Effect of SlSAHH2 on metabolites in over-expressed and wild-type tomato fruit.

Background: Tomato (Solanum lycopersicum) is an annual or perennial herb that occupies an important position in daily agricultural production. It is an essential food crop for humans and its ripening process is regulated by a number of genes. S-adenosyl-l-homocysteine hydrolase (AdoHcyase, EC 3.3.1.1) is widespread in organisms and plays an important role in regulating biological methylation reactions. Previous studies have revealed that transgenic tomato that over-express SlSAHH2 ripen earlier than the wild-type (WT). However, the differences in metabolites and the mechanisms driving how these differences affect the ripening cycle are unclear. Objective: To investigate the effects of SlSAHH2 on metabolites in over-expressed tomato and WT tomato. Methods: SlSAHH2 over-expressed tomato fruit (OE-5# and OE-6#) and WT tomato fruit at the breaker stage (Br) were selected for non-targeted metabolome analysis. Results: A total of 733 metabolites were identified by mass spectrometry using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and the Human Metabolome database (HMDB). The metabolites were divided into 12 categories based on the superclass results and a comparison with the HMDB. The differences between the two databases were analyzed by PLS-DA. Based on a variable important in projection value >1 and P < 0.05, 103 differential metabolites were found between tomato variety OE-5# and WT and 63 differential metabolites were found between OE-6# and WT. These included dehydrotomatine, L-serine, and gallic acid amongst others. Many metabolites are associated with fruit ripening and eight common metabolites were found between the OE-5# vs. WT and OE-6# vs. WT comparison groups. The low L-tryptophan expression in OE-5# and OE-6# is consistent with previous reports that its content decreases with fruit ripening. A KEGG pathway enrichment analysis of the significantly different metabolites revealed that in the OE-5# and WT groups, up-regulated metabolites were enriched in 23 metabolic pathways and down-regulated metabolites were enriched in 11 metabolic pathways. In the OE-6# and WT groups, up-regulated metabolites were enriched in 29 pathways and down-regulated metabolites were enriched in six metabolic pathways. In addition, the differential metabolite changes in the L-serine to flavonoid transformation metabolic pathway also provide evidence that there is a phenotypic explanation for the changes in transgenic tomato. Discussion: The metabolomic mechanism controlling SlSAHH2 promotion of tomato fruit ripening has been further elucidated.

Adenosylhomocysteinase plays multiple roles in maintaining the identity and pluripotency of mouse embryonic stem cells†.

Adenosylhomocysteinase (AHCY), a key enzyme in the methionine cycle, is essential for the development of embryos and the maintenance of mouse embryonic stem cells (mESCs). However, the precise underlying mechanism of Ahcy in regulating pluripotency remains unclear. As the only enzyme that can hydrolyze S-adenosylhomocysteine in mammals, AHCY plays a critical role in the metabolic homeostasis, epigenetic remodeling, and transcriptional regulation. Here, we identified Ahcy as a direct target of OCT4 and unveiled that AHCY regulates the self-renewal and differentiation potency of mESCs through multiple mechanisms. Our study demonstrated that AHCY is required for the metabolic homeostasis of mESCs. We revealed the dual role of Ahcy in both transcriptional activation and inhibition, which is accomplished via the maintenance of H3K4me3 and H3K27me3, respectively. We found that Ahcy is required for H3K4me3-dependent transcriptional activation in mESCs. We also demonstrated that AHCY interacts with polycomb repressive complex 2 (PRC2), thereby maintaining the pluripotency of mESCs by sustaining the H3K27me3-regulated transcriptional repression of related genes. These results reveal a previously unrecognized OCT4-AHCY-PRC2 axis in the regulation of mESCs' pluripotency and provide insights into the interplay between transcriptional factors, cellular metabolism, chromatin dynamics and pluripotency regulation.

Inhibition of AHCY impedes proliferation and differentiation of mouse and human adipocyte progenitor cells.

