A Landscape of Pharmacogenomic Interactions in Cancer.

Systematic studies of cancer genomes have provided unprecedented insights into the molecular nature of cancer. Using this information to guide the development and application of therapies in the clinic is challenging. Here, we report how cancer-driven alterations identified in 11,289 tumors from 29 tissues (integrating somatic mutations, copy number alterations, DNA methylation, and gene expression) can be mapped onto 1,001 molecularly annotated human cancer cell lines and correlated with sensitivity to 265 drugs. We find that cell lines faithfully recapitulate oncogenic alterations identified in tumors, find that many of these associate with drug sensitivity/resistance, and highlight the importance of tissue lineage in mediating drug response. Logic-based modeling uncovers combinations of alterations that sensitize to drugs, while machine learning demonstrates the relative importance of different data types in predicting drug response. Our analysis and datasets are rich resources to link genotypes with cellular phenotypes and to identify therapeutic options for selected cancer sub-populations.

A synthetic mRNA cell reprogramming method using CYCLIN D1 promotes DNA repair, generating improved genetically stable human induced pluripotent stem cells.

A key challenge for clinical application of induced pluripotent stem cells (iPSC) to accurately model and treat human pathologies depends on developing a method to generate genetically stable cells to reduce long-term risks of cell transplant therapy. Here, we hypothesized that CYCLIN D1 repairs DNA by highly efficient homologous recombination (HR) during reprogramming to iPSC that reduces genetic instability and threat of neoplastic growth. We adopted a synthetic mRNA transfection method using clinically compatible conditions with CYCLIN D1 plus base factors (OCT3/4, SOX2, KLF4, LIN28) and compared with methods that use C-MYC. We demonstrate that CYCLIN D1 made iPSC have (a) lower multitelomeric signal, (b) reduced double-strand DNA breaks, (c) correct nuclear localization of RAD51 protein expression, and (d) reduced single-nucleotide polymorphism (SNP) changes per chromosome, compared with the classical reprogramming method using C-MYC. CYCLIN D1 iPSC have reduced teratoma Ki67 cell growth kinetics and derived neural stem cells successfully engraft in a hostile spinal cord injury (SCI) microenvironment with efficient survival, differentiation. We demonstrate that CYCLIN D1 promotes double-stranded DNA damage repair predominantly through HR during cell reprogramming to efficiently produce iPSC. CYCLIN D1 reduces general cell stress associated with significantly lower SIRT1 gene expression and can rescue Sirt1 null mouse cell reprogramming. In conclusion, we show synthetic mRNA transfection of CYCLIN D1 repairs DNA during reprogramming resulting in significantly improved genetically stable footprint in human iPSC, enabling a new cell reprogramming method for more accurate and reliable generation of human iPSC for disease modeling and future clinical applications.

Global Proteomic and Methylome Analysis in Human Induced Pluripotent Stem Cells Reveals Overexpression of a Human TLR3 Affecting Proper Innate Immune Response Signaling.

When considering the clinical applications of autologous cell replacement therapy of human induced pluripotent stem cells (iPSC)-derived cells, there is a clear need to better understand what the immune response will be before we embark on extensive clinical trials to treat or model human disease. We performed a detailed assessment comparing human fibroblast cell lines (termed F1) reprogrammed into human iPSC and subsequently differentiated back to fibroblast cells (termed F2) or other human iPSC-derived cells including neural stem cells (NSC) made from either retroviral, episomal, or synthetic mRNA cell reprogramming methods. Global proteomic analysis reveals the main differences in signal transduction and immune cell protein expression between F1 and F2 cells, implicating wild type (WT) toll like receptor protein 3 (TLR3). Furthermore, global methylome analysis identified an isoform of the human TLR3 gene that is not epigenetically reset correctly upon differentiation to F2 cells resulting in a hypomethylated transcription start site in the TLR3 isoform promoter and overexpression in most human iPSC-derived cells not seen in normal human tissue. The human TLR3 isoform in human iPSC-NSC functions to suppress NF-KB p65 signaling pathway in response to virus (Poly IC), suggesting suppressed immunity of iPSC-derived cells to viral infection. The sustained WT TLR3 and TLR3 isoform overexpression is central to understanding the altered immunogenicity of human iPSC-derived cells calling for screening of human iPSC-derived cells for TLR3 expression levels before applications. Stem Cells 2019;37:476-488.

