The methylenetetrahydrofolate dehydrogenase (MTHFD1) gene, as one of the key genes involved in the folate pathway, has been reported to play a critical role in the pathogenesis of neural tube defects (NTDs). However, the results of published studies are contradictory and inconclusive. Thus, this meta-analysis aimed to evaluate the effect of the common polymorphism in the MTHFD1 gene, the G1958A (R653Q, dbSNP ID: rs2236225) variant, on the risk of NTDs in all eligible studies.
Relevant literature published before January 3, 2014 was retrieved from the MEDLINE, EMBASE, Cochrane Library, and CBM databases. Pooled crude odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) were calculated to evaluate the association between the MTHFD1 G1958A polymorphism and NTDs risk.
We performed a meta-analysis of nine studies with a total of 4,302 NTDs patients and 4,238 healthy controls. Our results demonstrated a significant correlation between the MTHFD1 G1958A polymorphism and NTDs in an overall meta-analysis. For family-based studies, the study subjects were classified as NTD cases, mothers with NTDs offspring, and fathers with NTDs offspring. We found no association between any of the fathers’ genotypes and NTDs, whereas there was a clear excess of the 1958A allele in the mothers of children with NTDs compared with controls individuals.
In summary, our meta-analysis strongly suggests that the MTHFD1 G1958A polymorphism might be associated with maternal risk for NTDs in Caucasian populations. However, the evidence of this association should be interpreted with caution due to the selective nature of publication of genetic association studies.
Neural tube defects (NTDs) are common, serious malformations with a complex etiology that suggests involvement of both genetic and environmental factors. The authors evaluated maternal or offspring folate-related gene variants and interactions between the gene variants and maternal intake of folates on the risk of NTDs in their offspring. A case-control study was conducted on mothers and/or their fetuses and infants who were born in California from 1999–2003 with an NTD (cases n = 222, including 24 mother-infant pairs) or without a major malformation (controls n = 454, including 186 mother-infant pairs). Maternal intake of folates was assessed by food frequency questionnaire and genotyping was performed on samples from mothers and infants. For mothers in the lowest folate-intake group, risk of NTDs in offspring was significantly decreased for maternal MTHFR SNPs rs1476413, rs1801131 and rs1801133 (odds ratio (OR) = 0.55, 80% confidence interval (CI): 0.20, 1.48; OR = 0.58, 80% CI: 0.24, 1.43; OR = 0.69, 80% CI: 0.41, 1.17, respectively), and TYMS SNPs rs502396 and rs699517 (OR= 0.91, 80% CI: 0.53, 1.56; OR = 0.70, 80% CI: 0.38, 1.29). A gene-only effect was observed for maternal SHMT1 SNP rs669340 (OR = 0.69, 95% CI: 0.49, 0.96). When there was low maternal folate intake, risk of NTDs was significantly increased for infant MTHFD1 SNPs rs2236224, rs2236225 and rs11627387 (OR = 1.58, 80% CI: 0.99, 2.51; OR = 1.53, 80% CI: 0.95, 2.47; OR = 4.25, 80% CI: 2.33, 7.75, respectively) and SHMT1 SNP rs12939757 (OR = 2.01, 80% CI: 1.20, 3.37), but decreased for TYMS SNP rs2847153 (OR = 0.73, 80% CI: 0.37, 1.45). Although power to detect interaction effects was low for this birth defects association study, the gene-folate interactions observed in this study represent preliminary findings that will be useful for informing future studies on the complex etiology of NTDs.
