Specific Methylation Changes Research Articles

Cell-type specific methylation changes in the newborn child associated to obstetric pain relief

Although it is widely known that various pharmaceuticals affect the methylome, the knowledge of the effects from anesthesia is limited, and nearly nonexistent regarding the effects of obstetric anesthesia on the newborn child. Using sequencing based-methylation data and a reference-based statistical deconvolution approach we performed methylome-wide association studies (MWAS) of neonatal whole blood, and for each cell-type specifically, to detect methylation variations that are associated with the pain relief administered to the mother during delivery. Significant findings were replicated in a different dataset and followed-up with gene ontology analysis to pinpoint biological functions of potential relevance to these neonatal methylation alterations. The MWAS analyses detected methylome-wide significant (q<0.1) alterations in the newborn for laughing gas in granulocytes (two CpGs, p<5.50x10-9, q = 0.067), and for pudendal block in monocytes (five CpGs across three loci, p<1.51 x10-8, q = 0.073). Suggestively significant findings (p<1.00x10-6) were detected for both treatments for bulk and all cell-types, and replication analyses showed consistent significant enrichment (odds ratios ranging 3.47–39.02; p<4.00×10−4) for each treatment, suggesting our results are robust. In contrast, we did not observe any overlap across treatments, suggesting that the treatments are associated with different alterations of the neonatal blood methylome. Gene ontology analyses of the replicating suggestively significant results indicated functions related to, for example, cell differentiation, intracellular membrane-bound organelles and calcium transport. In conclusion, for the first time, we investigated and detected effect of obstetric pain-relief on the blood methylome in the newborn child. The observed differences suggest that anesthetic treatment, such as laughing gas or pudendal block, may alter the neonatal methylome in a cell-type specific manner. Some of the observed alterations are part of gene ontology terms that previously have been suggested in relation to anesthetic treatment, supporting its potential role also in obstetric anesthesia.

Blood methylation pattern reflects epigenetic remodelling in adipose tissue after bariatric surgery

Studies on DNA methylation following bariatric surgery have primarily focused on blood cells, while it is unclear to which extend it may reflect DNA methylation profiles in specific metabolically relevant organs such as adipose tissue. Here, we investigated whether adipose tissue depots specific methylation changes after bariatric surgery are mirrored in blood. Using Illumina 850K EPIC technology, we analysed genome-wide DNA methylation in paired blood, subcutaneous and omental visceral AT (SAT/OVAT) samples from nine individuals (N=6 female) with severe obesity pre- and post-surgery. The numbers and effect sizes of differentially methylated regions (DMRs) post-bariatric surgery were more pronounced in AT (SAT: 12,865 DMRs from-11.5 to 10.8%; OVAT: 14,632 DMRs from-13.7 to 12.8%) than in blood (9267 DMRs from-8.8 to 7.7%). Cross-tissue DMRs implicated immune-related genes. Among them, 49regions could be validated with similar methylation changes in blood from independent individuals. Fourteen DMRs correlated with differentially expressed genes in AT post bariatric surgery, including downregulation of PIK3AP1 in both SAT and OVAT. DNA methylation age acceleration was significantly higher in AT compared to blood, but remained unaffected after surgery. Concurrent methylation pattern changes in blood and AT, particularly in immune-related genes, suggest blood DNA methylation mirrors AT's inflammatory state post-bariatric surgery. The funding sources are listed in the Acknowledgments section.

DNA Methylation Landscape of Peripheral T-Cell Lymphomas Not Otherwise Specified

DNA methylation is an epigenetic modification catalyzed by DNA methyltransferases that regulate gene transcription in mammalian cells. DNA methylation in gene promoters is associated with repressive histone marks and contributes to gene repression. This process is involved in various physiological processes, including development, X chromosome inactivation, genomic imprinting, differentiation, and hematopoiesis. The global, genome-wide deregulation of methylation patterns contributes to the pathogenesis of immune disorders, hematologic malignancies, and cancer. Because aberrations in DNA methylation can deregulate spatiotemporal gene expression in a cell type-specific manner, genome-wide changes in this epigenetic modification may contribute to tumor development. Specifically, the downregulation of tumor suppressors through promoter hypermethylation is a widely accepted mechanism in cancer progression. Due to its distinct patterns in cancer, DNA methylation can be used as a diagnostic and predictive biomarker. Peripheral T-cell lymphomas, Not Otherwise Specified (PTCL-NOS), are a group of heterogeneous T-cell malignancies with diverse cytogenic, molecular, and morphological phenotypes. Despite large-scale gene expression profiling, the molecular features-and DNA methylation in particular-of these rare but aggressive malignancies remain poorly understood. To better understand the molecular landscape of PTCL-NOS, we performed global methylation profiling via Whole Genome Bisulfite Sequencing on 10 lymphomas and compared those to patterns seen in normal T-cells and Anaplastic Large Cell Lymphoma (ALCL). Our analysis revealed large-scale deregulation of methylation in all tumors at levels of both differentially methylated cytosines and regions. The dominant molecular change was hypomethylation, which was, on average, 7 times more frequent than hypermethylation. Hypomethylation was not limited to repetitive elements but was also seen in all genomic elements, including promoters, enhancers, gene bodies, and exons. Out of 1,286 genes whose promoters were consistently hypomethylated in Peripheral T-cell lymphomas (PTCL), 39 genes were associated with an increase in expression, suggesting that the loss of methylation may have contributed to their deregulation. This hypomethylated and overexpressed signature contained genes that might be involved in tumor progression or maintenance, such as Rac GTPase activating protein 1 ( RACGAP1), regulator of chromosome condensation 1 ( RCC1), anillin actin-binding protein ( ANLN), and others. Out of 578 genes whose promoters were consistently hypermethylated in PTCL, the expression of 56 genes was decreased. These included genes with tumor suppressor function, such as splicing factor 3b subunit 1 ( SF3B1) and lymphoid enhancer-binding factor 1 ( LEF1). Our analysis also identified differential methylation between PTCL-NOS and ALCL in various genomic features. For example, 71 hypo- and 59 hypermethylated promoters were found only in ALCL but not PTCL-NOS. Conversely, 326 hypo- and 254 hypermethylated promoters were observed only in PTCL-NOS. Analysis of gene expression also revealed specific changes in both PTCL subtypes, manifested by the activation of different signaling pathways as identified by Ingenuity Pathway Analysis. PTCL-NOS was characterized by the suppression of the SUMOylation pathway, PTEN, and EIF2 Signaling, and the activation of NF-κB and Notch signaling. ALCLs were characterized by the suppression of the Role of CHK Proteins in Cell Cycle Control, Erythropoietin, ERBB signaling, the Coronavirus Pathogenesis pathway, IGF-1 signaling, and the activation of Glutathione-mediated Detoxification and Triacylglycerol Biosynthesis. Altogether, we identified recurrent specific methylation and expression changes that could serve as the basis for the development of tools facilitating the diagnosis and prognosis of PTCL-NOS. In addition, our data may contribute to the identification of epigenetically-regulated drivers of tumor initiation, progression, and maintenance.

