DNA hydroxymethylation levels are altered in blood cells from Down syndrome persons enrolled in the MARK-AGE project

Down syndrome (DS) is caused by the presence of part or an entire extra copy of chromosome 21, a phenomenon that can cause a wide spectrum of clinically defined phenotypes of the disease. Most of the clinical signs of DS are typical of the ageing process including dysregulation of immune system. Beyond the causative genetic defect, DS persons display epigenetic alterations, particularly aberrant DNA methylation patterns that can contribute to the heterogeneity of the disease. In the present work we investigated the levels of 5-hydroxymethylcytosine (5hmC) and of the TET dioxygenase enzymes, which are involved in DNA demethylation processes and are often deregulated in pathological conditions as well as in ageing. Analyses were carried out on peripheral blood mononuclear cells of DS volunteers enrolled in the context of the MARK-AGE study, a large-scale cross-sectional population study with subjects representing the general population in eight European countries. We observed a decrease of 5hmC, TET1 and other components of the DNA methylation/demethylation machinery in DS subjects, indicating that aberrant DNA methylation patterns in DS, which may have consequences on the transcriptional status of immune cells, may be due to a global disturbance of methylation control in DS

Analysis of the machinery and intermediates of the 5hmC-mediated DNA demethylation pathway in aging on samples from the MARKAGE Study

Gradual changes in the DNA methylation landscape occur throughout aging virtually in all human tissues. A widespread reduction of 5-methylcytosine (5mC), associated with highly reproducible site-specific hypermethylation, characterizes the genome in aging. Therefore, an equilibrium seems to exist between general and directional deregulating events concerning DNA methylation controllers, which may underpin the age-related epigenetic changes. In this context, 5mC-hydroxylases (TET enzymes) are new potential players. In fact, TETs catalyze the stepwise oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), driving the DNA demethylation process based on thymine DNA glycosylase (TDG)-mediated DNA repair pathway. The present paper reports the expression of DNA hydroxymethylation components, the levels of 5hmC and of its derivatives in peripheral blood mononuclear cells of age-stratified donors recruited in several European countries in the context of the EU Project ‘MARK-AGE’. The results provide evidence for an age-related decline of TET1, TET3 and TDG gene expression along with a decrease of 5hmC and an accumulation of 5caC. These associations were independent of confounding variables, including recruitment center, gender and leukocyte composition. The observed impairment of 5hmC-mediated DNA demethylation pathway in blood cells may lead to aberrant transcriptional programs in the elderly

Age-dependent expression of DNMT1 and DNMT3B in PBMCs from a large European population enrolled in the MARK-AGE study

Aging is associated with alterations in the content and patterns of DNA methylation virtually throughout the entire human lifespan. Reasons for these variations are not well understood. However, several lines of evidence suggest that the epigenetic instability in aging may be traced back to the alteration of the expression of DNA methyltransferases. Here, the association of the expression of DNA methyltransferases DNMT1 and DNMT3B with age has been analysed in the context of the MARK-AGE study, a large-scale cross-sectional study of the European general population. Using peripheral blood mononuclear cells, we assessed the variation of DNMT1 and DNMT3B gene expression in more than two thousand age-stratified women and men (35-75 years) recruited across eight European countries. Significant age-related changes were detected for both transcripts. The level of DNMT1 gradually dropped with aging but this was only observed up to the age of 64 years. By contrast, the expression of DNMT3B decreased linearly with increasing age and this association was particularly evident in females. We next attempted to trace the age-related changes of both transcripts to the influence of different variables that have an impact on changes of their expression in the population, including demographics, dietary and health habits, and clinical parameters. Our results indicate that age affects the expression of DNMT1 and DNMT3B as an almost independent variable in respect of all other variables evaluated

Quality control data of physiological and immunological biomarkers measured in serum and plasma

In two work packages of the MARK-AGE project, 37 immunological and physiological biomarkers were measured in 3637 serum, plasma or blood samples in five batches during a period of 4 years. The quality of the serum and plasma samples was very good as judged by the low number of biomarker measurements (only 0.2%) that were rejected because of a high hemolysis, icteria or lipemia of the samples. Using quality control samples, day-to-day and batch variations were determined. The mean inter-assay variation of the five batches were all below 8%, with an average inter-assay coefficient of variation of all biomarkers of 4.0%. Also the precision of the measurements was very good, because all measurements were between 90% and 115% of the defined target values. A possible mix-up of samples was determined by comparison of the extreme testosterone levels of men and women. It was concluded that 3% of the sample identification could be mixed-up. Considering the complex procedure from collection to analysis, including preparation, handling, shipment and storage, of the samples in the MARK-AGE project, both the quality of the samples and the quality of the measurements are very good

Circulating cell-free DNA in health and disease - the relationship to health behaviours, ageing phenotypes and metabolomics