S-adenosyl-homocysteine-hydrolase (AHCY) plays an important role in the methionine cycle regulating cellular methylation levels. AHCY has been reported to influence proliferation and differentiation processes in different cell types, e.g. in cancer cells and mouse embryonic stem cells. In the development of adipose tissue, both the proliferation and differentiation of adipocyte progenitor cells (APCs) are important processes, which in the context of obesity are often dysregulated. To assess whether AHCY might also be involved in cell proliferation and differentiation of APCs, we investigated the effect of reduced AHCY activity on human and mouse APCs in vitro. We show that the inhibition of AHCY using adenosine dialdehyde (AdOx) and the knockdown of AHCY using gene-specific siRNAs reduced APC proliferation and number. Inhibition of AHCY further reduced APC differentiation into mature adipocytes and the expression of adipogenic differentiation markers. Global DNA methylation profiling in human APCs revealed that inhibition of AHCY is associated with alterations in CpG methylation levels of genes involved in fat cell differentiation and pathways related to cellular growth. Our findings suggest that AHCY is necessary for the maintenance of APC proliferation and differentiation and inhibition of AHCY alters DNA methylation processes leading to a dysregulation of the expression of genes involved in the regulation of these processes.

Trypanosoma brucei proliferates normally even after losing all S-adenosylhomocysteine hydrolase genes.

S-adenosylhomocysteine (SAH) hydrolase is the enzyme responsible for breaking down SAH into adenosine and homocysteine. It has long been believed that a deficiency of this enzyme leads to SAH accumulation, subsequently inhibiting methyltransferases responsible for nucleic acids and proteins, which severely affects cell proliferation. To investigate whether targeting this enzyme could be a viable strategy to combat Trypanosoma brucei, the causative agent of human African trypanosomiasis, we created a null mutant of the SAH hydrolase gene in T. brucei using the Cre/loxP system and conducted a phenotype analysis. Surprisingly, the null mutant, where all five SAH hydrolase gene loci were deleted, exhibited normal proliferation despite the observed SAH accumulation. These findings suggest that inhibiting SAH hydrolase may not be an effective approach to suppressing T. brucei proliferation, making the enzyme a less promising target for antitrypanosome drug development.

Effects of S-Adenosylhomocysteine Hydrolase Downregulation on Wnt Signaling Pathway in SW480 Cells.

S-adenosylhomocysteine hydrolase (AHCY) deficiency results mainly in hypermethioninemia, developmental delay, and is potentially fatal. In order to shed new light on molecular aspects of AHCY deficiency, in particular any changes at transcriptome level, we enabled knockdown of AHCY expression in the colon cancer cell line SW480 to simulate the environment occurring in AHCY deficient individuals. The SW480 cell line is well known for elevated AHCY expression, and thereby represents a suitable model system, in particular as AHCY expression is regulated by MYC, which, on the other hand, is involved in Wnt signaling and the regulation of Wnt-related genes, such as the ß-catenin co-transcription factor LEF1 (lymphoid enhancer-binding factor 1). We selected LEF1 as a potential target to investigate its association with S-adenosylhomocysteine hydrolase deficiency. This decision was prompted by our analysis of RNA-Seq data, which revealed significant changes in the expression of genes related to the Wnt signaling pathway and genes involved in processes responsible for epithelial-mesenchymal transition (EMT) and cell proliferation. Notably, LEF1 emerged as a common factor in these processes, showing increased expression both on mRNA and protein levels. Additionally, we show alterations in interconnected signaling pathways linked to LEF1, causing gene expression changes with broad effects on cell cycle regulation, tumor microenvironment, and implications to cell invasion and metastasis. In summary, we provide a new link between AHCY deficiency and LEF1 serving as a mediator of changes to the Wnt signaling pathway, thereby indicating potential connections of AHCY expression and cancer cell phenotype, as Wnt signaling is frequently associated with cancer development, including colorectal cancer (CRC).

Metabolic profiling stratifies colorectal cancer and reveals adenosylhomocysteinase as a therapeutic target.

