[Show abstract][Hide abstract] ABSTRACT: Neuroblastoma (NB) is one of the most frequently occurring extracranial solid tumors of childhood (Maris et al., 2007 ; Brodeur, 2003 ). Probability of cure varies according to patient's age, extent of disease and tumor biology (Maris et al., 2007 ; Brodeur, 2003 ; Cohn et al., 2009 ). However, the etiology of this developmental tumor is unknown. Recent evidence has shown that pediatric solid tumors, including NB, harbor a paucity of recurrent genetic mutations, with a significant proportion of recurrent events converging on epigenetic mechanisms (Cheung et al., 2012 ; Molenaar et al., 2012 ; Pugh et al., 2013 ; Sausen et al., 2013 .We have analyzed the DNA methylome of neuroblastoma using high-density microarrays (Infinium Human Methylation 450k BeadChip) to define the epigenetic landscape of this pediatric tumor and its potential clinicopathological impact. Here, we provide the detail of methods and quality control parameters of the microarray data used for the study. Methylation data has been deposited at NCBI Gene Expression Omnibus data repository, accession number GSE54719; superseries record GSE54721.
Genomics Data 09/2015; 5:360-363. DOI:10.1016/j.gdata.2015.07.016
[Show abstract][Hide abstract] ABSTRACT: Ageing constitutes a critical impediment to somatic cell reprogramming. We have explored the regulatory mechanisms that constitute age-associated barriers, through derivation of induced pluripotent stem cells (iPSCs) from individuals with premature or physiological ageing. We demonstrate that NF-κB activation blocks the generation of iPSCs in ageing. We also show that NF-κB repression occurs during cell reprogramming towards a pluripotent state. Conversely, ageing-associated NF-κB hyperactivation impairs the generation of iPSCs by eliciting the reprogramming repressor DOT1L, which reinforces senescence signals and downregulates pluripotency genes. Genetic and pharmacological NF-κB inhibitory strategies significantly increase the reprogramming efficiency of fibroblasts from Néstor-Guillermo progeria syndrome and Hutchinson-Gilford progeria syndrome patients, as well as from normal aged donors. Finally, we demonstrate that DOT1L inhibition in vivo extends lifespan and ameliorates the accelerated ageing phenotype of progeroid mice, supporting the interest of studying age-associated molecular impairments to identify targets of rejuvenation strategies.
[Show abstract][Hide abstract] ABSTRACT: Chronic lymphocytic leukaemia (CLL) is a frequent disease in which the genetic alterations determining the clinicobiological behaviour are not fully understood. Here we describe a comprehensive evaluation of the genomic landscape of 452 CLL cases and 54 patients with monoclonal B-lymphocytosis, a precursor disorder. We extend the number of CLL driver alterations, including changes in ZNF292, ZMYM3, ARID1A and PTPN11. We also identify novel recurrent mutations in non-coding regions, including the 3' region of NOTCH1, which cause aberrant splicing events, increase NOTCH1 activity and result in a more aggressive disease. In addition, mutations in an enhancer located on chromosome 9p13 result in reduced expression of the B-cell-specific transcription factor PAX5. The accumulative number of driver alterations (0 to ≥4) discriminated between patients with differences in clinical behaviour. This study provides an integrated portrait of the CLL genomic landscape, identifies new recurrent driver mutations of the disease, and suggests clinical interventions that may improve the management of this neoplasia.
[Show abstract][Hide abstract] ABSTRACT: To define the DNA methylation landscape of neuroblastoma and its clinicopathological impact.
Microarray DNA methylation data were analyzed and associated with functional/regulatory genome annotation data, transcriptional profiles and clinicobiological parameters.
DNA methylation changes in neuroblastoma affect not only promoters but also intragenic and intergenic regions at cytosine-phosphate-guanine (CpG) and non-CpG sites, and target functional chromatin domains of development and cancer-related genes such as CCND1. Tumors with diverse clinical risk showed differences affecting CpG and, remarkably, non-CpG sites. Non-CpG methylation observed essentially in clinically favorable cases was associated with the differentiation status of neuroblastoma and expression of key genes such as ALK.
This epigenetic fingerprint of neuroblastoma provides new insights into the pathogenesis and clinical behavior of this pediatric tumor.
[Show abstract][Hide abstract] ABSTRACT: We analyzed the DNA methylome of ten subpopulations spanning the entire B cell differentiation program by whole-genome bisulfite sequencing and high-density microarrays. We observed that non-CpG methylation disappeared upon B cell commitment, whereas CpG methylation changed extensively during B cell maturation, showing an accumulative pattern and affecting around 30% of all measured CpG sites. Early differentiation stages mainly displayed enhancer demethylation, which was associated with upregulation of key B cell transcription factors and affected multiple genes involved in B cell biology. Late differentiation stages, in contrast, showed extensive demethylation of heterochromatin and methylation gain at Polycomb-repressed areas, and genes with apparent functional impact in B cells were not affected. This signature, which has previously been linked to aging and cancer, was particularly widespread in mature cells with an extended lifespan. Comparing B cell neoplasms with their normal counterparts, we determined that they frequently acquire methylation changes in regions already undergoing dynamic methylation during normal B cell differentiation.
