Search

Abstract Parkinson’s disease is increasingly prevalent. It progresses from the pre-motor stage (characterised by non-motor symptoms like REM sleep behaviour disorder), to the disabling motor stage. We need objective biomarkers for early/pre-motor disease stages to be able to intervene and slow the underlying neurodegenerative process. Here, we validate a targeted multiplexed mass spectrometry assay for blood samples from recently diagnosed motor Parkinson’s patients (n = 99), pre-motor individuals with isolated REM sleep behaviour disorder (two cohorts: n = 18 and n = 54 longitudinally), and healthy controls (n = 36). Our machine-learning model accurately identifies all Parkinson patients and classifies 79% of the pre-motor individuals up to 7 years before motor onset by analysing the expression of eight proteins—Granulin precursor, Mannan-binding-lectin-serine-peptidase-2, Endoplasmatic-reticulum-chaperone-BiP, Prostaglaindin-H2-D-isomaerase, Interceullular-adhesion-molecule-1, Complement C3, Dickkopf-WNT-signalling pathway-inhibitor-3, and Plasma-protease-C1-inhibitor. Many of these biomarkers correlate with symptom severity. This specific blood panel indicates molecular events in early stages and could help identify at-risk participants for clinical trials aimed at slowing/preventing motor Parkinson’s disease.

Down syndrome (DS) is a segmental progeroid genetic disorder associated with multi-systemic precocious aging phenotypes, which are particularly evident in the immune and nervous systems. Accordingly, people with DS show an increased biological age as measured by epigenetic clocks. The Ts65Dn trisomic mouse, which harbors extra-numerary copies of chromosome 21 (Hsa21)-syntenic regions, was shown to recapitulate several progeroid features of DS, but no biomarkers of age have been applied to it so far. In this pilot study, we used a mouse-specific epigenetic clock to measure the epigenetic age of hippocampi from Ts65Dn and euploid mice at 20 weeks. Ts65Dn mice showed an increased epigenetic age in comparison with controls, and the observed changes in DNA methylation partially recapitulated those observed in hippocampi from people with DS. Collectively, our results support the use of the Ts65Dn model to decipher the molecular mechanisms underlying the progeroid DS phenotypes.

Subcutaneous adipocytes are crucial for mammary gland epithelial development during pregnancy. Our and others’ previous data have suggested that adipo-epithelial transdifferentiation could play a key role in the mammary gland alveolar development. In this study, we tested whether adipo-epithelial transdifferentiation occurs in vitro. Data show that, under appropriate co-culture conditions with mammary epithelial organoids (MEOs), mature adipocytes lose their phenotype and acquire an epithelial one. Interestingly, even in the absence of MEOs, extracellular matrix and diffusible growth factors are able to promote adipo-epithelial transdifferentiation. Gene and protein expression studies indicate that transdifferentiating adipocytes exhibit some characteristics of milk-secreting alveolar glands, including significantly higher expression of milk proteins such as whey acidic protein and β-casein. Similar data were also obtained in cultured human multipotent adipose-derived stem cell adipocytes. A miRNA sequencing experiment on the supernatant highlighted mir200c, which has a well-established role in the mesenchymal–epithelial transition, as a potential player in this phenomenon. Collectively, our data show that adipo-epithelial transdifferentiation can be reproduced in in vitro models where this phenomenon can be investigated at the molecular level.

Abstract Isolated REM Sleep Behavior Disorder (iRBD) is the strongest prodromal marker for α-synucleinopathies. Overt α-synucleinopathies and aging share several mechanisms, but this relationship has been poorly investigated in prodromal phases. Using DNA methylation-based epigenetic clocks, we measured biological aging in videopolysomnography confirmed iRBD patients, videopolysomnography-negative and population-based controls. We found that iRBDs tended to be epigenetically older than controls, suggesting that accelerated aging characterizes prodromal neurodegeneration.

