Methionine Synthase Research Articles

52 Young Scholar Presentation: Maternal supply of methionine during late-pregnancy alters in utero and neonatal development, hepatic one-carbon metabolism,and innate immune response in Holstein calves

Abstract Pregnancy and early life are critical periods of plasticity during which the fetus and neonate may be influenced by environmental factors such as nutrition. Objectives were to investigate if increasing methionine (Met) supply during late-pregnancy affects developmental, metabolic, and immune parameters of the calf at birth and during the neonatal period. Calves born to Holstein cows individually-fed a basal control (CON; 1.47 Mcal/kg dry matter [DM] and 15.3% crude protein) diet with no added Met or CON plus rumen-protected Met (MET; Mepron® at 0.09% of diet DM) during the last 28±2 d of pregnancy were used. Liver biopsies were harvested at 4, 14, 28, and 50 d of age and used for metabolomics via GS-MS and activity of hepatic 1-carbon metabolism enzymes. Blood for biomarker analyses and innate immune function assays was harvested at birth (before colostrum feeding), 7, 21, 42 and 50 d of age. Whole blood was challenged with enteropathogenic bacteria (E. coli 0118:H8), and phagocytosis and oxidative burst of neutrophils and monocytes quantified by flow cytometry. At birth, MET calves had greater (P≤0.05) body weight (BW, 44.1 vs. 42.1±0.70 kg), hip height (HH, 81.3 vs. 79.6±0.53 cm), and wither height (WH, 77.8 vs. 75.9±0.47 cm). Differences persisted through 9 wk of age, resulting in average responses of +3.1 kg BW, +1.9 cm HH, and+1.8 cm WH in MET compared with CON. Average daily gain during the 9 wk was (P<0.05) 0.72±0.02 kg/d in MET and 0.67±0.02 kg/d in CON calves. Despite similar rates of daily DMI, maternal supplementation with Met led to greater (P ≤ 0.05) overall hepatic concentrations of the 1-carbon metabolism intermediates adenosine, betaine, choline, glycine, and N,N-dimethylglycine in liver tissue. Among transsulfuration pathway metabolites, concentrations of cystathionine, cysteinesulfinic acid, hypotaurine, serine, and taurine were greater (P ≤ 0.05) in MET calves. There was a treatment × day effect for activity of betaine-homocysteine S-methyltransferase (BHMT), methionine synthase (MTR) and cystathionine-beta-synthase (CBS) in liver tissue. Activity of BHMT and CBS increased in MET calves between d 4 and 14, with a peak at 28 d. Despite a linear increase from d 4 to 28, activity of MTR in MET calves was lower on d 4 and 50. Plasma concentrations of haptoglobin and activity of myeloperoxidase in both maternal groups increased (P ≤ 0.05) markedly between 0 and 7 d of age followed by a decrease to baseline at d 21 with responses being lower in MET compared with CON calves. In vitro phagocytosis in stimulated neutrophils was greater overall (P ≤ 0.05) in MET calves. Overall, data indicate that increasing the maternal supply of Met during late-pregnancy benefits calf growth in part through alterations in hepatic metabolism and innate immune response.

Cysteine and methionine contribute differentially to regulate alternative oxidase in leaves of poplar (Populus deltoides x Populus euramericana ‘Nanlin 895’) seedlings exposed to different salinity

The effects of different doses of NaCl on the expression profiles of genes involved in the mitochondrial electron transport chain (miETC), H2O2 and O2− levels, and antioxidant enzymes and amino acid metabolism were investigated in the leaves of poplar (Populus deltoides x Populus euramericana 'Nanlin 895'). In the miETC, complexes II and III and bypasses of the cytochrome c pathway including AOX and UCP displayed higher transcript abundance, whereas COX6b encoding cytochrome c oxidase were suppressed at 200 and 400 mM. H2O2 accumulated at 200 mM NaCl but O2− was generated at 400 mM. Accordingly, CAT was enhanced at 200 and 400 mM, while G-POD strengthened only at 400 mM. In addition, cysteine was reduced at 400 mM but did not change at 200 mM, although methionine was accumulated at 200 mM but not altered at 400 mM. Exogenous cysteine accumulated H2S and methionine increased ACC at 200 mM NaCl. At 400 mM NaCl, cysteine elevated the expression of CGS encoding cystathionine gamma-synthase and MS2 encoding methionine synthase as well as ACC and H2S levels, and methionine increased ACC content with repressed CGS and MS2. Moreover, exogenous KCN decreased cysteine levels, with an augment in H2S and up-regulation of CYS C1 encoding β-cyanoalanine synthase at all salinity conditions, whereas antimycin A (AA) and salicylhydroxamic acid (SHAM) affected neither the levels of cysteine or H2S, nor the CYS C1 expression. However, neither KCN, AA nor SHAM affected ACC content. AOX1b was induced both by exogenous cysteine and methionine as well as KCN and AA but suppressed by SHAM at 200 and 400 mM NaCl, in negative correlation with MDA content. These results suggest that poplar leaf evolved diverse strategies in amino acid metabolism of manipulating the AOX pathway to defend against different levels of salt stress.

