Investigation of MLH1, MGMT, CDKN2A, and RASSF1A Gene Methylation in Thymomas From Patients With Myasthenia Gravis

A feature of thymomas is their frequent association with myasthenia gravis (MG), an autoimmune disease characterized by the production of autoantibodies directed to different targets at the neuromuscular junction. Indeed, almost 30–40% of thymomas are found in patients with a type of MG termed thymoma-associated MG (TAMG). Recent studies suggest that TAMG-associated thymomas could represent a molecularly distinct subtype of thymic epithelial tumors (TETs), but few data are still available concerning the epigenetic modifications occurring in TAMG tissues. The promoter methylation levels of DNA repair (MLH1 and MGMT) and tumor suppressor genes (CDKN2A and RASSF1A) have been frequently investigated in TETs, but methylation data in TAMG tissues are scarce and controversial. To further address this issue, we investigated MLH1, MGMT, CDKN2A, and RASSF1A methylation levels in blood samples and surgically resected thymomas from 69 patients with TAMG and in the adjacent normal thymus available from 44 of them. Promoter methylation levels of MLH1, MGMT, CDKN2A, and RASSF1A genes were not increased in cancer with respect to healthy tissues and did not correlate with the histological or pathological features of the tumor or with the MG symptoms. The present study suggests that hypermethylation of these genes is not frequent in TAMG tissues

Reduced mitochondrial D-loop methylation levels in sporadic amyotrophic lateral sclerosis

Background: Mitochondrial dysregulation and aberrant epigenetic mechanisms have been frequently reported in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), and several researchers suggested that epigenetic dysregulation in mitochondrial DNA (mtDNA) could contribute to the neurodegenerative process. We recently screened families with mutations in the major ALS causative genes, namely C9orf72, SOD1, FUS, and TARDBP, observing reduced methylation levels of the mtDNA regulatory region (D-loop) only in peripheral lymphocytes of SOD1 carriers. However, until now no studies investigated the potential role of mtDNA methylation impairment in the sporadic form of ALS, which accounts for the majority of disease cases. The aim of the current study was to investigate the D-loop methylation levels and the mtDNA copy number in sporadic ALS patients and compare them to those observed in healthy controls and in familial ALS patients. Pyrosequencing analysis of D-loop methylation levels and quantitative analysis of mtDNA copy number were performed in peripheral white blood cells from 36 sporadic ALS patients, 51 age- and sex-matched controls, and 27 familial ALS patients with germinal mutations in SOD1 or C9orf72 that represent the major familial ALS forms. Results: In the total sample, D-loop methylation levels were significantly lower in ALS patients compared to controls, and a significant inverse correlation between D-loop methylation levels and the mtDNA copy number was observed. Stratification of ALS patients into different subtypes revealed that both SOD1-mutant and sporadic ALS patients showed lower D-loop methylation levels compared to controls, while C9orf72-ALS patients showed similar D-loop methylation levels than controls. In healthy controls, but not in ALS patients, D-loop methylation levels decreased with increasing age at sampling and were higher in males compared to females. Conclusions: Present data reveal altered D-loop methylation levels in sporadic ALS and confirm previous evidence of an inverse correlation between D-loop methylation levels and the mtDNA copy number, as well as differences among the major familial ALS subtypes. Overall, present results suggest that D-loop methylation and mitochondrial replication are strictly related to each other and could represent compensatory mechanisms to counteract mitochondrial impairment in sporadic and SOD1-related ALS forms

Preferential X Chromosome Inactivation as a Mechanism to Explain Female Preponderance in Myasthenia Gravis

Myasthenia gravis (MG) is a neuromuscular autoimmune disease characterized by preva- lence in young women (3:1). Several mechanisms proposed as explanations for gender bias, including skewed X chromosome inactivation (XCI) and dosage or sex hormones, are often involved in the development of autoimmunity. The skewed XCI pattern can lead to an unbalanced expression of some X-linked genes, as observed in several autoimmune disorders characterized by female predominance. No data are yet available regarding XCI and MG. We hypothesize that the preferential XCI pattern may contribute to the female bias observed in the onset of MG, especially among younger women. XCI analysis was performed on blood samples of 284 women between the ages of 20 and 82. XCI was tested using the Human Androgen Receptor Assay (HUMARA). XCI patterns were classified as random (XCI < 75%) and preferential (XCI 75%). In 121 informative patients, the frequency of skewed XCI patterns was 47%, significantly higher than in healthy controls (17%; p 0.00001). Interestingly, the phenomenon was observed mainly in younger patients (<45 years; p 0.00001). Furthermore, considering the XCI pattern and the other clinical characteristics of patients, no signif- icant differences were found. In conclusion, we observed preferential XCI in MG female patients, suggesting its potential role in the aetiology of MG, as observed in other autoimmune diseases in women

Comparison Study of MS-HRM and Pyrosequencing Techniques for Quantification of APC and CDKN2A Gene Methylation

There is increasing interest in the development of cost-effective techniques for the quantification of DNA methylation biomarkers. We analyzed 90 samples of surgically resected colorectal cancer tissues for APC and CDKN2A promoter methylation using methylation sensitive-high resolution melting (MS-HRM) and pyrosequencing. MS-HRM is a less expensive technique compared with pyrosequencing but is usually more limited because it gives a range of methylation estimates rather than a single value. Here, we developed a method for deriving single estimates, rather than a range, of methylation using MS-HRM and compared the values obtained in this way with those obtained using the gold standard quantitative method of pyrosequencing. We derived an interpolation curve using standards of known methylated/ unmethylated ratio (0%, 12.5%, 25%, 50%, 75%, and 100% of methylation) to obtain the best estimate of the extent of methylation for each of our samples. We observed similar profiles of methylation and a high correlation coefficient between the two techniques. Overall, our new approach allows MS-HRM to be used as a quantitative assay which provides results which are comparable with those obtained by pyrosequencing

