Mice lacking the folic acid-binding protein Folbp1 are defective in early embryonic development.

Periconceptional folic acid supplementation reduces the occurrence of several human congenital malformations, including craniofacial, heart and neural tube defects. Although the underlying mechanism is unknown, there may be a maternal-to-fetal folate-transport defect or an inherent fetal biochemical disorder that is neutralized by supplementation. Previous experiments have identified a folate-binding protein (Folbp1) that functions as a membrane receptor to mediate the high-affinity internalization and delivery of folate to the cytoplasm of the cell. In vitro, this receptor facilitates the accumulation of cellular folate a thousand-fold relative to the media, suggesting that it may be essential in cytoplasmic folate delivery in vivo. The importance of an adequate intracellular folate pool for normal embryogenesis has long been recognized in humans and experimental animals. To determine whether Folbp1 is involved in maternal-to-fetal folate transport, we inactivated Folbp1 in mice. We also produced mice lacking Folbp2, another member of the folate receptor family that is GPI anchored but binds folate poorly. Folbp2-/- embryos developed normally, but Folbp1-/- embryos had severe morphogenetic abnormalities and died in utero by embryonic day (E) 10. Supplementing pregnant Folbp1+/- dams with folinic acid reversed this phenotype in nullizygous pups. Our results suggest that Folbp1 has a critical role in folate homeostasis during development, and that functional defects in the human homologue (FOLR1) of Folbp1 may contribute to similar defects in humans.

Association between inhibited binding of folic acid to folate receptor alpha in maternal serum and folate-related birth defects in Norway.

BACKGROUND: Folic acid intake during pregnancy can reduce the risk of neural tube defects (NTDs) and perhaps also oral facial clefts. Maternal autoantibodies to folate receptors can impair folic acid binding. We explored the relationship of these birth defects to inhibition of folic acid binding to folate receptor α (FRα), as well as possible effects of parental demographics or prenatal exposures. METHODS: We conducted a nested case-control study within the Norwegian Mother and Child Cohort Study. The study included mothers of children with an NTD (n = 11), cleft lip with or without cleft palate (CL/P, n= 72), or cleft palate only (CPO, n= 27), and randomly selected mothers of controls (n = 221). The inhibition of folic acid binding to FRα was measured in maternal plasma collected around 17 weeks of gestation. On the basis of prior literature, the maternal age, gravidity, education, smoking, periconception folic acid supplement use and milk consumption were considered as potential confounding factors. RESULTS: There was an increased risk of NTDs with increased binding inhibition [adjusted odds ratio (aOR) = 1.4, 95% confidence interval (CI) 1.0-1.8]. There was no increased risk of oral facial clefts from inhibited folic acid binding to FRα (CL/P aOR = 0.7, 95% CI 0.6-1.0; CPO aOR = 1.1, 95% CI 0.8-1.4). No association was seen between smoking, folate supplementation or other cofactors and inhibition of folic acid binding to FRα. CONCLUSIONS: Inhibition of folic acid binding to FRα in maternal plasma collected during pregnancy was associated with increased risk of NTDs but not oral facial clefts.

De novo ALX4 variant detected in child with non-syndromic craniosynostosis.

