Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that recruits monocytes into the subendothelial cell layer in atherosclerotic lesions. Elevated homocysteine (hyper-homocysteinemia), which is usually associated with low-folate status, is a known risk factor for many pathologies with inflammatory etiologies. The present study was undertaken to examine whether there are associations between MCP-1 concentrations and folate/Hcy phenotype or methylenetetrahydrofolate reductase (MTHFR) 677C>T genotype in healthy young adults. In females, MCP-1 concentrations were positively correlated with Hcy and negatively correlated with both serum and red blood cell folate; female smokers and MTHFR 677T carriers had particularly elevated MCP-1 concentrations. Similar relationships were not seen in males. These findings may have implications for understanding the female predominance observed for a range of autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis.
MCP-1; Folate; Homocysteine; Autoimmune
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.
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.
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)
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.
ferritin; iron; hemoglobin; neural tube defects
Neural tube defects (NTDs), which are among the most common congenital malformations, are influenced by environmental and genetic factors. Low maternal folate is the strongest known contributing factor, making variants in genes in the folate metabolic pathway attractive candidates for NTD risk. Multiple studies have identified nominally significant allelic associations with NTDs. We tested whether associations detected in a large Irish cohort could be replicated in an independent population.
Replication tests of 24 nominally significant NTD associations were performed in racially/ethnically matched populations. Family-based tests of fifteen nominally significant single nucleotide polymorphisms (SNPs) were repeated in a cohort of NTD trios (530 cases and their parents) from the United Kingdom, and case–control tests of nine nominally significant SNPs were repeated in a cohort (190 cases, 941 controls) from New York State (NYS). Secondary hypotheses involved evaluating the latter set of nine SNPs for NTD association using alternate case–control models and NTD groupings in white, African American and Hispanic cohorts from NYS.
Of the 24 SNPs tested for replication, ADA rs452159 and MTR rs10925260 were significantly associated with isolated NTDs. Of the secondary tests performed, ARID1A rs11247593 was associated with NTDs in whites, and ALDH1A2 rs7169289 was associated with isolated NTDs in African Americans.
We report a number of associations between SNP genotypes and neural tube defects. These associations were nominally significant before correction for multiple hypothesis testing. These corrections are highly conservative for association studies of untested hypotheses, and may be too conservative for replication studies. We therefore believe the true effect of these four nominally significant SNPs on NTD risk will be more definitively determined by further study in other populations, and eventual meta-analysis.
Electronic supplementary material
The online version of this article (doi:10.1186/s12881-014-0102-9) contains supplementary material, which is available to authorized users.
Neural tube defects; Spina bifida; Folate; Folic acid; One-carbon metabolism; Replication
Folic acid fortification has reduced neural tube defect prevalence by 50% to 70%. It is unlikely that fortification levels will be increased to reduce neural tube defect prevalence further. Therefore, it is important to identify other modifiable risk factors. Vitamin B12 is metabolically related to folate; moreover, previous studies have found low B12 status in mothers of children affected by neural tube defect. Our objective was to quantify the effect of low B12 status on neural tube defect risk in a high-prevalence, unfortified population.
We assessed pregnancy vitamin B12 status concentrations in blood samples taken at an average of 15 weeks’ gestation from 3 independent nested case-control groups of Irish women within population-based cohorts, at a time when vitamin supplementation or food fortification was rare. Group 1 blood samples were from 95 women during a neural tube defect–affected pregnancy and 265 control subjects. Group 2 included blood samples from 107 women who had a previous neural tube defect birth but whose current pregnancy was not affected and 414 control subjects. Group 3 samples were from 76 women during an affected pregnancy and 222 control subjects.
Mothers of children affected by neural tube defect had significantly lower B12 status. In all 3 groups those in the lowest B12 quartiles, compared with the highest, had between two and threefold higher adjusted odds ratios for being the mother of a child affected by neural tube defect. Pregnancy blood B12 concentrations of <250 ng/L were associated with the highest risks.
