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1.  IS LOW IRON STATUS A RISK FACTOR FOR NEURAL TUBE DEFECTS? 
Background
Folic acid supplements can protect against neural tube defects (NTDs). Low folate and low vitamin B12 status may be maternal risk factors for having an NTD affected pregnancy. However, not all NTDs are preventable by having an adequate folate/ B12 status and other potentially modifiable factors may be involved. Folate and vitamin B12 status have important links to iron metabolism. Animal studies support an association between poor iron status and NTDs but human data are scarce. We examined the relevance of low iron status in a nested NTD case-control study of women within a pregnant population-based cohort.
Methods
Pregnant women were recruited between 1986 and 1990, when vitamin or iron supplementation in early pregnancy was rare. Blood samples, taken at an average of 14 weeks gestation, were used to measure ferritin and hemoglobin in 64 women during an NTD affected pregnancy and 207 women with unaffected pregnancies.
Results
No significant differences in maternal ferritin or hemoglobin concentrations were observed between NTD affected and non-affected pregnancies (case median ferritin 16.8μg/L and hemoglobin 12.4g/dL versus 15.4μg/L and 12.3g/dL in controls). As reported previously, red cell folate and vitamin B12 concentrations were significantly lower in cases. Furthermore, there was no significant association of iron status with type of NTD lesion (anencephaly or spina bifida)
Conclusions
We conclude that low maternal iron status during early pregnancy is not an independent risk factor for NTDs. Adding iron to folic acid for periconceptional use may improve iron status but is not likely to prevent NTDs.
doi:10.1002/bdra.23223
PMCID: PMC4018583  PMID: 24535840
ferritin; iron; hemoglobin; neural tube defects
2.  Conotruncal Heart Defects and Common Variants in Maternal and Fetal Genes in Folate, Homocysteine and Transsulfuration Pathways 
Background
We investigated the association between conotruncal heart defects (CTDs) and maternal and fetal single nucleotide polymorphisms (SNPs) in 60 genes in the folate, homocysteine and pathways. We also investigated whether periconceptional maternal folic acid supplementation modified associations between CTDs and SNPs.
Methods
Participants were enrolled in the National Birth Defects Prevention Study between 1997 and 2007. DNA samples from 616 case-parental triads affected by CTDs and 1,645 control-parental triads were genotyped using an Illumina® Golden Gate custom SNP panel. A hybrid design analysis, optimizing data from case and control trios, was used to identify maternal and fetal SNPs associated with CTDs.
Results
Among 921 SNPs, 17 maternal and 17 fetal SNPs had a Bayesian false-discovery probability (BFDP) of <0.8. Ten of the 17 maternal SNPs and 2 of the 17 fetal SNPs were found within the glutamate-cysteine ligase, catalytic subunit (GCLC) gene. Fetal SNPs with the lowest BFDP (rs2612101, rs2847607, rs2847326, rs2847324) were found within the thymidylate synthetase (TYMS) gene. Additional analyses indicated that the risk of CTDs associated with candidate SNPs was modified by periconceptional folic acid supplementation. Nineteen maternal and 9 fetal SNPs had BFDP <0.8 for gene-by-environment (GxE) interactions with maternal folic acid supplementation.
Conclusions
These results support previous studies suggesting that maternal and fetal SNPs within folate, homocysteine and transsulfuration pathways are associated with CTD risk. Maternal use of supplements containing folic acid may modify the impact of SNPs on the developing heart.
doi:10.1002/bdra.23225
PMCID: PMC4118819  PMID: 24535845
conotruncal heart defects; single nucleotide polymorphisms; oxidative stress; genetics; folic acid; gene X environment interaction
3.  One-carbon metabolite levels in mid-pregnancy and risks of conotruncal heart defects 
Background
Evidence exists for an association between use of vitamin supplements with folic acid in early pregnancy and reduced risk for offspring with conotruncal heart defects. A few observations have been made about nutrients related to one-carbon metabolism other than folate. Our prospective study attempted to extend information on nutrition and conotruncal heart defects by measuring analytes in mid-pregnancy sera.
Methods
This study included data from a repository of women’s mid-pregnancy serum specimens based on screened pregnancies in California from 2002–07. Each woman’s specimen was linked with delivery information to determine whether her fetus had a conotruncal heart defect or another structural malformation, or was nonmalformed. We identified 140 conotruncal cases and randomly selected 280 specimens as nonmalformed controls. Specimens were tested for a variety of analytes including: homocysteine, methylmalonic acid, folate, vitamin B12, pyridoxal phosphate, pyridoxal, pyridoxic acid, riboflavin, total choline, betaine, methionine, cysteine, cystathionine, arginine, asymmetric and symmetric dimethylarginine.