The genomic landscape of colorectal cancer (CRC) is shaped by inactivating mutations in tumour suppressors such as APC, and oncogenic mutations such as mutant KRAS. Here we used genetically engineered mouse models, and multimodal mass spectrometry-based metabolomics to study the impact of common genetic drivers of CRC on the metabolic landscape of the intestine. We show that untargeted metabolic profiling can be applied to stratify intestinal tissues according to underlying genetic alterations, and use mass spectrometry imaging to identify tumour, stromal and normal adjacent tissues. By identifying ions that drive variation between normal and transformed tissues, we found dysregulation of the methionine cycle to be a hallmark of APC-deficient CRC. Loss of Apc in the mouse intestine was found to be sufficient to drive expression of one of its enzymes, adenosylhomocysteinase (AHCY), which was also found to be transcriptionally upregulated in human CRC. Targeting of AHCY function impaired growth of APC-deficient organoids in vitro, and prevented the characteristic hyperproliferative/crypt progenitor phenotype driven by acute deletion of Apc in vivo, even in the context of mutant Kras. Finally, pharmacological inhibition of AHCY reduced intestinal tumour burden in ApcMin/+ mice indicating its potential as a metabolic drug target in CRC.

Epigenetic modulation of Drp1-mediated mitochondrial fission by inhibition of S-adenosylhomocysteine hydrolase promotes vascular senescence and atherosclerosis.

AIMS: Vascular senescence, which is closely related to epigenetic regulation, is an early pathological condition in cardiovascular diseases including atherosclerosis. Inhibition of S-adenosylhomocysteine hydrolase (SAHH) and the consequent increase of S-adenosylhomocysteine (SAH), a potent inhibitor of DNA methyltransferase, has been associated with an elevated risk of cardiovascular diseases. This study aimed to investigate whether the inhibition of SAHH accelerates vascular senescence and the development of atherosclerosis. METHODS AND RESULTS: The case-control study related to vascular aging showed that increased levels of plasma SAH were positively associated with the risk of vascular aging, with an odds ratio (OR) of 3.90 (95% CI, 1.17-13.02). Elevated pulse wave velocity, impaired endothelium-dependent relaxation response, and increased senescence-associated ß-galactosidase staining were observed in the artery of SAHH+/- mice at 32 weeks of age. Additionally, elevated expression of p16, p21, and p53, fission morphology of mitochondria, and over-upregulated expression of Drp1 were observed in vascular endothelial cells with SAHH inhibition in vitro and in vivo. Further downregulation of Drp1 using siRNA or its specific inhibitor, mdivi-1, restored the abnormal mitochondrial morphology and rescued the phenotypes of vascular senescence. Furthermore, inhibition of SAHH in APOE-/- mice promoted vascular senescence and atherosclerosis progression, which was attenuated by mdivi-1 treatment. Mechanistically, hypomethylation over the promoter region of DRP1 and downregulation of DNMT1 were demonstrated with SAHH inhibition in HUVECs. CONCLUSIONS: SAHH inhibition epigenetically upregulates Drp1 expression through repressing DNA methylation in endothelial cells, leading to vascular senescence and atherosclerosis. These results identify SAHH or SAH as a potential therapeutic target for vascular senescence and cardiovascular diseases.

Inhibition of S-adenosylhomocysteine hydrolase induces endothelial senescence via hTERT downregulation.

BACKGROUND AND AIMS: It has been established that endothelial senescence plays a critical role in the development of atherosclerosis. Elevated S-adenosylhomocysteine (SAH) level induced by inhibition of S-adenosylhomocysteine hydrolase (SAHH) is one of the risk factors of atherosclerosis; however, the interplay between endothelial senescence and inhibition of SAHH is largely unknown. METHODS: Human umbilical vein endothelial cells (HUVECs) after serial passage were used. SAHH-specific inhibitor adenosine dialdehyde (ADA) and SAHH siRNA treated HUVECs and SAHH+/-mice were used to investigate the effect of SAHH inhibition on endothelial senescence. RESULTS: HUVECs exhibited distinct senescence morphology as HUVECs were passaged, together with a decrease in intracellular SAHH expression and an increase in intracellular SAH levels. SAHH inhibition by ADA or SAHH siRNA elevated SA ß-gal activity, arrested proliferation, and increased the expression of p16, p21 and p53 in HUVECs and the aortas of mice. In addition, decreased expression of hTERT and reduced occupancy of H3K4me3 over the hTERT promoter region were observed following SAHH inhibition treatment. To further verify the role of hTERT in the endothelial senescence induced by SAHH inhibition, hTERT was overexpressed with a plasmid vector under CMV promoter. hTERT overexpression rescued the senescence phenotypes in endothelial cells induced by SAHH inhibition. CONCLUSIONS: SAHH inhibition induces endothelial senescence via downregulation of hTERT expression, which is associated with attenuated histone methylation over the hTERT promoter region.