Deficient or inadequate maternal vitamin B12 status is associated with a significantly increased risk for neural tube defects. We suggest that women have vitamin B12 levels of >300 ng/L (221 pmol/L) before becoming pregnant. Improving B12 status beyond this level may afford a further reduction in risk, but this is uncertain.
vitamin B12; cobalamin; neural tube defects; folic acid fortification; folate
A previous report described the presence of autoantibodies against folate
receptors in 75% of serum samples from women with a history of pregnancy complicated by
a neural-tube defect, as compared with 10% of controls. We sought to confirm this
finding in an Irish population, which traditionally has had a high prevalence of
We performed two studies. Study 1 consisted of analysis of stored frozen blood
samples collected from 1993 through 1994 from 103 mothers with a history of pregnancy
complicated by a neural-tube defect (case mothers), 103 mothers with a history of
pregnancy but no complication by a neural-tube defect (matched with regard to number of
pregnancies and sampling dates), 58 women who had never been pregnant, and 36 men. Study
2, conducted to confirm that the storage of samples did not influence the
folate-receptor autoantibodies, included fresh samples from 37 case mothers, 22 control
mothers, 10 women who had never been pregnant, and 9 men. All samples were assayed for
blocking and binding autoantibodies against folate receptors.
In Study 1, blocking autoantibodies were found in 17% of case mothers, as
compared with 13% of control mothers (odds ratio, 1.54; 95% confidence interval [CI],
0.70 to 3.39), and binding autoantibodies in 29%, as compared with 32%, respectively
(odds ratio, 0.82; 95% CI, 0.44 to 1.50). Study 2 showed similar results, indicating
that sample degradation was unlikely.
The presence and titer of maternal folate-receptor autoantibodies were not
significantly associated with a neural-tube defect–affected pregnancy in this
Women who have low cobalamin (vitamin B12) levels are at increased risk for having children with neural tube defects (NTDs). The transcobalamin II receptor (TCblR) mediates uptake of cobalamin into cells. We evaluated inherited variants in the TCblR gene as NTD risk factors.
Case-control and family-based tests of association were used to screen common variation in TCblR as genetic risk factors for NTDs in a large Irish group. A confirmatory group of NTD triads was used to test positive findings.
We found two tightly linked variants associated with NTDs in a recessive model: TCblR rs2336573 (G220R) (pcorr=0.0080, corrected for multiple hypothesis testing) and TCblR rs9426 (pcorr =0. 0279). These variants were also associated with NTDs in a family-based test prior to multiple test correction (log-linear analysis of a recessive model: rs2336573 (G220R) (RR=6.59, p=0.0037) and rs9426 (RR=6.71, p=0.0035)). We describe a copy number variant (CNV) distal to TCblR and two previously unreported exonic insertion-deletion polymorphisms.
TCblR rs2336573 (G220R) and TCblR rs9426 represent a significant risk factor in NTD cases in the Irish population. The homozygous risk genotype was not detected in nearly one thousand controls, indicating this NTD risk factor may be of low frequency and high penetrance. Nine other variants are in perfect LD with the associated SNPs. Additional work is required to identify the disease-causing variant. Our data suggest that variation in TCblR plays a role in NTD risk and that these variants may modulate cobalamin metabolism.
neural tube defects; spina bifida; transcobalamin II receptor (TCblR); cobalamin; vitamin B12; copy number variant (CNV)
Individual studies of the genetics of neural tube defects (NTDs) contain results on a small number of genes in each report. To identify genetic risk factors for NTDs, we evaluated potentially functional single nucleotide polymorphisms (SNPs) that are biologically plausible risk factors for NTDs but that have never been investigated for an association with NTDs, examined SNPs that previously showed no association with NTDs in published studies, and tried to confirm previously reported associations in folate-related and non-folate-related genes. We investigated 64 SNPs in 34 genes for association with spina bifida in up to 558 case-families (520 cases, 507 mothers, 457 fathers) and 994 controls in Ireland. Case-control and mother-control comparisons of genotype frequencies, tests of transmission disequilibrium, and log-linear regression models were used to calculate effect estimates. Spina bifida was associated with over-transmission of the LEPR (leptin receptor) rs1805134 minor C allele (genotype relative risk (GRR): 1.5; 95% confidence interval (CI): 1.0, 2.1; P = 0.0264) and the COMT (catechol-O-methyltransferase) rs737865 major T allele (GRR: 1.4; 95% CI: 1.1, 2.0; P = 0.0206). After correcting for multiple comparisons, these individual test P-values exceeded 0.05. Consistent with previous reports, spina bifida was associated with MTHFR 677C>T, T (Brachyury) rs3127334, LEPR K109R, and PDGFRA promoter haplotype combinations. The associations between LEPR SNPs and spina bifida suggest a possible mechanism for the finding that obesity is a NTD risk factor. The association between a variant in COMT and spina bifida implicates methylation and epigenetics in NTDs.
