Tissue factor pathway-related biomarkers in liver cancer: activated factor VII-antithrombin complex and tissue factor mRNA levels are associated with mortality.

Background: Tissue factor (TF), the main initiator of the coagulation cascade, plays a role in cancer progression and prognosis. Activated factor VII-antithrombin complex (FVIIa-AT) is considered an indirect marker of TF exposure by reflecting TF-FVIIa interaction. Objectives: To assess the link between FVIIa-AT plasma levels, TF messenger RNA (mRNA) expression, and survival in cancer. Methods: TF pathway-related coagulation biomarkers were assessed in 136 patients with cancer (52 with hepatocellular carcinoma, 41 with cholangiocarcinoma, and 43 with colon cancer) undergoing surgical intervention with curative intent. TF mRNA expression analysis in neoplastic vs nonneoplastic liver tissues was evaluated in a subgroup of 91 patients with primary liver cancer. Results: FVIIa-AT levels were higher in patients with cancer than in 136 sex- and age-matched cancer-free controls. In patients with cancer, high levels of FVIIa-AT and total TF pathway inhibitor were associated with an increased mortality risk after adjustment for confounders, but only FVIIa-AT remained a predictor of mortality by including both FVIIa-AT and total TF pathway inhibitor in Cox regression (hazard ratio, 2.80; 95% CI, 1.23-6.39; the highest vs the lowest quartile). This association remained significant even after adjustment for extracellular vesicle-associated TF-dependent procoagulant activity. In the subgroup of patients with primary liver cancer, patients with high TF mRNA levels had an increased mortality risk compared with that for those with low TF mRNA levels (hazard ratio, 1.92; 95% CI, 1.03-3.57), and there was a consistent correlation among high FVIIa-AT levels, high TF mRNA levels, and increased risk of mortality. Conclusion: High FVIIa-AT levels may allow the identification of patients with cancer involving high TF expression and predict a higher mortality risk in liver cancer.

Changes of gene expression in peripheral blood mononuclear cells of lung cancer patients with or without anorexia.

BACKGROUND & AIMS: Anorexia is a disabling symptom in cancer and we aimed at investigating the role of changes in gene expression in lung cancer patients presenting with anorexia. METHODS: Genome-wide transcriptomic profiling was assessed in PBMCs RNA from newly diagnosed lung cancer patients and in a control group. RT-qPCR was used for selected genes. RESULTS: RNA-Seq analysis revealed among groups a large number of differentially expressed genes mainly implicated in immune system regulation, oxidative stress and cytokine-mediated inflammation signaling pathways. In particular, we identified a total of 983 DEGs (843 up-regulated; 140 down-regulated) in anorexic cancer compared to controls. A selected number of DEGs including ADAM8, SMAD4, CCR4 and CLU were differentially expressed within cancer group according to the presence/absence of anorexia. In terms of RT-qPCR, ADAM8 was less expressed in cancer patients than controls (p < 0.001), and in anorexic patients vs controls (p = 0.001). The expression of SMAD4 was lower in cancer vs controls (p = 0.005), and in anorexic patients vs controls (p = 0.009). We observed lower CCR4 expression in both anorexic and non-anorexic vs control (p = 0.004, p = 0.011, respectively) and a similar trend was present for CLU. CONCLUSIONS: Our data shed new light on the role of specific genes and their associated molecular pathways as potential key mechanisms for the development of anorexia and may represent a novel landmark for understanding the complex pathophysiology of impaired appetite in cancer.

