Aberrant Methylation Patterns Research Articles

A DNA Polymerase Variant Senses the Epigenetic Marker 5-Methylcytosine by Increased Misincorporation.

Dysregulation of DNA methylation is associated with human disease, particularly cancer, and the assessment of aberrant methylation patterns holds great promise for clinical diagnostics. However, DNA polymerases do not effectively discriminate between processing 5-methylcytosine (5 mC) and unmethylated cytosine, resulting in the silencing of methylation information during amplification or sequencing. As a result, current detection methods require multi-step DNA conversion treatments or careful analysis of sequencing data to decipher individual 5 mC bases. To overcome these challenges, we propose a novel DNA polymerase-mediated 5 mC detection approach. Here, we describe the engineering of a thermostable DNA polymerase variant derived from Thermus aquaticus with altered fidelity towards 5 mC. Using a screening-based evolutionary approach, we have identified a DNA polymerase that exhibits increased misincorporation towards 5 mC during DNA synthesis. This DNA polymerase generates mutation signatures at methylated CpG sites, allowing direct detection of 5 mC by reading an increased error rate after sequencing without prior treatment of the sample DNA.

Epigenetic changes driven by environmental pollutants in lung carcinogenesis: a comprehensive review.

Lung cancer remains the leading cause of cancer-related mortality globally, with environmental pollutants identified as significant risk factors, especially for nonsmokers. The intersection of these pollutants with epigenetic mechanisms has emerged as a critical area of interest for understanding the etiology and progression of lung cancer. Epigenetic changes, including DNA methylation, histone modifications, and non-coding RNAs, can induce alterations in gene expression without affecting the DNA sequence and are influenced by environmental factors, contributing to the transformation of normal cells into malignant cells. This review assessed the literature on the influence of environmental pollutants on lung cancer epigenetics. A comprehensive search across databases such as PubMed, Web of Science, Cochrane Library, and Embase yielded 3,254 publications, with 22 high-quality papers included for in-depth analysis. These studies demonstrated the role of epigenetic markers, such as DNA methylation patterns of genes like F2RL3 and AHRR and alterations in the miRNA expression profiles, as potential biomarkers for lung cancer diagnosis and treatment. The review highlights the need to expand research beyond homogenous adult male groups typically found in high-risk occupational environments to broader population demographics. Such diversification can reduce biases and enhance the relevance of findings to various clinical contexts, fostering the development of personalized preventive and therapeutic measures. In conclusion, our findings underscore the potential of innovative epigenetic therapies, such as DNA demethylating drugs and histone modification agents, to counter environmental toxins' carcinogenic effects. The growing interest in miRNA therapies and studies aiming to correct aberrant methylation patterns indicate significant strides toward better lung cancer management and a healthier future for global communities.

Comprehensive molecular and clinical findings in 29 patients with multi-locus imprinting disturbance

BackgroundMulti-locus imprinting disturbance (MLID) with methylation defects in various differentially methylated regions (DMRs) has recently been identified in approximately 150 cases with imprinting disorders (IDs), and deleterious variants have been found in genes related to methylation maintenance of DMRs, such as those encoding proteins constructing the subcortical maternal complex (SCMC), in a small fraction of patients and/or their mothers. However, integrated methylation analysis for DMRs and sequence analysis for MLID-causative genes in MLID cases and their mothers have been performed only in a single study focusing on Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS) phenotypes.ResultsOf 783 patients with various IDs we have identified to date, we examined a total of 386 patients with confirmed epimutation and 71 patients with epimutation or uniparental disomy. Consequently, we identified MLID in 29 patients with epimutation confirmed by methylation analysis for multiple ID-associated DMRs using pyrosequencing and/or methylation-specific multiple ligation-dependent probe amplification. MLID was detected in approximately 12% of patients with BWS phenotype and approximately 5% of patients with SRS phenotype, but not in patients with Kagami-Ogata syndrome, Prader-Willi syndrome, or Angelman syndrome phenotypes. We next conducted array-based methylation analysis for 78 DMRs and whole-exome sequencing in the 29 patients, revealing hypomethylation-dominant aberrant methylation patterns in various DMRs of all the patients, eight probably deleterious variants in genes for SCMC in the mothers of patients, and one homozygous deleterious variant in ZNF445 in one patient. These variants did not show gene-specific methylation disturbance patterns. Clinically, neurodevelopmental delay and/or intellectual developmental disorder (ND/IDD) was observed in about half of the MLID patients, with no association with the identified methylation disturbance patterns and genetic variants. Notably, seven patients with BWS phenotype were conceived by assisted reproductive technology (ART).ConclusionsThe frequency of MLID was 7.5% (29/386) in IDs caused by confirmed epimutation. Furthermore, we revealed diverse patterns of hypomethylation-dominant methylation defects, nine deleterious variants, ND/IDD complications in about half of the MLID patients, and a high frequency of MLID in ART-conceived patients.