Abstract Transcriptomics in Parkinson’s disease (PD) offers new insights into the molecular mechanism of PD pathogenesis. Several pathways, such as inflammation and protein degradation, have been identified by differential gene expression analysis. Our aim was to identify gene expression differences underlying the disease etiology and the discovery of pre-symptomatic risk biomarkers for PD from a multicenter study in the context of the PROPAG-AGEING project. We performed RNA sequencing from 47 patients with de novo PD, 10 centenarians, and 65 healthy controls. Using identified differentially expressed genes, functional annotations were assigned using gene ontology to unveil significant enriched biological processes. The expression of 16 selected genes was validated using OpenArray® assays and samples from independent cohorts of 201 patients with advanced PD, 340 healthy siblings of PD patients, and 177 healthy controls. Differential gene expression analysis identified higher FCGBP expression in patients with de novo PD compared with healthy controls and compared with centenarians. Furthermore, FCGBP showed no differences in terms of population origin or aging process. The increased FCGBP expression was validated in patients with advanced PD and their siblings. Thus, we provided evidence for an upregulation of FCGBP mRNA levels not only in patients with PD but also in individuals at putative higher risk of PD, suggesting that it could be important in gut–brain PD interaction, mediating the connection between microbiota and intestinal inflammatory processes, as well as neuroinflammation and neurodegeneration.

Abstract Parkinson’s disease (PD) is the neurological disorder showing the greatest rise in prevalence from 1990 to 2016. Despite clinical definition criteria and a tremendous effort to develop objective biomarkers, precise diagnosis of PD is still unavailable at early stage. In recent years, an increasing number of studies have used omic methods to unveil the molecular basis of PD, providing a detailed characterization of potentially pathological alterations in various biological specimens. Metabolomics could provide useful insights to deepen our knowledge of PD aetiopathogenesis, to identify signatures that distinguish groups of patients and uncover responsive biomarkers of PD that may be significant in early detection and in tracking the disease progression and drug treatment efficacy. The present work is the first large metabolomic study based on nuclear magnetic resonance (NMR) with an independent validation cohort aiming at the serum characterization of de novo drug-naive PD patients. Here, NMR is applied to sera from large training and independent validation cohorts of German subjects. Multivariate and univariate approaches are used to infer metabolic differences that characterize the metabolite and the lipoprotein profiles of newly diagnosed de novo drug-naive PD patients also in relation to the biological sex of the subjects in the study, evidencing a more pronounced fingerprint of the pathology in male patients. The presence of a validation cohort allowed us to confirm altered levels of acetone and cholesterol in male PD patients. By comparing the metabolites and lipoproteins levels among de novo drug-naive PD patients, age- and sex-matched healthy controls, and a group of advanced PD patients, we detected several descriptors of stronger oxidative stress.

Abstract Advanced age represents one of the major risk factors for Parkinson’s Disease. Recent biomedical studies posit a role for microRNAs, also known to be remodelled during ageing. However, the relationship between microRNA remodelling and ageing in Parkinson’s Disease, has not been fully elucidated. Therefore, the aim of the present study is to unravel the relevance of microRNAs as biomarkers of Parkinson’s Disease within the ageing framework. We employed Next Generation Sequencing to profile serum microRNAs from samples informative for Parkinson’s Disease (recently diagnosed, drug-naïve) and healthy ageing (centenarians) plus healthy controls, age-matched with Parkinson’s Disease patients. Potential microRNA candidates markers, emerging from the combination of differential expression and network analyses, were further validated in an independent cohort including both drug-naïve and advanced Parkinson’s Disease patients, and healthy siblings of Parkinson’s Disease patients at higher genetic risk for developing the disease. While we did not find evidences of microRNAs co-regulated in Parkinson’s Disease and ageing, we report that hsa-miR-144-3p is consistently down-regulated in early Parkinson’s Disease patients. Moreover, interestingly, functional analysis revealed that hsa-miR-144-3p is involved in the regulation of coagulation, a process known to be altered in Parkinson’s Disease. Our results consistently show the down-regulation of hsa-mir144-3p in early Parkinson’s Disease, robustly confirmed across a variety of analytical and experimental analyses. These promising results ask for further research to unveil the functional details of the involvement of hsa-mir144-3p in Parkinson’s Disease.