Walking pathways with positive feedback loops reveal DNA methylation biomarkers of colorectal cancer.

BACKGROUND: The search for molecular biomarkers of early-onset colorectal cancer (CRC) is an important but still quite challenging and unsolved task. Detection of CpG methylation in human DNA obtained from blood or stool has been proposed as a promising approach to a noninvasive early diagnosis of CRC. Thousands of abnormally methylated CpG positions in CRC genomes are often located in non-coding parts of genes. Novel bioinformatic methods are thus urgently needed for multi-omics data analysis to reveal causative biomarkers with a potential driver role in early stages of cancer. METHODS: We have developed a method for finding potential causal relationships between epigenetic changes (DNA methylations) in gene regulatory regions that affect transcription factor binding sites (TFBS) and gene expression changes. This method also considers the topology of the involved signal transduction pathways and searches for positive feedback loops that may cause the carcinogenic aberrations in gene expression. We call this method "Walking pathways", since it searches for potential rewiring mechanisms in cancer pathways due to dynamic changes in the DNA methylation status of important gene regulatory regions ("epigenomic walking"). RESULTS: In this paper, we analysed an extensive collection of full genome gene-expression data (RNA-seq) and DNA methylation data of genomic CpG islands (using Illumina methylation arrays) generated from a sample of tumor and normal gut epithelial tissues of 300 patients with colorectal cancer (at different stages of the disease) (data generated in the EU-supported SysCol project). Identification of potential epigenetic biomarkers of DNA methylation was performed using the fully automatic multi-omics analysis web service "My Genome Enhancer" (MGE) (my-genome-enhancer.com). MGE uses the database on gene regulation TRANSFAC®, the signal transduction pathways database TRANSPATH®, and software that employs AI (artificial intelligence) methods for the analysis of cancer-specific enhancers. CONCLUSIONS: The identified biomarkers underwent experimental testing on an independent set of blood samples from patients with colorectal cancer. As a result, using advanced methods of statistics and machine learning, a minimum set of 6 biomarkers was selected, which together achieve the best cancer detection potential. The markers include hypermethylated positions in regulatory regions of the following genes: CALCA, ENO1, MYC, PDX1, TCF7, ZNF43.

Molecular portrait of high alpha-fetoprotein in hepatocellular carcinoma: implications for biomarker-driven clinical trials.

The clinical utility of serum alpha-fetoprotein (AFP) in patients with hepatocellular carcinoma (HCC) is widely recognised. However, a clear understanding of the mechanisms of AFP overexpression and the molecular traits of patients with AFP-high tumours are not known. We assessed transcriptome data, whole-exome sequencing data and DNA methylome profiling of 520 HCC patients from two independent cohorts to identify distinct molecular traits of patients with AFP-high tumours (serum concentration > 400 ng/ml), which represents an accepted prognostic cut-off and a predictor of response to ramucirumab. Those AFP-high tumours (18% of resected cases) were characterised by significantly lower AFP promoter methylation (p < 0.001), significant enrichment of progenitor-cell features (CK19, EPCAM), higher incidence of BAP1 oncogene mutations (8.5% vs 1.6%) and lower mutational rates of CTNNB1 (14% vs 30%). Specifically, AFP-high tumours displayed significant activation of VEGF signalling (p < 0.001), which might provide the rationale for the reported benefit of ramucirumab in this subgroup of patients.

Identification of an episignature of human colorectal cancer associated with obesity by genome-wide DNA methylation analysis.

