Betaine-homocysteine Methyltransferase Gene Research Articles

BHMT polymorphism and susceptibility to PTE in Chinese patients.

Pulmonary thromboembolism (PTE) is as a common form of venous thrombosis and a potentially fatal cardiovascular disorder, which has become a severe clinical problem with high incidence and mortality. The PTE has a strong genetic basis, which contributes up to half of the variance in PTE incidence and susceptibility single-nucleotide polymorphisms (SNPs) is associated with PTE. Betaine homocysteine methyltransferase (BHMT) is an essential enzyme that catalyzes the remethylating reaction from homocysteine to methionine and participates in conserving methionine and detoxifying homocysteine. In this work, we aimed to explore BHMT polymorphism and susceptibility to PTE in Chinese patients. Variant loci of the BHMT gene were screened in serum samples of PTE patients, followed by verification using Sanger sequencing. These polymorphic loci were validated in 16 PTE patients and 16 matched normal patients. The frequency differences between the allele and genotypes were compared using the Hardy-Weinberg equilibrium test and Chi-square test. A SNP was identified in PTE patients and a heterozygous transition of G>A (Arg239Gln) in rs3733890 was found. The variance difference at rs3733890 between normal patients (2/16, 0.125) and PTE patients (9/16, 0.5625) was significant (p<0.01). Therefore, we concluded that the BHMT polymorphism, rs3733890 may be a susceptibility SNP for PTE.

Association and Interaction Effect of BHMT Gene Polymorphisms and Maternal Dietary Habits with Ventricular Septal Defect in Offspring.

This study attempted to learn the association between maternal betaine-homocysteine methyltransferase (BHMT) gene polymorphisms, maternal dietary habits, and their interactions with the risk of ventricular septal defects (VSD) in offspring. A total of 426 mothers of VSD children and 740 control mothers were included in the study. Logistic regression was used to evaluate the level of associations and interaction effects. Our study suggested that mothers reporting excessive intake of smoked foods (aOR = 2.44, 95%CI: 1.89–3.13), barbecued foods (aOR = 1.86, 95%CI: 1.39–2.48), fried foods (aOR = 1.93, 95%CI: 1.51–2.46), and pickled vegetables (aOR = 2.50, 95%CI: 1.92–3.25) were at a significantly higher risk of VSD in offspring, instead, mothers reporting regular intake of fresh fruits (aOR = 0.47, 95%CI: 0.36–0.62), fish and shrimp (aOR = 0.35, 95%CI: 0.28–0.44), fresh eggs, (aOR = 0.56, 95%CI: 0.45–0.71), beans (aOR = 0.68, 95%CI: 0.56–0.83), and milk products (aOR = 0.67, 95%CI: 0.56–0.80) were at a lower risk of VSD in offspring. In addition, maternal BHMT gene polymorphisms at rs1316753 (CG vs. CC: aOR = 2.01, 95%CI: 1.43–2.83) and rs1915706 (CT vs. TT: (aOR = 1.81, 95%CI: 1.33–2.46) were significantly associated with increased risk of VSD in offspring. Furthermore, a significant interaction between BHMT polymorphisms and maternal bean intake was identified in the study. In conclusion, Maternal BHMT polymorphisms at rs1316753 and rs1915706, dietary habits as well as their interaction were observed to be significantly associated with the risk of VSD in offspring.

Association of Maternal Betaine-Homocysteine Methyltransferase (BHMT) and BHMT2 Genes Polymorphisms with Congenital Heart Disease in Offspring.

