Specific Methylation Patterns Research Articles

Factors Affecting Neutrophil Functions During Sepsis: Human Microbiome and Epigenetics.

Sepsis is a severe disease that occurs when the body's immune system reacts excessively to infection. The body's response, which includes an intense anti-bacterial reaction, can damage its tissues and organs. Neutrophils are the major components of white blood cells in circulation and play a vital role in innate immunity while fighting against infections, and are considered a feature determining sepsis classification. There's a plethora of basic research detailing neutrophil functioning, among which, the study of neutrophil extracellular traps (NETs) is providing novel insights into mechanisms and treatments of sepsis. This review explores their functions, dysfunctions, and influences in the context of sepsis. The interplay between neutrophils and the human microbiome and the impact of DNA methylation on neutrophil function in sepsis are crucial areas of study. The interaction between neutrophils and the human microbiome is complex, particularly in the context of sepsis where dysbiosis may occur. We highlight the importance of deciphering neutrophil's functional alterations and their epigenetic features in sepsis because it is critical for defining sepsis endotypes and opening up the possibility for novel diagnostic methods and therapy. Specifically, epigenetic signatures are pivotal since they will provide a novel implication for sepsis diagnostic method when used in combination with the cell-free DNA (cfDNA). Research is exploring how specific patterns of DNA methylation in neutrophils, detectable in cfDNA, could serve as biomarkers for the early detection of sepsis.

Epigenome-Wide Meta-Analysis of 11 Military and Civilian Cohorts Reveals Cell-Type Specific and Inflammatory DNA Methylation Patterns Associated With PTSD

Epigenome-Wide Meta-Analysis of 11 Military and Civilian Cohorts Reveals Cell-Type Specific and Inflammatory DNA Methylation Patterns Associated With PTSD

Lithium response in bipolar disorder: Epigenome-wide DNA methylation signatures and epigenetic aging

Lithium (Li) is the first-line treatment for bipolar disorder (BD) even though only 30 % of BD patients are considered excellent responders. The mechanisms by which Li exerts its action are not clearly understood, but it has been suggested that specific epigenetic mechanisms, such as methylation processes, may play a role. In this regard, DNA methylation patterns can be used to estimate epigenetic age (EpiAge), which is accelerated in BD patients and reversed by Li treatment. Our first aim was to compare the DNA methylation profile in peripheral blood between BD patients categorized as excellent responders to Li (Ex-Rp) and non-responders (N-Rp). Secondly, EpiAge was estimated to detect differential age acceleration between the two groups.A total of 130 differentially methylated positions (DMPs) and 16 differentially methylated regions (DMRs) between Ex-Rp (n = 26) and N-Rp (n = 37) were identified (FDR adjusted p-value < 0.05). We found 122 genes mapping the DMPs and DMRs, nine of which (HOXB6, HOXB3, HOXB-AS3, TENM2, CACNA1B, ANK3, EEF2K, CYP1A1, and SORCS2) had previously been linked to Li response. We found genes related to the GSK3β pathway to be highly represented. Using FUMA, we found enrichment in Gene Ontology Cell Component for the synapse. Gene network analysis highlighted functions related to the cell cycle, nervous system development and function, and gene expression. No significant differences in age acceleration were found between Ex-Rp and N-Rp for any of the epigenetic clocks analysed.Our findings indicate that a specific methylation pattern could determine the response to Li in BD patients. We also found that a significant portion of the differentially methylated genes are closely associated with the GSK3β pathway, reinforcing the role of this system in Li response. Future longitudinal studies with larger samples will help to elucidate the epigenetic mechanisms underlying Li response.

Hypomethylation of ATP1A1 Is Associated with Poor Prognosis and Cancer Progression in Triple-Negative Breast Cancer.

