Role of the Protein Tyrosine Kinase 7 gene in human neural tube defects

Les anomalies du tube neural (ATN) sont des anomalies développementales où le tube neural reste ouvert (1-2/1000 naissances). Afin de prévenir cette maladie, une connaissance accrue des processus moléculaires est nécessaire. L’étiologie des ATN est complexe et implique des facteurs génétiques et environnementaux. La supplémentation en acide folique est reconnue pour diminuer les risques de développer une ATN de 50-70% et cette diminution varie en fonction du début de la supplémentation et de l’origine démographique. Les gènes impliqués dans les ATN sont largement inconnus. Les études génétiques sur les ATN chez l’humain se sont concentrées sur les gènes de la voie métabolique des folates du à leur rôle protecteur dans les ATN et les gènes candidats inférés des souris modèles. Ces derniers ont montré une forte association entre la voie non-canonique Wnt/polarité cellulaire planaire (PCP) et les ATN. Le gène Protein Tyrosine Kinase 7 est un membre de cette voie qui cause l’ATN sévère de la craniorachischisis chez les souris mutantes. Ptk7 interagit génétiquement avec Vangl2 (un autre gène de la voie PCP), où les doubles hétérozygotes montrent une spina bifida. Ces données font de PTK7 comme un excellent candidat pour les ATN chez l’humain. Nous avons re-séquencé la région codante et les jonctions intron-exon de ce gène dans une cohorte de 473 patients atteints de plusieurs types d’ATN. Nous avons identifié 6 mutations rares (fréquence allélique <1%) faux-sens présentes chez 1.1% de notre cohorte, dont 3 sont absentes dans les bases de données publiques. Une variante, p.Gly348Ser, a agi comme un allèle hypermorphique lorsqu'elle est surexprimée dans le modèle de poisson zèbre. Nos résultats impliquent la mutation de PTK7 comme un facteur de risque pour les ATN et supporte l'idée d'un rôle pathogène de la signalisation PCP dans ces malformations.

Neural tube defects in Mexican-Americans living on the US-Mexico border: The effects of folic acid and dietary folate

Neural tube defects (NTDs) are malformations of the developing brain and spinal cord; the most common are anencephaly and spina bifida. Evidence from many populations suggests that 50% of NTDs can be prevented through daily consumption of folic acid. A recent study has reported that folic acid may not protect populations of Mexican descent. This finding has serious implications for women living along the US-Mexico border. Not only is risk high in these Mexican American women compared with other US women; they also differ markedly in supplemental folic acid and dietary folate consumption, and in NTD-related risks (e.g., obesity, diabetes). This case-control study investigated whether folic acid supplements and dietary folate reduces NTDs in Mexican Americans. Cases included liveborn, stillborn, electively and spontaneously aborted NTD-affected fetuses and infants occurring in the 14-county Texas-Mexico border. Controls were randomly selected from unaffected live births, frequency matched to cases by hospital and year. An in-person interview of 110 case and 113 control mothers solicited data on folic acid supplements, dietary folate, and other covariates. Consumption of folic acid-containing vitamins before conception was only 5% for both case and control women. Taking vitamins the trimester before conception had no apparent effect, after adjusting for covariates [odds ratio (OR) = 1.0, 95% confidence interval (CI) = 0.3–3.4]. Combining folate from vitamins and diet showed a 20% risk reduction for women consuming at least 400 μg of folate daily [OR = 0.8, 95% CI = 0.5–1.5]; however, this estimate is statistically indistinguishable from the null. Although consistent with an inherent ineffectiveness of supplemental folic acid, that so few women consumed multivitamins during the critical time severely limited the assessment of folic acid in this population. A reduced folate response in Mexican descent women may be due to a genetic heterogeneity for metabolizing folate. Alternatively, folate intakes may be insufficient to overcome other underlying risk factors. In conclusion, determining whether folic acid reduces NTD risk in Mexican American women requires further study in populations with higher folic acid exposures. Meanwhile, we should pursue all recommended prevention strategies to reduce risk, including motivating Mexican American women of childbearing age to take folic acid routinely. ^

