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
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
Methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR), riboflavin-dependent enzymes, participate in homocysteine metabolism. Reported effects of riboflavin status on the association between the MTHFR 677C>T polymorphism and homocysteine vary, and the effects of the MTRR 66A>G or MTRR 524C>T polymorphisms on homocysteine are unclear. We tested the hypothesis that the effects of the MTHFR 677C>T, MTRR 66A>G and MTRR 524C>T polymorphisms on fasting plasma total homocysteine (tHcy) depend on riboflavin status (erythrocyte glutathionine reductase activation coefficient, optimum: <1.2; marginally deficient: 1.2–1.4; deficient: ≥1.4) in 771 adults aged 18–75 years. MTHFR 677T allele carriers with middle or low tertile plasma folate (<14.7 nmol/L) had 8.2 % higher tHcy compared to the 677CC genotype (p < 0.01). This effect was eliminated when riboflavin status was optimal (p for interaction: 0.048). In the lowest cobalamin quartile (≤273 pmol/L), riboflavin status modifies the relationship between the MTRR 66 A>G polymorphism and tHcy (p for interaction: 0.034). tHcy was 6.6 % higher in MTRR 66G allele carriers compared to the 66AA genotype with marginally deficient or optimal riboflavin status, but there was no difference when riboflavin status was deficient (p for interaction: 0.059). tHcy was 13.7 % higher in MTRR 524T allele carriers compared to the 524CC genotype when cobalamin status was low (p < 0.01), but no difference was observed when we stratified by riboflavin status. The effect of the MTHFR 677C>T polymorphism on tHcy depends on riboflavin status, that of the MTRR 66A>G polymorphism on cobalamin and riboflavin status and that of the MTRR 524C>T polymorphism on cobalamin status.
Homocysteine; MTHFR; MTRR; Riboflavin; EGRAC; Vitamin B6; EASTAC
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
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
Although methylenetetrahydrofolate reductase, a folate enzyme gene, has been associated with idiopathic male infertility, few studies have examined other folate-related metabolites and genes. We investigated whether idiopathic male infertility is associated with variants in folate, vitamin B12 (B12) and total homocysteine (tHcy)-related genes and measured these metabolites in blood. We conducted a case–control study that included 153 men with idiopathic infertility and 184 fertile male controls recruited at the Fertility Center and Antenatal Care Center, University Hospital, Malmö and Lund, Sweden. Serum folate, red cell folate (RCF), serum B12, plasma tHcy and semen quality were measured. Subjects were genotyped for 20 common variants in 12 genes related to folate/B12/homocysteine metabolism. Metabolite concentrations and genotype distributions were compared between cases and controls using linear and logistic regression with adjustment for covariates. The phosphatidylethanolamine N-methyltransferase (PEMT) M175V and TCblR rs173665 polymorphisms were significantly associated with infertility (P = 0.01 and P = 0.009, respectively), but not with semen quality. Among non-users of supplements, infertile men had lower serum folate concentrations than fertile men (12.89 vs. 14.73 nmol l−1; P = 0.02), but there were no significant differences in RCF, B12 or tHcy. Folate, B12 and tHcy concentrations were not correlated with any semen parameters. This study provides little support for low folate or B12 status in the pathogenesis of idiopathic male infertility. Although additional data are needed to confirm these initial findings, our results suggest that PEMT and TCblR, genes involved in choline and B12 metabolism, merit further investigation in idiopathic male infertility.
folate; idiopathic male infertility; semen quality; vitamin B12
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.
Although methylenetetrahydrofolate reductase, a folate enzyme gene, has been associated with idiopathic male infertility, few studies have examined other folate-related metabolites and genes. We investigated whether idiopathic male infertility is associated with variants in folate, vitamin B12 (B12) and total homocysteine (tHcy)-related genes and measured these metabolites in blood. We conducted a case–control study that included 153 men with idiopathic infertility and 184 fertile male controls recruited at the Fertility Center and Antenatal Care Center, University Hospital, Malmö and Lund, Sweden. Serum folate, red cell folate (RCF), serum B12, plasma tHcy and semen quality were measured. Subjects were genotyped for 20 common variants in 12 genes related to folate/B12/homocysteine metabolism. Metabolite concentrations and genotype distributions were compared between cases and controls using linear and logistic regression with adjustment for covariates. The phosphatidylethanolamine N-methyltransferase (PEMT) M175V and TCblR rs173665 polymorphisms were significantly associated with infertility (P=0.01 and P=0.009, respectively), but not with semen quality. Among non-users of supplements, infertile men had lower serum folate concentrations than fertile men (12.89 vs. 14.73 nmol l−1; P=0.02), but there were no significant differences in RCF, B12 or tHcy. Folate, B12 and tHcy concentrations were not correlated with any semen parameters. This study provides little support for low folate or B12 status in the pathogenesis of idiopathic male infertility. Although additional data are needed to confirm these initial findings, our results suggest that PEMT and TCblR, genes involved in choline and B12 metabolism, merit further investigation in idiopathic male infertility.
folate; idiopathic male infertility; semen quality; vitamin B12
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.
