Mitochondria are central organelles in regulating apoptosis, cellular metabolism, metabolite biosynthesis, energy production, and overall cellular homeostasis. Over the past years, abundant evidence has shown that mitochondrial dysfunction and the resulting metabolic reprogramming profoundly influence key hallmarks of tumor development, including initiation, progression, angiogenesis, and metastasis, playing a role also in therapeutic resistance. Consequently, mitochondria have emerged as a promising target for anticancer therapy. Beyond well-known mutational abnormalities in the mitochondrial genome, recent studies indicate that altered mitochondrial epigenetic mechanisms could also contribute to cancer etiology. In the current review, we present a brief, up-to-date overview of the literature on mitochondrial epigenetic regulation in cancer. We will focus on the main characterized mitoepigenetic mechanisms, namely mitochondrial DNA (mtDNA) methylation and activity of mtDNA-encoded non-coding RNAs. We also consider bidirectional epigenetic crosstalk between the nucleus and mitochondria, whereby metabolites and signaling pathways coordinate chromatin states and mitochondrial function. Collectively, available evidence links mitoepigenetic alterations to tumor progression and pharmacoresistance, nominating these pathways as tractable targets for pharmacological intervention.

PURPOSE:Thymomas are rare organotypic tumors frequently associated with immune-related paraneoplastic syndromes. The genetic alterations of thymomas are largely unknown and rely on general transcription factor I (GTF2I) and Harvey rat sarcoma (HRAS) mutations. Glutathione transferase omega-1-1 (GSTO1-1) is an atypical member of the glutathione transferase superfamily involved in regulating pro-survival and anti-apoptotic pathways. The association between GSTO1-1 polymorphisms and the increased risk of different types of cancer has been investigated, but no study has focused on thymomas to date. Therefore, we have investigated whether two major GSTO1-1 genetic variants, namely GSTO1∗A140D and GSTO1∗E155del polymorphisms, might represent a risk for thymoma development. PATIENTS AND METHODS:Genotyping was performed in 47 patients diagnosed with thymoma and myasthenia gravis by using a PCR-RFLP assay. RESULTS:The results of our study show no significant correlation between the two polymorphisms and thymoma histotype, stage, disease progression, and myasthenia gravis symptoms. A worse progression-free survival (PFS; 66.8 vs 236.6 months) was found, however, in patients bearing the homozygous GSTO1∗A140D polymorphism, suggesting a potential role for GSTO1-1 in thymoma. Indeed, enzymes with a regulatory function, such as GSTO1-1, could produce very different effects depending on both their activity and the relevance of their substrates in the risk/progression of a specific neoplasm. CONCLUSIONS:GSTO1∗A140D polymorphism deserves to be further explored as a possible risk factor associated with a worse PFS in thymoma. Specific targets of GSTO1-1 in thymomas need to be determined.

Background and objectives: Myasthenia gravis associated with antibodies against muscle-specific kinase (MuSK-MG) is a well-characterized IgG4-autoimmune disease, however, the mechanisms driving IgG4 predominance remain poorly understood. This study investigated whether promoter DNA methylation of cytokine genes involved in IgG4 class switching is associated with this immune response. Methods: Peripheral blood mononuclear cells were isolated from MuSK-MG patients (n=36), acetylcholine receptor myasthenia gravis (AChR-MG) patients as disease controls (n=7), and sex-matched healthy controls (n=12). Promoter DNA methylation of IL4 , IL10 , and IL13 was assessed by methylation-sensitive high-resolution melting and relative cytokine mRNA expression by qPCR. Associations with clinical variables, and antibody levels were subsequently evaluated. Results: MuSK-MG patients showed lower median IL13 promoter methylation compared with healthy controls (p = 0.004). Median IL4 promoter methylation was also reduced in MuSK-MG compared with healthy controls (p < 0.001) and AChR-MG disease controls (p < 0.001), whereas no differences were observed for IL10 promoter methylation. Relative mRNA expression of IL4 (p = 0.0005), IL10 (p = 0.0462), and IL13 (p = 0.0002) was increased in MuSK-MG compared with AChR-MG. Compared with healthy controls, only IL4 expression remained significantly increased (p < 0.0001). Promoter methylation was inversely correlated with relative mRNA expression for IL4 (p < 0.0001), while IL13 showed a similar but non-significant trend (p = 0.054), no association was observed for IL10 . Multivariable analysis demonstrated that treatment at sampling was independently associated with lower IL10 and IL13 promoter methylation, whereas no associations were observed with age, sex, disease phase, or disease duration. Promoter methylation did not correlate with total serum IgG4 or anti-MuSK IgG4 levels. Discussion: MuSK-MG is associated with selective hypomethylation of IL4 and IL13 promoters accompanied by increased cytokine gene expression, while IL10 promoter methylation remains unchanged. The association between treatment and IL10 and IL13 promoter methylation suggests that immunosuppressive therapy may influence epigenetic regulation in MuSK-MG. Together, these findings support a role for epigenetic dysregulation of Th2-associated cytokines in the immunological environment associated with IgG4 subclass switch. To our knowledge, this is the first study investigating IL4 , IL10 , and IL13 promoter DNA methylation in MuSK-MG.