High Plasma Levels of Activated Factor VII-Antithrombin Complex Point to Increased Tissue Factor Expression in Patients with SARS-CoV-2 Pneumonia: A Potential Link with COVID-19 Prothrombotic Diathesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causal agent of coronavirus disease 2019 (COVID-19), in which coagulation abnormalities and endothelial dysfunction play a key pathogenic role. Tissue factor (TF) expression is triggered by endothelial dysfunction. Activated factor VII-antithrombin (FVIIa-AT) complex reflects indirectly FVIIa-TF interaction and has been proposed as a potential biomarker of prothrombotic diathesis. FVIIa-AT plasma concentration was measured in 40 patients (30 males and 10 females; 64.8 ± 12.3 years) admitted with SARS-CoV-2 pneumonia during the first pandemic wave in Italy. Two sex- and age-matched cohorts without COVID-19, with or without signs of systemic inflammation, were used to compare FVIIa-AT data. The FVIIa-AT plasma levels in COVID-19 patients were higher than those in non-COVID-19 subjects, either with or without inflammation, while no difference was observed among non-COVID-19 subjects. The association between COVID-19 and FVIIa-AT levels remained significant after adjustment for sex, age, C-reactive protein, renal function, fibrinogen, prothrombin time and activated partial thromboplastin time. Our results indicate that SARS-CoV-2 infection, at least during the first pandemic wave, was characterized by high FVIIa-AT levels, which may suggest an enhanced FVIIa-TF interaction in COVID-19, potentially consistent with SARS-CoV-2-induced endotheliopathy.

B vitamin blood concentrations and one-carbon metabolism polymorphisms in a sample of Italian women and men attending a unit of transfusion medicine: a cross-sectional study.

PURPOSE: To define blood status of folate, vitamin B12, vitamin B6, homocysteine, and major one-carbon metabolism-related polymorphisms in healthy, males and females blood donors, aged 18-65 years were evaluated. General characteristics and lifestyle factors were also investigated. METHODS: An explorative cross-sectional study design was used to evaluate a sample of blood donors attending the Unit of Transfusion Medicine of the Verona University Hospital, Italy. From April 2016 to May 2018, 499 subjects were enrolled (255 men, 244 women of whom 155 of childbearing age). Major clinical characteristics including lifestyle and dietary habits, B vitamins and homocysteine were analyzed. The MTHFR 677 C>T, cSHMT 1420 C>T, DHFR 19 bp ins/del, RFC1 80 G>A polymorphisms were also determined. RESULTS: Mean plasma concentrations of folate, vitamin B12, vitamin B6 and homocysteine were 14.2 nmol/L (95% CI 13.7-14.8), 271.9 pmol/L (95% CI 262.6-281.5), 51.0 nmol/L (95% CI 48.7-53.4) and 13.5 µmol/L (95% CI 13.1-14.0), respectively. Plasma folate, was adequate (> 15 nmol/L) in 44.7% of all subjects, 39.0% of males and 42.5% of women < 45 years. Similarly, vitamin B12 was adequate (> 350 pmol/L) in 25.1% of all subjects and in 20.3% of men ≥ 45 years. The rare allele frequencies were 0.21 for MTHFR 677TT, 0.11 for cSHMT 1420TT, 0.18 for DHFR 19 bp del/del, 0.20 for RFC1 80AA, and a gene-nutrient interaction was confirmed for folate concentrations according to MTHFR 677C>T and DHFR 19 bp del/del. CONCLUSION: An Italian sample of healthy blood donors shows that an adequate concentration of plasma folate and vitamin B12 is reached only in a limited percentage of subjects, thus encouraging consideration for specific public health strategies.

The Positive Association between Plasma Myristic Acid and ApoCIII Concentrations in Cardiovascular Disease Patients Is Supported by the Effects of Myristic Acid in HepG2 Cells.

BACKGROUND: In the settings of primary and secondary prevention for coronary artery disease (CAD), a crucial role is played by some key molecules involved in triglyceride (TG) metabolism, such as ApoCIII. Fatty acid (FA) intake is well recognized as a main determinant of plasma lipids, including plasma TG concentration. OBJECTIVES: The aim was to investigate the possible relations between the intakes of different FAs, estimated by their plasma concentrations, and circulating amounts of ApoCIII. METHODS: Plasma samples were obtained from 1370 subjects with or without angiographically demonstrated CAD (mean ± SD age: 60.6 ± 11.0 y; males: 75.8%; BMI: 25.9 ± 4.6 kg/m2; CAD: 73.3%). Plasma lipid, ApoCIII, and FA concentrations were measured. Data were analyzed by regression models adjusted for FAs and other potential confounders, such as sex, age, BMI, diabetes, smoking, and lipid-lowering therapies. The in vitro effects of FAs were tested by incubating HepG2 hepatoma cells with increasing concentrations of selected FAs, and the mRNA and protein contents in the cells were quantified by real-time RT-PCR and LC-MS/MS analyses. RESULTS: Among all the analyzed FAs, myristic acid (14:0) showed the most robust correlations with both TGs (R = 0.441, P = 2.6 × 10-66) and ApoCIII (R = 0.327, P = 1.1 × 10-31). By multiple regression analysis, myristic acid was the best predictor of both plasma TG and ApoCIII variability. Plasma TG and ApoCIII concentrations increased progressively at increasing concentrations of myristic acid, independently of CAD diagnosis and gender. Consistent with these data, in the in vitro experiments, an ∼2-fold increase in the expression levels of the ApoCIII mRNA and protein was observed after incubation with 250 µM myristic acid. A weaker effect (∼30% increase) was observed for palmitic acid, whereas incubation with oleic acid did not affect ApoCIII protein or gene expression. CONCLUSIONS: Plasma myristic acid is associated with increased ApoCIII concentrations in cardiovascular patients. In vitro experiments indicated that myristic acid stimulates ApoCIII expression in HepG2 cells.