[Show abstract][Hide abstract] ABSTRACT: While analyzing the DNA methylome of multiple myeloma (MM), a plasma cell neoplasm, by whole-genome bisulfite sequencing and high-density arrays, we observed a highly heterogeneous pattern globally characterized by regional DNA hypermethylation embedded in extensive hypomethylation. In contrast to the widely reported DNA hypermethylation of promoter-associated CpG islands (CGIs) in cancer, hypermethylated sites in MM, as opposed to normal plasma cells, were located outside CpG islands and were unexpectedly associated with intronic enhancer regions defined in normal B cells and plasma cells. Both RNA-seq and in vitro reporter assays indicated that enhancer hypermethylation is globally associated with downregulation of its host genes. ChIP-seq and DNase-seq further revealed that DNA hypermethylation in these regions is related to enhancer decommissioning. Hypermethylated enhancer regions overlapped with binding sites of B cell-specific transcription factors (TFs) and the degree of enhancer methylation inversely correlated with expression levels of these TFs in MM. Furthermore, hypermethylated regions in MM were methylated in stem cells and gradually became demethylated during normal B-cell differentiation, suggesting that MM cells either reacquire epigenetic features of undifferentiated cells or maintain an epigenetic signature of a putative myeloma stem cell progenitor. Overall, we have identified DNA hypermethylation of developmentally-regulated enhancers as a new type of epigenetic modification associated with the pathogenesis of MM.
Published by Cold Spring Harbor Laboratory Press.
Genome Research 02/2015; 25(4). DOI:10.1101/gr.180240.114 · 14.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Prospective identification of patients with chronic lymphocytic leukemia (CLL) destined to progress would greatly facilitate their clinical management. Recently, whole-genome DNA methylation analyses identified three clinico-biological CLL subgroups with an epigenetic signature related to different normal B-cell counterparts. Here, we developed a clinically-applicable method to identify these subgroups and to study their clinical relevance. Using a support vector machine approach, we built a prediction model using five epigenetic biomarkers that was able to accurately classify CLL patients into the three subgroups, namely naive B cell-like, intermediate and memory B cell-like CLL. DNA methylation was quantified by highly reproducible bisulfite pyrosequencing (BPS) assays in two independent CLL series. In the initial series (n=211), the three subgroups showed differential levels of IGHV mutation (P<0.001) and VH usage (P<0.03), as well as different clinical features and outcome in terms of time-to-first-treatment (TTT) and overall survival (P<0.001). A multivariate Cox model showed that epigenetic classification was the strongest predictor of TTT (P<0.001) along with Binet stage (P<0.001). These findings were corroborated in a validation series (n=97). In this study, we developed a simple and robust method using epigenetic biomarkers to categorize CLLs into three subgroups with different clinico-biological features and outcome.Leukemia accepted article preview online, 25 August 2014 doi:10.1038/leu.2014.252.
[Show abstract][Hide abstract] ABSTRACT: In a subset of imprinting disorders caused by epimutations, multiple imprinted loci are affected. Familial occurrence of multilocus imprinting disorders is rare.
We have investigated the clinical and molecular features of a familial DNA-methylation disorder.
Tissues of affected individuals and blood samples of family members were investigated by conventional and molecular karyotyping. Sanger sequencing and RT-PCR of imprinting-associated genes (NLRP2, NLRP7, ZFP57, KHDC3L, DNMT1o), exome sequencing and locus-specific, array-based and genome-wide technologies to determine DNA-methylation were performed.
In three offspring of a healthy couple, we observed prenatal onset of severe growth retardation and dysmorphism associated with altered DNA-methylation at paternally and maternally imprinted loci. Array-based analyses in various tissues of the offspring identified the DNA-methylation of 2.1% of the genes in the genome to be recurrently altered. Despite significant enrichment of imprinted genes (OR 9.49), altered DNA-methylation predominately (90.2%) affected genes not known to be imprinted. Sequencing of genes known to cause comparable conditions and exome sequencing in affected individuals and their ancestors did not unambiguously point to a causative gene.
The family presented herein suggests the existence of a familial disorder of DNA-methylation affecting imprinted but also not imprinted gene loci potentially caused by a maternal effect mutation in a hitherto not identified gene.