Hepatic betaine-homocysteine methyltransferase and methionine synthase activity and intermediates of the methionine cycle are altered by choline supply during negative energy balance in Holstein cows

Although choline requirements are unknown, enhanced postruminal supply may decrease liver triacylglycerol (TAG) storage and increase flux through the methionine cycle, helping cows during a negative energy balance (NEB). The objective was to investigate effects of postruminal choline supply during NEB on hepatic activity of betaine-homocysteine methyltransferase (BHMT), methionine synthase (MTR), methionine adenosyltransferase, transcription of enzymes, and metabolite concentrations in the methionine cycle. Ten primiparous rumen-cannulated Holstein cows (158 ± 24 d postpartum) were used in a replicated 5 × 5 Latin square design with 4-d treatment periods and 10 d of recovery (14 d/period). Treatments were unrestricted intake with abomasal infusion of water (A0), restricted intake (R; 60% of net energy for lactation requirements to induce NEB) with abomasal infusion of water (R0) or R plus abomasal infusion of 6.25, 12.5, or 25 g/d of choline ion. Liver tissue was collected on d 5 after the infusions ended, blood on d 1 to 5, and milk on d 1 to 4. Statistical contrasts were A0 versus R0 (CONT1) and tests of linear (L), quadratic (Q), and cubic (C) effects of choline dose. Plasma choline increased with R (CONT1) and choline (L). Although R decreased milk yield (CONT1), choline increased milk yield and liver phosphatidylcholine (PC), but decreased TAG (L). No differences were observed in plasma PC or very-low-density lipoprotein concentrations with R or choline. Activity and mRNA abundance of BHMT were greater with R (CONT1) and increased with choline (L). Although activity of MTR was lower with R (CONT1), it tended to increase with choline (L). No effect of R was detected for activity of methionine adenosyltransferase, but it changed cubically across dose of choline. Those responses were associated with linear increases in the concentrations of liver tissue (+13%) and plasma methionine concentrations. The mRNA abundance of CPT1A, SLC22A5, APOA5, and APOB, genes associated with fatty acid oxidation and lipoprotein metabolism, was upregulated by choline (Q). Overall, enhanced supply of choline during NEB increases hepatic activity of BHMT and MTR to regenerate methionine and PC, partly to help clear TAG. The relevance of these effects during the periparturient period merits further research.

Metabolic effects of vitamin B12 on physiology, stress resistance, growth rate and biomass productivity of Cyanobacterium stanieri planktonic and biofilm cultures

Although synthesized only by bacteria and archaea, cobalamin (vitamin B12) is essential for virtually all living cells. One major function is its role in methionine synthesis, as a co-factor for the B12-dependent methionine synthase MetH. However, a large number of microbes avoid requirements for B12 by encoding cobalamin-independent enzymes, such as the B12-independent methionine synthase MetE. Interestingly, many such microbes retain transporters for exogenous B12, produced by neighboring microbes. We hypothesize that selection for retention of B12 transport suggests preservation of unrevealed but critical roles for cobalamin in photoautotroph fitness. To identify the impacts of B12 on photoautotrophic metabolism, we studied the physiological and transcriptional adaptation of Cyanobacterium stanieri HL-69 to varying irradiance and oxidative stress in the presence and absence of B12. The metabolic flexibility of C. stanieri, which possesses both MetH and MetE, allows comparative analysis of cobalamin impacts on its global metabolism. As anticipated, B12 availability governed transcription of cobalamin transporter btuB, metH and a number of genes involved in the methionine-folate cycle. Surprisingly, however, B12 impacted the cell integrity, growth rate and biomass productivity of C. stanieri under conditions of likely oxidative stress due to biofilm growth or under high partial pressures of O2. Furthermore, C. stanieri response to B12 globally rewired cellular metabolic networks, including nitrogen metabolism, energy metabolism, redox homeostasis and oxidative stress response. These findings demonstrate previously-unappreciated roles for B12 metabolism beyond methionine synthesis and reveal how interactions with cobalamin-producing heterotrophs may affect phytoplankton function and dynamics in natural microbial communities. Further comprehension and mastering of the natural oxidative stress resistance mechanisms modulated by cobalamin could be used for the design and implementation of more robust algal bioprocesses retaining high biomass productivities especially under stress conditions.