Abstract A prodromal phase of Parkinson’s disease (PD) may precede motor manifestations by decades. PD patients’ siblings are at higher risk for PD, but the prevalence and distribution of prodromal symptoms are unknown. The study objectives were (1) to assess motor and non-motor features estimating prodromal PD probability in PD siblings recruited within the European PROPAG-AGEING project; (2) to compare motor and non-motor symptoms to the well-established DeNoPa cohort. 340 PD siblings from three sites (Bologna, Seville, Kassel/Goettingen) underwent clinical and neurological evaluations of PD markers. The German part of the cohort was compared with German de novo PD patients (dnPDs) and healthy controls (CTRs) from DeNoPa. Fifteen (4.4%) siblings presented with subtle signs of motor impairment, with MDS-UPDRS-III scores not clinically different from CTRs. Symptoms of orthostatic hypotension were present in 47 siblings (13.8%), no different to CTRs (p = 0.072). No differences were found for olfaction and overall cognition; German-siblings performed worse than CTRs in visuospatial-executive and language tasks. 3/147 siblings had video-polysomnography-confirmed REM sleep behavior disorder (RBD), none was positive on the RBD Screening Questionnaire. 173/300 siblings had

A large body of evidence indicates that environmental agents can induce alterations in DNA methylation (DNAm) profiles. Radiofrequency electromagnetic fields (RF-EMFs) are radiations emitted by everyday devices, which have been classified as “possibly carcinogenic”; however, their biological effects are unclear. As aberrant DNAm of genomic repetitive elements (REs) may promote genomic instability, here, we sought to determine whether exposure to RF-EMFs could affect DNAm of different classes of REs, such as long interspersed nuclear elements-1 (LINE-1), Alu short interspersed nuclear elements and ribosomal repeats. To this purpose, we analysed DNAm profiles of cervical cancer and neuroblastoma cell lines (HeLa, BE(2)C and SH-SY5Y) exposed to 900 MHz GSM-modulated RF-EMF through an Illumina-based targeted deep bisulfite sequencing approach. Our findings showed that radiofrequency exposure did not affect the DNAm of Alu elements in any of the cell lines analysed. Conversely, it influenced DNAm of LINE-1 and ribosomal repeats in terms of both average profiles and organisation of methylated and unmethylated CpG sites, in different ways in each of the three cell lines studied.

Control of ribosome biogenesis is a critical aspect of the regulation of cell metabolism. As ribosomal genes (rDNA) are organized in repeated clusters on chromosomes 13, 14, 15, 21, and 22, trisomy of chromosome 21 confers an excess of rDNA copies to persons with Down syndrome (DS). Previous studies showed an alteration of ribosome biogenesis in children with DS, but the epigenetic regulation of rDNA genes has not been investigated in adults with DS so far. In this study, we used a targeted deep-sequencing approach to measure DNA methylation (DNAm) of rDNA units in whole blood from 69 adults with DS and 95 euploid controls. We further evaluated the expression of the precursor of ribosomal RNAs (RNA45S) in peripheral blood mononuclear cells (PBMCs) from the same subjects. We found that the rDNA promoter tends to be hypermethylated in DS concerning the control group. The analysis of epihaplotypes (the combination of methylated and unmethylated CpG sites along the same DNA molecule) showed a significantly lower intra-individual diversity in the DS group, which at the same time was characterized by a higher interindividual variability. Finally, we showed that RNA45S expression is lower in adults with DS. Collectively, our results suggest a rearrangement of the epigenetic profile of rDNA in DS, possibly to compensate for the extranumerary rDNA copies. Future studies should assess whether the regulation of ribosome biogenesis can contribute to the pathogenesis of DS and explain the clinical heterogeneity characteristic of the syndrome.