Background/Objectives: Although the etiopathogenesis of autism spectrum disorder (ASD) remains incompletely elucidated, current evidence supports a multifactorial model involving genetic and environmental factors that interact to induce a heterogeneous range of symptoms. In recent years, epigenetic mechanisms, particularly DNA methylation, have been recognized as key contributors to ASD pathophysiology. Alterations in mitochondrial DNA (mtDNA) methylation are also emerging as relevant contributors in several human conditions. The mitochondrial D-loop, a non-coding control region essential for mtDNA replication and transcription, is considered a hotspot for epigenetic regulation and its methylation levels have been found altered in various diseases, such as cancer, metabolic disorders, and neurological illness. However, to date, no studies have investigated mtDNA methylation changes in ASD. Methods: We analyzed the average methylation levels of a fragment containing ten CpG sites within the D-loop region and the mtDNA copy number in peripheral blood samples from 49 children with ASD and 50 neurotypically developing (NT) controls using Methylation-Sensitive High-Resolution Melting and quantitative PCR. Results: No significant differences in D-loop methylation levels were observed between ASD and NT children. Similarly, the mtDNA copy number did not differ between the two groups. No significant correlations were found between D-loop methylation or mtDNA copy number and either ASD severity or age. Conclusions: This is the first study investigating mtDNA methylation in ASD. Our results indicate that methylation of the D-loop region and the mtDNA copy number are not altered in ASD children. Further studies including larger cohorts and extended mtDNA regions are warranted to confirm and expand these findings.

BACKGROUND:The molecular landscape of thymic epithelial tumors has been partially elucidated. GTF2I mutation drives the pathogenesis in approximately 50% of tumors; however, the key molecular aberrations in the other cases remain unclear. METHODS:We designed a panel including the most frequently mutated genes in thymic epithelial tumors and sequenced tumor and normal DNA from 70 patients prospectively accrued at a single institution in the Thymogene trial. Moreover, 19 neoplastic samples were dissociated to isolate tumor cells using flow cytometry. RESULTS:GTF2I mutations were the most common, being present in 41% of patients. GTF2I mutations were prevalent in type A and AB thymomas, in Stage I-II tumors, and in patients without myasthenia gravis. The unique pattern of mutually exclusive and co-occurring mutations suggests a distinct pathogenesis for thymomas with and without GTF2I mutation. In 39% of patients, no mutations were found in the 77 genes evaluated. The absence of epithelial cells in some dissociated tumors highlights the challenge of identifying mutations in a subset of thymic epithelial tumors that lack the GTF2I mutation. Mutational signatures, including COSMIC 1, 19, and 25, were enriched, possibly linked to 5'-methylcytosine deamination and the effects of chemotherapy. CONCLUSIONS:GTF2I mutations drive the growth of a significant portion of thymic epithelial tumors, often in conjunction with other gene mutations. Somatic mutations are not commonly found in many GTF2I wild-type tumors, where the underlying genomic abnormalities remain elusive, even when using a dedicated tool for sequencing thymic epithelial tumors.

Down syndrome (DS) is the most common chromosomal disorder associated with intellectual disability and is characterized by multiple clinical features affecting the neurological, musculoskeletal, and cardiovascular systems. Among these, congenital heart defects (CHD) occur in nearly half of individuals with DS. While the role of nuclear epigenetics in DS has been well characterized, particularly concerning clinical outcomes, mitochondrial epigenetic changes have yet to be thoroughly investigated. Recent evidence suggests that alterations in the methylation pattern of mitochondrial DNA (mtDNA), especially in the regulatory D-loop region, may play a role in various human diseases, including cardiovascular and neurological conditions. This study aimed to investigate D-loop methylation and mtDNA copy number in DS. Peripheral blood DNA samples were collected from 59 individuals with DS and 59 age- and sex-matched controls, ranging in age from newborns to 55 years. Additionally, comparisons were made between DS newborns with CHD (DS-CHD) and without CHD (DS-nonCHD). No significant differences were found in D-loop methylation pattern or mtDNA amount between DS and controls. Similarly, no differences were observed between DS-CHD, DS-nonCHD, and control neonates. Age showed no significant correlation with either biomarker, and only a slight, non-significant increase in mtDNA copy number was observed in males. In conclusion, despite the known mitochondrial dysfunction in DS, particularly in CHD cases, our results suggest that such dysfunction is not associated with changes in D-loop methylation or mtDNA copy number. Further research with larger cohorts is needed to clarify these findings.

