Pan-cancer multi-omics analysis and orthogonal experimental assessment of epigenetic driver genes.

The recent identification of recurrently mutated epigenetic regulator genes (ERGs) supports their critical role in tumorigenesis. We conducted a pan-cancer analysis integrating (epi)genome, transcriptome, and DNA methylome alterations in a curated list of 426 ERGs across 33 cancer types, comprising 10,845 tumor and 730 normal tissues. We found that, in addition to mutations, copy number alterations in ERGs were more frequent than previously anticipated and tightly linked to expression aberrations. Novel bioinformatics approaches, integrating the strengths of various driver prediction and multi-omics algorithms, and an orthogonal in vitro screen (CRISPR-Cas9) targeting all ERGs revealed genes with driver roles within and across malignancies and shared driver mechanisms operating across multiple cancer types and hallmarks. This is the largest and most comprehensive analysis thus far; it is also the first experimental effort to specifically identify ERG drivers (epidrivers) and characterize their deregulation and functional impact in oncogenic processes.

Epigenetic remodelling of enhancers in response to estrogen deprivation and re-stimulation.

Estrogen hormones are implicated in a majority of breast cancers and estrogen receptor alpha (ER), the main nuclear factor mediating estrogen signaling, orchestrates a complex molecular circuitry that is not yet fully elucidated. Here, we investigated genome-wide DNA methylation, histone acetylation and transcription after estradiol (E2) deprivation and re-stimulation to better characterize the ability of ER to coordinate gene regulation. We found that E2 deprivation mostly resulted in DNA hypermethylation and histone deacetylation in enhancers. Transcriptome analysis revealed that E2 deprivation leads to a global down-regulation in gene expression, and more specifically of TET2 demethylase that may be involved in the DNA hypermethylation following short-term E2 deprivation. Further enrichment analysis of transcription factor (TF) binding and motif occurrence highlights the importance of ER connection mainly with two partner TF families, AP-1 and FOX. These interactions take place in the proximity of E2 deprivation-mediated differentially methylated and histone acetylated enhancers. Finally, while most deprivation-dependent epigenetic changes were reversed following E2 re-stimulation, DNA hypermethylation and H3K27 deacetylation at certain enhancers were partially retained. Overall, these results show that inactivation of ER mediates rapid and mostly reversible epigenetic changes at enhancers, and bring new insight into early events, which may ultimately lead to endocrine resistance.

Lentinula edodes Cultured Extract and Rouxiella badensis subsp. acadiensis (Canan SV-53) Intake Alleviates Immune Deregulation and Inflammation by Modulating Signaling Pathways and Epigenetic Mechanisms.

Puberty is a critical developmental period of life characterized by marked physiological changes, including changes in the immune system and gut microbiota development. Exposure to inflammation induced by immune stressors during puberty has been found to stimulate central inflammation and lead to immune disturbance at distant sites from the gut; however, its enduring effects on gut immunity are not well explored. Therefore, in this study, we used a pubertal lipopolysaccharides (LPS)-induced inflammation mouse model to mimic pubertal exposure to inflammation and dysbiosis. We hypothesized that pubertal LPS-induced inflammation may cause long-term dysfunction in gut immunity by enduring dysregulation of inflammatory signaling and epigenetic changes, while prebiotic/probiotic intake may mitigate the gut immune system deregulation later in life. To this end, four-week-old female Balb/c mice were fed prebiotics/probiotics and exposed to LPS in the pubertal window. To better decipher the acute and enduring immunoprotective effects of biotic intake, we addressed the effect of treatment on interleukin (IL)-17 signaling related-cytokines and pathways. In addition, the effect of treatment on gut microbiota and epigenetic alterations, including changes in microRNA (miRNA) expression and DNA methylation, were studied. Our results revealed a significant dysregulation in selected cytokines, proteins, and miRNAs involved in key signaling pathways related to IL-17 production and function, including IL-17A and F, IL-6, IL-1ß, transforming growth factor-ß (TGF-ß), signal transducer and activator of transcription-3 (STAT3), p-STAT3, forkhead box O1 (FOXO1), and miR-145 in the small intestine of adult mice challenged with LPS during puberty. In contrast, dietary interventions mitigated the lasting adverse effects of LPS on gut immune function, partly through epigenetic mechanisms. A DNA methylation analysis demonstrated that enduring changes in gut immunity in adult mice might be linked to differentially methylated genes, including Lpb, Rorc, Runx1, Il17ra, Rac1, Ccl5, and Il10, involved in Th17 cell differentiation and IL-17 production and signaling. In addition, prebiotic administration prevented LPS-induced changes in the gut microbiota in pubertal mice. Together, these results indicate that following a healthy diet rich in prebiotics and probiotics is an optimal strategy for programming immune system function in the critical developmental windows of life and controlling inflammation later in life.

