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Allogeneic hematopoietic cell transplantation (HCT) is used to treat many blood-based disorders and malignancies, however it can also result in serious adverse events, such as the development of acute graft-versus-host disease (aGVHD). This study aimed to develop a donor-specific epigenetic classifier to reduce incidence of aGVHD by improving donor selection. Genome-wide DNA methylation was assessed in a discovery cohort of 288 HCT donors selected based on recipient aGVHD outcome; this cohort consisted of 144 cases with aGVHD grades III-IV and 144 controls with no aGVHD. We applied a machine learning algorithm to identify CpG sites predictive of aGVHD. Receiver operating characteristic (ROC) curve analysis of these sites resulted in a classifier with an encouraging area under the ROC curve (AUC) of 0.91. To test this classifier, we used an independent validation cohort (n = 288) selected using the same criteria as the discovery cohort. Attempts to validate the classifier failed with the AUC falling to 0.51. These results indicate that donor DNA methylation may not be a suitable predictor of aGVHD in an HCT setting involving unrelated donors, despite the initial promising results in the discovery cohort. Our work highlights the importance of independent validation of machine learning classifiers, particularly when developing classifiers intended for clinical use., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Webster, Ecker, Moghul, Liu, Dhami, Marzi, Paul, Kuxhausen, Lee, Spellman, Wang, Feber, Rakyan, Peggs and Beck.)

Periodontal disease is a chronic inflammatory disease in which the oral pathogen Porphyromonas gingivalis plays an important role. Porphyromonas gingivalis expresses virulence determinants in response to higher hemin concentrations, but the underlying regulatory processes remain unclear. Bacterial DNA methylation has the potential to fulfil this mechanistic role. We characterized the methylome of P. gingivalis , and compared its variation to transcriptome changes in response to hemin availability. Porphyromonas gingivalis W50 was grown in chemostat continuous culture with excess or limited hemin, prior to whole-methylome and transcriptome profiling using Nanopore and Illumina RNA-Seq. DNA methylation was quantified for Dam/Dcm motifs and all-context N6-methyladenine (6mA) and 5-methylcytosine (5mC). Of all 1,992 genes analyzed, 161 and 268 were respectively over- and under-expressed with excess hemin. Notably, we detected differential DNA methylation signatures for the Dam "GATC" motif and both all-context 6mA and 5mC in response to hemin availability. Joint analyses identified a subset of coordinated changes in gene expression, 6mA, and 5mC methylation that target genes involved in lactate utilization and ABC transporters. The results identify altered methylation and expression responses to hemin availability in P. gingivalis , with insights into mechanisms regulating its virulence in periodontal disease. IMPORTANCE DNA methylation has important roles in bacteria, including in the regulation of transcription. Porphyromonas gingivalis , an oral pathogen in periodontitis, exhibits well-established gene expression changes in response to hemin availability. However, the regulatory processes underlying these effects remain unknown. We profiled the novel P. gingivalis epigenome, and assessed epigenetic and transcriptome variation under limited and excess hemin conditions. As expected, multiple gene expression changes were detected in response to limited and excess hemin that reflect health and disease, respectively. Notably, we also detected differential DNA methylation signatures for the Dam "GATC" motif and both all-context 6mA and 5mC in response to hemin. Joint analyses identified coordinated changes in gene expression, 6mA, and 5mC methylation that target genes involved in lactate utilization and ABC transporters. The results identify novel regulatory processes underlying the mechanism of hemin regulated gene expression in P. gingivalis, with phenotypic impacts on its virulence in periodontal disease., Competing Interests: The authors declare no conflict of interest.

Vitamin B12 has multiple biochemical functions including in the one-carbon cycle generating a methyl group for DNA methylation, and metabolism of fatty acids and amino acids to generate energy via the citric acid cycle. The aim of our study was to use a combined epigenomic and transcriptomic approach to identify novel genes mediating the effect of B12 on adipogenesis.Human pre-adipocytes (CHUB-S7) were treated with a range of B12 (0-500 nM) concentrations from the day of cell seeding until harvesting in discovery and validation experiments prior to genome-wide methylation analysis using the Illumina HumanMethylation 450Beadchip. For transcriptomic analysis, RNA-seq libraries were run on the Illumina HiSeq 2500. To further investigate the expression of any genes on human adipogenesis, a second human preadipocyte strain was studied (SGBS) by real-time quantitative PCR (qRT-PCR).A combined epigenetic and transcriptomic approach in differentiated human pre-adipocyte cell line, CHUB-S7, identified that the Human cartilage chitinase 3-like protein 2 ( CHI3L2) gene was hypo-methylated and had increased expression in low B12 conditions. Furthermore, there was an approximately 1000-fold increase in CHI3L2 expression in the early days of adipocyte differentiation, which paralleled an increase of lipid droplets in differentiated SGBS cells and an increased expression level of markers of mature adipocytes.In summary, we have identified a potential role of the human cartilage chitinase 3-like protein 2 ( CHI3L2) in adipocyte function in the presence of low B12 levels.