congenital abnormalities; folic acid; neural tube defects; single nucleotide polymorphism; spina bifida
Suggestive, but not conclusive, studies implicate many genetic variants in oral cleft etiology. We used a large, ethnically homogenous study population to test whether reported associations between nonsyndromic oral clefts and 12 genes (CLPTM1, CRISPLD2, FGFR2, GABRB3, GLI2, IRF6, PTCH1, RARA, RYK, SATB2, SUMO1, TGFA) could be confirmed.
Thirty-one single nucleotide polymorphisms (SNPs) in exons, splice sites, and conserved non-coding regions were studied in 509 patients with cleft lip with or without cleft palate (CLP), 383 with cleft palate only (CP), 838 mothers and 719 fathers of patients with oral clefts, and 902 controls from Ireland. Case-control and family-based statistical tests were performed using isolated oral clefts for the main analyses.
In case-control comparisons, the minor allele of PTCH1 A562A (rs2066836) was associated with reduced odds of CLP (OR: 0.29, 95% CI: 0.13–0.64 for homozygotes) whereas the minor allele of PTCH1 L1315P (rs357564) was associated with increased odds of CLP (OR: 1.36, 95% CI: 1.07–1.74 for heterozygotes and OR: 1.56, 95% CI: 1.09–2.24 for homozygotes). The minor allele of one SUMO1 SNP, rs3769817 located in intron 2, was associated with increased odds of CP (OR: 1.45, 95% CI: 1.06–1.99 for heterozygotes). Transmission disequilibrium was observed for the minor allele of TGFA V159V (rs2166975) which was over-transmitted to CP cases (P=0.041).
For 10 of the 12 genes, this is the largest candidate gene study of nonsyndromic oral clefts to date. The findings provide further evidence that PTCH1, SUMO1, and TGFA contribute to nonsyndromic oral clefts.
cleft lip; cleft palate; congenital abnormalities
Neural tube defects (NTDs) are common birth defects (~1 in 1000 pregnancies in the US and Europe) that have complex origins, including environmental and genetic factors. A low level of maternal folate is one well-established risk factor, with maternal periconceptional folic acid supplementation reducing the occurrence of NTD pregnancies by 50-70%. Gene variants in the folate metabolic pathway (e.g., MTHFR rs1801133 (677 C > T) and MTHFD1 rs2236225 (R653Q)) have been found to increase NTD risk. We hypothesized that variants in additional folate/B12 pathway genes contribute to NTD risk.
A tagSNP approach was used to screen common variation in 82 candidate genes selected from the folate/B12 pathway and NTD mouse models. We initially genotyped polymorphisms in 320 Irish triads (NTD cases and their parents), including 301 cases and 341 Irish controls to perform case–control and family based association tests. Significantly associated polymorphisms were genotyped in a secondary set of 250 families that included 229 cases and 658 controls. The combined results for 1441 SNPs were used in a joint analysis to test for case and maternal effects.
Nearly 70 SNPs in 30 genes were found to be associated with NTDs at the p < 0.01 level. The ten strongest association signals (p-value range: 0.0003–0.0023) were found in nine genes (MFTC, CDKN2A, ADA, PEMT, CUBN, GART, DNMT3A, MTHFD1 and T (Brachyury)) and included the known NTD risk factor MTHFD1 R653Q (rs2236225). The single strongest signal was observed in a new candidate, MFTC rs17803441 (OR = 1.61 [1.23-2.08], p = 0.0003 for the minor allele). Though nominally significant, these associations did not remain significant after correction for multiple hypothesis testing.
To our knowledge, with respect to sample size and scope of evaluation of candidate polymorphisms, this is the largest NTD genetic association study reported to date. The scale of the study and the stringency of correction are likely to have contributed to real associations failing to survive correction. We have produced a ranked list of variants with the strongest association signals. Variants in the highest rank of associations are likely to include true associations and should be high priority candidates for further study of NTD risk.
Neural tube defects; Spina bifida; Folic acid; One-carbon metabolism; Candidate gene
Periconceptional use of folic acid prevents most neural tube defects (NTDs). Whether folic acid and/or multivitamins can prevent other congenital anomalies is not clear. This study tested whether maternal blood levels of folate and vitamin B12 in pregnancies affected by congenital malformations excluding NTDs are lower when compared to non-affected pregnancies.
