Genome-wide DNA Methylation Patterns Research Articles

Practical implementation of DNA methylation and copy-number-based CNS tumor diagnostics: the Heidelberg experience

Recently, we described a machine learning approach for classification of central nervous system tumors based on the analysis of genome-wide DNA methylation patterns [6]. Here, we report on DNA methylation-based central nervous system (CNS) tumor diagnostics conducted in our institution between the years 2015 and 2018. In this period, more than 1000 tumors from the neurosurgical departments in Heidelberg and Mannheim and more than 1000 tumors referred from external institutions were subjected to DNA methylation analysis for diagnostic purposes. We describe our current approach to the integrated diagnosis of CNS tumors with a focus on constellations with conflicts between morphological and molecular genetic findings. We further describe the benefit of integrating DNA copy-number alterations into diagnostic considerations and provide a catalog of copy-number changes for individual DNA methylation classes. We also point to several pitfalls accompanying the diagnostic implementation of DNA methylation profiling and give practical suggestions for recurring diagnostic scenarios.

Genome-wide DNA methylation patterns of bovine blastocysts derived from in vivo embryos subjected to in vitro culture before, during or after embryonic genome activation

BackgroundAberrant DNA methylation patterns of genes required for development are common in in vitro produced embryos. In this regard, we previously identified altered DNA methylation patterns of in vivo developed blastocysts from embryos which spent different stages of development in vitro, indicating carryover effects of suboptimal culture conditions on epigenetic signatures of preimplantation embryos. However, epigenetic responses of in vivo originated embryos to suboptimal culture conditions are not fully understood. Therefore, here we investigated DNA methylation patterns of in vivo derived bovine embryos subjected to in vitro culture condition before, during or after major embryonic genome activation (EGA). For this, in vivo produced 2-, 8- and 16-cell stage embryos were cultured in vitro until the blastocyst stage and blastocysts were used for genome-wide DNA methylation analysis.ResultsThe 2- and 8-cell flushed embryo groups showed lower blastocyst rates compared to the 16-cell flush group. This was further accompanied by increased numbers of differentially methylated genomic regions (DMRs) in blastocysts of the 2- and 8-cell flush groups compared to the complete in vivo control ones. Moreover, 1623 genomic loci including imprinted genes were hypermethylated in blastocyst of 2-, 8- and 16-cell flushed groups, indicating the presence of genomic regions which are sensitive to the in vitro culture at any stage of embryonic development. Furthermore, hypermethylated genomic loci outnumbered hypomethylated ones in blastocysts of 2- and 16-cell flushed embryo groups, but the opposite occurred in the 8-cell group. Moreover, DMRs which were unique to blastocysts of the 2-cell flushed group and inversely correlated with corresponding mRNA expression levels were involved in plasma membrane lactate transport, amino acid transport and phosphorus metabolic processes, whereas DMRs which were specific to the 8-cell group and inversely correlated with corresponding mRNA expression levels were involved in several biological processes including regulation of fatty acids and steroid biosynthesis processes.ConclusionIn vivo embryos subjected to in vitro culture before and during major embryonic genome activation (EGA) are prone to changes in DNA methylation marks and exposure of in vivo embryos to in vitro culture during the time of EGA increased hypomethylated genomic loci in blastocysts.

DNA methylation and inflammation marker profiles associated with a history of depression

Depression is a common and disabling disorder, representing a major social and economic health issue. Moreover, depression is associated with the progression of diseases with an inflammatory etiology including many inflammatory-related disorders. At the molecular level, the mechanisms by which depression might promote the onset of these diseases and associated immune-dysfunction are not well understood. In this study we assessed genome-wide patterns of DNA methylation in whole blood-derived DNA obtained from individuals with a self-reported history of depression (n = 100) and individuals without a history of depression (n = 100) using the Illumina 450K microarray. Our analysis identified six significant (Šidák corrected P < 0.05) depression-associated differentially methylated regions (DMRs); the top-ranked DMR was located in exon 1 of the LTB4R2 gene (Šidák corrected P = 1.27 × 10-14). Polygenic risk scores (PRS) for depression were generated and known biological markers of inflammation, telomere length (TL) and IL-6, were measured in DNA and serum samples, respectively. Next, we employed a systems-level approach to identify networks of co-methylated loci associated with a history of depression, in addition to depression PRS, TL and IL-6 levels. Our analysis identified one depression-associated co-methylation module (P = 0.04). Interestingly, the depression-associated module was highly enriched for pathways related to immune function and was also associated with TL and IL-6 cytokine levels. In summary, our genome-wide DNA methylation analysis of individuals with and without a self-reported history of depression identified several candidate DMRs of potential relevance to the pathogenesis of depression and its associated immune-dysfunction phenotype.