Association of MTHFR C677T, MTHFR A1298C, and MTRR A66G Polymorphisms with Neural Tube Defects in Tunisian Parents

Objective: This study aims to investigate the association of 5,10-methylenetetrahydrofolate reductase (MTHFR C677T and A1298C) and methionine synthase reductase (MTRR A66G) gene polymorphisms with neural tube defects (NTDs) in a Tunisian population. Methods: Genotyping was performed by polymerase chain reaction with restriction fragment length polymorphisms (PCR-RFLPs) using the restriction enzymes. Allele and genotype frequencies were compared between mothers and fathers of fetuses with NTDs with matched controls based on an association analysis using SPSS software. Results: MTHFR (C677T, A1298C) and MTRR A66G polymorphisms were found to be protector factors for NTD fetuses in the mother group. In addition, a combination of the three wild-type alleles C677/A1298/A66 has increased four-fold the incidence of NTDs (p = 0.004, OR = 3.96, 95% CI: 1.53–10.23). In the father group, MTHFR C677T was a risk factor for NTDs. However, no association was found between MTHFR A1298C, MTRR A66G, and the occurrence of this anomaly. The analysis of MTHFR C677T and MTRR A66G polymorphisms has demonstrated a significant difference in vitamin B₁₂ levels between recessive and dominant genotypes in case mothers (p < 0.05). Conclusion: Additional studies are required to better understand the roles of parental gene polymorphisms related to folate-homocysteine metabolism in the pathogenesis of NTD.

Associations of MTRR and TSER polymorphisms related to folate metabolism with susceptibility to metabolic syndrome.

Hyperhomocysteinemia is a potential risk factor for the development of metabolic syndrome (MetS). Among genes involved in homocysteine metabolism, polymorphisms of methylenetetrahydrofolate reductase (MTHFR) gene are known to be associated with MetS incidence. However, effects of polymorphisms of other folate metabolism-related genes on MetS susceptibility are not well known yet. This study was to determine whether methionine synthase (MTR) 2756A > G, methionine synthase reductase (MTRR) 66A > G, and thymidylate synthase enhancer region (TSER) 2R/3R polymorphisms might be associated with risks of MetS development in the Korean population. Genotype analysis of the three polymorphisms was performed for a total of 483 subjects including 236 MetS patients and 247 unrelated healthy controls using polymerase chain reaction-restriction fragment length polymorphism technique. The present study revealed that MTRR and TSER polymorphisms were associated with susceptibility to MetS. Several genotypes and allele combinations from the three polymorphisms were also related to the MetS prevalence. When polymorphism data were stratified according to the risk components of MetS, MTR polymorphism was significantly associated with an increased risk of MetS in subjects with systolic blood pressure < 132.7mmHg (AOR 1.842, 95% CI 1.039-3.266, P = 0.037) and fasting blood glucose level < 106.3mg/dL (AOR 1.772, 95% CI 1.069-2.937, P = 0.027). MTRR polymorphism was significantly associated with a decreased risk of MetS in subjects with triglyceride level < 216.3mg/dL (AOR 0.616, 95% CI 0.399-0.951, P = 0.029). To the best of our knowledge, this is the first to provide reliable evidence about the association of other folate metabolism-related gene polymorphisms besides MTHFR with MetS susceptibility and its risk factors. Results of this study suggest that MTRR and TSER polymorphisms might be potential genetic markers for the risk of MetS development in Korean population.

Interactions between genetic variants involved in the folate metabolic pathway and serum lipid, homocysteine levels on the risk of recurrent spontaneous abortion