Trace Elements Status and Metallothioneins DNA Methylation Influence Human Hepatocellular Carcinoma Survival Rate.

BACKGROUND: Mechanisms underlying hepatocellular carcinoma (HCC) development are largely unknown. The role of trace elements and proteins regulating metal ions homeostasis, i.e. metallothioneins (MTs), recently gained an increased interest. Object of the study was to investigate the role of promoter DNA methylation in MTs transcriptional regulation and the possible prognostic significance of serum trace elements in HCC. METHODS: Forty-nine HCC patients were enrolled and clinically characterized. Cu, Se, and Zn contents were measured by Inductively Coupled Plasma Mass Spectrometry in the serum and, for a subset of 27 patients, in HCC and homologous non-neoplastic liver (N) tissues. MT1G and MT1H gene expression in hepatic tissues was assessed by Real-Time RT-PCR and the specific promoter DNA methylation by Bisulfite-Amplicon Sequencing. RESULTS: Patients with Cu serum concentration above the 80th percentile had a significantly decreased survival rate (P < 0.001) with a marked increased hazard ratio for mortality (HR 6.88 with 95% CI 2.60-18.23, P < 0.001). Se and Zn levels were significantly lower in HCC as compared to N tissues (P < 0.0001). MT1G and MT1H gene expression was significantly down-regulated in HCC as compared to N tissues (P < 0.05). MTs promoter was hypermethylated in 9 out of the 19 HCC tissues showing MTs down-regulation and methylation levels of three specific CpGs paralleled to an increased mortality rate among the 23 patients analyzed (P = 0.015). CONCLUSIONS: MT1G and MT1H act as potential tumor suppressor genes regulated through promoter DNA methylation and, together with serum Cu concentrations, be related to survival rate in HCC.

Hepcidin and DNA promoter methylation in hepatocellular carcinoma.

BACKGROUND: The liver hormone hepcidin regulates iron homoeostasis that is often altered in hepatocellular carcinoma (HCC). Epigenetic phenomena control gene expression through a dynamic fashion; therefore, considering the plasticity of both iron homoeostasis and epigenetic mechanisms and their role in liver carcinogenesis, we investigated whether hepcidin gene (HAMP) expression is modulated by DNA methylation, thus affecting iron status in human HCC. MATERIALS AND METHODS: Thirty-two patients affected by nonviral HCC were enrolled, and their main clinical and biochemical characteristics were obtained. Neoplastic and homologous non-neoplastic liver tissues were analysed for HAMP promoter DNA methylation, for HAMP gene expression and for iron content. An in vitro demethylation assay with a human hepatocarcinoma cell line was performed to evaluate the role of DNA methylation on HAMP transcriptional repression. RESULTS: Gene expression and DNA methylation analyses on tissues showed that HAMP was transcriptionally repressed in HCC tissues consensually with a promoter hypermethylation. Furthermore, patients with HCC had low serum hepcidin concentrations, and HCC tissues had relative iron depletion as compared to non-neoplastic liver tissues. The cell culture model showed the functional role of DNA hypermethylation by downregulating HAMP gene expression. Through a quantitative methylation analysis on HCC tissues, we then proved that methylation at definite CpG sites within consensus sequences for specific transcription factors is possibly the mechanism underlying HAMP repression. CONCLUSIONS: This study highlights a novel role for HAMP downregulation through DNA promoter hypermethylation and emphasises the significance of epigenetics in the regulation of iron metabolism in HCC.