The aim of the present study was to provide a comprehensive characterization of whole genome DNA methylation patterns in replicative and ionizing irradiation- or doxorubicin-induced premature senescence, exhaustively exploring epigenetic modifications in three different human cell types: in somatic diploid skin fibroblasts and in bone marrow- and adipose-derived mesenchymal stem cells. With CpG-wise differential analysis, three epigenetic signatures were identified: (a) cell type- and treatment-specific signature; (b) cell type-specific senescence-related signature; and (c) cell type-transversal replicative senescence-related signature. Cluster analysis revealed that only replicative senescent cells created a distinct group reflecting notable alterations in the DNA methylation patterns accompanying this cellular state. Replicative senescence-associated epigenetic changes seemed to be of such an extent that they surpassed interpersonal dissimilarities. Enrichment in pathways linked to the nervous system and involved in the neurological functions was shown after pathway analysis of genes involved in the cell type-transversal replicative senescence-related signature. Although DNA methylation clock analysis provided no statistically significant evidence on epigenetic age acceleration related to senescence, a persistent trend of increased biological age in replicative senescent cultures of all three cell types was observed. Overall, this work indicates the heterogeneity of senescent cells depending on the tissue of origin and the type of senescence inducer that could be putatively translated to a distinct impact on tissue homeostasis.

Abstract Background The cline of human genetic diversity observable across Europe is recapitulated at a micro-geographic scale by variation within the Italian population. Besides resulting from extensive gene flow, this might be ascribable also to local adaptations to diverse ecological contexts evolved by people who anciently spread along the Italian Peninsula. Dissecting the evolutionary history of the ancestors of present-day Italians may thus improve the understanding of demographic and biological processes that contributed to shape the gene pool of European populations. However, previous SNP array-based studies failed to investigate the full spectrum of Italian variation, generally neglecting low-frequency genetic variants and examining a limited set of small effect size alleles, which may represent important determinants of population structure and complex adaptive traits. To overcome these issues, we analyzed 38 high-coverage whole-genome sequences representative of population clusters at the opposite ends of the cline of Italian variation, along with a large panel of modern and ancient Euro-Mediterranean genomes. Results We provided evidence for the early divergence of Italian groups dating back to the Late Glacial and for Neolithic and distinct Bronze Age migrations having further differentiated their gene pools. We inferred adaptive evolution at insulin-related loci in people from Italian regions with a temperate climate, while possible adaptations to pathogens and ultraviolet radiation were observed in Mediterranean Italians. Some of these adaptive events may also have secondarily modulated population disease or longevity predisposition. Conclusions We disentangled the contribution of multiple migratory and adaptive events in shaping the heterogeneous Italian genomic background, which exemplify population dynamics and gene-environment interactions that played significant roles also in the formation of the Continental and Southern European genomic landscapes.

Homo sapiens have been exposed to various toxins and harmful compounds that change according to various phases of human evolution. Population genetics studies showed that such exposures lead to adaptive genetic changes; while observing present exposures to different toxicants, the first molecular mechanism that confers plasticity is epigenetic remodeling and, in particular, DNA methylation variation, a molecular mechanism proposed for medium-term adaptation. A large amount of scientific literature from clinical and medical studies revealed the high impact of such exposure on human biology; thus, in this review, we examine and infer the impact that different environmental toxicants may have in shaping human evolution. We first describe how environmental toxicants shape natural human variation in terms of genetic and epigenetic diversity, and then we describe how DNA methylation may influence mutation rate and, thus, genetic variability. We describe the impact of these substances on biological fitness in terms of reproduction and survival, and in conclusion, we focus on their effect on brain evolution and physiology.

