Folate intake, markers of folate status and oral clefts: An updated set of systematic reviews and meta-analyses.

BACKGROUND: There has been a longstanding debate about the role of folate in the etiology of orofacial clefts (OFCs). Studies of different measures of nutritional intake or folate status have been done to investigate the possible role of folate in the prevention of OFC. Only one knowledge synthesis has attempted to bring together different types of evidence. The aim of the present work was to update it. METHODS: Evidence for associations between OFC and dietary folate, supplement use, folic acid fortification, biomarkers of folate status, and variants of MTHFR (C677T and A1298C) were included. Potentially eligible articles were systematically identified from PubMed, Medline, Embase, and Web of Science (2007-2020) and combined using random-effects meta-analysis when appropriate. Quality assessments were conducted using the Newcastle-Ottawa scale and Cochrane's risk of bias tool. RESULTS: Sixty-four studies published since the previous knowledge synthesis were identified, with eight of these identified through a supplementary search from October, 2018 to August, 2020. There was an inverse association between folic acid-containing supplement use before or during pregnancy and cleft lip with or without cleft palate (CL/P) (OR 0.60, 95% CI 0.51-0.69), with considerable between-study heterogeneity. The prevalence of CL/P showed a small decline post-folic acid fortification in seven studies (OR 0.94, 95% CI 0.86-1.02). No association was found between OFC and genetic markers of folate status. The coronavirus-19 pandemic has threatened food availability globally and therefore there is a need to maintain and even enhance surveillance concerning maternal intake of folate and related vitamins. CONCLUSIONS: The risk of non-syndromic OFC was reduced among pregnant women with folic acid-containing supplements during the etiologically relevant period. However, high heterogeneity between included studies, incomplete reporting of population characteristics and variation in timing of exposure and supplement types mean that conclusions should be drawn with caution.

Protective effect of folic acid on vulnerability to oxidative stress in dental pulp stem cells of deciduous teeth from children with orofacial clefts.

Orofacial clefts (OFCs) are among the most common congenital craniofacial malformations, including cleft lip with or without cleft palate as the core symptoms. Developmental or functional defects in neural crest cells (NCCs) that contribute to craniofacial morphogenesis are involved in OFC development. Previous studies have suggested that oxidative stress in NCCs is involved in the development of OFCs, suggesting that the anti-oxidative activity of folic acid (FA) could have protective effects. However, studies of human-derived NCCs are limited, as these cells are predominantly active during the embryonic stage. In this study, the effects of oxidative stress and FA were evaluated in human OFCs. In particular, NCC-derived stem cells from human exfoliated deciduous teeth (SHEDs) were obtained from 3 children with non-syndromic cleft lip with cleft palate (CLPs) and from 3 healthy children (CTRLs). Mitochondrial reactive oxygen species (ROS) levels were significantly higher in CLPs than in CTRLs and were associated with lower mRNA expression levels of superoxide dismutase 1 (SOD1) and decreased cell mobility. In addition, significantly greater vulnerability to pyocyanin-induced ROS, mimicking exogenous ROS, was observed in CLPs than in CTRLs. These vulnerabilities to endogenous and exogenous ROS in CLPs were significantly improved by FA. These results indicated that the transcriptional dysregulation of SOD1 in NCCs is an oxidative stress-related pathological factor in OFCs, providing novel evidence for the benefits of perinatal anti-oxidant supplementation, including FA, for the management of these common deformities.

Involvement of RBP4 in all­trans retinoic acid induced cleft palate.

The current study was designed to elucidate the mechanism of retinol binding protein 4 (RBP4) in cleft palate induced by all­trans retinoic acid (atRA). To establish a cleft palate model in C57BL/6J mice, pregnant mice were administered atRA (100 mg/kg) by gavage at the tenth embryonic stage (E10.0). Control groups were given the equivalent volume of corn oil. Pregnant mice were dissected at E12.5, E13.5 and E14.5 to obtain the embryonic palates. The expression levels of RBP4 in the embryonic palatal mesenchyme (EPM) were determined by immunohistochemistry, reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and western blotting. Human embryonic palatal mesenchymal cells were exposed to atRA to detect the variation in RBP4 induced by atRA in vitro. Small interfering RNA was used to suppress the expression of RBP4, and a plasmid overexpressing RBP4 was used to examine upregulated expression. The cell counting kit­8 assay was used to evaluate the effect of RBP4 on cell proliferation. The expression levels of p27 and cyclin D1 were determined by RT­qPCR and western blotting, while the expression levels of extracellular signal­related kinase (ERK) 1/2 and protein kinase B (AKT) were assessed by western blotting. At E14.5, RBP4 was strongly expressed in the EPM, while it was downregulated following atRA treatment, which induced cleft palate in vivo. In vitro experiments indicated that atRA suppressed the expression of RBP4 and altered the expression of p27 and cyclin D1 to cause growth inhibition. Knockdown of RBP4 resulted in decreased expression of cyclin D1 and increased p27, and suppressed proliferation. Overexpression of RBP4 reversed the inhibitory effect of atRA and promoted proliferation via the ERK1/2 and AKT signaling pathways. These results suggested that RBP4 was involved in cleft palate induced by atRA and it can be suppressed by atRA to cause growth inhibition in the embryonic palate.