Dysregulated DNA methylation in cancer is critical in the transcription machinery associated with cancer progression. Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, but no treatment targeting TNBC biomarkers has yet been developed. To identify specific DNA methylation patterns in TNBC, methyl-binding domain protein 2 (MBD) sequencing data were compared in TNBC and the three other major breast cancer subtypes. Integrated analysis of DNA methylation and gene expression identified a gene set showing a correlation between DNA methylation and gene expression. ATPase Na+/K+-transporting subunit alpha 1 (ATP1A1) was found to be specifically hypomethylated in the coding sequence (CDS) region and to show increased expression in TNBC. The Cancer Genome Atlas (TCGA) database also showed that hypomethylation and high expression of ATP1A1 were strongly associated with poor survival in patients with TNBC. Furthermore, ATP1A1 knockdown significantly reduced the viability and tumor-sphere formation of TNBC cells. These results suggest that the hypomethylation and overexpression of ATP1A1 could be a prognostic marker in TNBC and that the manipulation of ATP1A1 expression could be a therapeutic target in this disease.

Abstract LB197: Methylation patterns at adjacent CpG sites within enhancers are part of cell identity and are disrupted during malignant transformation

Abstract Background: Following the principles of physiology of DNA methylation, it is generally accepted that methylation status of CpG sites spaced up to 50 bp is correlated and accumulation of locally disordered methylation at CpG sites is a part of neoplastic transformation, acting in similar way as stochastic accumulation of mutations during malignant transformation. Methods: We developed a new algorithm allowing to analyze methylation status at the adjacent CpG sites. We then, used EPIC microarray data to analyze genome wide methylation patterns at the adjacent CpG sites in almost 600 samples, representing 12 healthy tissues and cell types as well as 572 cancer specimens. We also used bisulfite next generation sequencing data and Sanger sequencing to validate our findings. Results: Our analysis, identified in human genome a subset of loci with adjacent CpG sites spaced less than 50bp (average 28bp), harboring one cytosine methylated and the other devoid of methyl group. These loci are enriched in enhancers that are targeted by families of transcription factors involved in cell differentiation. The methylation patterns at these loci can differ between alleles within a cell, what allows for remarkable level of heterogeneity of methylation patterns. However, our analysis also showed that, different types of specialized cells, acquire during differentiation only one specific and remarkably stable pattern of methylation at each of these loci and that pattern is to a large extent lost during neoplastic transformation. Conclusions: Contrary to generally accepted principle of identical methylation status of the adjacent CpG sites being essential for regulatory function of DNA methylation. We have shown that methylation at a subset of adjacent CpG sites in human genome located within important functional genomic elements differs in healthy cells. These CpG sites acquire remarkably stable and tissue specific patterns of methylation during cell differentiation. Moreover, the methylation patterns at these CpG sites, are lost during malignant transformation suggesting the involvement of identified phenomenon in cancer pathology. Funding: This work was funded by grant OPUS22 from Polish National Science Centre (grant number: 2021/43/B/NZ2/02979) and Polish National Agency for Academic Exchange (grant number: PPN/PPO/2018/1/00088/U). Citation Format: Olga Taryma-Leśniak, Jan Bińkowski, Patrycja K. Przybylowicz, Katarzyna E. Sokolowska, Konrad Borowski, Tomasz K. Wojdacz. Methylation patterns at adjacent CpG sites within enhancers are part of cell identity and are disrupted during malignant transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB197.

Effect of CHST11, a novel biomarker, on the biological functionalities of clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is a common malignant tumor, and the role of carbohydrate sulfotransferase 11 (CHST11) in this cancer remains unclear. Here, by using bioinformatics methods, we comprehensively analyzed the relationship between CHST11 and clinical significance, immune infiltration, functional enrichment, m6A methylation, and protein–protein interaction networks. We found that CHST11 expression was significantly higher in ccRCC samples than in normal tissues. Additionally, CHST11 levels correlated with the clinicopathological features of ccRCC patients and functioned as a prognostic factor for patient survival. Functional analysis revealed the involvement of CHST11 in metabolic pathways. Immune infiltration and m6A methylation analysis suggested the association of CHST11 with immune cell abundance in the tumor microenvironment and specific methylation patterns in ccRCC. The in vitro analysis of the clinical samples and ccRCC cell lines demonstrated that the overexpression of CHST11 promotes ccRCC cell proliferation, migration, and invasion, while its suppression has the opposite effect. Thus, CHST11 may play a remarkable role in the occurrence and progression of ccRCC. Functionally, CHST11 promotes the aggressiveness of ccRCC cells. These findings provide insights into the role of CHST11 in ccRCC progression.Registry and the Registration No. of the study/trial: No. 2021K034.

