Association of Tissue-Specific DNA Methylation Alterations with α-Thalassemia Southeast Asian Deletion

Hypermethylation of the ribosomal DNA coding sequence and disordered transcription factor binding as hallmarks of acute myeloid leukemia

Distinct patterns of DNA methylation in conventional adenomas involving the right and left colon

Study question Do DNA methylation changes occur in testicular germ cells (TGCs) from patients with impaired spermatogenesis? Summary answer TGCs from men with cryptozoospermia exhibit altered DNA methylation levels at several genomic regions, many of which are associated with genes involved in spermatogenesis. What is known already In the last 15 years, several studies have described DNA methylation changes in sperm of infertile men. More recently, using whole genome bisulfite sequencing (WGBS) we were able to refute these findings by demonstrating that somatic DNA contamination and genetic variation confound methylation studies in swim-up purified sperm of severely oligozoospermic men. However, it remains unknown whether altered DNA methylation plays a role during the development of the germ cells in the testes of these patients. Study design, size, duration For identifying DNA methylation differences associated with impaired spermatogenesis, we compared the TGC methylomes of men with cryptozoospermia (CZ) and men with obstructive azoospermia (n = 4 each), who had normal spermatogenesis and served as controls (CTR). Study participants were selected among an age-matched cohort of 24 CTR and 10 CZ. The selection was based on similar composition of the TGC suspension evaluated by ploidy analysis and absence of somatic DNA. Participants/materials, setting, methods TGCs were isolated from biopsies after short-term cell culture. Presence of somatic DNA was evaluated by analyzing the DNA methylation levels of H19, MEST, DDX4 and XIST. WGBS was performed at ∼14× coverage. Bioinformatic tools were used to compare global DNA methylation levels, identify differentially methylated regions (DMRs) and functionally annotate the DMRs. Single-cell RNA sequencing (scRNA-seq) was used to associate the DNA methylation changes to gene expression. Main results and the role of chance We could not identify any difference in the global DNA methylation level or at imprinted regions between CZ and CTR samples. However, using stringent filters to identify group-specific methylation differences, we detected 271 DMRs, 238 of which were hypermethylated in CZ (binominal test, p < 2.2 × 10–16). The DMRs are associated with 132 genes, 61 of which are known to be differentially expressed at various stages of spermatogenesis according to scRNA-seq studies. Almost all of the DMRs associated with the 61 genes are hypermethylated in CZ (63/67, p = 1.107 × 10–14). As assessed by scRNA-seq, 13 DMR-associated genes, which were mainly expressed during meiosis and spermiogenesis, show a significantly different pattern of expression in CZ patients. In four of these genes, the promoter was hypermethylated in CZ men, which correlates with a lower expression level in these patients. In the other nine genes, most of which downregulated in CZ, germ cell-specific enhancers may be affected. Limitations, reasons for caution The small sample size constitutes a limitation of this study. Furthermore, even though the cellular composition of samples was similar by ploidy analysis, we cannot rule out that the observed DNA methylation changes might be due to differences in the relative proportion of different germ cell types. Wider implications of the findings: Impaired spermatogenesis is associated with DNA methylation changes in testicular germ cells at functionally relevant regions of the genome, which points to an important role of DNA methylation in normal spermatogenesis. The DNA methylation changes may contribute to premature abortion of spermatogenesis and therefore not appear in mature sperm. Trial registration number N/A

Effects of DNA Methylation on Progression to Interstitial Fibrosis and Tubular Atrophy in Renal Allograft Biopsies: A Multi-Omics Approach.