Circulating cell-free DNA (cf-DNA) has emerged as a promising biomarker of ageing, tissue damage and cellular stress. However, less is known about health behaviours, ageing phenotypes and metabolic processes that lead to elevated cf-DNA levels. We sought to analyse the relationship of circulating cf-DNA level to age, sex, smoking, physical activity, vegetable consumption, ageing phenotypes (physical functioning, the number of diseases, frailty) and an extensive panel of biomarkers including blood and urine metabolites and inflammatory markers in three human cohorts (N = 5385; 17–82 years). The relationships were assessed using correlation statistics, and linear and penalised regressions (the Lasso), also stratified by sex.cf-DNA levels were significantly higher in men than in women, and especially in middle-aged men and women who smoke, and in older more frail individuals. Correlation statistics of biomarker data showed that cf-DNA level was higher with elevated inflammation (C-reactive protein, interleukin-6), and higher levels of homocysteine, and proportion of red blood cells and lower levels of ascorbic acid. Inflammation (C-reactive protein, glycoprotein acetylation), amino acids (isoleucine, leucine, tyrosine), and ketogenesis (3-hydroxybutyrate) were included in the cf-DNA level-related biomarker profiles in at least two of the cohorts.In conclusion, circulating cf-DNA level is different by sex, and related to health behaviour, health decline and metabolic processes common in health and disease. These results can inform future studies where epidemiological and biological pathways of cf-DNA are to be analysed in details, and for studies evaluating cf-DNA as a potential clinical marker.</p

Fatty Acid Status and Its Relationship to Cognitive Decline and Homocysteine Levels in the Elderly

Polyunsaturated fatty acids (PUFAs), especially the n-3 series, are known for their protective effects. Considering that cardiovascular diseases are risk factors for dementia, which is common at aging, the aim of this study was to evaluate whether fatty acid status in the elderly was associated with cognitive function and cardiovascular risk. Forty-five elderly persons (age ≥60 years) were included and divided into two groups based on their Mini-Mental Status Examination score adjusted for educational level: the case group (n = 12) and the control group (n = 33). Serum fatty acid composition, homocysteine (Hcy), hs-CRP, lipid profile and different cognitive domains were evaluated. The case group, characterized by reduced cognitive performance, showed higher levels of 14:0, 16:0, 16:1n-7 fatty acids and lower levels of 22:0, 24:1n-9, 22:6n-3 (DHA) and total PUFAs compared to the control group (p &lt; 0.05). The n-6/n-3 ratio was elevated in both study groups, whereas alterations in Hcy, hs-CRP and lipid profile were observed in the case group. Cognitive function was positively associated with the 24:1n-9, DHA and total n-3 PUFAs, while 14:0, 16:0 and 16:1n-7 fatty acids, the n-6/n-3 ratio and Hcy were inversely associated. In addition, n-3 PUFAs, particularly DHA, were inversely associated with cardiovascular risk, assessed by Hcy levels in the elderly

MARK-AGE standard operating procedures (SOPs): A successful effort

Within the MARK-AGE project, a population study (3337 subjects) was conducted to identify a set of biomarkers of ageing which, as a combination of parameters with appropriate weighting, would measure biological age better than any single marker. The MARK-AGE project involves 14 European countries and a total of 26 research centres. In such a study, standard operating procedures (SOPs) are an essential task, which are binding for all MARK-AGE Beneficiaries. The SOPs cover all aspects of subject’s recruitment, collection, shipment and distribution of biological samples (blood and its components, buccal mucosa cells or BMC and urine) as well as the anthropometric measurements and questionnaires

The Proteasome Is an Integral Part of Solar Ultraviolet A Radiation-induced Gene Expression

Solar ultraviolet (UV) A radiation is a well known trigger of signaling responses in human skin fibroblasts. One important consequence of this stress response is the increased expression of matrix metalloproteinase-1 (MMP-1), which causes extracellular protein degradation and thereby contributes to photoaging of human skin. In the present study we identify the proteasome as an integral part of the UVA-induced, intracellular signaling cascade in human dermal fibroblasts. UVA-induced singlet oxygen formation was accompanied by protein oxidation, the cross-linking of oxidized proteins, and an inhibition of the proteasomal system. This proteasomal inhibition subsequently led to an accumulation of c-Jun and phosphorylated c-Jun and activation of activator protein-1, i.e. transcription factors known to control MMP-1 expression. Increased transcription factor activation was also observed if the proteasome was inhibited by cross-linked proteins or lactacystin, indicating a general mechanism. Most importantly, inhibition of the proteasome was of functional relevance for UVA-induced MMP-1 expression, because overexpression of the proteasome or the protein repair enzyme methionine sulfoxide reductase prevented the UVA-induced induction of MMP-1. These studies show that an environmentally relevant stimulus can trigger a signaling pathway, which links intracellular and extracellular protein degradation. They also identify the proteasome as an integral part of the UVA stress response