BACKGROUND: Obesity was established as a relevant modifiable risk factor in the onset and progression of colorectal cancer (CRC). This relationship could be mediated by an epigenetic regulation. OBJECTIVES: The current work aimed to explore the effects of excess body weight on the DNA methylation profile of CRC using a genome-wide DNA methylation approach and to identify an epigenetic signature of obesity-related CRC. METHODS: Fifty-six CRC-diagnosed patients (50 years) were included in the study and categorized according to their body mass index (BMI) as non-obese (BMI ≤ 25 kg/m2) or overweight/obese (BMI > 25 kg/m2). Data from Infinium 450k array-based methylomes of 28 CRC tumor samples were coupled with information on BMI categories. Additionally, DNA methylation results were validated in 28 CRC tumor samples. RESULTS: The analysis revealed statistically significant differences at 299 CpG sites, and they were mostly characterized as changes towards CpG hypermethylation occurring in the obese group. The 152 identified genes were involved in inflammatory and metabolic functional processes. Among these genes, novel genes were identified as epigenetically regulated in CRC depending on adiposity. ZNF397OS and ZNF543 represented the top scoring associated events that were further validated in an independent cohort and exhibited strong correlation with BMI and excellent and statistically significant efficiency in the discrimination of obese from non-obese CRC patients (area under the curve >0.80; p < 0.05). CONCLUSIONS: The present study identifies a potential epigenome mark of obesity-related CRC that could be useful for precision medicine in the management of this disease taking into account adiposity as a relevant risk factor.

Epigenetic loss of RNA-methyltransferase NSUN5 in glioma targets ribosomes to drive a stress adaptive translational program.

Tumors have aberrant proteomes that often do not match their corresponding transcriptome profiles. One possible cause of this discrepancy is the existence of aberrant RNA modification landscapes in the so-called epitranscriptome. Here, we report that human glioma cells undergo DNA methylation-associated epigenetic silencing of NSUN5, a candidate RNA methyltransferase for 5-methylcytosine. In this setting, NSUN5 exhibits tumor-suppressor characteristics in vivo glioma models. We also found that NSUN5 loss generates an unmethylated status at the C3782 position of 28S rRNA that drives an overall depletion of protein synthesis, and leads to the emergence of an adaptive translational program for survival under conditions of cellular stress. Interestingly, NSUN5 epigenetic inactivation also renders these gliomas sensitive to bioactivatable substrates of the stress-related enzyme NQO1. Most importantly, NSUN5 epigenetic inactivation is a hallmark of glioma patients with long-term survival for this otherwise devastating disease.

Discovery of methylated circulating DNA biomarkers for comprehensive non-invasive monitoring of treatment response in metastatic colorectal cancer.

OBJECTIVE: Mutations in cell-free circulating DNA (cfDNA) have been studied for tracking disease relapse in colorectal cancer (CRC). This approach requires personalised assay design due to the lack of universally mutated genes. In contrast, early methylation alterations are restricted to defined genomic loci allowing comprehensive assay design for population studies. Our objective was to identify cancer-specific methylated biomarkers which could be measured longitudinally in cfDNA (liquid biopsy) to monitor therapeutic outcome in patients with metastatic CRC (mCRC). DESIGN: Genome-wide methylation microarrays of CRC cell lines (n=149) identified five cancer-specific methylated loci (EYA4, GRIA4, ITGA4, MAP3K14-AS1, MSC). Digital PCR assays were employed to measure methylation of these genes in tumour tissue DNA (n=82) and cfDNA from patients with mCRC (n=182). Plasma longitudinal assessment was performed in a patient subset treated with chemotherapy or targeted therapy. RESULTS: Methylation in at least one marker was detected in all tumour tissue samples and in 156 mCRC patient cfDNA samples (85.7%). Plasma marker prevalence was 71.4% for EYA4, 68.5% for GRIA4, 69.7% for ITGA4, 69.1% for MAP3K14-AS1% and 65.1% for MSC. Dynamics of methylation markers was not affected by treatment type and correlated with objective tumour response and progression-free survival. CONCLUSION: This five-gene methylation panel can be used to circumvent the absence of patient-specific mutations for monitoring tumour burden dynamics in liquid biopsy under different therapeutic regimens. This method might be proposed for assessing pharmacodynamics in clinical trials or when conventional imaging has limitations.

