Epigenetic changes in DNA methylation contribute to the development of many diseases, including cancer. In glioblastoma multiforme, the most prevalent primary brain cancer and an incurable tumor with a median survival time of 15 months, a single epigenetic modification, the methylation of the O⁶-Methylguanine-DNA Methyltransferase (MGMT) gene, is a valid biomarker for predicting response to therapy with alkylating agents and also, independently, prognosis. More recently, the progress from single gene to whole-genome analysis of DNA methylation has allowed a better subclassification of glioblastomas. Here, we review the clinically relevant information that can be obtained by studying MGMT gene and whole-genome DNA methylation changes in glioblastomas, also highlighting benefits, including those of liquid biopsy, and pitfalls of the different detection methods. Finally, we discuss how changes in DNA methylation, especially in glioblastomas bearing mutations in the Isocitrate Dehydrogenase (IDH) 1 and 2 genes, can be exploited as targets for tailoring therapy.

Recessive X-linked disorders may occasionally evolve in clinical manifestations of variable severity also in female carriers. For some of such diseases, the frequency of the symptoms’ appearance during women’s life may be particularly relevant. This phenomenon has been largely attributed to the potential skewness of the X-inactivation process leading to variable phenotypes. Nonetheless, in many cases, no correlation with X-inactivation unbalance was demonstrated. However, methods for analyzing skewness have been mainly limited to Human Androgen Receptor methylation analysis (HUMARA). Recently, the X-inactivation process has been largely revisited, highlighting the heterogeneity existing among loci in the epigenetic state within inactive and, possibly, active X-chromosomes. We reasoned that gene-specific and ultra-deep DNA methylation analyses could greatly help to unravel details of the X-inactivation process and the roles of specific X genes inactivation in disease manifestations. We recently provided evidence that studying DNA methylation at specific autosomic loci at a single-molecule resolution (epiallele distribution analysis) allows one to analyze cell-to-cell methylation differences in a given cell population. We here apply the epiallele analysis at two X-linked loci to investigate whether females show allele-specific epiallelic patterns. Due to the high potential of this approach, the method allows us to obtain clearly distinct allele-specific epiallele profiles.

Diagnoses of primary malignant mesenchymal brain tumors are a challenge for pathologists. Here, we report the case of a 52-year-old man with a primary brain tumor, histologically diagnosed as a high-grade glioma, not otherwise specified (NOS). The patient underwent two neurosurgeries in several months, followed by radiotherapy and chemotherapy. We re-examined the tumor samples by methylome profiling. Methylome analysis revealed an epi-signature typical of a primary intracranial sarcoma, DICER1-mutant, an extremely rare tumor. The diagnosis was confirmed by DNA sequencing that revealed a mutation in DICER1 exon 25. DICER1 mutations were not found in the patient’s blood cells, thus excluding an inherited DICER1 syndrome. The methylome profile of the DICER1 mutant sarcoma was then compared with that of a high-grade glioma, a morphologically similar tumor type. We found that several relevant regions were differentially methylated. Taken together, we report the morphological, epigenetic, and genetic characterization of the sixth described case of an adult primary intracranial sarcoma, DICER1-mutant to-date. Furthermore, this case report underscores the importance of methylome analysis to refine primary brain tumor diagnosis and to avoid misdiagnosis among morphologically similar subtypes.

Anderson–Fabry disease (FD) is an X-linked disease caused by a functional deficit of the α-galactosidase A enzyme. FD diagnosis relies on the clinical manifestations and research of GLA gene mutations. However, because of the lack of a clear genotype/phenotype correlation, FD diagnosis can be challenging. Recently, several studies have highlighted the importance of investigating DNA methylation patterns for confirming the correct diagnosis of different rare Mendelian diseases, but to date, no such studies have been reported for FD. Thus, in the present investigation, we analyzed for the first time the genome-wide methylation profile of a well-characterized cohort of patients with Fabry disease. We profiled the methylation status of about 850,000 CpG sites in 5 FD patients, all carrying the same mutation in the GLA gene (exon 6 c.901C>G) and presenting comparable low levels of α-Gal A activity. We found that, although the whole methylome profile did not discriminate the FD group from the unaffected one, several genes were significantly differentially methylated in Fabry patients. Thus, we provide here a proof of concept, to be tested in patients with different mutations and in a larger cohort, that the methylation state of specific genes can potentially identify Fabry patients and possibly predict organ involvement and disease evolution.