Synaptic plasticity in hippocampal CA1 neurons of mice lacking inositol-1,4,5-trisphosphate receptor-binding protein released with IP3 (IRBIT).

In hippocampal CA1 neurons of wild-type mice, a short tetanus (15 or 20 pulses at 100 Hz) or a standard tetanus (100 pulses at 100 Hz) to a naive input pathway induces long-term potentiation (LTP) of the responses. Low-frequency stimulation (LFS; 1000 pulses at 1 Hz) 60 min after the standard tetanus reverses LTP (depotentiation [DP]), while LFS applied 60 min prior to the standard tetanus suppresses LTP induction (LTP suppression). We investigated LTP, DP, and LTP suppression of both field excitatory postsynaptic potentials and population spikes in CA1 neurons of mice lacking the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)-binding protein released with IP3 (IRBIT). The mean magnitudes of LTP induced by short and standard tetanus were not different in mutant and wild-type mice. In contrast, DP and LTP suppression were attenuated in mutant mice, whereby the mean magnitude of responses after LFS or tetanus were significantly greater than in wild-type mice. These results suggest that, in hippocampal CA1 neurons, IRBIT is involved in DP and LTP suppression, but is not essential for LTP. The attenuation of DP and LTP suppression in mice lacking IRBIT indicates that this protein, released during or after priming stimulations, determines the direction of LTP expression after the delivery of subsequent stimulations.

Betaine Supplementation Attenuates S-Adenosylhomocysteine Hydrolase-Deficiency-Accelerated Atherosclerosis in Apolipoprotein E-Deficient Mice.

S-adenosylhomocysteine (SAH) is a risk factor of cardiovascular diseases and atherosclerosis. However, the causal association between SAH and atherosclerosis is still uncertain. In the present study, heterozygous SAH hydrolase (SAHH+/-) knockout mice were bred with apolipoprotein E-deficient mice to produce ApoE-/-/SAHH+/- mice. At 8 weeks of age, these mice were fed on AIN-93G diets added with or without betaine (4 g betaine/100 g diet) for 8 weeks. Compared with ApoE-/-/SAHHWT mice, SAHH deficiency caused an accumulation of plasma SAH concentration and a decrease in S-adenosylmethionine (SAM)/SAH ratio as well as plasma homocysteine levels. Betaine supplementation lowered SAH levels and increased SAM/SAH ratio and homocysteine levels in ApoE-/-/SAHH+/- mice. Furthermore, SAHH deficiency promoted the development of atherosclerosis, which was reduced by betaine supplementation. The atheroprotective effects of betaine on SAHH-deficiency-promoted atherosclerosis were associated with inhibition of NFκB inflammation signaling pathway and inhibition of proliferation and migration of smooth muscle cells. In conclusion, our results suggest that betaine supplementation lowered plasma SAH levels and protected against SAHH-deficiency-promoted atherosclerosis through repressing inflammation and proliferation and migration of smooth muscle cells.

Genomic and transcriptomic profiling of hepatoid adenocarcinoma of the stomach.