We measured pregnancy red cell folate (RCF), vitamin B12, and homocysteine (tHcy) concentrations in blood samples taken at the first antenatal clinic in Dublin maternity hospitals in 1986–1990 when vitamin supplementation was rare. The cases were mothers who delivered a baby with a congenital malformation other than NTD identified by the Dublin EUROCAT Registry; controls were a systematic sample of mothers of offspring without congenital malformations from the same hospitals in the same time period.
The median maternal levels of RCF and tHcy did not differ significantly between cases and controls for any of the congenital malformation groups examined (RCF: all malformations 275.9 ug/L v controls 271.2; p=0.77; tHcy: all malformations 7.5 umol/L v controls 7.6; p=0.57). In an unadjusted analysis vitamin B12 was significantly higher in case-mothers whose babies had cleft palate only (p=0.006), musculoskeletal malformations (p=0.034) and midline defects (p=0.039) but not after adjustment for multiple testing.
Our data suggest that low maternal folate and B12 levels or high tHcy levels in early pregnancy are not associated with all congenital malformations excluding NTDs. Fortification with folic acid or B12 may not have a beneficial effect in the prevention of these anomalies.
Polymorphisms within the MTHFD1L gene were previously associated with risk of neural tube defects in Ireland. We sought to test the most significant MTHFD1L polymorphisms for an association with risk of cleft in an Irish cohort. This required the development of a new melting curve assay to genotype the technically challenging MTHFD1L triallelic deletion/insertion polymorphism (rs3832406).
Melting curve analysis was used to genotype the MTHFD1L triallelic deletion/insertion polymorphism (rs3832406) and a Single Nucleotide Polymorphism rs17080476 in an Irish cohort consisting of 981 Irish case-parent trios and 1,008 controls. Tests for association with nonsyndromic cleft lip with or without cleft palate and cleft palate included case/control analysis, mother/control analysis and Transmission Disequilibrium Tests of case-parent trios.
A successful melting curve genotyping assay was developed for the deletion/insertion polymorphism (rs3832406). The TDT analysis initially showed that the rs3832406 polymorphism was associated with isolated cleft lip with or without cleft palate. However, corrected p-values indicated that this association was not significant.
Melting Curve Analysis can be employed to successfully genotype challenging polymorphisms such as the MTHFD1L triallelic deletion/insertion polymorphism (DIP) reported here (rs3832406) and is a viable alternative to capillary electrophoresis. Corrected p-values indicate no association between MTHFD1L and risk of cleft in an Irish cohort.
One-carbon metabolism (OCM) is linked to DNA synthesis and methylation, amino acid metabolism and cell proliferation. OCM dysfunction has been associated with increased risk for various diseases, including cancer and neural tube defects. MicroRNAs (miRNAs) are ∼22 nt RNA regulators that have been implicated in a wide array of basic cellular processes, such as differentiation and metabolism. Accordingly, mis-regulation of miRNA expression and/or activity can underlie complex disease etiology. We examined the possibility of OCM regulation by miRNAs. Using computational miRNA target prediction methods and Monte-Carlo based statistical analyses, we identified two candidate miRNA “master regulators” (miR-22 and miR-125) and one candidate pair of “master co-regulators” (miR-344-5p/484 and miR-488) that may influence the expression of a significant number of genes involved in OCM. Interestingly, miR-22 and miR-125 are significantly up-regulated in cells grown under low-folate conditions. In a complementary analysis, we identified 15 single nucleotide polymorphisms (SNPs) that are located within predicted miRNA target sites in OCM genes. We genotyped these 15 SNPs in a population of healthy individuals (age 18–28, n = 2,506) that was previously phenotyped for various serum metabolites related to OCM. Prior to correction for multiple testing, we detected significant associations between TCblR rs9426 and methylmalonic acid (p = 0.045), total homocysteine levels (tHcy) (p = 0.033), serum B12 (p < 0.0001), holo transcobalamin (p < 0.0001) and total transcobalamin (p < 0.0001); and between MTHFR rs1537514 and red blood cell folate (p < 0.0001). However, upon further genetic analysis, we determined that in each case, a linked missense SNP is the more likely causative variant. Nonetheless, our Monte-Carlo based in silico simulations suggest that miRNAs could play an important role in the regulation of OCM.