Epigenetic aging of human hematopoietic cells is not accelerated upon transplantation into mice

BackgroundTransplantation of human hematopoietic stem cells into immunodeficient mice provides a powerful in vivo model system to gain functional insights into hematopoietic differentiation. So far, it remains unclear if epigenetic changes of normal human hematopoiesis are recapitulated upon engraftment into such “humanized mice.” Mice have a much shorter life expectancy than men, and therefore, we hypothesized that the xenogeneic environment might greatly accelerate the epigenetic clock.ResultsWe demonstrate that genome-wide DNA methylation patterns of normal human hematopoietic development are indeed recapitulated upon engraftment in mice—particularly those of normal early B cell progenitor cells. Furthermore, we tested three epigenetic aging signatures, and none of them indicated that the murine environment accelerated age-associated DNA methylation changes.ConclusionsEpigenetic changes of human hematopoietic development are recapitulated in the murine transplantation model, whereas epigenetic aging is not accelerated by the faster aging environment and seems to occur in the cell intrinsically.

Whole genome bisulfite sequencing reveals unique adaptations to high-altitude environments in Tibetan chickens.

BackgroundTibetan chickens living at high altitudes show specific adaptations to high-altitude conditions, but the epigenetic modifications associated with these adaptations have not been characterized.ResultsWe investigated the genome-wide DNA methylation patterns in Tibetan chicken blood by using whole genome bisulfite sequencing. Generally, Tibetan chickens exhibited analogous methylation patterns to that of lowland chickens. A total of 3.92% of genomic cytosines were methylcytosines and 51.22% of cytosines in CG contexts were methylated, which was less than those in lowland chicken (55.69%). Moreover, the base adjacent to the methylcytosines of mCHGs in Tibetan chickens had a preference for T, which was different from that in lowland chickens. In Tibetan chickens, the methylation levels in the promoter were relatively low, while the gene body was also maintained in a hypomethylated state. DNA methylation levels in regions upstream of the transcription start site of genes were negatively correlated with the level of gene expression, and DNA methylation of gene body regions was also negatively related to gene expression.ConclusionsWe generated the genome-wide DNA methylation patterns in Tibetan chickens and our results will be helpful for future epigenetic studies related to adaptations to high-altitude conditions.

The DNA Methylation Landscape of Stickleback Reveals Patterns of Sex Chromosome Evolution and Effects of Environmental Salinity.

Epigenetic mechanisms such as DNA methylation are a key component of dosage compensation on sex chromosomes and have been proposed as an important source of phenotypic variation influencing plasticity and adaptive evolutionary processes, yet little is known about the role of DNA methylation in an ecological or evolutionary context in vertebrates. The threespine stickleback (Gasterosteus aculeatus) is an ecological and evolutionary model system that has been used to study mechanisms involved in the evolution of adaptive phenotypes in novel environments as well as the evolution heteromorphic sex chromosomes and dosage compensation in vertebrates. Using whole genome bisulfite sequencing, we compared genome-wide DNA methylation patterns between threespine stickleback males and females and between stickleback reared at different environmental salinities. Apparent hypermethylation of the younger evolutionary stratum of the stickleback X chromosome in females relative to males suggests a potential role of DNA methylation in the evolution of heteromorphic sex chromosomes. We also demonstrate that rearing salinity has genome-wide effects on DNA methylation levels, which has the potential to lead to the accumulation of epigenetic variation between natural populations in different environments.

