One-carbon genetic variants and the role of MTHFD1 1958G>A in liver and colon cancer risk according to global DNA methylation

Several polymorphic gene variants within one-carbon metabolism, an essential pathway for nucleotide synthesis and methylation reactions, are related to cancer risk. An aberrant DNA methylation is a common feature in cancer but whether the link between one-carbon metabolism variants and cancer occurs through an altered DNA methylation is yet unclear. Aims of the study were to evaluate the frequency of one-carbon metabolism gene variants in hepatocellular-carcinoma, cholangiocarcinoma and colon cancer, and their relationship to cancer risk together with global DNA methylation status. Genotyping for BHMT 716A>G, DHFR 19bp ins/del, MTHFD1 1958G>A, MTHFR 677C>T, MTR 2756A>G, MTRR 66A>G, RFC1 80G>A, SHMT1 1420C>T, TCII 776C>G and TS 2rpt-3rpt was performed in 102 cancer patients and 363 cancer-free subjects. Methylcytosine (mCyt) content was measured by LC/MS/MS in peripheral blood mononuclear cells (PBMCs) DNA. The MTHFD1 1958AA genotype was significantly less frequent among cancer patients as compared to controls (p = 0.007) and related to 63% reduction of overall cancer risk (p = 0.003) and 75% of colon cancer risk (p = 0.006). When considering PBMCs mCyt content, carriers of the MTHFD1 1958GG genotype showed a lower DNA methylation as compared to carriers of the A allele (p = 0.048). No differences were highlighted by evaluating a possible relationship between the other polymorphisms analyzed with cancer risk and DNA methylation. The MTHFD1 1958AA genotype is linked to a significantly reduced cancer risk. The 1958GG genotype is associated to PBMCs DNA hypomethylation as compared to the A allele carriership that may exert a protective effect for cancer risk by preserving from DNA hypomethylation

DNA Methylation and Hydroxymethylation in Primary Colon Cancer and Synchronous Hepatic Metastasis

Colon cancer is one of the most frequent solid tumor and simultaneous diagnosis of primary colon cancer and liver metastases occurs in about one fourth of cases. The current knowledge on epigenetic signatures, especially those related to hydroxymethylation in primary cancer tissue, synchronous metastasis, and blood circulating cells is lacking. This study aimed to investigate both methylcytosine (mCyt) and hydroxymethylcytosine (hmCyt) status in the DNA of individual patients from colon cancer tissue, synchronous liver metastases, and in cancer-free colon and liver tissues and leukocytes. Patients undergoing curative surgery (n= 16) were enrolled and their laboratory and clinical history data collected. The contents of mCyt and hmCyt were determined by a liquid chromatography/mass spectrometry (LC/MS/MS) method in DNA extracted from primary colon cancer, synchronous hepatic metastatic tissues and homologous cancer-free tissues, i.e., colon and liver tissues as well as leukocytes. The mCyt and hmCyt levels were compared between cancerous and cancer-free tissues, and correlations between leukocytes and colon/liver tissues for both the mCyt and hmCyt levels were evaluated. The mCyt levels were similar in primary colon cancer and liver metastasis tissues (4.69 \ub1 0.37% vs. 4.77 \ub1 0.38%, respectively,p= 0.535), and both primary and metastatic tissues were hypomethylated compared to cancer-free colon (4.98 \ub1 0.26%). The difference in the mCyt content between cancerous and cancer-free colon tissues was significantly lower in primary colon cancer (p= 0.004), but not in liver metastasis (p= 0.148). The hmCyt content was similar in primary colon cancer compared to liver metastasis (0.035%, C.I. 0.024-0.052% versus 0.035%, C.I. 0.021-0.058%, respectively,p =0.905) and markedly depleted compared to the cancer-free colon (0.081%, C.I. 0.055-0.119%) with a statistically significant difference (p< 0.05) for both comparisons. The mCyt levels showed a borderline correlation between leukocytes and colon cancer tissue (Pearson's correlation coefficient = 0.51,p= 0.052) while no correlations were detected for the hmCyt levels. In conclusion, primary colon cancer and synchronous liver metastasis tissues showed a similar epigenetic status but were significantly hypomethylated and hypohydroxymethylated as compared to homologous cancer-free colon tissues

The RFC1 80G>A, among Common One-Carbon Polymorphisms, Relates to Survival Rate According to DNA Global Methylation in Primary Liver Cancers