Journal of Medical Genetics 04/2014; 51(6). DOI:10.1136/jmedgenet-2013-102149 · 6.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The precise regulation of S-phase-specific genes is critical for cell proliferation. How the repressive chromatin configuration mediated by the retinoblastoma protein and repressor E2F factors changes at the G1/S transition to allow transcription activation is unclear. Here we show ChIP-on-chip studies that reveal that the chromatin remodeller CHD8 binds ∼2000 transcriptionally active promoters. The spectrum of CHD8 target genes was enriched in E2F-dependent genes. We found that CHD8 binds E2F-dependent promoters at the G1/S transition but not in quiescent cells. Consistently, CHD8 was required for G1/S-specific expression of these genes and for cell cycle re-entry on serum stimulation of quiescent cells. We also show that CHD8 interacts with E2F1 and, importantly, loading of E2F1 and E2F3, but not E2F4, onto S-specific promoters, requires CHD8. However, CHD8 recruiting is independent of these factors. Recruiting of MLL histone methyltransferase complexes to S-specific promoters was also severely impaired in the absence of CHD8. Furthermore, depletion of CHD8 abolished E2F1 overexpression-dependent S-phase stimulation of serum-starved cells, highlighting the essential role of CHD8 in E2F-dependent transcription activation.
Nucleic Acids Research 11/2013; 42(4). DOI:10.1093/nar/gkt1161 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic lymphocytic leukemia (CLL) has heterogeneous clinical and biological behavior. Whole-genome and -exome sequencing has contributed to the characterization of the mutational spectrum of the disease, but the underlying transcriptional profile is still poorly understood. We have performed deep RNA sequencing in different subpopulations of normal B-lymphocytes and CLL cells from a cohort of 98 patients, and characterized the CLL transcriptional landscape with unprecedented resolution. We detected thousands of transcriptional elements differentially expressed between the CLL and normal B cells, including protein coding genes, non-coding RNAs and pseudogenes. Transposable elements are globally de-repressed in CLL cells. In addition, two thousand genes - most of which are not differentially expressed - exhibit CLL-specific splicing patterns. Genes involved in metabolic pathways showed higher expression in CLL, while genes related to spliceosome, proteasome and ribosome were among the most downregulated in CLL. Clustering of the CLL samples according to RNA-seq derived gene expression levels unveiled two robust molecular subgroups, C1 and C2. C1/C2 subgroups and the mutational status of the immunoglobulin heavy variable (IGHV) region were the only independent variables in predicting time to treatment in a multivariate analysis with main clinico-biological features. This subdivision was validated in an independent cohort of patients monitored through DNA microarrays. Further analysis shows that B-cell receptor (BCR) activation in the microenvironment of the lymph node may be at the origin of the C1/C2 differences.
Genome Research 11/2013; 24(2). DOI:10.1101/gr.152132.112 · 14.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mantle cell lymphoma (MCL) is an aggressive tumor, but a subset of patients may follow an indolent clinical course. To understand the mechanisms underlying this biological heterogeneity, we performed whole-genome and/or whole-exome sequencing on 29 MCL cases and their respective matched normal DNA, as well as 6 MCL cell lines. Recurrently mutated genes were investigated by targeted sequencing in an independent cohort of 172 MCL patients. We identified 25 significantly mutated genes, including known drivers such as ataxia-telangectasia mutated (ATM), cyclin D1 (CCND1), and the tumor suppressor TP53; mutated genes encoding the anti-apoptotic protein BIRC3 and Toll-like receptor 2 (TLR2); and the chromatin modifiers WHSC1, MLL2, and MEF2B. We also found NOTCH2 mutations as an alternative phenomenon to NOTCH1 mutations in aggressive tumors with a dismal prognosis. Analysis of two simultaneous or subsequent MCL samples by whole-genome/whole-exome (n = 8) or targeted (n = 19) sequencing revealed subclonal heterogeneity at diagnosis in samples from different topographic sites and modulation of the initial mutational profile at the progression of the disease. Some mutations were predominantly clonal or subclonal, indicating an early or late event in tumor evolution, respectively. Our study identifies molecular mechanisms contributing to MCL pathogenesis and offers potential targets for therapeutic intervention.
Proceedings of the National Academy of Sciences 10/2013; 110(45). DOI:10.1073/pnas.1314608110 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The diagnosis of mantle cell lymphoma (MCL) can be difficult, especially when no t(11;14) translocation and cyclin D1 overexpression can be detected. In such cases, the transcription factor SOX11 represents an important diagnostic marker, as it is expressed in most MCLs and, in particular, in all cyclin D1-negative MCLs reported so far. A reliable anti-SOX11 antibody is therefore a very useful tool for routine diagnosis. Here, we characterize the new monoclonal anti-SOX11 antibodies, suitable for Western blot assay and immunohistochemistry (IHC) on formalin-fixed paraffin-embedded tissue; we tested them on a large series of primary lymphoid tumors and compared these results with those of other routinely used antibodies. Moreover, we show that IHC results depend on transcription levels of SOX11, which suggests that posttranscriptional and posttranslational modifications do not significantly affect cutoff levels for IHC detection of SOX11.