Hepatoid adenocarcinoma of the stomach (HAS), a rare subtype of gastric cancer (GC), has a low incidence but a high mortality rate. Little is known about the molecular features of HAS. Here we applied whole-exome sequencing (WES) on 58 tumours and the matched normal controls from 54 HAS patients, transcriptome sequencing on 30 HAS tumours, and single-cell RNA sequencing (scRNA-seq) on one HAS tumour. Our results reveal that the adenocarcinomatous component and hepatocellular-like component of the same HAS tumour originate monoclonally, and HAS is likely to initiate from pluripotent precursor cells. HAS has high stemness and high methionine cycle activity compared to classical GC. Two genes in the methionine cycle, MAT2A, and AHCY are potential targets for HAS treatments. We provide the first integrative genomic profiles of HAS, which may facilitate its diagnosis, prognosis, and treatment.

Interaction analysis of gene variants related to one-carbon metabolism with chronic hepatitis B infection in Chinese patients.

BACKGROUND: The risk of chronic hepatitis B (CHB) infection is influenced by aberrant DNA methylation and altered nucleotide synthesis and repair, possibly caused by polymorphic variants in one-carbon metabolism genes. In the present study, we investigated the relationship between polymorphisms belonging to the one-carbon metabolic pathway and CHB infection. METHODS: A case-control study using 230 CHB patients and 234 unrelated healthy controls was carried out to assess the genetic association of 24 single nucleotide polymorphisins (SNPs) determined by mass spectrometry. RESULTS: Three SNPs, comprising rs10717122 and rs2229717 in serine hydroxymethyltransferase1/2 (SHMT2) and rs585800 in betaine-homocysteine S-methyltransferase (BHMT), were associated with the risk of CHB. Patients with DEL allele, DEL.DEL and DEL.T genotypes of rs10717122 had a 1.40-, 2.00- and 1.83-fold increased risk for CHB, respectively. Cases inheriting TA genotype of rs585800 had a 2.19-fold risk for CHB infection. The T allele of rs2229717 was less represented in the CHB cases (odds ratio = 0.66, 95% confidence interval = 0.48-0.92). The T allele of rs2229717 was less in patients with a low hepatitis B virus-DNA level compared to the control group (odds ratio = 0.49, 95% confidence interval = 0.25-0.97) and TT genotype of rs2229717 had a significant correlation with hepatitis B surface antigen level (p = 0.0195). Further gene-gene interaction analysis showed that subjects carrying the rs10717122 DEL.DEL/DEL.T and rs585800 TT/TA genotypes had a 2.74-fold increased risk of CHB. CONCLUSIONS: The results of the present study suggest that rs10717122, rs585800 and rs2229717 and gene-gene interactions of rs10717122 and rs585800 affect the outcome of CHB infection, at the same time as indicating their usefulness as a predictive and diagnostic biomarker of CHB infection.

Coordinated expression of Jumonji and AHCY under OCT transcription factor control to regulate gene methylation in wood frogs during anoxia.

Wood frogs (Rana sylvatica) can survive extended periods of whole body freezing. Freezing imparts multiple stresses on cells that include anoxia and dehydration, but these can also be experienced as independent stresses. Under anoxia stress, energy metabolism is suppressed, and pro-survival pathways are prioritized to differentially regulate some transcription factors including OCT1 and OCT4. Jumonji C domain proteins (JMJD1A and JMJD2C) are hypoxia responsive demethylases whose expression is accelerated by OCT1 and OCT4 which act to demethylate genes related to the methionine cycle. The responses by these factors to 24 h anoxia exposure and 4 h aerobic recovery was analyzed in liver and skeletal muscle of wood frogs to assess their involvement in metabolic adaptation to oxygen limitation. Immunoblot results showed a decrease in JMJD1A levels under anoxia in liver and muscle, but an increase was observed in JMJD2C demethylase protein in anoxic skeletal muscle. Protein levels of adenosylhomocysteinase (AHCY) and methionine adenosyl transferase (MAT), enzymes of the methionine cycle, also showed an increase in the reoxygenated liver, whereas the levels decreased in muscle. A transcription factor ELISA showed a decrease in DNA binding by OCT1 in the reoxygenated liver and anoxic skeletal muscle, and transcript levels also showed tissue specific gene expression. The present study provides the first analysis of the role of the OCT1 transcription factor, associated proteins, and lysine demethylases in mediating responses to anoxia by wood frog tissues.