Genome-wide DNA methylation profiling in whole blood reveals epigenetic signatures associated with migraine

BackgroundMigraine is a common heritable neurovascular disorder typically characterised by episodic attacks of severe pulsating headache and nausea, often accompanied by visual, auditory or other sensory symptoms. Although genome-wide association studies have identified over 40 single nucleotide polymorphisms associated with migraine, there remains uncertainty about the casual genes involved in disease pathogenesis and how their function is regulated.ResultsWe performed an epigenome-wide association study, quantifying genome-wide patterns of DNA methylation in 67 migraine cases and 67 controls with a matching age and sex distribution. Association analyses between migraine and methylation probe expression, after adjustment for cell type proportions, indicated an excess of small P values, but there was no significant single-probe association after correction for multiple testing (P < 1.09 × 10− 7). However, utilising a 1 kb sliding window approach to combine adjacent migraine-methylation association P values, we identified 62 independent differentially methylated regions (DMRs) underlying migraine (false discovery rate < 0.05). Migraine association signals were subtle but consistent in effect direction across the length of each DMR. Subsequent analyses showed that the migraine-associated DMRs were enriched in regulatory elements of the genome and were in close proximity to genes involved in solute transportation and haemostasis.ConclusionsThis study represents the first genome-wide analysis of DNA methylation in migraine. We have identified DNA methylation in the whole blood of subjects associated with migraine, highlighting novel loci that provide insight into the biological pathways and mechanisms underlying migraine pathogenesis.

Integrative Analysis Identifies Potential DNA Methylation Biomarkers for Pan-Cancer Diagnosis and Prognosis

Background: DNA methylation status is closely associated with diverse diseases, and could be more stable than gene expression, thus DNA methylation could be important biomarkers for tumor diagnosis, treatment and prognosis. However, the signatures regarding DNA methylation changes for pan-cancer diagnosis and prognosis are less explored. Methods: Here we systematically analyzed the genome-wide DNA methylation patterns in diverse TCGA cancers with machine learning. Findings: We identified seven CpG sites that could effectively separate tumor samples from adjacent normal tissue samples from 12 main cancers (1216 samples, AUC > 0.99). Those seven potential diagnostic biomarkers were further validated in the other 9 different TCGA cancers and 4 independent datasets (AUC > 0.92). Three out of the seven CpG sites are correlated with cell division, DNA replication and cell cycle. We also identified 12 CpG sites, that can effectively distinguish 26 different cancers (7605 samples), and the result was repeated in independent datasets of 6 cancers as well as two disparate tumors with metastases (micro-average AUC > 0.89). Furthermore, a series of potential signatures that could significantly predict the prognosis of tumor patients for 7 different cancer were identified via survival analysis (P-value < 1e-4). Interpretation: DNA methylation patterns vary greatly between tumor and adjacent normal tissues, as well as different types of cancers. The results demonstrate that our identified signatures may aid the decision of clinical diagnosis and prognosis for pan-cancer and the potential tissue-specific biomarkers could be used to predict the primary site of metastatic breast and prostate cancers. Funding: This work was supported by the National High Technology Research and Development Program of China (2015AA020108), National Key Research and Development Program of China (2016YFC0902100), National Science Foundation of China (31671377, 31771460, 91629103) and Shanghai 111 Project (B14019). Declaration of Interest: The authors declare no potential conflicts of interest. Ethical Approval: Not applicable

Unlinking the methylome pattern from nucleotide sequence, revealed by large-scale in vivo genome engineering and methylome editing in medaka fish.