Mitochondrial dna methylation and human diseases

Epigenetic modifications of the nuclear genome, including DNA methylation, histone modifications and non-coding RNA post-transcriptional regulation, are increasingly being involved in the pathogenesis of several human diseases. Recent evidence suggests that also epigenetic modifications of the mitochondrial genome could contribute to the etiology of human diseases. In particular, altered methylation and hydroxymethylation levels of mitochondrial DNA (mtDNA) have been found in animal models and in human tissues from patients affected by cancer, obesity, diabetes and cardiovascular and neurodegenerative diseases. Moreover, environmental factors, as well as nuclear DNA genetic variants, have been found to impair mtDNA methylation patterns. Some authors failed to find DNA methylation marks in the mitochondrial genome, suggesting that it is unlikely that this epigenetic modification plays any role in the control of the mitochondrial function. On the other hand, several other studies successfully identified the presence of mtDNA methylation, particularly in the mitochondrial displacement loop (D-loop) region, relating it to changes in both mtDNA gene transcription and mitochondrial replication. Overall, investigations performed until now suggest that methylation and hydroxymethylation marks are present in the mtDNA genome, albeit at lower levels compared to those detectable in nuclear DNA, potentially contributing to the mitochondria impairment underlying several human diseases

Epigenetic effects of nano-sized materials.

The term epigenetics includes several phenomena such as DNA methylation, histone tail modifications, and microRNA mediated mechanisms, which are able to mold the chromatin structure and/or gene expression levels, without altering the primary DNA sequence. Environmental agents can exert epigenetic properties and there is increasing evidence of epigenetic deregulation of gene expression in several human diseases, including cancer, cardiovascular diseases, autism spectrum disorders, autoimmune diseases, and neurodegeneration, among others. Given the widespread use and dispersion in the environment of nano-sized materials, this article summarizes the studies performed so far to evaluate their potential epigenetic properties. Those studies highlight the ability of certain nano-sized compounds to induce an impaired expression of genes involved in DNA methylation reactions leading to global DNA methylation changes, as well as changes of gene specific methylation of tumor suppressor genes, inflammatory genes, and DNA repair genes, all potentially involved in cancer development. Moreover, some nano-sized compounds are able to induce changes in the acetylation and methylation of histone tails, as well as microRNA deregulated expression. We also provided a detailed description of currently available methodologies to evaluate epigenetic modifications. Standard protocols are currently available to evaluate cytotoxic and genotoxic effects of nano-sized materials. By contrast, there are at present no available standard protocols to evaluate the epigenetic potential of any given compound. The currently available methodologies offer different, but often complementary information to characterize potential epigenetic changes induced by exposure to nano-sized compounds. Given the widespread use and dispersion in the environment of nano-sized materials, at present and foreseeable in the near future, and in light of the indication of potential epigenetic properties here reviewed, more attention should be paid to unravel the consequences of such effects in future studies

Plasma homocysteine and polymorphisms of genes involved in folate metabolism correlate with DNMT1 gene methylation levels

DNA methyltransferase 1 (DNMT1) is responsible for the maintenance of DNA methylation patterns during cell division. Several human diseases are characterized by impaired DNMT1 gene methylation, but less is known about the factors that regulate DNMT1 promoter methylation levels. Dietary folates and related B-vitamins are essential micronutrients for DNA methylation processes, and we performed the present study to investigate the contribution of circulating folate, vitamin B12, homocysteine, and common polymorphisms in folate pathway genes to the DNMT1 gene methylation levels. We investigated DNMT1 gene methylation levels in peripheral blood DNA samples from 215 healthy individuals. All the DNA samples were genotyped for MTHFR 677C > T (rs1801133) and 1298A > C (rs1801131), MTRR 66A > G (rs1801394), MTR 2756A > G (rs1805087), SLC19A1 (RFC1) 80G > A (rs1051266), TYMS 28-bp tandem repeats (rs34743033) and 1494 6-bp insertion/deletion (indel) (rs34489327), DNMT3A-448A > G (rs1550117), and DNMT3B-149C > T (rs2424913) polymorphisms. Circulating homocysteine, folate, and vitamin B12 levels were available from 158 of the recruited individuals. We observed an inverse correlation between plasma homocysteine and DNMT1 methylation levels. Furthermore, both MTR rs1805087 and TYMS rs34743033 polymorphisms showed a statistically significant effect on DNMT1 methylation levels. The present study revealed several correlations between the folate metabolic pathway and DNMT1 promoter methylation that could be of relevance for those disorders characterized by altered DNA methylation

Polymorphisms of genes required for methionine synthesis and DNA methylation influence mitochondrial DNA methylation

Aim: Impaired methylation of the mitochondrial DNA and particularly in the regulatory displacement loop (D-loop) region, is increasingly observed in patients with neurodegenerative disorders. The present study aims to investigate if common polymorphisms of genes required for one-carbon metabolism (MTHFR, MTRR, MTR and RFC-1) and DNA methylation reactions (DNMT1, DNMT3A and DNMT3B) influence D-loop methylation levels. Materials & methods: D-loop methylation data were available from 133 late-onset Alzheimer's disease patients and 130 matched controls. Genotyping was performed with PCR-RFLP or high resolution melting techniques. Results: Both MTRR 66A > G and DNMT3A -448A > G polymorphisms were significantly associated with D-loop methylation levels. Conclusion: This exploratory study suggests that MTRR and DNMT3A polymorphisms influence mitochondrial DNA methylation; further research is required to better address this issue