Abstract 6009: An end-to-end workflow for improved methylation detection

Abstract Introduction: DNA methylation at CpG nucleotide sites in eukaryotes is a key epigenetic mark that can help regulate gene expression. Specific changes in CpG methylation occur in many human cancers, making them a promising biomarker for early cancer detection. However, existing assays can be costly, lack specificity to regions of interest and often provide only semi-quantitative estimates of the methylation fraction. Here, we present a targeted methylome panel to decrease costs associated with next generation sequencing (NGS), methylation controls to calibrate quantitative assays and UMIs for accurate deduplication in low-diversity samples. Experimental Procedures: Genomic DNA (gDNA) was prepared for sequencing using the Twist Methylation Detection System consisting of enzymatic methylation conversion and hybrid capture using the Twist Human Methylome Panel. Twist’s synthetic CpG methylation level controls were spiked-in to gDNA and taken through the Twist Methylation Detection System to demonstrate their utility in calibrating methylation assays. Additionally, libraries were generated using cell free DNA (cfDNA) and either conventional or UMI-containing adapters to investigate the impact on quantitative detection and total unique coverage. Results: Using the Twist Human Methylome Panel at 150x raw coverage achieves uniform coverage with low off-bait Picard metrics. 6.59 million CpG sites were detected using a minimum depth of 10X. Target capture with the Human Methylome Panel allows for informative CpG calling of up to 82 samples on a single Illumina Novaseq S4 flowcell, compared to 3 samples per flowcell with a tradition whole-genome bisulfite sequencing (WGBS) assay. The Twist CpG methylation specific controls are constructed of 48 unique contrived sequences that contain a total of 8 different levels of methylation, ranging from 100% to 0%. Including these controls allows for quantitation of methylation levels in the experimental samples and qualification of the enzymatic conversion process. Conclusions: Our study leverages the Twist methylation detection portfolio to interrogate genome-wide methylation patterns for various applications. In combination, these tools can be used to design cost effective end-to-end assays. *For Research Use Only. Not for use in diagnostic procedures. Citation Format: Lydia Bonar, Kristin Butcher, Michael Bocek, Holly Corbitt, Bryan Hoglund, Cibelle Nassif, Patrick Cherry, Derek Murphy, Jean Challacombe, Esteban Toro. An end-to-end workflow for improved methylation detection. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6009.

Genome-Wide DNA Methylation Dynamics During Drought Responsiveness in Tibetan Hulless Barley

Differences in drought stress tolerance within diverse grass genotypes have been attributed to epigenetic modifications. DNA methylation is an important epigenetic alteration regulating responses to drought stress. However, its effects on drought tolerance are poorly understood in Tibetan hulless barley. Here, bisulfite sequencing was conducted to profile the DNA methylation patterns of drought-tolerant variety (XL) and drought-sensitive (DQ) under drought and control conditions. A total of 5843 million reads were generated. We found the significant genome-wide changes in CHH methylation rates between XL and DQ, while CG or CHG methylation rates did not. Besides that, the two contrasting varieties do reveal distinct responses to drought stress in antioxidant activities and differentially methylated regions (DMRs). Genes in drought-tolerant varieties XL are rapidly and significantly methylated when exposed to drought stimulus. These DMRs-related genes in XL are significantly enriched in defense response and response to stimuli via gene-ontology enrichment analysis. Then, we focused on 1003 transcription factors and identified 15 specific DMRs-related transcription factors exhibiting specific methylation changes under drought stimuli. Finally, we identified three DMRs-related TFs (HvRR12, HvRR2, and HvCSP41B), where Arabidopsis homologs involve in responses to drought conditions. Altogether, abiotic stresses could be rapidly respond and mediated by methylation of transcription factors in hulless barely.