Polymorphisms in folate-related genes have emerged as important risk factors in a range of diseases including neural tube defects (NTDs), cancer and coronary artery disease (CAD). Having previously identified a polymorphism within the cytoplasmic folate enzyme, MTHFD1, as a maternal risk factor for NTDs; we considered the more recently identified mitochondrial paralogue, MTHFD1L as a candidate gene for NTD association. We identified a common deletion/insertion polymorphism, rs3832406, c.781-6823ATT(7-9), that influences splicing efficiency and is strongly associated with NTD risk. Three alleles of rs3832406 were detected in the Irish population with varying number of ATT repeats; Allele 1 consists of ATT7, while Alleles 2 and 3 consist of ATT8 and ATT9 respectively. Allele 2 of this triallelic polymorphism showed a decreased case risk as demonstrated by case-control logistic regression (P= 0.002) and by transmission disequilibrium test (TDT) (P= 0.001); while Allele 1 showed an increased case risk. Allele 3 showed no influence on NTD risk and represents the lowest frequency allele (0.15). Additional SNP genotyping in the same genomic region provides additional supportive evidence of an association. We demonstrate that two of the three alleles of rs3832406 are functionally different and influence the splicing efficiency of the alternate MTHFD1L mRNA transcripts.
MTHFD1L; NTD; Splicing; Polymorphism; Association; Folate; Mitochondria
Case-parent trios were used in a genome wide association study of cleft lip with/without cleft palate (CL/P). SNPs near two genes not previously associated with CL/P [MAFB: most significant SNP rs13041247, with odds ratio per minor allele OR=0.704; 95%CI=0.635,0.778; p=2.05*10−11; and ABCA4: most significant SNP rs560426, with OR=1.432; 95%CI=1.292,1.587; p=5.70*10−12] and two previously identified regions (chr. 8q24 and IRF6) attained genome wide significance. Stratifying trios into European and Asian ancestry groups revealed differences in statistical significance, although estimated effect sizes were similar. Replication studies from several populations showed confirming evidence, with families of European ancestry giving stronger evidence for markers in 8q24 while Asian families showed stronger evidence for MAFB and ABCA4. Expression studies support a role for MAFB in palate development.
Genetic variants in MTHFD1 (5,10-methylenetetrahydrofolate dehydrogenase/ 5,10-methenyltetrahydrofolate cyclohydrolase/ 10-formyltetrahydrofolate synthetase), an important folate metabolic enzyme, are associated with a number of common diseases, including neural tube defects (NTDs). This study investigates the promoter of the human MTHFD1 gene in a bid to understand how this gene is controlled and regulated. Following a combination of in silico and molecular approaches, we report that MTHFD1 expression is controlled by a TATA-less, Initiator-less promoter and transcription is initiated at multiple start sites over a 126bp region. We confirmed the presence of three database polymorphisms (dbSNP) by direct sequencing of the upstream region (rs1076991 C>T, rs8010584 G>A, rs4243628 G>T), with a fourth (dbSNP rs746488 A>T) not found to be polymorphic in our population and no novel polymorphisms identified. We demonstrate that a common SNP rs1076991 C>T within the window of transcriptional initiation exerts a significant effect on promoter activity in vitro. We investigated this SNP as a potential risk factor for NTDs in a large homogenous Irish population and determined that it is not an independent risk factor, but, it does increase both case (χ2 = 11.06, P = 0.001) and maternal (χ2 = 6.68, P = 0.01) risk when allele frequencies were analysed in combination with the previously identified disease-associated p.R653Q (c.1958 G>A; dbSNP rs2236225) polymorphism. These results provide the first insight into how MTHFD1 is regulated and further emphasise its importance during embryonic development.