Congenital Abnormalities; Folic Acid; Genetic Association Studies; Molecular Epidemiology; Neural Tube Defects; Maternal Nutritional Physiological Phenomena; Nervous System Malformations; Nutrigenomics
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
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
Periconceptional folic acid use can often prevent neural tube defects (NTDs). Variants of genes involved in folate metabolism in mothers and children have been associated with occurrence of NTDs. We identified Irish families with individuals affected by neural tube defects. In these families, we observed that neural tube defects and birth defects overall occurred at a higher rate in the maternal lineage compared with the paternal lineage. The goal of this study was to look for evidence for genetic effects that could explain the discrepancy in the occurrence of these birth defects in the maternal vs. paternal lineage. We genotyped blood samples from 322 individuals from NTD-affected Irish families, identified through their membership in spina bifida associations. We looked for differences in distribution in maternal vs. paternal lineages of five genetic polymorphisms: the DHFR 19 bp deletion, MTHFD1 1958G>A, MTHFR 1298A>C, MTHFR 677C>T, and SLC19A1 80A>G. In addition to looking at genotypes individually, we determined the number of genotypes associated with decreased folate metabolism in each relative (“risk genotypes”) and compared the distribution of these genotypes in maternal vs. paternal relatives. Overall, maternal relatives had a higher number of genotypes associated with lower folate metabolism than paternal relatives (p = 0.017). We expected that relatives would share the same risk genotype as the individuals with NTDs and/or their mothers. However, we observed that maternal relatives had an over-abundance of any risk genotype, rather than one specific genotype. The observed genetic effects suggest an epigenetic mechanism in which decreased folate metabolism results in epigenetic alterations related to the increased rate of NTDs and other birth defects seen in the maternal lineage. Future studies on the etiology of NTDs and other birth defects could benefit from including multigenerational extended families, in order to explore potential epigenetic mechanisms.
neural tube defects; folate metabolism; DHFR 19bp deletion; MTHFD1 1958G>A; MTHFR 1298A>C; MTHFR 677C>T; SLC19A1 80A>G; maternal inheritance
The polymorphism of methylenetetrahydrofolate dehydrogenase (MTHFD1) has been reported as a risk factor for neural tube defects (NTDs). In the present study, we aimed to investigate whether the single-nucleotide polymorphisms (SNPs) of MTHFD1 gene are associated with NTDs in a Chinese population and to determine their mechanism of action.
MTHFD1 gene was scanned in a total of 270 NTDs cases and 192 healthy controls by using next-generation sequencing (NGS) method. After quality control procedures, 208 selected SNP sites in MTHFD1 gene were enrolled for follow-up statistical association analyses. Functional analyses were also performed for significant SNPs through bioinformatics analysis. Folic acid levels of brain tissue in available NTDs cases and healthy controls (113 and 123, respectively) were measured. Statistical and bioinformatics analyses were performed to investigate the relationship between SNPs in MTHFD1 and susceptibility to NTDs.
Statistical analysis showed that 2 independent SNPs, rs1956545 and rs56811449, confer the risk of NTDs (P value=0.0195, OR (odds ratio)=1.41, 95% CI (confidence interval)=1.06–1.88; P value=0.0107, OR=0.56, 95% CI=0.36–0.87). The haplotype GGGG, which consists of 4 SNPs (rs2236225, rs2236224, rs1256146, and rs6573559), is also associated with risk of NTDs (P value=0.0438, OR=0.7180, 95% CI=0.5214–0.9888). The risk allele C of rs1956545 is also associated with decreased folic acid levels in the brain (P value=0.0222, standard beta=−0.2238, 95% CI=−0.4128 – −0.0349) according to analysis in the subset of NTDs cases and healthy controls. Bioinformatics analysis indicates that rs1956545 and rs56811449 are within ENCODE regulatory regions, the open chromatin regions of blastula Trophoblast cell line, and histone-marked region of brain astrocyte cell line.
The polymorphism of SNP loci rs1956545 and rs56811449 as well as a haplotype in MTHFD1 gene could serve as an indicator for the occurrence of NTDs in Chinese population and some specific genotypes of the loci may have lower risk of developing NTDs.
Folic Acid Deficiency; Neural Tube Defects; Polymorphism, Single Nucleotide
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
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
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
Objective: To investigate the contribution of polymorphic variation in genes involved in the folate-dependent homocysteine pathway in the aetiology of neural tube defects (NTD).
Design: Case-control association study.
Subjects: A total of 530 individuals from families affected by NTD, 645 maternal controls, and 602 healthy newborn controls from the northern UK.
Main outcome measures: Seven polymorphisms in six genes coding for proteins in the folate-dependent homocysteine pathway (MTHFR 677C→T, MTHFR 1298A→C, MTRR 66A→G, SHMT 1420C→T, CßS 844ins68, GCPII 1561C→T, RFC-1 80G→A). The impact of each polymorphism and the effect of gene–gene interactions (epistasis) upon risk of NTD were assessed using logistic regression analysis.