Blood levels of T-Cell Receptor Excision Circles (TRECs) provide an index of exposure to traumatic stress in mice and humans

Exposure to stress triggers biological changes throughout the body. Accumulating evidence indicates that alterations in immune system function are associated with the development of stress-associated illnesses such as major depressive disorder and post-traumatic stress disorder, increasing interest in identifying immune markers that provide insight into mental health. Recombination events during T-cell receptor rearrangement and T-cell maturation in the thymus produce circular DNA fragments called T-cell receptor excision circles (TRECs) that can be utilized as indicators of thymic function and numbers of newly emigrating T-cells. Given data suggesting that stress affects thymus function, we examined whether blood levels of TRECs might serve as a quantitative peripheral index of cumulative stress exposure and its physiological correlates. We hypothesized that chronic stress exposure would compromise thymus function and produce corresponding decreases in levels of TRECs. In male mice, exposure to chronic social defeat stress (CSDS) produced thymic involution, adrenal hypertrophy, and decreased levels of TRECs in blood. Extending these studies to humans revealed robust inverse correlations between levels of circulating TRECs and childhood emotional and physical abuse. Cell-type specific analyses also revealed associations between TREC levels and blood cell composition, as well as cell-type specific methylation changes in CD4T + and CD8T + cells. Additionally, TREC levels correlated with epigenetic age acceleration, a common biomarker of stress exposure. Our findings demonstrate alignment between findings in mice and humans and suggest that blood-borne TRECs are a translationally-relevant biomarker that correlates with, and provides insight into, the cumulative physiological and immune-related impacts of stress exposure in mammals.

Pseudohypoparathyroidism and variants: A translational medicine success story

Pseudohypoparathyroidism (PHP) is an uncommon disorder which is characterized by end-organ PTH resistance. The genetic defect is located at the GNAS locus that encodes the alpha-subunit of the stimulatory G protein (Gαs) and several splice variants thereof. This complex locus undergoes parental specific methylation changes that result in tissue-specific silencing of the paternal allele. Heterozygous inactivating mutations that disrupt Gαs function or epigenetics changes that impair Gαs expression contribute to a wide clinical spectrum of the disease: PHP1A, PHP1B, osseous heteroplasia, osteodystrophy, obesity, intrauterine growth retardation… whose mechanisms at the molecular level remain unresolved.

Long-Term Treatment with Bortezomib Induces Specific Methylation Changes in Differentiated Neuronal Cells.

Simple SummaryWe exposed LUHMES cells, differentiated into mature neurons, to bortezomib (BTZ) in two treatment cycles and analyzed the methylomes of these cells after each cycle, controlling the analysis for the methylation changes potentially induced by the long-term culture. Our results show that BTZ induces methylation changes that may affect cell morphogenesis, neurogenesis, and neurotransmission. These changes are specifically enriched within transcription factor binding sites of EBF, PAX, DLX, LHX, and HNF family members, which have been shown to regulate neurogenesis and neuronal differentiation. We further show that the observed methylation changes are not present in the SH-SY5Y cells that we used to study mechanisms of development of BTZ resistance. Altogether, our results show that BTZ treatment induces very specific changes in the methylomes of neuronal cells.Bortezomib (BTZ) is proteasome inhibitor, effectively used in the treatment of multiple myeloma, but frequently discontinued due to peripheral neuropathy, which develops in patients after consecutive treatment cycles. The molecular mechanisms affected by BTZ in neuronal cells, which result in neuropathy, remain unknown. However, BTZ is unlikely to lead to permanent morphological nerve damage, because neuropathy reverses after discontinuation of treatment, and nerve cells have very limited renewal capacity. We have previously shown that BTZ induces methylation changes in SH-SY5Y cells, which take part in the development of treatment resistance. Here, we hypothesized that BTZ affects the methylomes of mature neurons, and these changes are associated with BTZ neurotoxicity. Thus, we studied methylomes of neuronal cells, differentiated from the LUHMES cell line, after cycles of treatment with BTZ. Our results show that BTZ induces specific methylation changes in mature neurons, which are not present in SH-SY5Y cells after BTZ treatment. These changes appear to affect genes involved in morphogenesis, neurogenesis, and neurotransmission. Furthermore, identified methylation changes are significantly enriched within binding sites of transcription factors previously linked to neuron physiology, including EBF, PAX, DLX, LHX, and HNF family members. Altogether, our results indicate that methylation changes are likely to be involved in BTZ neurotoxicity.

Abstract 5152: Mutation-agnostic detection of colorectal cancer-specific cell-free DNA using targeted methylation sequencing

