Impact of the common MTHFR 677C→T polymorphism on blood pressure in adulthood and role of riboflavin in modifying the genetic risk of hypertension: evidence from the JINGO project.

BACKGROUND: Genome-wide and clinical studies have linked the 677C→T polymorphism in the gene encoding methylenetetrahydrofolate reductase (MTHFR) with hypertension, whilst limited evidence shows that intervention with riboflavin (i.e. the MTHFR co-factor) can lower blood pressure (BP) in hypertensive patients with the variant MTHFR 677TT genotype. We investigated the impact of this common polymorphism on BP throughout adulthood and hypothesised that riboflavin status would modulate the genetic risk of hypertension. METHODS: Observational data on 6076 adults of 18-102 years were drawn from the Joint Irish Nutrigenomics Organisation project, comprising the Trinity-Ulster Department of Agriculture (TUDA; volunteer sample) and the National Adult Nutrition Survey (NANS; population-based sample) cohorts. Participants were recruited from the Republic of Ireland and Northern Ireland (UK) in 2008-2012 using standardised methods. RESULTS: The variant MTHFR 677TT genotype was identified in 12% of adults. From 18 to 70 years, this genotype was associated with an increased risk of hypertension (i.e. systolic BP ≥ 140 and/or a diastolic BP ≥ 90 mmHg): odds ratio (OR) 1.42, 95% confidence interval (CI) 1.07 to 1.90; P = 0.016, after adjustment for antihypertensive drug use and other significant factors, namely, age, male sex, BMI, alcohol and total cholesterol. Low or deficient biomarker status of riboflavin (observed in 30.2% and 30.0% of participants, respectively) exacerbated the genetic risk of hypertension, with a 3-fold increased risk for the TT genotype in combination with deficient riboflavin status (OR 3.00, 95% CI, 1.34-6.68; P = 0.007) relative to the CC genotype combined with normal riboflavin status. Up to 65 years, we observed poorer BP control rates on antihypertensive treatment in participants with the TT genotype (30%) compared to those without this variant, CT (37%) and CC (45%) genotypes (P < 0.027). CONCLUSIONS: The MTHFR 677TT genotype is associated with higher BP independently of homocysteine and predisposes adults to an increased risk of hypertension and poorer BP control with antihypertensive treatment, whilst better riboflavin status is associated with a reduced genetic risk. Riboflavin intervention may thus offer a personalised approach to prevent the onset of hypertension in adults with the TT genotype; however, this requires confirmation in a randomised trial in non-hypertensive adults.

Evidence from a Randomized Trial That Exposure to Supplemental Folic Acid at Recommended Levels during Pregnancy Does Not Lead to Increased Unmetabolized Folic Acid Concentrations in Maternal or Cord Blood.

BACKGROUND: Exposure to higher intakes of folic acid (FA) from fortified foods and supplements, although largely considered beneficial, is associated with unmetabolized FA in the circulation, which has raised some health concerns. OBJECTIVE: The effect of supplemental FA at a dose of 400 µg/d during pregnancy on unmetabolized FA concentrations in maternal plasma and newborn cord blood plasma was investigated. METHODS: A new analysis was performed of blood samples from participants in a randomized trial in pregnancy. Women aged 18-35 y, who had taken 400 µg FA/d as recommended in the first trimester, were recruited at the start of trimester 2 and randomly allocated to receive either 400 µg FA/d (n = 59) or a placebo (n = 67) throughout the second and third trimesters until delivery. Unmetabolized FA concentrations in maternal and cord blood samples were measured by LC-tandem MS analysis. RESULTS: In response to the intervention from gestational week 14 through delivery, a higher proportion of women in the FA compared with the placebo group had detectable FA (≥0.27 nmol/L) in plasma, but the difference in concentrations was not statistically significant (mean ± SD: 0.44 ± 0.80 compared with 0.13 ± 0.49 nmol/L, P = 0.38). FA treatment throughout pregnancy resulted in higher cord blood plasma total folate (50.6 ± 20.1 compared with 34.5 ± 14.4 nmol/L; P = 0.004) and 5-methyltetrahydrofolate (50.4 ± 20.3 compared with 34.5 ± 14.4 nmol/L; P = 0.005) concentrations, but FA was detected only in 8 of 53 available cord blood samples, and the proportion of samples with detectable FA concentrations was similar in FA-treated and placebo groups. CONCLUSIONS: Plasma concentrations of unmetabolized FA arising from supplemental FA at a dose of 400 µg/d, in addition to FA from fortified foods, were low or undetectable in mothers and newborns. The benefits for mothers and offspring of continuing FA supplementation beyond the first trimester of pregnancy can be achieved without posing any risk of increasing unmetabolized circulating FA, even in those already exposed to FA from fortified foods.

