Characteristic DNA Methylation Research Articles

The analysis of the genome-wide dna methylation profile of human articular chondrocytes reveals different forms of OA

Purpose: To identify and analyze the genome-wide DNA methylation profiles of human articular chondrocytes from a population-based case-control study of OA. Methods: DNA methylation profiling was performed using the Infinium HumanMethylation27 beadchip (Illumina Inc.), which allows interrogation of 27,578 highly informative CpG loci. Previously, cartilage isolated DNA from 23 OA patients and 19 healthy controls was bisulfite-modified, using the EZ DNA methylation kit (Zymo Research) and hybridized according to the manufacturer's instructions. DNA methylation β-values were normalized using GenomeStudio v3.0 (Illumina Inc.). Appropriate bioinformatics analyses were carried out using both R bioconductor software packages and Babelomics suite v 4.2 (babelomics.bioinfo.cipf.es). Results: A first approach based on an unsupervised clustering method for the most variable CpG loci (n=508) showed three distinct groups of samples, called cluster 1 (5 OA patiens), cluster 2 (6 OA patients) and cluster 3 (12 OA patients and 17 healthy controls). Specifically, cluster 2 formed a particularly tight cluster with a characteristic DNA methylation profile (Figure 1). The analyses of the biological relevance of the differentially methylated genes in cluster 2 compared with non-cluster 2 by means of a gene set enrichment approach, showed that the biological processes significantly altered were those related to the superoxide metabolic process, morphogenesis/angiogenesis and regulation of cell proliferation, all of them hypermethylated in cluster 2; on the contrary, those mechanisms related to both IL-8 biosynthetic process and apoptosis appeared significantly hypomethylated in cluster 2. Conclusions: The genome-wide methylation analysis shows a clearly distinct epigenetic profile for OA. The DNA methylation profile could be one of the reasons of the existence of different forms of OA and could also be related to both the prevalence and progression of this disease.

Abstract 4724: Identification of a CpG-island methylator phenotype (CIMP) in a subgroup of pilocytic astrocytoma with favorable prognosis

Abstract Pilocytic astrocytomas (PA) are classified as WHO grade I tumors, and comprise the most frequent brain tumor in childhood. The most common tumor location is the cerebellum, and these tumors can typically be completely resected. However, for tumors in other locations such as the diencephalon, brain stem, or optic tract, complete tumor resection is often challenging. Recently, we were able to identify BRAF as a centrally important oncogene in these tumors, showing duplication or activating mutation in >70% of primary PAs, with KIAA1549:BRAF fusion genes being the most frequent genetic mechanism of BRAF activation. Approximately 90% of tumors show some form of MAPK activation as assessed by detection of ERK phosphorylation. Thus, alternative mechanisms of MAPK activation are yet to be discovered in 10-20% of cases. Here we describe an integrative approach combining genome-wide DNA methylation and mRNA expression analyses. In total, we studied 80 pilocytic astrocytomas, with a tissue microarray containing cores from 70 of these samples available for immunohistochemical validation. To identify novel subgroups and critical genes in PA pathogenesis, we performed methylation profiling and compared the resulting subgroups with transcriptome-based subgroups of overlapping tumors. Both experiments were performed on Illumina platforms, HumanWG-6 (expression profiling) and HumanMethylation27 BeadChip. Two CpG sites were analyzed for each of ∼14,000 promoters per sample. The 1000 most variant probes were selected for hierarchical consensus clustering for each platform. Identified molecular subgroups were investigated using Significance Analysis of Microarrays (SAM). Furthermore, to identify genes whose expression was potentially regulated by aberrant methylation, we performed an anti-correlation approach. By methylation profiling we could identify three subgroups in the 80 PA tumors that showed characteristic DNA methylation patterns. One subgroup that could also be derived by transcriptome analysis showed evidence of a CpG island methylator phenotype (PA-CIMP), since it displayed markedly elevated methylation of multiple genes compared with the other two groups. PA-CIMP tumors showed a lower frequency of MAPK alterations, including BRAF fusions or activating mutations, compared with the other subgroups. Patients with PA-CIMP tumors also showed an excellent prognosis, as none of these patients relapsed, despite 50% of the cases being non-cerebellar PAs. Thus, our PA-CIMP signature is potentially useful to identify tumors with a good prognosis, independent of location. This hypothesis is currently being validated in an independent series of 50 PAs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4724. doi:10.1158/1538-7445.AM2011-4724

066 IDENTIFICATION AND CHARACTERISATION OF MICRORNAS INVOLVED IN CHONDROCYTE DIFFERENTIATION AND OSTEOARTHRITIS

066 IDENTIFICATION AND CHARACTERISATION OF MICRORNAS INVOLVED IN CHONDROCYTE DIFFERENTIATION AND OSTEOARTHRITIS

The Epigenetic Landscape of Latent Kaposi Sarcoma-Associated Herpesvirus Genomes

Herpesvirus latency is generally thought to be governed by epigenetic modifications, but the dynamics of viral chromatin at early timepoints of latent infection are poorly understood. Here, we report a comprehensive spatial and temporal analysis of DNA methylation and histone modifications during latent infection with Kaposi Sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi Sarcoma and primary effusion lymphoma (PEL). By use of high resolution tiling microarrays in conjunction with immunoprecipitation of methylated DNA (MeDIP) or modified histones (chromatin IP, ChIP), our study revealed highly distinct landscapes of epigenetic modifications associated with latent KSHV infection in several tumor-derived cell lines as well as de novo infected endothelial cells. We find that KSHV genomes are subject to profound methylation at CpG dinucleotides, leading to the establishment of characteristic global DNA methylation patterns. However, such patterns evolve slowly and thus are unlikely to control early latency. In contrast, we observed that latency-specific histone modification patterns were rapidly established upon a de novo infection. Our analysis furthermore demonstrates that such patterns are not characterized by the absence of activating histone modifications, as H3K9/K14-ac and H3K4-me3 marks were prominently detected at several loci, including the promoter of the lytic cycle transactivator Rta. While these regions were furthermore largely devoid of the constitutive heterochromatin marker H3K9-me3, we observed rapid and widespread deposition of H3K27-me3 across latent KSHV genomes, a bivalent modification which is able to repress transcription in spite of the simultaneous presence of activating marks. Our findings suggest that the modification patterns identified here induce a poised state of repression during viral latency, which can be rapidly reversed once the lytic cycle is induced.