Epigenetic mechanisms which play an essential role in normal developmental processes, such as self-renewal and fate specification of neural stem cells (NSC) are also responsible for some of the changes in the glioblastoma (GBM) genome. Here we develop a strategy to compare the epigenetic and transcriptional make-up of primary GBM cells (GIC) with patient-matched expanded potential stem cell (EPSC)-derived NSC (iNSC). Using a comparative analysis of the transcriptome of syngeneic GIC/iNSC pairs, we identify a glycosaminoglycan (GAG)-mediated mechanism of recruitment of regulatory T cells (Tregs) in GBM. Integrated analysis of the transcriptome and DNA methylome of GBM cells identifies druggable target genes and patient-specific prediction of drug response in primary GIC cultures, which is validated in 3D and in vivo models. Taken together, we provide a proof of principle that this experimental pipeline has the potential to identify patient-specific disease mechanisms and druggable targets in GBM., (© 2021. The Author(s).)

Motivation: The Oxford Nanopore Technologies (ONT) MinION is used for sequencing a wide variety of sample types with diverse methods of sample extraction. Nanopore sequencers output FAST5 files containing signal data subsequently base called to FASTQ format. Optionally, ONT devices can collect data from all sequencing channels simultaneously in a bulk FAST5 file enabling inspection of signal in any channel at any point. We sought to visualize this signal to inspect challenging or difficult to sequence samples., Results: The BulkVis tool can load a bulk FAST5 file and overlays MinKNOW (the software that controls ONT sequencers) classifications on the signal trace and can show mappings to a reference. Users can navigate to a channel and time or, given a FASTQ header from a read, jump to its specific position. BulkVis can export regions as Nanopore base caller compatible reads. Using BulkVis, we find long reads can be incorrectly divided by MinKNOW resulting in single DNA molecules being split into two or more reads. The longest seen to date is 2 272 580 bases in length and reported in eleven consecutive reads. We provide helper scripts that identify and reconstruct split reads given a sequencing summary file and alignment to a reference. We note that incorrect read splitting appears to vary according to input sample type and is more common in 'ultra-long' read preparations., Availability and Implementation: The software is available freely under an MIT license at https://github.com/LooseLab/bulkvis., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2018. Published by Oxford University Press.)

The interaction between the (epi)genetic makeup of an individual and his/her environmental exposure record (exposome) is accepted as a determinant factor for a significant proportion of human malignancies. Recent evidence has highlighted the key role of epigenetic mechanisms in mediating gene-environment interactions and translating exposures into tumorigenesis. There is also growing evidence that epigenetic changes may be risk factor-specific ("fingerprints") that should prove instrumental in the discovery of new biomarkers in cancer. Here, we review the state of the science of epigenetics associated with environmental stimuli and cancer risk, highlighting key developments in the field. Critical knowledge gaps and research needs are discussed and advances in epigenomics that may help in understanding the functional relevance of epigenetic alterations. Key elements required for causality inferences linking epigenetic changes to exposure and cancer are discussed and how these alterations can be incorporated in carcinogen evaluation and in understanding mechanisms underlying epigenome deregulation by the environment., (© 2017 International Agency for Research on Cancer (IARC/WHO); licensed by UICC.)

Aim: Epidemiological studies of deoxyribonucleic acid (DNA) methylation in airway disease have largely been conducted using blood or buccal samples. However, given tissue specificity of DNA methylation, these surrogate tissues may not allow reliable inferences about methylation in the lung. We sought to compare the pattern of DNA methylation in blood, buccal and nasal epithelial cells to that in airway epithelial cells from children., Methods: Samples of blood, and buccal, nasal and airway epithelium were obtained from six children undergoing elective anaesthesia for adenotonsillectomy. DNA methylation was assessed at 450 000 5'-C-phosphate-G-3' (CpG) sites using the Illumina HumanMethylation450 array., Results: Eighteen samples from all sites were suitable for analysis. Hierarchical clustering demonstrated that the methylation profile in nasal epithelium was most representative of that in airway epithelium; the profile in buccal cells was moderately similar and that in blood was least similar., Conclusion: DNA methylation in blood poorly reflects methylation in airway epithelium. Future epidemiological studies of DNA methylation and airway diseases should consider measurement of methylation either in buccal cells or, preferably, in nasal epithelial cells., (©2017 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.)

Many human diseases are multifactorial, involving multiple genetic and environmental factors impacting on one or more biological pathways. Much of the environmental effect is believed to be mediated through epigenetic changes. Although many genome-wide genetic and epigenetic association studies have been conducted for different diseases and traits, it is still far from clear to what extent the genomic loci and biological pathways identified in the genetic and epigenetic studies are shared. There is also a lack of statistical tools to assess these important aspects of disease mechanisms. In the present study, we describe a protocol for the integrated analysis of genome-wide genetic and epigenetic data based on permutation of a sum statistic for the combined effects in a locus or pathway. The method was then applied to published type 1 diabetes (T1D) genome-wide- and epigenome-wide-association studies data to identify genomic loci and biological pathways that are associated with T1D genetically and epigenetically. Through combined analysis, novel loci and pathways were also identified, which could add to our understanding of disease mechanisms of T1D as well as complex diseases in general.