To systematically explore the association of single nucleotide polymorphisms (SNPs) of maternal BHMT and BHMT2 genes with the risk of congenital heart disease (CHD) and its three subtypes including atrial septal defect (ASD), ventricular septal defect (VSD), and patent ductus arteriosus (PDA) in offspring. A hospital-based case-control study involving 683 mothers of CHD children and 740 controls was performed. Necessary exposure information was captured through epidemiological investigation. Totally twelve SNPs of maternal BHMT and BHMT2 genes were detected and analyzed systematically. The study showed that maternal BHMT gene polymorphisms at rs1316753 (CG vs. CC: OR = 1.96 [95% CI 1.41-2.71]; GG vs. CC: OR = 1.99 [95% CI 1.32-3.00]; dominant model: OR = 1.97 [95% CI 1.44-2.68]) and rs1915706 (TC vs. TT: OR = 1.93 [95% CI 1.44-2.59]; CC vs. TT: OR = 2.55 [95% CI 1.38-4.72]; additive model: OR = 1.77 [95% CI 1.40-2.24]) were significantly associated with increased risk of total CHD in offspring. And two haplotypes were observed to be significantly associated with risk of total CHD, including C-C haplotype involving rs1915706 and rs3829809 in BHMT gene (OR = 1.30 [95% CI 1.07-1.58]) and C-A-A-C haplotype involving rs642431, rs592052, rs626105, and rs682985 in BHMT2 gene (OR = 0.71 [95% CI 0.58-0.88]). Besides, a three-locus model involving rs1316753 (BHMT), rs1915706 (BHMT), and rs642431 (BHMT2) was identified through gene-gene interaction analyses (P < 0.01). As for three subtypes including ASD, VSD, and PDA, significant SNPs and haplotypes were also identified. The results indicated that maternal BHMT gene polymorphisms at rs1316753 and rs1915706 are significantly associated with increased risk of total CHD and its three subtypes in offspring. Besides, significant interactions between different SNPs do exist on risk of CHD. Nevertheless, studies with larger sample size in different ethnic populations and involving more SNPs in more genes are expected to further define the genetic contribution underlying CHD and its subtypes.

Association between gene promoter methylation of the one-carbon metabolism pathway and serum folate among patients with hyperhomocysteinemia.

Age and lower folate concentrations are well-known risk factors for cardiovascular disease (CVD), but the potential roles of age and folate deficiency in hyperhomocysteinemia (HHcy), especially in HHcy patients with abnormal methylation levels of key enzyme genes promoter in homocysteinemia (Hcy) pathway, have not been thoroughly evaluated. The purpose of this study was to evaluate the relationship between the promoter methylation levels of six key enzyme genes and age and serum folate level to better understand the pathogenesis of HHcy. In 299 HHcy patients, six key enzyme genes promoter methylation was analyzed by PCR amplification and MethylTargetTM methods. The betaine homocysteine methyltransferase (BHMT), Cystathionine β-synthase (CBS), and Methionine synthasegene (MTR) promoter methylation levels were positively associated with age and a negative correlation was found between CBS promoter methylation level and folate levels. However, these associations were not significant after Bonferroni correction. The stratified analysis showed that the methylation level of CBS gene promoter was positively correlated with age in males, and a positive correlation was also found between BHMT gene promoter methylation level and age in HHcy patients with a history of diabetes or hypertension. Moreover, stratified analysis according to sex revealed that the methylation levels of three CpG regions of BHMT_2, CBS_2, and CBS_3 were positively correlated with age in males after Bonferroni correction. Our data suggested that age and folate deficiency may increase the risk of HHcy by mediating methylation of the promoter regions of key enzyme genes in the one-carbon metabolism pathway.

Exploring effects of betaine-homocysteine methyltransferase (BHMT) gene polymorphisms on fatty acid traits and cholesterol in sheep

This study was aimed to explore the effects of Betaine-Homocysteine Methyltransferase (BHMT) gene polymorphisms on fatty acid traits and cholesterol in lambs. This study used a total of 147 blood samples for genotyping including 19 Javanese Fat-Tailed (JFT), 16 Javanese Thin-Tailed (JTT), 41 Composite Garut (CG), 35 Compass Agrinak (CA) and 36 Barbados Black Belly Cross (BC). A total of 61 rams as representative from five breed of sheep were selected for association study. Identification of BHMT single nucleotide polymorphisms was analyzed by PCR-RFLP method. Association of BHMT genotypes with fatty acid traits and cholesterol was performed by T-TEST. BHMT genotyping resulted into three genotypes (CC, CT and TT). Gene frequency of BHMT (g. 9947372 C&gt;T) was in Hardy-Weinberg Equilibrium, excluding Javanese Fat-Tailed sheep. Association of BHMT genotypes with fatty acid traits resulted into a significant association (P&lt;0.05) with C14:1, C17:1 and C18:0 fatty acids but not with cholesterol in sheep. SNP g. 9947372 (C&gt;T) of BHMT gene might be a useful marker for selecting and producing sheep meat with desirable fatty acids.