Folic Acid Supplementation in Postpolypectomy Patients in a Randomized Controlled Trial Increases Tissue Folate Concentrations and Reduces Aberrant DNA Biomarkers in Colonic Tissues Adjacent to the Former Polyp Site.

BACKGROUND: Low folate status is associated with an increased risk of colorectal carcinogenesis. Optimal folate status may be genoprotective by preventing uracil misincorporation into DNA and DNA hypomethylation. Adenomatous polyps have low folate status compared with normal colonic mucosa, and they are surrounded by histologically normal mucosa that also is of low folate status. OBJECTIVE: In a randomized controlled trial conducted at a single Dublin hospital between April 2002 and March 2004, we assessed the effect of folic acid supplementation on tissue folate, uracil misincorporation into DNA, and global DNA hypomethylation in colonocytes isolated from sites of adenomatous polyps and from histologically normal tissue adjacent and 10-15 cm distal to them. METHODS: Twenty patients with adenomatous polyps on initial colonoscopy and polypectomy were randomly assigned to receive either 600 µg folic acid/d [n = 12, 38% men, mean age 64.3 y, and body mass index (BMI, in kg/m(2)) 26.6] or placebo (n = 8, 50% men, mean age 68.4 y, and BMI 27.2) for 6 mo, and then repeat the colonoscopy. Blood and colonocyte tissue folate concentrations were measured with the use of a microbiological assay. Uracil misincorporation and global DNA hypomethylation were measured in colonocytes with the use of modified comet assays. RESULTS: Over time, folic acid supplementation, compared with placebo, increased tissue folate (mean ± SEM) from 15.6 ± 2.62 pg/10(5) cells to 18.1 ± 2.12 pg/10(5) cells (P < 0.001) and decreased the global DNA hypomethylation ratio from 1.7 ± 0.1 to 1.0 ± 0.1 (P < 0.001). The uracil misincorporation ratio decreased by 0.5 ± 0.1 for the site adjacent to the polyp over time (P = 0.05). CONCLUSION: A response to folic acid supplementation, which increased colonocyte folate and improved folate-related DNA biomarkers of cancer risk, was seen in the participants studied. Exploratory analysis points toward the area formerly adjacent to polyps as possibly driving the response. That these areas persist after polypectomy in the absence of folate supplementation is consistent with a potentially carcinogenic field's causing the appearance of the polyp.

Linkage analysis identifies a locus for plasma von Willebrand factor undetected by genome-wide association.

The plasma glycoprotein von Willebrand factor (VWF) exhibits fivefold antigen level variation across the normal human population determined by both genetic and environmental factors. Low levels of VWF are associated with bleeding and elevated levels with increased risk for thrombosis, myocardial infarction, and stroke. To identify additional genetic determinants of VWF antigen levels and to minimize the impact of age and illness-related environmental factors, we performed genome-wide association analysis in two young and healthy cohorts (n = 1,152 and n = 2,310) and identified signals at ABO (P < 7.9E-139) and VWF (P < 5.5E-16), consistent with previous reports. Additionally, linkage analysis based on sibling structure within the cohorts, identified significant signals at chromosome 2q12-2p13 (LOD score 5.3) and at the ABO locus on chromosome 9q34 (LOD score 2.9) that explained 19.2% and 24.5% of the variance in VWF levels, respectively. Given its strong effect, the linkage region on chromosome 2 could harbor a potentially important determinant of bleeding and thrombosis risk. The absence of a chromosome 2 association signal in this or previous association studies suggests a causative gene harboring many genetic variants that are individually rare, but in aggregate common. These results raise the possibility that similar loci could explain a significant portion of the "missing heritability" for other complex genetic traits.