Results: The MTHFR 677C→T polymorphism was shown to represent a risk factor in NTD cases (CC v CT+TT odds ratio (OR) 2.03 [95% confidence interval (CI) 1.09, 3.79] p = 0.025) and the MTRR 66A→G polymorphism was shown to exert a protective effect in NTD cases (AA v AG+GG OR 0.31 [95% CI 0.10, 0.94] p = 0.04). When statistical tests for interaction were conducted, three genotype combinations in cases (MTRR/GCPII; MTHFR 677/CßS; MTHFR 677/MTRR) and one combination in case mothers (CßS/RFC-1) were shown to elevate NTD risk. Maternal–fetal interaction was also detected when offspring carried the MTHFR 677C→T variant and mothers carried the MTRR 66A→G variant, resulting in a significantly elevated risk of NTD.
Conclusion: Both independent genetic effects and gene–gene interaction were observed in relation to NTD risk. Multi-locus rather than single locus analysis might be preferable to gain an accurate assessment of genetic susceptibility to NTD.
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
Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme in folate metabolism and is involved in DNA methylation, DNA synthesis, and DNA repair. In addition, it is a possible risk factor in neural tube defects (NTDs). The association of the C677T polymorphism in the MTHFR gene and NTD susceptibility has been widely demonstrated, but the results remain inconclusive. In this study, we performed a meta-analysis with 2429 cases and 3570 controls to investigate the effect of the MTHFR C677T polymorphism on NTDs.
An electronic search of PubMed and Embase database for papers on the MTHFR C677T polymorphism and NTD risk was performed. All data were analysed with STATA (version 11). Odds ratios (ORs) with 95% confidence intervals (CIs) were estimated to assess the association. Sensitivity analysis, test of heterogeneity, cumulative meta-analysis, and assessment of bias were performed in our meta-analysis.
A significant association between the MTHFR C677T polymorphism and NTD susceptibility was revealed in our meta-analysis ( TT versus CC: OR = 2.022, 95% CI: 1.508, 2.712; CT+TT versus CC: OR = 1.303, 95% CI: 1.089, 1.558; TT versus CC+CT: OR = 1.716, 95% CI: 1.448, 2.033; 2TT+CT versus 2CC+CT: OR = 1.330, 95% CI: 1.160, 1.525). Moreover, an increased NTD risk was found after stratification of the MTHFR C677T variant data by ethnicity and source of controls.
The results suggested the maternal MTHFR C677T polymorphism is a genetic risk factor for NTDs. Further functional studies to investigate folate-related gene polymorphisms, periconceptional multivitamin supplements, complex interactions, and the development of NTDs are warranted.
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)
Folate deficiency is implicated in the causation of neural tube defects (NTDs). The preventive effect of periconceptional folic acid supplement use is partially explained by the treatment of a deranged folate-dependent one carbon metabolism, which provides methyl groups for DNA-methylation as an epigenetic mechanism. Here, we hypothesize that variations in DNA-methylation of genes implicated in the development of NTDs and embryonic growth are part of the underlying mechanism. In 48 children with a neural tube defect and 62 controls from a Dutch case-control study and 34 children with a neural tube defect and 78 controls from a Texan case-control study, we measured the DNA-methylation levels of imprinted candidate genes (IGF2-DMR, H19, KCNQ1OT1) and non-imprinted genes (the LEKR/CCNL gene region associated with birth weight, and MTHFR and VANGL1 associated with NTD). We used the MassARRAY EpiTYPER assay from Sequenom for the assessment of DNA-methylation. Linear mixed model analysis was used to estimate associations between DNA-methylation levels of the genes and a neural tube defect. In the Dutch study group, but not in the Texan study group we found a significant association between the risk of having an NTD and DNA methylation levels of MTHFR (absolute decrease in methylation of −0.33% in cases, P-value = 0.001), and LEKR/CCNL (absolute increase in methylation: 1.36% in cases, P-value = 0.048), and a borderline significant association for VANGL (absolute increase in methylation: 0.17% in cases, P-value = 0.063). Only the association between MTHFR and NTD-risk remained significant after multiple testing correction. The associations in the Dutch study were not replicated in the Texan study. We conclude that the associations between NTDs and the methylation of the MTHFR gene, and maybe VANGL and LEKKR/CNNL, are in line with previous studies showing polymorphisms in the same genes in association with NTDs and embryonic development, respectively.