Association between the BHMT gene rs3733890 polymorphism and the efficacy of oral folate therapy in patients with hyperhomocysteinemia.

Oral folate is currently the most common treatment for hyperhomocysteinemia (HHcy), which seriously threatens human health, but its efficacy is unsatisfactory. Betaine-homocysteine methyltransferase (BHMT) is a key enzyme that regulates Hcy metabolism. We investigated the association between the BHMT rs3733890 and the efficacy of oral folate therapy for HHcy in the Chinese Han population and analysed the effects of gene-environmental interactions on the efficacy. Blood samples were collected from 1071 eligible patients at baseline, and these individuals received subsequent folate treatment for 90 days. A total of 638 patients included in the final analysis were grouped into the treatment success group or the treatment failure group based on posttreatment Hcy levels. Hcy concentrations were measured by fluorescence polarization immunoassay. Time-of-flight mass spectrometry (MassArray system) was used to assess the genotype of BHMT rs3733890. Stratified analyses based on additive models and generalized multifactor dimensionality reduction were used to explore gene-environmental interactions. The genotype distribution presented distinct differences in the two groups. The mutant genotype and allele had significantly increased risk of treatment failure (p<0.05). Furthermore, synergistic effects of the BHMT rs3733890 polymorphism with environmental risk factors (smoking, drinking, past history) on the efficacy of therapy were also found. However, future, large well-designed studies, as well as mechanistic studies, are still needed to validate our findings.

Evidence of interaction between genes in the folate/homocysteine metabolic pathway in controlling risk of non-syndromic oral cleft.

Little consistent evidence is available for the association between the risk of non-syndromic cleft lip with or without cleft palate (NSCL/P) and any of the individual genes in the folate/homocysteine metabolic pathway. We investigated the genes in the folate pathway to further clarify its potential influence on the risk of NSCL/P considering gene-gene (G×G) interaction. We selected markers in 18 genes from the pathway and applied Cordell's method to test for G×G interaction using 1,908 NSCL/P case-parent trios ascertained in an international consortium where a genomewide association study (GWAS) of oral clefts was conducted. We found intriguing signals among Asian and European ancestry groups for G×G interaction between markers in betaine-homocysteine methyltransferase gene (BHMT/BHMT2) and dimethylglycine dehydrogenase gene (DMGDH) attaining genomewide significance. In the pooled data, the top significant interaction was found between rs13158309 (BHMT) and rs10514154 (DMGDH, p=1.45×10-12 ). Our study illustrated the importance of taking into account potential G×G interaction for genetic association analysis in NSCL/P, and this study suggested both BHMT/BHMT2 and DMGDH should be considered as candidate genes for NSCL/P in future studies.

Association of neural tube defects with gene polymorphisms in one-carbon metabolic pathway.

Neural tube defects (NTDs) are common congenital malformations. In this study, we aimed to explore the association between single nucleotide polymorphisms (SNPs) related to one-carbon metabolism (OCM) and NTDs in Han population of Northern China. A case-control study was conducted in 152 children with NTDs and 169 controls. Twenty-nine SNPs in five genes were genotyped by Sequenom MassARRAY technology, and haplotype analysis was done by Haploview4.2 software. The allele frequency of rs3733890 in betaine-homocysteine methyltransferase (BHMT) gene was statistically different between NTDs and control groups (P=0.041), and the children with A allele had higher risk for NTDs than G allele (OR=1.408, 95%CI 1.013-1.956). In addition, there was a statistical difference in the allele and genotype frequencies of rs1051266 in reduced folate carrier1 (RFC1) gene between cases and controls (P=0.013, 0.034), and the risk for NTDs was also higher in children with G allele and GG genotype, compared with A allele and AA genotype, respectively (OR=1.492, 95%CI 1.089-2.044; OR=2.020, 95%CI 1.081-3.780). The statistical significant difference was also found in allele frequency of rs1805087 in methionine synthetase (MTR) gene between cases and controls (P=0.031), and the children with G allele were associated with an increased NTDs risk, compared with A allele (OR=1.664, 95%CI 1.045-2.647). Meanwhile, haplotype analysis showed C-A-A-A haplotype of BHMT, and G-G-G-T haplotype of RFC1 was correlated with an increased risk of NTDs, but C-G-A-A haplotype of BHMT and G-G-C-A haplotype of MTR might decrease the risk of NTDs. The BHMT gene rs3733890, RFC1 gene rs1051266 and MTR gene rs1805087 were associated with the occurrence of NTDs in Han population of Northern China. It was confirmed that the gene variation related to OCM was one of the susceptibility factors for NTDs.