Results and Conclusions
We did not observe statistical evidence for substantial differences between cases and controls for any of the measured analytes. Analyses specifically targeting B-vitamins also did not reveal differences between cases and controls.
doi:10.1002/bdra.23224
PMCID: PMC4156397  PMID: 24532477
heart defects; nutrition; pregnancy; folic acid; B vitamins
4.  Oral Cleft Recurrence Risk and Subsequent Maternal Fertility Preferences and Behavior in Brazil 
Background
Oral clefts are among the most common birth defects with numerous impacts on affected individuals and families. However, little is known about how being at a greater risk of having an affected child affects subsequent maternal fertility decisions. We investigated differences in fertility preferences and behavior between mothers who are themselves affected with cleft lip with/without cleft palate but have had no affected children and unaffected mothers of an affected child. We also compared these outcomes between unaffected mothers of a first versus another affected child.
Methods
The sample included 1475 Brazilian women interviewed between 2004 and 2009. The outcomes were wanting more children, contraceptive use and type, and maternal age at first child. Comparisons between the various maternal groups were performed using regression analysis adjusting for conceptually relevant demographic, socioeconomic, and geographic factors.
Results
Affected mothers of unaffected children were less likely to use contraceptives than unaffected mothers of affected children by 31% [95% CI: 1–53%]. Among unaffected mothers, those who had a first affected child were 67% [95% CI: 15–144%] more likely to use contraceptives.
Conclusions
The results suggest that having an affected child represents a stronger signal of recurrence risk to the mother than her own cleft status, and that cleft status of the first child is especially important in influencing subsequent maternal fertility decisions in affected families. These findings highlight the importance of adequate counseling of at-risk women about recurrence risks and available care resources and policies that improve access to quality cleft care.
doi:10.1002/bdra.23214
PMCID: PMC4103876  PMID: 24382743
birth defects; oral clefts; fertility; contraception; family planning
5.  Epigenomic Reprogramming of the Developing Reproductive Tract and Disease Susceptibility in Adulthood 
During development, epigenetic programs are “installed” on the genome that direct differentiation and normal tissue and organ function in adulthood. Consequently, development is also a period of susceptibility to reprogramming of the epigenome. Developmental reprogramming occurs when an adverse stimulus or insult interrupts the proper “install” of epigenetic programs during development, reprogramming normal physiological responses in such a way as to promote disease later in life. Some of the best examples of developmental reprogramming involve the reproductive tract, where early life exposures to environmental estrogens can increase susceptibility to benign and malignant tumors in adulthood including leiomyoma (fibroids), endometrial and prostate cancer. Although specific mechanism(s) by which environmental estrogens reprogram the developing epigenome were unknown, both DNA and histone methylation were considered likely targets for epigenetic reprogramming. We have now identified a mechanism by which developmental exposures to environmental estrogens reprogram the epigenome by inducing inappropriate activation of nongenomic estrogen receptor (ER) signaling. Activation of non-genomic ER signaling via the PI3K pathway activates the kinase AKT/PKB in the developing reproductive tract, which phosphorylates the histone lysine methyltransferase (HKMT) EZH2, the key “installer” of epigenetic histone H3 lysine 27 trimethylation (H3K27me3). AKT phosphorylation inactivates EZH2, decreasing levels of H3K27 methylation, a repressive mark that inhibits gene expression, in the developing uterus. As a result of this developmental reprogramming, many estrogen-responsive genes become hypersensitive to estrogen in adulthood, exhibiting elevated expression throughout the estrus cycle, and resulting in a “hyper-estrogenized” phenotype in the adult uterus that promotes development of hormone dependent tumors.
doi:10.1002/bdra.20827
PMCID: PMC4266586  PMID: 21656660
Epigenetics; Leiomyoma; Uterus; Prostate; reproductive tract; environmental exposures; estrogen; histone methyltransferase
6.  Traffic-Related Air Pollution and Selected Birth Defects in the San Joaquin Valley of California 
BACKGROUND
Birth defects are a leading cause of infant morbidity and mortality. Studies suggest associations between environmental contaminants and some structural anomalies, although evidence is limited and several anomalies have not been investigated previously.
METHODS
We used data from the California Center of the National Birth Defects Prevention Study and the Children's Health and Air Pollution Study to estimate the odds of 26 congenital birth defect phenotypes with respect to quartiles of seven ambient air pollutant and traffic exposures in California during the first 2 months of pregnancy, 1997 to 2006 (874 cases and 849 controls). We calculated odds ratios (adjusted for maternal race/ethnicity, education, and vitamin use; aOR) for 11 phenotypes that had at least 40 cases.
RESULTS
Few odds ratios had confidence intervals that did not include 1.0. Odds of esophageal atresia were increased for the highest versus lowest of traffic density (aOR = 2.8, 95% confidence interval [CI], 1.1–7.4) and PM10 exposure (aOR 4.9; 95% CI, 1.4–17.2). PM10 was associated with a decreased risk of hydrocephaly (aOR= 0.3; 95% CI, 0.1–0.9) and CO with decreased risk of anotia/microtia (aOR = 0.4; 95% CI, 0.2–0.8) and transverse limb deficiency (aOR = 0.4; 95% CI, 0.2–0.9), again reflecting highest versus lowest quartile comparisons.