Computationally inferred cell-type specific epigenome-wide DNA methylation analysis unveils distinct methylation patterns among immune cells for HIV infection in three cohorts.

Epigenome-wide association studies (EWAS) have identified CpG sites associated with HIV infection in blood cells in bulk, which offer limited knowledge of cell-type specific methylation patterns associated with HIV infection. In this study, we aim to identify differentially methylated CpG sites for HIV infection in immune cell types: CD4+ T-cells, CD8+ T-cells, B cells, Natural Killer (NK) cells, and monocytes. Applying a computational deconvolution method, we performed a cell-type based EWAS for HIV infection in three independent cohorts (Ntotal = 1,382). DNA methylation in blood or in peripheral blood mononuclear cells (PBMCs) was profiled by an array-based method and then deconvoluted by Tensor Composition Analysis (TCA). The TCA-computed CpG methylation in each cell type was first benchmarked by bisulfite DNA methylation capture sequencing in a subset of the samples. Cell-type EWAS of HIV infection was performed in each cohort separately and a meta-EWAS was conducted followed by gene set enrichment analysis. The meta-analysis unveiled a total of 2,021 cell-type unique significant CpG sites for five inferred cell types. Among these inferred cell-type unique CpG sites, the concordance rate in the three cohorts ranged from 96% to 100% in each cell type. Cell-type level meta-EWAS unveiled distinct patterns of HIV-associated differential CpG methylation, where 74% of CpG sites were unique to individual cell types (false discovery rate, FDR <0.05). CD4+ T-cells had the largest number of unique HIV-associated CpG sites (N = 1,624) compared to any other cell type. Genes harboring significant CpG sites are involved in immunity and HIV pathogenesis (e.g. CD4+ T-cells: NLRC5, CX3CR1, B cells: IFI44L, NK cells: IL12R, monocytes: IRF7), and in oncogenesis (e.g. CD4+ T-cells: BCL family, PRDM16, monocytes: PRDM16, PDCD1LG2). HIV-associated CpG sites were enriched among genes involved in HIV pathogenesis and oncogenesis that were enriched among interferon-α and -γ, TNF-α, inflammatory response, and apoptotic pathways. Our findings uncovered computationally inferred cell-type specific modifications in the host epigenome for people with HIV that contribute to the growing body of evidence regarding HIV pathogenesis.

EBV DNA methylation profiles and its application in distinguishing nasopharyngeal carcinoma and nasal NK/T-cell lymphoma

BackgroundAs an oncovirus, EBV is associated with multiple cancers, including solid tumors and hematological malignancies. EBV methylation plays an important role in regulating tumor occurrence. However, the EBV methylation profiles in EBV-associated tumor tissues are poorly understood.ResultsIn this study, EBV methylation capture sequencing was conducted in several different tumor tissue samples, including NPC, EBVaGC, lung LELC and parotid LELC. Besides, EBV capture sequencing and following qMSP were performed on nasopharyngeal brushing samples from NPC and nasal NKTCL patients. Our results showed that the EBV genome among different types of tumors displayed specific methylation patterns. Among the four types of tumors from epithelial origin (NPC, EBVaGC, lung LELC and parotid LELC), the most significant differences were found between EBVaGC and the others. For example, in EBVaGC, all CpG sites within 1,44,189–1,45,136 bp of the EBV genome sequence on gene RPMS1 were hyper-methylated compared to the others. Differently, significant differences of EBV CpG sites, particularly those located on gene BILF2, were observed between NPC and nasal NKTCL patients in nasopharyngeal brushing samples. Further, the methylated level of BILF2 was further detected using qMSP, and a diagnostic model distinguishing NPC and nasal NKTCL was established. The AUC of the model was 0.9801 (95% CI 0.9524–1.0000), with the sensitivity and specificity of 98.81% (95% CI 93.63–99.94%) and 76.92% (95% CI 49.74–91.82%), respectively.ConclusionsOur study reveals more clues for further understanding the pathogenesis of EBV, and provides a possibility for distinguishing EBV-related tumor by detecting specific EBV CpG sites.