Acute lymphoblastic leukemia and genetic variations in BHMT gene: Case-control study and computational characterization.

Remethylation of homocysteine is catalyzed by B12 dependent methionine synthase (MTR) in all types of cells and by B12 non-dependent betaine homocysteine methyltransferase (BHMT) in liver and kidney cells. Of many etiologies of cancer, an unexplored area is the variations of genes implicated in methylation reaction. The study evaluated the association of BHMT (rs3733890) with acute lymphoblastic leukemia (ALL), followed by in-silico characterization of variations in BHMT gene. BHMT [rs3733890; c.742G > A, which substitutes an arginine by a glutamine at codon 239 (R239Q)] was screened by Tetra-primer Amplification Refractory Mutation System PCR (T-ARMS-PCR) and confirmed using DNA sequencing. In-silico analysis was conducted using bioinformatics tools. BHMT (rs3733890) showed an insignificant association with both childhood and adult ALL. Bioinformatics analysis showed that 18 nsSNPs are deleterious, 3 SNPs in 3'-UTR (rs59109725, rs116634518 and rs138578732) alter the miRNA-binding site, and 11 CNVs are present in the BHMT gene. As consequence of BHMT (rs3733890) polymorphism the free energy changes from -101210.1kJ/mol to -200021.8kJ/mol. BHMT (rs3733890) polymorphism showed no association with ALL. Hence this investigation needs further evaluation in larger sample size and effect of other SNPs, CNVs and miRNA's is required to elucidate the role of BHMT gene in ALL development.

Effects of dietary methionine and betaine on slaughter performance, biochemical and enzymatic parameters in goose liver and hepatic composition

The effects of dietary methionine (Met) and betaine (Bet) on slaughter performance, serum biochemical parameters, and liver betaine-homocysteine methyltransferase (BHMT) were investigated in geese. Six Met-deficient diets were prepared according to a 2×3 factorial design that included two levels of Bet (0 and 600mg/kg) and three levels of Met (0, 600, and 1200mg/kg). Three hundred 21-d-old healthy male Yangzhou geese with similar body weight were randomly distributed into 6 groups with 5 replicates per treatment and 10 geese per replicate. All geese had free access to diets and water for 49 d. slaughter performance were recorded at 70 d of age. Results showed that increasing dietary levels of methionine gave a linear (P<0.05) increase in body weight and average daily gain. No significant effect of dietary Met level was found on slaughter performance (P>0.05). Geese that received 600mg/kg Bet had a higher percentage of eviscerated yield than those that did not (P<0.05). There were significant interaction effects between Met and Bet in dressing percentage, half-eviscerated yield, and eviscerated yield (P<0.05). Increasing supplemental Met led to linear increases in serum concentrations of total protein (TP), albumin (ALB), and globulin (GLOB) and hepatic crude protein levels (P<0.05). Bet supplementation also increased TP and ALB plus high- and low-density lipoprotein in serum and the mRNA level of BHMT (P<0.05) in livers. In contrast, triglyceride and homocysteine were higher in geese that did not receive additional Bet than those in the Bet supplementation group (P<0.05). The hepatic crude fat contents in the Bet supplementation group also decreased (P<0.05). Significant interactions between Met and Bet supplementation (P<0.05) were detected in several amino acids. In conclusion, optimal Met dietary supplementation could increase growth performance, serum TP, ALB, and GLOB and hepatic protein synthesis in growing goslings. Furthermore, dietary supplementation with Bet could possibly replace Met for slaughter performance through upregulation of BHMT gene expression.