Metformin pharmacogenomics: a genome-wide association study to identify genetic and epigenetic biomarkers involved in metformin anticancer response using human lymphoblastoid cell lines.

Metformin is currently considered as a promising anticancer agent in addition to its anti-diabetic effect. To better individualize metformin therapy and explore novel molecular mechanisms in cancer treatment, we conducted a pharmacogenomic study using 266 lymphoblastoid cell lines (LCLs). Metformin cytotoxicity assay was performed using the MTS assay. Genome-wide association (GWA) analyses were performed in LCLs using 1.3 million SNPs, 485k DNA methylation probes, 54k mRNA expression probe sets, and metformin cytotoxicity (IC50s). Top candidate genes were functionally validated using siRNA screening, followed by MTS assay in breast cancer cell lines. Further study of one top candidate, STUB1, was performed to elucidate the mechanisms by which STUB1 might contribute to metformin action. GWA analyses in LCLs identified 198 mRNA expression probe sets, 12 SNP loci, and 5 DNA methylation loci associated with metformin IC50 with P-values <10−4 or <10−5. Integrated SNP/methylation loci-expression-IC50 analyses found 3 SNP loci or 5 DNA methylation loci associated with metformin IC50 through trans-regulation of expression of 11 or 26 genes with P-value <10−4. Functional validation of top 61 candidate genes in 4 IPA networks indicated down regulation of 14 genes significantly altered metformin sensitivity in two breast cancer cell lines. Mechanistic studies revealed that the E3 ubiquitin ligase, STUB1, could influence metformin response by facilitating proteasome-mediated degradation of cyclin A. GWAS using a genomic data-enriched LCL model system, together with functional and mechanistic studies using cancer cell lines, help us to identify novel genetic and epigenetic biomarkers involved in metformin anticancer response.

H3K4me1 marks DNA regions hypomethylated during aging in human stem and differentiated cells.

In differentiated cells, aging is associated with hypermethylation of DNA regions enriched in repressive histone post-translational modifications. However, the chromatin marks associated with changes in DNA methylation in adult stem cells during lifetime are still largely unknown. Here, DNA methylation profiling of mesenchymal stem cells (MSCs) obtained from individuals aged 2 to 92 yr identified 18,735 hypermethylated and 45,407 hypomethylated CpG sites associated with aging. As in differentiated cells, hypermethylated sequences were enriched in chromatin repressive marks. Most importantly, hypomethylated CpG sites were strongly enriched in the active chromatin mark H3K4me1 in stem and differentiated cells, suggesting this is a cell type-independent chromatin signature of DNA hypomethylation during aging. Analysis of scedasticity showed that interindividual variability of DNA methylation increased during aging in MSCs and differentiated cells, providing a new avenue for the identification of DNA methylation changes over time. DNA methylation profiling of genetically identical individuals showed that both the tendency of DNA methylation changes and scedasticity depended on nongenetic as well as genetic factors. Our results indicate that the dynamics of DNA methylation during aging depend on a complex mixture of factors that include the DNA sequence, cell type, and chromatin context involved and that, depending on the locus, the changes can be modulated by genetic and/or external factors.

Correction to: DNA methylation-based reclassification of olfactory neuroblastoma.

In the original publication, the second name of the twentieth author was incorrect. It should read as 'Miguel Sáinz-Jaspeado'. The original publication of the article has been updated to reflect the change. This correction was authored by Ulrich Schüller on behalf of all authors of the original publication.

DNA methylation-based reclassification of olfactory neuroblastoma.