Fine-Scale Map Reveals Highly Variable Recombination Rates Associated with Genomic Features in the Eurasian Blackcap.

Recombination is responsible for breaking up haplotypes, influencing genetic variability, and the efficacy of selection. Bird genomes lack the protein PR domain-containing protein 9, a key determinant of recombination dynamics in most metazoans. Historical recombination maps in birds show an apparent stasis in positioning recombination events. This highly conserved recombination pattern over long timescales may constrain the evolution of recombination in birds. At the same time, extensive variation in recombination rate is observed across the genome and between different species of birds. Here, we characterize the fine-scale historical recombination map of an iconic migratory songbird, the Eurasian blackcap (Sylvia atricapilla), using a linkage disequilibrium-based approach that accounts for population demography. Our results reveal variable recombination rates among and within chromosomes, which associate positively with nucleotide diversity and GC content and negatively with chromosome size. Recombination rates increased significantly at regulatory regions but not necessarily at gene bodies. CpG islands are associated strongly with recombination rates, though their specific position and local DNA methylation patterns likely influence this relationship. The association with retrotransposons varied according to specific family and location. Our results also provide evidence of heterogeneous intrachromosomal conservation of recombination maps between the blackcap and its closest sister taxon, the garden warbler. These findings highlight the considerable variability of recombination rates at different scales and the role of specific genomic features in shaping this variation. This study opens the possibility of further investigating the impact of recombination on specific population-genomic features.

Abstract C058: Differential DNA methylation in HPV+ and HPV- penile tumors from Puerto Rican men

Abstract The incidence of penile squamous cell carcinoma (PSCC) in Puerto Rico is nearly three times higher when compared with other racial and ethnic groups in the United States. Mortality rate for PSCC is also significantly higher among Puerto Rican men when compared to the US population. Human papillomavirus (HPV) is a common virus, and its infection may increase the risk of developing PeCa. Despite the high incidence, the molecular mechanism of PSCC remains poorly understood. Our laboratory found that the prevalence of HPV in PSCC in Puerto Rican men is 49%. Due to the high rates of HPV infection and increased incidence of penile cancer in Puerto Rican men, there is a need to understand the pathogenesis of penile cancer. DNA Methylation is an epigenetic alteration that can be found in cancer cells, which can lead to changes in gene expression and cellular function. This study aims to analyze the DNA methylation patterns in HPV-positive and HPV-negative PSCC tissues from Puerto Rican men. DNA was extracted from twenty-four fresh PSCC tissue samples from Puerto Rican patients. HPV status and genotyping were determined using the DNA ELISA kit HPV SPF10 protocol. DNA was amplified and hybridized using PCR. We perform a DNA methylation assay using the Infinium Methylation EPIC v2.0 BeadChip Kit to identify and compare average DNA methylation levels (%) in the 9 HPV-positive and 15 HPV-negative penile cancer samples. Our results demonstrated 43 loci significantly different between HPV-positive and HPV-negative samples, with an adjusted p-value &amp;lt;0.1 (Benjamini-Hochberg method). Most of the hypermethylated loci were present in HPV-negative tumor samples. Additionally, we identified four methylated-differentially expressed genes: ANXA4, CACNA1C-AS1, UBXN2A, and DUOX1, which are found as uncharacterized proteins (open reading frames, ORFs) of the human genome with adjusted p-value &amp;lt;0.05. Studies have revealed that specific DNA methylation patterns could be linked with cancer development and progression. Identification of differentially methylated regions in HPV positive (HPV+) PSCC compared with HPV negative (HPV-) cases will provide much needed data to aid studies on early detection, prognosis, and development of therapeutic agents to reduce the morbidity and mortality from PeCa. Identification of differentially expressed genes and pathways in HPV positive (HPV+) PeCa compared with HPV negative (HPV-) cases will provide much needed data to aid studies on prevention, early detection, prognosis, and development of therapeutic agents to reduce the morbidity and mortality from PeCa. Citation Format: Yarelis M. Roque Reyes, María Sánchez-Vázquez, Josué Perez, Luis R. Llanos, Ingrid Montes, Jasmine Figueroa-Díaz, Carlos Rivera-Lopez, María Marcos, Antonio Puras, Magaly Martínez-Ferrer. Differential DNA methylation in HPV+ and HPV- penile tumors from Puerto Rican men [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C058.