Current understanding of the genetic factors contributing to the etiology of non-syndromic craniosynostosis (NSC) remains scarce. The present work investigated the presence of variants in ALX4, EFNA4, and TWIST1 genes in children with NSC to verify if variants within these genes may contribute to the occurrence of these abnormal phenotypes. A total of 101 children (aged 45.07±40.94 months) with NSC participated in this cross-sectional study. Parents and siblings of the probands were invited to participate. Medical and family history of craniosynostosis were documented. Biological samples were collected to obtain genomic DNA. Coding exons of human TWIST1, ALX4, and EFNA4 genes were amplified by polymerase chain reaction and Sanger sequenced. Five missense variants were identified in ALX4 in children with bilateral coronal, sagittal, and metopic synostosis. A de novo ALX4 variant, c.799G>A: p.Ala267Thr, was identified in a proband with sagittal synostosis. Three missense variants were identified in the EFNA4 gene in children with metopic and sagittal synostosis. A TWIST1 variant occurred in a child with unilateral coronal synostosis. Variants were predicted to be among the 0.1% (TWIST1, c.380C>A: p. Ala127Glu) and 1% (ALX4, c.769C>T: p.Arg257Cys, c.799G>A: p.Ala267Thr, c.929G>A: p.Gly310Asp; EFNA4, c.178C>T: p.His60Tyr, C.283A>G: p.Lys95Glu, c.349C>A: Pro117Thr) most deleterious variants in the human genome. With the exception of ALX4, c.799G>A: p.Ala267Thr, all other variants were present in at least one non-affected family member, suggesting incomplete penetrance. Thus, these variants may contribute to the development of craniosynostosis, and should not be discarded as potential candidate genes in the diagnosis of this condition.

Importance of gene-environment interactions in the etiology of selected birth defects.

It is generally understood that both genetic and environmental factors contribute to the highly complex etiology of structural birth defects, including neural tube defects, oral clefts and congenital heart defects, by disrupting highly regulated embryonic developmental processes. The intrauterine environment of the developing embryo/fetus is determined by maternal factors such as health/disease status, lifestyle, medication, exposure to environmental teratogens, as well as the maternal genotype. Certain genetic characteristics of the embryo/fetus also predispose it to developmental abnormalities. Epidemiologic and animal studies conducted over the last few decades have suggested that the interplay between genes and environmental factors underlies the etiological heterogeneity of these defects. It is now widely believed that the study of gene-environment interactions will lead to better understanding of the biological mechanisms and pathological processes that contribute to the development of complex birth defects. It is only through such an understanding that more efficient measures will be developed to prevent these severe, costly and often deadly defects. In this review, we attempt to summarize the complex clinical and experimental literature on current hypotheses of interactions between several select environmental factors and those genetic pathways in which they are most likely to have significant modifying effects. These include maternal folate nutritional status, maternal diabetes/obesity-related conditions, and maternal exposure to selected medications and environmental contaminants. Our goal is to highlight the potential gene-environment interactions affecting early embryogenesis that deserve comprehensive study.

Phenytoin-induced teratogenesis: a mouse model.

Malformations associated with the fetal hydantoin syndrome have been reproduced in a mouse model. The occurrence of these defects was correlated with maternal serum concentrations, but not with maternal or fetal genotype or the presence of a seizure disorder.

De novo ALX4 variant detected in child with non-syndromic craniosynostosis

Current understanding of the genetic factors contributing to the etiology of non-syndromic craniosynostosis (NSC) remains scarce. The present work investigated the presence of variants in ALX4, EFNA4, and TWIST1 genes in children with NSC to verify if variants within these genes may contribute to the occurrence of these abnormal phenotypes. A total of 101 children (aged 45.07±40.94 months) with NSC participated in this cross-sectional study. Parents and siblings of the probands were invited to participate. Medical and family history of craniosynostosis were documented. Biological samples were collected to obtain genomic DNA. Coding exons of human TWIST1, ALX4, and EFNA4 genes were amplified by polymerase chain reaction and Sanger sequenced. Five missense variants were identified in ALX4 in children with bilateral coronal, sagittal, and metopic synostosis. A de novo ALX4 variant, c.799G>A: p.Ala267Thr, was identified in a proband with sagittal synostosis. Three missense variants were identified in the EFNA4 gene in children with metopic and sagittal synostosis. A TWIST1 variant occurred in a child with unilateral coronal synostosis. Variants were predicted to be among the 0.1% (TWIST1, c.380C>A: p. Ala127Glu) and 1% (ALX4, c.769C>T: p.Arg257Cys, c.799G>A: p.Ala267Thr, c.929G>A: p.Gly310Asp; EFNA4, c.178C>T: p.His60Tyr, C.283A>G: p.Lys95Glu, c.349C>A: Pro117Thr) most deleterious variants in the human genome. With the exception of ALX4, c.799G>A: p.Ala267Thr, all other variants were present in at least one non-affected family member, suggesting incomplete penetrance. Thus, these variants may contribute to the development of craniosynostosis, and should not be discarded as potential candidate genes in the diagnosis of this condition.