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.

Increased urinary excretion of the epithelial Na channel activator prostasin in patients with primary aldosteronism.

OBJECTIVES: Prostasin is a glycosylphosphatidylinositol-anchored serine protease that is released in urine and is involved in epithelial Na channel activation. A direct association between urinary prostasin (u-prostasin) concentration and activation of the aldosterone-driven pathway has been suggested; however, in previous studies on primary aldosteronism, a semiquantitative evaluation, rather than a precise quantification, of prostasin was performed. We aim to investigate if u-prostasin concentrations are higher in patients with primary aldosteronism than in patients with essential hypertension and whether u-prostasin measurements could be a useful marker for diagnosing primary aldosteronism in hypertensive patients. METHODS: A total of 62 primary aldosteronism and 56 essential hypertension patients were enrolled. Biochemical and hormonal parameters were measured by applying routine laboratory methods, and u-prostasin levels were assessed by ELISA. RESULTS: Primary aldosteronism patients had higher u-prostasin levels than did essential hypertension patients. Prostasin levels were positively correlated with the aldosterone-to-renin ratio and inversely correlated with plasma K and urinary Na levels. In the highest concentration quartile, u-prostasin levels were associated with a several-fold higher probability of primary aldosteronism diagnosis in hypertensive patients. Receiver operating characteristic curve analysis showed that prostasin was specific but poorly sensitive as a diagnostic marker for primary aldosteronism. CONCLUSIONS: The study shows that an elevated u-prostasin concentration in humans is a specific marker for primary aldosteronism, which involves the classical model of epithelial Na channel activation. There was no statistically significant difference in prostasin concentrations among patients with different primary aldosteronism subtypes. Studies with a larger series of patients are necessary to clarify the clinical usefulness of the prostasin assay.

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 ± 0.37% vs. 4.77 ± 0.38%, respectively, p = 0.535), and both primary and metastatic tissues were hypomethylated compared to cancer-free colon (4.98 ± 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 (≤5.34%), the concomitant presence of AA genotype and low mCyt level led to a significantly worse survival rate as compared to the G allele carriership (p<0.0001) with a higher Hazard Ratio (HR = 6.62, p = 0.001). The subjects carrying the AA genotype in association with high mCyt did not show a significant difference in survival rate as compared with the G allele carriers (p = 0.919). The RFC1 80G>A 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.

DNA methylation and gene expression profiles show novel regulatory pathways in hepatocellular carcinoma.

BACKGROUND: Alcohol is a well-known risk factor for hepatocellular carcinoma (HCC), but the mechanisms underlying the alcohol-related hepatocarcinogenesis are still poorly understood. Alcohol alters the provision of methyl groups within the hepatic one-carbon metabolism, possibly inducing aberrant DNA methylation. Whether specific pathways are epigenetically regulated in alcohol-associated HCC is, however, unknown. The aim of the present study was to investigate the genome-wide promoter DNA methylation and gene expression profiles in non-viral, alcohol-associated HCC. From eight HCC patients undergoing curative surgery, array-based DNA methylation and gene expression data of all annotated genes were analyzed by comparing HCC tissue and homologous cancer-free liver tissue. RESULTS: After merging the DNA methylation with gene expression data, we identified 159 hypermethylated-repressed, 30 hypomethylated-induced, 49 hypermethylated-induced, and 56 hypomethylated-repressed genes. Notably, promoter DNA methylation emerged as a novel regulatory mechanism for the transcriptional repression of genes controlling the retinol metabolism (ADH1A, ADH1B, ADH6, CYP3A43, CYP4A22, RDH16), iron homeostasis (HAMP), one-carbon metabolism (SHMT1), and genes with a putative, newly identified function as tumor suppressors (FAM107A, IGFALS, MT1G, MT1H, RNF180). CONCLUSIONS: A genome-wide DNA methylation approach merged with array-based gene expression profiles allowed identifying a number of novel, epigenetically regulated candidate tumor-suppressor genes in alcohol-associated hepatocarcinogenesis. Retinol metabolism genes and SHMT1 are also epigenetically regulated through promoter DNA methylation in alcohol-associated HCC. Due to the reversibility of epigenetic mechanisms by environmental/nutritional factors, these findings may open up to novel interventional strategies for hepatocarcinogenesis prevention in HCC related to alcohol, a modifiable dietary component.