DDX41-Low Expression Mimics Germline DDX41-Phenotype in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a complex disease characterized by diverse molecular pathogenesis. Genetic alterations, including germline and somatic variants in the DEAD box helicase 41 gene ( DDX41) located on chromosome 5 play an increasingly recognized role. Recent reports indicate that 5% of intensively treated adult AML patients harbor DDX41 germline mutations ( DDX41MutGL), and their precise impact remains incompletely understood. These studies suggest that DDX41MutGL may define a distinct biological subgroup, associated with e.g. older age, male gender, low blast, and low white blood cell count (WBC). To further elucidate the role of DDX41 in AML, we performed a retrospective analysis of 906 unselected adult AML patients from the AML Cooperative Group (AMLCG) registry (2015-2022), by targeted sequencing. As DDX41MutGL are typically “null mutations”, leading to reduced DDX41 expression, we additionally investigated whether DDX41 gene expression correlates or resembles the observed germline phenotype. Our analysis encompassed &amp;gt;1000 independent gene expression profiles (GSE37642, GSE14468, and GSE106291) of intensively treated adult AML patients. We identified a sub-cohort with low DDX41 ( DDX41-low) expression and re-analyzed them with next-generation sequencing to detect DDX41MutGL. Additionally, we correlated gene expression data with 198 DNA methylation profiles (from patients who had undergone both analyses) to identify potential epigenetic mechanisms underlying DDX41-low expression. Among the 906 patients (median age 61 years; range 18-98 years), we identified 11 unrelated individuals with suspected DDX41MutGL (VAF &amp;gt; 40%). Notably, the overall frequency of DDX41MutGL in the German unselected AML patient population was merely 1%, considerably lower than previously reported (p&amp;lt;0.0001 in comparison to Duployez et al. 2022). Characteristically, DDX41MutGL patients in this cohort were mostly male (73%), with a median age of 67 years displaying low WBC (average 1,3G/l) and a normal karyotype (80%) at initial diagnosis. Remarkably, our analysis of independent large gene expression cohorts revealed a pattern of phenotypic association in patients with decreased DDX41 expression ( DDX41-low) closely resembling those with DDX41MutGL. These associations were consistent across different datasets and included e.g. older age, low WBC, and low blast count. Furthermore, DDX41-low patients had poor overall survival. The DDX41-low subgroup constituted almost 10% of all AML patients, surpassing the reported frequencies of DDX41MutGL. In an exploratory pilot study, we screened 48 patients exhibiting the lowest DDX41 gene expression by sequencing the coding regions for DDX41 mutations but found none, suggesting alternative mechanisms, such as copy number changes, non-coding alterations or aberrant DNA methylation patterns that may replicate the phenotypic effects associated with DDX41MutGL. To further elucidate this mechanism, we correlated the gene expression of DDX41 with all DNA methylation loci (CpG sites covered by the EPIC array) on chromosome 5 using Spearman correlation analysis (GSE106291). We plotted the correlations against the sorted chromosome length, identifying a peak at a specific location corresponding to the PCDH (Protocadherin) cluster (Figure). Thus, DDX41-low was associated with reduced methylation at the PCDH loci and linked to higher PCDH gene expression. Across several data sets, we confirmed the association between higher PCDH cluster gene expression and low DDX41 expression. In summary, our study reveals that the frequency of DDX41MutGL in an unselected population of German AML patients is considerably lower than previously reported. Additionally, we found that apart from DDX41MutGL, DDX41-low exhibits a comparable clinical profile and is linked to specific methylation and gene expression patterns, with a notable emphasis on the PCDH complex located on chromosome 5. Understanding the functional implications of PCDH genes and their interaction with DDX41 in AML may significantly advance our knowledge of AML pathogenesis. Gaining insights into DDX41 alterations may open avenues for personalized therapeutic approaches and further prognostic stratification for AML patients with distinct molecular characteristics.

Short report: Plasma based biomarkers detect radiation induced brain injury in cancer patients treated for brain metastasis: A pilot study.