Olfactory neuroblastoma/esthesioneuroblastoma (ONB) is an uncommon neuroectodermal neoplasm thought to arise from the olfactory epithelium. Little is known about its molecular pathogenesis. For this study, a retrospective cohort of n = 66 tumor samples with the institutional diagnosis of ONB was analyzed by immunohistochemistry, genome-wide DNA methylation profiling, copy number analysis, and in a subset, next-generation panel sequencing of 560 tumor-associated genes. DNA methylation profiles were compared to those of relevant differential diagnoses of ONB. Unsupervised hierarchical clustering analysis of DNA methylation data revealed four subgroups among institutionally diagnosed ONB. The largest group (n = 42, 64%, Core ONB) presented with classical ONB histology and no overlap with other classes upon methylation profiling-based t-distributed stochastic neighbor embedding (t-SNE) analysis. A second DNA methylation group (n = 7, 11%) with CpG island methylator phenotype (CIMP) consisted of cases with strong expression of cytokeratin, no or scarce chromogranin A expression and IDH2 hotspot mutation in all cases. T-SNE analysis clustered these cases together with sinonasal carcinoma with IDH2 mutation. Four cases (6%) formed a small group characterized by an overall high level of DNA methylation, but without CIMP. The fourth group consisted of 13 cases that had heterogeneous DNA methylation profiles and strong cytokeratin expression in most cases. In t-SNE analysis, these cases mostly grouped among sinonasal adenocarcinoma, squamous cell carcinoma, and undifferentiated carcinoma. Copy number analysis indicated highly recurrent chromosomal changes among Core ONB with a high frequency of combined loss of chromosome 1-4, 8-10, and 12. NGS sequencing did not reveal highly recurrent mutations in ONB, with the only recurrently mutated genes being TP53 and DNMT3A. In conclusion, we demonstrate that institutionally diagnosed ONB are a heterogeneous group of tumors. Expression of cytokeratin, chromogranin A, the mutational status of IDH2 as well as DNA methylation patterns may greatly aid in the precise classification of ONB.

Effects of APOE-ε4 allele load on brain morphology in a cohort of middle-aged healthy individuals with enriched genetic risk for Alzheimer's disease.

INTRODUCTION: Apolipoprotein E (APOE)-ε4 is the major genetic risk factor for Alzheimer's disease. However, the dose-dependent impact of this allele on brain morphology of healthy individuals remains unclear. METHODS: We analyzed gray matter volumes (GMvs) in a sample of 533 healthy middle-aged individuals with a substantial representation of ε4-carriers (207 heterozygotes and 65 homozygotes). RESULTS: We found APOE-ε4 additive GMv reductions in the right hippocampus, caudate, precentral gyrus, and cerebellar crus. In these regions, the APOE genotype interacted with age, with homozygotes displaying lower GMv after the fifth decade of life. APOE-ε4 was also associated to greater GMv in the right thalamus, left occipital gyrus, and right frontal cortex. DISCUSSION: Our data indicate that APOE-ε4 exerts additive effects on GMv in regions relevant for Alzheimer's disease pathophysiology already in healthy individuals. These findings elucidate the mechanisms underlying the increased Alzheimer's disease risk in ε4-carriers, suggesting a dose-dependent disease vulnerability on the brain structure level.

Epigenetic Homogeneity Within Colorectal Tumors Predicts Shorter Relapse-Free and Overall Survival Times for Patients With Locoregional Cancer.

BACKGROUND & AIMS: There are few validated biomarkers that can be used to predict outcomes for patients with colorectal cancer. Part of the challenge is the genetic and molecular heterogeneity of colorectal tumors not only among patients, but also within tumors. We have explored intratumor heterogeneity at the epigenetic level, due to its dynamic nature. We analyzed DNA methylation profiles of the digestive tract surface and the central bulk and invasive front regions of colorectal tumors. METHODS: We determined the DNA methylation profiles of >450,000 CpG sites in 3 macrodissected regions of 79 colorectal tumors and 23 associated liver metastases, obtained from 2 hospitals in Spain. We also analyzed samples for KRAS and BRAF mutations, 499,170 single nucleotide polymorphisms, and performed immunohistochemical analyses. RESULTS: We observed differences in DNA methylation among the 3 tumor sections; regions of tumor-host interface differed the most from the other tumor sections. Interestingly, tumor samples collected from areas closer to the gastrointestinal transit most frequently shared methylation events with metastases. When we calculated individual coefficients to quantify heterogeneity, we found that epigenetic homogeneity was significantly associated with short time of relapse-free survival (log-rank P = .037) and short time of overall survival (log-rank P = .026) in patients with locoregional colorectal cancer. CONCLUSIONS: In an analysis of 79 colorectal tumors, we found significant heterogeneity in patterns of DNA methylation within each tumor; the level of heterogeneity correlates with times of relapse-free and overall survival.

