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.
Arsenic is a human carcinogen with weak mutagenic properties that induces tumors through mechanisms not yet completely understood. People worldwide are exposed to arsenic-contaminated drinking water, and epidemiological studies showed a high percentage of lung, bladder, liver, and kidney cancer in these populations. Several mechanisms by which arsenical compounds induce tumorigenesis were proposed including genotoxic damage and chromosomal abnormalities. Over the past decade, a growing body of evidence indicated that epigenetic modifications have a role in arsenic-inducing adverse effects on human health. The main epigenetic mechanisms are DNA methylation in gene promoter regions that regulate gene expression, histone tail modifications that regulate the accessibility of transcriptional machinery to genes, and microRNA activity (noncoding RNA able to modulate mRNA translation). The "double capacity" of arsenic to induce mutations and epimutations could be the main cause of arsenic-induced carcinogenesis. The aim of this review is to better clarify the mechanisms of the initiation and/or the promotion of arsenic-induced carcinogenesis in order to understand the best way to perform an early diagnosis and a prompt prevention that is the key point for protecting arsenic-exposed population. Studies on arsenic-exposed population should be designed in order to examine more comprehensively the presence and consequences of these genetic/epigenetic alterations.
Mitochondrial impairment is a feature of neurodegeneration and many investigators have suggested that epigenetic modifications of the mitochondrial DNA (mtDNA) might be involved in late-onset Alzheimer's disease (LOAD), but evidence in humans is limited. We assessed the methylation levels of the mtDNA D-loop region in blood DNA from 133 LOAD patients and 130 controls, observing a significant 25% reduction of DNA methylation levels in the first group (2.3 versus 3.1%). Overall, the present data indicate that there is a decreased methylation of the D-loop region in LOAD peripheral blood DNA, suggesting that mtDNA epimutations deserve further investigations in AD pathogenesis.
Demethylation of the D-loop region could represent a compensatory mechanism for mtDNA upregulation in carriers of ALS-linked SOD1 mutations.
The wide spectrum of unique needs and strengths of Autism Spectrum Disorders (ASD) is a challenge for the worldwide healthcare system. With the plethora of information from research, a common thread is required to conceptualize an exhaustive pathogenetic paradigm. The epidemiological and clinical findings in ASD cannot be explained by the traditional linear genetic model, hence the need to move towards a more fluid conception, integrating genetics, environment, and epigenetics as a whole. The embryo-fetal period and the first two years of life (the so-called ‘First 1000 Days’) are the crucial time window for neurodevelopment. In particular, the interplay and the vicious loop between immune activation, gut dysbiosis, and mitochondrial impairment/oxidative stress significantly affects neurodevelopment during pregnancy and undermines the health of ASD people throughout life. Consequently, the most effective intervention in ASD is expected by primary prevention aimed at pregnancy and at early control of the main effector molecular pathways. We will reason here on a comprehensive and exhaustive pathogenetic paradigm in ASD, viewed not just as a theoretical issue, but as a tool to provide suggestions for effective preventive strategies and personalized, dynamic (from womb to adulthood), systemic, and interdisciplinary healthcare approach.
Mitochondrial impairment and increased oxidative stress are common features in neurodegenerative disorders, leading researchers to speculate that epigenetic changes in the mitochondrial DNA (mitoepigenetics) could contribute to neurodegeneration. The few studies performed so far to address this issue revealed impaired methylation levels of the mitochondrial regulatory region (D-loop region) in both animal models, postmortem brain regions, or circulating blood cells of patients with Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Those studies also revealed that mtDNA D-loop methylation levels are subjected to a dynamic regulation within the progression of the neurodegenerative process, could be affected by certain neurodegenerative disease-causative mutations, and are inversely correlated with the mtDNA copy number. The methylation levels of other mtDNA regions than the D-loop have been scarcely investigated in human specimens from patients with neurodegenerative disorders or in animal models of the disease, and evidence of impaired methylation levels is often limited to a single study, making it difficult to clarify their correlation with mitochondrial dynamics and gene expression levels in these disorders. Overall, the preliminary results of the studies performed so far are encouraging making mitoepigenetics a timely and attractive field of investigation, but additional research is warranted to clarify the connections among epigenetic changes occurring in the mitochondrial genome, mitochondrial DNA dynamics and gene expression, and the neurodegenerative process.
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