Abstract BACKGROUND: Tumor-specific methylation changes in DNA CpG sites commonly occur in cancer and are believed to drive oncogenesis through gene silencing. Detection of methylation changes in circulating cell-free DNA (cfDNA) can offer a novel approach for cancer diagnostics. METHODS: Plasma samples from healthy controls and from patients with advanced colorectal and non-colorectal cancers were included in the study. Bisulfite conversion of cfDNA extracted from plasma was performed using EZ DNA Methylation Lightning Kit (Zymo Research) and was followed by library preparation using Accel-NGS Methyl-Seq DNA Library Kit (Swift Biosciences) and target enrichment using xGen Hybridization Capture for NGS Kit (IDT). Targeted methylation sequencing was done using NextSeq500 mid-output flow cell (300 cycles) (Illumina). Detection rates of methylation ratios in colorectal cancer samples were compared to non-colorectal cancers and healthy controls. RESULTS: First, we reviewed methylation changes in nearly 9,000 CpG sites in colorectal cancer (through TCGA database) and healthy controls. Subsequently, 32 CpG sites with greater than 50% methylation ratio in colorectal cancer and less than 1% methylation ratio in healthy controls were selected to develop targeted methylation sequencing based cfDNA assay. The assay was performed in 32 plasma samples from 20 individuals with advanced colorectal cancer who had tumor KRAS mutation, 8 individuals with advanced non-colorectal cancer who had tumor KRAS mutation (ovarian, n=2; endometrial, n=2; pancreatic, n=2; and lung cancer, n=2), and 4 healthy controls. Colorectal cancer specific methylation changes in cfDNA were detected in 85% (17/20) of colorectal cancer patients with a specificity of 92%. In colorectal cancer patients with confirmed KRAS mutation in cfDNA, methylation changes were detected in 92% (11/12) in comparison to 75% (6/8) in colorectal cancer patients without KRAS mutation in cfDNA. Median methylation ratio for target CpG sites was higher in colorectal cancer patients compared to patients with non-colorectal cancers and healthy controls (p<0.001). In 17 colorectal cancer patients with plasma samples collected before initiation of systemic cancer therapy, detection of methylation changes in cfDNA was associated with a shorter median progression-free survival compared to no detection (PFS; 8 weeks versus 54 weeks; p=0.027). CONCLUSIONS: Targeted methylation sequencing of cfDNA demonstrated high sensitivity and specificity for detection of colorectal cancer-specific cfDNA. Colorectal cancer patients with methylated cfDNA had shorter PFS while on cancer therapy. Citation Format: Mohamed A. Gouda, Dzifa Y. Duose, Morten Lapin, Stephanie Zalles, Helen J. Huang, Yuanxin Xi, Xiaofeng Zheng, Amira I. Aldesoky, Alshimaa M. Alhanafy, Mohamed A. Shehata, Jing Wang, Scott Kopetz, Funda Meric-Bernstam, Ignacio I. Wistuba, Rajyalakshmi Luthra, Filip Janku. Mutation-agnostic detection of colorectal cancer-specific cell-free DNA using targeted methylation sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5152.

A role for epigenetic mechanisms in Lewy body dementias.

The Lewy body diseases, Dementia with Lewy bodies (DLB), Parkinson's disease (PD) and Parkinson's disease dementia (PDD) are all neurodegenerative diseases classified by the accumulation of alpha-synuclein in neurons, forming Lewy bodies (LB). We hypothesise that these LBs cause epigenetic changes within neurons and surrounding cells and that these changes can be used to distinguish the different LB diseases from one another. Bulk tissue from the cingulate gyrus and prefrontal cortex will be as analysed for DNA methylation levels using the Illumina Infinium Methylation EPIC array to generate quantitative methylation data for over 850,000 CpG sites across the genome (n=∼100/disease group). Linear regression and pathway analyses will be used to identify loci that are significantly different or specific to each disease. Following this we will validate loci and determine their cellular specificity using a subset of samples (15 DLB, 15 PDD, 15 PD only, 15 controls) using fluorescence activated cell sorting (FACS). In each sample we will isolate various different cellular populations, including neurons, microglia, oligodendrocytes and astrocytes before profiling these using the EPIC array. Study groups have been sourced consisting of cases with PD, PDD and DLB based on LB deposition and clinical symptom staging. Control cases have been selected for matched age and levels of concomitant AD pathology. Cases for FACS (n=15/group) have been selected to allow where possible a high base RIN, pH and minimal post-mortem interval. We are collating a well powered study cohort to interrogate the epigenetic basis of neuropathological progression and clinical staging of LB disease, controlling for levels of concomitant AD pathology. Follow up FACS sorting and analysis will allow for the cell specific methylation changes occurring in each of the LB diseases.

Abstract 480: Aberrant promoter hypermethylation of HIC1 and SOX1 in circulating cell free DNA as a potential biomarker for noninvasive early detection of ovarian cancer