MTHFD1; NTD; Functional; SNP; R653Q; Promoter
Genetic and environmental factors contribute to the etiology of neural tube defects (NTDs). While periconceptional folic acid supplementation is known to significantly reduce the risk of NTDs, folate metabolic pathway related factors do not account for all NTDs. Evidence from mouse models indicates that the tumor protein p53 (TP53) is involved in implantation and normal neural tube development. To determine whether genetic variation in the TP53 might contribute to NTD risk in humans, we constructed a high resolution linkage disequilibrium (LD) map of the TP53 genomic region based on genotyping 21 markers in an Irish population. We found that nine of these variants can be used to capture the majority of common variation in the TP53 genomic region. In contrast, the 3-marker haplotype commonly reported in the TP53 literature offers limited coverage of the variation in the gene. We used the expanded set of polymorphisms to measure the influence of TP53 on NTDs using both case-control and family-based tests of association. We also assayed a functional variant in the p53 regulator MDM2 (rs2279744). Alleles of three noncoding TP53 markers were associated with NTD risk. A case effect was seen with the GG genotype of rs1625895 in intron 6 (OR = 1.37 [1.04-1.79], p=0.02). A maternal effect was seen with the 135/135 genotype of the intron 1 VNTR (OR = 1.86 [1.16-2.96], p=0.01) and the TT genotype of rs1614984 (RR = 0.58 [0.37-0.91], p=0.02). As multiple comparisons were made, these cannot be considered definitive positive findings and additional investigation is required.
neural tube defects; spina bifida; p53; TP53; MDM2; linkage disequilibrium
Both environmental and genetic factors are involved in the etiology of neural tube defects (NTDs). Inadequate folate intake and obesity are important environmental risk factors. Several folate-related genetic variants have been identified as risk factors; however, little is known about how genetic variants relate to the increased risk seen in obese women. Uncoupling Protein 2 (UCP2) is an attractive candidate to screen for NTD risk because of its possible role in obesity as well as energy metabolism, type-2 diabetes, and the regulation of reactive oxygen species. Interestingly, a previous study found that a common UCP2 compound homozygous genotype was associated with a threefold increase in NTD risk.
We evaluated three polymorphisms, −866G>A, A55V, and the 3′UTR 45bp insertion/deletion, as risk factors for NTDs in Irish NTD cases (N=169), their mothers (N=163), their fathers (N=167) and normal control subjects (N=332).
Allele and genotype frequencies were not significantly different when comparing NTD mothers, NTD fathers, or affected children to controls. Additionally, the previously reported risk genotype (combined homozygosity of 55VV and 3′UTR 45bp deletion/deletion) was not present at a higher frequency in any NTD group when compared to controls.
In our Irish study population, UCP2 polymorphisms do not influence NTD risk. Moreover, the prevalence of this allele in other populations was similar to the Irish prevalence but far lower than reported in the previous NTD study, suggesting that this previous finding of an association with NTDs might have been due to an unrepresentative study sample.
neural tube defects; spina bifida; UCP2; obesity
Risk of neural tube defects (NTDs) is determined by genetic and environmental factors, among which folate status appears to play a key role. However, the precise nature of the link between low folate status and NTDs is poorly understood, and it remains unclear how folic acid prevents NTDs. We investigated the effect of folate level on risk of NTDs in splotch (Sp2H) mice, which carry a mutation in Pax3. Dietary folate restriction results in reduced maternal blood folate, elevated plasma homocysteine and reduced embryonic folate content. Folate deficiency does not cause NTDs in wild-type mice, but causes a significant increase in cranial NTDs among Sp2H embryos, demonstrating a gene–environment interaction. Control treatments, in which intermediate levels of folate are supplied, suggest that NTD risk is related to embryonic folate concentration, not maternal blood folate concentration. Notably, the effect of folate deficiency appears more deleterious in female embryos than males, since defects are not prevented by exogenous folic acid. Folate-deficient embryos exhibit developmental delay and growth retardation. However, folate content normalized to protein content is appropriate for developmental stage, suggesting that folate availability places a tight limit on growth and development. Folate-deficient embryos also exhibit a reduced ratio of s-adenosylmethionine (SAM) to s-adenosylhomocysteine (SAH). This could indicate inhibition of the methylation cycle, but we did not detect any diminution in global DNA methylation, in contrast to embryos in which the methylation cycle was specifically inhibited. Hence, folate deficiency increases the risk of NTDs in genetically predisposed splotch embryos, probably via embryonic growth retardation.
Ireland is an example of a country that has extensive voluntary fortification with folic acid. After a public consultation process, in 2006, the Food Safety Authority in Ireland FSAI  recommended mandatory fortification. However due to safety considerations this decision is now on hold. Before mandatory fortification goes ahead, existing levels of unmetabolised folic acid and their anticipated increase after fortification needs investigation because of the potential of folic acid to mask pernicious anaemia and possibly accelerate the growth of existing cancers. The aim of this study was to examine the levels of circulatory unmetabolised folic acid in Irish adults (both fasted and un-fasted) and new-born infants (fasted) before the proposed implementation of mandatory folic acid fortification. A secondary aim was to predict the increase in circulatory unmetabolised folic acid levels after fortification.