CONCLUSION
Most analyses showed no substantive association between air pollution and the selected birth defects with few exceptions of mixed results.
doi:10.1002/bdra.23175
PMCID: PMC4264633  PMID: 24108522
congenital anomalies; air pollution; traffic; birth outcomes
7.  Dermatan sulfate epimerase 1 deficient mice as a model for human abdominal wall defects 
Background
Dermatan sulfate (DS) is a highly sulfated polysaccharide with a variety of biological functions in extracellular matrix organization and processes such as tumorigenesis and wound healing. A distinct feature of DS is the presence of iduronic acid, produced by the two enzymes, DS-epimerase 1 and 2, which are encoded by Dse and Dsel, respectively.
Methods
We have previously shown that Dse knockout (KO) mice in a mixed C57BL/6–129/SvJ background have an altered collagen matrix structure in skin. In the current work we studied Dse KO mice in a pure NFR genetic background.
Results
Dse KO embryos and newborns had kinked tails and histological staining revealed significantly thicker epidermal layers in Dse KO mice when compared with heterozygote (Het) or wild-type (WT) littermates. Immunochemical analysis of the epidermal layers in newborn pups showed increased expression of keratin 5 in the basal layer and keratin 1 in the spinous layer. In addition, we observed an abdominal wall defect with herniated intestines in 16% of the Dse KO embryos. Other, less frequent, developmental defects were exencephaly and spina bifida.
Conclusion
We conclude that the combination of defective collagen structure in the dermis and imbalanced keratinocyte maturation could be responsible for the observed developmental defects in Dse KO mice. In addition, we propose that Dse KO mice could be used as a model in pathogenetic studies of human fetal abdominal wall defects. Birth Defects Research (Part A) 100:712–720, 2014. © 2014 Wiley Periodicals, Inc.
doi:10.1002/bdra.23300
PMCID: PMC4233991  PMID: 25186462
abdominal wall defect; neural tube defects; embryonic development; dermatan sulfate epimerase 1; epidermis
8.  AGRIN FUNCTION ASSOCIATED WITH OCULAR DEVELOPMENT IS A TARGET OF ETHANOL EXPOSURE IN EMBRYONIC ZEBRAFISH 
BACKGROUND
Alcohol (ethanol) is a teratogen known to affect the developing eyes, face and brain. Among the ocular defects in fetal alcohol spectrum disorder (FASD) are microphthalmia and optic nerve hypoplasia. Employing zebrafish as an FASD model provides an excellent system to analyze the molecular basis of prenatal ethanol exposure-induced defects since embryos can be exposed to ethanol at defined developmental stages and affected genetic pathways can be examined. We have previously shown that disruption of agrin function in zebrafish embryos produces microphthalmia and optic nerve hypoplasia.
METHODS
Zebrafish embryos were exposed to varying concentrations of ethanol in the absence or presence of morpholino oligonucleotides (MOs) that disrupt agrin function. In situ hybridization was employed to analyze ocular gene expression as a consequence of ethanol exposure and agrin knockdown. Morphological analysis of zebrafish embryos was also conducted.
RESULTS
Acute ethanol exposure induces diminished agrin gene expression in zebrafish eyes and, importantly, combined treatment with subthreshold levels of agrin MO and ethanol produces pronounced microphthalmia, markedly reduces agrin gene expression, and perturbs Pax6a andMbx gene expression. Microphthalmia produced by combined agrin MO and ethanol treatment was rescued by sonic hedgehog (Shh) mRNA overexpression, suggesting that ethanol-mediated disruption of agrin expression results in disrupted Shh function.
CONCLUSIONS
These studies illustrate the strong potential for using zebrafish as a model to aid in defining the molecular basis for ethanol's teratogenic effects. The results of this work suggest that agrin expression and function may be a target of ethanol exposure during embryogenesis.
doi:10.1002/bdra.20766
PMCID: PMC4230012  PMID: 21308976
heparan sulfate proteoglycan; zebrafish; fetal alcohol spectrum disorder; ocular development; sonic hedgehog; Mbx; Pax6
9.  FOREBRAIN AND HINDBRAIN DEVELOPMENT IN ZEBRAFISH IS SENSITIVE TO ETHANOL EXPOSURE INVOLVING AGRIN, FGF AND SONIC HEDGEHOG FUNCTION 
BACKGROUND
Ethanol is a teratogen that affects numerous developmental processes in the nervous system, which includes development and survival of GABAergic and glutamatergic neurons. Possible molecular mechanisms accounting for ethanol’s effects on nervous system development include perturbed fibroblast growth factor (Fgf) and Sonic hedgehog (Shh) signaling. In zebrafish, forebrain GABAergic neuron development is dependent on Fgf19 and Shh signaling. The present study was conducted to test the hypothesis that ethanol affects GABAergic and glutamatergic neuron development by disrupting Fgf, Shh, and agrin function.