Abstract Background: Aberrant CpG island hypermethylation is an early event during carcinogenesis and a significant mechanism of tumor suppressor gene silencing. Assessment of tumor specific methylation changes in tissue or body fluids might provide insights into disease mechanisms and discovery of biomarkers. Ovarian cancer remains the most lethal gynaecological malignancy. Non-specific symptoms at early stage, presentation of disease at advanced stage and lack of sufficiently sensitive screening modalities have rendered significant challenge to its prompt diagnosis. To date, no single biomarker is able to accurately detect early OC in either tissue or body fluid. Aberrant promoter hypermethylation in cell free tumor-specific DNA can be indicative of highly specific cancer signals. Methods: Herein, we concurrently evaluated the promoter methylation of HIC1 and SOX1 gene in 120 ovarian tissue samples and validated in 70 matched serum cell free DNA of cancerous and non cancerous samples by multiplex MethyLight assay (a high throughput quantitative, TaqMan based real time PCR technique). Additionally, promoter methylation pattern of these genes were validated by clonal bisulfite sequencing. The ROC (Receiver-operator characteristic) curves were constructed to evaluate the diagnostic performances of both individual and combined gene panel. Results: In singleplex assay, high methylation sensitivity and specificity was evident for SOX1 (78.8% and 80.0%, respectively; AUC= 0.88) and HIC1 (80.0% and 77.14%, respectively; AUC=0.85) in tissue samples. In the multiplex assay, when either or both gene promoter showed methylation, the highly sensitivity and specific discrimination of EOC patients from normal controls was observed for SOX1 + HIC1 (83.53% and 88.57%, respectively; AUC=0.89). We identified DNA methylation of HIC1 and SOX1 as best discriminator between cancer and non-neoplastic tissue. Methylated SOX1 and HIC1 occurred in 53.3% and 71.1% of OC serum samples when their promoter methylation was analyzed in singleplex assay (AUC=0.81 and 0.88, respectively). In cell free DNA, this novel panel achieved a sensitivity of 80.0% and a specificity of 96.0% with an area under the ROC curve of 0.93 for discriminating OC samples from normal control. The results of MethyLight were highly concordant with those of clonal bisulfite sequencing. Methylated Individual gene as well as in combined panel exhibited better discriminatory efficiencies to identify ovarian cancer at various stages of disease when analyzed in tissue and serum cell free DNA. Conclusion: We report a QPCR based non-invasive epigenetic biomarker assay with high sensitivity and specificity for OC screening. Our findings also reveal the potential utility of methylation based detection of circulating cell free tumor DNA in clinical management of ovarian cancer. Citation Format: Alka Singh, Manisha Sachan. Aberrant promoter hypermethylation of HIC1 and SOX1 in circulating cell free DNA as a potential biomarker for noninvasive early detection of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 480.

Clinically relevant aberrant Filip1l DNA methylation detected in a murine model of cutaneous squamous cell carcinoma.

BackgroundCutaneous squamous cell carcinomas (cSCC) are among the most common and highly mutated human malignancies. Understanding the impact of DNA methylation in cSCC may provide avenues for new therapeutic strategies.MethodsWe used reduced-representation bisulfite sequencing for DNA methylation analysis of murine cSCC. Differential methylation was assessed at the CpG level using limma. Next, we compared with human cSCC Infinium HumanMethylation BeadArray data. Genes were considered to be of major relevance when they featured at least one significantly differentially methylated CpGs (RRBS) / probes (Infinium) with at least a 30% difference between tumour vs. control in both a murine gene and its human orthologue. The human EPIC Infinium data were used to distinguish two cSCC subtypes, stem-cell-like and keratinocyte-like tumours.FindingsWe found increased average methylation in mouse cSCC (by 12.8%, p = 0.0011) as well as in stem-cell like (by 3.1%, p=0.002), but not keratinocyte-like (0.2%, p = 0.98), human cSCC. Comparison of differentially methylated genes revealed striking similarities between human and mouse cSCC. Locus specific methylation changes in mouse cSCC often occurred in regions of potential regulatory function, including enhancers and promoters. A key differentially methylated region was located in a potential enhancer of the tumour suppressor gene Filip1l and its expression was reduced in mouse tumours. Moreover, the FILIP1L locus showed hypermethylation in human cSCC and lower expression in human cSCC cell lines.InterpretationDeregulation of DNA methylation is an important feature of murine and human cSCC that likely contributes to silencing of tumour suppressor genes, as shown for Filip1l.FundingBritish Skin Foundation, Cancer Research UK

DNA-methylation for the detection and distinction of 19 human malignancies

ABSTRACT The contribution of DNA-methylation based gene silencing to carcinogenesis is well established. Increasingly, DNA-methylation is examined using genome-wide techniques, with recent public efforts yielding immense data sets of diverse malignancies representing the vast majority of human cancer related disease burden. Whereas mutation events may group preferentially or in high frequency with a given histology, mutations are poor classifiers of tumour type. Here we examine the hypothesis that cancer-specific DNA-methylation reflects the tissue of origin or carcinogenic risk factor, and these methylation abnormalities may be used to faithfully classify tumours according to histology. We present an analysis of 7427 tumours representing 19 human malignancies and 708 normal samples demonstrating that specific tumour changes in methylation can correctly determine site of origin and tumour histology with 86% overall accuracy. Examination of misclassified tumours reveals underlying shared biology as the source of misclassifications, including common cell of origin or risk factors.

Integrative DNA Methylation and Gene Expression Analysis Reveals Candidate Biomarkers Associated with Dichotomized Response to Chemoimmunotherapy in Diffuse Large B-Cell Lymphoma