Aims: MTHFR is a key enzyme in the one-carbon metabolic pathway, whose activity has been implicated in Down syndrome (DS) and in the development of congenital heart defects (CHDs). The main aim was to assess promoter methylation levels of the MTHFR gene in DS individuals, including those with congenital heart defects (DS-CHD+), and those without (DS-CHD-), as well as control subjects. We also investigated if common MTHFR polymorphisms, namely 677C > T and 1298A > C correlate with MTHFR promoter methylation levels. Patients and Methods: The study included 118 participants: 59 individuals with DS, 25 of which with CHD, and 59 age and gender matched controls. Genomic DNA was extracted from peripheral blood. Methylation-sensitive high-resolution melting and PCR - RFLP were used to assess methylation and genotyping. Results: DS individuals showed significantly higher MTHFR methylation levels than controls (p < 0.0001). No difference in MTHFR methylation levels between DS-CHD+ and DS-CHD- individuals was observed (p = 0.38). MTHFR 677TT carriers showed higher mean MTHFR methylation levels than 677CC carriers (p < 0.05). Conclusion: We observed a significant increase in MTHFR promoter methylation levels in DS individuals compared to controls. Folate metabolism could influence MTHFR methylation levels as shown indirectly by the association of the MTHFR 677C > T polymorphism.

Epigenetic changes affecting genes in the glucocorticoid pathway have been studied as biomarkers for major depressive disorder (MDD). The aim of this cross-sectional study was to evaluate glucocorticoid receptor (GR) gene promoter methylation levels in depressed workers exposed to occupational stress. Nuclear receptor subfamily 3 group C member 1 (NR3C1) promoter methylation levels were measured by methylation-sensitive high-resolution melting (MS-HRM) in 70 patients with MDD and 40 healthy controls. Occupational stress was evaluated in patients and controls using the Job Content Questionnaire (JCQ). NR3C1 promoter methylation levels were found to be significantly higher in MDD patients than in controls (p = 0.0001). A multiple regression analysis revealed a significant positive association between NR3C1 methylation levels and MDD diagnosis (r = 0.507, p < 0.0001), and a negative association with occupational stress (r = -0.218, p = 0.03). No differences in NR3C1 methylation levels were found between depressed patients exposed and non-exposed to previous traumatic events and the history of trauma was not a significant independent predictor of NR3C1 methylation levels. Hypothalamic-pituitary-adrenal (HPA) axis dysregulation through GR gene hypermethylation could play a key role in the pathophysiology of occupational stress-related disorders. Occupational stress could independently contribute to the epigenetic mechanisms underlying vulnerability to psychopathology. Further research is needed focusing on biomarkers for stress-related disorders as a potential tool for the diagnosis and prevention of occupational diseases.

Individuals affected by neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS), are dramatically increasing worldwide. Thus, several efforts are being made to develop strategies for stopping or slowing the spread of these illnesses. Although causative genetic variants linked to the onset of these diseases are known, they can explain only a small portion of cases. The etiopathology underlying the neurodegenerative process in most of the patients is likely due to the interplay between predisposing genetic variants and environmental factors. Epigenetic mechanisms, including DNA methylation, are central candidates in translating the effects of environmental factors in genome modulation, and they play a critical role in the etiology of AD, PD, and ALS. Among the main environmental exposures that have been linked to an increased risk for these diseases, accumulating evidence points to the role of heavy metals, pesticides, and air pollutants. These compounds could trigger neurodegeneration through different mechanisms, mainly neuroinflammation and the induction of oxidative stress. However, increasing evidence suggests that they are also capable of inducing epigenetic alterations in neurons. In this article, we review the available literature linking exposure to metals, pesticides, and air pollutants to DNA methylation changes relevant to neurodegeneration.

Aim: To investigate DNA methylation levels of a panel of genes in thymic epithelial tumors (TETs).Materials & methods: We selected 15 genes among the most promising epigenetic biomarkers of TETs and evaluated their methylation levels in 71 TET samples.Results: thymic carcinomas (TCs) showed hypermethylation of GHSR and ELF3 genes and reduced IL1RN methylation levels compared with thymomas (TMs) and healthy thymic tissues. RAG1 was hypomethylated in TMs compared with healthy thymic tissues. No difference in the methylation levels of the investigated genes was seen among TM stages and subtypes. No changes in blood methylation levels of the investigated genes were seen among TET subtypes.Conclusion: The present study confirms GHSR, ELF3, IL1RN and RAG1 as TET epigenetic biomarkers.