BackgroundThe interaction between folate pathway gene polymorphisms and homocysteine, serum lipid leverls are poorly understood in patients with recurrent spontaneous abortion (RSA). The aim of this study is to explore the effects of folate pathway gene polymorphisms (the 5–10-methylenetetrahydrofolate reductase, MTHTR C677T, MTHFR A1298C and the methionine synthase reductase, MTRR A66G) and their interactions with homocysteine on serum lipid levels in patients with RSA.MethodsA total of 403 RSA women and 342 healthy women were randomly selected. Genotyping of the MTHFR C677T, A1298C and MTRR A66G were performed by TaqMan-MGB technique. Serum homocysteine, folate, fasting glucose, fasting insulin, Interleukin 6, Tumor necrosis factorα (TNFα) and lipid profiles were measured according to the kits. Continuous variables were analyzed using 2-sample t-tests. Categorical variables were analyzed and compared by χ2 or Fisher’s exact tests. Unconditional logistic regression model was applied to test the interactions of gene polymorphisms on RSA.ResultsThe distribution of genotype of CC, CT TT and T allele of MTHFR C677T, genotype of AA and C allele of MTHFR A1298C, and genotype of AA, AG and G allele of MTRR A66G were different between cases and controls (all p were < 0.05). There were significant interactions between MTHFR C677T-A1298C and MTHFR A1298C-MTRR A66G in RSA group and control group, with ORs of 1.62 (95%CI: 1.28–2.04, p < 0.001) and 1.55 (95%CI: 1.27–1.88, p < 0.001), respectively. Serum TNFα level and insulin resistant status (HOMR-IR) were higher in RSA group than in control group (p = 0.038, 0.001, respectively). All the three gene SNPs except MTRR 66AG gene variant had detrimental effects on HOMA-IR (all p were < 0.05). RSA group who carried the MTHFR 677CT, TT, CT/TT genotypes and MTRR 66AG, AG/GG genotypes had detrimental effects on serum homocysteine levels, the MTHFR 677CT, CT/TT genotype carriers had favorable effects on serum folate levels, the MTHFR 677TT, CT/TT, 1298 AC, AC/CC genotype carriers had detrimental effects on serum low-density lipoprotein cholesterol (LDL-C) levels, and the MTRR 66AG genotype carriers had lower high-density lipoprotein cholesterol (HDL-C) levels than the AA genotype carriers (all p were < 0.05).ConclusionsInteraction between the MTHFR C677T, A1298C and MTHFR A1298C, MTRR A66G are observed in our RSA group. Besides, all the three gene SNPs except MTRR 66AG gene variant had detrimental effects on HOMA-IR. MTHFR C677T and MTRR A66G gene variants had detrimental effects on serum homocysteine levels and insulin resistance status, while MTHFR C677T, A1298C and MTRR A66G gene variants had detrimental effects on certain serum lipid profiles.

How haptophytes microalgae mitigate vitamin B12 limitation

Vitamin B12 (cobalamin) can control phytoplankton development and community composition, with around half of microalgal species requiring this vitamin for growth. B12 dependency is determined by the absence of cobalamin-independent methionine synthase and is unrelated across lineages. Despite their important role in carbon and sulphur biogeochemistry, little is known about haptophytes utilization of vitamin B12 and their ability to cope with its limitation. Here we report the first evaluation of B12 auxotrophy among this lineage based on molecular data of 19 species from 9 families. We assume that all species encode only a B12-dependent methionine synthase, suggesting ubiquitous B12 auxotrophy in this phylum. We further address the effect of different B12 limitations on the molecular physiology of the model haptophyte Tisochrysis lutea. By coupling growth assays in batch and chemostat to cobalamin quantification and expression analyses, we propose that haptophytes use three strategies to cope with B12 limitation. Haptophytes may assimilate dissolved methionine, finely regulate genes involved in methionine cycle and B12 transport and/or limit B12 transport to the mitochondrion. Taken together, these results provide better understanding of B12 metabolism in haptophytes and represent valuable data for deciphering how B12-producing bacteria shape the structure and dynamics of this important phytoplankton community.

Influence of genetic factors on long-term treatment related neurocognitive complications, and on anxiety and depression in survivors of childhood acute lymphoblastic leukemia: The Petale study.

BackgroundA substantial number of survivors of childhood acute lymphoblastic leukemia suffer from treatment-related late adverse effects including neurocognitive impairment. While multiple studies have described neurocognitive outcomes in childhood acute lymphoblastic leukemia (ALL) survivors, relatively few have investigated their association with individual genetic constitution.MethodsTo further address this issue, genetic variants located in 99 genes relevant to the effects of anticancer drugs and in 360 genes implicated in nervous system function and predicted to affect protein function, were pooled from whole exome sequencing data of childhood ALL survivors (PETALE cohort) and analyzed for an association with neurocognitive complications, as well as with anxiety and depression. Variants that sustained correction for multiple testing were genotyped in entire cohort (n = 236) and analyzed with same outcomes.ResultsCommon variants in MTR, PPARA, ABCC3, CALML5, CACNB2 and PCDHB10 genes were associated with deficits in neurocognitive tests performance, whereas a variant in SLCO1B1 and EPHA5 genes was associated with anxiety and depression. Majority of associations were modulated by intensity of treatment. Associated variants were further analyzed in an independent SJLIFE cohort of 545 ALL survivors. Two variants, rs1805087 in methionine synthase, MTR and rs58225473 in voltage-dependent calcium channel protein encoding gene, CACNB2 are of particular interest, since associations of borderline significance were found in replication cohort and remain significant in combined discovery and replication groups (OR = 1.5, 95% CI, 1–2.3; p = 0.04 and; OR = 3.7, 95% CI, 1.25–11; p = 0.01, respectively). Variant rs4149056 in SLCO1B1 gene also deserves further attention since previously shown to affect methotrexate clearance and short-term toxicity in ALL patients.ConclusionsCurrent findings can help understanding of the influence of genetic component on long-term neurocognitive impairment. Further studies are needed to confirm whether identified variants may be useful in identifying survivors at increased risk of these complications.