Association between BHMT gene rs3733890 polymorphism and cancer risk: evidence from a meta-analysis.

Background and objectiveThe gene betaine-homocysteine methyltransferase (BHMT) has drawn much attention during the past decades. An increasing number of clinical and genetic investigations have supposed that BHMT rs3733890 polymorphism might be associated with risk of breast cancer and ovarian cancer. As no consistent conclusion has been achieved, we conducted an up-to-date summary of BHMT rs3733890 polymorphism and cancer risk through a meta-analysis.Materials and methodsThe articles were collected from PubMed, Google Scholar, and CNKI (Chinese) databases up to December 2015. Then, the correlations were determined by reading the titles and abstracts and by further reading the full text to filter the unqualified articles. Odds ratio (OR) and the corresponding 95% confidence intervals (CI) were used to assess the results.ResultsAmong 187 articles collected in the analysis, seven studies with a total of 2,832 cases and 3,958 controls were included for evaluation of the association between BHMT rs3733890 polymorphism and susceptibility of cancer risk. The heterogeneity test showed no significant differences. Furthermore, we found that BHMT −742G>A polymorphism in case and control groups showed no statistically significant association with susceptibility in various cancer types except for uterine cervical cancer (A vs G: OR =0.641, 95% CI =0.445–0.923, P=0.017; AA+AG vs GG: OR =0.579, 95% CI =0.362–0.924, P=0.022). In addition, no statistically significant association was uncovered when stratification analyses were conducted by ethnicity and genotyping methods.ConclusionOur results have shown no obvious evidence that rs3733890 polymorphism in BHMT gene affected the susceptibility of head and neck squamous cell carcinoma, breast cancer, ovarian cancer, colorectal adenoma, and liver cancer. In contrast, we found the protective role of BHMT −742G>A polymorphism in uterine cervical cancer incidence. Future well-designed studies comprising larger sample size are warranted to verify our findings.

Folate Gene Polymorphisms MTR A2756G, MTRR A66G, and BHMT G742A and Risk for Coronary Artery Disease: A Meta-Analysis

Folate pathway gene polymorphisms may be a risk factor for coronary artery disease (CAD). However, studies of the association between these polymorphisms and CAD have reported conflicting results. Therefore, we performed a meta-analysis to better assess the association. To investigate the association between 3 major polymorphisms in genes encoding enzymes involved in remethylation of homocysteine to methionine--methionine synthase (MTR) A2756G, methionine synthase reductase (MTRR) A66G, and betaine homocysteine methyltransferase (BHMT) G742A--and CAD, with assessment of small-study bias and differences between studies. Case-control studies were identified by searching electronic literature databases for relevant reports published before February 2011. Data on genotype frequency were extracted, and 4 genetic models were applied. Heterogeneity was explored with stratification by ethnicity of the study sample. We found weak evidence of a recessive effect of the G allele in MTR A2756G (odds ratio, 1.61 [95% confidence interval, 0.98-2.66]; p=0.06). No effect of MTRR A66G and BHMT G742A in dominant, recessive, homozygous, and contrast allele genetic models was observed. Known common single-nucleotide polymorphisms in MTRR and BHMT genes may not be significant risk factors for CAD. The functional impact of these polymorphisms on enzyme activity is still unknown. Before additional epidemiologic studies are done, the functional impact of these polymorphisms, if any, should be established.

Homocysteine homeostasis in the rat is maintained by compensatory changes in cystathionine β-synthase, betaine-homocysteine methyltransferase, and phosphatidylethanolamine N-methyltransferase gene transcription occurring in response to maternal protein and folic acid intake during pregnancy and fat intake after weaning