Replication and exploratory analysis of 24 candidate risk polymorphisms for neural tube defects.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Is low iron status a risk factor for neural tube defects?

BACKGROUND: 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. METHODS: 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. RESULTS: No significant differences in maternal ferritin or hemoglobin concentrations were observed between NTD affected and nonaffected pregnancies (case median ferritin 16.9 µg/L and hemoglobin 12.4 g/dl versus 15.4 µg/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). CONCLUSION: 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.

Low colonocyte folate is associated with uracil misincorporation and global DNA hypomethylation in human colorectum.

Low folate status is a risk factor for colon carcinogenesis; mechanisms proposed to account for this relationship include uracil misincorporation into DNA and global DNA hypomethylation. We investigated whether such biomarkers are related to folate status in isolated colonocytes from colonoscopy patients. In cases with adenomatous polyps (n = 40) or hyperplastic polyps (n = 16), colonocytes were isolated from biopsies from the polyp, from a site adjacent to the polyp, and from normal mucosa 10-15 cm distal to the polyp. In polyp-free controls (n = 53), biopsies were taken from ascending, transverse, and descending areas of colon. Within adenoma cases, there was a trend (P-trend < 0.001) of decreasing colonocyte folate (pg/105 cells, mean ± CI) from the site distal to the polyp (16.9 ± 2.4), to the site adjacent to the polyp (14.7 ± 2.3), to the polyp (12.8 ± 2.0). Correspondingly, there were increases in uracil misincorporation (P-trend < 0.001) and global DNA hypomethylation (P-trend = 0.012) across the 3 sites. Colonocyte folate concentrations were significantly correlated with RBC folate concentrations, but only in individuals with generally lower (≤484 µg/L) RBC folate status (r = 0.54; P = 0.006; n = 24), and were also significantly lower in normal mucosa of cases with adenomatous polyps than in controls matched for colonic segment. In conclusion, localized folate deficiency in specific areas of colon might create carcinogenic fields and affect the development of colorectal polyps through uracil misincorporation and DNA hypomethylation; alternatively, the polyp itself might deplete folate in the surrounding tissue. Folate supplementation trials aimed at colon cancer prevention should target individuals with suboptimal folate status.

Lack of association between folate-receptor autoantibodies and neural-tube defects.

BACKGROUND: 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 neural-tube defects. METHODS: 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. RESULTS: 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. CONCLUSIONS: The presence and titer of maternal folate-receptor autoantibodies were not significantly associated with a neural-tube defect-affected pregnancy in this Irish population.

Genotyping of a tri-allelic polymorphism by a novel melting curve assay in MTHFD1L: an association study of nonsyndromic Cleft in Ireland.

BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Evaluation of common genetic variants in 82 candidate genes as risk factors for neural tube defects.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Choline supplementation and measures of choline and betaine status: a randomised, controlled trial in postmenopausal women.