CBS 844ins68, MTR A2756G and MTHFR C677T gene polymorphisms in hyperhomocysteinemia: A study among a non-tribal population group with East Asian ancestry (India)

Hyperhomocysteinemia (Hhcy) is reported to be one of the non-traditional risk factors for cardiovascular diseases. Mutation of genes in one-carbon metabolic pathway (homocysteine metabolism pathway) like methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR) and cystathionine beta synthase (CBS) have been implicated in hyperhomocysteinemia. The present study is an attempt to understand the association of CBS 844ins68, MTR A2756G and MTHFR C677T gene polymorphisms with hyperhomocysteinemia among a non-tribal population of Manipur, India. A total of 200 individuals from both sexes age ranging between 35 yrs. to 65 yrs. were randomly recruited for the present study. Genotyping of MTHFR C677T, MTR A2756G and CBS 844ins68 gene polymorphisms and analysis of homocysteine levels were done using standard protocols. Of the recruited individuals, 70% were classified as hyperhomocysteinemia cases and 30% were classified as normal homocysteine controls. Results indicate that none of the selected gene polymorphisms were found to be associated with hyperhomocysteinemia in the presently studied population group. However, individuals with TT genotype of MTHFR C677T gene polymorphism are bound to have hyperhomocysteinemia. Further, this TT genotype mediated hyperhomocysteinemia could be lethal among the individuals who have NI genotype of CBS 844ins68 gene polymorphism.

Dietary Intakes of Folic Acid During Pregnancy Determines Maternal Re-Set of Metabolism Post-Birth in Wistar Rat Dams (FS08-06-19)

ObjectivesNutrition during pregnancy alters the “re-set” of maternal metabolism and in turn the mother’s metabolic phenotype later in life. Folic acid (FA, synthetic folate) consumed at intakes above requirements during pregnancy by rats leads to increased weight gain and altered DNA methylation in central and peripheral pathways regulating food intake. The objectives of this study were to examine the effects of intakes below and above FA dietary requirements on the re-set of energy metabolic pathways in Wistar rat mothers early post-birth. MethodsPregnant Wistar rats (n = 12/group) were fed an AIN93G diet with 5 levels of FA: 0X, 1X (control, 2 mg FA/kg), 2.5X, 5X or 10X. Dams were fed 1X-FA during lactation up to 1-week post-weaning (PW) when maternal metabolism is thought to re-set to homeostasis and then terminated. Weekly body weight, food intake, expression of hypothalamic food-intake neurons, mRNA and protein expression of folate-related and energy metabolic genes, and glucoregulatory hormones were measured. The homeostatic model assessment of insulin resistance (HOMA-IR) was used as a surrogate index of insulin resistance. ResultsBelow (0X) and above (5X and 10X) FA requirements during pregnancy suppressed expression of hepatic folate metabolism (methyltetrahydrofolate (MTHF) reductase, and methionine synthase; P < 0.05) genes and led to higher 5-MTHF (P < 0.005) in blood compared to control suggesting dysregulation of 1-carbon pathways. Dams fed 0X- and 5X-FA also had higher plasma insulin and HOMA-IR than controls and changes in glucose and lipid metabolism-regulating genes in muscle (Glucose transporter-4, and Peroxisome-proliferator activated receptors; P < 0.05) but not liver or adipose at 1-week PW. The diets did not affect expression of hypothalamic food intake neurons nor body weight or food intake of the dams from birth to 1-week PW. ConclusionsFA below (0X) or above (5X, 10X) requirements during pregnancy induce dysregulation of 1-carbon pathways and delay re-set of energy metabolic pathways in Wistar rat dams by 4-weeks after birth, potentially programming long-term negative metabolic effects. Funding SourcesThis research was supported by: Canadian Institute of Health Research, Institute of Nutrition, Metabolism and Diabetes (CIHR-INMD); EP supported by NSERC Alexander Graham Bell Canada Graduate Scholarships-Doctoral Program (CGS D).

Reverse Erythroblastosis Virus α Antagonism Promotes Homocysteine Catabolism and Ammonia Clearance.

