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1.  Transcobalamin II Receptor Polymorphisms Are Associated with Increased Risk for Neural Tube Defects 
Journal of medical genetics  2010;47(10):677-685.
Objective:
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.
Methods:
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.
Results:
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.
Conclusions:
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.
doi:10.1136/jmg.2009.073775
PMCID: PMC4112773  PMID: 20577008
neural tube defects; spina bifida; transcobalamin II receptor (TCblR); cobalamin; vitamin B12; copy number variant (CNV)
2.  Neural Tube Defects and Folate Pathway Genes: Family-Based Association Tests of Gene–Gene and Gene–Environment Interactions 
Environmental Health Perspectives  2006;114(10):1547-1552.
Background
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.
Objectives
Our study evaluates several folate pathway genes for association with human NTDs, incorporating an environmental cofactor: maternal folate supplementation.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1289/ehp.9166
PMCID: PMC1626421  PMID: 17035141
folate; folic acid supplementation; genetic association; neural tube defects
3.  Vitamin B12 deficiency in the brain leads to DNA hypomethylation in the TCblR/CD320 knockout mouse 
Background
DNA methylation is an epigenetic phenomenon that can modulate gene function by up or downregulation of gene expression. Vitamin B12 and folate pathways are involved in the production of S-Adenosylmethionine, the universal methyl donor.
Findings
Brain vitamin B12 concentration and global DNA methylation was determined in transcobalamin receptor (TCblR/CD320) knock out (KO) (n = 4) and control mice (n = 4) at 20–24 weeks of age. Median [IQR] brain vitamin B12 concentrations (pg/mg) in TCblR/CD320 KO mice compared with control mice was 8.59 [0.52] vs 112.42 [33.12]; p < 0.05. Global DNA methylation levels in brain genomic DNA were lower in TCblR/CD320 KO compared with control mice (Median [IQR]: 0.31[0.16] % vs 0.55[0.15] %; p < 0.05.).
Conclusions
In TCblR/CD320 KO mice, brain vitamin B12 drops precipitously by as much as 90% during a 20 week period. This decrease is associated with a 40% decrease in global DNA methylation in the brain. Future research will reveal whether the disruption in gene expression profiles due to changes in DNA hypomethylation contribute to central nervous system pathologies that are frequently seen in vitamin B12 deficiency.
doi:10.1186/1743-7075-9-41
PMCID: PMC3433370  PMID: 22607050
Vitamin B12 deficiency; Brain; Global DNA methylation; Transcobalamin receptor knock out mice
4.  Bioinformatic and Genetic Association Analysis of MicroRNA Target Sites in One-Carbon Metabolism Genes 
PLoS ONE  2011;6(7):e21851.
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.
doi:10.1371/journal.pone.0021851
PMCID: PMC3134459  PMID: 21765920
5.  Folate and vitamin B12 in idiopathic male infertility 
Asian Journal of Andrology  2011;13(6):856-861.
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.
doi:10.1038/aja.2011.96
PMCID: PMC3372894  PMID: 21857689
folate; idiopathic male infertility; semen quality; vitamin B12
6.  Folate and vitamin B12 in idiopathic male infertility 
Asian Journal of Andrology  2011;13(6):856-861.
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.
doi:10.1038/aja.2011.96
PMCID: PMC3372894  PMID: 21857689
folate; idiopathic male infertility; semen quality; vitamin B12
7.  Characterization of the promoter region of TCblR/CD320 gene, the receptor for cellular uptake of transcobalamin-bound cobalamin 
Gene  2010;466(1-2):49-55.
Cellular uptake of cobalamin (Cbl) is mediated by the transcobalamin receptor (TCblR) that binds and internalizes transcobalamin (TC) saturated with Cbl. These receptors are expressed in actively proliferating cells and are down regulated in quiescent cells. The 5′ region of TCblR gene was analyzed for promoter activity to determine transcriptional regulation of TCblR expression. The region −668 to −455 appears to regulate TCblR expression. We have identified transcription factors MZF-1 (myeloid zinc finger 1) / RREB-1 (Ras-responsive element binding protein 1), C/EBP (CCAAT/enhancer binding protein) / HNF-3ß (hepatocyte nuclear factor 3) and AP-1(activator protein 1) as proteins likely to be involved in this regulation with the former region primarily involved in up regulation and the latter two regions involved in suppression of TCblR expression. These transcription factors are involved in cell proliferation and differentiation. Thus the cell cycle associated expression of TCblR appears to be tightly regulated in synchrony with the proliferative phase of the cell cycle.
doi:10.1016/j.gene.2010.07.004
PMCID: PMC2947791  PMID: 20627121
Transcobalamin; Receptor; TCblR/CD320; vitamin B12; Cobalamin; Promoter; Transcription
8.  Maternal Risk for Down Syndrome Is Modulated by Genes Involved in Folate Metabolism 
Disease markers  2012;32(2):73-81.
