commonly seen in human craniofacial syndromes•In ciliopathic mice, Fgf8 overexpression leads to cranial neural crest hyperplasia•Enlargement of the maxillary primordia underlies high arched palate in Fuz mutants•An etiological link between ciliopathies and FGF-hyperactivation syndromes
High arched palate is common to many human disorders, including ciliopathies and craniosynostosis syndromes. Tabler et al. develop and analyze a genetic model of high arched palate; they conclude that embryonic changes in neural crest and fibroblast growth factor signaling underlie this unusual phenotype.
PMCID: PMC3697100  PMID: 23806618

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