Choline is an essential nutrient and can also be obtained by de novo synthesis via an oestrogen responsive pathway. Choline can be oxidised to the methyl donor betaine, with short-term supplementation reported to lower plasma total homocysteine (tHcy); however, the effects of longer-term choline supplementation are less clear. We investigated the effect of choline supplementation on plasma concentrations of free choline, betaine and tHcy and B-vitamin status in postmenopausal women, a group more susceptible to low choline status. We also assessed whether supplementation altered plasma lipid profiles. In this randomised, double-blinded, placebo-controlled study, forty-two healthy postmenopausal women received 1 g choline per d (as choline bitartrate), or an identical placebo supplement with their habitual diet. Fasting blood samples were collected at baseline, week 6 and week 12. Administration of choline increased median choline and betaine concentrations in plasma, with significant effects evident after 6 weeks of supplementation (P<0·001) and remaining significant at 12 weeks (P<0·001); no effect was observed on folate status or on plasma lipids. Choline supplementation induced a median (25th, 75th percentile) change in plasma tHcy concentration at week 6 of -0·9 (-1·6, 0·2) µmol, a change which, when compared to that observed in the placebo group 0·6 (-0·4, 1·9) µmol, approached statistical significance (P=0·058). Choline supplementation at a dose of 1 g/d significantly increases the circulating concentration of free choline, and can also significantly increase the concentration of the methyl donor, betaine, thereby potentially enhancing the betaine-homocysteine methyltransferase-mediated remethylation of tHcy.

The discovery of vitamin B(12).

The discovery of vitamin B(12), the elucidation of its role in metabolism, and the effects and treatment of its deficiency occurred in distinct phases over more than 100 years, and it was the subject of two separate Nobel Prizes. The valuable contribution of clinical reports and studies of patients with pernicious anemia throughout the 19th century resulted in enough clinical definition to allow Minot and Murphy to put together the first hallmark study on treatment of the condition, leading them to a Nobel Prize. These researchers were not the first to suggest that an inadequacy of nutrients was the cause of pernicious anemia, but their particular input was a carefully designed intervention in well-characterized pernicious anemia patients, of a special diet containing large amounts of liver. They found consistent improvement in the clinical and blood status of all subjects, most of whom remained on remission indefinitely. After the successful intervention studies, the next advance was made by Castle who discovered that a gastric component, which he called intrinsic factor, was missing in pernicious anemia. Many years later, intrinsic factor was found to be a glycoprotein that formed a complex with vitamin B(12), promoting its absorption through ileal receptors. The vitamin was isolated by two groups simultaneously and was crystallized and characterized in the laboratory of Dorothy Hodgkin, contributing to her Nobel Prize in 1964. Subsequently, the various biochemical roles of vitamin B(12) were elucidated, including its important interaction with folate and their common link with megaloblastic anemia. Many of the early clinical studies recognized that vitamin B(12) deficiency also caused a severe neuropathy leading to paralysis and death, while post mortem analysis demonstrated spinal cord demyelination. Vitamin B(12) is still the subject of intense research and, in particular, its role in preventing these irreversible neurological lesions remains unclear.

Diagnostic accuracy of holotranscobalamin, methylmalonic acid, serum cobalamin, and other indicators of tissue vitamin B12 status in the elderly.

BACKGROUND: Vitamin B12 deficiency is common among the elderly, and early detection is clinically important. However, clinical signs and symptoms have limited diagnostic accuracy and there is no accepted reference test method. METHODS: In elderly subjects (n = 700; age range 63-97 years), we investigated the ability of serum cobalamin, holotranscobalamin (holoTC), total homocysteine (tHcy), methylmalonic acid (MMA), serum and erythrocyte folate, and other hematologic variables to discriminate cobalamin deficiency, defined as red blood cell cobalamin <33 pmol/L. RESULTS: Serum holoTC was the best predictor, with area under the ROC curve (95% CI) 0.90 (0.86-0.93), and this was significantly better (P ≤ 0.0002) than the next best predictors; serum cobalamin, 0.80 (0.75-0.85), and MMA, 0.78 (0.72-0.83). For these 3 analytes, we constructed a 3-zone partition of positive and negative zones and a deliberate indeterminate zone between. The boundaries were values of each test that resulted in a posttest probability of deficiency of 60% and a posttest probability of no deficiency of 98%. The proportion of indeterminate observations for holoTC, cobalamin, and MMA was 14%, 45%, and 50%, respectively. Within the holoTC indeterminate zone (defined as 20-30 pmol/L), discriminant analysis selected only erythrocyte folate, which correctly allocated 65% (58/89) of the observations. Renal dysfunction compromised the diagnostic accuracy of MMA but not holoTC or serum cobalamin. CONCLUSIONS: This study supports the use of holoTC as the first-line diagnostic procedure for vitamin B12 status.