METHODS
Zebrafish embryos were exposed to varying concentrations of ethanol during a range of developmental stages, in the absence or presence of morpholino oligonucleotides (MOs) that disrupt agrin or Shh function. In situ hybridization was employed to analyze glutamic acid decarboxylase (GAD1) gene expression, as well as markers of glutamatergic neurons.
RESULTS
Acute ethanol exposure results in marked reduction in GAD1 gene expression in forebrain and hindbrain, and reduction of glutamatergic neuronal markers in hindbrain. Subthreshold ethanol exposure, combined with agrin or Shh MO treatment, produces a similar diminution in expression of markers for GABAergic and glutamatergic neurons. Consistent with the ethanol effects on Fgf and Shh pathways, Fgf19, Fgf8 or Shh mRNA overexpression rescues ethanol-induced decreases in GAD1 and atonal1a gene expression.
CONCLUSIONS
These studies demonstrate that GABAergic and glutamatergic neuron development in zebrafish forebrain or cerebellum is sensitive to ethanol exposure, and provides additional evidence that a signaling pathway involving agrin, Fgfs and Shh may be a critical target of ethanol exposure during zebrafish embryogenesis.
doi:10.1002/bdra.23099
PMCID: PMC4230296  PMID: 23184466
heparan sulfate proteoglycan; fetal alcohol spectrum disorder; GAD1; Sonic hedgehog; Fgf; alcohol
10.  USE OF A MURINE EMBRYONIC STEM CELL LINE THAT IS SENSITIVE TO HIGH GLUCOSE ENVIRONMENT TO MODEL NEURAL TUBE DEVELOPMENT IN DIABETIC PREGNANCY 
Background
Neural tube defects (NTDs) are significantly increased by maternal diabetes. Embryonic stem cells (ESC) that can differentiate into neuroepithelium and can sense supraphysiological glucose concentrations would be very valuable to simulate the effects of maternal diabetes on molecular and cellular processes during neural tube formation.
Methods
LG-ESC, a recently established ESC line that expresses the glucose transporter, Scl2a2, and is sensitive to elevated glucose concentrations, were grown for up to 8 days in a 3-dimensional culture to form neural cysts (NC). We tested whether high glucose media inhibits expression of Pax3, a gene that is required for neural tube closure and whose expression is inhibited in embryos of diabetic mice, and inhibits formation of NC.
Results
Pax3 expression was detected after 4 days of culture and increased with time. Pax3 expression was inhibited by high glucose media, but not if cells had been cultured in low glucose media for the first 4 days of culture. Pax7, which is also expressed in dorsal neural tube, was not detected. Pax6, which is expressed in the ventral neural tube, was detected only after 8 days of culture, but was not inhibited by high glucose. High glucose media did not inhibit formation of NC.
Conclusions
LG-ESC can be used as a model of embryonic exposure to a diabetic environment during neural tube development. While high glucose exposure inhibits expression of a gene required for neural tube closure, it may not inhibit all of the processes involved in formation of a neural tube-like structure.
doi:10.1002/bdra.23281
PMCID: PMC4220291  PMID: 25124397
Embryonic stem cells; Scl2a2; Glut2; hyperglycemia; neural tube defect; pregestational diabetic pregnancy; Pax3
11.  Age of Onset and Effect Size in Genome-Wide Association Studies 
BACKGROUND
Genome-wide association studies (GWAS) have identified many susceptibility loci for complex traits, but have not identified the majority of the genetic contribution to common diseases. We explored whether the magnitude of associations detected in GWAS and, therefore, the likelihood of detecting a significant association for a given sample size, is generally greater for childhood-onset traits (e.g., birth defects) than for traits with onset in adulthood.
METHODS
Data were obtained from the National Human Genome Research Institute Catalog of Published GWAS. Traits were categorized as having an average age of onset in childhood (<18 years, n = 15 traits), early adulthood (18–54 years, n = 32 traits), or late adulthood (≥55 years, n = 31 traits). The relationship between age of onset category and the magnitude of significant associations detected by GWAS was assessed using logistic regression.
RESULTS
Associations characterized by an odds ratio (OR) ≥1.5 were significantly more common for GWAS of childhood traits than for late adulthood-onset traits after adjustment for several covariates (adjusted OR, 2.55; 95% confidence interval, 1.37–4.73). Results in subgroup analyses using more stringent inclusion criteria (based on sample size, effect size, p value threshold for inclusion, and novel variant-trait associations) were similar.
CONCLUSIONS
These findings suggest that, on average, marker-trait associations detected in GWAS for traits with young onset may have a larger magnitude of effect than those for traits with adult onset. Therefore, GWAS for young-onset traits, such as birth defects, may be more likely than those for adult-onset traits to identify major genetic risk factors.
doi:10.1002/bdra.23066
PMCID: PMC4219508  PMID: 22933422
age of onset; genome-wide association study; epidemiology; genetics; polymorphism; single nucleotide
12.  Risk Factors and Demographics for Microtia in South America: a Case-Control Analysis 
BACKGROUND
The etiopathogenesis of microtia is still unknown in the majority of the cases, particularly for individuals presenting with isolated microtia. Our aim was to evaluate potential risk factors for this condition using a case-control approach.