An integrative analysis to identify pancancer epigenetic biomarkers

Integrating and analyzing the pancancer data collected from different experiments is crucial for gaining insights into the common mechanisms in the molecular level underlying the development and progression of cancers. Epigenetic study of the pancancer data can provide promising results in biomarker discovery. The genes that are epigenetically dysregulated in different cancers are powerful biomarkers for drug-related studies. This paper identifies the genes having altered expression due to aberrant methylation patterns using differential analysis of TCGA pancancer data of 12 different cancers. We identified a comprehensive set of 115 epigenetic biomarker genes out of which 106 genes having pancancer properties. The correlation analysis, gene set enrichment, protein-protein interaction analysis, pancancer characteristics analysis, and diagnostic modeling were performed on these biomarkers to illustrate the power of this signature and found to be important in different molecular operations related to cancer. An accuracy of 97.56% was obtained on TCGA pancancer gene expression dataset for predicting the binary class tumor or normal. The source code and dataset of this work are available at https://github.com/panchamisuneeth/EpiPanCan.git.

The Epigenetic Contribution to the Pathogenesis of Psoriasis: Recent Advances.

Psoriasis is defined as a chronic autoimmune disorder of the skin in which abnormal proliferation and differentiation of keratinocytes are blamed as the central culprit of disease etiopathogenesis. A complex interplay between environmental factors and genetic risk factors has been suggested to trigger the disease. However, epigenetic regulation appears to connect external stimuli and genetic abnormalities in the development of psoriasis. The discordance in the prevalence of psoriasis between monozygotic twins and environmental factors that contribute to its onset have caused a paradigm shift regarding the mechanisms underlying the pathogenesis of this disease. Epigenetic dysregulation may be involved in aberrancies of keratinocyte differentiation, T-cell activation, and other plausible cells, leading to the initiation and perpetuation of psoriasis. Epigenetics is characterized by heritable alterations in the transcription of genes without nucleotide change and is commonly considered at three levels, i.e., DNA methylation, histone modifications, and microRNAs. To date, scientific evidence has indicated abnormal DNA methylation, histone modifications, and non-coding RNA transcription in psoriatic patients. In order to reverse aberrant epigenetic changes in psoriasis patients, several compounds and drugs (epi-drugs) have been developed to affect the major enzymes involved in the methylation of DNA, or the acetylation of histones, which aim to correct the aberrant methylation and acetylation patterns. A number of clinical trials have suggested the therapeutic potential of such drugs in the treatment of psoriasis. In the present review, we attempt to clarify recent findings with respect to epigenetic irregularities in psoriasis and discuss future challenges.

Altered methylation of imprinted genes in neuroblastoma: implications for prognostic refinement