The heavily methylated vertebrate genomes are punctuated by stretches of poorly methylated DNA sequences that usually mark gene regulatory regions. It is known that the methylation state of these regions confers transcriptional control over their associated genes. Given its governance on the transcriptome, cellular functions and identity, genome-wide DNA methylation pattern is tightly regulated and evidently predefined. However, how is the methylation pattern determined in vivo remains enigmatic. Based on in silico and in vitro evidence, recent studies proposed that the regional hypomethylated state is primarily determined by local DNA sequence, e.g., high CpG density and presence of specific transcription factor binding sites. Nonetheless, the dependency of DNA methylation on nucleotide sequence has not been carefully validated in vertebrates in vivo. Herein, with the use of medaka (Oryzias latipes) as a model, the sequence dependency of DNA methylation was intensively tested in vivo. Our statistical modeling confirmed the strong statistical association between nucleotide sequence pattern and methylation state in the medaka genome. However, by manipulating the methylation state of a number of genomic sequences and reintegrating them into medaka embryos, we demonstrated that artificially conferred DNA methylation states were predominantly and robustly maintained in vivo, regardless of their sequences and endogenous states. This feature was also observed in the medaka transgene that had passed across generations. Thus, despite the observed statistical association, nucleotide sequence was unable to autonomously determine its own methylation state in medaka in vivo. Our results apparently argue against the notion of the governance on the DNA methylation by nucleotide sequence, but instead suggest the involvement of other epigenetic factors in defining and maintaining the DNA methylation landscape. Further investigation in other vertebrate models in vivo will be needed for the generalization of our observations made in medaka.

Developmental dynamics of the epigenome: A longitudinal study of three toddlers

Epigenetic regulation plays an important role in development, at the embryonic stages and later during the lifespan. Some epigenetic marks are highly conserved throughout the lifespan whereas others are closely associated with specific age periods and/or particular environmental factors. Little is known about the dynamics of epigenetic regulation during childhood, especially during the period of rapid early development. Our study was aimed to determine whether the developmental program at the early stages of human development is accompanied by significant changes in the systems of genome regulation, specifically, by genome-wide changes in DNA methylation. Using a sequencing approach (MBD-seq) we investigated genome-wide DNA methylation patterns in the T-lymphocytes of three healthy toddlers at two timepoints within the second year of life. Pairwise comparison of the methylation patterns across the individuals and time points was conducted to determine common longitudinal changes in the DNA methylation patterns. Despite relatively high interindividual variability in their epigenetic profiles and the dynamics of these profiles during the second year of life, all children showed consistent changes in the DNA methylation patterns of genes involved in the control of the immune system and genes related to the development of the CNS. Thereby, we provide evidence that early development might be accompanied by epigenetic changes in specific functional groups of genes; many such epigenetic changes appear to be related to the rapid development of the CNS.

A genome-wide DNA methylation analysis in peripheral blood from patients identifies risk loci associated with Graves' orbitopathy.

Graves' orbitopathy (GO) is an inflammatory orbital disease of autoimmune origin with the potential to cause severe functional and psychosocial effects. The pathogenesis has not been fully elucidated. We investigated whether DNA methylation was associated with GO incidence in Chinese patients. Six GO patients and six age-matched controls were recruited, and genome-wide DNA methylation patterns were analyzed in their peripheral blood. t tests were performed to determine differential methylated sites in genomic regions and the univariable logistic regression analyses was performed to evaluate their risk with GO incidence. Cluster analysis and principal component analysis (PCA) were performed to determine the effects of the extracted differentially methylated sites. One hundred and forty-eight differentially methylated sites were identified, including CD14 (fold change=4.31, p=0.005), IL17RE (fold change=2.128, p=0.005), and DRD4 (fold change=0.25, p=0.004), and were supported by cluster and PCA analyses. Univariable logistic regression analyses showed that the methylation patterns at 12 loci were associated with GO incidence. The relative risk per 1% decrease in methylation at ZCCHC6 and GLI3 was 0.15 (95% CI 0.03-0.91; p=0.039) and 0.65 (95% CI 0.42-0.98; p=0.042), respectively. Pearson correlation analyses demonstrated that methylation levels at IL17RE were positively associated with Clinical Activity Score (CAS) (r=0.967, p<0.05). Our results demonstrate that differential methylation levels at analyzed sites (genes) may be risk markers of GO. DNA methylation analysis could provide new insights into understanding the disease and provide new treatment strategies for GO in Chinese patients.