Metabolic homeostasis of amino acids is essential for human health. Here, we aimed to investigate a potential role for the clock component reverse erythroblastosis virus α (Rev-erbα) in circadian regulation of amino acid metabolism. RNA-seq with Rev-erbα-/- mice showed expression changes in genes involved in amino acid metabolism, particularly, the urea cycle and methionine metabolism. Rev-erbα ablation increased hepatic mRNA, protein, and enzymatic activity of betaine homocysteine methyltransferase (Bhmt), cystathionine β-synthase (Cbs), and cystathionine γ-lyase (Cth) and decreased the levels of plasma and liver homocysteine in mice. Cell-based assays confirmed negative regulation of these three genes by Rev-erbα. Combined luciferase reporter, mobility-shift, and chromatin immunoprecipitation assays identified Rev-erbα as a transcriptional repressor of Bhmt, Cbs, and Cth. Rev-erbα ablation or antagonism alleviated chemical-induced hyperhomocysteinemia in mice. This was accompanied by elevated expressions of Bhmt, Cbs, and Cth. Moreover, Rev-erbα ablation or antagonism promoted urea production and ammonia clearance. Of urea cycle-related genes, arginase 1 (Arg1), ornithine transcarbamylase (Otc), and carbamoyl-phosphate synthase 1 (Cps1) expressions were up-regulated in Rev-erbα-/- mice. Negative regulation of these urea cycle genes by Rev-erbα was validated using cell-based experiments. Mechanistic studies revealed that Rev-erbα inhibited CCAAT-enhancer-binding protein α transactivation to repress the transcription of Arg1, Cps1, and Otc. Conclusion: Rev-erbα antagonism alleviates hyperhomocysteinemia and promotes ammonia clearance. Targeting Rev-erbα represents an approach for the management of homocysteine- and ammonia-related diseases.

One-carbon metabolism gene polymorphisms are associated with cognitive trajectory among African-American adults

The sex-specific link between longitudinal annual rate of cognitive change (LARCC) and polymorphisms in one-carbon metabolism enzymatic genes remains unclear, particularly among African-American adults. We tested associations of 14 single nucleotide polymorphisms (SNPs) from MTHFR, MTRR, MTR, and SHMT genes and select MTHFR haplotypes and latent classes (SNPHAP/SNPLC) with LARCC. Up to 797 African-American participants in the Healthy Aging in Neighborhoods of Diversity across the Life Span study (age: 30–64 y, 52% women) had 1.6–1.7 (i.e., 1 or 2) repeated measures (follow-up time, mean = 4.69 y) on 9 cognitive test scores, reflecting verbal and visual memory, verbal fluency, psychomotor speed, attention, and executive function: California Verbal Learning Test–immediate recall (CVLT-List A), CVLT-DFR (delayed free recall), Benton Visual Retention Test (BVRT), Animal Fluency (AF), Digits Span Forward and Backward tests, and Trail Making Test parts A and B (Trails A and B). Multiple linear mixed-effects and multiple linear regression models were conducted. Overall, MTHFR SNPs rs4846051(A1317G, G>A) and rs1801131(A1298C, G>T) were associated with slower and faster declines on AF, respectively, whereas rs2066462(C1056T, A>G) was related to slower decline on Trails B (executive function). Among men, rs4846051(A1317G, G>A) was linked to faster decline on BVRT (visual memory), whereas rs2066462(C1056T, A>G) and rs9651118(C>T) were associated with slower decline on CVLT-List A and rs9651118(C>T) with faster decline on CVLT-DFR. Among women, a slower decline on the domain “verbal memory/fluency” was observed with rs1801133(C677T, A>G). MTHFR2SNPHAP [rs1801133(C677T, A>G)/rs1801131(A1298C, G>T): GG] was associated with slower decline on AF among women, whereas MTHFR3SNPHAP(AT) was linked with slower decline on CVLT-List A among men but faster decline on “verbal memory/fluency” among women. Similar patterns were observed for MTHFR SNPLCs. In sum, MTHFR gene variations can differentially impact longitudinal changes in multiple cognitive domains among African-American adults.

Association of methionine synthase (rs1805087), methionine synthase reductase (rs1801394), and methylenetetrahydrofolate dehydrogenase 1 (rs2236225) genetic polymorphisms with recurrent implantation failure

One-carbon metabolism, in which folate plays an essential role, is important for maintaining pregnancy and foetal development. Here, polymorphisms in three genes involved in the methylation of homocysteine were examined: methionine synthase (MTR), methionine synthase reductase (MTRR), and methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), each of which is involved in methionine metabolism, a component of the one-carbon metabolism process. This involved a case–control study of 343 Korean women: 118 patients with RIF and 225 controls with at least one live birth and no history of pregnancy loss. The MTHFD1 1958GA genotype was observed less frequently than the MTHFD1 1958GG genotype (IF ≥ 4, p = 0.015) in women with RIF. In addition, the MTRR 66A > G polymorphism was associated with increased plasma homocysteine levels (p = 0.019). Based on these results, we propose that the MTRR 66A > G and MTHFD1 1958G > A polymorphisms are predisposing factors for RIF development. This study is the first to examine a potential association between the MTHFD1 and MTRR polymorphisms and RIF susceptibility in Korean patients.