BackgroundNeuroblastoma (NB) is a complex disease, and the current understanding of NB biology is limited. Deregulation in genomic imprinting is a common event in malignancy. Since imprinted genes play crucial roles in early fetal growth and development, their role in NB pathogenesis could be suggested.MethodsWe examined alterations in DNA methylation patterns of 369 NB tumours at 49 imprinted differentially methylated regions (DMRs) and assessed its association with overall survival probabilities and selected clinical and genomic features of the tumours. In addition, an integrated analysis of DNA methylation and allele-specific copy number alterations (CNAs) was performed, to understand the correlation between the two molecular events.ResultsSeveral imprinted regions with aberrant methylation patterns in NB were identified. Regions that underwent loss of methylation in > 30% of NB samples were DMRs annotated to the genes NDN, SNRPN, IGF2, MAGEL2 and HTR5A and regions with gain of methylation were NNAT, RB1 and GPR1. Methylation alterations at six of the 49 imprinted DMRs were statistically significantly associated with reduced overall survival: MIR886, RB1, NNAT/BLCAP, MAGEL2, MKRN3 and INPP5F. RB1, NNAT/BLCAP and MKRN3 were further able to stratify low-risk NB tumours i.e. tumours that lacked MYCN amplification and 11q deletion into risk groups. Methylation alterations at NNAT/BLCAP, MAGEL2 and MIR886 predicted risk independently of MYCN amplification or 11q deletion and age at diagnosis. Investigation of the allele-specific CNAs demonstrated that the imprinted regions that displayed most alterations in NB tumours harbor true epigenetic changes and are not result of the underlying CNAs.ConclusionsAberrant methylation in imprinted regions is frequently occurring in NB tumours and several of these regions have independent prognostic value. Thus, these could serve as potentially important clinical epigenetic markers to identify individuals with adverse prognosis. Incorporation of methylation status of these regions together with the established risk predictors may further refine the prognostication of NB patients.

Phaser-Trim: A Phase Separation Based Genetically Encoded Reporter for H3K9 Trimethylation in Living Cells.

Histone methylation is a key epigenetic modification that regulates the chromatin structure and gene expression for proper cellular and physiological processes. Aberrant histone methylation patterns are implicated in many diseases. Therefore, monitoring histone methylation dynamics in living cells and species is essential for elucidating its regulatory mechanisms and identifying potential therapeutic targets. However, current methods for detecting histone methylation are limited by their low sensitivity and specificity. To overcome this challenge, we have developed a genetically encoded biosensor named Phaser-Trim (Phase separation based genetically encoded reporter for H3K9 Trimethylation) to detect the dynamic changes of H3K9me3 in living cells and species through the generation and disappearance of phase-separated droplets. Phaser-Trim demonstrates advantages of clear phenotypic characteristics, convenient operation, quantitative accuracy, biocompatibility, high specificity, and superior imaging performance with high signal-to-background ratio (SBR) for in vivo animal imaging. Using Phaser-Trim, we have successfully detected the dynamics of the H3K9me3 level during the differentiation of neural stem cells in Drosophila. Furthermore, Phaser-Trim also holds promise for application in high-throughput screening systems to facilitate the discovery of novel anticancer drugs.

Methylome analysis of endothelial cells suggests new insights on sporadic brain arteriovenous malformation

Arteriovenous malformation of the brain (bAVM) is a vascular phenotype related to brain defective angiogenesis. Involved vessels show impaired expression of vascular differentiation markers resulting in the arteriolar to venule direct shunt. In order to clarify aberrant gene expression occurring in bAVM, here we describe results obtained by methylome analysis performed on endothelial cells (ECs) isolated from bAVM specimens, compared to human cerebral microvascular ECs. Results were validated by quantitative methylation-specific PCR and quantitative realtime-PCR. Differential methylation events occur in genes already linked to bAVM onset, as RBPJ and KRAS. However, among differentially methylated genes, we identified EPHB1 and several other loci involved in EC adhesion as well as in EC/vascular smooth muscle cell (VSMC) crosstalk, suggesting that only endothelial dysfunction might not be sufficient to trigger the bAVM phenotype. Moreover, aberrant methylation pattern was reported for many lncRNA genes targeting transcription factors expressed during neurovascular development. Among these, the YBX1 that was recently shown to target the arteridin coding gene. Finally, in addition to the conventional CpG methylation, we further considered the role of impaired CHG methylation, mainly occurring in brain at embryo stage. We showed as differentially CHG methylated genes are clustered in pathways related to EC homeostasis, as well as to VSMC-EC crosstalk, suggesting as impairment of this interaction plays a prominent role in loss of vascular differentiation, in bAVM phenotype.