Requirement of GCN5 histone acetyltransferase in mouse neural tube closure and skeletal patterning

Histone acetylation plays an essential role in many DNA-related processes such as transcriptional regulation via modulation of chromatin structure. Many histone acetytransferases have been discovered and studied in the past few years, but the roles of different histone acetyltransferases (HAT) during mammalian development are not well defined at present. Gcn5 histone acetyltransferase is highly expressed until E16.5 during development. Previous studies in our lab using a constitutive null allele demonstrated that Gcn5 knock out mice are embryonic lethal, precluding the study of Gcn5 functions at later developmental stages. The creation of a conditional Gcn5 null allele, Gcn5flox allele, bypasses the early lethality. Mice homozygous for this allele are viable and appear healthy. In contrast, mice homozygous for a Gcn5 Δex3-18 allele created by Cre-loxP mediated deletion display a phenotype identical to our original Gcn5 null mice. Strikingly, a Gcn5flox(neo) allele, which contain a neomycin cassette in the second intron of Gcn5 is only partially functional and gives rise to a hypomorphic phenotype. Initiation of cranial neural tube closure at forebrain/midbrain boundary fails, resulting in an exencephaly in some Gcn5flox(neo)/flox(neo) embryos. These defects were found at an even greater penetrance in Gcn5flox(neo)/Δ embryos and become completely penetrant in the 129Sv genetic background, suggesting that Gcn5 controls mouse neural tube closure in a dose dependent manner. Furthermore, both Gcn5flox(neo)/flox(neo) and Gcn5 flox(neo)/Δ embryos exhibit anterior homeotic transformations in lower thoracic and lumbar vertebrae. These defects are accompanied by decreased expression levels and a shift in anterior expression boundary of Hoxc8 and Hoxc9. This study provides the first evidence that Gcn5 regulates Hox gene expression and is required for normal axial skeletal patterning in mice. ^

Folic acid fortification, maternal obesity and neural tube defects: Policy implications

In 1996, the Food and Drug Administration (FDA) mandated that beginning in January 1998, flour and other enriched grain products be fortified with 140 μg of folic acid per 100 g of grain to prevent neural tube defects (NTDs) that occur in approximately 1 in 1,000 pregnancies in the United States (U.S.). Although this program has demonstrated important public health effects, it is argued that current fortification levels may not be enough to prevent all folic acid-preventable NTD cases. This study reviews published literature, on folic acid fortification in the U.S. and countries with mandatory folic acid fortification programs reported after 1992 and through January 2008. Published studies are evaluated to determine if the current level of folic acid fortification in the U.S. is adequate to prevent the most common forms of NTDs (spina bifida and anencephaly), particularly among overweight and obese women. ^ Although consistent improvement in blood folate levels of child bearing age women is reported in almost all studies, the RBC folate concentration has not reached the level associated with the most significant reduction of risk for NTDs (906 nmol/L); approximately half of the potentially preventable NTDs are prevented by fortification at the current U.S. level. Furthermore, the blood folate status of women in higher BMI categories (obese or overweight) has not improved as much as among women in lower BMI categories. Therefore, women classified as overweight or obese have not benefited from the preventive effects of folic acid fortification as much as normal or underweight women. ^ To reduce risk of folate preventable NTDs, especially in overweight and obese women, it may be necessary to increase the current level of folic acid fortification. However, further research is required to determine the optimal levels of fortification to achieve this goal without causing adverse health effects in the general population. ^

Determining associations and assessing methodological issues in a case-control study of hazardous air pollutants and neural tube defects