Plasma concentrations of biologically active vitamin D (1,25-(OH)(2)D) are tightly controlled via feedback regulation of renal 1alpha-hydroxylase (CYP27B1; positive) and 24-hydroxylase (CYP24A1; catabolic) enzymes. In pregnancy, this regulation is uncoupled, and 1,25-(OH)(2)D levels are significantly elevated, suggesting a role in pregnancy progression. Epigenetic regulation of CYP27B1 and CYP24A1 has previously been described in cell and animal models, and despite emerging evidence for a critical role of epigenetics in placentation generally, little is known about the regulation of enzymes modulating vitamin D homeostasis at the fetomaternal interface. In this study, we investigated the methylation status of genes regulating vitamin D bioavailability and activity in the placenta. No methylation of the VDR (vitamin D receptor) and CYP27B1 genes was found in any placental tissues. In contrast, the CYP24A1 gene is methylated in human placenta, purified cytotrophoblasts, and primary and cultured chorionic villus sampling tissue. No methylation was detected in any somatic human tissue tested. Methylation was also evident in marmoset and mouse placental tissue. All three genes were hypermethylated in choriocarcinoma cell lines, highlighting the role of vitamin D deregulation in this cancer. Gene expression analysis confirmed a reduced capacity for CYP24A1 induction with promoter methylation in primary cells and in vitro reporter analysis demonstrated that promoter methylation directly down-regulates basal promoter activity and abolishes vitamin D-mediated feedback activation. This study strongly suggests that epigenetic decoupling of vitamin D feedback catabolism plays an important role in maximizing active vitamin D bioavailability at the fetomaternal interface.

The use of epigenetic differences between maternal whole blood and fetal (placental) DNA is one of the main areas of interest for the development of noninvasive prenatal diagnosis of aneuploidies. However, the lack of detailed chromosome-wide identification of differentially methylated sites has limited the application of this approach. In this study, we describe an analysis of chromosome-wide methylation status using methylation DNA immunoprecipitation coupled with high-resolution tiling oligonucleotide array analysis specific for chromosomes 21, 18, 13, X, and Y using female whole blood and placental DNA. We identified more than 2000 regions of differential methylation between female whole blood and placental DNA on each of the chromosomes tested. A subset of the differentially methylated regions identified was validated by real-time quantitative polymerase chain reaction. Additionally, correlation of these regions with CpG islands, genes, and promoter regions was investigated. Between 56 to 83% of the regions were located within nongenic regions whereas only 1 to 11% of the regions overlapped with CpG islands; of these, up to 65% were found in promoter regions. In summary, we identified a large number of previously unreported fetal epigenetic molecular markers that have the potential to be developed into targets for noninvasive prenatal diagnosis of trisomy 21 and other common aneuploidies. In addition, we demonstrated the effectiveness of the methylation DNA immunoprecipitation approach in the enrichment of hypermethylated fetal DNA.

It is well recognized that there is a surprising degree of phenotypic variation among genetically identical individuals, even when the environmental influences, in the strict sense of the word, are identical. Genetic textbooks acknowledge this fact and use different terms, such as 'intangible variation' or 'developmental noise', to describe it. We believe that this intangible variation results from the stochastic establishment of epigenetic modifications to the DNA nucleotide sequence. These modifications, which may involve cytosine methylation and chromatin remodelling, result in alterations in gene expression which, in turn, affects the phenotype of the organism. Recent evidence, from our work and that of others in mice, suggests that these epigenetic modifications, which in the past were thought to be cleared and reset on passage through the germline, may sometimes be inherited to the next generation. This is termed epigenetic inheritance, and while this process has been well recognized in plants, the recent findings in mice force us to consider the implications of this type of inheritance in mammals. At this stage we do not know how extensive this phenomenon is in humans, but it may well turn out to be the explanation for some diseases which appear to be sporadic or show only weak genetic linkage.

Priming of NB4 promyelocytic cells with all-trans retinoic acid, followed by extracellular ATP in the presence of a phosphodiesterase inhibitor, elevated cAMP and activated protein kinase A. The order of potency for cAMP production was ATP (EC50 = 95 +/- 13 micromol/L) > ADP > AMP = adenosine. The order of potency of ATP analogues was 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (EC50 = 54 +/- 15 micromol/L) = adenosine 5'-O-(3-thio) triphosphate (EC50 = 66 +/- 4 micromol/L) > ATP > beta,gamma-methylene ATP (EC50 = 200 +/- 55 micromol/L). Adenosine 5'-O-thiomonophosphate and adenosine 5'-O-(2-thio) diphosphate inhibited ATP-induced cAMP production. Differentiation also occurred as measured by increased expression of CD11b and N-formyl peptide receptor and changes in cell morphology. UTP did not elevate cAMP or induce differentiation, indicating that P2Y2, P2Y4, and P2Y6 receptors were not involved. The P2Y11 receptor, a cAMP-linked receptor on promyelocytic HL-60 cells, was detected in NB4 cells by reverse transcription-polymerase chain reaction and northern blotting. This receptor has the same order of potency with respect to cAMP production as that observed in HL-60 cells.