The role of folate metabolism in orofacial development and clefting.

Folate deficiency has been associated with numerous diseases and birth defects including orofacial defects. However, whether folate has a role in the face during early orofacial development has been unclear. The present study reveals that pharmacological and antisense oligonucleotide mediated inhibition of DHFR, an integral enzyme in the folate pathway, results in specific changes in the size and shape of the midface and embryonic mouth. Such defects are accompanied by a severe reduction in the muscle and cartilage jaw elements without significant change in neural crest pattern or global levels of methylation. We propose that the orofacial defects associated with DHFR deficient function are the result of decreased cell proliferation and increased cell death via DNA damage. In particular, localized apoptosis may also be depleting the cells of the face that express crucial genes for the differentiation of the jaw structures. Folate supplementation is widely known to reduce human risk for orofacial clefts. In the present study, we show that activating folate metabolism can reduce median oral clefts in the primary palate by increasing cell survival. Moreover, we demonstrate that a minor decrease in DHFR function exacerbates median facial clefts caused by RAR inhibition. This work suggests that folate deficiencies could be a major contributing factor to multifactorial orofacial defects.

Association between C677T and A1298C MTHFR gene polymorphism and nonsyndromic orofacial clefts in the Turkish population: a case-parent study.

Two common MTHFR gene polymorphisms (C677T and A1298C) have been implicated in the etiology of nonsyndromic cleft lip/palate (nsCL/P). To investigate the genotype association among nsCL/P in the Turkish population, 56 case-parent trios were recruited into the study. Genotype frequencies were compared to two groups of controls from the same population. A total of 46 case-parent trios were included in transmission disequilibrium test (TDT) analysis. The mothers of the study group had a higher frequency of 677TT genotype, with a three-fold increased risk of having nsCL/P offspring (odds ratio [OR]: 3.14, p=0.03). The combined 677CT/1298AC genotype was also common among these mothers (28%), but it did not reach statistical significance (OR: 2.27, p=0.07). TDT analysis for (C677T) T allele transmission did not reveal a significant association. In conclusion, mothers carrying 677TT genotype or with 677CT/1298AC combined genotype have increased risk of having nsCL/P offspring; therefore, higher periconceptional folic acid supplementation should be advised for decreasing the recurrence risk.

Nested case-control study of one-carbon metabolites in mid-pregnancy and risks of cleft lip with and without cleft palate.

Evidence exists for an association between use of vitamin supplements with folic acid in early pregnancy and reduced risk for offspring with cleft lip with/without cleft palate (CLP). 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 CLP by measuring nutrient analytes in mid-pregnancy sera. This study included data from a repository of women's mid-pregnancy serum specimens collected in California from 2003-04. Each woman's specimen was linked with delivery information to determine whether her fetus had CLP or another structural malformation, or was nonmalformed. We identified 89 CLP cases. We randomly selected 409 specimens as controls. Specimens were tested for homocysteine, methylmalonic acid, folate, vitamin B12, pyridoxal phosphate, pyridoxal, pyridoxic acid, riboflavin, choline, betaine, methionine, methionine sulfoxide, cysteine, cystathionine, arginine, and asymmetric and symmetric dimethylarginine. We observed three analytes with odds ratios unlikely to be explained by random variation, i.e., elevated CLP risks were observed for low levels and for high levels of pyridoxal phosphate (vitamin B6), higher levels of choline, and low levels of symmetric dimethylarginine. These data did not show meaningful differences between cases and controls for any other analytes.

Genome-wide pathway analysis of folate-responsive genes to unravel the pathogenesis of orofacial clefting in man.

BACKGROUND: A cleft of the lip with or without the palate (CLP) is a frequent congenital malformation with a heterogeneous etiology, for which folic acid supplementation has a protective effect. To gain more insight into the molecular pathways affected by natural folate, we examined gene expression profiles of cultured B-lymphoblasts from CLP patients before and after the addition of 5-methyltetrahydrofolate (5-mTHF) to the cultures. METHODS: Immortalized B-lymphoblasts from five children with CLP were cultured in folate-deficient medium for 5 days. 5-mTHF was added to a concentration of 30 nM. Gene expression patterns were then evaluated before and after supplementation using Human Genome U133 Plus 2.0 arrays. Data analysis was performed with Omniviz and the GEPAS analysis suite. Differential genes were categorized into biological pathways with Ingenuity Pathway systems. Differential expression was validated by quantitative RT-PCR. RESULTS: Using supervised clustering, with a false discovery rate <1%, we identified 144 and 409 significantly up-regulated and down-regulated probesets, respectively, after 5-mTHF addition. The regulated genes were involved in a variety of biological pathways, including one carbon pool and cell cycle regulation, biosynthesis of amino acids and DNA/RNA nucleotides, protein processing, apoptosis, and DNA repair. CONCLUSIONS: The large variety of the identified folate responsive pathways fits with the modifying role of folate via the methylation pathway. From the present data we may conclude that folate deficiency deranges normal cell development, which might contribute to the development of CLP. The role of these folate responsive genes in CLP development is intriguing and needs further investigation.