Epigenome-wide studies revealed hundreds of differentially methylated or hydroxymethylated regions in postmortem brains of individuals deceased from neurodegenerative diseases, and those changes were coupled to genome-wide modifications of the tales of flanking histones. The analysis of postmortem neurons of individuals affected by Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis or Huntington's disease, however, did not allow discriminating early epigenetic modifications likely contributing to disease onset from those resulting from the neurodegenerative process itself. Therefore, researchers are currently searching for peripheral epigenetic biomarkers that could represent early indicators of an ongoing neurodegenerative process or linked to the exposure to certain neurotoxic compounds. Intense investigation is carried out to test the beneficial effects of either natural or synthetic compounds exerting epigenetic properties to counteract the neurodegenerative process in animal models of major neurodegenerative disorders. Recent studies suggest that also epigenetic modifications of the mitochondrial DNA could contribute to neurodegeneration, warranting further investigation.

This comprehensive review provides insights and suggested strategies for the analysis of germline variants using second- and third-generation sequencing technologies (SGS and TGS). It addresses the critical stages of data processing, starting from alignment and preprocessing to quality control, variant calling, and the removal of artifacts. The document emphasized the importance of meticulous data handling, highlighting advanced methodologies for annotating variants and identifying structural variations and methylated DNA sites. Special attention is given to the inspection of problematic variants, a step that is crucial for ensuring the accuracy of the analysis, particularly in clinical settings where genetic diagnostics can inform patient care. Additionally, the document covers the use of various bioinformatics tools and software that enhance the precision and reliability of these analyses. It outlines best practices for the annotation of variants, including considerations for problematic genetic alterations such as those in the human leukocyte antigen region, runs of homozygosity, and mitochondrial DNA alterations. The document also explores the complexities associated with identifying structural variants and copy number variations, underscoring the challenges posed by these large-scale genomic alterations. The objective is to offer a comprehensive framework for researchers and clinicians, ensuring that genetic analyses conducted with SGS and TGS are both accurate and reproducible. By following these best practices, the document aims to increase the diagnostic accuracy for hereditary diseases, facilitating early diagnosis, prevention, and personalized treatment strategies. This review serves as a valuable resource for both novices and experts in the field, providing insights into the latest advancements and methodologies in genetic analysis. It also aims to encourage the adoption of these practices in diverse research and clinical contexts, promoting consistency and reliability across studies.

Background/Objectives: One-carbon metabolism is a critical pathway for epigenetic mechanisms. Circulating biomarkers of one-carbon metabolism have been associated with changes in nuclear DNA methylation levels in individuals affected by age-related diseases. More and more studies are showing that even mitochondrial DNA (mtDNA) could be methylated. In particular, methylation of the mitochondrial displacement (D-loop) region modulates the gene expression and replication of mtDNA and, when altered, can contribute to the development of human illnesses. However, no study until now has demonstrated an association between circulating biomarkers of one-carbon metabolism and D-loop methylation levels. Methods: In the study presented herein, we searched for associations between circulating one-carbon metabolism biomarkers, including folate, homocysteine, and vitamin B12, and the methylation levels of the D-loop region in DNA obtained from the peripheral blood of 94 elderly voluntary subjects. Results: We observed a positive correlation between D-loop methylation and vitamin B12 (r = 0.21; p = 0.03), while no significant correlation was observed with folate (r = 0.02; p = 0.80) or homocysteine levels (r = 0.02; p = 0.82). Moreover, D-loop methylation was increased in individuals with high vitamin B12 levels compared to those with normal vitamin B12 levels (p = 0.04). Conclusions: This is the first study suggesting an association between vitamin B12 circulating levels and mtDNA methylation in human subjects. Given the potential implications of altered one-carbon metabolism and mitochondrial epigenetics in human diseases, a deeper understanding of their interaction could inspire novel interventions with beneficial effects for human health.

Genes involved in immune response, inflammation and metabolism are among those most likely affected by changes in DNA methylation (DNAm) and expression levels in amyotrophic lateral sclerosis (ALS) tissues. Unfortunately, it is still largely unclear whether any of these changes precede the onset of disease symptoms or whether most of them are the result of the muscular and metabolic changes that follow symptoms onset. In this article the author discusses the strengths and limitations of the available studies of DNAm in ALS and provides some suggestions on what, in his opinion, could be done in the near future for a better understanding of the DNAm changes occurring in ALS, their link with environmental exposures and their potential clinical utility.