Folate metabolism pathway genes have been examined for association with neural tube defects (NTDs) because folic acid supplementation reduces the risk of this debilitating birth defect. Most studies addressed these genes individually, often with different populations providing conflicting results.
Our study evaluates several folate pathway genes for association with human NTDs, incorporating an environmental cofactor: maternal folate supplementation.
In 304 Caucasian American NTD families with myelomeningocele or anencephaly, we examined 28 polymorphisms in 11 genes: folate receptor 1, folate receptor 2, solute carrier family 19 member 1, transcobalamin II, methylenetetrahydrofolate dehydrogenase 1, serine hydroxymethyl-transferase 1, 5,10-methylenetetrahydrofolate reductase (MTHFR), 5-methyltetrahydrofolate-homo-cysteine methyltransferase, 5-methyltetrahydrofolate-homocysteine methyltransferase reductase, betaine-homocysteine methyltransferase (BHMT), and cystathionine-beta-synthase.
Only single nucleotide polymorphisms (SNPs) in BHMT were significantly associated in the overall data set; this significance was strongest when mothers took folate-containing nutritional supplements before conception. The BHMT SNP rs3733890 was more significant when the data were stratified by preferential transmission of the MTHFR rs1801133 thermolabile T allele from parent to offspring. Other SNPs in folate pathway genes were marginally significant in some analyses when stratified by maternal supplementation, MTHFR, or BHMT allele transmission.
BHMT rs3733890 is significantly associated in our data set, whereas MTHFR rs1801133 is not a major risk factor. Further investigation of folate and methionine cycle genes will require extensive SNP genotyping and/or resequencing to identify novel variants, inclusion of environmental factors, and investigation of gene–gene interactions in large data sets.
folate; folic acid supplementation; genetic association; neural tube defects
Neural tube defects (NTDs) are the most common congenital defects of the central nervous system among neonates and the folate status during pregnancy was considered as the most important etiopathogenesis of NTDs. Besides, methionine synthase (MTR) gene and methionine synthase reductase (MTRR) gene were folate metabolism involved genes and had been investigated in several previous studies with inconsistent results. Hence, we aimed to explore the association of 4 selected single-nucleotide polymorphisms (SNPs) on MTRR/MTR gene and the susceptibility of NTDs in a Chinese population.
Seven SNPs were selected from HapMap databases with Haploview 4.2 software. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to genotype the polymorphisms from blood samples of 165 NTDs patients and 280 healthy controls. The correlation between these SNPs and NTDs risk was tested by Student t test and Chi-square test by STATA 11.0 software. Furthermore, we performed a meta-analysis of relevant studies to investigate the association between the SNPs MTRR 66A>G and MTR 2756A>G and the susceptibility of NTDs.
An increased risk of NTDs was verified to be significantly associated with MTRR 66A>G (G allele vs. A allele: OR = 1.36 (1.03–1.80), P = 0.028; GG + AG vs. AA: OR = 1.60 (1.05–2.43), P = 0.027) and MTR 2756A>G (G allele vs. A allele: OR = 1.45 (1.06–1.98), P = 0.021; GG + AG vs. AA: OR = 1.51 (1.02–2.23), P = 0.038) in our study. However, the other SNPs in our analysis showed no significant association with NTDs risk (all P > 0.05). Furthermore, the result of the meta-analysis supported the association between MTRR 66A>G and NTDs risk (G allele vs. A allele: OR = 1.32, 95% CI = 1.09–1.61, GG + GA vs. AA: OR = 1.49, 95% CI = 1.06–2.09, GG vs. AA: OR = 1.61, 95% CI = 1.04–2.49).