Studies have shown that the maternal risk for Down syndrome (DS) may be modulated by alterations in folate metabolism. The aim of this study was to evaluate the influence of 12 genetic polymorphisms involved in folate metabolism on maternal risk for DS. In addition, we evaluated the impact of these polymorphisms on serum folate and plasma methylmalonic acid (MMA, an indicator of vitamin B12 status) concentrations. The polymorphisms transcobalamin II (TCN2) c.776C>G, betaine-homocysteine S-methyltransferase (BHMT) c.742A>G, methylenetetrahydrofolate reductase (NAD(P)H) (MTHFR) c.677 C>T and the MTHFR 677C-1298A-1317T haplotype modulate DS risk. The polymorphisms MTHFR c.677C>T and solute carrier family 19 (folate transporter), member 1 (SLC19A1) c.80 A>G modulate folate concentrations, whereas the 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR) c.66A>G polymorphism affects the MMA concentration. These results are consistent with the modulation of the maternal risk for DS by these polymorphisms.
doi:10.3233/DMA-2011-0869
PMCID: PMC3826801  PMID: 22377700
Down syndrome; genetic polymorphism; folate metabolism
9.  The Folate Pathway and Nonsyndromic Cleft Lip and Palate 
Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth malformation caused by genetic, environmental and gene-environment interactions. Periconceptional supplementation with folic acid, a key component in DNA synthesis and cell division, has reduced the birth prevalence of neural tube defects (NTDs) and may similarly reduce the birth prevalence of other complex birth defects including NSCLP. Past studies investigating the role of two common methylenetetrahydrofolate reductase (MTHFR) SNP polymorphisms, C677T (rs1801133) and A1298C (rs1801131), in NSCLP have produced conflicting results. Most studies of folate pathway genes have been limited in scope, as few genes/SNPs have been interrogated. In this study, we asked whether variations in a more comprehensive group of folate pathway genes were associated with NSCLP and, if so, were there detectable interactions between these genes and environmental exposures. In addition, we evaluated the data for a sex effect. Fourteen folate metabolism related genes were interrogated using eighty-nine SNPs in multiplex and simplex non-Hispanic White (NHW) (317) and Hispanic (128) NSCLP families. Evidence for a risk association between NSCLP and SNPs in nitrous oxide 3 (NOS3) and thymidylate synthetase (TYMS) was detected in the NHW group, whereas associations with methionine synthase (MTR), betaine-homocysteine methyltransferase (BHMT2), MTHFS and SLC19A1 were detected in the Hispanic group. Evidence for over-transmission of haplotypes and gene interactions in the methionine arm was detected. These results suggest that perturbations of the genes in the folate pathway may contribute to NSCLP. There was evidence for an interaction between several SNPs and maternal smoking, and for one SNP with sex of the offspring. These results provide support for other studies that suggest that high maternal homocysteine levels may contribute to NSCLP and should be further investigated.
doi:10.1002/bdra.20740
PMCID: PMC4098909  PMID: 21254359
Nonsyndromic cleft lip and palate; NSCLP; folate metabolism; association; genetics; homocysteine; methionine
10.  Transcobalamin 2 variant associated with poststroke homocysteine modifies recurrent stroke risk 
Neurology  2011;77(16):1543-1550.
Objectives:
The Vitamin Intervention for Stroke Prevention trial found an association between baseline poststroke homocysteine (Hcy) and recurrent stroke. We investigated genes for enzymes and cofactors in the Hcy metabolic pathway for association with Hcy and determined whether associated single nucleotide polymorphisms (SNPs) influenced recurrent stroke risk.
Methods:
Eighty-six SNPs in 9 candidate genes (BHMT1, BHMT2, CBS, CTH, MTHFR, MTR, MTRR, TCN1, and TCN2) were genotyped in 2,206 subjects (83% European American). Associations with Hcy measures were assessed using linear regression models assuming an additive genetic model, adjusting for age, sex, and race and additionally for baseline Hcy when postmethionine load change was assessed. Associations with recurrent stroke were evaluated using survival analyses.
Results:
Five SNPs in the transcobalamin 2 (TCN2) gene were associated with baseline Hcy (false discovery rate [FDR]–adjusted p = 0.049). TCN2 SNP rs731991 was associated with recurrent stroke risk in the low-dose arm of the trial under a recessive model (log-rank test p = 0.009, hazard ratio 0.34). Associations with change in postmethionine load Hcy levels were found with 5 SNPs in the cystathionine β-synthase (CBS) gene (FDR-adjusted p < 0.031).
Conclusions:
TCN2 variants contribute to poststroke Hcy levels, whereas variants in the CBS gene influence Hcy metabolism. Variation in the TCN2 gene also affects recurrent stroke risk in response to cofactor therapy.
doi:10.1212/WNL.0b013e318233b1f9
PMCID: PMC3198974  PMID: 21975197
11.  Targeted Delivery of Saporin Toxin by Monoclonal Antibody to the Transcobalamin Receptor, TCblR/CD320 
Molecular cancer therapeutics  2010;9(11):3033-3040.