Radiotherapy has an important role in the treatment of brain metastases but carries risk of short and/or long-term toxicity, termed radiation-induced brain injury (RBI). As the diagnosis of RBI is crucial for correct patient management, there is an unmet need for reliable biomarkers for RBI. The aim of this proof-of concept study is to determine the utility of brain-derived circulating free DNA (BncfDNA), identified by specific methylation patterns for neurons, astrocytes, and oligodendrocytes, as biomarkers brain injury induced by radiotherapy. Twenty-four patients with brain metastases were monitored clinically and radiologically before, during and after brain radiotherapy, and blood for BncfDNA analysis (98 samples) was concurrently collected. Sixteen patients were treated with whole brain radiotherapy and eight patients with stereotactic radiosurgery. During follow-up nine RBI events were detected, and all correlated with significant increase in BncfDNA levels compared to baseline. Additionally, resolution of RBI correlated with a decrease in BncfDNA. Changes in BncfDNA were independent of tumor response. Elevated BncfDNA levels reflects brain cell injury incurred by radiotherapy. further research is needed to establish BncfDNA as a novel plasma-based biomarker for brain injury induced by radiotherapy.

Mucosal DNA methylome alteration in Crohn's disease: surgical and non-surgical groups.

Crohn's disease (CD) is characterized as a chronic, relapsing, and progressive disorder with a complex etiology involving interactions between host, microbiome, and the external environment. Genome wide association studies (GWAS) suggest several genetic variations in the diseased individuals but that explains only a small proportion of susceptibility to disease conditions. This indicates the possible role of epigenome which links environmental factors to the genetic variation in the disease etiology. The current study is focused on the DNA methylome evolution with disease progression. We performed Reduced Representation Bisulfite Sequencing (RRBS) to analyze differential DNA methylation in the diseased and healthy mucosal tissues of 2 different groups of CD patients: non-surgical and surgical, categorized based on the severity of disease and standard of care needed. Patients in both groups have unique DNA methylation signature compared to the healthy tissue. After removing single nucleotide polymorphisms (SNPs), 1,671 differentially methylated loci were found in the non-surgical and 3,334 in the surgical group of which only 206 were found overlapping in both groups. Furthermore, differential DNA methylation was noted in some of the GWAS associated genes implicated in CD. Also, functional enrichment analysis showed high representation of several key pathways where differential methylations were observed, and these can be implicated in CD pathogenesis. We identified specific DNA methylation patterns in the mucosal DNA of surgical and non-surgical CD patients which indicates evolution of the methylome as the disease progresses from initial to the advance stage. These unique patterns can be used as DNA methylation signatures to identify different stages of the disease.

On latent idealized models in symbolic datasets: unveiling signals in noisy sequencing data.

Data taking values on discrete sample spaces are the embodiment of modern biological research. "Omics" experiments based on high-throughput sequencing produce millions of symbolic outcomes in the form of reads (i.e., DNA sequences of a few dozens to a few hundred nucleotides). Unfortunately, these intrinsically non-numerical datasets often deviate dramatically from natural assumptions a practitioner might make, and the possible sources of this deviation are usually poorly characterized. This contrasts with numerical datasets where Gaussian-type errors are often well-justified. To overcome this hurdle, we introduce the notion of latent weight, which measures the largest expected fraction of samples from a probabilistic source that conform to a model in a class of idealized models. We examine various properties of latent weights, which we specialize to the class of exchangeable probability distributions. As proof of concept, we analyze DNA methylation data from the 22 human autosome pairs. Contrary to what is usually assumed in the literature, we provide strong evidence that highly specific methylation patterns are overrepresented at some genomic locations when latent weights are taken into account.

Applying single cell multi-omic analyses to understand treatment resistance in pediatric high grade glioma.