EZH2 mutations in follicular lymphoma distort H3K27me3 profiles and alter transcriptional responses to PRC2 inhibition

Mutations in chromatin regulators are widespread in cancer. Among them, the histone H3 lysine 27 methyltransferase Polycomb Repressive Complex 2 (PRC2) shows distinct alterations according to tumor type. This specificity is poorly understood. Here, we model several PRC2 alterations in one isogenic system to reveal their comparative effects. Focusing then on lymphoma-associated EZH2 mutations, we show that Ezh2Y641F induces aberrant H3K27 methylation patterns even without wild-type Ezh2, which are alleviated by partial PRC2 inhibition. Remarkably, Ezh2Y641F rewires the response to PRC2 inhibition, leading to induction of antigen presentation genes. Using a unique longitudinal follicular lymphoma cohort, we further link EZH2 status to abnormal H3K27 methylation. We also uncover unexpected variability in the mutational landscape of successive biopsies, pointing to frequent co-existence of different clones and cautioning against stratifying patients based on single sampling. Our results clarify how oncogenic PRC2 mutations disrupt chromatin and transcription, and the therapeutic vulnerabilities this creates.

Abstract 4415: Utilization of MBD-seq to elucidate differentially methylated regions in cfDNA across healthy and cancer patients

Abstract 5-Methylcytosine (5-mC) is the most abundant epigenetic DNA modification in eukaryotes. The presence of 5-mC alters gene expression and genome metabolism, and its deregulation is associated with many human diseases. Cell-free DNA (cfDNA) released from apoptotic cells has recently gained attention, as mutations or epimutations (e.g. 5-mC variations) detected in cfDNA were shown to have high diagnostic potential to assess the presence, stage and outcome of several cancers. However, 5-mC detection in cfDNA remains a challenge as input material is often very scarce. MBD-Seq is a method that leverages the ability of Methyl-CpG-binding domain protein 2 (MBD2) to capture and detect highly methylated regions of DNA genome-wide. We evaluated MBD-Seq for the detection of aberrant methylation patterns in cfDNA from healthy and diseased patients. We found MBD-seq on cfDNA to be specific (over 100-fold mC:C detection ratio) and sensitive (5 ng of input cfDNA). Applying MBD-seq to samples from healthy vs colorectal and breast cancer patients showed striking differences in methylation of key tumor-associated genes. Hence, MBD-Seq can elucidate aberrant methylation patterns from cfDNA, highlighting its potential as a diagnostics tool for the detection of cancer. Citation Format: Camille Michelon, Brian Egan, Benjamin Delatte. Utilization of MBD-seq to elucidate differentially methylated regions in cfDNA across healthy and cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4415.

Abstract 280: Aberrant β-catenin signaling from the bone marrow niche leads to transformation of IDH1/2-mutant hematopoietic stem cells and compromises response to IDH1/2 inhibitors

Abstract Our purpose is to identify stromal-derived signals required for IDH1/2-mut hematopoietic stem cells (HSCs) to become leukemogenic. Mutations in IDH genes are found in 20% of acute myeloid leukemia (AML) patients and are not transformative. They produce the oncometabolite 2HG which inhibits key epigenetic regulators. We explored whether epigenetic changes in the bone marrow (BM) niche could provide cooperative signals to IDH-mut HSCs leading to their transformation and disease development. We found that 2HG is elevated in the BM plasma of MDS/AML patients with IDH1/2 mutations as compared to IDH-wt patients. Interestingly, 2HG is taken up by BM stromal cells (BMSCs) suggesting it may induce aberrant methylation patterns in them. Indeed, methylome analysis showed hypermethylation in BMSCs of AML patients with IDH1/2 mutations compared to wt. Among the most hypermethylated genes in the IDH-mut stroma are AXIN1, AXIN2 and NDK2, all negative regulators of β-catenin; this pathway activation was previously shown to cause MDS/AML in mice and present in 38% of patients. In agreement with these findings βcat activation in the stroma of IDH-mut vs IDH-wt AML patients showed a strong positive correlation as 50% of IDH-mut patients had activated βcat in their BMSCs. Inquiring whether stroma methylation precedes HSC transformation, we analyzed samples from healthy individuals with IDH-mut clonal hematopoiesis. They exhibited increased global and AXIN1 promoter methylation compared to samples without such clones, but not active βcat in osteoblasts, indicating this might be a later step along with disease development. In fact, transplantation of HSCs from Idh2-mut mice to recipients expressing active βcat in osteoblasts led to increase in blasts and shortened survival compared to wt mice transplanted with Idh2-mut HSCs. As many patients relapse following treatment with IDH inhibitors, we investigated a potential role for βcat signaling in treatment outcome. IDH-mut AML patients who do not respond to treatment with the IDHi present more frequently with activated βcat in osteoblasts compared to responders. Analyzing samples from the same patient pre- and post-treatment with IDHi, we found before treatment most patients were positive for βcat stromal activation, whereas after treatment, all patients with complete response were negative, while patients with progressive disease remained positive. Our model suggests IDH-mut HSCs secrete 2HG inducing epigenetic alterations in the niche, some of which cause activation of the βcat pathway. In turn, this additional signal leads to the transformation of HSCs to dysplastic cells and MDS/AML development. This work uncovers a new mechanism for transformation to MDS/AML and resistance to IDHi treatment. Thus, targeting βcat activation in the stroma, in combination with IDHi, could improve treatment outcome. Citation Format: Paraskevi Vgenopoulou, Luis Flores, Ioanna Mosialou, Junfei Zhao, X. Shawn Liu, Pamela Sung, Virginie Penard-Lacronique, Martin Carroll, Stavroula Kousteni. Aberrant β-catenin signaling from the bone marrow niche leads to transformation of IDH1/2-mutant hematopoietic stem cells and compromises response to IDH1/2 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 280.