Cellular uptake of cobalamin (Cbl) occurs by endocytosis of transcobalamin (TC) saturated with Cbl by the transcobalamin receptor (TCblR/CD320). The cell cycle associated over expression of this receptor in many cancer cells, provides a suitable target for delivering chemotherapeutic drugs and cytotoxic molecules to these cells while minimizing the effect on the normal cell population. We have used monoclonal antibodies to the extracellular domain of TCblR to deliver saporin conjugated secondary antibody to various cell lines propagating in culture. A molar ratio of 2.5:10nM of primary:secondary antibody concentration was identified as the lowest concentration needed to produce the optimum cytotoxic effect. The effect was more pronounced when cells were seeded at lower density suggesting lack of cell division in a fraction of the cells at higher density as the likely explanation. Cells in suspension culture such as K562 and U266 cells were more severely affected than adherent cultures such as SW48 and KB cells. This differential effect of the anti TCblR-saporin antibody conjugate and the ability of an anti TCblR antibody to target proliferating cells was further evident by the virtual lack of any effect on primary skin fibroblasts and minimal effect on bone marrow cells. These results indicate that preferential targeting of some cancer cells could be accomplished via the TCblR receptor.
doi:10.1158/1535-7163.MCT-10-0513
PMCID: PMC2978776  PMID: 20858723
Transcobalamin; CD320; monoclonal antibodies; vitamin B12
12.  Homocysteine and Coronary Heart Disease: Meta-analysis of MTHFR Case-Control Studies, Avoiding Publication Bias 
PLoS Medicine  2012;9(2):e1001177.
Robert Clarke and colleagues conduct a meta-analysis of unpublished datasets to examine the causal relationship between elevation of homocysteine levels in the blood and the risk of coronary heart disease. Their data suggest that an increase in homocysteine levels is not likely to result in an increase in risk of coronary heart disease.
Background
Moderately elevated blood levels of homocysteine are weakly correlated with coronary heart disease (CHD) risk, but causality remains uncertain. When folate levels are low, the TT genotype of the common C677T polymorphism (rs1801133) of the methylene tetrahydrofolate reductase gene (MTHFR) appreciably increases homocysteine levels, so “Mendelian randomization” studies using this variant as an instrumental variable could help test causality.
Methods and Findings
Nineteen unpublished datasets were obtained (total 48,175 CHD cases and 67,961 controls) in which multiple genetic variants had been measured, including MTHFR C677T. These datasets did not include measurements of blood homocysteine, but homocysteine levels would be expected to be about 20% higher with TT than with CC genotype in the populations studied. In meta-analyses of these unpublished datasets, the case-control CHD odds ratio (OR) and 95% CI comparing TT versus CC homozygotes was 1.02 (0.98–1.07; p = 0.28) overall, and 1.01 (0.95–1.07) in unsupplemented low-folate populations. By contrast, in a slightly updated meta-analysis of the 86 published studies (28,617 CHD cases and 41,857 controls), the OR was 1.15 (1.09–1.21), significantly discrepant (p = 0.001) with the OR in the unpublished datasets. Within the meta-analysis of published studies, the OR was 1.12 (1.04–1.21) in the 14 larger studies (those with variance of log OR<0.05; total 13,119 cases) and 1.18 (1.09–1.28) in the 72 smaller ones (total 15,498 cases).
Conclusions
The CI for the overall result from large unpublished datasets shows lifelong moderate homocysteine elevation has little or no effect on CHD. The discrepant overall result from previously published studies reflects publication bias or methodological problems.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Coronary heart disease (CHD) is the leading cause of death among adults in developed countries. With age, fatty deposits (atherosclerotic plaques) coat the walls of the coronary arteries, the blood vessels that supply the heart with oxygen and nutrients. The resultant restriction of the heart's blood supply causes shortness of breath, angina (chest pains that are usually relieved by rest), and sometimes fatal heart attacks. Many established risk factors for CHD, including smoking, physical inactivity, being overweight, and eating a fat-rich diet, can be modified by lifestyle changes. Another possible modifiable risk factor for CHD is a high blood level of the amino acid homocysteine. Methylene tetrahydofolate reductase, which is encoded by the MTHFR gene, uses folate to break down and remove homocysteine so fortification of cereals with folate can reduce population homocysteine blood levels. Pooled results from prospective observational studies that have looked for an association between homocysteine levels and later development of CHD suggest that the reduction in homocysteine levels that can be achieved by folate supplementation is associated with an 11% lower CHD risk.
Why Was This Study Done?
Prospective observational studies cannot prove that high homocysteine levels cause CHD because of confounding, the potential presence of other unknown shared characteristics that really cause CHD. However, an approach called “Mendelian randomization” can test whether high blood homocysteine causes CHD. A common genetic variant of the MTHFR gene—the C677T polymorphism—reduces MTHFR efficiency so TT homozygotes (individuals in whom both copies of the MTHFR gene have the nucleotide thymine at position 677; the human genome contains two copies of most genes) have 25% higher blood homocysteine levels than CC homozygotes. In meta-analyses (statistical pooling of the results of several studies) of published Mendelian randomized studies, TT homozygotes have a higher CHD risk than CC homozygotes. Because gene variants are inherited randomly, they are not subject to confounding, so this result suggests that high blood homocysteine causes CHD. But what if only Mendelian randomization studies that found an association have been published? Such publication bias would affect this aggregate result. Here, the researchers investigate the association of the MTHFR C677T polymorphism with CHD in unpublished datasets that have analyzed this polymorphism incidentally during other genetic studies.