Whole genome grey and white matter DNA methylation profiles in dorsolateral prefrontal cortex.

The brain's neocortex is anatomically organized into grey and white matter, which are mainly composed by neuronal and glial cells, respectively. The neocortex can be further divided in different Brodmann areas according to their cytoarchitectural organization, which are associated with distinct cortical functions. There is increasing evidence that brain development and function are governed by epigenetic processes, yet their contribution to the functional organization of the neocortex remains incompletely understood. Herein, we determined the DNA methylation patterns of grey and white matter of dorsolateral prefrontal cortex (Brodmann area 9), an important region for higher cognitive skills that is particularly affected in various neurological diseases. For avoiding interindividual differences, we analyzed white and grey matter from the same donor using whole genome bisulfite sequencing, and for validating their biological significance, we used Infinium HumanMethylation450 BeadChip and pyrosequencing in ten and twenty independent samples, respectively. The combination of these analysis indicated robust grey-white matter differences in DNA methylation. What is more, cell type-specific markers were enriched among the most differentially methylated genes. Interestingly, we also found an outstanding number of grey-white matter differentially methylated genes that have previously been associated with Alzheimer's, Parkinson's, and Huntington's disease, as well as Multiple and Amyotrophic lateral sclerosis. The data presented here thus constitute an important resource for future studies not only to gain insight into brain regional as well as grey and white matter differences, but also to unmask epigenetic alterations that might underlie neurological and neurodegenerative diseases.

Technologies for Deciphering Epigenomic DNA Patterns.

DNA methylation, consisting on the covalent addition of a methyl group in cytosines, plays a vital role for the development and correct functioning of cells. It constitutes a mechanism by which cell genome is regulated, allowing from a common genome of an individual to obtain all the different cell types that constitute the individual. Nowadays, we understand how the epigenetic machinery works; however, this critical mechanism might promote the appearance of certain diseases if dysregulated, thus the importance of studying the epigenetic patterns on both normal and disease tissues. During the last decades, huge advances on techniques to measure the level of DNA methylation have occurred; we have passed from measuring it with more rudimentary and expensive techniques to nowadays the ability to measure DNA methylation at a single-base resolution in an affordable manner. In this chapter we will cover all the main technologies available, with a special emphasis on the microarray technology, as it supposes a perfect choice taking into account its price as well as the amount of cytosines interrogated, the compatibility with formalin-fixed paraffin-embedded samples, and its standardized procedure.

Epigenetic profiling to classify cancer of unknown primary: a multicentre, retrospective analysis.