Background: Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma. While 60% of DLBCL patients achieve complete remission with frontline therapy, relapsed/refractory (R/R) DLBCL patients have a poor prognosis with median overall survival below one year, necessitating investigation into the biological principles that distinguish cured from R/R DLBCL. Recent analyses have identified unfavorable molecular signatures when accounting for gene expression, copy number alterations and mutational profiles in R/R DLBCL. However, an integrative analysis of the relationship between epigenetic and transcriptomic changes has yet to be described. In this study, we compared baseline methylation and gene expression profiles of DLBCL patients with dichotomized clinical outcomes. Methods: Diagnostic DLBCL biopsies were obtained from two patient cohorts: patients who relapsed or were refractory following chemoimmunotherapy ("R/R"), and patients who entered durable clinical remission following therapy ("cured"). The median age for R/R and cured cohorts were 62 (range 35-86) years vs. 64 (range 28-83) years (P= 0.27). High-intermediate or high IPI scores were present in 14 vs. 6 patients (P= 0.08) in the R/R and cured cohorts, respectively. All patients were treated with frontline R-CHOP or R-EPOCH. DNA and RNA were extracted simultaneously from formalin-fixed, paraffin embedded biopsy samples. An Illumina 850k Methylation Array was used to identify DNA methylation levels in 29 R/R patients and 20 cured patients. RNA sequencing was performed on 9 R/R patients and 7 cured patients at diagnosis using Illumina HiSeq4000. Differentially methylated probes were identified using the DMRcate package, and differentially expressed genes were identified using the DESeq2 package. Gene set enrichment analysis was performed using canonical pathway gene sets from MSigDB. Results: At the time of diagnosis, we found significant epigenetic and transcriptomic differences between cured and R/R patients. Comparing cured to R/R samples, there were 8,159 differentially methylated probes (FDR<0.05). Differentially methylated regions between R/R and cured cohorts overlap with genes previously identified as mutation hotspots in DLBCL. Upon comparing transcriptomic profiles between R/R and cured, 267 genes were found to be differentially expressed (Log2FC>|1| and FDR<0.05). Gene set enrichment analysis revealed gene sets related to cell cycle, membrane trafficking, Rho and Rab family GTPase function, and transcriptional regulation were upregulated in the R/R samples. Gene sets related to innate immune signaling, Type I and II interferon signaling, fatty acid and carbohydrate metabolism were upregulated in the cured samples. To identify genes likely to be regulated by specific changes in methylation, we selected genes that were both differentially expressed and differentially methylated between the R/R and cured cohorts. In the R/R samples, 13 genes (ARMC5, ARRDC1, C12orf57, CCSER1, D2HGDH, DUOX2, FAM189B, FKBP2, KLF5, MFSD10, NEK8, NT5C, and WDR18) were significantly hypermethylated and underexpressed when compared to cured specimens, suggesting that epigenetic silencing of these genes is associated with lack of response to chemoimmunotherapy. In contrast, 12 genes (ATP2B1, C15orf41, FAM102B, FAM3C, FHOD3, FYTTD1, GPR180, KIAA1841, LRMP, MEF2A, RRAS2, and TPD52) were significantly hypermethylated and underexpressed in cured patients, suggesting that epigenetic silencing of these genes is favorable for treatment response. Many of these epigenetically modified genes have been previously implicated in cancer biology, including roles in NOTCH signaling, chromosomal instability, and biomarkers of prognosis. Conclusions: This is the first integrative epigenetic and transcriptomic analysis of diagnostic biopsies from cured and R/R DLBCL patients following chemoimmunotherapy. At the time of diagnosis, both the methylation and gene expression profiles significantly differ between patients that enter durable remission as opposed to those who are R/R to therapy. Soon, the hypomethylating agent CC-486 (i.e. oral azacitidine) will be explored in combination with mini-R-CHOP for older DLBCL patients in whom DNA methylation is likely increased. These data support the use of hypomethylating agents to potentially restore sensitivity of DLBCL to chemoimmunotherapy. Disclosures Hsi: Eli Lilly: Research Funding; Abbvie: Research Funding; Miltenyi: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; CytomX: Consultancy, Honoraria. Hill:Celgene: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Kite, a Gilead Company: Consultancy, Honoraria, Research Funding; AstraZenica: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Takeda: Research Funding; Beigene: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding.

Associations between the development of PTSD symptoms and longitudinal changes in the DNA methylome of deployed military servicemen: A comparison with polygenic risk scores

ObjectiveMilitary servicemen deployed to war zones are at increased risk of developing posttraumatic stress disorder (PTSD) and successful adaptation to stress is important. Epigenetic alterations in response to trauma have been identified as mechanism of adaptation and may therefore predict deployment-related PTSD symptoms. To date, human studies of epigenetic marks for traumatic stress have been largely constrained by short-term analyses of one or two time points. MethodThis study in a prospective Dutch military cohort (N ​= ​125) examined longitudinal changes of DNA methylation profiles before, as well as one and six months after deployment-related combat exposure in relation to the development of PTSD symptoms over a period of up to five years after deployment. We investigated the predictive value of specific methylation changes for immediate and delayed-onset PTSD symptoms and recovery. This epigenetic prediction was compared to polygenic risk score predictions obtained from the currently available largest genome-wide association study of PTSD. ResultsA total of fourteen genomic regions were identified in which PTSD symptom levels were associated with methylation changes over time (pre-deployment, one, and six months post-deployment). Of these regions, four were significant determinants of longitudinal development of PTSD symptoms. In addition, we observed that, together with risk level during deployment (operating inside or outside the military base) and physical childhood trauma, post-deployment decreases in methylation at a genomic region in EP300/miRNA1281 was associated with a delayed onset of PTSD compared to a resilient profile. Polygenic risk, in contrast, was related to PTSD onset within six months after deployment but was not associated with long term outcomes. ConclusionThe present study suggests predictive utility of changes in DNA methylation for the subsequent development of PTSD symptoms and showed that the currently available measure of polygenic risk is primarily related to non-delayed disease onset.

