IntroductionSex-based differences in immune responses to vaccination are well-documented, yet the underlying epigenetic mechanisms remain poorly understood. This study investigates DNA methylation profiles in B cells following hepatitis B virus (HBV) vaccination, with a focus on sex-specific patterns.MethodsUsing high-resolution genome-wide methylation analysis, we examined post-vaccination samples from healthy male and female health care workers.ResultsOur results reveal distinct methylation signatures associated with vaccine response, with several loci showing sex-dependent differential methylation. Pathway analysis identified immune-related genes and regulatory elements potentially involved in B cell activation and memory formation. Our findings show that DNA methylation levels differ between responders versus non-responders to HBV vaccination and these alterations vary with biological sex.DiscussionUnderstanding these epigenetic variations may open new perspective on vaccination practice. Collecting data on B cell epigenetics in different vaccination protocols could improve our knowledge on immunization function and contribute to more personalized vaccination strategies.

Medication overuse headache (MOH) causes substantial disability in suffering patients, significantly reducing the quality of life. It may lead to structural and functional brain changes detectable by neuroimaging. Successful and effective treatments can alleviate headache burden, reduce consumption of abused drugs and reverse the biological alterations. Therefore, improvement of therapeutic strategies and optimization of patient stratification to match the individuals who can benefit with certain approaches, is essential. This study investigates DNA methylation (DNAm) associated with response to MOH treatment. This analysis was performed within the frame of Epimode project - prospective quantitative longitudinal observational study of genome-wide DNA methylation in neurological cohort. 18 MOH patients, in age range between 33 and 66 years, received treatment which included education on diagnosis, recommendation to stop overused drugs and eventually 5-day inpatient withdrawal program. The effectiveness of MOH therapy was evaluated based on (a) overuse outcome (remission or persistence of MOH diagnosis) and (b) mitigating effect (reversal from chronic to episodic headache attacks), both assessed after 5 months from the enrolment. Genome-wide DNA methylation assay was performed (Infinium Human MethylationEPIC BeadChip, Illumina) in whole blood samples collected at baseline and after 3, 5 and 9 months from enrolment. Performed DNAm differential analysis identified methylation signatures linked to MOH treatment and revealed genes involved in epigenetic regulation of response. Among the others, we found PTPRN2, RHOJ, PCDH-γs, CACNA family and SLC38A4 genes as associated with MOH overuse outcome; MUC4 and FKBP11 - as related to mitigating effect of the intervention; and FMOD or ZDHHC14 which resulted linked to both endpoints. We observed that differential methylation signal seemed to predominantly capture the changes associated to excessive use of acute medications. Pathway enrichment analysis demonstrated that the genes with differential methylation signal might be involved in pathways related to (i) metabolic detoxification and (ii) neuropsychiatric/behavioral regulation. Our findings describe methylation patterns, genes and pathways that may be associated with response to MOH intervention.

Background Circulating microRNAs (miRNAs) have been suggested as candidate biomarkers of frailty, a complex multidomain geriatric syndrome associated with a higher risk of poor outcomes. However, few studies have adopted an unbiased approach to screen for plasma miRNAs associated with frailty independently of common risk factors and little is known relatively to their putative etiopathogenic role. Methods Leveraging a cohort recruited for a randomized trial testing the effect a remote rehabilitation intervention on frailty in elderlies with cardiovascular diseases (CVD), we tested plasma samples from 24 people, 12 frail and 12 not frail according to the EFT scale and matched for all the other clinical characteristics, to search for miRNAs associated with frailty through small RNA-Sequencing. From this analysis, we selected the 11 most significantly deregulated miRNAs and quantified them in the entire cohort (n = 197, frail = 57, non-frail = 140) through quantitative RT-PCR. Then, we explored the candidate pathways targeted by miRNAs significantly deregulated in frailty through bioinformatics analyses and tested the effect of miR-181b-3p and miR-490-5p overexpression on cellular senescence and inflammation in endothelial cells. Results We found that plasma levels of miR-181b-3p, miR-490-5p, and miR-500a-5p were increased, while miR-511-5p was reduced, in people with frailty compared with non-frail individuals, also after adjustment for age. Bioinformatics analysis of the frailty-upregulated miRNAs suggested cell cycle and cellular response to DNA damage, two recognized drivers of aging, as regulated biological processes for both miR-181b-3p and miR-490-5p. Overexpression of these two miRNAs through transient transfection in endothelial cells promoted a prototypical cellular senescence response accompanied by a pro-inflammatory phenotype, as assessed through SA-Beta Gal staining and increased expression of multiple senescence and inflammatory markers at both mRNA and protein levels. Conclusions Overall, these data suggest that circulating levels of miR-181b-3p, miR-490-5p, miR-500a-5p and miR-511-5p might serve as markers of frailty in old people with CVD and that miR-181b-3p and miR-490-5p might be functionally linked to the etiopathogenesis of the syndrome through their effects on cellular senescence and inflammation.