What Did the Researchers Do and Find?
The researchers obtained 19 unpublished datasets that contained data on the MTHFR C677T polymorphism in thousands of people with and without CHD. Meta-analysis of these datasets indicates that the excess CHD risk in TT homozygotes compared to CC homozygotes was 2% (much lower than predicted from the prospective observational studies), a nonsignificant difference (that is, it could have occurred by chance). When the probable folate status of the study populations (based on when national folic acid fortification legislation came into effect) was taken into account, there was still no evidence that TT homozygotes had an excess CHD risk. By contrast, in an updated meta-analysis of 86 published studies of the association of the polymorphism with CHD, the excess CHD risk in TT homozygotes compared to CC homozygotes was 15%. Finally, in a meta-analysis of randomized trials on the use of vitamin B supplements for homocysteine reduction, folate supplementation had no significant effect on the 5-year incidence of CHD.
What Do These Findings Mean?
These analyses of unpublished datasets are consistent with lifelong moderate elevation of homocysteine levels having no significant effect on CHD risk. In other words, these findings indicate that circulating homocysteine levels within the normal range are not causally related to CHD risk. The meta-analysis of the randomized trials of folate supplementation also supports this conclusion. So why is there a discrepancy between these findings and those of meta-analyses of published Mendelian randomization studies? The discrepancy is too large to be dismissed as a chance finding, suggest the researchers, but could be the result of publication bias—some studies might have been prioritized for publication because of the positive nature of their results whereas the unpublished datasets used in this study would not have been affected by any failure to publish null results. Overall, these findings reveal a serious example of publication bias and argue against the use of folate supplements as a means of reducing CHD risk.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001177.
The American Heart Association provides information about CHD and tips on keeping the heart healthy; it also provides information on homocysteine, folic acid, and CHD, general information on supplements and heart health, and personal stories about CHD
The UK National Health Service Choices website provides information about CHD, including personal stories about CHD
Information is available from the British Heart Foundation on heart disease and keeping the heart healthy
The US National Heart Lung and Blood Institute also provides information on CHD (in English and Spanish)
MedlinePlus provides links to many other sources of information on CHD (in English and Spanish)
Wikipedia has a page on Mendelian randomization (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
doi:10.1371/journal.pmed.1001177
PMCID: PMC3283559  PMID: 22363213
13.  The Effect of Multiple Single Nucleotide Polymorphisms in the Folic Acid Pathway Genes on Homocysteine Metabolism 
BioMed Research International  2014;2014:560183.
Objective. To investigate the joint effects of the single nucleotide polymorphisms (SNPs) of genes in the folic acid pathway on homocysteine (Hcy) metabolism. Methods. Four hundred women with normal pregnancies were enrolled in this study. SNPs were identified by MassARRAY. Serum folic acid and Hcy concentration were measured. Analysis of variance (ANOVA) and support vector machine (SVM) regressions were used to analyze the joint effects of SNPs on the Hcy level. Results. SNPs of MTHFR (rs1801133 and rs3733965) were significantly associated with maternal serum Hcy level. In the different genotypes of MTHFR (rs1801133), SNPs of RFC1 (rs1051266), TCN2 (rs9606756), BHMT (rs3733890), and CBS (rs234713 and rs2851391) were linked with the Hcy level adjusted for folic acid concentration. The integrated SNPs scores were significantly associated with the residual Hcy concentration (RHC) (r = 0.247). The Hcy level was significantly higher in the group with high SNP scores than that in other groups with SNP scores of less than 0.2 (P = 0.000). Moreover, this difference was even more significant in moderate and high levels of folic acid. Conclusion. SNPs of genes in the folic acid pathway possibly affect the Hcy metabolism in the presence of moderate and high levels of folic acid.
doi:10.1155/2014/560183
PMCID: PMC3913508  PMID: 24524080
14.  118 SNPs of folate-related genes and risks of spina bifida and conotruncal heart defects 
BMC Medical Genetics  2009;10:49.
Background
Folic acid taken in early pregnancy reduces risks for delivering offspring with several congenital anomalies. The mechanism by which folic acid reduces risk is unknown. Investigations into genetic variation that influences transport and metabolism of folate will help fill this data gap. We focused on 118 SNPs involved in folate transport and metabolism.
Methods
Using data from a California population-based registry, we investigated whether risks of spina bifida or conotruncal heart defects were influenced by 118 single nucleotide polymorphisms (SNPs) associated with the complex folate pathway. This case-control study included 259 infants with spina bifida and a random sample of 359 nonmalformed control infants born during 1983–86 or 1994–95. It also included 214 infants with conotruncal heart defects born during 1983–86. Infant genotyping was performed blinded to case or control status using a designed SNPlex assay. We examined single SNP effects for each of the 118 SNPs, as well as haplotypes, for each of the two outcomes.