BACKGROUND: Cancer of unknown primary ranks in the top ten cancer presentations and has an extremely poor prognosis. Identification of the primary tumour and development of a tailored site-specific therapy could improve the survival of these patients. We examined the feasability of using DNA methylation profiles to determine the occult original cancer in cases of cancer of unknown primary. METHODS: We established a classifier of cancer type based on the microarray DNA methylation signatures (EPICUP) in a training set of 2790 tumour samples of known origin representing 38 tumour types and including 85 metastases. To validate the classifier, we used an independent set of 7691 known tumour samples from the same tumour types that included 534 metastases. We applied the developed diagnostic test to predict the tumour type of 216 well-characterised cases of cancer of unknown primary. We validated the accuracy of the predictions from the EPICUP assay using autopsy examination, follow-up for subsequent clinical detection of the primary sites months after the initial presentation, light microscopy, and comprehensive immunohistochemistry profiling. FINDINGS: The tumour type classifier based on the DNA methylation profiles showed a 99·6% specificity (95% CI 99·5-99·7), 97·7% sensitivity (96·1-99·2), 88·6% positive predictive value (85·8-91·3), and 99·9% negative predictive value (99·9-100·0) in the validation set of 7691 tumours. DNA methylation profiling predicted a primary cancer of origin in 188 (87%) of 216 patients with cancer with unknown primary. Patients with EPICUP diagnoses who received a tumour type-specific therapy showed improved overall survival compared with that in patients who received empiric therapy (hazard ratio [HR] 3·24, p=0·0051 [95% CI 1·42-7·38]; log-rank p=0·0029). INTERPRETATION: We show that the development of a DNA methylation based assay can significantly improve diagnoses of cancer of unknown primary and guide more precise therapies associated with better outcomes. Epigenetic profiling could be a useful approach to unmask the original primary tumour site of cancer of unknown primary cases and a step towards the improvement of the clinical management of these patients. FUNDING: European Research Council (ERC), Cellex Foundation, the Institute of Health Carlos III (ISCIII), Cancer Australia, Victorian Cancer Agency, Samuel Waxman Cancer Research Foundation, the Health and Science Departments of the Generalitat de Catalunya, and Ferrer.

DNA methylation contributes to natural human variation.

DNA methylation patterns are important for establishing cell, tissue, and organism phenotypes, but little is known about their contribution to natural human variation. To determine their contribution to variability, we have generated genome-scale DNA methylation profiles of three human populations (Caucasian-American, African-American, and Han Chinese-American) and examined the differentially methylated CpG sites. The distinctly methylated genes identified suggest an influence of DNA methylation on phenotype differences, such as susceptibility to certain diseases and pathogens, and response to drugs and environmental agents. DNA methylation differences can be partially traced back to genetic variation, suggesting that differentially methylated CpG sites serve as evolutionarily established mediators between the genetic code and phenotypic variability. Notably, one-third of the DNA methylation differences were not associated with any genetic variation, suggesting that variation in population-specific sites takes place at the genetic and epigenetic levels, highlighting the contribution of epigenetic modification to natural human variation.

DNA methylation dynamics in human carotid plaques after cerebrovascular events.

OBJECTIVE: To understand whether cerebrovascular events, a major complication of atherosclerosis, are associated with any specific DNA methylation changes in the carotid plaque. APPROACH AND RESULTS: We profiled the DNA methylomes of human symptomatic carotid plaques obtained from patients who had cerebrovascular events (n=19) and asymptomatic counterparts (n=19) with a high-density microarray (≈485 000 CpG sites, Illumina), and crossed DNA methylation data with RNAseq-based expression data from an independent symptomatic carotid plaque set (n=8). Few (30) CpGs showed a significant (P<0.05; absolute Delta-Beta, >0.20) differential methylation between the 2 groups. Within symptomatic carotid plaques, DNA methylation correlated significantly with postcerebrovascular event time (range, 3-45 days; r-value range, -0.926 to 0.857; P<0.05) for ≈45 000 CpGs, the vast majority of which became hypomethylated with increasing postcerebrovascular event time. Hypomethylation was not due to erasure of the gene-body and CG-poor region hypermethylation that accompany the progression of stable lesions, but rather targeted promoters and CpG islands. Noticeably, promoter hypomethylation and increased expression of genes involved in the inhibition of the inflammatory response, defense against oxidative stress, and active DNA demethylation were observed with increasing postcerebrovascular event time. Concomitantly, histological changes consistent with phagocyte-driven plaque healing were observed. CONCLUSIONS: Weak changes in the DNA methylome distinguish symptomatic from asymptomatic plaques, but a widespread demethylation resulting in permissive transcriptional marks at atheroprotective gene promoters is established in plaques after a cerebrovascular event, thus mirroring previous observations that ruptured plaques tend to revert to a stable structure. The identified loci are candidate targets to accelerate the pace of carotid plaque stabilization.