Disentangling age-dependent DNA methylation: deterministic, stochastic, and nonlinear.

DNA methylation variability arises due to concurrent genetic and environmental influences. Each of them is a mixture of regular and noisy sources, whose relative contribution has not been satisfactorily understood yet. We conduct a systematic assessment of the age-dependent methylation by the signal-to-noise ratio and identify a wealth of "deterministic" CpG probes (about 90%), whose methylation variability likely originates due to genetic and general environmental factors. The remaining 10% of "stochastic" CpG probes are arguably governed by the biological noise or incidental environmental factors. Investigating the mathematical functional relationship between methylation levels and variability, we find that in about 90% of the age-associated differentially methylated positions, the variability changes as the square of the methylation level, whereas in the most of the remaining cases the dependence is linear. Furthermore, we demonstrate that the methylation level itself in more than 15% cases varies nonlinearly with age (according to the power law), in contrast to the previously assumed linear changes. Our findings present ample evidence of the ubiquity of strong DNA methylation regulation, resulting in the individual age-dependent and nonlinear methylation trajectories, whose divergence explains the cross-sectional variability. It may also serve a basis for constructing novel nonlinear epigenetic clocks.

Genome-wide DNA methylation analysis in blood cells from patients with Werner syndrome.

BACKGROUND: Werner syndrome is a progeroid disorder characterized by premature age-related phenotypes. Although it is well established that autosomal recessive mutations in the WRN gene is responsible for Werner syndrome, the molecular alterations that lead to disease phenotype remain still unidentified. RESULTS: To address whether epigenetic changes can be associated with Werner syndrome phenotype, we analysed genome-wide DNA methylation profile using the Infinium MethylationEPIC BeadChip in the whole blood from three patients affected by Werner syndrome compared with three age- and sex-matched healthy controls. Hypermethylated probes were enriched in glycosphingolipid biosynthesis, FoxO signalling and insulin signalling pathways, while hypomethylated probes were enriched in PI3K-Akt signalling and focal adhesion pathways. Twenty-two out of 47 of the differentially methylated genes belonging to the enriched pathways resulted differentially expressed in a publicly available dataset on Werner syndrome fibroblasts. Interestingly, differentially methylated regions identified CERS1 and CERS3, two members of the ceramide synthase family. Moreover, we found differentially methylated probes within ITGA9 and ADAM12 genes, whose methylation is altered in systemic sclerosis, and within the PRDM8 gene, whose methylation is affected in dyskeratosis congenita and Down syndrome. CONCLUSIONS: DNA methylation changes in the peripheral blood from Werner syndrome patients provide new insight in the pathogenesis of the disease, highlighting in some cases a functional correlation of gene expression and methylation status.

Metformin improves putative longevity effectors in peripheral mononuclear cells from subjects with prediabetes. A randomized controlled trial.

BACKGROUND AND AIMS: Prediabetes increases cardiovascular risk and is associated with excess mortality. In preclinical models, metformin has been shown to exert anti-ageing effects. In this study, we sought to assess whether metformin modulates putative effector longevity programs in prediabetic subjects. METHODS AND RESULTS: In a randomized, single-blind, placebo-controlled trial, 38 prediabetic subjects received metformin (1500 mg/day) or placebo for 2 months. At baseline and after treatment, we collected anthropometric and metabolic parameters. Gene and protein levels of SIRT1, mTOR, p53, p66Shc, SIRT1 activity, AMPK activation, telomere length, and SIRT1 promoter chromatin accessibility were determined in peripheral blood mononuclear cells (PBMCs). Plasma N-glycans, non-invasive surrogate markers of ageing, were also analysed. Compared to baseline, metformin significantly improved metabolic parameters and insulin sensitivity, increased SIRT1 gene/protein expression and SIRT1 promoter chromatin accessibility, elevated mTOR gene expression with concomitant reduction in p70S6K phosphorylation in subjects' PBMCs, and modified the plasma N-glycan profile. Compared to placebo, metformin increased SIRT1 protein expression and reduced p70S6K phosphorylation (a proxy of mTOR activity). Plasma N-glycans were also favourably modified by metformin compared to placebo. CONCLUSION: In individuals with prediabetes, metformin ameliorated effector pathways that have been shown to regulate longevity in animal models. ClinicalTrials. gov identifier: NCT01765946 - January 2013.

TET2 gene expression and 5-hydroxymethylcytosine level in multiple sclerosis peripheral blood cells.

Aberrant DNA methylation can lead to genome destabilization and to deregulated gene expression. Recently, 5-hydroxymethylcytosine (5hmC), derived from oxidation of 5-methylcytosine (5mC) by the Ten-Eleven Translocation (TET) enzymes, has been detected. 5hmC is now considered as a new epigenetic DNA modification with relevant roles in cell homeostasis regulating DNA demethylation and transcription. Our aim was to investigate possible changes in the DNA methylation/demethylation machinery in MS. We assessed the expression of enzymes involved in DNA methylation/demethylation in peripheral blood mononuclear cells (PBMCs) from 40 subjects with MS and 40 matched healthy controls. We performed also, DNA methylation analysis of specific promoters and analysis of global levels of 5mC and 5hmC. We show that TET2 and DNMT1 expression is significantly down-regulated in MS PBMCs and it is associated with aberrant methylation of their promoters. Furthermore, 5hmC is decreased in MS PBMCs, probably as a result of the diminished TET2 level.

Validation of suitable internal control genes for expression studies in aging.

Quantitative data from experiments of gene expression are often normalized through levels of housekeeping genes transcription by assuming that expression of these genes is highly uniform. This practice is being questioned as it becomes increasingly clear that the level of housekeeping genes expression may vary considerably in certain biological samples. To date, the validation of reference genes in aging has received little attention and suitable reference genes have not yet been defined. Our aim was to evaluate the expression stability of frequently used reference genes in human peripheral blood mononuclear cells with respect to aging. Using quantitative RT-PCR, we carried out an extensive evaluation of five housekeeping genes, i.e. 18s rRNA, ACTB, GAPDH, HPRT1 and GUSB, for stability of expression in samples from donors in the age range 35-74 years. The consistency in the expression stability was quantified on the basis of the coefficient of variation and two algorithms termed geNorm and NormFinder. Our results indicated GUSB be the most suitable transcript and 18s the least for accurate normalization in PBMCs. We also demonstrated that aging is a confounding factor with respect to stability of 18s, HPRT1 and ACTB expression, which were particularly prone to variability in aged donors.