Understanding Mitochondrial Polymorphisms in Cancer.

Alterations in mitochondrial DNA (mtDNA) were once thought to be predominantly innocuous to cell growth. Recent evidence suggests that mtDNA undergo naturally occurring alterations, including mutations and polymorphisms, which profoundly affect the cells in which they appear and contribute to a variety of diseases, including cardiovascular disease, diabetes, and cancer. Furthermore, interplay between mtDNA and nuclear DNA has been found in cancer cells, necessitating consideration of these complex interactions for future studies of cancer mutations and polymorphisms. In this issue of Cancer Research, Vivian and colleagues utilize a unique mouse model, called Mitochondrial Nuclear eXchange mice, that contain the nuclear DNA from one inbred mouse strain, and the mtDNA from a different inbred mouse strain to examine the genome-wide nuclear DNA methylation and gene expression patterns of brain tissue. Results demonstrated there were alterations in nuclear DNA expression and DNA methylation driven by mtDNA. These alterations may impact disease pathogenesis. In light of these results, in this review, we highlight alterations in mtDNA, with a specific focus on polymorphisms associated with cancer susceptibility and/or prognosis, mtDNA as cancer biomarkers, and considerations for investigating the role of mtDNA in cancer progression for future studies. Cancer Res; 77(22); 6051-9. ©2017 AACR.

DNA methylation at enhancers identifies distinct breast cancer lineages

Breast cancers exhibit genome-wide aberrant DNA methylation patterns. To investigate how these affect the transcriptome and which changes are linked to transformation or progression, we apply genome-wide expression–methylation quantitative trait loci (emQTL) analysis between DNA methylation and gene expression. On a whole genome scale, in cis and in trans, DNA methylation and gene expression have remarkably and reproducibly conserved patterns of association in three breast cancer cohorts (n = 104, n = 253 and n = 277). The expression–methylation quantitative trait loci associations form two main clusters; one relates to tumor infiltrating immune cell signatures and the other to estrogen receptor signaling. In the estrogen related cluster, using ChromHMM segmentation and transcription factor chromatin immunoprecipitation sequencing data, we identify transcriptional networks regulated in a cell lineage-specific manner by DNA methylation at enhancers. These networks are strongly dominated by ERα, FOXA1 or GATA3 and their targets were functionally validated using knockdown by small interfering RNA or GRO-seq analysis after transcriptional stimulation with estrogen.

P1.02-070 Identification of Lung Cancer Specific Differentially Methylated Regions Using Genome-Wide DNA Methylation Study

The development of lung cancer is resulted by the interaction between genetic mutations and dynamic epigenetic alterations, although its mechanisms are not fully understood. Among the epigenetic alterations, DNA methylation is the most studied epigenetic regulatory mechanism. DNA methylation changes may be promising biomarker for early detection and prognosis in lung cancer. We evaluated serial changes of genome wide DNA methylation pattern in blood of lung cancer patients.

Genome-wide DNA methylome alterations in acute coronary syndrome.

Acute coronary syndrome (ACS) is a common disease with high mortality and morbidity rates. The methylation status of blood DNA may serve as a potential early diagnosis and prevention biomarker for numerous diseases. The present study was designed to explore novel genome-wide aberrant DNA methylation patterns associated with ACS. The Infinium HumanMethylation450 assay was used to examine genome-wide DNA methylation profiles in 3 pairs of ACS and control group samples. Epigenome-wide DNA methylation, genomic distribution, Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed. The results were confirmed using methylation-specific polymerase chain reaction (MSP) and Sequenom MassARRAY analyses in ACS, stable coronary artery disease (SCAD) and control samples. A total of 11,342 differentially methylated (DM) 5′-C-phosphate-G-3′ (CpG) sites were identified, including 8,865 hypomethylated and 2,477 hypermethylated CpG sites in the ACS group compared with the control samples. They varied in frequency across genomic compartments, but were particularly notable in gene bodies and shores. The results of GO term and KEGG pathway enrichment analyses revealed that the methylated genes were associated with certain biological processes and pathways. Despite the considerable variability in methylation data, the candidate selected possessed significant methylation alteration in mothers against decapentaplegic homolog 3 (SMAD3) transcription start site 155 (Chr1:67356838-Chr1:67356942). MSP analysis from 81 ACS samples, 74 SCAD samples and 53 healthy samples, and Sequenom MassARRAY analysis, confirmed that differential CpG methylation of SMAD3 was significantly corrected with the reference results of the HumanMethylation450 array. The data identified an ACS-specific DNA methylation profile with a large number of novel DM CpG sites, some of which may serve as candidate markers for the early diagnosis of ACS.

