Epigenetic Heterogeneity in Friedreich Ataxia Underlies Variable FXN Reactivation

Friedreich ataxia (FRDA) is typically caused by homozygosity for an expanded GAA triplet-repeat in intron 1 of the FXN gene. The expanded repeat induces repressive histone changes and DNA hypermethylation, which result in epigenetic silencing and FXN transcriptional deficiency. A class I histone deacetylase inhibitor (HDACi-109) reactivates the silenced FXN gene, although with considerable inter-individual variability, which remains etiologically unexplained. Because HDAC inhibitors work by reversing epigenetic silencing, we reasoned that epigenetic heterogeneity among patients may help to explain this inter-individual variability. As a surrogate measure for epigenetic heterogeneity, a highly quantitative measurement of DNA hypermethylation via bisulfite deep sequencing, with single molecule resolution, was used to assess the prevalence of unmethylated, partially methylated, and fully methylated somatic FXN molecules in PBMCs from a prospective cohort of 50 FRDA patients. Treatment of the same PBMCs from this cohort with HDACi-109 significantly increased FXN transcript to levels seen in asymptomatic heterozygous carriers, albeit with the expected inter-individual variability. Response to HDACi-109 correlated significantly with the prevalence of unmethylated and partially methylated FXN molecules, supporting the model that FXN reactivation involves a proportion of genes that are amenable to correction in non-dividing somatic cells, and that heavily methylated FXN molecules are relatively resistant to reactivation. FXN reactivation is a promising therapeutic strategy in FRDA, and inter-individual variability is explained, at least in part, by somatic epigenetic heterogeneity.

The MGMT (O6-methylguanine–DNA methyltransferase) promotes methylation and clinical outcomes of salvage temozolomide and irinotecan chemotherapy in progressive Ewing sarcoma: A preliminary report.

e23507 Background: There remains an unmet need to identify prognostic and predictive molecular biomarkers in advance Ewing sarcoma (ES). We sought to assess the influence of MGMT promoter methylation status on response rate, time to progression (TTP), and overall survival (OS) following salvage irinotecan and temozolomide (IT) chemotherapy. Methods: Data of advanced ES patients, treated with IT chemotherapy from Jan, 2014 to Jan, 2020 were retrospectively collected. Patients were required to have previously received and progressed after vincristine, doxorubicin, and cyclophosphamide alternating with ifosfamide and etoposide (VDC-IE). MGMT promoter methylation status was assessed by Methylation Sensitive Restriction Enzyme quantitative-PCR (MSRE-qPCR) on non-decalcified Formalin-fixed paraffin embedded (FFPE) tissue using internally developed primers and the OneStep qMethyl Kit (ZYMO RESEARCH CORP). Responses were assessed by response evaluation criteria in solid tumors (RECIST v. 1.1). TTP and OS were assessed by the Kaplan-Meier method. Survival comparisons were performed by the Log-rank test. Results: Herein, we present data of the preliminary analysis of the first 18 patients who underwent MGMT promoter methylation testing. Patients had a median age of 18 years (range: 5-34 years), and were predominantly male ( n=11; 61%). The primary tumor was located in the pelvis in 9 patients (50%), femur in 3 (17%), tibia in 2 (11%), kidney in 2 (11%), chest wall in one (6%), and scapula in one patient (6%). IT was given in a second ( n=15) or third-line setting ( n=3). At the time of initiation of IT, 13 patients (72%) had distant metastasis, and 5 (28%) had unresectable local progression in the pelvis ( n=4) or chest wall ( n=1). The mean percentage of MGMT promoter methylation was 29% (range: 3-98%). Five patients (28%) had methylated MGMT promoter, whereas the remaining had partially methylated ( n=6; 33%) or unmethylated ( n=7;39%) promoter. Five patients (28%) had objective response, with no observed difference according to MGMT promoter methylation ( p=0.58 for comparison between methylated and unmethylated/ partially methylated). Median TTP was 4.9 and 2.2 months for patients with methylated and partially methylated/ unmethylated MGMT respectively; p=0.76. The corresponding median OS was 69.4 and 14.3 months in favor of the methylated group; p=0.3. Conclusions: This preliminary data suggests a possible prognostic role for MGMT promoter methylation, with a markedly extended median OS for the methylated group. Nevertheless, the median OS difference did not reach statistical significance in this preliminary analysis. We plan to report data of the final analysis after finalizing MGMT testing for the rest of our study patients.

Physical Activity Level Influences MTHFR Gene Methylation Profile in Diabetic Patients

IntroductionMTHFR methylation status is associated with microvascular complications in diabetes, but the factors influencing this profile remain unknown.ObjectiveThe aim of this study was to evaluate the influence of physical activity level and nutritional status on the methylation profile of the MTHFR gene in patients with type 2 diabetes mellitus (T2DM).MethodsA total of 111 patients, 43 men and 68 women diagnosed with DM (7.0 ± 2.3 years), answered the International Physical Activity Questionnaire (IPAQ) and underwent blood collection for biochemical analysis, DNA extraction, and MTHFR gene methylation profile determination.ResultThe comparison of the methylation pattern showed that the partially methylated profile predominates in the insufficiently active group (85%), which does not occur in the sufficiently active group (54%) (p = 0.012). No differences were found in the nutritional status comparison. Logistic regression including overweight, waist circumference, gender, age, time of DM, hypertension, dyslipidemia, smoking, alcoholism, and family DM revealed that the association of the level of physical activity with methylation profile proved to be independent of these confounding variables. Considering the partially methylated profile as a result, being physically inactive favors the partially methylated MTHFR pattern in patients with DM.ConclusionWe concluded that insufficient physical activity is associated with partially methylated pattern of MTHFR promoter.