Global DNA methylation and hydroxymethylation differ in hepatocellular carcinoma and cholangiocarcinoma and relate to survival rate.

UNLABELLED: In addition to DNA methylation, hydroxymethylation of DNA is recognized as a novel epigenetic mark. Primary liver cancers, i.e., hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC), are highly prevalent but epigenetically poorly characterized, so far. In the present study we measured global methylcytosine (mCyt) and hydroxymethylcytosine (hmCyt) in HCC and CC tissues and in peripheral blood mononuclear cell (PBMC) DNA to define mCyt and hmCyt status and, accordingly, the survival rate. Both mCyt and hmCyt were measured by a liquid chromatography/tandem mass spectrometry method in neoplastic and homologous nonneoplastic tissues, i.e., liver and gallbladder, and in PBMCs of 31 HCC and 16 CC patients. Content of mCyt was notably lower in HCC than in CC tissues (3.97% versus 5.26%, respectively; P < 0.0001). Significantly reduced mCyt was also detected in HCC compared to nonneoplastic tissue (3.97% versus 4.82% mCyt, respectively; P < 0.0001), but no such difference was found for CC versus homologous nonneoplastic tissue. Hydroxymethylation was significantly decreased in HCC versus nonneoplastic liver tissue (0.044 versus 0.128, respectively; P < 0.0001) and in CC versus both liver and gallbladder nonneoplastic tissue (0.030 versus 0.124, P = 0.026, and 0.030 versus 0.123, P = 0.006, respectively). When the survival rate was evaluated according to mCyt PBMC content by Kaplan-Meier analysis, patients with mCyt ≥5.59% had a significantly higher life expectancy than those with mCyt <5.59% (P = 0.034) at a follow-up period up to 48 months. CONCLUSION: A significant DNA hypomethylation distinguishes HCC from CC, while DNA hypo-hydroxymethylation characterizes both HCC and CC, and a PBMC DNA mCyt content ≥5.59% relates to a favorable outcome in primary liver cancers.

Global DNA hypomethylation in peripheral blood mononuclear cells as a biomarker of cancer risk.

BACKGROUND: Global DNA hypomethylation is an early molecular event in carcinogenesis. Whether methylation measured in peripheral blood mononuclear cells (PBMCs) DNA is a clinically reliable biomarker for early detection or cancer risk assessment is to be established. METHODS: From an original sample-set of 753 male and female adults (ages 64.8 ± 7.3 years), PBMCs DNA methylation was measured in 68 subjects with history of cancer at time of enrollment and 62 who developed cancer during follow-up. Age- and sex-matched controls for prevalent and incident cancer cases (n = 68 and 58, respectively) were also selected. Global DNA methylation was assessed by liquid chromatography/mass spectrometry (LC/MS). Methylenetetrahydrofolate reductase (MTHFR) 677C>T genotype and plasma folate concentrations were also determined for the known gene-nutrient interaction affecting DNA methylation. RESULTS: Cancer subjects had significantly lower PBMCs-DNA methylation than controls [4.39 (95% confidence intervals (CI), 4.25-4.53) vs. 5.13 (95% CI, 5.03-5.21) %mCyt/(mCyt+Cyt); P < 0.0001]. A DNA methylation threshold of 4.74% clearly categorized patients with cancer from controls so that those with DNA methylation less than 4.74% showed an increased prevalence of cancer than those with higher levels (91.5% vs. 19%; P < 0.001). Subjects with cancer at follow-up had, already at enrollment, reduced DNA methylation as compared with controls [4.34 (95% CI, 4.24-4.51) vs. 5.08 (95% CI, 5.05-5.22) %mCyt/(mCyt+Cyt); P < 0.0001]. Moreover, MTHFR677C>T genotype and folate interact for determining DNA methylation, so that MTHFR677TT carriers with low folate had the lowest DNA methylation and concordantly showed a higher prevalence of cancer history (OR, 7.04; 95% CI, 1.52-32.63; P = 0.013). CONCLUSIONS: Genomic PBMCs-DNA methylation may be a useful epigenetic biomarker for early detection and cancer risk estimation. IMPACT: This study identifies a threshold for PBMCs-DNA methylation to detect cancer-affected from cancer-free subjects and an at-risk condition for cancer based on genomic DNA methylation and MTHFR677C>T-folate status.