Results
Few odds ratios (ORs) revealed sizable departures from 1.0. With respect to spina bifida, we observed ORs with 95% confidence intervals that did not include 1.0 for the following SNPs (heterozygous or homozygous) relative to the reference genotype: BHMT (rs3733890) OR = 1.8 (1.1–3.1), CBS (rs2851391) OR = 2.0 (1.2–3.1); CBS (rs234713) OR = 2.9 (1.3–6.7); MTHFD1 (rs2236224) OR = 1.7 (1.1–2.7); MTHFD1 (hcv11462908) OR = 0.2 (0–0.9); MTHFD2 (rs702465) OR = 0.6 (0.4–0.9); MTHFD2 (rs7571842) OR = 0.6 (0.4–0.9); MTHFR (rs1801133) OR = 2.0 (1.2–3.1); MTRR (rs162036) OR = 3.0 (1.5–5.9); MTRR (rs10380) OR = 3.4 (1.6–7.1); MTRR (rs1801394) OR = 0.7 (0.5–0.9); MTRR (rs9332) OR = 2.7 (1.3–5.3); TYMS (rs2847149) OR = 2.2 (1.4–3.5); TYMS (rs1001761) OR = 2.4 (1.5–3.8); and TYMS (rs502396) OR = 2.1 (1.3–3.3). However, multiple SNPs observed for a given gene showed evidence of linkage disequilibrium indicating that the observed SNPs were not individually contributing to risk. We did not observe any ORs with confidence intervals that did not include 1.0 for any of the studied SNPs with conotruncal heart defects. Haplotype reconstruction showed statistical evidence of nonrandom associations with TYMS, MTHFR, BHMT and MTR for spina bifida.
Conclusion
Our observations do not implicate a particular folate transport or metabolism gene to be strongly associated with risks for spina bifida or conotruncal defects.
doi:10.1186/1471-2350-10-49
PMCID: PMC2700092  PMID: 19493349
15.  Down-regulation of Transcobalamin Receptor TCblR/CD320 by siRNA Inhibits Cobalamin uptake and Proliferation of Cells in Culture 
Experimental cell research  2011;317(11):1603-1607.
The clinical phenotype of cobalamin (Cbl) deficiency is dictated by the essential role of this vitamin in two key enzymatic reactions. Multiple proteins and receptors participate in the absorption, transport and delivery of this vitamin to tissue cells. Cellular uptake of Cbl is mediated by transcobalamin (TC), a plasma protein and a transmembrane receptor (TCblR) with high affinity for TC saturated with Cbl. Knockdown of TCblR with siRNA results in decreased TC-Cbl uptake. The ensuing Cbl deficiency leads to an increase in doubling time and decreased proliferation of these cells. The study confirms the seminal role of this receptor in the cellular uptake of Cbl and its down-regulation as a potential strategy to inhibit proliferation of cancer cells.
doi:10.1016/j.yexcr.2011.02.016
PMCID: PMC3103659  PMID: 21377459
16.  Environmental influence on the worldwide prevalence of a 776C→G variant in the transcobalamin gene (TCN2) 
Journal of Medical Genetics  2007;44(6):363-367.
Background
A 776C→G variant (dbSNP ID: rs1801198) in the transcobalamin gene (TCN2; MIM# 275350) decreases the cellular and plasma concentration of transcobalamin and thereby influences the cellular availability of vitamin B12.
Objective
To evaluate the worldwide prevalence of this variant and its association with homocysteine plasma level.
Methods
The study was performed in 1433 apparently healthy subjects, including Afro‐Americans and Afro‐Africans and in 251 Afro‐Africans participants with severe malaria.
Results
The frequencies of the 776G allele were the highest in China (0.607; 95% CI 0.554 to 0.659), low in West Africa (Bénin and Togo, 0.178; 0.154 to 0.206), and intermediate in France (0.445; 0.408 to 0.481), Italy (0.352; 0.299 to 0.409), Morocco (0.370; 0.300 to 0.447) and Mexico (0.374; 0.392 to 0.419). The 776G genotype was more frequent in Afro‐Americans from New York (16.7; 8.4 to 30.7) and in Afro‐African patients with severe malaria (6.0%; 95% CI 3.7 to 9.6) than in healthy Afro‐African volunteers (p = 0.0004 and p = 0.033, respectively), while no difference was observed for MTHFR 677TT and 677T alleles. A disequilibrium of TCN2 genotype distribution was recorded in patients with severe malaria, with a twofold higher GG genotype than expected (p = 0.010). An association between the TCN2 polymorphism and homocysteine was observed only in Mexico and France, the two countries with the highest rate of low plasma concentration of vitamin B12 (<100 pmol/l).
Conclusion
Given the dramatic heterogeneity of the 776G allele frequency worldwide, this polymorphism may be prone to a selective pressure or confers an evolutionary advantage in confronting environmental factors, one of which is malaria.
doi:10.1136/jmg.2006.048041
PMCID: PMC2740879  PMID: 17220211
17.  Genetic Associations with Plasma B12, B6, and Folate Levels in an Ischemic Stroke Population from the Vitamin Intervention for Stroke Prevention (VISP) Trial 
Background: B vitamins play an important role in homocysteine metabolism, with vitamin deficiencies resulting in increased levels of homocysteine and increased risk for stroke. We performed a genome-wide association study (GWAS) in 2,100 stroke patients from the Vitamin Intervention for Stroke Prevention (VISP) trial, a clinical trial designed to determine whether the daily intake of high-dose folic acid, vitamins B6, and B12 reduce recurrent cerebral infarction.