Proliferation-Dependent Alterations of the DNA Methylation Landscape Underlie Hematopoietic Stem Cell Aging

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Recently, more evidences of epigenetic impact on the male fertility, particularly on sperm DNA methylation have been reported. Data related to this issue in livestock males is still limited. The present study analyzed the DNA methylation status of the important gene for spermatogenesis, SIRT1, in ram sperm and its correspondence with semen quality and fertilizing ability. The ejaculates of 10 rams (5 rams - 1.5 years old, and 5 rams - 4 years old) from Synthetic Population Bulgarian Milk breed were evaluated and used for the artificial insemination of 174 ewes in breeding season. Two semen samples from each animal were used for DNA extraction followed by bisulfite conversion. The DNA methylation status of SIRT1 was detected through quantitative methylation-specific PCR using two sets of primers designed specifically for bisulfite-converted DNA sequences to attach methylated and unmethylated sites. On the base of age and conception rate the rams were divided in different groups. Data of semen quality, DNA methylation status of SIRT1 and reproductive performances of each group were statistically processed. Results showed a high average value of DNA methylation of SIRT1 in ram sperm (78.5±23.9%) and wide individual variability among investigated animals, with a coefficient of variation of 34.4%. The 1.5 years old animals tended to have a higher level of SIRT1 methylation than 4 years old animals. The rams in group with high fertilizing ability had significantly higher DNA methylation of SIRT1 in sperm than those with low fertilizing ability. In conclusion, results of this study provided evidence that the alteration of sperm SIRT1 methylation is associated with fertility performances of the rams and, probably, with their age.

Infants undergo extensive developments during their first year of life. Although the biological mechanisms involved are not yet fully understood, changes in the DNA methylation in mammals are believed to play a key role. This study was designed to investigate changes in infant DNA methylation that occurs between 6 and 52 weeks. A total of 214 infant saliva samples from 6 or 52 weeks were assessed using principal component analyses and t-distributed stochastic neighbor-embedding algorithms. Between the two time points, there were clear differences in DNA methylation. To further investigate these findings, paired two-sided student’s t-tests were performed. Differently methylated regions were defined as at least two consecutive probes that showed significant differences, with a q-value < 0.01 and a mean difference > 0.2. After correcting for false discovery rates, changes in the DNA methylation levels were found in 42 genes. Of these, 36 genes showed increased and six decreased DNA methylation. The overall DNA methylation changes indicated decreased gene expression. This was surprising because infants undergo such profound developments during their first year of life. The results were evaluated by taking into consideration the extensive development that occurs during pregnancy. During the first year of life, infants have an overall three-fold increase in weight, while the fetus develops from a single cell into a viable infant in 9 months, with an 875-million-fold increase in weight. It is possible that the findings represent a biological slowing mechanism in response to extensive fetal development. In conclusion, our study provides evidence of DNA methylation changes during the first year of life, representing a possible biological slowing mechanism. We encourage future studies of DNA methylation changes in infants to replicate the findings by using a repeated measures model and less stringent criteria to see if the same genes can be found, as well as investigating whether other genes are involved in development during this period.