METHODS
We analyzed data from 1,194 livebirths with isolated microtia enrolled in the ECLAMC study (Estudio Colaborativo Latino Americano de Malformaciones Congénitas) from 1982 to 2011 and their respective controls. Odds ratios were estimated with conditional logistic regression models along with 95% confidence intervals for the resulting odds ratio estimates controlling for the effects of potential confounders (sex, maternal age, hospital and year of birth) for an adjusted OR (aOR).
RESULTS
Multiparity was associated with a higher risk of microtia compared to primiparity (aOR 1.5, 95%CI 1.2–1.8), with women who had eight or more prior pregnancies having the highest risk (aOR 2.8, 95%CI 1.6–5.2). Women who presented with cold-like symptoms were at higher risk for microtia (aOR 2.2, 95%CI 1.2–3.9) as well as those that used tobacco or alcohol during pregnancy (aOR 1.7, 95%CI 1.1–2.5 and aOR 1.4, 95%CI 0.9–2.1, respectively). The association with alcohol use appeared to be limited to those women who reported binge drinking during pregnancy (aOR 1.4, 95% 0.7–2.9). Cases from hospitals at low altitude (< 2,500 m) tended to have more severe types of microtia than those from hospitals at high altitude.
CONCLUSIONS
These results support the hypothesis that in addition to teratogens other non-genetic risk factors contribute to the occurrence of isolated microtia.
doi:10.1002/bdra.23193
PMCID: PMC4098829  PMID: 24265127
microtia; anotia; ear; epidemiology; non-genetic risk factors
13.  Missing Genetic Risk in Neural Tube Defects: Can Exome Sequencing Yield an Insight? 
Background
Neural tube defects (NTD) have a strong genetic component, with up to 70% of variance in human prevalence determined by heritable factors. Although the identification of causal DNA variants by sequencing candidate genes from functionally relevant pathways and model organisms has provided some success, alternative approaches are demanded.
Methods
Next generation sequencing platforms are facilitating the production of massive amounts of sequencing data, primarily from the protein coding regions of the genome, at a faster rate and cheaper cost than has previously been possible. These platforms are permitting the identification of variants (de novo, rare, and common) that are drivers of NYTD etiology, and the cost of the approach allows for the screening of increased numbers of affected and unaffected individuals from NTD families and in simplex cases.
Conclusion
The next generation sequencing platforms represent a powerful tool in the armory of the genetics researcher to identify the causal genetic basis of NTDs.
doi:10.1002/bdra.23276
PMCID: PMC4169137  PMID: 25044326
next generation exome sequencing; de novo; rare and common variation; compound heterozygosity
14.  High Density SNP Screen in A Large Multiplex Neural Tube Defect Family Refines Linkage to Loci at 7p21-Pter And 2q33.1-35 
BACKGROUND
Neural tube defects (NTDs) are considered complex with both genetic and environmental factors implicated. To date, no major causative genes have been identified in humans despite several investigations. The first genomewide screen in NTDs (Rampersaud et al. 2005) demonstrated evidence of linkage to chromosomes 7 and 10. This screen included forty-four multiplex families and consisted of 402 microsatellite markers spaced approximately 10 cM apart. Further investigation of the genomic screen data identified a single large multiplex family, pedigree 8776, as primarily driving the linkage results on chromosome 7.
METHODS
To investigate this family more thoroughly, a high-density single nucleotide polymorphism (SNP) screen was performed. Two-point and multipoint linkage analyses were performed using both parametric and nonparametric methods.
RESULTS
For both the microsatellite and SNP markers, linkage analysis suggested the involvement of a locus or loci proximal to the telomeric regions of chromosomes 2q and 7p, with both regions having nonparametric lod* scores of ~3.0, yielding very similar evidence in favor of linkage.
CONCLUSIONS
The regions of strongest evidence for linkage map proximal to the telomeres on these two chromosomes. In addition to mutations and/or variants in a major gene, these loci may harbor a microdeletion and/or translocation; potentially, polygenic factors may also be involved. This single family may be promising for narrowing the search for NTD susceptibility genes.