Polymorphisms within one-carbon metabolism genes have been largely studied in relation to cancer risk for the function of this pathway in nucleotide synthesis and DNA methylation. Aims of this study were to explore the possible link among several common functional gene polymorphisms within one-carbon metabolism and survival rate in primary liver cancers, i.e., hepatocellular carcinoma and cholangiocarcinoma, and to assess the additional effect of global DNA methylation on survival rate and mortality risk. Forty-seven primary liver cancer patients were genotyped for ten polymorphisms: DHFR 19bp ins/del, TS 2rpt-3rpt, MTHFD1 1958G>A, MTHFR 677C>T, MTR 2756A>G, MTRR 66A>G, RFC1 80G>A, SHMT1 1420C>T, BHMT 716 A>G, TC II 776C>G. Methylation was determined in peripheral blood mononuclear cells (PBMCs) DNA as methylcytosine (mCyt) content using LC/MS/MS. Among the polymorphisms analysed, the RFC1 80G>A (rs1051266) influenced the survival rate in primary liver cancers. The RFC1 80AA was associated to a significantly reduced survival rate (22.2%) as compared to both GG and GA genotypes (61.5% and 76% respectively, p = 0.005). When the cancer patients were stratified according to the mCyt median value as high (>5.34%) or low ( 645.34%), the concomitant presence of AA genotype and low mCyt level led to a significantly worse survival rate as compared to the G allele carriership (pA polymorphism influenced the survival rate, and the presence of RFC1 80AA genotype with low global methylation in PBMCs DNA was associated with poorer prognosis and higher mortality risk, therefore highlighting novel molecular signatures potentially helpful to define prognostic markers for primary liver cancers

Preliminary Evidence for Cell Membrane Amelioration in Children with Cystic Fibrosis by 5-MTHF and Vitamin B12 Supplementation: A Single Arm Trial

Cystic fibrosis (CF) is one of the most common fatal autosomal recessive disorders in the Caucasian population caused by mutations of gene for the cystic fibrosis transmembrane conductance regulator (CFTR). New experimental therapeutic strategies for CF propose a diet supplementation to affect the plasma membrane fluidity and to modulate amplified inflammatory response. The objective of this study was to evaluate the efficacy of 5-methyltetrahydrofolate (5-MTHF) and vitamin B12 supplementation for ameliorating cell plasma membrane features in pediatric patients with cystic fibrosis.A single arm trial was conducted from April 2004 to March 2006 in an Italian CF care centre. 31 children with CF aged from 3 to 8 years old were enrolled. Exclusion criteria were diabetes, chronic infections of the airways and regular antibiotics intake. Children with CF were supplemented for 24 weeks with 5-methyltetrahydrofolate (5-MTHF, 7.5 mg /day) and vitamin B12 (0.5 mg/day). Red blood cells (RBCs) were used to investigate plasma membrane, since RBCs share lipid, protein composition and organization with other cell types. We evaluated RBCs membrane lipid composition, membrane protein oxidative damage, cation content, cation transport pathways, plasma and RBCs folate levels and plasma homocysteine levels at baseline and after 24 weeks of 5-MTHF and vitamin B12 supplementation. In CF children, 5-MTHF and vitamin B12 supplementation (i) increased plasma and RBC folate levels; (ii) decreased plasma homocysteine levels; (iii) modified RBC membrane phospholipid fatty acid composition; (iv) increased RBC K(+) content; (v) reduced RBC membrane oxidative damage and HSP70 membrane association.5-MTHF and vitamin B12 supplementation might ameliorate RBC membrane features of children with CF.ClinicalTrials.gov NCT00730509

High ferritin and low folate increases PBMCs genomic DNA methylation in association with SHMT1-1420TT variant.