Migraine is the most frequent primary disorder of medication overuse headache (MOH). As MOH progresses, patients tend to increase medication doses, which paradoxically results in more frequent headaches and reduced efficacy of analgesics. Epigenetic age, which can be evaluated by DNA methylation (DNAm)-based models, has been associated with disease risk, morbidity, and mortality in several health conditions. The principal aim of this work was to study epigenetic aging-related mechanisms in MOH. This study is part of Epimode project in which MOH (n = 23), episodic migraineurs (EM, n = 18) and healthy controls (HC, n = 13) were recruited. MOH group received therapy including education and detoxification. Responsiveness to MOH intervention was evaluated based on a reduction in headache frequency from chronic to episodic, consistent with established diagnostic thresholds, and did not imply complete cure of MOH. Six subsets of biomarkers derived from DNA methylation were calculated to evaluate changes in epigenetic aging: DNAm clocks, PC-clocks, components of GrimAge and FitAge clocks, DNAm-estimated blood cell composition measures and EpiScores. We used two‑stage residual‑outcome regression (2SR) approach to perform disease-related analysis comparing MOH patients with HC, and intervention-related analysis comparing non-responders (NR) with responders (R) before and after the treatment. We observed epigenetic age deceleration (GrimAge, HannumAge and ZhangAge) in MOH. Compared to controls, MOH showed decreased DNAm-based surrogates of PAI1 and CCL11, and increased estimates of VO2max, B2M, cystatin C and C9. At baseline, there were no differences in epigenetic clocks between R and NR but increased estimated levels of cystatin C, ADM and GDF15, and decreased NEP and CXCL10 were found in MOH NR group. After detoxification, intervention-related differences were observed in DNAmAge, DunedInPoAm, GrimAge, surrogates of ADM, GDF15, TARC, OSM, VCAM1 and estimated counts of natural killer cells (NK). Longitudinal exploration of three timepoints (baseline, after 5 and 9 months) demonstrated that alterations tended to be preserved over time. Our findings indicate that the DNA methylation aging-related mechanisms may be involved in the MOH pathology as well as in MOH treatment.

Drug Combinations offer increased therapeutic efficacy and reduced toxicity compared with single agents. Understanding a drug combination's mechanisms of action (MoA) can provide important insights into therapeutic efficacy. The MoA of many FDA-approved drugs, however, often remains unclear. To decipher the underlying molecular mechanisms of drugs used alone and in combination, we investigated the combination of a statin (atorvastatin or simvastatin) plus ezetimibe using drug-treated RNA-seq transcriptome data from the human hepatocyte-like SOAT2-only-HepG2 cells and from liver biopsies of non-obese normolipidemic patients with uncomplicated cholesterol gallstone disease in the Stockholm Study. We proposed a novel Boolean logical modeling framework to simulate the MoA of a drug combination using fourteen two-variable Boolean models. Thereafter, a pattern matching approach was applied to associate drug-induced differentially expressed genes with the idealized differential expression templates derived from Boolean models. We found 1560 and 565 genes differentially expressed in at least one treatment condition in SOAT2-only-HepG2 cells and liver biopsies, respectively. Our analysis revealed both expected and novel combinatorial modes of the statins and ezetimibe. We mapped the downstream genes of each combinatorial mode to the human protein-protein interactome and obtained underlying pathways, which are important for understanding the therapeutic effects of the drug combinations. Functional enrichment and disease-association analyses of the downstream genes also provide critical insights into the additional therapeutic actions of the drugs. Our study demonstrates that drug-induced transcriptomes, integrated with the human interactome, are informative in deciphering the MoA of drug combinations using Boolean logical modeling.