Genetic differences in the duration of the lymphocyte heat shock response in mice.

Lymphocytes from adult mice bearing a known difference in genetic susceptibility to teratogen-induced exencephaly (SWV/SD, and DBA/2J) were evaluated for changes in protein synthesis following an in vivo heat treatment. Particular attention was paid to changes indicative of the heat shock response, a highly conserved response to environmental insult consisting of induction of a few, highly conserved proteins with simultaneous decreases in normal protein synthesis. The duration of heat shock protein induction in lymphocytes was found to be increased by 1 hr in the teratogen-sensitive SWV/SD strain as compared to the resistant DBA/2J strain. Densitometric analysis revealed a significant decrease in the relative synthesis of at least two non-heat shock proteins (36 kD and 45 kD) in the SWV/SD lymphocytes as compared to DBA/2J cells. The increased sensitivity of protein synthesis to hyperthermia in the SWV/SD lymphocytes were lost in the F1 progeny of reciprocal crosses between SWV/SD and DBA/2J mouse strains. Sensitivity to hyperthermia-induced exencephaly is recessive to resistance in these crosses. The relationship between altered protein synthesis and teratogen susceptibility is discussed.

Identification of two putative novel folate receptor genes in humans and mouse.

Utilizing a 'database mining' strategy to detect novel folate receptors (FR), we identified two potential novel members in the mouse and human. The mouse gene (Folbp3) was sequenced and found to predict a 28.2 kDa protein that consists of 244 amino acids that is highly expressed in both the thymus and spleen, suggesting a potential role in the immune system. The human gene (FR-delta) is mapped to chromosome 11q14, and predicts a 27.7 kDa protein that is comprised of 241 amino acids. However, expression of the human gene was not detected in 59 samples from both adult and embryonic tissue sources, suggesting a highly restricted spatial/temporal expression pattern, an alternatively spliced variant or an additional FR pseudogene. Using T31 mouse radiation hybrid mapping, Folbp3 was mapped to a region on mouse chromosome 9 that is syntenic to human chromosome 19p13. As the chromosomal locations of Folbp1 murine and Folbp2 genes were previously unknown, we utilized the same approach and mapped both genes to a region of mouse chromosome 7 that is syntenic to the human FR loci on chromosome 11q13.

Interleukin-1beta upregulates MMP-9 expression in stromal cells of human giant cell tumor of bone.

Giant cell tumor (GCT) of bone is a progressive, potentially malignant process that destroys skeletal tissue. It consists of multinucleated giant cells, which are hypothesized to be derived from a monocyte/macrophage lineage and mononuclear stromal cells, and the precise relationship of these cells is not fully understood. Recently, we demonstrated that the production of matrix metalloproteinase-9 (MMP-9) in GCT stromal cells is regulated by certain factor(s) secreted by the multinucleated giant cells. In the present study, we evaluated for the presence of interleukin-1beta (IL-1beta) and attempted to establish its possible role for the induction of MMP-9 in GCT stromal cells. Using enzyme-linked immunosorbent assay (ELISA), we have demonstrated that the primary GCT cultures secrete both IL-1beta and MMP-9. The addition of monoclonal antibody (mAb) against IL-1beta partially abrogated, but did not abolish, MMP-9 expression. Our results on gelatin zymography, reverse transcriptase-polymerase chain reaction (RT-PCR), and immunofluorescence showed that GCT stromal cells did not express MMP-9, although treatment with IL-1beta induced MMP-9 expression in a dose-dependent manner, and the secretion peaked 24 h after stimulation and then plateaued. Studies with cycloheximide, a protein synthesis inhibitor, demonstrated that de novo protein synthesis is required for IL-1beta induced MMP-9 expression. Moreover, nuclear run-on analysis has revealed that IL-1beta significantly increased MMP-9 gene transcription in GCT stromal cells. The data suggest that IL-1beta secreted by the multinucleated giant cells in GCT may be one of the factors responsible for the induction of MMP-9 at the transcriptional level in GCT stromal cells in vivo. We conclude that GCT has a self-stimulatory system for the production of MMP-9, and the ability of stromal cells to produce MMP-9 with appropriate stimuli, such as IL-1beta, and possibly in concert with other cytokines may contribute to the aggressive and potentially malignant behavior of GCT.