The reactions of the methionine/homocysteine pathway are mediated by several enzymes, including phosphatidylethanolamine N-methyltransferase, cystathionine β-synthase, and betaine-homocysteine methyltransferase. Homocysteine homeostasis is regulated by these enzymes. We hypothesized here that the protein and folic acid content in the maternal diet affects methionine/homocysteine metabolism in the progeny. To test this hypothesis, pregnant rats were fed a diet with normal protein and normal folic acid levels (a modified casein-based AIN-93G diet), a protein-restricted and normal folic acid diet, a protein-restricted and folic acid-supplemented diet, or a normal protein and folic acid-supplemented diet. The progeny were fed either the modified AIN-93G diet or a high-fat lard-based diet. Progeny were analyzed for expression of the phosphatidylethanolamine N-methyltransferase, cystathionine β-synthase, and betaine-homocysteine methyltransferase genes in the liver and for serum homocysteine concentration. Interactions between prenatal and postnatal nutrition were also determined. The progeny of the dams fed the diets supplemented with folic acid showed decreased expression of all 3 genes (P < .001). An interaction effect between the protein and folic acid content in the maternal diet contributed to this down-regulation (P < .001), and the postweaning diet modified these effects. Serum homocysteine concentrations were approximately 15% higher in the male rats (P < .01), but neither prenatal nutrition nor the postweaning diet affected it significantly. We conclude that maternal diet during gestation has an important effect on the transcription level of these 3 genes, but changes in gene expression were not associated with significant changes in progeny homocysteine concentrations.

BHMT Gene Polymorphisms as Risk Factors for Cleft Lip and Cleft Palate in a Chinese Population

Objective Convincing evidence suggests a link between increased risk of nonsyndromic cleft lip with or without cleft palate (NSCL/P) and low intake of folic acid by the mother during pregnancy. The present study was designed to explore if genetic variation in the betaine-homocysteine methyltransferase (BHMT) gene contributes to NSCL/P. Methods DNA was obtained from 166 individuals with NSCL/P and 285 healthy subjects. Three known single nucleotide polymorphisms (SNPs) present in the BHMT gene (rs651852, rs3797546, and rs3733890) were investigated by real-time PCR-based TaqMan genotyping. Results Neither allelic nor genotypic association was found between NSCL/P and SNPs rs651852 and rs3733890. SNP rs3797546 did not show allelic association with NSCL/P; however, a higher proportion of NSCL/P patients carry the CC genotype compared with the TT+CT genotype ( P=0.020, OR=2.10, 95% CI=1.11-3.95). Conclusion Our study suggests that polymorphism rs3797546 in the BHMT gene may confer genetic risk of NSCL/P in a recessive manner.

Molecular characterization and analysis of the porcine betaine homocysteine methyltransferase and betaine homocysteine methyltransferase-2 genes

Betaine homocysteine methyltransferase (BHMT) and BHMT-2 enzymes methylate homocysteine to form methionine using betaine and S-methylmethionine, respectively. These activities are observed only in the liver of adult rodents, but in adult humans and pigs these activities are detected in both the liver and kidney, indicating the pig is a more appropriate model for studying the biochemical and physiological roles of these enzymes in human biology. Porcine BHMT and BHMT-2 cDNAs were cloned and sequenced, and their 5′ and 3′ UTR were amplified using RLM-RACE. The BHMT transcript had significantly longer 5′ and 3′ UTRs than BHMT-2. The pig BHMT and BHMT-2 genes span approximately 26 and 16 kb, respectively, and both genes have 8 exons. The deduced amino acid sequences of BHMT and BHMT-2 contain 407 and 363 amino acids, respectively, and shared 78% amino acid identity. No promoter element (TATA or CAAT box) was observed for either BHMT or BHMT-2, although a CpG island surrounding the promoter and transcriptional start site was observed in both genes implying that methylation could regulate their expression. Using qPCR, it was determined that BHMT and BHMT-2 transcripts are very abundant in liver and kidney cortex, whereas the expression is significantly less in other tissues. These findings confirm that the expression pattern of BHMT and BHMT-2 genes in pigs is similar to humans, supporting the use of the pig as an animal model to study the genetics and regulation of BHMT and BHMT-2 expression.