Despite improvements in cancer patient outcomes seen in the past decade, tumor resistance to therapy remains a major impediment to achieving durable clinical responses. Intratumoral heterogeneity related to genetic, epigenetic, transcriptomic, proteomic, and metabolic differences between individual cancer cells has emerged as a driver of therapeutic resistance. This cell to cell heterogeneity can be assessed using single cell profiling technologies that enable the identification of tumor cell clones that exhibit similar defining features like specific mutations or patterns of DNA methylation. Single cell profiling of tumors before and after treatment can generate new insights into the cancer cell characteristics that confer therapeutic resistance by identifying intrinsically resistant sub-populations that survive treatment and by describing new cellular features that emerge post-treatment due to tumor cell evolution. Integrative, single cell analytical approaches have already proven advantageous in studies characterizing treatment-resistant clones in cancers where pre- and post-treatment patient samples are readily available, such as leukemia. In contrast, little is known about other cancer subtypes like pediatric high grade glioma, a class of heterogeneous, malignant brain tumors in children that rapidly develop resistance to multiple therapeutic modalities, including chemotherapy, immunotherapy, and radiation. Leveraging single cell multi-omic technologies to analyze naïve and therapy-resistant glioma may lead to the discovery of novel strategies to overcome treatment resistance in brain tumors with dismal clinical outcomes. In this review, we explore the potential for single cell multi-omic analyses to reveal mechanisms of glioma resistance to therapy and discuss opportunities to apply these approaches to improve long-term therapeutic response in pediatric high grade glioma and other brain tumors with limited treatment options.

Epigenome-Wide Meta-Analysis of 13 Military and Civilian Cohorts Reveals Common and Cell-Type Specific DNA Methylation Patterns Associated With PTSD

Epigenome-Wide Meta-Analysis of 13 Military and Civilian Cohorts Reveals Common and Cell-Type Specific DNA Methylation Patterns Associated With PTSD

Nrf2-/- regulated lung DNA demethylation and CYP2E1 DNA methylation under PM2.5 exposure.

Cytochrome P450 (CYP450) can mediate fine particulate matter (PM2.5) exposure leading to lung injury. Nuclear factor E2-related factor 2 (Nrf2) can regulate CYP450 expression; however, the mechanism by which Nrf2-/- (KO) regulates CYP450 expression via methylation of its promoter after PM2.5 exposure remains unclear. Here, Nrf2-/- (KO) mice and wild-type (WT) were placed in a PM2.5 exposure chamber (PM) or a filtered air chamber (FA) for 12weeks using the real-ambient exposure system. The CYP2E1 expression trends were opposite between the WT and KO mice following PM2.5 exposure. After exposure to PM2.5, CYP2E1 mRNA and protein levels were increased in WT mice but decreased in KO mice, and CYP1A1 expression was increased after exposure to PM2.5 in both WT and KO mice. CYP2S1 expression decreased after exposure to PM2.5 in both the WT and KO groups. We studied the effect of PM2.5 exposure on CYP450 promoter methylation and global methylation levels in WT and KO mice. In WT and KO mice in the PM2.5 exposure chamber, among the methylation sites examined in the CYP2E1 promoter, the CpG2 methylation level showed an opposite trend with CYP2E1 mRNA expression. The same relationship was evident between CpG3 unit methylation in the CYP1A1 promoter and CYP1A1 mRNA expression, and between CpG1 unit methylation in the CYP2S1 promoter and CYP2S1 mRNA expression. This data suggests that methylation of these CpG units regulates the expression of the corresponding gene. After exposure to PM2.5, the expression of the DNA methylation markers ten-eleven translocation 3 (TET3) and 5-hydroxymethylcytosine (5hmC) was decreased in the WT group but significantly increased in the KO group. In summary, the changes in CYP2E1, CYP1A1, and CYP2S1 expression in the PM2.5 exposure chamber of WT and Nrf2-/- mice might be related to the specific methylation patterns in their promoter CpG units. After exposure to PM2.5, Nrf2 might regulate CYP2E1 expression by affecting CpG2 unit methylation and induce DNA demethylation via TET3 expression. Our study revealed the underlying mechanism for Nrf2 to regulate epigenetics after lung exposure to PM2.5.