Clinical and experimental studies linking oxidative metabolism to phenytoin-induced teratogenesis.

The research presented in this article concerns the proposed mechanism of phenytoin-induced teratogenicity that focuses on oxidative metabolites as sources of reactive species in clinical studies and by testing paradigms in animal models. The clinical aspect involved determining whether at-risk fetuses could be detected prenatally on the basis of low or deficient epoxide hydrolase activity. In 19 pregnancies monitored by amniocentesis, we predicted an adverse outcome in four infants on the basis of low enzyme activity. When examined neonatally, all four infants had the dysmorphic features of the "fetal hydantoin syndrome." In an animal model of phenytoin-induced teratogenesis, the level of fetal exposure to oxidative metabolites was decreased by coadministration of the cytochrome P-450-inhibiting antiepileptic drug stiripentol, which significantly reduced the incidence of phenytoin-induced congenital malformations in two of the three inbred mouse strains, thus providing support for the hypothesis that oxidative metabolites are critical in mediating phenytoin teratogenesis.

Variable patterns of malformation in the mouse fetal hydantoin syndrome.

Controversy over the existence of a fetal hydantoin syndrome continues in medical literature despite numerous recent clinical studies describing additional patients with a characteristic pattern of abnormalities. Resistance to its acceptance as a clinical entity seems to stem from the variability of the component malformations seen in this syndrome. To examine this variability in a controlled experimental situation, we utilized data obtained in previously reported studies of a mouse model of the fetal hydantoin syndrome. In the mouse, prenatally exposed fetuses had congenital anomalies similar to those observed in the human syndrome. In terms of overall frequency of malformation there were no differences among three inbred mouse strains. However, when considering the individual rates of the 11 most common malformations, considerable differences were noted among the strains. These strain differences in the pattern of malformations appear to be related to genotypic differences in susceptibility to specific malformations. These results provide one possible explanation for the variability observed in the human fetal hydantoin syndrome.

Strain-dependent alterations in the expression of folate pathway genes following teratogenic exposure to valproic acid in a mouse model.

The molecular basis for the well-established hierarchy of susceptibility to valproic acid-induced neural tube defects in inbred mouse strains was examined using in situ transcription and anti-sense RNA amplification methodologies with both univariate and multivariate analyses of the resulting gene expression data. The highly sensitive SWV strain demonstrated a significant reduction in the expression of the folate binding protein (FBP-1) following the teratogenic insult at gestational day 8:18, while the more resistant LM/Bc embryos were up-regulating this gene in response to valproic acid treatment. More importantly, at all 3 gestational timepoints spanning the period of murine neural tube closure examined in this study, the LM/Bc embryos had significantly higher MTHFR (5,10-methylenetetrahydrofolate reductase) gene expression levels compared to the SWV embryos. As this folate pathway enzyme is important in homocysteine and methionine metabolism, it suggests that the SWV embryos may be hypomethylated, and essential gene expression during critical periods of neural tube closure is compromised by the teratogenic exposure to valproic acid. This study represents the first evidence of a strain difference in transcriptional activity in response to a teratogenic exposure that might be causally related to the development of the teratogen-induced congenital malformations.

Infant C677T mutation in MTHFR, maternal periconceptional vitamin use, and cleft lip.