Abstract 3669: Clinical application of urinary DNA methylation biomarkers for identifying patients with non-muscle invasive bladder cancer

Abstract Introduction Differences in DNA methylation along cell-free DNA (cfDNA) fragments are known to exist between cancer patients and healthy individuals. Profiling these aberrant methylation patterns in urinary cfDNA (ucfNDA) holds great promise for the non-invasive detection and monitoring of bladder tumors, including non-muscle invasive bladder cancer (NMIBC). Here, we analyzed fragment-level DNA methylation patterns in urine samples obtained prior to repeat transurethral resection of a bladder tumor (reTURBT) from a cohort of NMIBC patients. The results were compared to traditional clinical disease assessment and mutation-based tumor fractions. We found a strong concordance between DNA methylation abnormality and clinical diagnosis, as well as tumor fractions, highlighting the potential of epigenetic alterations as a non-invasive indicator of bladder cancer status. Methods Patients with high-risk NMIBC (n = 30) were prospectively enrolled prior to reTURBT. The PredicineEPIC™ genome-wide methylation analysis and PredicineBEACON™ MRD profiling was performed on pre-repeat TURBT urine samples. Abnormally methylated genomic fragments were detected against a background model built from a panel of plasma samples from healthy donors. Results DNA methylation abnormality was significantly higher (p = 4.8 × 10−6) in samples classified as NIMBC positive by a clinical pathologist (n = 19) compared to negative samples (n = 11), as well as compared to hold-out healthy donor samples (n=24; p = 7.0 × 10−9). Classifying the samples based on a DNA methylation score cut-off derived from healthy donor samples, 24/30 samples showed concordant disease status. Furthermore, DNA methylation abnormality scores correlated positively with mutation-based tumor fractions (r = 0.62), with higher grade samples showing a stronger correlation (r = 0.81; n = 13) than lower grade samples (r = 0.54; n = 17). Conclusion We demonstrate the utility of methylation patterns extracted ucfDNA samples obtained prior to reTURBT to detect and monitor NMIBC. Urinary DNA methylation patterns were highly concordant with clinical pathology status and as well as tumor fractions determined from mutation analysis. Citation Format: Giancarlo Bonora, Ziqi Zhu, Billie Gould, Binggang Xiang, Kemin Zhou, Shidong Jia, Roger Li, Pan Du. Clinical application of urinary DNA methylation biomarkers for identifying patients with non-muscle invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3669.

Exploring the Diagnostic and Prognostic Predictive Values of Ferroptosis- Related Markers in Lung Adenocarcinoma.