Our study confirmed that the MTRR 66A>G and MTR 2756A>G were significantly associated with the increased NTDs risk in a Chinese population. The further meta-analysis enhance that MTRR 66A>G was connected with the susceptibility of NTDs widely. Further investigations based on more detailed stratification were recommended.
The PCMT1 gene encodes the protein repair enzyme protein-l-isoaspartate (d-aspartate) O-methyltransferase, which is known to protect certain neural cells against Bax-induced apoptosis. Previous studies have produced inconsistent results regarding the effects of PCMT1 (rs4816 and rs4552) polymorphisms on neural tube defects (NTDs). Reduced maternal plasma folate levels and/or elevated homocysteine (Hcy) levels are considered to be risk factors for NTDs. In order to clarify the key factors contributing to the apparent discrepancy and investigate gene–environment interaction, we conducted a case–control study including 121 cases and 146 matched controls to investigate the association between the two PCMT1 polymorphisms in fetuses and the risk of NTDs in the Chinese population of Lvliang, which has low folate intake. Maternal plasma folate and Hcy levels were also measured, and the interaction between fetal PCMT1 gene status and maternal folate metabolites was assessed. Maternal plasma folate concentrations in the NTD group were lower than in controls (10.23 vs. 13.08 nmol/L, adjusted P = 0.059), and Hcy concentrations were significantly higher (14.46 vs. 11.65 μmol/L, adjusted P = 0.026). Fetuses carrying the rs4816 AG + GG genotype, combined with higher maternal plasma Hcy, had a 6.46-fold (95 % CI 1.15–36.46) increased risk of anencephaly. The results of this study imply that the fetal PCMT1 rs4816 polymorphism may play only a weak role in NTD formation and that gene–environment interactions might be more significant.
Association study; PCMT1; Homocysteine; Neural tube defect; Gene–environment interaction
Folate hydrolase 1 (FOLH1) gene encodes intestinal folate hydrolase, which regulates intestinal absorption of dietary folate. Previous studies on the association between polymorphisms rs202676 and rs61886492 and the risk of neural tube defects (NTDs) were inconclusive. A case–control study of women with NTD-affected pregnancies (n = 160) and controls (n = 320) was conducted in the Chinese population of Lvliang, a high-risk area for NTDs. We genotyped the polymorphic sites rs202676 and rs61886492 and assessed maternal plasma folate and total homocysteine (tHcy). Our results showed that in case group, plasma folate concentrations were 18 % lower compared with those of control group (8.32 vs. 6.79 nmol/L, p = 0.033) and tHcy concentrations were 17 % higher (10.47 vs. 12.65 μmol/L, p = 0.047). Almost all samples had the rs61886492 GG genotype (99.78 %). The result showed that the frequency of GG genotype in rs202676 was significantly higher in group with multiple NTDs than in controls (p = 0.030, OR = 2.157, 95 % CI, 1.06–4.38). The multiple-NTD group showed higher maternal plasma concentrations of tHcy (10.47 vs. 13.96 μmol/L, p = 0.024). The GG genotype of rs202676 had a lower maternal folate and higher tHcy concentrations than other genotypes with no significant differences. The result of structural prediction indicated that this variation might change the spatial structure of the protein. These results suggested that the maternal polymorphism rs202676 was a potential risk factor for multiple NTDs in this Chinese population. The allele G might affect maternal plasma folate and tHcy concentration.