Studies have reported an association between homozygosity for a variant form of the methylenetetrahydrofolate reductase (MTHFR) gene and risk for neural tube defects. Because of MTHFR's involvement with folate metabolism and evidence that maternal use of a multivitamin with folic acid in early pregnancy reduces risk for cleft lip with or without cleft palate (CLP), we hypothesized that infants homozygous for the C677T genotype would be at increased risk for CLP because of lower MTHFR enzymatic activity. Data were derived from a large population-based, case-control study of fetuses and liveborn infants among a cohort of 1987 to 1989 California births. The analyses involved 310 infants with isolated CLP whose mothers completed a telephone interview and whose DNA was available from newborn screening blood specimens and involved 383 control infants without a congenital anomaly whose mothers completed a telephone interview and whose DNA was available. Cases and controls were genotyped TT if homozygous for the C677T allele, CT if heterozygous for the C677T allele, and CC if homozygous for the C677 (wild-type) allele. Odds ratios for CLP were 0.89 (0.55 to 1.4) and 0.78 (0.56 to 1.1) for infants with TT versus CC and infants with CT versus CC genotypes, respectively. Compared with the CC genotype, the odds ratios for CLP among infants with the TT genotype were 0.74 (0.39 to 1.4) for those infants whose mothers were users and 1.4 (0.54 to 3.6) for those infants whose mothers were not users of multivitamins containing folic acid periconceptionally. The two estimates were not statistically heterogeneous (P = 0.30). Our results did not indicate increased risks for CLP among infants homozygous for the C677T genotype, nor do they indicate an interaction between infant C677T genotype and maternal multivitamin use on the occurrence of CLP.

Folate pathway gene alterations in patients with neural tube defects.

Periconceptional folate supplementation reduces the recurrence and occurrence risk of neural tube defects (NTD) by as much as 70%, yet the protective mechanism remains unknown. Inborn errors of folate and homocysteine metabolism may be involved in the aetiology of NTDs. Previous studies have demonstrated that both homozygosity for the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene, and combined heterozygosity for the C677T and for another mutation in the same gene, the A1298C polymorphism, represent genetic risk factors for NTDs. In an attempt to identify additional folate related genes that contribute to NTD pathogenesis, we performed molecular genetic analysis of folate receptors (FRs). We identified 4 unrelated patients out of 50 with de novo insertions of pseudogene (PS)-specific mutations in exon 7 and 3'UTR of the FRalpha gene, arising by microconversion events. All of the substitutions affect the carboxy-terminal amino acid membrane tail, or the GPI anchor region of the nascent protein. Furthermore, among 150 control individuals, we also identified one infant with a gene conversion event within the FRalpha coding region. This study, though preliminary, provides the first genetic association between molecular variations of the FRalpha gene and NTDs and suggests that this gene can act as a risk factor for human NTD.

Lack of association between mutations in the folate receptor-alpha gene and spina bifida.

Defects of neural tube closure are among the most common of all human malformations. Epidemiological and genetic studies indicate that most of these defects are multifactorial in origin with genetic and environmental causes. Although periconceptional supplementation of the maternal diet with folic acid has been shown to reduce the recurrence and occurrence of neural tube defects (NTDs) by up to 70%, the underlying mechanism remains unknown. Folic acid enters cells of certain tissues via a receptor-mediated process known as potocytosis. The folate receptor alpha (FR-alpha) gene codes for the protein responsible for binding folate, which is the first, and only, folate-dependent step in folate transport. The FR-alpha exons, which code for mature protein and the intron-exon boundaries, were examined for mutations in three separate studies. Initial screening was performed by single-stranded conformational polymorphism (SSCP) analysis in a subset of 1,688 samples obtained from a population-based case-control study of NTDs in California. In the second study, the DNA sequence of exons 5 and 6 was determined in a group of 50 NTD affected individuals. The final experiment involved using dideoxy fingerprinting (ddF) to screen a population-based case-control sample of 219 individuals who were stratified into four sub-groups on the basis of folate intake and pregnancy outcome. No polymorphism was detected in any of the four exons examined. It is unlikely that the beneficial effects of maternal folate supplementation in preventing NTDs acts through a mechanism involving pharmacological correction of a variant form of folate receptor alpha.

Transcriptional regulation of MMP-9 expression in stromal cells of human giant cell tumor of bone by tumor necrosis factor-alpha.