[Effects of hyperbaric oxygen treatment on healing of maxillary distraction osteogenesis in beagle dogs].

Distraction osteogenesis has been widely used even in the craniofacial region. A long fixation time during the consolidation period, however, is a major clinical disadvantage. Hyperbaric oxygen (HBO) has been used to improve healing in ischemic wounds. We have recently started applying hyperbaric oxygen to cleft palate patients after maxillary distraction, but there is little basic evidence. We hypothesized that hyperbaric oxygen would enhance the healing of distraction osteogenesis in the cleft palate model in dogs. A bony segment including a canine was transported proximally into an artificial bone defect in the left palate. Three dogs were treated with hyperbaric oxygen for 20 days just after the distraction and three other dogs underwent only the distraction process (control group). Blood flow of the canine pulp in the bone segment was monitored using a laser Doppler flowmeter throughout the experiment. All the dogs were sacrificed on day 100, and radiological analysis using peripheral quantitative CT and histomorphometric evaluations were performed. Blood flow in the HBO-treated group recovered to the original level about 30 days faster than in the control group (p<0.05). Cortical bone mineral density was significantly higher at the distraction site in the HBO-treated group than in the control group (p<0.05). The histomorphometric analysis revealed that the newly formed bone area was also larger in the HBO-treated group than in the control group (p<0.05). These results suggest that hyperbaric oxygen treatment could be useful for early removal of the distraction device in distraction osteogenesis.

An in vitro screening system for characterizing the cleft palate-inducing potential of chemicals and underlying mechanisms.

An in vitro organ culture system with developing mouse palates was improved to characterize the cleft palate (CP)-inducing potential of chemicals and underlying mechanisms. Palatal explants collected from gestation day 12 mouse fetuses were cultured with various concentrations of teratogens and examined for palatal development after 48 and 72 h of culture to assess effects of the chemicals on growth and/or fusion of palatal shelves. When the explants were exposed to diphenylhydantoin or 5-fluorouracil, palatal growth was inhibited in a concentration-dependent manner at 48 h. Suppression of the expression of proliferative cell nuclear antigen revealed poor cell proliferation. At 72 h, the incidence of explants with CP was significantly increased in the high-dose groups, suggesting that CP induction is mainly attributable to inhibition of palatal growth. By contrast, retinoic acid and hydrocortisone significantly lowered the rates of fused palates at 72 h in all treated groups, while they exhibited no effects on palatal growth at 48 h even at the highest concentration. Because no apoptosis was found in the epithelial cells at the tip of these palates, these chemicals are suggested to inhibit palatal fusion process by preventing apoptosis.

[Immunological and biochemical indices during the use of hyperbaric oxygenation for treating patients with jaw deformities]. / Immunologicheskie i biokhimicheskie pokazateli pri ispol'zovanii giperbaricheskoi oksigenatsii dlia lecheniia bol'nykh s deformatsiiami cheliustei.

Analysis of changes in immunological and biochemical parameters in patients operated on for congenital deformations of the jaw bones and improperly grown fractures showed that postoperative therapy including hyperbaric oxygenation sessions was conducive to increase of immunological reactivity in patients with initially reduced immunological reactivity. No changes in urinary excretion of hydroxyproline were observed in the patients with deformations of the jaws.

Mechanisms of teratogenesis: folic acid and antiepileptic therapy.

Gestational folate deficiency has been associated with abnormal growth and development in both experimental animal and human studies and has been postulated as a putative mechanism for the teratogenic effects of antiepileptic drugs (AEDs). Animal studies have shown that the administration of AEDs results in folate depletion and teratogenic effects. Attempts to prevent the teratogenic effects of AEDs by coadministration of folate have shown variable results, perhaps because of a lack of understanding about the specific effects of AEDs on folate metabolism. Our prospective study of women with epilepsy showed that blood folate levels decreased with increasing plasma AED levels and with the number of AEDs. Low blood folate levels before and/or early in pregnancy were significantly associated with spontaneous abortion and the occurrence of developmental anomalies in the offspring. These findings suggest that folate supplementation might be one means of preventing the occurrence of abnormal pregnancy outcome in women with epilepsy, including neural-tube defects in the offspring.