Lung Adenocarcinoma (LUAD), a common and aggressive form of lung cancer, poses significant treatment challenges due to its low survival rates. To better understand the role of ferroptosis driver genes in LUAD, this study aimed to explore their diagnostic and prognostic significance, as well as their impact on treatment approaches and tumor immune function in LUAD. To accomplish the defined goals, a comprehensive methodology incorporating both in silico and wet lab experiments was employed. A comprehensive analysis was conducted on a total of 233 ferroptosis driver genes obtained from the FerrDB database. Utilizing various TCGA databases and the RT-qPCR technique, the expression profiles of 233 genes were examined. Among them, TP53, KRAS, PTEN, and HRAS were identified as hub genes with significant differential expression. Notably, TP53, KRAS, and HRAS exhibited substantial up-regulation, while PTEN demonstrated significant down-regulation at both the mRNA and protein levels in LUAD samples. The dysregulation of hub genes was further associated with poor overall survival in LUAD patients. Additionally, targeted bisulfite-sequencing (bisulfite-seq) analysis revealed aberrant promoter methylation patterns linked to the dysregulation of hub genes. Furthermore, hub genes were found to participate in diverse oncogenic pathways, highlighting their involvement in LUAD tumorigenesis. By leveraging the diagnostic and prognostic potential of ferroptosis driver hub genes (TP53, KRAS, PTEN, and HRAS), significant advancements can be made in the understanding and management of LUAD pathogenesis. Therapeutic targeting of these genes using specific drugs holds great promise for revolutionizing drug discovery and improving the overall survival of LUAD patients.

DNMT1/DNMT3a-mediated promoter hypermethylation and transcription activation of ICAM5 augments thyroid carcinoma progression.

Promoter methylation is one of the most studied epigenetic modifications and it is highly relevant to the onset and progression of thyroid carcinoma (THCA). This study investigates the promoter methylation and expression pattern of intercellular adhesion molecule 5 (ICAM5) in THCA. CpG islands with aberrant methylation pattern in THCA, and the expression profiles of the corresponding genes in THCA, were analyzed using bioinformatics. ICAM5 was suggested to have a hypermethylation status, and it was highly expressed in THCA tissues and cells. Its overexpression promoted proliferation, mobility, and tumorigenic activity of THCA cells. As for the downstream signaling, ICAM5 was found to activate the MAPK/ERK and MAPK/JNK signaling pathways. Either inhibition of ERK or JNK blocked the oncogenic effects of ICAM5. DNA methyltransferases 1 (DNMT1) and DNMT3a were found to induce promoter hypermethylation of ICAM5 in THCA cells. Knockdown of DNMT1 or DNMT3a decreased the ICAM5 expression and suppressed malignant properties of THCA cells in vitro and in vivo, which were, however, restored by further artificial ICAM5 overexpression. Collectively, this study reveals that DNMT1 and DNMT3a mediates promoter hypermethylation and transcription activation of ICAM5 in THCA, which promotes malignant progression of THCA through the MAPK signaling pathway.

Protocol for Analyzing Epigenetic Regulation Mechanisms in Breast Cancer.

DNA methylation and gene expression are two critical aspects of the epigenetic landscape that contribute significantly to cancer pathogenesis. Analysis of aberrant genome-wide methylation patterns can provide insights into how these affect the cancer transcriptome and possible clinical implications for cancer diagnosis and treatment. The role of tumor suppressors and oncogenes is well known in tumorigenesis. Epigenetic alterations can significantly impact the expression and function of these critical genes, contributing to the initiation and progression of cancer. This protocol chapter presents a unified workflow to explore the role of DNA methylation in gene expression regulation in breast cancer by identifying differentially expressed genes whose promoter or gene body regions are differentially methylated using various Bioconductor packages in R environment. Functional enrichment analysis of these genes can help in understanding the mechanisms leading to tumorigenesis due to epigenetic alterations.

In Silico Gene Prioritization Highlights the Significance of Bone Morphogenetic Protein 4 (BMP4) Promoter Methylation across All Methylation Clusters in Colorectal Cancer.

The cytosine-phosphate-guanine (CpG) island methylator phenotype (CIMP) represents one of the pathways involved in the development of colorectal cancer, characterized by genome-wide hypermethylation. To identify samples exhibiting hypermethylation, we used unsupervised hierarchical clustering on genome-wide methylation data. This clustering analysis revealed the presence of four distinct subtypes within the tumor samples, namely, CIMP-H, CIMP-L, cluster 3, and cluster 4. These subtypes demonstrated varying levels of methylation, categorized as high, intermediate, and very low. To gain further insights, we mapped significant probes from all clusters to Ensembl Regulatory build 89, with a specific focus on those located within promoter regions or bound regions. By intersecting the methylated promoter and bound regions across all methylation subtypes, we identified a total of 253 genes exhibiting aberrant methylation patterns in the promoter regions across all four subtypes of colorectal cancer. Among these genes, our comprehensive genome-wide analysis highlights bone morphogenic protein 4 (BMP4) as the most prominent candidate. This significant finding was derived through the utilization of various bioinformatics tools, emphasizing the potential role of BMP4 in colorectal cancer development and progression.