We determined whether certain factor(s) secreted by multinucleated giant cells, which is of monocyte/macrophage lineage in giant cell tumor of bone (GCT), regulate the induction of matrix metalloproteinase (MMP)-9 expression in mononucleated stromal cells. Our data derived using enzyme linked immunosorbant assays (ELISAs) suggest that the GCT cells in primary culture produce both MMP-9 and tumor necrosis factor-alpha (TNF-alpha). Further, the MMP-9 expression in GCT primary cultures was partially abrogated by neutralizing antibody to TNF-alpha, suggesting that TNF-alpha secretion by the multinucleated giant cells may be one of the factors responsible for the production of MMP-9 by the stromal cells in vivo. In order to confirm this we examined the role of TNF-alpha on the induction of MMP-9 expression in bone GCT stromal cells. These cells express MMP-2, but not MMP-9. However, treatment of these cells with TNF-alpha induced the expression of MMP-9 in a concentration-dependent manner. Kinetic experiments revealed that the secretion of MMP-9 peaked 12 h post TNF-alpha stimulation. Immunofluorescence studies confirmed the expression of MMP-9 after stimulation of GCT stromal cells with TNF-alpha. Further, TNF-alpha-induced MMP-9 expression was completely blocked with neutralizing antibody to TNF-alpha, thereby demonstrating the specificity. In addition, the induction of MMP-9 expression by TNF-alpha was completely abrogated in the presence of cycloheximide, a protein synthesis inhibitor, suggesting that de novo protein synthesis may be required. Nuclear run-on analysis demonstrated that treatment of GCT stromal cells significantly enhanced the MMP-9 gene transcription. Together, our data suggest that TNF-alpha secreted by the multinucleated giant cells up-regulates MMP-9 expression in GCT stromal cells by the induction of certain transcription factors, which in turn enhanced the rate of transcription of MMP-9 gene. These studies also suggest the existence of an essential cell-cell interaction in the regulation of MMP-9 expression in GCT.

Genetic basis of susceptibility to environmentally induced neural tube defects.

Neural tube defects (NTDs) are among the most common of all human congenital defects, with multifactorial etiologies comprising both environmental and genetic components. Several murine model systems have been developed in an effort to elucidate genetic factors regulating expression of NTDs. Strain-dependent differences in susceptibility to teratogenic insults and altered patterns of gene expression observed within the neuroepithelium of affected embryos support the hypothesis that subtle genetic changes can result in NTDs. Since several affected genes are folate-regulated, transgenic knockout mice lacking a functional folate receptor were developed. Nullizygous embryos died in utero with significant morphological defects, supporting the critical role of folic acid in early embryogenesis. While epidemiological studies have not established an association between polymorphisms in the human folate receptor gene and NTDs, it is known that folate supplementation reduces infant NTD risk. Continued efforts are therefore necessary to reveal the mechanism by which folate works and the nature of the gene(s) responsible for human NTDs.

Seizure control following administration of anticonvulsant drugs in the quaking mouse.

The effectiveness of a variety of clinical anticonvulsant drugs was evaluated in the quaking mutant mouse model of epilepsy. In this model, tonic-clonic seizures are easily elicited by handling and the effects of administration of carbamazepine (CBZ), phenytoin (DPH), phenobarbital (PB), diazepam, valproic acid (VPA) and ethosuximide were quantitatively evaluated. Chronic oral administration of CBZ, DPH and PB reduced the frequency of seizures and this was positively correlated with plasma levels of the drugs. The plasma levels of the 10,11-epoxide metabolite of CBZ were found to be approximately 3-5 times that of the parent compound with chronic oral administration. Acute intraperitoneal administration of the other drugs revealed VPA to be an effective anticonvulsant agent, whereas ethosuximide and diazepam were ineffective at dosage levels that are normally effective in mice as determined by classical testing methods such as electroshock and chemoshock. The results of the present study suggest that the quaking mouse may be a simple, reliable and inexpensive animal model for the evaluation of agents effective against focal motor seizures in humans.