The Co-mutational Spectrum Determines the Therapeutic Response in Murine FGFR2 Fusion-Driven Cholangiocarcinoma.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer and a highly lethal malignancy. Chemotherapeutic options are limited, but a considerable subset of patients harbors genetic lesions for which targeted agents exist. Fibroblast growth factor receptor 2 (FGFR2) fusions belong to the most frequent and therapeutically relevant alterations in ICC, and the first FGFR inhibitor was recently approved for the treatment of patients with progressed, fusion-positive ICC. Response rates of up to 35% indicate that FGFR-targeted therapies are beneficial in many but not all patients. Thus far, no established biomarkers exist that predict resistance or response to FGFR-targeted therapies in patients with ICC. APPROACH AND RESULTS: In this study, we use an autochthonous murine model of ICC to demonstrate that FGFR2 fusions are potent drivers of malignant transformation. Furthermore, we provide preclinical evidence that the co-mutational spectrum acts not only as an accelerator of tumor development, but also modifies the response to targeted FGFR inhibitors. Using pharmacologic approaches and RNA-interference technology, we delineate that Kirsten rat sarcoma oncogene (KRAS)-activated mitogen-activated protein kinase signaling causes primary resistance to FGFR inhibitors in FGFR2 fusion-positive ICC. The translational relevance is supported by the observation that a subset of human FGFR2 fusion patients exhibits transcriptome profiles reminiscent of KRAS mutant ICC. Moreover, we demonstrate that combination therapy has the potential to overcome primary resistance and to sensitize tumors to FGFR inhibition. CONCLUSIONS: Our work highlights the importance of the co-mutational spectrum as a significant modifier of response in tumors that harbor potent oncogenic drivers. A better understanding of the genetic underpinnings of resistance will be pivotal to improve biomarker-guided patient selection and to design clinically relevant combination strategies.

Novel candidate factors predicting the effect of S-1 adjuvant chemotherapy of pancreatic cancer.

The collagen gel droplet-embedded drug sensitivity test (CD-DST) was revealed to be useful for predicting the effect of S-1 adjuvant chemotherapy for pancreatic ductal adenocarcinoma (PDAC). However, collection of an adequate number of PDAC cells is difficult due to the surrounding fibroblasts. Thus, the aim of this study was to discover novel biomarkers to predict chemosensitivity based on the CD-DST results. Proteomics analysis was performed using liquid chromatography tandem mass spectrometry (LC-MS/MS). Candidate proteins were validated in patients with 5-FU CD-DST results via immunohistochemistry (IHC). The relationships between the candidate proteins and the effect of the adjuvant S-1 were investigated via IHC. Among the 2696 proteins extracted by LC-MS/MS, C1TC and SAHH could accurately predict the CD-DST results. Recurrence-free survival (RFS) was significantly improved in the IHC-positive group compared with the IHC-negative group in both factors. The negative group did not show a significant difference from the group that did not receive S-1. The double-positive group was associated with significantly prolonged RFS compared to the no adjuvant chemotherapy group. C1TC and SAHH have been shown to be useful biomarkers for predicting 5-FU sensitivity as a substitute for the CD-DST in adjuvant chemotherapy for PDAC.

Derangement of hepatic polyamine, folate, and methionine cycle metabolism in cystathionine beta-synthase-deficient homocystinuria in the presence and absence of treatment: Possible implications for pathogenesis.