Association study; Chinese population; FOLH1; Neural tube defects; Single-nucleotide polymorphism
Neural tube defects (NTDs), including spina bifida and anencephaly, are common birth defects whose complex multigenic causation has hampered efforts to delineate their molecular basis. The effect of putative modifier genes in determining NTD susceptibility may be investigated in mouse models, particularly those that display partial penetrance such as curly tail, a strain in which NTDs result from a hypomorphic allele of the grainyhead-like-3 gene. Through proteomic analysis, we found that the curly tail genetic background harbours a polymorphic variant of lamin B1, lacking one of a series of nine glutamic acid residues. Lamins are intermediate filament proteins of the nuclear lamina with multiple functions that influence nuclear structure, cell cycle properties, and transcriptional regulation. Fluorescence loss in photobleaching showed that the variant lamin B1 exhibited reduced stability in the nuclear lamina. Genetic analysis demonstrated that the variant also affects neural tube closure: the frequency of spina bifida and anencephaly was reduced three-fold when wild-type lamin B1 was bred into the curly tail strain background. Cultured fibroblasts expressing variant lamin B1 show significantly increased nuclear dysmorphology and diminished proliferative capacity, as well as premature senescence, associated with reduced expression of cyclins and Smc2, and increased expression of p16. The cellular basis of spinal NTDs in curly tail embryos involves a proliferation defect localised to the hindgut epithelium, and S-phase progression was diminished in the hindgut of embryos expressing variant lamin B1. These observations indicate a mechanistic link between altered lamin B1 function, exacerbation of the Grhl3-mediated cell proliferation defect, and enhanced susceptibility to NTDs. We conclude that lamin B1 is a modifier gene of major effect for NTDs resulting from loss of Grhl3 function, a role that is likely mediated via the key function of lamin B1 in maintaining integrity of the nuclear envelope and ensuring normal cell cycle progression.
Failure of early development of the central nervous system leads to severe malformations termed neural tube defects (NTDs), including spina bifida and anencephaly. Inherited genetic risk factors play a major role in determining susceptibility to NTDs, but causative genes have proven difficult to identify. In this study we investigated genetic factors that could alter the risk of NTDs in an established mouse model, curly tail, in which defects result from partial loss of function of the grainyhead-like-3 (Grhl3) gene. We identified a variant of lamin B1, a key protein component of the envelope that surrounds the cell nucleus. The protein alteration reduces the structural integrity of the nuclear envelope, causes the nuclei to have altered shape, and reduces the rate of cell division. Curly tail embryos that carry the “abnormal” lamin B1 variant develop NTDs at three times the rate of those that carry the normal version. We conclude that lamin B1 function influences risk of NTDs due to effects on cell proliferation.
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
Few studies have evaluated genetic susceptibility related to diabetes and obesity as a risk factor for neural tube defects (NTDs). The authors investigated 23 single nucleotide polymorphisms among 9 genes (ADRB3, ENPP1, FTO, LEP, PPARG, PPARGC1A, SLC2A2, TCF7L2, and UCP2) associated with type 2 diabetes or obesity. Samples were obtained from 737 NTD case-parent triads included in the National Birth Defects Prevention Study during 1999–2007. Log-linear models were used to evaluate maternal and offspring genetic effects. After application of the false discovery rate, there were 5 significant maternal genetic effects. The less common alleles at the 4 FTO single nucleotide polymorphisms showed a reduction of NTD risk (for rs1421085, relative risk (RR) = 0.73 (95% confidence interval (CI): 0.62, 0.87); for rs8050136, RR = 0.79 (95% CI: 0.67, 0.93); for rs9939609, RR = 0.79 (95% CI: 0.67, 0.94); and for rs17187449, RR = 0.80 (95% CI: 0.68, 0.95)). Additionally, maternal LEP rs2071045 (RR = 1.31, 95% CI: 1.08, 1.60) and offspring UCP2 rs660339 (RR = 1.32, 95% CI: 1.06, 1.64) were associated with NTD risk. Furthermore, the maternal genotype for TCF7L2 rs3814573 suggested an increased NTD risk among obese women. These findings indicate that maternal genetic variants associated with glucose homeostasis may modify the risk of having an NTD-affected pregnancy.
case-parent triads; diabetes; genetics; neural tube defects; obesity
Neural tube defects (NTDs) are one of the most common birth defects caused by a combination of genetic and environmental factors. Currently, little is known about the genetic basis of NTDs although up to 70% of human NTDs were reported to be attributed to genetic factors. Here we performed genome-wide copy number variants (CNVs) detection in a cohort of Chinese NTD patients in order to exam the potential role of CNVs in the pathogenesis of NTDs.
The genomic DNA from eighty-five NTD cases and seventy-five matched normal controls were subjected for whole genome CNVs analysis. Non-DGV (the Database of Genomic Variants) CNVs from each group were further analyzed for their associations with NTDs. Gene content in non-DGV CNVs as well as participating pathways were examined.