Nutrient-gene interactions within one-carbon metabolism modulate DNA methylation, the major potentially reversible epigenetic modification in eukaryotic cells. The cytosolic serine hydroxymethyltransferase (SHMT1) regulates the metabolic balance between nucleotide synthesis and methylation in one-carbon pathway. The SHMT1-1420T allele has been associated with a reduced enzyme activity and a decreased risk of cancer. By enhancing SHMT1 expression, ferritin affects folate-mediated one-carbon metabolism. Aim of this study was to analyze how the interaction among ferritin, folate and SHMT1-1420C>T polymorphism may affect peripheral blood mononuclear cells (PBMCs) DNA methylation (LC/ESI/MS method) in 537 subjects enrolled in the Verona Heart Studyto identify a possible biomarker for cancer. Results showed that SHMT1-TT carriers, under a high ferritin/low folate condition, show significantly increased PBMCs genomic DNA methylation than SHMT1-CC subjects (P=0.01). Since cancer is usually associated with genomic hypomethylation, the increased genomic methylation in SHMT1-1420TT genotypes in presence of high ferritin/low folate, could be potentially protective for cancer risk

11beta-hydroxysteroid dehydrogenase type 2 promoter methylation is increased in adults essential hypertensive patients

Role of promoter methylation in regulating 11beta-hydroxysteroid dehydrogenase type 2 enzyme in essential hypertensive adult patients

CpG methylation of 11betaHydroxysteroid dehydrogenase type 2 promoter is increased in adult essential hypertension.

We reported that 15% of essential hypertensives may suffer from impairment of the enzyme 11beta-hydroxysteroid-dehydrogenase type 2 (11BHSD2). 11BHSD2 activity and expression can be affected by mutations, polymorphisms, and lately, epigenetic modifications. Aim: To evaluate the HSD11B2 promoter methylation in adults and pediatric essential hypertensives (HT). Material and Methods: We recruited 64 patients, grouped in 16 HT and 16 Normotensive (NT) adults; 16 HT and 16 NT pediatrics. We measured serum aldosterone, plasma renin activity (PRA), cortisol (F), cortisone (E) and free urinary cortisol metabolites (THF, aTHF, and THE). PBMC bisulfite\u2013treated DNA was used to perform the methylation-specific PCR (MS-PCR) and calculated the methylation index in HSD11B2 promoter. Results: HT adults have higher methylation index compared with NT adults (0.154\ub10.031 vs. 0.072\ub10.011, p<0.05). HT and NT children have low and similar methylation (0.021\ub10.005 vs. 0.052\ub10.008, p NS). Methylation in HT adults was higher than either HT or NT children (p<0.05), and negatively associated with lower urinary cortisone levels. Conclusions: CpG methylation of HSD11B2 promoter is increased in adult esential hypertensives compared to NT adults and either HT/NT children. A high cortisol/cortisone ratio is in agreement with previously reported low expression of renal HSD11B2. Further studies would support the HSD11B2 methylation index in PBMC as potential molecular biomarker of mineralocorticoid activity and essential hypertension

11beta-hydroxysteroid dehydrogenase type2 promoter methylation is increased in adults essential hypertensive patients

11beta-hydroxysteroid dehydrogenase type2 promoter methylation is increased in adults essential hypertensive patient

Array-based genome-wide DNA methylation profiles and gene expression analyses show novel regulatory pathways in alcohol-related hepatocellular carcinoma

Increasing interest has been given to the epigenetic regulation by DNA methylation in cancer development including hepatocellular carcinoma (HCC). Alcohol is a major risk factor for HCC although the mechanisms underlying the alcohol-related liver carcinogenesis are still incompletely understood. Alcohol is linked both to carcinogenesis and to aberrant DNA methylation by interfering with methyl group transfer within one-carbon metabolism through reactions mainly occurring in the liver. The effort to identify epigenetically-regulated pathways in alcohol-related HCC is therefore of great interest. Aim of the present study was to investigate the genome-wide promoter DNA methylation patterns together with array-based, gene expression profiles of non-viral, alcohol-related HCC. The methylation and gene expression profiles of all annotated genes were assessed in HCC tissue compared to tumor-free tissue, using a genome-wide, array-based approach in liver samples of eight patients undergoing curative surgery. The merging of DNA methylation and gene expression data allowed the detection of 160 hypermethylated-repressed, 31 hypomethylated -induced, 50 hypermethylated-induced and 56 hypomethylated-repressed genes. The analysis of transcriptionally-repressed genes associated with promoter hypermethylation enabled to identify several candidate tumor-suppressor genes, among which also a number of gene belonging to retinol metabolism (ADH1A, ADH1B, ADH6, CYP3A43, CYP4A22 and RDH16), and SHMT1 a key gene of one-carbon metabolism. DNA methylation at promoter site appears to regulate the expression of genes involved in retinol and one-carbon metabolism in alcohol-related HCC