Methods: Extensive quality control (QC) measures resulted in a total of 737,081 SNPs for analysis. Genome-wide association analyses for baseline quantitative measures of folate, Vitamins B12, and B6 were completed using linear regression approaches, implemented in PLINK.
Results: Six associations met or exceeded genome-wide significance (P ≤ 5 × 10−08). For baseline Vitamin B12, the strongest association was observed with a non-synonymous SNP (nsSNP) located in the CUBN gene (P = 1.76 × 10−13). Two additional CUBN intronic SNPs demonstrated strong associations with B12 (P = 2.92 × 10−10 and 4.11 × 10−10), while a second nsSNP, located in the TCN1 gene, also reached genome-wide significance (P = 5.14 × 10−11). For baseline measures of Vitamin B6, we identified genome-wide significant associations for SNPs at the ALPL locus (rs1697421; P = 7.06 × 10−10 and rs1780316; P = 2.25 × 10−08). In addition to the six genome-wide significant associations, nine SNPs (two for Vitamin B6, six for Vitamin B12, and one for folate measures) provided suggestive evidence for association (P ≤ 10−07).
Conclusion: Our GWAS study has identified six genome-wide significant associations, nine suggestive associations, and successfully replicated 5 of 16 SNPs previously reported to be associated with measures of B vitamins. The six genome-wide significant associations are located in gene regions that have shown previous associations with measures of B vitamins; however, four of the nine suggestive associations represent novel finding and warrant further investigation in additional populations.
doi:10.3389/fpubh.2014.00112
PMCID: PMC4123605  PMID: 25147783
VISP; association; GWAS; one-carbon metabolism; B12; B6; folate
18.  Response of serum and red blood cell folate concentrations to folic acid supplementation depends on methylenetetrahydrofolate reductase C677T genotype: Results from a crossover trial 
Scope
By increasing blood folate concentrations, folic acid supplementation reduces risk for neural tube defect-affected pregnancies, and lowers homocysteine concentrations. We assessed response of red blood cell (RBC) and serum folate to folic acid supplementation, and examined association of response with the genetic polymorphism C677T of the methylenetetrahydrofolate NAD(P)H (MTHFR) gene.
Methods and Results
Randomized, controlled, crossover trial with two folic acid supplement treatment periods and a 30-week washout period. The primary outcome is blood folate (serum and RBC) concentrations. Volunteers (n=142) aged 18-69 were randomized to two of three doses (0, 200, and 400 μg) of folic acid for twelve weeks. Serum folate response depended on treatment period with significant responses to 200 μg seen only in the second treatment periods (4.4 ng/mL or 3.4 ng/mL). Additionally, serum folate increased as folic acid dose increased to 400 μg (p< 0.01) and response was greater after the washout period (8.7 ng/mL), than after a 6-week run-in (2.3 ng/mL). The differential change attributable to a daily supplement of 400 μg compared to 200 μg was 96.8 ng/mL; while the change attributable to 400 μg compared to 0 μg was 121.4. Increases in RBC folate concentrations with 400 μg occurred within MTHFR gene mutation (C677T); and in the African American group.
Conclusions
Serum folate concentration is responsive to modest increases in folic acid intake. Red blood cell folate increases only with higher additional doses of folic acid supplementation, and this is true for each MTHFR C677T genotype.
doi:10.1002/mnfr.201200108
PMCID: PMC4132693  PMID: 23456769
Folic acid; fortification; methylenetetrahydrofolate reductase; supplementation
19.  Positive Newborn Screen for Methylmalonic Aciduria Identifies the First Mutation in TCblR/CD320, the Gene for Cellular Uptake of Transcobalamin-bound Vitamin B12 
Human mutation  2010;31(8):924-929.
Elevated methylmalonic acid in five asymptomatic newborns whose fibroblasts showed decreased uptake of transcobalamin-bound cobalamin (holo-TC), suggested a defect in the cellular uptake of cobalamin. Analysis of TCblR/CD320, the gene for the receptor for cellular uptake of holo-TC, identified a homozygous single codon deletion, c.262_264GAG (p.E88del), resulting in the loss of a glutamic acid residue in the low-density lipoprotein receptor type A-like domain. Inserting the codon by site-directed mutagenesis fully restored TCblR function.
doi:10.1002/humu.21297
PMCID: PMC2909035  PMID: 20524213
methylmalonic acid; homocysteine; transcobalamin-receptor; cobalamin; vitamin B12; TCblR; CD320
20.  Markers of B-vitamin deficiency and frailty in older women 
Objective
This project was designed to follow-up prior evidence that demonstrated a significant association between vitamin B12 transport and metabolism and the frailty syndrome in community-dwelling older women. The cross-sectional relationship between genetic variants within six candidate genes along this pathway with serum methylmalonic acid (MMA) levels and frailty was evaluated in this same population of older women.