Enzymatic basis for C-lignin monomer biosynthesis in the seed coat of Cleome hassleriana.

C-lignin is a linear polymer of caffeyl alcohol, found in the seed coats of several exotic plant species, with promising properties for generation of carbon fibers and high value chemicals. In the ornamental plant Cleome hassleriana, guaiacyl (G) lignin is deposited in the seed coat for the first 6-12days after pollination, after which G-lignin deposition ceases and C-lignin accumulates, providing an excellent model system to study C-lignin biosynthesis. We performed RNA sequencing of seed coats harvested at 2-day intervals throughout development. Bioinformatic analysis identified a complete set of lignin biosynthesis genes for Cleome. Transcript analysis coupled with kinetic analysis of recombinant enzymes in Escherichia coli revealed that the switch to C-lignin formation was accompanied by down-regulation of transcripts encoding functional caffeoyl CoA- and caffeic acid 3-O-methyltransferases (CCoAOMT and COMT) and a form of cinnamyl alcohol dehydrogenase (ChCAD4) with preference for coniferaldehyde as substrate, and up-regulation of a form of CAD (ChCAD5) with preference for caffealdehyde. Based on these analyses, blockage of lignin monomer methylation by down-regulation of both O-methyltransferases (OMTs) and methionine synthase (for provision of C1 units) appears to be the major factor in diversion of flux to C-lignin in the Cleome seed coat, although the change in CAD specificity also contributes based on the reduction of C-lignin levels in transgenic Cleome with down-regulation of ChCAD5. Structure modeling and mutational analysis identified amino acid residues important for the preference of ChCAD5 for caffealdehyde.

Folate Metabolism Interferes with Plant Immunity through 1C Methionine Synthase-Directed Genome-wide DNA Methylation Enhancement

Plants rely on primary metabolism for flexible adaptation to environmental changes. Here, through a combination of chemical genetics and forward genetic studies in Arabidopsis plants, we identified that the essential folate metabolic pathway exerts a salicylic acid-independent negative control on plant immunity. Disruption of the folate pathway promotes enhanced resistance to Pseudomonas syringae DC3000 via activation of a primed immune state in plants, whereas its implementation results in enhanced susceptibility. Comparative proteomics analysis using immune-defective mutants identified a methionine synthase (METS1), in charge of the synthesis of Met through the folate-dependent 1C metabolism, acting as a nexus between the folate pathway and plant immunity. Overexpression of METS1 represses plant immunity and is accompanied by genome-wide global increase in DNA methylation, revealing that imposing a methylation pressure at the genomic level compromises plant immunity. Take together, these results indicate that the folate pathway represents a new layer of complexity in the regulation of plant defense responses.

CBS 844INS68 and MS D919G polymorphism: early pregnancy loss risk

Cystathionine beta synthase (CBS) and Methionine synthase (MS) maintains the level of homocysteine in blood. Our objective was to study the genetic association of CBS 844ins68 and MS D919G polymorphism with early pregnancy loss (EPL) and Recurrent pregnancy loss (RPL). We investigated 174 EPL patients in which 130 were RPL patients and 180 healthy controls. Genotyping was done through PCR-RFLP for MS D919G polymorphism whereas that for CBS 844ins68 was performed in duplex PCR reaction. Age adjusted odds ratios (AORs) were calculated by logistic regression analysis. The CBS 844ins68 was found to have protective effect rather than having risk for RPL (AORs = 0.49, 95% CI=0.22-1.08, p=0.07). The MS D919G polymorphism also did not differ significantly between patients and controls. This is the first study to see the association of CBS and MS with EPL and more study in ethically different population is needed to confirm its association with EPL.