Cystathionine beta-synthase deficient homocystinuria (HCU) is a life-threatening disorder of sulfur metabolism. Our knowledge of the metabolic changes induced in HCU are based almost exclusively on data derived from plasma. In the present study, we present a comprehensive analysis on the effects of HCU upon the hepatic metabolites and enzyme expression levels of the methionine-folate cycles in a mouse model of HCU. HCU induced a 10-fold increase in hepatic total homocysteine and in contrast to plasma, this metabolite was only lowered by approximately 20% by betaine treatment indicating that this toxic metabolite remains unacceptably elevated. Hepatic methionine, S-adenosylmethionine, S-adenosylhomocysteine, N-acetlymethionine, N-formylmethionine, methionine sulfoxide, S-methylcysteine, serine, N-acetylserine, taurocyamine and N-acetyltaurine levels were also significantly increased by HCU while cysteine, N-acetylcysteine and hypotaurine were all significantly decreased. In terms of polyamine metabolism, HCU significantly decreased spermine and spermidine levels while increasing 5'-methylthioadenosine. Betaine treatment restored normal spermine and spermidine levels but further increased 5'-methylthioadenosine. HCU induced a 2-fold induction in expression of both S-adenosylhomocysteine hydrolase and methylenetetrahydrofolate reductase. Induction of this latter enzyme was accompanied by a 10-fold accumulation of its product, 5-methyl-tetrahydrofolate, with the potential to significantly perturb one­carbon metabolism. Expression of the cytoplasmic isoform of serine hydroxymethyltransferase was unaffected by HCU but the mitochondrial isoform was repressed indicating differential regulation of one­carbon metabolism in different sub-cellular compartments. All HCU-induced changes in enzyme expression were completely reversed by either betaine or taurine treatment. Collectively, our data show significant alterations of polyamine, folate and methionine cycle metabolism in HCU hepatic tissues that in some cases, differ significantly from those observed in plasma, and have the potential to contribute to multiple aspects of pathogenesis.

Genetic variants in S-adenosyl-methionine synthesis pathway and nonsyndromic cleft lip with or without cleft palate in Chile.

BACKGROUND: The S-adenosyl-methionine (SAM) availability is crucial for DNA methylation, an epigenetic mechanism involved in nonsyndromic cleft lip with or without cleft palate (NSCL/P) expression. The aim of this study was to assess the association between single-nucleotide polymorphisms (SNPs) of genes involved in SAM synthesis and NSCL/P in a Chilean population. METHODS: In 234 cases and 309 controls, 18 SNPs in AHCY, MTR, MTRR, and MAT2A were genotyped, and the association between them and the phenotype was evaluated based on additive (allele), dominant, recessive and haplotype models, by odds ratio (OR) computing. RESULTS: Three deep intronic SNPs of MTR showed a protective effect on NSCL/P expression: rs10925239 (OR 0.68; p = 0.0032; q = 0.0192), rs10925254 (OR 0.66; p = 0.0018; q = 0.0162), and rs3768142 (OR 0.66; p = 0.0015; q = 0.0162). Annotations in expression database demonstrate that the protective allele of the three SNPs is associated with a reduction of MTR expression summed to the prediction by bioinformatic tools of its potentiality to modify splicing sites. CONCLUSIONS: The protective effect against NSCL/P of these intronic MTR SNPs seems to be related to a decrease in MTR enzyme expression, modulating the SAM availability for proper substrate methylation. However, functional analyses are necessary to confirm our findings. IMPACT: SAM synthesis pathway genetic variants are factors associated to NSCL/P. This article adds new evidence for folate related genes in NSCL/P in Chile. Its impact is to contribute with potential new markers for genetic counseling.

A comprehensive association analysis between homocysteine metabolic pathway gene methylation and ischemic stroke in a Chinese hypertensive population.

BACKGROUND: Ischemic stroke (IS) is a serious global health burden. In order to improve our understanding of the risk factors associated with IS, we investigated the combined effect of the methylation of five genes related to the metabolism of homocysteine on developing IS. METHODS: Quantitative methylation-specific PCR was used to measure the levels of promoter methylation in hypertensive and stroke patients. The cutoff value calculated by the maximum Youden index was used to classify the levels of gene methylation as hypomethylation and hypermethylation. Logistic regression was used to explore the relationship between gene methylation and IS. RESULTS: The methylation levels of the genes encoding methylenetetrahydrofolate dehydrogenase 1 [MTHFD1], cystathionine ß-synthase [CBS], and dihydrofolate reductase [DHFR] in hypertensive patients were higher than those in stroke patients (all p < 0.01). MTHFD1 hypermethylation, CBS hypermethylation, and DHFR hypermethylation were protective factors for stroke after adjustment for confounding factors. Compared with individuals carrying none of the biomarkers, the ORs [95% CIs] for stroke of those with 1 and 2 elevated biomarkers were 4.068 [1.670-9.913] and 15.345 [6.198-37.994] after adjustment for confounding factors. The participants with a larger number of biomarkers had an increased risk of stroke (p for trend <0.001). For the combination biomarkers, the area under the curve of the receiver operating characteristic was 0.716. CONCLUSION: A significant linear relationship between the number of elevated biomarkers and the risk of stroke has been observed, suggesting that elevations of these biomarkers could be used for potentially predicting the disease.