Hemicastration induced spermatogenesis-related DNA methylation and gene expression changes in mice testis.

ObjectiveHemicastration is a unilateral orchiectomy to remove an injured testis, which can induce hormonal changes and compensatory hypertrophy of the remaining testis, and may influence spermatogenesis. However, the underlying molecular mechanisms are poorly understood. Here, we investigated the impact of hemicastration on remaining testicular function.MethodsPrepubertal mice (age 24 days) were hemicastrated, and their growth was monitored until they reached physical maturity (age 72 days). Subsequently, we determined testis DNA methylation patterns using reduced representation bisulfite sequencing of normal and hemicastrated mice. Moreover, we profiled the testicular gene expression patterns by RNA sequencing (RNA-seq) to examine whether methylation changes affected gene expression in hemicastrated mice.ResultsHemicastration did not significantly affect growth or testosterone (p>0.05) compared with control. The genome-wide DNA methylation pattern of remaining testis suggested that substantial genes harbored differentially methylated regions (1,139) in gene bodies, which were enriched in process of protein binding and cell adhesion. Moreover, RNA-seq results indicated that 46 differentially expressed genes (DEGs) involved in meiotic cell cycle, synaptonemal complex assembly and spermatogenesis were upregulated in the hemicastration group, while 197 DEGs were downregulated, which were related to arachidonic acid metabolism. Integrative analysis revealed that proteasome 26S subunit ATPase 3 interacting protein gene, which encodes a protein crucial for homologous recombination in spermatocytes, exhibited promoter hypomethylation and higher expression level in hemicastrated mice.ConclusionGlobal profiling of DNA methylation and gene expression demonstrated that hemicastration-induced compensatory response maintained normal growth and testicular morphological structure in mice.

DNA Methylation and Transcription Patterns in Intestinal Epithelial Cells From Pediatric Patients With Inflammatory Bowel Diseases Differentiate Disease Subtypes and Associate With Outcome

Background & AimsWe analyzed DNA methylation patterns and transcriptomes of primary intestinal epithelial cells (IEC) of children newly diagnosed with inflammatory bowel diseases (IBD) to learn more about pathogenesis.MethodsWe obtained mucosal biopsies (N = 236) collected from terminal ileum and ascending and sigmoid colons of children (median age 13 years) newly diagnosed with IBD (43 with Crohn’s disease [CD], 23 with ulcerative colitis [UC]), and 30 children without IBD (controls). Patients were recruited and managed at a hospital in the United Kingdom from 2013 through 2016. We also obtained biopsies collected at later stages from a subset of patients. IECs were purified and analyzed for genome-wide DNA methylation patterns and gene expression profiles. Adjacent microbiota were isolated from biopsies and analyzed by 16S gene sequencing. We generated intestinal organoid cultures from a subset of samples and genome-wide DNA methylation analysis was performed.ResultsWe found gut segment-specific differences in DNA methylation and transcription profiles of IECs from children with IBD vs controls; some were independent of mucosal inflammation. Changes in gut microbiota between IBD and control groups were not as large and were difficult to assess because of large amounts of intra-individual variation. Only IECs from patients with CD had changes in DNA methylation and transcription patterns in terminal ileum epithelium, compared with controls. Colon epithelium from patients with CD and from patients with ulcerative colitis had distinct changes in DNA methylation and transcription patterns, compared with controls. In IECs from patients with IBD, changes in DNA methylation, compared with controls, were stable over time and were partially retained in ex-vivo organoid cultures. Statistical analyses of epithelial cell profiles allowed us to distinguish children with CD or UC from controls; profiles correlated with disease outcome parameters, such as the requirement for treatment with biologic agents.ConclusionsWe identified specific changes in DNA methylation and transcriptome patterns in IECs from pediatric patients with IBD compared with controls. These data indicate that IECs undergo changes during IBD development and could be involved in pathogenesis. Further analyses of primary IECs from patients with IBD could improve our understanding of the large variations in disease progression and outcomes.