Methionine Synthase Reductase-A66G and -C524T Single Nucleotide Polymorphisms and Prostate Cancer: A Case-Control Trial

Purpose:Some variations in the sequence of methionine synthase reductase (MTRR) gene can increase the risk of various cancers such as prostate cancer. The aim of this study was to investigate the association between prostate cancer and the MTRR A66G and C524T gene single nucleotide polymorphisms (SNPs) using an in silico analysis.Methods:In this case-control study, 218 Iranian men, including 108 men with prostate cancer and 110 prostate cancer-free men, were enrolled. The MTRR A66G and C524T genotyping was performed by PCR-RFLP. Some of the bioinformatics tools were employed for the evaluation of polymorphism on the molecular aspects of the MTRR.Results:With regard to the MTRR A66G polymorphism, the genotype AG (OR: 0.85, 95% CI: 0.47-1.54, p= 0.6014), genotype GG (OR: 0.89, 95% CI: 0.42-1.87, p= 0.7512), and allele G (OR: 0.92, 95% CI: 0.63-1.35, p= 0.6686) were not associated with prostate cancer risk. However, the data for C524T SNP showed that the genotype CT was associated with prostate cancer risk (OR: 1.92, 95% CI: 1.06-3.47, p= 0.0308). Further, carriers of the allele T (OR: 1.80, 95% CI: 1.04-3.13, p= 0.0358) were associated with high risk of prostate cancer. In addition, bioinformatics analysis revealed that C524T SNP could affect some molecular aspects of the protein structure, while having no effect on the mRNA structure.Conclusion:The MTRR C524T is a genetic risk factor for prostate cancer; however, the MTRR A66G is not suggested as a suitable biomarker for prostate cancer. To obtain more reliable results, further studies are recommended to use larger sample sizes and investigate the effects of environmental factors.

Folic acid and cyanocobalamin effects on the expression of genes involved in DNA methylation of the oyster Crassostrea gigas

The microalga Chaetoceros calcitrans was cultured with three concentrations of vitamin B12 (cyanocobalamin) to supplement the feed for the oyster Crassostrea gigas. In two additional assays, vitamins B12 and B9 (folic acid) were dissolved in the water to increase the methyl donors necessary for the one‐carbon cycle and DNA methylation. Microalgae did not present differences in their concentration of vitamins, and no correlation was found between the concentration of vitamin supplement and the vitamin concentration in oysters; however, changes in the expression of genes involved in the DNA methylation were found. The expression of proton‐coupled folate transporter (PCFT) was lower in oysters supplemented with Vit. B9. The expression of proton‐coupled folate transporter (PCFT) was lower in oysters supplemented with Vit. B9., however, the expression of genes implicated in the release of the methyl used to maintain methylation such as methionine synthase (MS) and DNMT1 expression was high, suggesting faster transportation of Vit. B9. The methylenetetrahydrofolate reductase (MTHFR), implicated in the reduction of folic acid, was lower in oysters supplemented with Vit. 9 and B12 dissolved in the water. DNA methyltransferase DNMT1A, involved in DNA maintenance, was higher in oyster supplemented with Vit. B9, but the expression of DNMT1B was not different. In conclusion, the supplementation with folic acid increases the expression of genes implicated in the maintenance of DNA methylation in C. gigas.

807 Homocysteine induces apoptosis of melanocytes via PERK-eif2α-CHOP pathway in vitiligo

Hyperhomocysteinemia and folic acid deficiencies have been reported in patients with vitligo, while the mechanism underlying this process is unclear. This study aims to investigate the effect of homocysteine (Hcy) on vitiligo pathogenesis and the underlying mechanisms. Our results demonstrated that Hcy was highly expressed in the serum of patients with vitiligo and associated with the degree of disease progression. In vitro study confirmed that oxidative stress can promote the accumulation of Hcy in melanocytes. Hcy treatment can inhibit melanocyte growth and induce cell apoptosis in a dose-dependent manner. Hcy treatment can upregulate glucose-regulated protein 78(GRP78), activate endoplasmic reticulum stress and induce the expression of C/EBP homologous protein(CHOP). Knockout the IRE1 pathway has no effect on apoptosis, whereas, the level of apoptosis is reduced by knockout the PERK pathway. We also observed activation of pPERK in peripheral melanocytes of vitiligo patient. Folic acid serves as cofactors of homocysteine methyltransferase of methionine from homocysteine. Folic acid supplementation can reduce intracellular Hcy levels, inhibit the activation of the perk-eif2a-CHOP pathway, reduce cell apoptosis rate and promote melanocyte surivival. Our study also showed that homosysteine disrupts melanogenesis via inhibition of MITF TYR TYRP1 and melanA experssion, supplementation of folic acid can restore above molecular expression and restore melanin synthesis. Our data suggest that an elevated Hcy level may be a precipitating factor for vitiligo in predisposed individuals. In addition, Hcy facilitate melanocyte apoptosis through perk-eif2a-CHOP pathway, and decrease melanin synthesis. Importantly, folic acid supplementation can reduce the Hcy toxicity to melanocyte and restore melanin synthesis.