doi:10.1002/bdra.20272
PMCID: PMC4169147  PMID: 16933213
Neural tube defects (NTDs); spina bifida; genetic mapping; linkage; genome screen
15.  Malformations in Infants of Diabetic Mothers 
Maternal insulin-dependent diabetes has long been associated with congenital malformations. As other causes of mortality and morbidity have been eliminated or reduced, malformations have become increasingly prominent. Although there is not universal agreement, the great majority of investigators find a two- to threefold increase in malformations in infants of insulin-dependent diabetic mothers. This increase is not seen in infants of gestational diabetics. It probably is not present in women whose diabetes can be controlled by diet or oral hypoglycemic agents. The risk does not appear to be primarily genetic since diabetic fathers do not have an increased number of malformed offspring. Most studies show a generalized increase in malformations involving multiple organ systems. Multiple malformations seem to be more common in diabetic than non-diabetic infants. Caudal regression has the strongest association with diabetes, occurring roughly 200 times more frequently in infants of diabetic mothers than in other infants. The teratogenic mechanism in diabetes is not known. Hyperglycemia may be important but human studies focusing on the period of organogenesis are lacking. Hypoglycemia has also been suggested based mainly on animal experiments. Insulin appears unlikely. Numerous other factors including vascular disease, hypoxia, ketone and amino acid abnormalities, glycosylation of proteins, or hormone imbalances could be teratogenic. None has been studied in sufficient detail to make a judgment. A large-scale prospective study is required to determine early fetal loss rates, correlate metabolic status during organogenesis with outcome, and assess the effect of diabetic control on malformation rates.
doi:10.1002/bdra.20757
PMCID: PMC4158942  PMID: 20973049
16.  Exon Sequencing of PAX3 and T (Brachyury) in Cases with Spina Bifida 
BACKGROUND
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.
METHODS
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.
RESULTS
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.
CONCLUSION
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.
doi:10.1002/bdra.23163
PMCID: PMC3877942  PMID: 23913553
spina bifida; myelomeningocele; genetic epidemiology; sequencing; PAX3; T locus
17.  Nonsyndromic cleft lip and palate: CRISPLD Genes and the Folate Gene Pathway Connection 
Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect that has a multifactorial etiology. Despite having substantial genetic liability, less than 15% of the genetic contribution to NSCLP has been delineated. In our efforts to dissect the genetics of NSCLP, we found that variation in the CRISPLD2 (cysteine rich secretory protein LCCL domain containing 2) gene is associated with NSCLP and that the protein is expressed in the developing murine craniofacies. In addition, we found suggestive linkage of NSCLP (LOD>1.0) to the chromosomal region on 8q13.2-21.13 that contains the CRISPLD1 gene. The protein products of both CRISPLD1 and CRISPLD2 contain more cysteine residues than comparably sized proteins. Interestingly, the folic acid pathway produces endogenous cysteines, and variation in genes in this pathway is associated with NSCLP. Based on these observations, we hypothesized that variation in CRISPLD1 contributes to NSCLP and that both CRISPLD genes interact with each other and genes in the folic acid pathway. SNPs in CRISPLD1 were genotyped in our nonHispanic white and Hispanic multiplex and simplex NSCLP families. There was little evidence for a role of variation for CRISPLD1 alone in NSCLP. However, interactions were detected between CRISPLD1/CRISPLD2 SNPs and variation in folate pathway genes. Altered transmission of one CRISPLD1 SNP was detected in the nonHispanic white simplex families. Importantly, interactions were detected between SNPs in CRISPLD1 and CRISPLD2 (15 interactions, 0.0031≤p<0.05). These novel findings suggest that CRISPLD1 plays a role in NSCLP through the interaction with CRISPLD2 and folate pathway genes.
doi:10.1002/bdra.20737
PMCID: PMC4142894  PMID: 21254358
18.  Folic Acid Supplementation Use and the MTHFR C677T Polymorphism in Orofacial Clefts Etiology: An Individual Participant Data Pooled-Analysis 
Background
This study examines gene-environment interaction (GEI) between the MTHFR C667T polymorphism and folic acid in the etiology of orofacial clefts (OFC). We used a pooled-analyticapproach on four studies that used similar methods.
Methods
We used logistic regression to analyse the pooled sample of 1149 isolated cases and 1161 controls. Fetal and maternal MTHFR C677T genotypes, and maternal periconceptional exposure to smoking, alcohol, vitamin containing folic acid and folic acid supplements were contrasted between the cleft types [non-syndromic clefts lip or without cleft palate (CL(P)) and non syndromic cleft palate (CP)] and control groups.
Results
There was a reduced risk of CL(P) with maternal folic acid use (p=0.008; OR=0.70, 95% CI: 0.65–0.94) and with supplements containing folic acid (p=0.028, OR=0.80, 95% CI: 0.65–0.94). Maternal smoking increased the risk of both CL(P) (p<10e−3; OR=1.62, 95% CI: 1.35–1.95) and CP (p=0.028; OR=1.38, 95% CI: 1.04–1.83). No significant risk was observed with either maternal or fetal MTHFR C677T genotypes.