Epigenetic modifier alpha-ketoglutarate modulates aberrant gene body methylation and hydroxymethylation marks in diabetic heart

BackgroundDiabetic cardiomyopathy (DCM) is a leading cause of death in diabetic patients. Hyperglycemic myocardial microenvironment significantly alters chromatin architecture and the transcriptome, resulting in aberrant activation of signaling pathways in a diabetic heart. Epigenetic marks play vital roles in transcriptional reprogramming during the development of DCM. The current study is aimed to profile genome-wide DNA (hydroxy)methylation patterns in the hearts of control and streptozotocin (STZ)-induced diabetic rats and decipher the effect of modulation of DNA methylation by alpha-ketoglutarate (AKG), a TET enzyme cofactor, on the progression of DCM.MethodsDiabetes was induced in male adult Wistar rats with an intraperitoneal injection of STZ. Diabetic and vehicle control animals were randomly divided into groups with/without AKG treatment. Cardiac function was monitored by performing cardiac catheterization. Global methylation (5mC) and hydroxymethylation (5hmC) patterns were mapped in the Left ventricular tissue of control and diabetic rats with the help of an enrichment-based (h)MEDIP-sequencing technique by using antibodies specific for 5mC and 5hmC. Sequencing data were validated by performing (h)MEDIP-qPCR analysis at the gene-specific level, and gene expression was analyzed by qPCR. The mRNA and protein expression of enzymes involved in the DNA methylation and demethylation cycle were analyzed by qPCR and western blotting. Global 5mC and 5hmC levels were also assessed in high glucose-treated DNMT3B knockdown H9c2 cells.ResultsWe found the increased expression of DNMT3B, MBD2, and MeCP2 with a concomitant accumulation of 5mC and 5hmC, specifically in gene body regions of diabetic rat hearts compared to the control. Calcium signaling was the most significantly affected pathway by cytosine modifications in the diabetic heart. Additionally, hypermethylated gene body regions were associated with Rap1, apelin, and phosphatidyl inositol signaling, while metabolic pathways were most affected by hyperhydroxymethylation. AKG supplementation in diabetic rats reversed aberrant methylation patterns and restored cardiac function. Hyperglycemia also increased 5mC and 5hmC levels in H9c2 cells, which was normalized by DNMT3B knockdown or AKG supplementation.ConclusionThis study demonstrates that reverting hyperglycemic damage to cardiac tissue might be possible by erasing adverse epigenetic signatures by supplementing epigenetic modulators such as AKG along with an existing antidiabetic treatment regimen.

Direct and high-throughput detection of DNA methylation and demethylation intermediates using biological nanopore.

This paper describes a nanopore sensing approach for 5-methylcytosine (5mC) positions with numbers and demethylation intermediates directly in a high-throughput way. Methylation-demethylation processes are crucial for mammalian development and aberrant methylation patterns could lead to human diseases. The general method of 5mC detection, the bisulfite conversion, requires multiple procedures, and the incomplete conversion suggests the inappropriateness of reliable epigenetic analysis. For detecting demethylation intermediates, restriction enzymes and antibodies are used. However, these methods are hard to recognize the intermediaries at a single molecular level. Here, we report a nanopore-based electrical sensor that utilized epigenetic modulation of cytosine dynamics in dsDNA, which destabilizes the Watson-Crick base pair to allow modified cytosine to interact with the acidic anion acids in nanopore for directly detecting 5mC, accurately to its position, number and variants without chemical conversion and modification, or enzyme amplification.A probe DNA hybridizes with the target DNA, then the dsDNA is grasped in nanopore, which reconstitutes the artificial bilayers, and passes through the pore under an applied voltage with unzipping strands electrophoretically. Furthermore, to realize high throughput determination, we mounted this system on MinION, a commercial device measuring more than 2,000 nanopores simultaneously.To determine 5mC information, we analyzed the unzipping behaviors, including unzipping time and blocking ratio, then shown in scatter plot graph. Moreover, to improve the detection accuracy of 5mC variants, we changed K+ to the lower mobility ion Li+ to decrease the speed of DNA translocation. As a result, the scatter plot graph showed the possibility of detecting 5mC and its variants using our method. Using MinION showed that our system could work as a high-throughput nanopore epigenetic detection.Our method offers a direct and high-throughput tool for detecting 5mC position, number, and demethylation intermediates.