Biomarkers of ageing are defined as age-related changes in body function or composition that could serve as a measure of 'biological' age and predict the onset of age-related diseases and/or residual life expectancy. We conducted the MARK-AGE Study, a European population study (3300 subjects aged 35-74) to identify a powerful set of biomarkers of ageing. A total of 362 clinical-chemistry, genetic, cellular or molecular biomarkers were analysed for each subject. Using statistical models as well as machine learning we derived mathematical formulas for females and for males that yield a 'bioage score' of an individual, based on sets of 10 biomarkers for females and 10 for males. Collectively, these biomarkers model chronological age of our study population and, thus yield the 'biological' age of a certain person. 'Age difference' (defined as biological minus chronological age) should then identify biologically older or younger individuals. Using our set of biomarkers, subjects with Down Syndrome and smoking females are biologically older, whereas postmenopausal females taking hormone replacement therapy are biologically younger. Strikingly, our data reveal that age difference of MARK-AGE subjects, but not chronological age, is linearly correlated with levels of HDL, 25-hydroxy-Vitamin D, and CD3+ CD4+/CD45+ ratio in such a way that biologically younger subjects display values that are favourable to good health, whereas other markers such as glucose and HbA1c are correlated with chronological age, but not age difference. This dichotomy of correlations may point to different roles of such markers, that is, drivers of the ageing process versus bystanders of ageing.

Changes in gene function or expression caused by epigenetic modifications may play a role in painful diabetic neuropathy. Two independent cohorts of patients deeply phenotyped for painful diabetic neuropathy underwent whole genome DNA methylation data analysis. Burden of rare site events at the global, chromosomal and gene level; epigenetic homogeneity for regions enriched in epivariants (epilesions) and functional analysis of the genes with stochastic phenomena was undertaken. This revealed significant involvement of the SLIT/ROBO signaling axis-engaged in peripheral nerve regeneration after injury, among several molecular pathways, making it an attractive therapeutic target in patients with diabetic painful neuropathy.

About one out of two diabetic patients develop diabetic neuropathy (DN), of these 20% experience neuropathic pain (NP) leading to individual, social, and health-economic burden. Risk factors for NP are largely unknown; however, premature aging was recently associated with several chronic pain disorders. DNA methylation-based biological age (DNAm) is associated with disease risk, morbidity, and mortality in different clinical settings. The purpose of this work was to study, for the first time, whether biological age is involved in pain development in a huge cohort of DN patients with neuropathy assessed by anatomopathological assay (99 painful (PDN), 132 painless (PLDN) patients, 84 controls (CTRL)). Six subsets of DNAm biomarkers were calculated to evaluate NP-associated changes in epigenetic aging, telomere shortening, blood cell count estimates, and plasma protein surrogates. We observed pain-related acceleration of epigenetic age (DNAmAgeHannum, DNAmGrimAgeBasedOnPredictedAge, DNAmAgeSkinBloodClock), pace of aging (DunedinPoAm), and shortening of telomeres between PDN and PLDN patients. PDN showed decreased predicted counts of B lymphocytes, naive and absolute CD8 T cells, and increased granulocyte counts. Several surrogates of plasma proteins were significantly different (GHR, MMP1, THBS2, PAPPA, TGF-α, GDF8, EDA, MPL, CCL21) in PDNs compared to PLDNs. These results provide the first evidence of an acceleration of biological aging in patients with painful compared to painless DN. This achievement has been possible thanks to the state of the art clinical phenotyping of the enrolled patients. Our findings indicate that the aging process may be directly involved in the PDN progression and in general health degeneration in the T2DM patients. Therefore, it is possible to hypothesize that the administration of effective antiaging drugs could slow down or even block the disease advancement.