S-adenosyl-l-homocysteine hydrolase links methionine metabolism to the circadian clock and chromatin remodeling.

Circadian gene expression driven by transcription activators CLOCK and BMAL1 is intimately associated with dynamic chromatin remodeling. However, how cellular metabolism directs circadian chromatin remodeling is virtually unexplored. We report that the S-adenosylhomocysteine (SAH) hydrolyzing enzyme adenosylhomocysteinase (AHCY) cyclically associates to CLOCK-BMAL1 at chromatin sites and promotes circadian transcriptional activity. SAH is a potent feedback inhibitor of S-adenosylmethionine (SAM)-dependent methyltransferases, and timely hydrolysis of SAH by AHCY is critical to sustain methylation reactions. We show that AHCY is essential for cyclic H3K4 trimethylation, genome-wide recruitment of BMAL1 to chromatin, and subsequent circadian transcription. Depletion or targeted pharmacological inhibition of AHCY in mammalian cells markedly decreases the amplitude of circadian gene expression. In mice, pharmacological inhibition of AHCY in the hypothalamus alters circadian locomotor activity and rhythmic transcription within the suprachiasmatic nucleus. These results reveal a previously unappreciated connection between cellular metabolism, chromatin dynamics, and circadian regulation.

The values of AHCY and CBS promoter methylation on the diagnosis of cerebral infarction in Chinese Han population.

BACKGROUND: The goal of our study is to investigate whether the methylation levels of AHCY and CBS promoters are related to the risk of cerebral infarction by detecting the methylation level of AHCY and CBS genes. METHODS: We extracted peripheral venous blood from 152 patients with cerebral infarction and 152 gender- and age-matched healthy controls, and determined methylation levels of AHCY and CBS promoters using quantitative methylation-specific polymerase chain reaction. We used the percentage of methylation reference (PMR) to indicate gene methylation level. RESULTS: We compared the promoter methylation levels of two genes (AHCY and CBS) in peripheral blood DNA between the cerebral infarction case group and the control group. Our study showed no significant difference in AHCY promoter methylation between case and control. Subgroup analysis by gender showed that the methylation level of AHCY in males in the case group was lower than that in the control group, but the difference was not statistically significant in females. In a subgroup analysis by age, there was no significant difference in the AHCY methylation level between the case and control in the young group (≤44 years old). However, the level of AHCY gene methylation in the middle-aged group (45-59 years old) was significantly higher and the aged group (≥60 years old) was significantly lower than that in the control groups. However, CBS promoter methylation levels were significantly lower in the case group than in the control group (median PMR: 70.20% vs 104.10%, P = 3.71E-10). In addition, the CBS methylation levels of males and females in the case group were significantly lower than those in the control group (male: 64.33% vs 105%, P = 2.667E-08; female: 78.05% vs 102.8%, P = 0.003). We also found that the CBS levels in the young (23-44), middle-aged (45-59), and older (60-90) groups were significantly lower than those in the control group (young group: 69.97% vs 114.71%; P = 0.015; middle-aged group: 56.04% vs 91.71%; P = 6.744E-06; older group: 81.6% vs 119.35%; P = 2.644E-04). Our ROC curve analysis of CBS hypomethylation showed an area under the curve of 0.713, a sensitivity of 67.4%, and a specificity of 74.0%. CONCLUSION: Our study suggests that hypomethylation of the CBS promoter may be closely related to the risk of cerebral infarction and may be used as a non-invasive diagnostic biomarker for cerebral infarction.