Betaine-homocysteine methyltransferase: Human liver genotype–phenotype correlation

Betaine-homocysteine methyltransferase (BHMT) catalyzes the remethylation of homocysteine. BHMT is highly expressed in the human liver. In the liver, BHMT catalyzes up to 50% of homocysteine metabolism. Understanding the relationship between BHMT genetic polymorphisms and function might increase our understanding of the role of this reaction in homocysteine remethylation and in S-adenosylmethionine-dependent methylation. To help achieve those goals, we measured levels of BHMT enzyme activity and immunoreactive protein in 268 human hepatic surgical biopsy samples from adult subjects as well as 73 fetal hepatic tissue samples obtained at different gestational ages. BHMT protein levels were correlated significantly ( p < 0.001) with levels of enzyme activity in both fetal and adult tissues, but both were decreased in fetal tissue when compared with levels in the adult hepatic biopsies. To determine possible genotype–phenotype correlations, 12 tag SNPs for BHMT and the closely related BHMT2 gene were selected from SNPs observed during our own gene resequencing studies as well as from HapMap. These SNPs data were used to genotype DNA from the adult hepatic surgical biopsy samples, and genotype–phenotype association analysis was performed. Three SNPs (rs41272270, rs16876512, and rs6875201), located 28 kb upstream, in the 5′-UTR and in intron 1 of BHMT, respectively, were significantly correlated with both BHMT activity ( p = 3.41E-8, 2.55E-9 and 2.46E-10, respectively) and protein levels ( p = 5.78E-5, 1.08E-5 and 6.92E-6, respectively). We also imputed 230 additional SNPs across the BHMT and BHMT2 genes, identifying an additional imputed SNP, rs7700790, that was also highly associated with hepatic BHMT enzyme activity and protein. However, none of the 3 genotyped or one imputed SNPs displayed a “shift” during electrophoretic mobility shift assays. These observations may help us to understand individual variation in the regulation of BHMT in the human liver and its possible relationship to variation in methylation.

Polymorphisms located in the region containing BHMT and BHMT2 genes as maternal protective factors for orofacial clefts

Nonsyndromic cleft lip with or without cleft palate (NCL/P) is one of the most common craniofacial malformations; however, its aetiology is still unclear. Because the effects of maternal nutrition on fetal development are well known, we decided to pursue the question of whether polymorphic variants of genes encoding enzymes involved in choline metabolism might be associated with the maternal risk of having a baby with NCL/P. Analysis of 18 single nucleotide polymorphisms (SNPs) of betaine-homocysteine methyltransferase (BHMT), betaine-homocysteine methyltransferase-2 (BHMT2), choline dehydrogenase (CHDH), choline kinase (CHKA), dimethylglycine dehydrogenase (DMGDH), choline-phosphate cytidylyltransferase A (PCYT1A), and phosphatidylethanolamine N-methyltransferase (PEMT) provided evidence that polymorphisms located in the region containing BHMT and BHMT2 were protective factors against NCL/P affected pregnancies in our population. The strongest signal was found for the SNP located in the intronic sequence of BHMT2. Women carrying two copies of the rs625879 T allele had a significantly decreased risk of having offspring with orofacial clefts. These results were significant, even after correction for multiple comparisons. Moreover, the gene-gene interaction analysis revealed a significant epistatic interaction of BHMT2 (rs673752), PEMT (rs12325817), and PCYT1A (rs712012) with maternal NCL/P susceptibility. Altogether, our study identified a novel gene, the nucleotide variants of which were be associated with a decreased risk of having a baby with NCL/P.

High intakes of choline and betaine reduce breast cancer mortality in a population‐based study

Choline and betaine provide methyl groups for one-carbon metabolism. Humans obtain these nutrients from a wide range of foods. Betaine can also be synthesized endogenously from its precursor, choline. Although animal studies have implied a causal relationship between choline deficiency and carcinogenesis, the role of these two nutrients in human carcinogenesis and tumor progression is not well understood. We investigated the associations of dietary intakes of choline and betaine and breast cancer risk and mortality in the population-based Long Island Breast Cancer Study Project. Among the 1508 case-group women, 308 (20.2%) deaths occurred, among whom 164 (53.2%) died of breast cancer by December 31, 2005. There was an indication that a higher intake of free choline was associated with reduced risk of breast cancer (P(trend)=0.04). Higher intakes of betaine, phosphocholine, and free choline were associated with reduced all-cause as well as breast cancer-specific mortality in a dose-dependent fashion. We also explored associations of polymorphisms of three key choline- and betaine-metabolizing genes and breast cancer mortality. The betaine-homocysteine methyltransferase gene (BHMT) rs3733890 polymorphism was associated with reduced breast cancer-specific mortality (hazard ratio, 0.64; 95% confidence interval, 0.42-0.97). Our study supports the important roles of choline and betaine in breast carcinogenesis. It suggests that high intake of these nutrients may be a promising strategy to prevent the development of breast cancer and to reduce its mortality.