Fifty-five and twenty-six non-DGV CNVs were detected in cases and controls respectively. Among them, forty and nineteen CNVs involve genes (genic CNV). Significantly more non-DGV CNVs and non-DGV genic CNVs were detected in NTD patients than in control (41.2% vs. 25.3%, p<0.05 and 37.6% vs. 20%, p<0.05). Non-DGV genic CNVs are associated with a 2.65-fold increased risk for NTDs (95% CI: 1.24–5.87). Interestingly, there are 41 cilia genes involved in non-DGV CNVs from NTD patients which is significantly enriched in cases compared with that in controls (24.7% vs. 9.3%, p<0.05), corresponding with a 3.19-fold increased risk for NTDs (95% CI: 1.27–8.01). Pathway analyses further suggested that two ciliogenesis pathways, tight junction and protein kinase A signaling, are top canonical pathways implicated in NTD-specific CNVs, and these two novel pathways interact with known NTD pathways.
Evidence from the genome-wide CNV study suggests that genic CNVs, particularly ciliogenic CNVs are associated with NTDs and two ciliogenesis pathways, tight junction and protein kinase A signaling, are potential pathways involved in NTD pathogenesis.
Neural tube defects (NTDs) are common, severe congenital malformations whose causation involves multiple genes and environmental factors. Although more than 200 genes are known to cause NTDs in mice, there has been rather limited progress in delineating the molecular basis underlying most human NTDs. Numerous genetic studies have been carried out to investigate candidate genes in cohorts of patients, with particular reference to those that participate in folate one-carbon metabolism. Although the homocysteine remethylation gene MTHFR has emerged as a risk factor in some human populations, few other consistent findings have resulted from this approach. Similarly, attention focused on the human homologues of mouse NTD genes has contributed only limited positive findings to date, although an emerging association between genes of the non-canonical Wnt (planar cell polarity) pathway and NTDs provides candidates for future studies. Priorities for the next phase of this research include: (i) larger studies that are sufficiently powered to detect significant associations with relatively minor risk factors; (ii) analysis of multiple candidate genes in groups of well-genotyped individuals to detect possible gene–gene interactions; (iii) use of high throughput genomic technology to evaluate the role of copy number variants and to detect ‘private’ and regulatory mutations, neither of which have been studied to date; (iv) detailed analysis of patient samples stratified by phenotype to enable, for example, hypothesis-driven testing of candidates genes in groups of NTDs with specific defects of folate metabolism, or in groups of fetuses with well-defined phenotypes such as craniorachischisis.
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
Tetrahydrobiopterin (BH4) is an essential cofactor and an important cellular antioxidant. BH4 deficiency has been associated with diseases whose etiologies stem from excessive oxidative stress. GTP cyclohydrolase I (GCH1) catalyzes the first and rate-limiting step of de novo BH4 synthesis. A 3-SNP haplotype in GCH1 (rs8007267, rs3783641, and rs10483639) is known to modulate GCH1 gene expression levels and has been suggested as a major determinant of plasma BH4 bioavailability. As plasma BH4 bioavailability has been suggested as a mechanism of neural tube defect (NTD) teratogenesis, we evaluated the association between this GCH1 haplotype and the risk of NTDs. Samples were obtained from 760 NTD case-parent triads included in the National Birth Defects Prevention Study (NBDPS). The three SNPs were genotyped using TaqMan® SNP assays. An extension of the log-linear model was used to assess the association between NTDs and both offspring and maternal haplotypes. Offspring carrying two copies of haplotype C-T-C had a significantly increased NTD risk (risk ratio [RR] = 3.40, 95% confidence interval [CI]: 1.02–11.50), after adjusting for the effect of the maternal haplotype. Additionally, mothers carrying two copies of haplotype C-T-C had a significantly increased risk of having an NTD-affected offspring (RR = 3.46, 95% CI: 1.05–11.00), after adjusting for the effect of the offspring haplotype. These results suggest offspring and maternal variation in the GCH1 gene and altered BH4 biosynthesis may contribute to NTD risk.
GCH1 gene; GTP cyclohydrolase I; haplotype; neural tube defects; tetrahydrobiopterin (BH4)