Induction and recovery of CpG site specific methylation changes in human bronchial cells after long-term exposure to carbon nanotubes and asbestos

IntroductionInhalation of asbestos induces lung cancer via different cellular mechanisms. Together with the increased production of carbon nanotubes (CNTs) grows the concern about adverse effects on the lungs given the similarities with asbestos. While it has been established that CNT and asbestos induce epigenetic alterations, it is currently not known whether alterations at epigenetic level remain stable after withdrawal of the exposure. Identification of DNA methylation changes after a low dose of CNT and asbestos exposure and recovery can be useful to determine the fibre/particle toxicity and adverse outcome. MethodsHuman bronchial epithelial cells (16HBE) were treated with a low and non-cytotoxic dose (0.25 µg/ml) of multi-walled carbon nanotubes (MWCNTs-NM400) or single-walled carbon nanotubes (SWCNTs-SRM2483) and 0.05 µg/ml amosite (brown) asbestos for the course of four weeks (sub-chronic exposure). After this treatment, the cells were further incubated (without particle/fibre) for two weeks, allowing recovery from the exposure (recovery period). Nuclear depositions of the CNTs were assessed using femtosecond pulsed laser microscopy in a label-free manner. DNA methylation alterations were analysed using microarrays that assess more than 850 thousand CpG sites in the whole genome. ResultsAt non-cytotoxic doses, CNTs were noted to be incorporated with in the nucleus after a four weeks period. Exposure to MWCNTs induced a single hypomethylation at a CpG site and gene promoter region. No change in DNA methylation was observed after the recovery period for MWCNTs. Exposure to SWCNTs or amosite induced hypermethylation at CpG sites after sub-chronic exposure which may involve in ‘transcription factor activity’ and ‘sequence-specific DNA binding’ gene ontologies. After the recovery period, hypermethylation and hypomethylation were noted for both SWCNTs and amosite. Hippocalcinlike 1 (HPCAL1), protease serine 3 (PRSS3), kallikrein-related peptidase 3 (KLK3), kruppel like factor 3 (KLF3) genes were hypermethylated at different time points in either SWCNT-exposed or amosite-exposed cells. ConclusionThese results suggest that the specific SWCNT (SRM2483) and amosite fibres studied induce hypo- or hypermethylation on CpG sites in DNA after very low-dose exposure and recovery period. This effect was not seen for the studied MWCNT (NM400).

Whole Genome Bisulfite Sequencing of 63 Primary AML Samples Identifies a Unique DNA Hypermethylation Signature for Mutant IDH1/2 Cases That Is Different from That of TET2 Mutant AML

Mutations in IDH1, IDH2, and TET2 occur in more than 30% of AML patients, and are thought to contribute to leukemogenesis by disrupting TET2-mediated DNA demethylation, either directly or indirectly. While IDH1/2 and TET2 mutations are both predicted to result in hypermethylation, the specific methylation changes associated with these mutations are poorly understood. Additionally, we recently showed that CpG island (CGI) hypermethylation is common across virtually all AML subtypes, and it has been yet unclear whether IDH or TET2 hypermethylation is distinct from these 'generic', AML-associated changes. To investigate genome-wide methylation in IDH and TET2 mutated AML samples, we performed whole genome bisulfite sequencing (WBGS) on 15 primary AML samples with canonical IDH mutations (IDH1R132C, N=6; IDH1R132G, N=1; IDH2R140Q, N=7; and IDH2R172K, N=1) and 9 samples with biallelic loss-of-function mutations in TET2. These data were analyzed with WGBS data from primary CD34+ hematopoietic stem/progenitor cells (HSPCs) from 6 normal adult bone marrow donors, and 39 additional primary AML samples (representing 9 AML mutational categories) to define the methylation phenotypes specifically associated with IDH and/or TET2 mutations. Differential methylation analysis of each AML subtype vs. HSPCs identified a mean of 8,622 (53,73-13,773) AML-associated Differentially Methylated Regions (DMRs) in each mutational category (mean size: 986 bp), of which 6% to 97% were hypermethylated in the AML samples vs. HSPCs. IDH-mutant AML samples were the most hypermethylated subtype, with 97% of the 6,392 identified DMRs having increased methylation compared to HSPCs. The TET2-mutant samples were hypermethylated at fewer loci, representing 71% of the 5,892 identified DMRs vs HSPCs. Analysis of all AML subtypes demonstrated that 55% (16,275/29,534) of identified DMRs were shared across multiple subtypes, with more pronounced overlap among hypermethylated DMRs (>70%). To distinguish mutation-specific changes in IDH and TET2 mutated AMLs from commonly hypermethylated loci across AML subtypes, we conducted a joint analysis of either IDH or TET2 mutant AMLs with data from all other AML samples and normal HSPCs. We performed hierarchical clustering of the methylation values at each DMR, and used the clustering topology to identify DMRs where the single outlier branch represented either the IDH or TET2 AML samples. We identified 2,024 DMRs (mean size: 998 bp) specific to IDH mutant AML samples, of which 100% were hypermethylated with respect to normal HSPCs (mean DMR b value of 0.60 vs 0.35 in HSPCs). IDH outlier DMRs were enriched for functional genomic elements relative to the set of commonly hypermethylated loci, including CGIs (65% of DMRs; 2.4-fold enrichment) and potential enhancers marked by H3K27ac in HSPCs (82% of DMRs; 1.4-fold enrichment); overlap with promoter regions was reduced (20% of DMRs; 0.77-fold decrease). Outlier analysis of TET2 mutated samples demonstrated that most changes were also present in other AML types, with only 93 unique DMRs (mean size 552 bp), and a subtler hypermethylation phenotype (83% of DMRs were hypermethylated, with a mean b value of 0.51 vs. 0.32 in HSPCs). TET2 DMRs were also enriched for overlap with CGIs with respect to background, but to a lesser extent than IDH outliers (52% of DMRs; 1.9-fold). Intersection of IDH and TET2-associated DMRs showed that TET2-specific DMRs represented a subset of IDH hypermethylated regions, with 60% of TET2 DMRs overlapping a DMR independently identified in the IDH samples. Conversely, the vast majority of IDH outliers (97%) were identified only in IDH mutated AMLs. This comprehensive, genome-wide analysis demonstrates that IDH mutations result in a clear hypermethylation phenotype in primary AML samples that is far more expansive than the observed TET2-associated methylation changes. IDH-specific methylation changes were enriched for CGIs located outside of promoters and were distinct from the AML-associated CGI hypermethylation that occurs in other AML subtypes. TET2 mutations were associated with far fewer hypermethylated DMRs, and only a small fraction of mutation-specific changes occurred in bothIDH and TET2 samples. Our results demonstrate that the methylation phenotypes of IDH and TET2 mutant AML samples do not phenocopy, and suggest that IDH-associated hypermethylation must have a TET2-independent component. Disclosures No relevant conflicts of interest to declare.