Convergent adaptations represent paradigmatic examples of the capacity of natural selection to influence organisms' biology. However, the possibility to investigate the genetic determinants underpinning convergent complex adaptive traits has been offered only recently by methods for inferring polygenic adaptations from genomic data. Relying on this approach, we demonstrate how high-altitude Andean human groups experienced pervasive selective events at angiogenic pathways, which resemble those previously attested for Himalayan populations despite partial convergence at the single-gene level was observed. This provides additional evidence for the drivers of convergent evolution of enhanced blood perfusion in populations exposed to hypobaric hypoxia for thousands of years.

Environmental toxicants, including chlorinated solvents present in contaminated water sources, can modify human DNA methylation (DNAm), an epigenetic mechanism that underlies biological plasticity in response to environmental perturbations. Given the relevance of these epigenetic mechanisms, this study examines the DNAm profiles of communities near Bussi sul Tirino (Abruzzo, Italy), an area declared a Site of National Interest (SNI) in 2008 due to extensive environmental contamination, including chlorinated solvents. The aim was to determine whether long-standing groundwater contamination is associated with changes in DNAm variability. Specifically, buccal swabs were collected from 61 volunteers classified into high (HLE) and low (LLE) exposure groups based on proximity to contaminated water sources. Subsequently, bisulfite sequencing (MiSeq, Illumina) was used to assess DNAm in repetitive elements, while genome-wide DNAm and genotyping were performed on a subset of 32 individuals using Illumina MethylationEPIC and HumanOmniExpress 720k BeadChips, respectively. Genome-wide analysis identified differentially methylated positions enriched in genes related to embryonic/cellular development, nervous system development, and immune function. In particular, the strongest association was observed at cg04879348 (GCC2 gene; p = 6.52 ×10-5), previously linked to organochlorine exposure in a group of workers diagnosed with Parkinson's disease. Importantly, the HLE group displayed reduced DNAm variability, potentially reflecting environmental pressure on epigenetic regulation, and a trend toward LINE-1 hypomethylation, suggesting genomic instability. However, no differences were found in epimutation load and epigenetic aging in the oral tissue of this group. In conclusion, this study provides novel evidence that past long-term environmental contamination exposure can shape DNAm profiles in exposed populations, highlighting the relevance of epigenetic markers in environmental health research.

Cytomegalovirus (CMV) infection has been linked to accelerated biological aging, potentially increasing the risk of cardiovascular disease. DNA methylation of the gene Elongation Of Very Long Chain Fatty Acids-Like 2 (ELOVL2) is a molecular biomarker for aging, and its gene product is involved in polyunsaturated fatty acid synthesis, which impacts immune and inflammatory responses. This study, conducted in the MARK-AGE population, aimed to investigate the relationship between CMV infection and ELOVL2 methylation in adults aged 35-75, as well as the influence of CMV IgG levels on lipid metabolism, inflammation, DNA damage, and DNA repair. Our data revealed a higher prevalence of ischemic heart disease, atrial fibrillation, hypertension, and diabetes in CMV-positive individuals. CMV IgG levels were positively associated with ELOVL2 methylation at specific CpG sites and with increased expression of DNA methyltransferase-1 (DNMT1). CMV IgG was linked to lipid imbalances, such as increased BMI, VLDL-cholesterol, triglycerides, and HDL1-cholesterol. Additionally, ELOVL2 methylation was associated with systemic inflammation markers, lipid parameters and altered T-cell subsets. A negative correlation was observed between CMV IgG levels and both baseline DNA integrity and repair capacity. These results suggest that CMV infection might promote cardiovascular disease through ELOVL2 hypermethylation, lipid dysregulation, inflammation, and DNA damage.