Methods
Baseline measures were collected prior to folate fortification from 326 women in the Women's Health and Aging Studies I and II. Odds ratios and statistical tests were estimated for single SNP and haplotype via linear regression models for serum MMA, a marker for available vitamin B12, and in logistic regression models for frailty.
Results
Fifty-six SNPs from CBS, MTHFR, MTR, MTRR, TCN1 and TCN2 genes were genotyped. Several SNPs in MTHFR, MTR and MTRR demonstrated a modest association to elevated MMA, while SNPs in TCN2 showed significant association to the frailty syndrome. TCN2 polymorphisms, particularly one SNP reported to be in perfect LD with functional variant Pro259Arg, were significantly associated with increased odds of frailty, after adjustment for age, presence of cardiovascular disease and elevated MMA (OR = 2.25, p-value = 0.009).
Conclusions
Using MMA as a marker for vitamin B12, these results suggest that TCN2 gene variants may lead to decreased vitamin B12 availability, leading to reduced energy metabolism, ultimately contributing to frailty pathology. Further studies to determine the biological role of functional TCN2 polymorphisms in frailty are needed.
PMCID: PMC2739594  PMID: 18443711
transcobalamin-II; vitamin B12; genetic association
21.  TRANSCOBALAMIN-II VARIANTS, DECREASED VITAMIN B12 AVAILABILITY AND INCREASED RISK OF FRAILTY 
Objective
This project was designed to follow-up prior evidence that demonstrated a significant association between vitamin B12 transport and metabolism and the frailty syndrome in community-dwelling older women. The cross-sectional relationship between genetic variants within six candidate genes along this pathway with serum methylmalonic acid (MMA) levels and frailty was evaluated in this same population of older women.
Methods
Baseline measures were collected prior to folate fortification from 326 women in the Women’s Health and Aging Studies I and II. Odds ratios and statistical tests were estimated for single SNP and haplotype via linear regression models for serum MMA, a marker for available vitamin B12, and in logistic regression models for frailty.
Results
Fifty-six SNPs from CBS, MTHFR, MTR, MTRR, TCN1 and TCN2 genes were genotyped. Several SNPs in MTHFR, MTR and MTRR demonstrated a modest association to elevated MMA, while SNPs in TCN2 showed significant association to the frailty syndrome. TCN2 polymorphisms, particularly one SNP reported to be in perfect LD with functional variant Pro259Arg, were significantly associated with increased odds of frailty, after adjustment for age, presence of cardiovascular disease and elevated MMA (OR = 2.25, p-value =0.009).
Conclusions
Using MMA as a marker for vitamin B12, these results suggest that TCN2 gene variants may lead to decreased vitamin B12 availability, leading to reduced energy metabolism, ultimately contributing to frailty pathology. Further studies to determine the biological role of functional TCN2 polymorphisms in frailty are needed.
PMCID: PMC3042247  PMID: 20082058
Transcobalamin-II; vitamin B12; genetic association
22.  Serum Homocysteine, Vitamin B12, Folic Acid Levels and Methylenetetrahydrofolate Reductase (MTHFR) Gene Polymorphism in Vitiligo 
Disease markers  2012;33(2):85-89.
The aim of this study was to determine serum vitamin B12, folic acid and homocysteine (Hcy) levels as well as MTHFR (C677, A1298C) gene polymorphisms in patients with vitiligo, and to compare the results with healthy controls. Forty patients with vitiligo and 40 age and sex matched healthy subjects were studied. Serum vitamin B12 and folate levels were determined by enzyme-linked immunosorbent assay. Plasma Hcy levels and MTHFR polymorphisms were determined by chemiluminescence and real time PCR methods, respectively. Mean serum vitamin B12 and Hcy levels were not significantly different while folic acid levels were significantly lower in the control group. There was no significant relationship between disease activity and vitamin B12, folic acid and homocystein levels. No significant difference in C677T gene polymorphism was detected. Heterozygote A1298C gene polymorphism in the patient group was statistically higher than the control group. There was no significant relationship between MTHFR gene polymorphisms and vitamin B12, folic acid and homocysteine levels. In conclusion, vitamin B12, folate and Hcy levels are not altered in vitiligo and MTHFR gene mutations (C677T and A1298C) do not seem to create susceptibility for vitiligo.