Promoter methylation in coagulation F7 gene influences plasma FVII concentrations and relates to coronary artery disease.

BACKGROUND: Plasma factor VII concentrations (FVIIa), a marker of coronary artery disease (CAD) risk, are influenced by genetic markers at the promoter site: the A2 allele, due to a 10bp insertion at position -323, is a determinant of lower FVIIa concentrations and reduced CAD risk, while the -402A allele, due to a G>A substitution, confers increased transcriptional activity in vitro resulting in higher FVIIa. Transcriptional regulation of F7 by epigenetic features is, however, still unknown as is the inter-relationship of genetic and epigenetic modifications at the promoter site. OBJECTIVE: To investigate a possible epigenetic regulation of the F7 gene at the promoter region and its link with functional F7 polymorphisms at the same site. METHODS AND RESULTS: F7 promoter methylation and its relation to F7 promoter polymorphisms in modulating FVIIa and CAD risk were evaluated by methyl-specific PCR and bisulfite sequencing techniques in 253 subjects, of whom 168 had CAD and 88 were CAD-free. Plasma FVIIa was inversely related to methylation in A1A1 and -402GG, that is in the absence of the rare A2 and -402A allele. The higher FVIIa paralleled the lower methylation in A1A1 compared to A2A2 (p=0.035), while no variation in methylation was associated with the different -402G>A genotypes. The modulation of methylation-induced FVIIa concentrations was observed only in A1A1 where the higher methylation resulting in lower FVIIa was prevalent within the CAD-free group compared to the CAD group (p=0.011). CONCLUSIONS: Epigenetic regulation through methylation of F7 promoter is associated with CAD by affecting plasma FVIIa concentrations in A1A1 genotypes.

Preliminary evidence for cell membrane amelioration in children with cystic fibrosis by 5-MTHF and vitamin B12 supplementation: a single arm trial.

BACKGROUND: 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. METHODOLOGY AND PRINCIPAL FINDINGS: 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. CONCLUSION AND SIGNIFICANCE: 5-MTHF and vitamin B12 supplementation might ameliorate RBC membrane features of children with CF. TRIAL REGISTRATION: ClinicalTrials.gov NCT00730509.

Epigenetic control of 11 beta-hydroxysteroid dehydrogenase 2 gene promoter is related to human hypertension.

BACKGROUND: Lower activity of 11 beta-hydroxysteroid dehydrogenase 2 (11beta-HSD2) classically induces hypertension by leading to an altered tetrahydrocortisol- versus tetrahydrocortisone-metabolites (THFs/THE) shuttle. Recent cell culture and animal studies suggest a role for promoter methylation, a major epigenetic feature of DNA, in regulation of HSD11B2 expression. Little is known, however, of human HSD11B2 epigenetic control and its relationship with the onset of hypertension. OBJECTIVE: To explore the possible relevance of HSD11B2 promoter methylation, by examining human peripheral blood mononuclear cell (PBMC) DNA and urinary THFs/THE ratio as a biochemical indicator of 11beta-HSD2 activity, in blood pressure control. METHODS: Twenty-five essential hypertensives and 32 subjects on prednisone therapy were analyzed, the latter to investigate 11beta-HSD2 function in the development of hypertension. RESULTS: Elevated HSD11B2 promoter methylation was associated with hypertension developing in glucocorticoid-treated patients in parallel with a higher urinary THFs/THE ratio. Essential hypertensives with elevated urinary THFs/THE ratio also showed higher HSD11B2 promoter methylation. CONCLUSIONS: These results show a clear link between the epigenetic regulation through repression of HSD11B2 in PBMC DNA and hypertension.