Characterization of porcine betaine homocysteine methyltransferase (BHMT) and betaine homocysteine methyltransferase ‐2 (BHMT2) genes

Betaine homocysteine methyltransferase (BHMT) and BHMT‐2 methylate homocysteine using betaine or S‐methylmethionine, respectively. These enzyme activities are found only in the liver of adult rodents, but in the liver and kidney of adult humans and pigs. Using a pig model, we seek to understand the developmental expression pattern of these enzymes in different porcine organs. Immunohistochemical staining revealed the presence of BHMT in adult liver and kidney cortex, as reported earlier, and also in fetal lungs (aged days 30, 60, 84, 90, 105). Full length cDNA of BHMT and BHMT‐2 were cloned and sequenced, and the 5' and 3' UTRs were amplified using RLM‐RACE. BHMT has a longer 5' and 3' UTR, consisting of 77 and 1142 nucleotides, whereas BHMT‐2 consisted of 17 and 893 nucleotides respectively. The predicted primary sequences of BHMT and BHMT‐2 contain 407 and 363 amino acids, respectively, and share 78% amino acid identity. Relative to BHMT‐2, BHMT has two additional regions of amino acid sequence; a 9 amino acid sequence (86‐94) in the N‐terminal region, and 34 amino acid sequence (373‐407) at the carboxy terminus. Preliminary sequencing results identified two isoforms of liver BHMT gene resulting from alternative splicing of 5' UTR. BHMT enzymes are organ/developmental stage‐dependent and further sequence analysis might reveal new splice variants that result in different protein functions.Grant Funding SourceUSDA AG 2008‐34480‐19328 &amp; USDA‐ARS 538 AG58‐5438‐7‐317l

Inhibition of human betaine–homocysteine methyltransferase expression by S-adenosylmethionine and methylthioadenosine

BHMT (betaine-homocysteine methyltransferase) remethylates homocysteine to form methionine. SAM (S-adenosylmethionine) inhibits BHMT activity, but whether SAM modulates BHMT gene expression is unknown. Transcriptional regulation of the human BHMT is also unknown. The present study examined regulation of the human BHMT gene by SAM and its metabolite, MTA (5'-methylthioadenosine). To facilitate these studies, we cloned the 2.7 kb 5'-flanking region of the human BHMT gene (GenBank accession number AY325901). Both SAM and MTA treatment of HepG2 cells resulted in a dose- and time-dependent decrease in BHMT mRNA levels, which paralleled their effects on the BHMT promoter activity. Maximal suppression was observed with the BHMT promoter construct -347/+33, which contains a number of NF-kappaB (nuclear factor kappaB) binding sites. SAM and MTA treatment increased NF-kappaB nuclear binding and NF-kappaB-driven luciferase activities, and increased nuclear binding activity of multiple histone deacetylase co-repressors to the NF-kappaB sites. Overexpression of p50 and p65 decreased BHMT promoter activity, while blocking NF-kappaB activation increased BHMT expression and promoter activity, and prevented SAM but not MTA's ability to inhibit BHMT expression. The NF-kappaB binding site at -301 is responsible, at least in part, for this effect. Lower BHMT expression can impair homocysteine metabolism, which can induce ER (endoplasmic reticulum) stress. Indeed, MTA treatment resulted in increased expression ER stress markers. In conclusion, SAM and MTA down-regulate BHMT expression in HepG2 cells in part by inducing NF-kappaB, which acts as a repressor for the human BHMT gene. While SAM's mechanism is NF-kappaB-dependent, MTA has both NF-kappaB-dependent and -independent mechanisms.