Whole genome DNA methylation sequencing of the chicken retina, cornea and brain

Whole genome bisulfite sequencing (WGBS) analysis of DNA methylation uses massively parallel next generation sequencing technology to characterize global epigenetic patterns and fluctuations throughout a range of tissue samples. Development of the vertebrate retina is thought to involve extensive epigenetic reprogramming during embryogenesis. The chicken embryo (Gallus gallus) is a classic model system for studying developmental biology and retinogenesis, however, there are currently no publicly available data sets describing the developing chicken retinal methylome. Here we used Illumina WGBS analysis to characterize genome-wide patterns of DNA methylation in the developing chicken retina as well as cornea and brain in an effort to further our understanding of retina-specific epigenetic regulation. These data will be valuable to the vision research community for correlating global changes in DNA methylation to differential gene expression between ocular and neural tissues during critical developmental time points of retinogenesis in the chicken retina.

Global DNA methylation analysis reveals miR-214-3p contributes to cisplatin resistance in pediatric intracranial nongerminomatous malignant germ cell tumors.

Pediatric central nervous system germ cell tumors (CNSGCTs) are rare and heterogeneous neoplasms, which can be divided into germinomas and nongerminomatous germ cell tumors (NGGCTs). NGGCTs are further subdivided into mature teratomas and nongerminomatous malignant GCTs (NGMGCTs). Clinical outcomes suggest that NGMGCTs have poor prognosis and survival and that they require more extensive radiotherapy and adjuvant chemotherapy. However, the mechanisms underlying this difference are still unclear. DNA methylation alteration is generally acknowledged to cause therapeutic resistance in cancers. We hypothesized that the pediatric NGMGCTs exhibit a different genome-wide DNA methylation pattern, which is involved in the mechanism of its therapeutic resistance. We performed methylation and hydroxymethylation DNA immunoprecipitation sequencing, mRNA expression microarray, and small RNA sequencing (smRNA-seq) to determine methylation-regulated genes, including microRNAs (miRNAs). The expression levels of 97 genes and 8 miRNAs were correlated with promoter DNA methylation and hydroxymethylation status, such as the miR-199/-214 cluster, and treatment with DNA demethylating agent 5-aza-2'-deoxycytidine elevated its expression level. Furthermore, smRNA-seq analysis showed 27 novel miRNA candidates with differential expression between germinomas and NGMGCTs. Overexpresssion of miR-214-3p in NCCIT cells leads to reduced expression of the pro-apoptotic protein BCL2-like 11 and induces cisplatin resistance. We interrogated the differential DNA methylation patterns between germinomas and NGMGCTs and proposed a mechanism for chemoresistance in NGMGCTs. In addition, our sequencing data provide a roadmap for further pediatric CNSGCT research and potential targets for the development of new therapeutic strategies.

Exploring genome-wide DNA methylation patterns in Aicardi syndrome.

To explore differential DNA methylation (DNAm) in Aicardi syndrome (AIC), a severe neurodevelopmental disorder with largely unknown etiology. We characterized DNAm in AIC female patients and parents using the Illumina 450K array. Differential DNAm was assessed using the local outlier factor algorithm, and results were validated via qPCR in a larger set of AIC female patients, parents and unrelated young female controls. Functional epigenetic modules analysis was used to detect pathways integrating both genome-wide DNAm and RNA-seq data. We detected differential methylation patterns in AIC patients in several neurodevelopmental and/or neuroimmunological networks. These networks may be part of the underlying pathogenic mechanisms involved in the disease.