Epigenetic clocks were initially developed to track chronological age, but accumulating evidence indicates that they can also predict biological age. They are usually based on the analysis of DNA methylation by genome-wide methods, but targeted approaches, based on the assessment of a small number of CpG sites, are advisable in several settings. In this study, we developed a targeted epigenetic clock purposely optimized for the measurement of biological age. The clock includes six genomic regions mapping in ELOVL2, NHLRC1, AIM2, EDARADD, SIRT7 and TFAP2E genes, selected from a re-analysis of existing microarray data, whose DNA methylation is measured by EpiTYPER assay. In healthy subjects (n = 278), epigenetic age calculated using the targeted clock was highly correlated with chronological age (Spearman correlation = 0.89). Most importantly, and in agreement with previous results from genome-wide clocks, epigenetic age was significantly higher and lower than expected in models of increased (persons with Down syndrome, n = 62) and decreased (centenarians, n = 106; centenarians’ offspring, n = 143; nutritional intervention in elderly, n = 233) biological age, respectively. These results support the potential of our targeted epigenetic clock as a new marker of biological age and open its evaluation in large cohorts to further promote the assessment of biological age in healthcare practice.

Extreme longevity is the paradigm of healthy aging as individuals who reached the extreme decades of human life avoided or largely postponed all major age-related diseases. In this study, we sequenced at high coverage (90X) the whole genome of 81 semi-supercentenarians and supercentenarians [105+/110+] (mean age: 106.6 ± 1.6) and of 36 healthy unrelated geographically matched controls (mean age 68.0 ± 5.9) recruited in Italy. The results showed that 105+/110+ are characterized by a peculiar genetic background associated with efficient DNA repair mechanisms, as evidenced by both germline data (common and rare variants) and somatic mutations patterns (lower mutation load if compared to younger healthy controls). Results were replicated in a second independent cohort of 333 Italian centenarians and 358 geographically matched controls. The genetics of 105+/110+ identified DNA repair and clonal haematopoiesis as crucial players for healthy aging and for the protection from cardiovascular events.

Abstract Background Here, we isolated, expanded and functionally characterized regulatory T cells (Tregs) from patients with end stage kidney and liver disease, waiting for kidney/liver transplantation (KT/LT), with the aim to establish a suitable method to obtain large numbers of immunomodulatory cells for adoptive immunotherapy post-transplantation. Methods We first established a preclinical protocol for expansion/isolation of Tregs from peripheral blood of LT/KT patients. We then scaled up and optimized such protocol according to good manufacturing practice (GMP) to obtain high numbers of purified Tregs which were phenotypically and functionally characterized in vitro and in vivo in a xenogeneic acute graft-versus-host disease (aGVHD) mouse model. Specifically, immunodepressed mice (NOD-SCID-gamma KO mice) received human effector T cells with or without GMP-produced Tregs to prevent the onset of xenogeneic GVHD. Results Our small scale Treg isolation/expansion protocol generated functional Tregs. Interestingly, cryopreservation/thawing did not impair phenotype/function and DNA methylation pattern of FOXP3 gene of the expanded Tregs. Fully functional Tregs were also isolated/expanded from KT and LT patients according to GMP. In the mouse model, GMP Tregs from LT or KT patient proved to be safe and show a trend toward reduced lethality of acute GVHD. Conclusions These data demonstrate that expanded/thawed GMP-Tregs from patients with end-stage organ disease are fully functional in vitro. Moreover, their infusion is safe and results in a trend toward reduced lethality of acute GVHD in vivo, further supporting Tregs-based adoptive immunotherapy in solid organ transplantation.