Genetic and epigenetic characteristics of patients with colorectal cancer from Uruguay.

Colorectal cancer (CRC), the 3rd most frequent cancer worldwide, affects both men and women. This pathology arises from the progressive accumulation of genetic and epigenetic alterations. In this study, KRAS, NRAS, PIK3CA, and BRAF gene mutations, mismatch repair (MMR) genes methylation profile, microsatellite instability (MSI) and CpG Island Methylator Phenotype (CIMP) status were assessed. The associations of these molecular features with clinicopathological data were also investigated. A hundred and eight unselected CRC samples and their histological and clinical data, were gathered between 2017 and 2020. The prevalence of KRAS, NRAS and BRAF gene mutations was similar to that described in other populations. 28.7% of tumors were KRAS-mutated, mostly in men, distal location, with a CIMP-negative status. BRAFV600E frequency was 6.5% and associated with MSI (p=0.048), MLH1-methylated (p<0.001) and CIMP-High (p<0.001) status. We also confirmed that BRAFV600E tumors were more prevalent in older women and proximal location. A striking different result was the lack of most common variants in the PIK3CA gene. A complete absence of PIK3CA-mutated tumors in a population has not been previously reported. Among MMR genes, the only with an aberrant methylation pattern was MLH1 gene. Its frequency was 9.25%, lower than previously reported. Methylated tumors were most frequent in patients older than 70 years old and proximal tumor location. Finally, CIMP-High status was mainly observed in moderately differentiated tumors with a rate of 15.7%. Our findings were consistent with previous reports in other populations, but also showed some features unique to our cohort. This study is the first to report the analysis of a large number molecular biomarkers of CRC in Uruguay and one of the few performed in Latin-America.

Global and promoter specific hypermethylation of tumor suppressor genes P16, SOCS1, and SHP1 in oral squamous cell carcinoma and oral submucous fibrosis.

Aberrant methylation pattern leads to altered gene expression, that is, involved in the transformation of various cancers, including oral squamous cell carcinoma (OSCC). In the present study, an attempt has been made to examine the association of global and promoter-specific methylation of tumor suppressor genes in patients with OSCC and oral submucous fibrosis (OSMF). Promoter-specific methylation of tumor suppressor genes P16, SOCS1, and SHP1 had been studied earlier for their aberrant methylation patterns in other cancers; however, these studies were mainly conducted in-vitro or in animal models, and as such, only a few studies are available on human samples. In the present study evaluation of promoter-specific methylation of genes P16, SOCS1, and SHP1 in 76 patients' blood and tissue samples was done and compared with methylation of 35 healthy control samples using qPCR. Further, these samples were analyzed for global methylation patterns using ELISA. The results have shown a significant decreasing trend of promoter methylation (OSCC > OSMF > Controls); the methylation indices (MI) were significantly higher in OSCC than in the controls. The median MI of three genes for OSCC were P16MI (0.96), SHP1MI (0.79), and SOCS1 (0.80). Similarly, median MIs for OSMF were P16MI (0.18), SHP1 MI (0.19), and SOCS1 MI (0.5) against controls with MI (0) for each of the three genes. The global methylation %mC values were 1.9, 0.5, and 0.1, respectively. The values of MI and %mC were found to correlate with various risk factors such as tobacco, smoking, and alcohol consumption, which are positively involved in OSMF pathogenesis followed by oral cancer progression. Further, the methylation trend in tissue was reflected in blood samples, proving a window for methylation load to be used as a lesser invasive biomarker. The sensitivity and specificity of methylation load were also found reasonable. Therefore, the current study suggests that there may be a role of global and promoter-specific methylation load in the transition of OSMF to OSCC.