Brain region specific methylation and Sirt1 binding changes in MAOA promoter is associated with sexual dimorphism in early life stress induced aggressive behavior

The maladaptive form of aggressive behavior confers risk for violence and criminal incidences with profound impact on society. Although considerable research has been devoted to elucidate the etiology of aggression, molecular correlates of sex differences remains largely unexplored. Also, little attention has been given to whether males and females respond differently to similar causal factor of aggression. Here, we show the possible association of brain region specific neural activity (c-Fos expression) and monoamine oxidase A (MAOA) epigenetic state with sexual dimorphism in peripubertal stress (PPS) induced adulthood aggression. While PPS adult males exhibited escalated aggression, females spent maximal time in social exploration. c-Fos expression was brain region and sex specific. In the PPS adult cohort, only males showed elevated c-Fos expression in the prefrontal cortex, indicative of their hyper-responsive behavior. MAOA expression and enzyme activity was reduced in hypothalamus and increased in prefrontal cortex of hyper-aggressive male mice. Investigation into the underlying mechanisms revealed hypomethylation in prefrontal cortex and hypermethylation in hypothalamus of MAOA promoter negatively correlating with the expression pattern. On the other hand, binding of Sirt1 to MAOA promoter was diametrically opposite being increased in prefrontal cortex and reduced in hypothalamus. In females, neither expression nor epigenetic state of MAOA gene was significantly altered between control and PPS adult mice. Our study revealed novel epigenetic correlates of sexual dimorphism in stress induced aggressive psychopathology. However, given the multi-factorial nature with environmental influences, further studies are warranted to uncover the biological hub.

F8 Inversions at Xq28 Causing Hemophilia A Are Associated With Specific Methylation Changes: Implication for Molecular Epigenetic Diagnosis.

Diverse DNA structural variations (SVs) in human cancers and several other diseases are well documented. For genomic inversions in particular, the disease causing mechanism may not be clear, especially if the inversion border does not cross a coding sequence. Understanding about the molecular processes of these inverted genomic sequences, in a mainly epigenetic context, may provide additional information regarding sequence-specific regulation of gene expression in human diseases. Herein, we study one such inversion hotspot at Xq28, which leads to the disruption of F8 gene and results in hemophilia A phenotype. To determine the epigenetic consequence of this rearrangement, we evaluated DNA methylation levels of 12 CpG rich regions with the coverage of 550 kb by using bisulfite-pyrosequencing and next-generation sequencing (NGS)-based bisulfite re-sequencing enrichment assay. Our results show that this inversion prone area harbors widespread methylation changes at the studied regions. However, only 5/12 regions showed significant methylation changes, specifically in case of intron 1 inversion (two regions), intron 22 inversion (two regions) and one common region in both inversions. Interestingly, these aberrant methylated regions were found to be overlapping with the inversion proximities. In addition, two CpG sites reached 100% sensitivity and specificity to discriminate wild type from intron 22 and intron 1 inversion samples. While we found age to be an influencing factor on methylation levels at some regions, covariate analysis still confirms the differential methylation induced by inversion, regardless of age. The hemophilia A methylation inversion “HAMI” assay provides an advantage over conventional PCR-based methods, which may not detect novel rare genomic rearrangements. Taken together, we showed that genomic inversions in the F8 (Xq28) region are associated with detectable changes in methylation levels and can be used as an epigenetic diagnostic marker.

Investigating epigenetic effects of activation-induced deaminase in chronic lymphocytic leukemia.

Activation induced deaminase (AID) has two distinct and well defined roles, both relying on its deoxycytidine (dC) deaminating function: one as a DNA mutator and another in DNA demethylation. In chronic lymphocytic leukemia (CLL), AID was previously shown to be an independent negative prognostic factor. While there is substantial impact on DNA mutations, effects of AID on gene expression by promoter demethylation of disease related target genes in leukemia has not been addressed. To shed light on this question, we aimed at determining genome wide methylation changes as well as gene expression changes in response to AID expression in CLL. Although we found minor differences in individual methylation variable positions following AID expression, we could not find recurrent methylation changes of specific target sites or changes in global methylation.