BackgroundDNA methylation is a biomarker suited to investigate dynamic processes, such as treatment response. For this analysis we focus on DNA methylation changes during a randomised controlled trial (RCT) in treatment resistant depressed patients comparing racemic ketamine (s.c.) versus midazolam (s.c.) (the Ketamine for Adult Depression Study - KADS). This clinical trial showed a stronger clinical response for patients in the response-guided dosing paradigm. We investigated epigenomic correlates of these dosing paradigms. We expected a correlation between dosing paradigm and DNA methylation changes with a stronger effect on DNA methylation changes for the response-guided paradigm.Aims & ObjectivesWe performed a cross-sectional epigenome-wide association study (EWAS) at end of RCT treatment (after 4 weeks) comparing ketamine versus midazolam on the whole group and stratified by dosing paradigm. We also performed a paired longitudinal EWAS spanning from baseline to end of RCT treatment for the whole sample, then we repeated the analyses stratified by dosing paradigm (fixed and flexible dosing).MethodDNA methylation data from 113 MDD patients in the KADS trial, treated with ketamine or midazolam for four weeks, were analyzed. A protocol amendment led to fixed ‘lower’ and flexible ‘higher’ dose cohorts. Cross-sectional EWAS compared ketamine and midazolam treatments across the whole sample, stratified by dosing paradigm. Longitudinal DNA methylation changes from baseline to week 4 were also examined, stratified by treatment and dosing group. Methylation data were collected using the Illumina Infinium MethylationEPIC 850k BeadChip. Differential methylation analysis was performed using limma and corrected for confounders. Significance was set at p<9.42×10−8 or FDR-adjusted p<0.05.ResultsWhile the overall analysis comparing DNA methylation for the ketamine vs. midazolam group in the entire cohort did not return any epigenome wide significant CpGs, the most significant CpG (cg11159519; p=2.38x10-6) was in the KCNH1 gene, a potassium-channel gene.The stratified analysis in the flexible dosing sub-cohort comparing ketamine vs. midazolam showed one epigenome-wide significant CpG, associated with the PDCD6-gene (cg15945600: p= 7.28x10-8; fdr p-val < 0.05). The analysis comparing both fixed-dosing conditions did not return any epigenome-wide significant CpGs. The analyses comparing flexible dosing (midazolam and ketamine) vs. fixed dosing (midazolam and ketamine) returned a suggestive hit, linked to the NEDD9 gene (cg20023762; p=1.11x10-7; fdr p-val < 0.10). The longitudinal analyses stratified by dosing condition and therapy returned a statistically significant differentially methylated region (DMR) for the flexibly dosed ketamine condition only. This DMR is related to the CAPS2-gene (fdr p-val < 0.05).Discussion & ConclusionsThe results from these stratified analyses indicate that the clinically observed dose-response relationship for ketamine, with a higher anti-depressant effect for the flexible dosing paradigm, has a correlate at the DNA methylation level. DNA methylation changes were primarily observed in the higher-dose conditions, in particular for the patient group receiving high dose ketamine. Specifically, the genes PDCD6 and CAPS2contribute to known biological pathways related to ketamine, e.g., the mTOR/VEGF/BDNF system. These results contribute to the growing field of pharmaco-epigenomics in Psychiatry and help to understand the dynamics of DNA-methylation changes in the background of ketamine-related treatment response.

S-adenosylmethionine Levels Regulate the Schwann Cell DNA Methylome

Differential DNA methylation of the hypothalamic‐pituitary‐adrenal axis related gene FKBP5 has recently been shown to be associated with varying response to environmental influences and may play a role in how well people respond to psychological treatments. Participants (n = 111) received exposure‐based cognitive behavioural therapy (CBT) for agoraphobia with or without panic disorder, or specific phobias. Percentage DNA methylation levels were measured for the promoter region and intron 7 of FKBP5. The association between percentage reduction in clinical severity and change in DNA methylation was tested using linear mixed models. The effect of genotype (rs1360780) was tested by the inclusion of an interaction term. The association between change in DNA methylation and FKBP5 expression was examined. Change in percentage DNA methylation at one CpG site of intron 7 was associated with percentage reduction in severity (β = −4.26, p = 3.90 × 10−4), where a decrease in DNA methylation was associated with greater response to therapy. An interaction was detected between rs1360780 and changes in DNA methylation in the promoter region of FKBP5 on treatment outcome (p = .045) but did not survive correction for multiple testing. Changes in DNA methylation were not associated with FKBP5 expression. Decreasing DNA methylation at one CpG site of intron 7 of FKBP5 was strongly associated with decreasing anxiety severity following exposure‐based CBT. In addition, there was suggestive evidence that allele‐specific methylation at the promoter region may also be associated with treatment response. The results of this study add to the growing literature demonstrating the role of biological processes such as DNA methylation in response to environmental influences.

Persistent organic pollutants (POPs) such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorobiphenyl (PCB) 126 and 153, perfluorooctanesulfonic acid (PFOS), hexabromocyclododecane (HBCD), 2,2,4,4-tetrabromodiphenyl ether (BDE-47), tributyltin (TBT), and methylmercury (MeHg) can be accumulated in seafood and then form a main source for human exposure. Some POPs have been associated with changes in steroid hormone levels in both humans and animals. This study describes the in vitro effects of these POPs and mixtures thereof in H295R adrenocortical carcinoma cells.

Cross-Reactive DNA Microarray Probes Lead to False Discovery of Autosomal Sex-Associated DNA Methylation

Persistent Organic Pollutants