Conclusion
This individual paticipant data (IPD) meta-analysis affords greater statistical power and can help alleviate the problems associated with aggregate-level data-sharing. The result of this IPD meta-analysis is consistent with previous reports suggesting that folic acid and smoking influence OFC outcomes.
doi:10.1002/bdra.23133
PMCID: PMC3745533  PMID: 23670871
Cleft lip and palate; MTHFR; Folic acid; Individual patient data; pooled-analysis
19.  Evaluating the effects of maternal exposure to benzene, toluene, ethyl benzene, and xylene on oral clefts among offspring in Texas: 1999-2008 
BACKGROUND
There is evidence from previous studies that maternal occupational exposure to hazardous air pollutants is positively associated with oral clefts, however, studies evaluating the association between residential exposure to these toxicants and oral clefts are lacking. Therefore, our goal was to conduct a case-control study examining the association between estimated maternal residential exposure to benzene, toluene, ethyl benzene, and xylene (BTEX) and the risk of oral clefts among offspring.
METHODS
Data on 6,045 non-syndromic isolated oral cleft cases (3,915 cleft lip with or without cleft palate [CL±P] and 2,130 non-syndromic isolated cleft palate [CP] cases) delivered between 1999 and 2008 were obtained from the Texas Birth Defects Registry. The control group was a sample of unaffected live births, frequency matched to cases on year of birth. Census tract-level estimates of annual average exposures were obtained from the U.S. Environmental Protection Agency 2005 Hazardous Air Pollutant Exposure Model (HAPEM5) for each pollutant and assigned to each subject based on maternal residence during pregnancy. Logistic regression was used to assess the relationship between estimated maternal exposure to each pollutant (benzene, toluene, ethyl benzene, and xylene) separately and the risk of oral clefts in offspring.
RESULTS
High estimated maternal exposure to benzene was not associated with oral clefts, compared with low estimated exposure (CL±P adjusted OR=0.95; 95% CI=0.81-1.12; CP adjusted OR=0.85; 95% CI=0.67-1.09). Similar results were seen for the other pollutants.
CONCLUSION
In our study, there was no evidence that maternal exposure to environmental levels of BTEX was associated with oral clefts.
doi:10.1002/bdra.23139
PMCID: PMC3771492  PMID: 23893927
oral clefts; maternal exposure; hazardous air pollutants; benzene; epidemiology
20.  Commonality in Down and Fetal Alcohol Syndromes 
BACKGROUND
Down syndrome (DS) and Fetal Alcohol Syndrome (FAS) are two leading causes of birth defects with phenotypes ranging from craniofacial abnormalities to cognitive impairment. Despite different origins, we report that in addition to sharing many phenotypes, DS and FAS may have common underlying mechanisms of development.
METHODS
Literature was surveyed for DS and FAS as well as mouse models. Gene expression and apoptosis were compared in embryonic mouse models of DS and FAS by qPCR, immunohistochemical and immunoflurorescence analyses. The craniometry was examined using MicroCT at postnatal day 21.
RESULTS
A literature survey revealed over 20 comparable craniofacial and structural deficits in both humans with DS and FAS and corresponding mouse models. Similar phenotypes were experimentally found in pre- and postnatal craniofacial and neurological tissues of DS and FAS mice. Dysregulation of two genes, Dyrk1a and Rcan1, key to craniofacial and neurological precursors of DS, was shared in craniofacial precursors of DS and FAS embryos. Increased cleaved caspase 3 expression was also discovered in comparable regions of the craniofacial and brain precursors of DS and FAS embryos. Further mechanistic studies suggested overexpression of trisomic Ttc3 in DS embyros may influence nuclear pAkt localization and cell survival.
CONCLUSIONS
This first and initial study indicates that DS and FAS share common dysmorphologies in humans and animal models. This work also suggests common mechanisms at cellular and molecular levels that are disrupted by trisomy or alcohol consumption during pregnancy and lead to craniofacial and neurological phenotypes associated with DS or FAS.
doi:10.1002/bdra.23129
PMCID: PMC4096968  PMID: 23554291
Down syndrome; fetal alcohol syndrome; craniofacial; apoptosis; development; mouse models
21.  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.
doi:10.1002/bdra.20740
PMCID: PMC4098909  PMID: 21254359
Nonsyndromic cleft lip and palate; NSCLP; folate metabolism; association; genetics; homocysteine; methionine
22.  Red blood cell folate levels in pregnant women with a history of mood disorders: a case series 
Objective
Maternal folate supplementation reduces offspring risk for neural tube defects (NTDs) and other congenital abnormalities. Maternal red blood cell (RBC) folate concentrations of >906nmol/L have been associated with the lowest risk of having an NTD affected pregnancy. Mood disorders (e.g. depression, bipolar disorder) are common among women and can be associated with folate deficiency. Thus, pregnant women with histories of mood disorders may be prone to RBC folate levels insufficient to provide optimal protection against NTDs. While previous studies have assessed RBC folate concentrations in pregnant women from the general population, none have looked specifically at a group of pregnant women who have a history of a mood disorder.
Methods
We collected data about RBC folate concentrations and folic acid supplement intake during early pregnancy (<161days gestation) from n=24 women with histories of mood disorders. We also collected information about offspring congenital abnormalities and birthweight.