Women's compliance with current folic acid recommendations and achievement of optimal vitamin status for preventing neural tube defects.

BACKGROUND: The timing of folic acid supplement usage is critical to preventing pregnancies affected by neural tube defects (NTDs) because the neural tube closes by Day 28 post-conception. We investigated compliance of pregnant women with current folic acid recommendations (400 µg/day from preconception to 12 weeks) in relation to achieving a folate status associated with lowest risk of NTDs. METHODS: From a sample of 296 women with singleton uncomplicated pregnancies attending an antenatal clinic in Northern Ireland, those who reported taking folic acid in the first trimester (n = 226) were investigated. Samples were taken at 14 weeks gestation to measure serum concentrations of folate and vitamin B12 (related to folate and an independent predictor of NTD), and dietary B-vitamin intake and folic acid usage were investigated. RESULTS: Although the majority of the overall sample (84%) reported taking folic acid supplements in the first trimester, only 19% had started before conception, as recommended. Multigravidae compared with primigravidae women were less likely to have followed the recommendations correctly (P= 0.001). At 14 weeks, red cell folate (considered a reliable biomarker of previous 3 months, covering time of neural tube closure) was correlated (r = 0.320, P < 0.001) with the reported duration of folic acid usage, and was lower (P< 0.0001) in women who started folic acid after conception. CONCLUSIONS: Red cell folate concentrations in women not complying with recommendations were suboptimal in relation to NTD risk. The findings generally support the recent official recommendation to the Chief Medical Officer for mandatory fortification of food with folic acid in the UK.

Evaluation of 64 candidate single nucleotide polymorphisms as risk factors for neural tube defects in a large Irish study population.

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.

B-vitamins, methylenetetrahydrofolate reductase (MTHFR) and hypertension.

Hypertension is a leading risk factor for cardiovascular disease (CVD) and stroke. A common polymorphism in the gene encoding the enzyme methylenetetrahydrofolate reductase (MTHFR), previously identified as the main genetic determinant of elevated homocysteine concentration and also recognized as a risk factor for CVD, appears to be independently associated with hypertension. The B-vitamin riboflavin is required as a cofactor by MTHFR and recent evidence suggests it may have a role in modulating blood pressure, specifically in those with the homozygous mutant MTHFR 677 TT genotype. If studies confirm that this genetic predisposition to hypertension is correctable by low-dose riboflavin, the findings could have important implications for the management of hypertension given that the frequency of this polymorphism ranges from 3 to 32 % worldwide.

Gene-environment interactions in the causation of neural tube defects: folate deficiency increases susceptibility conferred by loss of Pax3 function.

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 (Sp(2)(H)) 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 Sp(2)(H) 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.

The genetic background of the curly tail strain confers susceptibility to folate-deficiency-induced exencephaly.

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.

Testing reported associations of genetic risk factors for oral clefts in a large Irish study population.

BACKGROUND: 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. METHODS: 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. RESULTS: In case-control comparisons, the minor allele of PTCH1 A562A (rs2066836) was associated with reduced odds of CLP (odds ratios [OR], 0.29; 95% confidence interval [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). CONCLUSIONS: 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.

A common variant in MTHFD1L is associated with neural tube defects and mRNA splicing efficiency.

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), which influences splicing efficiency and is strongly associated with NTD risk. Three alleles of rs3832406 were detected in the Irish population with varying numbers of ATT repeats: Allele 1 consists of ATT(7), whereas Alleles 2 and 3 consist of ATT(8) and ATT(9), 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), whereas Allele 1 showed an increased case risk. Allele 3 showed no influence on NTD risk and represents the lowest frequency allele (0.15). Additional single nucleotide polymorphism (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.