Molecular subtypes of breast cancer are associated with characteristic DNA methylation patterns.

IntroductionFive different molecular subtypes of breast cancer have been identified through gene expression profiling. Each subtype has a characteristic expression pattern suggested to partly depend on cellular origin. We aimed to investigate whether the molecular subtypes also display distinct methylation profiles.MethodsWe analysed methylation status of 807 cancer-related genes in 189 fresh frozen primary breast tumours and four normal breast tissue samples using an array-based methylation assay.ResultsUnsupervised analysis revealed three groups of breast cancer with characteristic methylation patterns. The three groups were associated with the luminal A, luminal B and basal-like molecular subtypes of breast cancer, respectively, whereas cancers of the HER2-enriched and normal-like subtypes were distributed among the three groups. The methylation frequencies were significantly different between subtypes, with luminal B and basal-like tumours being most and least frequently methylated, respectively. Moreover, targets of the polycomb repressor complex in breast cancer and embryonic stem cells were more methylated in luminal B tumours than in other tumours. BRCA2-mutated tumours had a particularly high degree of methylation. Finally, by utilizing gene expression data, we observed that a large fraction of genes reported as having subtype-specific expression patterns might be regulated through methylation.ConclusionsWe have found that breast cancers of the basal-like, luminal A and luminal B molecular subtypes harbour specific methylation profiles. Our results suggest that methylation may play an important role in the development of breast cancers.

Identification of a CpG Island Methylator Phenotype that Defines a Distinct Subgroup of Glioma

We have profiled promoter DNA methylation alterations in 272 glioblastoma tumors in the context of The Cancer Genome Atlas (TCGA). We found that a distinct subset of samples displays concerted hypermethylation at a large number of loci, indicating the existence of a glioma-CpG island methylator phenotype (G-CIMP). We validated G-CIMP in a set of non-TCGA glioblastomas and low-grade gliomas. G-CIMP tumors belong to the proneural subgroup, are more prevalent among lower-grade gliomas, display distinct copy-number alterations, and are tightly associated with IDH1 somatic mutations. Patients with G-CIMP tumors are younger at the time of diagnosis and experience significantly improved outcome. These findings identify G-CIMP as a distinct subset of human gliomas on molecular and clinical grounds.

Hypomethylation of Repeated Dna Sequences in Cancer

An important feature of cancer development and progression is the change in DNA methylation patterns, characterized by the hypermethylation of specific genes concurrently with an overall decrease in the level of 5-methylcytosine. Hypomethylation of the genome can affect both single-copy genes, repeat DNA sequences and transposable elements, and is highly variable among and within cancer types. Here, we review our current understanding of genome hypomethylation in cancer, with a particular focus on hypomethylation of the different classes and families of repeat sequences. The emerging data provide insights into the importance of methylation of different repeat families in the maintenance of chromosome structural integrity and the fidelity of normal transcriptional regulation. We also consider the events underlying cancer-associated hypomethylation and the potential for the clinical use of characteristic DNA methylation changes in diagnosis, prognosis or classification of tumors.

Epigenetic Profiles Distinguish Malignant Pleural Mesothelioma from Lung Adenocarcinoma

Malignant pleural mesothelioma (MPM) is a fatal thoracic malignancy, the epigenetics of which are poorly defined. We performed high-throughput methylation analysis covering 6,157 CpG islands in 20 MPMs and 20 lung adenocarcinomas. Newly identified genes were further analyzed in 50 MPMs and 56 adenocarcinomas via quantitative methylation-specific PCR. Targets of histone H3 lysine 27 trimethylation (H3K27me3) and genetic alterations were also assessed in MPM cells by chromatin immunoprecipitation arrays and comparative genomic hybridization arrays. An average of 387 genes (6.3%) and 544 genes (8.8%) were hypermethylated in MPM and adenocarcinoma, respectively. Hierarchical cluster analysis showed that the two malignancies have characteristic DNA methylation patterns, likely a result of different pathologic processes. In MPM, a separate subset of genes was silenced by H3K27me3 and could be reactivated by treatment with a histone deacetylase inhibitor alone. Integrated analysis of these epigenetic and genetic alterations revealed that only 11% of heterozygously deleted genes were affected by DNA methylation and/or H3K27me3 in MPMs. Among the DNA hypermethylated genes, three (TMEM30B, KAZALD1, and MAPK13) were specifically methylated only in MPM and could serve as potential diagnostic markers. Interestingly, a subset of MPM cases (4 cases, 20%) had very low levels of DNA methylation and substantially longer survival, suggesting that the epigenetic alterations are one mechanism affecting progression of this disease. Our findings show a characteristic epigenetic profile of MPM and uncover multiple distinct epigenetic abnormalities that lead to the silencing of tumor suppressor genes in MPM and could serve as diagnostic or prognostic targets.