Study 1. Setting: Irish Blood Transfusion Service (IBTS). Whole blood samples were collected from blood donors (n = 50) attending for routine blood donation sessions (representing the general population). Subjects were not fasted prior to sampling. Study 2. Setting: Coombe Women's and Infant's University Hospital, Dublin. Whole blood samples were collected by venipuncture from mothers (n = 20), and from their infant's umbilical-cords (n = 20) immediately after caesarean section. All women had been fasted for at least 8 hours prior to the surgery. A questionnaire on habitual and recent dietary intakes of folic acid was administered by an interviewer to all subjects. The data collection period was February to April 2006. Serum samples were analysed for plasma folate, plasma folic acid and red cell folate.
Blood Donor Group: Circulatory unmetabolised folic acid was present in 18 out of 20 mothers (fasted) (CI: 68.3%–99.8%) comprising 1.31% of total plasma folate, 17 out of 20 babies (fasted) (CI: 62.1%–96.8%), and 49 out of 50 blood donors (unfasted) (CI: 88.0%–99.9%), comprising 2.25% of total plasma folate,
While the levels of circulatory unmetabolised folic acid reported are low, it is persistently present in women immediately after caesarean section who were fasting indicating that there would be a constant/habitual exposure of existing tumours to folic acid, with the potential for accelerated growth. Mandatory fortification might exacerbate this. This has implications for those with responsibility for drafting legislating in this area.
Cleft lip with or without cleft palate (CLP) and cleft palate only (CPO) have an inherited component and, many studies suggest, a relationship with folate. Attempts to find folate-related genes associated with clefts have, however, often been inconclusive. This study examined four SNPs related to folate metabolism (MTHFR 677 C→T, MTHFR 1298 A→C, MTHFD1 1958 G→A, and TC II 776 C→G) in a large Irish population to clarify their relationship with clefts.
Cases and their parents were recruited from major surgical centers performing cleft repairs in Ireland and a support organization. Data on risk factors, medical history, and DNA were collected. Controls were pregnant women from the greater Dublin area (n = 1,599).
CLP cases numbered 536 and CPO cases 426 after exclusions. CPO mothers were significantly more likely than controls to be MTHFR 677 TT, OR 1.50 (95% CI: 1.05–2.16; p = .03). Log-linear analysis showed a borderline association (p = .07). Isolated CPO case mothers were significantly more likely than controls to be homozygous for the MTHFD1 1958 G→A variant, OR 1.50 (95%CI: 1.08–2.09; p = .02). When multiple cases were added, both CPO cases and case mothers were significantly more likely to be AA (p = .02 and p = .007, respectively). The CLP case-control and mother-control analyses also showed significant effects, ORs 1.38 (95% CI: 1.05–1.82; p = .03) and 1.39 (95% CI: 1.04–1.85; p = .03), respectively.
Associations were found for both CPO and CLP and MTHFD1 1958 G→A in cases and case mothers. MTHFR 677 C→T could be a maternal risk factor for clefts but the association was not strong. Because multiple comparisons were made, these findings require additional investigation. Given the known association between MTHFD1 1958 G→A and NTDs, these findings should be explored in more detail.
cleft lip; cleft palate; oral clefts; folate; folate genes; vitamin B12; transcobalamin gene
Both folate and betaine are important predictors of total homocysteine (tHcy) during pregnancy. However, studies to date have only been undertaken in populations with Western dietary patterns.
We investigated the predictors of tHcy in pregnant women recruited in the Seychelles, a population where access to fortified foods is limited and where women habitually consume diets rich in fish, eggs, rice, and fruit.
Pregnant women (n = 226) provided blood samples at enrollment, at week 28 of gestation, and at delivery. Cord blood was obtained from a subset of participants (n = 135).
As in other studies, maternal tHcy was lower during pregnancy than at delivery, whereas folate and vitamin B-12 status declined significantly to delivery. Despite low maternal folate status at delivery (median: 9.0 nmol/L), with 35% of women in the deficient range (serum folate: <6.8 nmol/L), cord blood folate status (median: 40.2 nmol/L) was similar to concentrations reported in Western populations. Folate was a significant predictor of tHcy at all time points (P < 0.001). In contrast with previous studies, betaine was only a significant predictor of maternal tHcy (P < 0.001) when the essential amino acid methionine was low.