Alzheimer's disease (AD) is characterized by specific alterations of brain DNA methylation (DNAm) patterns. Age and sex, two major risk factors for AD, are also known to largely affect the epigenetic profiles in brain, but their contribution to AD-associated DNAm changes has been poorly investigated. In this study we considered publicly available DNAm datasets of four brain regions (temporal, frontal, entorhinal cortex, and cerebellum) from healthy adult subjects and AD patients, and performed a meta-analysis to identify sex-, age-, and AD-associated epigenetic profiles. In one of these datasets it was also possible to distinguish 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) profiles. We showed that DNAm differences between males and females tend to be shared between the four brain regions, while aging differently affects cortical regions compared to cerebellum. We found that the proportion of sex-dependent probes whose methylation is modified also during aging is higher than expected, but that differences between males and females tend to be maintained, with only a few probes showing age-by-sex interaction. We did not find significant overlaps between AD- and sex-associated probes, nor disease-by-sex interaction effects. On the contrary, we found that AD-related epigenetic modifications are significantly enriched in probes whose DNAm varies with age and that there is a high concordance between the direction of changes (hyper or hypo-methylation) in aging and AD, supporting accelerated epigenetic aging in the disease. In summary, our results suggest that age-associated DNAm patterns concur to the epigenetic deregulation observed in AD, providing new insights on how advanced age enables neurodegeneration.

Chronic pain prevalence is high worldwide and increases at older ages. Signs of premature aging have been associated with chronic pain, but few studies have investigated aging biomarkers in pain-related conditions. A set of DNA methylation (DNAm)-based estimates of age, called “epigenetic clocks,” has been proposed as biological measures of age-related adverse processes, morbidity, and mortality. The aim of this study is to assess if different pain-related phenotypes show alterations in DNAm age. In our analysis, we considered three cohorts for which whole-blood DNAm data were available: heat pain sensitivity (HPS), including 20 monozygotic twin pairs discordant for heat pain temperature threshold; fibromyalgia (FM), including 24 cases and 20 controls; and headache, including 22 chronic migraine and medication overuse headache patients (MOH), 18 episodic migraineurs (EM), and 13 healthy subjects. We used the Horvath's epigenetic age calculator to obtain DNAm-based estimates of epigenetic age, telomere length, levels of 7 proteins in plasma, number of smoked packs of cigarettes per year, and blood cell counts. We did not find differences in epigenetic age acceleration, calculated using five different epigenetic clocks, between subjects discordant for pain-related phenotypes. Twins with high HPS had increased CD8+ T cell counts (nominal p = 0.028). HPS thresholds were negatively associated with estimated levels of GDF15 (nominal p = 0.008). FM patients showed decreased naive CD4+ T cell counts compared with controls (nominal p = 0.015). The severity of FM manifestations expressed through various evaluation tests was associated with decreased levels of leptin, shorter length of telomeres, and reduced CD8+ T and natural killer cell counts (nominal p < 0.05), while the duration of painful symptoms was positively associated with telomere length (nominal p = 0.034). No differences in DNAm-based estimates were detected for MOH or EM compared with controls. In summary, our study suggests that HPS, FM, and MOH/EM do not show signs of epigenetic age acceleration in whole blood, while HPS and FM are associated with DNAm-based estimates of immunological parameters, plasma proteins, and telomere length. Future studies should extend these observations in larger cohorts.

Malignant pleural mesothelioma (MPM) is a rare and aggressive neoplasm. Patients are usually diagnosed when current treatments have limited benefits, highlighting the need for noninvasive tests aimed at an MPM risk assessment tool that might improve life expectancy. Three hundred asbestos-exposed subjects (163 MPM cases and 137 cancer-free controls), from the same geographical region in Italy, were recruited. The evaluation of asbestos exposure was conducted considering the frequency, the duration and the intensity of occupational, environmental and domestic exposure. A genome-wide methylation array was performed to identify novel blood DNA methylation (DNAm) markers of MPM. Multiple regression analyses adjusting for potential confounding factors and interaction between asbestos exposure and DNAm on the MPM odds ratio were applied. Epigenome-wide analysis (EWAS) revealed 12 single-CpGs associated with the disease. Two of these showed high statistical power (99%) and effect size (>0.05) after false discovery rate (FDR) multiple comparison corrections: (i) cg03546163 in FKBP5, significantly hypomethylated in cases (Mean Difference in beta values (MD) = −0.09, 95% CI = −0.12|−0.06, p = 1.2 × 10−7), and (ii) cg06633438 in MLLT1, statistically hypermethylated in cases (MD = 0.07, 95% CI = 0.04|0.10, p = 1.0 × 10−6). Based on the interaction analysis, asbestos exposure and epigenetic profile together may improve MPM risk assessment. Above-median asbestos exposure and hypomethylation of cg03546163 in FKBP5 (OR = 20.84, 95% CI = 8.71|53.96, p = 5.5 × 10−11) and hypermethylation of cg06633438 in MLLT1 (OR = 11.71, 95% CI = 4.97|29.64, p = 5.9 × 10−8) genes compared to below-median asbestos exposure and hyper/hypomethylation of single-CpG DNAm, respectively. Receiver Operation Characteristics (ROC) for Case-Control Discrimination showed a significant increase in MPM discrimination when DNAm information was added in the model (baseline model, BM: asbestos exposure, age, gender and white blood cells); area under the curve, AUC = 0.75; BM + cg03546163 at FKBP5. AUC = 0.89, 2.1 × 10−7; BM + cg06633438 at MLLT1. AUC = 0.89, 6.3 × 10−8. Validation and replication procedures, considering independent sample size and a different DNAm analysis technique, confirmed the observed associations. Our results suggest the potential application of DNAm profiles in blood to develop noninvasive tests for MPM risk assessment in asbestos-exposed subjects.