Recent studies have reported positive associations between maternal exposures to air pollutants and several adverse birth outcomes. However, there have been no assessments of the association between environmental hazardous air pollutants (HAPs) such as benzene, toluene, ethylbenzene, and xylene (BTEX) and neural tube defects (NTDs) a common and serious group of congenital malformations. Before examining this association, two important methodological questions must be addressed: (1) is maternal residential movement likely to result in exposure misclassification and (2) is it appropriate to lump defects of the neural tube, such as anencephaly and spina bifida, into a composite disease endpoint (i.e., NTDs). ^ Data from the National Birth Defects Prevention Study and Texas Birth Defects Registry were used to: (1) assess the extent to which change of residence may result in exposure misclassification when exposure is based on the address at delivery; (2) formally assess heterogeneity of the associations between known risk factors for NTDs, using polytomous logistic regression; and (3) conduct a case-control study assessing the association between ambient air levels of BTEX and the risk of NTDs among offspring. ^ Regarding maternal residential mobility, this study suggests address at delivery was not significantly different from using address at conception when assigning quartile of benzene exposure (OR 1.0, 95% CI 0.9, 1.3). On the question of effect heterogeneity among NTDs, the effect estimates for infant sex P = 0.017), maternal body mass index P = 0.016), and folate supplementation P = 0.050) were significantly different for anencephaly and spina bifida, suggesting it is often more appropriate to assess potential risk factors among subgroups of NTDs. For the main study question on the association between environmental HAPs and NTDs, mothers who have offspring with isolated spina bifida are 2.4 times likely to live in areas with the highest benzene levels (95% CI 1.1, 5.0). However, no other significant associations were observed.^ This project is the first to include not only an assessment of the relationship between environmental levels of BTEX and NTDs, but also two separate studies addressing important methodological issues associated with this question. Our results contribute to the growing body of evidence regarding air pollutant exposure and adverse birth outcomes. ^

Association of maternal sphinganine:sphingosine ratio and folate levels with neural tube defects along the South Texas border

Neural tube defects (NTDs) remain elevated in Hispanic women along the South Texas Border, despite folate supplementation and folate fortification of cereal products. Missmer et al. examined the relationships between fumonisins, a class of corn mycotoxin, and NTDs in Hispanic women who ate corn tortillas and found increased odds ratios with increasing exposure, as measured by serum sphinganine:sphingosine (sa:so) ratios. This study examined the interactions between categorized maternal serum folate levels and stratified sa:so ratios and the resultant odds ratios of NTDs, stratified by type (anencephaly and spina bifida). The hypothesis was that the above normal folate category would have lower odds ratios of NTDs at given sa:so ratio categories and that there would be a difference in odds ratio patterns for anencephaly and spina bifida. Methods. Data for 406 Hispanic women were obtained from the Missmer case-control study. Sa:so ratios were calculated and subjects were stratified into “below normal,” “normal,” and above normal range for folate. A logistic regression model was applied, controlling for BMI, serum B12, lab batch, and conception date. Results. While OR’s of NTDs increased for increasing sa:so ratios, OR’s for “above normal” folate were not decreased at any sa:so ratio and there was no statistically significant difference between OR’s of anencephaly and spina bifida. Conclusion. Folate does not appear to be protective against the potential teratogenic effect of fumonisins and did not differ in effect on OR’s of NTD by type. More research is necessary to determine the extent of fumonisin exposure in Hispanic women along the South Texas Border.^

The use of the computerized and revised vital record in conducting etiologic studies on neural tube defects

The purpose of this study was to evaluate the adequacy of computerized vital records in Texas for conducting etiologic studies on neural tube defects (NTDs), using the revised and expanded National Centers for Health Statistics vital record forms introduced in Texas in 1989.^ Cases of NTDs (anencephaly and spina bifida) among Harris County (Houston) residents were identified from the computerized birth and death records for 1989-1991. The validity of the system was then measured against cases ascertained independently through medical records and death certificates. The computerized system performed poorly in its identification of NTDs, particularly for anencephaly, where the false positive rate was 80% with little or no improvement over the 3-year period. For both NTDs the sensitivity and predictive value positive of the tapes were somewhat higher for Hispanic than non-Hispanic mothers.^ Case control studies were conducted utilizing the tape set and the independently verified data set, using controls selected from the live birth tapes. Findings varied widely between the data sets. For example, the anencephaly odds ratio for Hispanic mothers (vs. non-Hispanic) was 1.91 (CI = 1.38-2.65) for the tape file, but 3.18 (CI = 1.81-5.58) for verified records. The odds ratio for diabetes was elevated for the tape set (OR = 3.33, CI = 1.67-6.66) but not for verified cases (OR = 1.09, CI = 0.24-4.96), among whom few mothers were diabetic. It was concluded that computerized tapes should not be solely relied on for NTD studies.^ Using the verified cases, Hispanic mother was associated with spina bifida, and Hispanic mother, teen mother, and previous pregnancy terminations were associated with anencephaly. Mother's birthplace, education, parity, and diabetes were not significant for either NTD.^ Stratified analyses revealed several notable examples of statistical interaction. For anencephaly, strong interaction was observed between Hispanic origin and trimester of first prenatal care.^ The prevalence was 3.8 per 10,000 live births for anencephaly and 2.0 for spina bifida (5.8 per 10,000 births for the combined categories). ^