The American journal of surgical pathology 10/2013; 38(1). DOI:10.1097/PAS.0b013e3182a43996 · 5.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The central nervous system has a pattern of gene expression that is closely regulated with respect to functional and anatomical regions. DNA methylation is a major regulator of transcriptional activity, and aberrations in the distribution of this epigenetic mark may be involved in many neurological disorders, such as Alzheimer's disease. Herein, we have analysed 12 distinct mouse brain regions according to their CpG 5'-end gene methylation patterns and observed their unique epigenetic landscapes. The DNA methylomes obtained from the cerebral cortex were used to identify aberrant DNA methylation changes that occurred in two mouse models of Alzheimer's disease. We were able to translate these findings to patients with Alzheimer's disease, identifying DNA methylation-associated silencing of three targets genes: thromboxane A2 receptor (TBXA2R), sorbin and SH3 domain containing 3 (SORBS3) and spectrin beta 4 (SPTBN4). These hypermethylation targets indicate that the cyclic AMP response element-binding protein (CREB) activation pathway and the axon initial segment could contribute to the disease.
[Show abstract][Hide abstract] ABSTRACT: Ever since the discovery of DNA methylation at cytosine residues, the role of this so called fifth base has been extensively studied and debated. Until recently, the majority of DNA methylation studies focused on the analysis of CpG islands associated to promoter regions. However, with the upcoming possibilities to study DNA methylation in a genome-wide context, this epigenetic mark can now be studied in an unbiased manner. As a result, recent studies have shown that not only promoters but also intragenic and intergenic regions are widely modulated during physiological processes and disease. In particular, it is becoming increasingly clear that DNA methylation in the gene body is not just a passive witness of gene transcription but it seems to be actively involved in multiple gene regulation processes. In this review we discuss the potential role of intragenic DNA methylation in alternative promoter usage, regulation of short and long non-coding RNAs, alternative RNA processing, as well as enhancer activity. Furthermore, we summarize how the intragenic DNA methylome is modified both during normal cell differentiation and neoplastic transformation.
[Show abstract][Hide abstract] ABSTRACT: Most DNA methylation studies in Classic Philadelphia-negative myeloproliferative neoplasms have been performed on a gene-by-gene basis. Therefore, a more comprehensive methylation profiling is needed to study the implications of this epigenetic mark in myeloproliferative neoplasms. Here, we have analyzed 71 chronic (24 polycythaemia vera, 23 essential thrombocythaemia and 24 primary myelofibrosis) and 13 transformed myeloproliferative neoplasms using genome-wide DNA methylation arrays. The three types of chronic Philadelphia-negative myeloproliferative neoplasms showed a similar aberrant DNA methylation pattern when compared to control samples. Differentially methylated regions were enriched in a gene network centered on the NF-κB pathway, indicating that they may be involved in the pathogenesis of these diseases. In the case of transformed myeloproliferative neoplasms we detected an increased number of differentially methylated regions with respect to chronic myeloproliferative neoplasms. Interestingly, these genes were enriched in a list of differentially methylated regions in primary acute myeloid leukemia and in a gene network centered around the IFN pathway. Our results suggest that alterations in the DNA methylation landscape play an important role in the pathogenesis and leukaemic transformation of myeloproliferative neoplasms. The therapeutically modulation of epigenetically-deregulated pathways may allow us to design targeted therapies for these patients.
[Show abstract][Hide abstract] ABSTRACT: Purpose of review:
Next-generation sequencing of whole genomes, exomes and DNA methylomes in chronic lymphocytic leukemia (CLL) has provided the first comprehensive view of somatic mutations and methylation changes in this disease. This review summarizes the recent findings in this field and their impact on our current understanding of this neoplasm.
Genomic studies have revealed a remarkable molecular heterogeneity of the disease, with only few genes mutated in up to 10-15% of the patients and a relatively large number of genes recurrently mutated at low frequency. The mutated genes tend to cluster in different pathways that include NOTCH1 signaling, RNA splicing, processing and transport machinery, innate inflammatory response, and DNA damage and cell cycle control, among others. NOTCH1 and SF3B1 mutations are emerging as new drivers of aggressive forms of the disease. Genome-wide methylation studies have shown that CLL transformation is associated with a massive hypomethylation phenomenon frequently affecting the enhancer regions. This epigenetic reprogramming maintains an imprint of the putative cell of origin from naïve and memory B-cells.
Genomic and epigenomic studies of CLL are reshaping our understanding of the disease and provide new perspective for a more individualized diagnosis and new potential therapeutic targets.
Current opinion in hematology 05/2013; 20(4). DOI:10.1097/MOH.0b013e32836235dc · 3.97 Impact Factor