Results
Among women with histories of mood disorders, the mean RBC folate concentration was 674 nmol/L (range: 362 –1105nmol/L). Only 12.5% (n=3) of the women had an RBC folate concentrations >906nmol/L, despite all participants reporting current daily use of folic acid supplements. Data regarding offspring were available for 22 women: birthweights ranged from 2296g to 4819g, and congenital abnormalities were identified in two (hypoplastic left heart, annular pancreas).
Conclusion
Data from this exploratory case series suggest a need for future larger scale controlled studies investigating RBC folate concentrations in early pregnancy and offspring outcomes among women with and without histories of mood disorders.
doi:10.1002/bdra.23144
PMCID: PMC3951991  PMID: 23760977 CAMSID: cams3096
folic acid; folate; pregnancy; mood disorders; depression; birth defects; congenital abnormalities
23.  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.
doi:10.1002/bdra.23177
PMCID: PMC4053177  PMID: 24078477
complement; C5a; neural tube defects; folate; neurulation
24.  Genetic and Lifestyle Variables Associated with Homocysteine Concentrations and the Distribution of Folate Derivatives in Healthy Premenopausal Women 
Background
Low folate and high homocysteine (Hcy) concentrations are associated with pregnancy-related pathologies such as spina bifida. Polymorphisms in folate/Hcy metabolic enzymes may contribute to this potentially pathogenic biochemical phenotype.
Methods
The study comprised 26 Caucasian and 23 African-American premenopausal women. Subjects gave fasting blood samples for biochemical phenotyping and genotyping. Total Hcy (tHcy) and both plasma and red blood cell (RBC) folate derivatives [i.e. tetrahydrofolate (THF), 5-methylTHF (5-MTHF), and 5,10-methenylTHF (5,10-MTHF)] were measured using stable isotope dilution liquid chromatography, multiple reaction monitoring, mass spectrometry. Eleven polymorphisms from nine folate/Hcy pathway genes were genotyped. Tests of association between genetic, lifestyle, and biochemical variables were applied.
Results
In African American women, tHcy concentrations were associated (p<0.05) with total RBC folate, RBC 5-MTHF, B12, and polymorphisms in methionine synthase (MTR) and thymidylate synthase (TYMS). In Caucasian women, tHcy concentrations were not associated with total folate levels, but were associated (p<0.05) with RBC THF, ratios of RBC 5-MTHF: THF, and polymorphisms in 5,10-methylenetetrahydrofolate reductase (MTHFR) and MTR . In African Americans, folate derivative levels were associated with smoking, B12, and polymorphisms in MTR, TYMS, methionine synthase reductase (MTRR), and reduced folate carrier1 (RFC1). In Caucasians, folate derivative levels were associated with vitamin use, B12, and polymorphisms in MTHFR, TYMS, and RFC1.
Conclusions
Polymorphisms in the folate/Hcy pathway are associated with tHcy and folate derivative levels. In African American and Caucasian women, different factors are associated with folate/Hcy phenotypes and may contribute to race-specific differences in the risks of a range of pregnancy-related pathologies.
doi:10.1002/bdra.20683
PMCID: PMC4051228  PMID: 20544798
Genetics; folate; homocysteine; women; reproductive age; spina bifida risk
25.  Gene expression profiling in the fetal cardiac tissue after folate and low dose trichloroethylene exposure 
Background
Previous studies show gene expression alterations in rat embryo hearts and cell lines that correspond to the cardio-teratogenic effects of trichloroethylene (TCE) in animal models. One potential mechanism of TCE teratogenicity may be through altered regulation of calcium homeostatic genes with a corresponding inhibition of cardiac function. It has been suggested that TCE may interfere with the folic acid/methylation pathway in liver and kidney and alter gene regulation by epigenetic mechanisms. According to this hypothesis, folate supplementation in the maternal diet should counteract TCE effects on gene expression in the embryonic heart.
Approach
To identify transcriptional targets altered in the embryonic heart after exposure to TCE, and possible protective effects of folate, we used DNA microarray technology to profile gene expression in embryonic mouse hearts with maternal TCE exposure and dietary changes in maternal folate.
Results
Exposure to low doses of TCE (10ppb) caused extensive alterations in transcripts encoding proteins involved in transport, ion channel, transcription, differentiation, cytoskeleton, cell cycle and apoptosis. Exogenous folate did not offset the effects of TCE exposure on normal gene expression and both high and low levels of folate produced additional significant changes in gene expression.
Conclusions
A mechanism where TCE induces a folate deficiency does not explain altered gene expression patterns in the embryonic mouse heart. The data further suggest that use of folate supplementation, in the presence of this toxin, may be detrimental and non-protective of the developing embryo.
doi:10.1002/bdra.20631
PMCID: PMC4045246  PMID: 19813261
Halogenated hydrocarbons; trichloroethylene; cardiac; folate; embryonic development; gene expression; ryanodine receptor

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