Region-specific differentiation of neural tube-derived neuronal restricted progenitor cells after heterotopic transplantation

Spinal cord neuronal restricted progenitor (NRP) cells, when transplanted into the neonatal anterior forebrain subventricular zone, migrate to distinct regions throughout the forebrain including the olfactory bulb, frontal cortex, and occipital cortex but not to the hippocampus. Their migration pattern and differentiation potential is distinct from anterior forebrain subventricular zone NRPs. Irrespective of their final destination, NRP cells do not differentiate into glia. Rather they synthesize neurotransmitters, acquire region-specific phenotypes, and receive synapses from host neurons after transplantation. Spinal cord NRPs express choline acetyl transferase even in regions where host neurons do not express this marker. The restricted distribution of transplanted spinal cord NRP cells and their acquisition of varied region-specific phenotypes suggest that their ultimate fate and phenotype is dictated by a combination of intrinsic properties and extrinsic cues from the host.

Regulation of patched by sonic hedgehog in the developing neural tube.

Ventral cell fates in the central nervous system are induced by Sonic hedgehog, a homolog of hedgehog, a secreted Drosophila protein. In the central nervous system, Sonic hedgehog has been identified as the signal inducing floor plate, motor neurons, and dopaminergic neurons. Sonic hedgehog is also involved in the induction of ventral cell type in the developing somites. ptc is a key gene in the Drosophila hedgehog signaling pathway where it is involved in transducing the hedgehog signal and is also a transcriptional target of the signal. PTC, a vertebrate homolog of this Drosophila gene, is genetically downstream of Sonic hedgehog (Shh) in the limb bud. We analyze PTC expression during chicken neural and somite development and find it expressed in all regions of these tissues known to be responsive to Sonic hedgehog signal. As in the limb bud, ectopic expression of Sonic hedgehog leads to ectopic induction of PTC in the neural tube and paraxial mesoderm. This conservation of regulation allows us to use PTC as a marker for Sonic hedgehog response. The pattern of PTC expression suggests that Sonic hedgehog may play an inductive role in more dorsal regions of the neural tube than have been previously demonstrated. Examination of the pattern of PTC expression also suggests that PTC may act in a negative feedback loop to attenuate hedgehog signaling.

Disruption of the MacMARCKS gene prevents cranial neural tube closure and results in anencephaly.

MacMARCKS is a member of the MARCKS family of protein kinase C (PKC) substrates. Biochemical evidence demonstrates that these proteins integrate calcium and PKC-dependent signals to regulate actin structure at the membrane. We report here that deletion of the MacMARCKS gene prevents cranial neural tube closure in the developing brain, resulting in anencephaly. This suggests a central role for MacMARCKS and the PKC signal transduction pathway in the folding of the anterior neural plate during the early phases of brain formation, and supports the hypothesis that actin-based motility directs cranial neural tube closure.

