A significant volume of research clearly shows that disease-related methylation changes can be used as biomarkers at all stages of clinical disease management, including risk assessment and ...predisposition screening through early diagnostics to personalization of patient care and monitoring of the relapse and chronic disease. Thus disease-related methylation changes are an attractive source of the biomarkers that can have significant impact on precision medicine. However, the translation of the research findings in methylation biomarkers field to clinical practice is at the very least not satisfactory. That is mainly because the evidence generated in research studies indicating the utility of the disease-related methylation change to predict clinical outcome is in majority of the cases not sufficient to postulate the diagnostic use of the biomarker. The research studies need to be followed by well-designed and systematic investigations of clinical utility of the biomarker that produce data of sufficient quality to meet regulatory approval for the test to be used to make clinically valid decision. In this review, we describe methylation-based IVD tests currently approved for IVD use or at the advanced stages of the development for the diagnostic use. For each of those tests, we analyze the technologies that the test utilizes for methylation detection as well as describe the types of the clinical studies that were performed to show clinical validity of the test and warrant regulatory approval. The examples reviewed here should help with planning of clinical investigations and delivery of the clinical evidence required for the regulatory approval of potential methylation biomarker based IVD tests.
Recent reports based on a substantial number of cases, warrant need for population-based research to determine implications of constitutional methylation of tumor suppressor genes such as BRCA1 ...occurring in healthy tissue in the prediction of cancer. However, the detection of the constitutional methylation in DNA extracted from blood has already been shown to be technologically challenging, mainly because epimutations appear to be present in blood at a very low level. The analytical sensitivity required for low-level methylation detection can be provided by NGS, but this technique is still labor and cost-intensive. We assessed if PCR-based MS-HRM and BeadChip microarray technologies, which are standardized and cost-effective technologies for methylation changes screening, provide a sufficient level of analytical sensitivity for constitutional BRCA1 methylation detection in blood samples. The study included whole blood samples from 67 healthy women, 35 with previously confirmed and 32 with no detectable BRCA1 promoter methylation for which we performed both MS-HRM based BRCA1 gene methylation screening and genome wide methylation profiling with EPIC microarray. Our results shown, that low-level BRCA1 methylation can be effectively detected in DNA extracted from blood by PCR-based MS-HRM. At the same time, EPIC microarray does not provide conclusive results to unambiguously determine the presence of BRCA1 constitutional methylation in MS-HRM epimutation positive samples. The analytical sensitivity of MS-HRM is sufficient to detect low level BRCA1 constitutional epimutation in DNA extracted from blood and BeadChip technology-based microarrays appear not to provide that level of analytical sensitivity.
Purpose: Fibromyalgia (FM) and Chronic Fatigue Syndrome (CFS) affect 0.4% and 1% of society, respectively, and the prevalence of these pain syndromes is increasing. To date, no strong association ...between these syndromes and the genetic background of affected individuals has been shown. Therefore, it is plausible that epigenetic changes might play a role in the development of these syndromes. Patients and Methods: Three previous studies have attempted to elaborate the involvement of genome-wide methylation changes in blood cells in the development of fibromyalgia and chronic fatigue syndrome. These studies included 22 patients with fibromyalgia and 127 patients with CFS, and the results of the studies were largely discrepant. Contradicting results of those studies may be attributed to differences in the omics data analysis approaches used in each study. We reanalyzed the data collected in these studies using an updated and coherent data-analysis framework. Results: Overall, the methylation changes that we observed overlapped with previous results only to some extent. However, the gene set enrichment analyses based on genes annotated to methylation changes identified in each of the analyzed datasets were surprisingly coherent and uniformly associated with the physiological processes that, when affected, may result in symptoms characteristic of fibromyalgia and chronic fatigue syndrome. Conclusion: Methylomes of the blood cells of patients with FM and CFS in three independent studies have shown methylation changes that appear to be implicated in the pathogenesis of these syndromes. Keywords: epigenetics, chronic pain, microarray studies, cytosine methylation
Publicly available repositories such as Genomic Data Commons or Gene Expression Omnibus are a valuable research resource useful for hypothesis driven research as well as validation of the results of ...new experiments. Frequently however, the use of those opulent resources is challenging because advanced computational skills are required to mine deposited data. To address this challenge, we have developed eDAVE, a user-friendly, web and desktop interface enabling intuitive and robust analysis of almost 12 000 methylomes and transcriptomes from over 200 types of cells and tissues deposited in the Genomic Data Commons repository. The application is implemented in Python, supported for major browsers and available at: https://edave.pum.edu.pl/.
Locus-specific methylation in blood differs between individuals. As those changes may represent de novo methylation, induced by environmental factors, we aimed to evaluate the biological and ...methodological limitations of detection of methylation in blood.
We used Methylation-Sensitive High Resolution Melting to analyze methylation at 21 gene loci in peripheral blood DNA samples from 203 healthy women.
Overall nine of the screened loci displayed marked inter-individual variation in methylation frequency with methylation levels predominantly around 1%. The methylation of specific loci showed different association with age and reproducibility of detection.
Our results allowed benchmarking of both technological and biological limitations that need to be accounted for when evaluating locus specific methylation in blood as potential biomarker.
High caloric diet and lack of physical activity are considered main causes of NAFLD, and a change in the diet is still the only effective treatment of this disease. However, molecular mechanism of ...the effectiveness of diet change in treatment of NAFLD is poorly understood. We aimed to assess the involvement of epigenetic mechanisms of gene expression regulation in treatment of NAFLD. Eighteen participants with medium- to high-grade steatosis were recruited and trained to follow the Mediterranean diet modified to include fibre supplements. At three timepoints (baseline, after 30 and 60 days), we evaluated adherence to the diet and measured a number of physiological parameters such as anthropometry, blood and stool biochemistry, liver steatosis and stiffness. We also collected whole blood samples for genome-wide methylation profiling and histone acetylation assessment.
The diet change resulted in a decrease in liver steatosis along with statistically significant, but a minor change in BMI and weight of our study participants. The epigenetic profiling of blood cells identified significant genome-wide changes of methylation and acetylation with the former not involving regions directly regulating gene expression. Most importantly, we were able to show that identified blood methylation changes occur also in liver cells of NAFLD patients and the machine learning-based classifier that we build on those methylation changes was able to predict the stage of liver fibrosis with ROC AUC = 0.9834.
Methylomes of blood cells from NAFLD patients display a number of changes that are most likely a consequence of unhealthy diet, and the diet change appears to reverse those epigenetic changes. Moreover, the methylation status at CpG sites undergoing diet-related methylation change in blood cells stratifies liver biopsies from NAFLD patients according to fibrosis grade.
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