Background/Objectives: Parkinson’s disease (PD) is an adult-onset neurodegenerative disorder whose pathogenesis is still not completely understood. Several lines of evidence suggest that alterations in epigenetic architecture may contribute to the development of this condition. Here, we present a pilot DNA methylation study from peripheral blood in a cohort of Sicilian PD patients and matched controls. Peripheral tissue analysis has previously been shown to reflect molecular and functional profiles relevant to neurological diseases, supporting their validity as a proxy for studying brain-related epigenetic mechanisms. Methods: We analyzed 20 PD patients and 20 healthy controls (19 males and 21 females overall), matched for sex, with an age range of 60–87 years (mean 72.3 years). Peripheral blood DNA was extracted and processed using the Illumina Infinium MethylationEPIC v2.0 BeadChip, which interrogates over 935,000 CpG sites across the genome, including promoters, enhancers, CpG islands, and other regulatory elements. The assay relies on sodium bisulfite conversion of DNA to detect methylation status at single-base resolution. Results: Epigenome-wide association study (EWAS) data allowed for multiple levels of analysis, including immune cell-type deconvolution, estimation of biological age (epigenetic clocks), quantification of stochastic epigenetic mutations (SEMs) as a measure of epigenomic stability, and differential methylation profiling. Immune cell-type inference revealed an increased but not significant proportion of monocytes in PD patients, consistent with previous reports. In contrast, epigenetic clock analysis did not reveal significant differences in biological age acceleration between cases and controls, partially at odds with earlier studies—likely due to the limited sample size. SEMs burden did not differ significantly between groups. Epivariations reveal genes involved in pathways known to be altered in dopaminergic neuron dysfunction and α-synuclein toxicity. Differential methylation analysis, however, yielded 167 CpG sites, of which 55 were located within genes, corresponding to 54 unique loci. Gene Ontology enrichment analysis highlighted significant overrepresentation of pathways with neurological relevance, including regulation of synapse structure and activity, axonogenesis, neuron migration, and synapse organization. Notably, alterations in KIAA0319, a gene involved in neuronal migration, synaptic formation, and cortical development, have previously been associated with Parkinson’s disease at the gene expression level, while methylation changes in FAM50B have been reported in neurotoxic and cognitive contexts; our data suggest, for the first time, a potential epigenetic involvement of both genes in Parkinson’s disease. Conclusions: This pilot study on a Sicilian population provides further evidence that DNA methylation profiling can yield valuable molecular insights into PD. Despite the small sample size, our results confirm previously reported findings and highlight biological pathways relevant to neuronal structure and function that may contribute to disease pathogenesis. These data support the potential of epigenetic profiling of peripheral blood as a tool to advance the understanding of PD and generate hypotheses for future large-scale studies.

The genetics of human longevity has been primarily studied using classical methods developed in genome-wide association studies. With the recent advances in paleogenomics, it is now possible to investigate to what extent ancient population ancestries contribute to complex traits. In this study, we explored the role of ancient genetic components in human longevity by focusing on the Italian Peninsula, whose genetic heritage includes several past genetic ancestries that have contributed to the current European genetic make-up. We analyzed genome-wide data of 333 Italian centenarians and 690 geographically matched healthy controls, and compared their genetic composition to 103 ancient genomes representative of the main past European population ancestries. Our findings indicate that Italian centenarians have a higher genetic affinity with Western Hunter-Gatherer (WHG)-related ancestry compared to controls, according to PCA and f4-statistics. Logistic regression models based on supervised admixture revealed a significant association between higher WHG ancestry and the centenarian status. Additionally, residual-based predictive analysis showed that centenarians exhibit a significantly higher WHG contribution independent of the genetic structuring of the general Italian population. By painting the chromosomes of modern Italians, we also showed a significantly higher number of WHG alleles at pro-longevity SNPs. In the present study, we demonstrate the contribution of ancient genetic components to the longevity phenotype. In particular, we showed a greater contribution from Western Hunter-Gatherer-related ancestry to Italian centenarians, thus suggesting that this pre-Neolithic genetic component, which has been linked to population shifts occurring within Europe after the Last Glacial Maximum, could be beneficial for longevity today.