Concerted involvement of Cdx/Hox genes and Wnt signaling in morphogenesis of the caudal neural tube and cloacal derivatives from the posterior growth zone

Decrease in Cdx dosage in an allelic series of mouse Cdx mutants leads to progressively more severe posterior vertebral defects. These defects are corrected by posterior gain of function of the Wnt effector Lef1. Precocious expression of Hox paralogous 13 genes also induces vertebral axis truncation by antagonizing Cdx function. We report here that the phenotypic similarity also applies to patterning of the caudal neural tube and uro-rectal tracts in Cdx and Wnt3a mutants, and in embryos precociously expressing Hox13 genes. Cdx2 inactivation after placentation leads to posterior defects, including incomplete uro-rectal septation. Compound mutants carrying one active Cdx2 allele in the Cdx4-null background (Cdx2/4), transgenic embryos precociously expressing Hox13 genes and a novel Wnt3a hypomorph mutant all manifest a comparable phenotype with similar uro-rectal defects. Phenotype and transcriptome analysis in early Cdx mutants, genetic rescue experiments and gene expression studies lead us to propose that Cdx transcription factors act via Wnt signaling during the laying down of uro-rectal mesoderm, and that they are operative in an early phase of these events, at the site of tissue progenitors in the posterior growth zone of the embryo. Cdx and Wnt mutations and premature Hox13 expression also cause similar neural dysmorphology, including ectopic neural structures that sometimes lead to neural tube splitting at caudal axial levels. These findings involve the Cdx genes, canonical Wnt signaling and the temporal control of posterior Hox gene expression in posterior morphogenesis in the different embryonic germ layers. They shed a new light on the etiology of the caudal dysplasia or caudal regression range of human congenital defects.

Prevalence of neural tube defects in Illinois, 1989-2002 /

"8/04"--Colophon.

Primary prevention of neural tube defects: data from the portuguese national registry of congenital anomalies (RENAC)

Background: In Portugal folic acid supplementation is recommended to start at least 2-3 months before conception for primary prevention of Neural Tube Defects. The aim of this study was to evaluate, within gestations with at least one congenital anomaly, possible association between maternal socio-demographic factors and the use of folic acid. Methods: Using data from the Portuguese national registry of congenital anomalies, for the 2004-2013 period, the association between folic acid use during pregnancy and maternal characteristics was studied using the chi-square test. Results: Considering all reported cases with congenital anomaly, the use of folic acid before conception was reported by 12.7% (n = 1233) of the women; 47.8% (n = 4623) started supplementation during the 1st trimester, 7% (n = 680) did not take folic acid and 32.5% (3143) of the records had no information on folic acid use. Women with professions that require higher academic differentiation started the use of supplements before pregnancy (p <0.001); women under 19 years old and with Arab ethnicity (p <0.001) did not take folic acid. Mothers with a previous pregnancy reported less use of folic acid (11.5% versus 14.7%) than mothers without a previous pregnancy (p <0.001). Conclusions: The results suggest some degree of association between maternal characteristics and use of folic acid. To increase the consumption of folic acid before pregnancy new measures are need to promote this primary prevention, among couples and health professionals. This study highlights some maternal characteristics and subgroups of mothers for who the measures should be reinforced.

Linkage to chromosome 2q36.1 in autosomal dominant Dandy-Walker malformation with occipital cephalocele and evidence for genetic heterogeneity

We previously reported a Vietnamese-American family with isolated autosomal dominant occipital cephalocele. Upon further neuroimaging studies, we have recharacterized this condition as autosomal dominant Dandy-Walker with occipital cephalocele (ADDWOC). A similar ADDWOC family from Brazil was also recently described. To determine the genetic etiology of ADDWOC, we performed genome-wide linkage analysis on members of the Vietnamese-American and Brazilian pedigrees. Linkage analysis of the Vietnamese-American family identified the ADDWOC causative locus on chromosome 2q36.1 with a multipoint parametric LOD score of 3.3, while haplotype analysis refined the locus to 1.1 Mb. Sequencing of the five known genes in this locus did not identify any protein-altering mutations. However, a terminal deletion of chromosome 2 in a patient with an isolated case of Dandy-Walker malformation also encompassed the 2q36.1 chromosomal region. The Brazilian pedigree did not show linkage to this 2q36.1 region. Taken together, these results demonstrate a locus for ADDWOC on 2q36.1 and also suggest locus heterogeneity for ADDWOC.