doi:10.3233/DMA-2012-0908
PMCID: PMC3810692  PMID: 22846211
Vitiligo; homocysteine; methylenetetrahydrofolate reductase; gene polymorphism; vitamin B12; folic acid
23.  Methylenetetrahydrofolate reductase and transcobalamin genetic polymorphisms in human spontaneous abortion: biological and clinical implications 
The pathogenesis of human spontaneous abortion involves a complex interaction of several genetic and environmental factors. The firm association between increased homocysteine concentration and neural tube defects (NTD) has led to the hypothesis that high concentrations of homocysteine might be embryotoxic and lead to decreased fetal viability. There are several genetic polymorphisms that are associated with defects in folate- and vitamin B12-dependent homocysteine metabolism. The methylenetetrahydrofolate reductase (MTHFR) 677C>T and 1298A>C polymorphisms cause elevated homocysteine concentration and are associated with an increased risk of NTD. Additionally, low concentration of vitamin B12 (cobalamin) or transcobalamin that delivers vitamin B12 to the cells of the body leads to hyperhomocysteinemia and is associated with NTD. This effect involves the transcobalamin (TC) 776C>G polymorphism. Importantly, the biochemical consequences of these polymorphisms can be modified by folate and vitamin B12 supplementation. In this review, I focus on recent studies on the role of hyperhomocysteinemia-associated polymorphisms in the pathogenesis of human spontaneous abortion and discuss the possibility that periconceptional supplementation with folate and vitamin B12 might lower the incidence of miscarriage in women planning a pregnancy.
doi:10.1186/1477-7827-2-7
PMCID: PMC356929  PMID: 14969589
24.  Associations between single nucleotide polymorphisms in folate uptake and metabolizing genes with blood folate, homocysteine, and DNA uracil concentrations1234 
Background
Folate is an essential nutrient that supports nucleotide synthesis and biological methylation reactions. Diminished folate status results in chromosome breakage and is associated with several diseases, including colorectal cancer. Folate status is also inversely related to plasma homocysteine concentrations—a risk factor for cardiovascular disease.
Objective
We sought to gain further understanding of the genetic determinants of plasma folate and homocysteine concentrations. Because folate is required for the synthesis of thymidine from uracil, the latter accumulating and being misincorporated into DNA during folate depletion, the DNA uracil content was also measured.
Design
Thirteen single nucleotide polymorphisms (SNPs) in genes involved in folate uptake and metabolism, including folate hydrolase (FOLH1), folate polyglutamate synthase (FPGS), γ-glutamyl hydrolase (GGH), methylene tetrahydrofolate reductase (MTHFR), methionine synthase (MTR), proton-coupled folate transporter (PCFT), and reduced folate carrier (RFC1), were studied in a cohort of 991 individuals.
Results
The MTHFR 677TT genotype was associated with increased plasma homocysteine and decreased plasma folate. MTHFR 1298A>C and RFC1 intron 5A>G polymorphisms were associated with significantly altered plasma homocysteine concentrations. The FOLH1 1561C>T SNP was associated with altered plasma folate concentrations. The MTHFR 677TT genotype was associated with a ≈34% lower DNA uracil content (P = 0.045), whereas the G allele of the GGH – 124T>G SNP was associated with a stepwise increase in DNA uracil content (P = 0.022).
Conclusion
Because the accumulation of uracil in DNA induces chromosome breaks, mutagenic lesions, we suggest that, as for MTHFR C677T, the GGH – 124 T>G SNP may modulate the risk of carcinogenesis and therefore warrants further attention.
PMCID: PMC2728423  PMID: 18842806
25.  Genetic and Lifestyle Variables Associated with Homocysteine Concentrations and the Distribution of Folate Derivatives in Healthy Premenopausal Women 
Background
Low folate and high homocysteine (Hcy) concentrations are associated with pregnancy-related pathologies such as spina bifida. Polymorphisms in folate/Hcy metabolic enzymes may contribute to this potentially pathogenic biochemical phenotype.
Methods
The study comprised 26 Caucasian and 23 African-American premenopausal women. Subjects gave fasting blood samples for biochemical phenotyping and genotyping. Total Hcy (tHcy) and both plasma and red blood cell (RBC) folate derivatives [i.e. tetrahydrofolate (THF), 5-methylTHF (5-MTHF), and 5,10-methenylTHF (5,10-MTHF)] were measured using stable isotope dilution liquid chromatography, multiple reaction monitoring, mass spectrometry. Eleven polymorphisms from nine folate/Hcy pathway genes were genotyped. Tests of association between genetic, lifestyle, and biochemical variables were applied.
Results
In African American women, tHcy concentrations were associated (p<0.05) with total RBC folate, RBC 5-MTHF, B12, and polymorphisms in methionine synthase (MTR) and thymidylate synthase (TYMS). In Caucasian women, tHcy concentrations were not associated with total folate levels, but were associated (p<0.05) with RBC THF, ratios of RBC 5-MTHF: THF, and polymorphisms in 5,10-methylenetetrahydrofolate reductase (MTHFR) and MTR . In African Americans, folate derivative levels were associated with smoking, B12, and polymorphisms in MTR, TYMS, methionine synthase reductase (MTRR), and reduced folate carrier1 (RFC1). In Caucasians, folate derivative levels were associated with vitamin use, B12, and polymorphisms in MTHFR, TYMS, and RFC1.
Conclusions
Polymorphisms in the folate/Hcy pathway are associated with tHcy and folate derivative levels. In African American and Caucasian women, different factors are associated with folate/Hcy phenotypes and may contribute to race-specific differences in the risks of a range of pregnancy-related pathologies.
doi:10.1002/bdra.20683
PMCID: PMC4051228  PMID: 20544798
Genetics; folate; homocysteine; women; reproductive age; spina bifida risk

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