The current study reports 2 important findings. First, fetal requirements for folate are paramount, such that cord blood folate status is maintained, even when maternal status is low. Second, betaine is a significant predictor of tHcy in pregnant women with low serum folate and low serum methionine concentrations.
Homocysteine; folate; betaine; vitamin B-12; pregnancy; maternal status
Folate has emerged as a key nutrient for optimising health. Impaired folate status has been identified as a risk factor for cardiovascular disease, various types of cancers, and neurocognitive disorders. The study aimed at examining the distribution and determinants of serum folate concentrations in a healthy adult population in Crete, Greece.
A cross-sectional sample of 486 healthy adults (250 men, 236 women) aged 39 ± 14 years, personnel of the Medical School and the University Hospital of Crete in Greece, was examined. Serum folate and vitamin B12 concentrations were measured by microbiological assay, and total homocysteine was determined fluorometrically and by high-pressure liquid chromatography. Lifestyle questionnaires were completed, and nutrient intakes and food consumption were assessed by 24-h dietary recalls. Multivariate analyses were performed using SPSS v10.1.
The geometric mean (95% confidence interval) concentrations of serum folate were 15.6 μmol/l (14.6–16.8) in men and 19.2 μmol/l (17.9–20.7) in women (p < 0.001). Inadequate folate levels (≤7 nmol/l) were present in 6.8% of men and 2.1% of women (p < 0.001). Approximately 76% of men and 87% of women did not meet the reference dietary intake for folate (400 μg/day). Serum folate was inversely related to total homocysteine levels (p < 0.001). Increased tobacco and coffee consumption were associated with lower folate concentrations (p < 0.05 for both) but these associations disappeared after controlling for nutrient intakes. In multivariate analysis, intakes of MUFA, fibre, calcium, magnesium, folate, and vitamins A, E, C, B1, and B6 were positively associated with serum folate. Consumption of potatoes, legumes, fruits, and vegetables were favourably related to the serum folate status.
Serum folate concentrations were associated with various demographic, lifestyle and dietary factors in healthy Cretan adults. Large-scale epidemiological studies should be conducted within the general Greek adult population to assess the prevalence of impaired folate status and further examine associations with dietary patterns and chronic disease risk. Considering the importance of folate in health maintenance, it is important to increase the public's awareness of modifiable lifestyle patterns and diet and tobacco use in particular, which may be associated with improved folate status.
BACKGROUND: Suboptimal maternal folate status is considered a risk factor for neural tube defects (NTDs). However, the relationship between dietary folate status and risk of NTDs appears complex, as experimentally induced folate deficiency is insufficient to cause NTDs in nonmutant mice. In contrast, folate deficiency can exacerbate the effect of an NTD-causing mutation, as in splotch mice. The purpose of the present study was to determine whether folate deficiency can induce NTDs in mice with a permissive genetic background which do not normally exhibit defects. METHODS: Folate deficiency was induced in curly tail and genetically matched wild-type mice, and we analyzed the effect on maternal folate status, embryonic growth and development, and frequency of NTDs. RESULTS: Folate-deficient diets resulted in reduced maternal blood folate, elevated homocysteine, and a diminished embryonic folate content. Folate deficiency had a deleterious effect on reproductive success, resulting in smaller litter sizes and an increased rate of resorption. Notably, folate deficiency caused a similar-sized, statistically significant increase in the frequency of cranial NTDs among both curly tail (Grhl3 mutant) embryos and background-matched embryos that are wild type for Grhl3. The latter do not exhibit NTDs under normal dietary conditions. Maternal supplementation with myo-inositol reduced the incidence of NTDs in the folate-deficient wild-type strain. CONCLUSIONS: Dietary folate deficiency can induce cranial NTDs in nonmutant mice with a permissive genetic background, a situation that likely parallels gene-nutrient interactions in human NTDs. Our findings suggest that inositol supplementation may ameliorate NTDs resulting from insufficient dietary folate. Birth Defects Research (Part A), 2010. © 2009 Wiley-Liss, Inc.
neural tube defects; folic acid; inositol; exencephaly; curly tail; diet