Malignant pleural mesothelioma (MPM) is an aggressive tumor with median survival of 12 months and limited effective treatments. The scope of this study was to study the relationship between blood DNA methylation (DNAm) and overall survival (OS) aiming at a noninvasive prognostic test. We investigated a cohort of 159 incident asbestos exposed MPM cases enrolled in an Italian area with high incidence of mesothelioma. Considering 12 months as a cut-off for OS, epigenome-wide association study (EWAS) revealed statistically significant (p value = 7.7 × 10−9) OS-related differential methylation of a single-CpG (cg03546163), located in the 5′UTR region of the FKBP5 gene. This is an independent marker of prognosis in MPM patients with a better performance than traditional inflammation-based scores such as lymphocyte-to-monocyte ratio (LMR). Cases with DNAm < 0.45 at the cg03546163 had significantly poor survival compared with those showing DNAm ≥ 0.45 (mean: 243 versus 534 days; p value< 0.001). Epigenetic changes at the FKBP5 gene were robustly associated with OS in MPM cases. Our results showed that blood DNA methylation levels could be promising and dynamic prognostic biomarkers in MPM.

A growing amount of evidence suggests that the downregulation of protein synthesis is an adaptive response during physiological aging, which positively contributes to longevity and can be modulated by nutritional interventions like caloric restriction (CR). The expression of ribosomal RNA (rRNA) is one of the main determinants of translational rate, and epigenetic modifications finely contribute to its regulation. Previous reports suggest that hypermethylation of ribosomal DNA (rDNA) locus occurs with aging, although with some species- and tissue- specificity. In the present study, we experimentally measured DNA methylation of three regions (the promoter, the 5′ of the 18S and the 5′ of 28S sequences) in the rDNA locus in liver tissues from rats at two, four, 10, and 18 months. We confirm previous findings, showing age-related hypermethylation, and describe, for the first time, that this gain in methylation also occurs in human hepatocytes. Furthermore, we show that age-related hypermethylation is enhanced in livers of rat upon CR at two and 10 months, and that at two months a trend towards the reduction of rRNA expression occurs. Collectively, our results suggest that CR modulates age-related regulation of methylation at the rDNA locus, thus providing an epigenetic readout of the pro-longevity effects of CR.