A roundtable to discuss the measurement of folate status biomarkers in NHANES took place in July 2010. NHANES has measured serum folate since 1974 and red blood cell (RBC) folate since 1978 with the use of several different measurement procedures. Data on serum 5-methyltetrahydrofolate (5MTHF) and folic acid (FA) concentrations in persons aged ≥60 y are available in NHANES 1999–2002. The roundtable reviewed data that showed that folate concentrations from the Bio-Rad Quantaphase II procedure (Bio-Rad Laboratories, Hercules, CA; used in NHANES 1991–1994 and NHANES 1999–2006) were, on average, 29% lower for serum and 45% lower for RBC than were those from the microbiological assay (MA), which was used in NHANES 2007–2010. Roundtable experts agreed that these differences required a data adjustment for time-trend analyses. The roundtable reviewed the possible use of an isotope-dilution liquid chromatography–tandem mass spectrometry (LC-MS/MS) measurement procedure for future NHANES and agreed that the close agreement between the MA and LC-MS/MS results for serum folate supported conversion to the LC-MS/MS procedure. However, for RBC folate, the MA gave 25% higher concentrations than did the LC-MS/MS procedure. The roundtable agreed that the use of the LC-MS/MS procedure to measure RBC folate is premature at this time. The roundtable reviewed the reference materials available or under development at the National Institute of Standards and Technology and recognized the challenges related to, and the scientific need for, these materials. They noted the need for a commutability study for the available reference materials for serum 5MTHF and FA.
A roundtable to discuss the measurement of vitamin B-12 (cobalamin) status biomarkers in NHANES took place in July 2010. NHANES stopped measuring vitamin B-12–related biomarkers after 2006. The roundtable reviewed 3 biomarkers of vitamin B-12 status used in past NHANES—serum vitamin B-12, methylmalonic acid (MMA), and total homocysteine (tHcy)—and discussed the potential utility of measuring holotranscobalamin (holoTC) for future NHANES. The roundtable focused on public health considerations and the quality of the measurement procedures and reference methods and materials that past NHANES used or that are available for future NHANES. Roundtable members supported reinstating vitamin B-12 status measures in NHANES. They noted evolving concerns and uncertainties regarding whether subclinical (mild, asymptomatic) vitamin B-12 deficiency is a public health concern. They identified the need for evidence from clinical trials to address causal relations between subclinical vitamin B-12 deficiency and adverse health outcomes as well as appropriate cutoffs for interpreting vitamin B-12–related biomarkers. They agreed that problems with sensitivity and specificity of individual biomarkers underscore the need for including at least one biomarker of circulating vitamin B-12 (serum vitamin B-12 or holoTC) and one functional biomarker (MMA or tHcy) in NHANES. The inclusion of both serum vitamin B-12 and plasma MMA, which have been associated with cognitive dysfunction and anemia in NHANES and in other population-based studies, was preferable to provide continuity with past NHANES. Reliable measurement procedures are available, and National Institute of Standards and Technology reference materials are available or in development for serum vitamin B-12 and MMA.
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
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
Objective To determine an optimal population red blood cell (RBC) folate concentration for the prevention of neural tube birth defects.
Design Bayesian model.
Setting Data from two population based studies in China.
Participants 247 831 participants in a prospective community intervention project in China (1993-95) to prevent neural tube defects with 400 μg/day folic acid supplementation and 1194 participants in a population based randomized trial (2003-05) to evaluate the effect of folic acid supplementation on blood folate concentration among Chinese women of reproductive age.
Intervention Folic acid supplementation (400 μg/day).
Main outcome measures Estimated RBC folate concentration at time of neural tube closure (day 28 of gestation) and risk of neural tube defects.
Results Risk of neural tube defects was high at the lowest estimated RBC folate concentrations (for example, 25.4 (95% uncertainty interval 20.8 to 31.2) neural tube defects per 10 000 births at 500 nmol/L) and decreased as estimated RBC folate concentration increased. Risk of neural tube defects was substantially attenuated at estimated RBC folate concentrations above about 1000 nmol/L (for example, 6 neural tube defects per 10 000 births at 1180 (1050 to 1340) nmol/L). The modeled dose-response relation was consistent with the existing literature. In addition, neural tube defect risk estimates developed using the proposed model and population level RBC information were consistent with the prevalence of neural tube defects in the US population before and after food fortification with folic acid.
Conclusions A threshold for “optimal” population RBC folate concentration for the prevention of neural tube defects could be defined (for example, approximately 1000 nmol/L). Population based RBC folate concentrations, as a biomarker for risk of neural tube defects, can be used to facilitate evaluation of prevention programs as well as to identify subpopulations at elevated risk for a neural tube defect affected pregnancy due to folate insufficiency.