Methylation Dynamics on 5′-UTR of DAT1 Gene as a Bio-Marker to Recognize Therapy Success in ADHD Children

Attention-deficit/hyperactivity disorder (ADHD), a neuropsychiatric condition characterized by inattention, hyperactivity, and impulsivity, afflicts 5% of children worldwide. Each ADHD patient presents with individual cognitive and motivational peculiarities. Furthermore, choice of appropriate therapy is still up to clinicians, who express somewhat qualitative advice on whether a child is being successfully cured or not: it would be more appropriate to use an objective biomarker to indicate whether a treatment led to benefits or not. The aim of our work is to search for such clinical biomarkers. We recruited 60 ADHD kids; psychopathological scales were administered at recruitment and after six weeks of therapy. Out of such a cohort of ADHD children, we rigorously extracted two specific subgroups; regardless of the initial severity of their disease, we compared those who obtained the largest improvement (ΔCGAS > 5) vs. those who were still characterized by a severe condition (CGAS < 40). After such a therapy, methylation levels of DNA extracted from buccal swabs were measured in the 5'-UTR of the DAT1 gene. CpGs 3 and 5 displayed, in relation to the other CpGs, a particular symmetrical pattern; for "improving" ADHD children, they were methylated together with CpG 2 and CpG 6; instead, for "severe" ADHD children, they accompanied a methylated CpG 1. These specific patterns of methylation could be used as objective molecular biomarkers of successful cures, establishing if a certain therapy is akin to a given patient (personalized medicine). Present data support the use of post-therapy molecular data obtained with non-invasive techniques.

Genetically unresolved case of Rauch-Steindl syndrome diagnosed by its wolf-hirschhorn associated DNA methylation episignature.

Wolf-Hirschhorn syndrome (WHS) is caused by deletion of a critical region of the short arm of chromosome 4. Clinical features of WHS include distinct dysmorphic facial features, growth restriction, developmental delay, intellectual disability, epilepsy, and other malformations. The NSD2 gene localizes within this critical region along with several other genes. Pathogenic variants in NSD2 cause Rauch-Steindl (RAUST) syndrome. Clinical features of RAUST syndrome partially overlap with WHS, however epilepsy and the recognizable facial gestalt are not observed. Here, we report a case of a young boy who presented with developmental delay, dysmorphic features and short stature. After negative chromosomal microarray and whole exome sequencing, genomic DNA methylation episignature analysis was performed. Episignatures are sensitive and specific genome-wide DNA methylation patterns associated with a growing number of rare disorders. The patient was positive for the WHS episignature. Reanalysis of the patient's exome data identified a previously undetected frameshift variant in NSD2, leading to a diagnosis of RAUST. This report demonstrates the clinical utility of DNA methylation episignature analysis for unresolved patients, and provides insight into the overlapping pathology between WHS and RAUST as demonstrated by the similarities in their genomic DNA methylation profiles.

The spectrum of tuberculosis described as differential DNA methylation patterns in alveolar macrophages and alveolar T cells

BackgroundHost innate immune cells have been identified as key players in the early eradication of Mycobacterium tuberculosis and in the maintenance of an anti-mycobacterial immune memory, which we and others have shown are induced through epigenetic reprogramming. Studies on human tuberculosis immunity are dominated by those using peripheral blood as surrogate markers for immunity. We aimed to investigate DNA methylation patterns in immune cells of the lung compartment by obtaining induced sputum from M. tuberculosis- exposed subjects including symptom-free subjects testing positively and negatively for latent tuberculosis as well as patients diagnosed with active tuberculosis. Alveolar macrophages and alveolar T cells were isolated from the collected sputum and DNA methylome analyses performed (Illumina Infinium Human Methylation 450 k).ResultsMultidimensional scaling analysis revealed that DNA methylomes of cells from the tuberculosis-exposed subjects and controls appeared as separate clusters. The numerous genes that were differentially methylated between the groups were functionally connected and overlapped with previous findings of trained immunity and tuberculosis. In addition, analysis of the interferon-gamma release assay (IGRA) status of the subjects demonstrated that the IGRA status was reflected in the DNA methylome by a unique signature.ConclusionsThis pilot study suggests that M. tuberculosis induces epigenetic reprogramming in immune cells of the lung compartment, reflected as a specific DNA methylation pattern. The DNA methylation signature emerging from the comparison of IGRA-negative and IGRA-positive subjects revealed a spectrum of signature strength with the TB patients grouping together at one end of the spectrum, both in alveolar macrophages and T cells. DNA methylation-based biosignatures could be considered for further development towards a clinically useful tool for determining tuberculosis infection status and the level of tuberculosis exposure.