The production of reactive oxygen species (ROS) may promote immunosenescence if not counterbalanced by the antioxidant systems. Cell membranes, proteins, and nucleic acids become the target of ROS and progressively lose their structure and functions. This process could lead to an impairment of the immune response. However, little is known about the capability of the immune cells of elderly individuals to dynamically counteract the oxidative stress. Here, the response of the main lymphocyte subsets to the induced oxidative stress in semisupercentenarians (CENT), their offspring (OFF), elderly controls (CTRL), and young individuals (YO) was analyzed using flow cytometry. The results showed that the ratio of the ROS levels between the induced and noninduced (I/NI) oxidative stress conditions was higher in CTRL and OFF than in CENT and YO, in almost all T, B, and NK subsets. Moreover, the ratio of reduced glutathione levels between I/NI conditions was higher in OFF and CENT compared to the other groups in almost all the subsets. Finally, we observed significant correlations between the response to the induced oxidative stress and the degree of methylation in specific genes on the oxidative stress pathway. Globally, these data suggest that the capability to buffer dynamic changes in the oxidative environment could be a hallmark of longevity in humans.

We developed a new statistical framework to find genetic variants associated with extreme longevity. The method, informed GWAS (iGWAS), takes advantage of knowledge from large studies of age-related disease in order to narrow the search for SNPs associated with longevity. To gain support for our approach, we first show there is an overlap between loci involved in disease and loci associated with extreme longevity. These results indicate that several disease variants may be depleted in centenarians versus the general population. Next, we used iGWAS to harness information from 14 meta-analyses of disease and trait GWAS to identify longevity loci in two studies of long-lived humans. In a standard GWAS analysis, only one locus in these studies is significant (APOE/TOMM40) when controlling the false discovery rate (FDR) at 10%. With iGWAS, we identify eight genetic loci to associate significantly with exceptional human longevity at FDR < 10%. We followed up the eight lead SNPs in independent cohorts, and found replication evidence of four loci and suggestive evidence for one more with exceptional longevity. The loci that replicated (FDR < 5%) included APOE/TOMM40 (associated with Alzheimer's disease), CDKN2B/ANRIL (implicated in the regulation of cellular senescence), ABO (tags the O blood group), and SH2B3/ATXN2 (a signaling gene that extends lifespan in Drosophila and a gene involved in neurological disease). Our results implicate new loci in longevity and reveal a genetic overlap between longevity and age-related diseases and traits, including coronary artery disease and Alzheimer's disease. iGWAS provides a new analytical strategy for uncovering SNPs that influence extreme longevity, and can be applied more broadly to boost power in other studies of complex phenotypes.

BackgroundAlzheimer's Disease (AD) is the most common neurodegenerative disease and the leading cause of dementia among senile subjects. It has been proposed that AD can be caused by defects in mitochondrial oxidative phosphorylation. Given the fundamental contribution of the mitochondrial genome (mtDNA) for the respiratory chain, there have been a number of studies investigating the association between mtDNA inherited variants and multifactorial diseases, however no general consensus has been reached yet on the correlation between mtDNA haplogroups and AD.Methodology/principal findingsWe applied for the first time a high resolution analysis (sequencing of displacement loop and restriction analysis of specific markers in the coding region of mtDNA) to investigate the possible association between mtDNA-inherited sequence variation and AD in 936 AD patients and 776 cognitively assessed normal controls from central and northern Italy. Among over 40 mtDNA sub-haplogroups analysed, we found that sub-haplogroup H5 is a risk factor for AD (OR=1.85, 95% CI:1.04-3.23) in particular for females (OR=2.19, 95% CI:1.06-4.51) and independently from the APOE genotype. Multivariate logistic regression revealed an interaction between H5 and age. When the whole sample is considered, the H5a subgroup of molecules, harboring the 4336 transition in the tRNAGln gene, already associated to AD in early studies, was about threefold more represented in AD patients than in controls (2.0% vs 0.8%; p=0.031), and it might account for the increased frequency of H5 in AD patients (4.2% vs 2.3%). The complete re-sequencing of the 56 mtDNAs belonging to H5 revealed that AD patients showed a trend towards a higher number (p=0.052) of sporadic mutations in tRNA and rRNA genes when compared with controls.ConclusionsOur results indicate that high resolution analysis of inherited mtDNA sequence variation can help in identifying both ancient polymorphisms defining sub-haplogroups and the accumulation of sporadic mutations associated with complex traits such as AD.