Sorption and Isomerselective Properties of Binary Sorbent Based on Supramolecular 4-(3-Hydroxypropyloxy)-4'-Formylazobenzene Liquid Crystal and Partially Methylated β-Cyclodextrin

The sorption and selective properties of a composite sorbent based on the supramolecular liquid crystal 4-(3-hydroxypropyloxy)-4'-formylazobenzene (HPOFAB) and partially methylated β-cyclodextrin heptakis-(2,6-di-O-methyl)-β-cyclodextrin (Me2,6-β-CD) have been studied by inverse gas chromatography. The standard thermodynamic functions of sorption on the smectic A phase of the sorbent for 29 volatile organic compounds of different classes were determined and compared with similar functions obtained on a column with “pure” HPOFAB. The reasons for the increase in the retention of all studied compounds (except for the optical isomers of butanediol-2,3) when Me2,6-β-CD (10 wt. %) added to HPOFAB are discussed. It was found that the mixed SA phase of the binary sorbent in conditions of gas-liquid chromatography has moderate values of structural selectivity and enantioselectivity to both low-polar terpene hydrocarbons and polar optical isomers (butanediols-2,3).

Characterization of universal features of partially methylated domains across tissues and species

Background Partially methylated domains (PMDs) are a hallmark of epigenomes in reproducible and specific biological contexts, including cancer cells, the placenta, and cultured cell lines. Existing methods for deciding whether PMDs exist in a sample, as well as their identification, are few, often tailored to specific biological questions, and require high coverage samples for accurate identification. Results In this study, we outline a set of axioms that take a step towards a functional definition for PMDs, describe an improved method for comparable PMD detection across samples with substantially differing sequencing depths, and refine the decision criteria for whether a sample contains PMDs using a data-driven approach. Applying our method to 267 methylomes from 7 species, we corroborated recent results regarding the general association between replication timing and PMD state, and report identification of several reproducibly “escapee” genes within late-replicating domains that escape the reduced expression and hypomethylation of their immediate genomic neighborhood. We also explored the discordant PMD state of orthologous genes between human and mouse, and observed a directional association of PMD state with gene expression and local gene density. Conclusions Our improved method makes low sequencing depth, population-level studies of PMD variation possible and our results further refine the model of PMD formation as one where sequence context and regional epigenomic features both play a role in gradual genome-wide hypomethylation.

Structural analysis of oligomeric fructans from excised leaves of Lolium temulentum

Individual fructan tri-, tetra- and pentasaccharide isomers in neutral, water-soluble extracts from Lolium temulentum were purified and the linkages present in these isomeric oligosaccharides were analysed by combined GC-mass spectrometry of partially methylated alditol acetates. 1-Kestose and neokestose were the most abundant trisaccharides with 6-kestose present in much lower amounts. Analysis of isomers of DP 4 and 5 showed that multiple linkage types were present with structures based on all three trisaccharides. Oligosaccharides based on neokestose but with 2,6 linkages between adjacent fructose residues have not been previously detected in higher plants. © 1992.

Structural analysis of oligomeric fructans from excised leaves of Lolium temulentum

Individual fructan tri-, tetra- and pentasaccharide isomers in neutral, water-soluble extracts from Lolium temulentum were purified and the linkages present in these isomeric oligosaccharides were analysed by combined GC-mass spectrometry of partially methylated alditol acetates. 1-Kestose and neokestose were the most abundant trisaccharides with 6-kestose present in much lower amounts. Analysis of isomers of DP 4 and 5 showed that multiple linkage types were present with structures based on all three trisaccharides. Oligosaccharides based on neokestose but with 2,6 linkages between adjacent fructose residues have not been previously detected in higher plants. © 1992.

Methylation analysis of polysaccharides: Technical advice

© 2018 Elsevier Ltd Glycosyl linkage (methylation) analysis is used widely for the structural determination of oligo- and poly-saccharides. The procedure involves derivatisation of the individual component sugars of a polysaccharide to partially methylated alditol acetates which are analysed and quantified by gas chromatography-mass spectrometry. The linkage positions for each component sugar can be determined by correctly identifying the partially methylated alditol acetates. Although the methods are well established, there are many technical aspects to this procedure and both careful attention to detail and considerable experience are required to achieve a successful methylation analysis and to correctly interpret the data generated. The aim of this article is to provide the technical details and critical procedural steps necessary for a successful methylation analysis and to assist researchers (a) with interpreting data correctly and (b) in providing the comprehensive data required for reviewers to fully assess the work.