The aim of this work was to describe the DNA methylation signature and to identify genes associated with neuropathic pain in type 2 diabetes mellitus. We analyzed two independent diabetic neuropathy cohorts: PROPGER consisting of 72 painful and 67 painless patients recruited at the German Diabetes Center in Düsseldorf (DE), and PROPENG comprising 27 painful and 65 painless diabetic neuropathy patients recruited at the University of Manchester (UK). Genome-wide methylation data was generated using Illumina Infinium Methylation EPIC v1.0 BeadChip. We used four different selection criteria to identify promising pain-related genes. Our findings revealed significant differences in methylation patterns between painful and painless diabetic neuropathy and identified a set of individual CpG sites of unique candidate genes associated with the painful phenotype. Several of these genes, including GCH1, MYT1L and MED16, have been previously linked to pain-related phenotypes or diabetes. Through pathway enrichment analysis, we demonstrated that specific epigenetic signatures could contribute to the complex phenotype of diabetic neuropathy and cluster analyses highlighted significant epigenetic dissimilarities between painful and painless phenotypes. Our results uncovered epigenetic differences between painful and painless diabetic neuropathy patients and identified targeted genes linked to neuropathic pain through DNA methylation mechanisms. This approach holds promise for investigating other chronic pain conditions, such as secondary chronic pain from cancer treatment, thoracic surgery, and various transplant settings.

In our recent research on AIDS Elite controllers, we demonstrated that the HLA-B*57:01 allele is part of an extensive MHC haplotype associated with impaired MICA/MICB function. Considering the pivotal role of natural killer (NK) cells and innate immunity in fighting cancer and infectious diseases, we investigated whether this allele could also influence human longevity. To this end, we performed a multinational European study including 2,597 centenarians and 9,973 controls. We found that HLA-B*57:01 was consistently less frequent in the European centenarian cohorts compared with the corresponding controls (OR ≈ 0.68, p &;lt 0.0005). These findings provide the first in vivo genetic evidence that innate immunity and NK cell function are key components of human longevity.

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.

Introduction: Drug combinations offer increased therapeutic efficacy and reduced toxicity and plays a vital role in treating chronic complex diseases. Understanding a drug combination's mechanism of action (MoA) can provide important insights into its therapeutic efficacy. The MoA of many FDA-approved drugs, however, often remains unclear. Methods: In this study, we investigated the combination of a statin [atorvastatin (ATO) or simvastatin (SIM) plus ezetimibe (EZE)] and utilized drug-treated RNA-seq transcriptome data from SOAT2-only-HepG2 cells (treated with ATO 5 umol/L, EZE, 25 umol/L, their combination or vehicle) and from liver biopsies of patients with uncomplicated cholesterol gallstone disease in the Stockholm Study (a single-blind, randomized trial with SIM 80 mg daily, EZE 10 mg daily, their combination, or placebo) to decipher the underlying molecular mechanisms of the drug combination. We proposed a Boolean logical modeling framework to simulate the MoA of a drug combination. Specifically, fourteen two-variable Boolean models were used to describe the combinatory relationships of ATO/SIM and EZE. Thereafter, a pattern matching approach was applied to associate drug-induced differentially expressed genes with the idealized differential expression templates derived from Boolean models. Results: We found 1,560 and 594 genes differentially expressed in at least one treatment condition in SOAT2-only-HepG2 cells and liver biopsies, respectively. Our analysis revealed both expected and novel combinatorial modes of ATO/SIM and EZE. For example, ATO independently activates downstream genes (i.e. B 4 (ATO,EZE)=ATO), and ATO and EZE synergistically inhibits downstream genes (i.e. B 15 (ATO,EZE)=NOT (ATO AND EZE)) , as the two most prevalent MoAs in SOAT2-only-HepG2 cells (Figure 1). Similarly, the combination of SIM and EZE synergistically inhibiting downstream genes was also the most prevalent MoA in the liver biopsies. We mapped the downstream genes of each combinatorial mode to the human interactome and obtained underlying subnetworks, which are important for understanding the therapeutic effects of the drug combination. Functional enrichment and disease-association analyses of the downstream suggest the additional therapeutic indications of the drugs. Conclusion: Drug-induced transcriptomes are informative in deciphering the MoA of drug combinations using Boolean logical modeling. This framework can be easily extended to the combinations of three drugs.