Experimental and pan-cancer genome analyses reveal widespread contribution of acrylamide exposure to carcinogenesis in humans.

Humans are frequently exposed to acrylamide, a probable human carcinogen found in commonplace sources such as most heated starchy foods or tobacco smoke. Prior evidence has shown that acrylamide causes cancer in rodents, yet epidemiological studies conducted to date are limited and, thus far, have yielded inconclusive data on association of human cancers with acrylamide exposure. In this study, we experimentally identify a novel and unique mutational signature imprinted by acrylamide through the effects of its reactive metabolite glycidamide. We next show that the glycidamide mutational signature is found in a full one-third of approximately 1600 tumor genomes corresponding to 19 human tumor types from 14 organs. The highest enrichment of the glycidamide signature was observed in the cancers of the lung (88% of the interrogated tumors), liver (73%), kidney (>70%), bile duct (57%), cervix (50%), and, to a lesser extent, additional cancer types. Overall, our study reveals an unexpectedly extensive contribution of acrylamide-associated mutagenesis to human cancers.

Human mitochondrial DNA is extensively methylated in a non-CpG context.

Mitochondrial dysfunction plays critical roles in cancer development and related therapeutic response; however, exact molecular mechanisms remain unclear. Recently, alongside the discovery of mitochondrial-specific DNA methyltransferases, global and site-specific methylation of the mitochondrial genome has been described. Investigation of any functional consequences however remains unclear and debated due to insufficient evidence of the quantitative degree and frequency of mitochondrial DNA (mtDNA) methylation. This study uses WGBS to provide the first quantitative report of mtDNA methylation at single base pair resolution. The data show that mitochondrial genomes are extensively methylated predominantly at non-CpG sites. Importantly, these methylation patterns display notable differences between normal and cancer cells. Furthermore, knockdown of DNA methyltransferase enzymes resulted in a marked global reduction of mtDNA methylation levels, indicating these enzymes may be associated with the establishment and/or maintenance of mtDNA methylation. DNMT3B knockdown cells displayed a comparatively pronounced global reduction in mtDNA methylation with concomitant increases in gene expression, suggesting a potential functional link between methylation and gene expression. Together these results demonstrate reproducible, non-random methylation patterns of mtDNA and challenge the notion that mtDNA is lowly methylated. This study discusses key differences in methodology that suggest future investigations must allow for techniques that assess both CpG and non-CpG methylation.

Genome-wide profiling of normal gastric mucosa identifies Helicobacter pylori- and cancer-associated DNA methylome changes.

The large geographic variations in the incidence of gastric cancer (GC) are likely due to differential environmental exposures, in particular to Helicobacter pylori (H. pylori) infection. We aimed to investigate the impact of H. pylori on the epigenome in normal gastric mucosa and methylation changes associated with cancer risk independent of H. pylori. A discovery set of normal gastric mucosa from GC cases (n = 42) and controls (n = 42), nested in a large case-control study and stratified by H. pylori status, were subjected to genome-wide methylation profiling. Single-nucleotide polymorphism arrays from peripheral blood leukocytes were used to conduct methylation quantitative trait loci (mQTL) analysis. A validation set of gastric mucosa samples (n = 180) was used in the replication phase. We found 1,924 differentially methylated positions (DMPs) and 438 differentially methylated regions (DMRs) associated with H. pylori infection, most of which were hypermethylated. Significant methylation alterations identified in the initial set were successfully replicated. Furthermore, the H. pylori-associated DMP/Rs showed marked stability ('epigenetic memory') after H. pylori clearance. Interestingly, we found 152 DMRs associated with cancer risk independent of the H. pylori status in normal gastric mucosa. The methylation score derived from three biomarkers was a strong predictor of GC. Finally, the mQTL analysis indicated that the H. pylori- and cancer-specific methylation signatures were minimally affected by genetic variation. The comprehensively characterized methylome changes associated with H. pylori infection and GC risk in our study might serve as potential biomarkers for early cancer progression in tumour-free gastric mucosa.

MutSpec: a Galaxy toolbox for streamlined analyses of somatic mutation spectra in human and mouse cancer genomes.

BACKGROUND: The nature of somatic mutations observed in human tumors at single gene or genome-wide levels can reveal information on past carcinogenic exposures and mutational processes contributing to tumor development. While large amounts of sequencing data are being generated, the associated analysis and interpretation of mutation patterns that may reveal clues about the natural history of cancer present complex and challenging tasks that require advanced bioinformatics skills. To make such analyses accessible to a wider community of researchers with no programming expertise, we have developed within the web-based user-friendly platform Galaxy a first-of-its-kind package called MutSpec. RESULTS: MutSpec includes a set of tools that perform variant annotation and use advanced statistics for the identification of mutation signatures present in cancer genomes and for comparing the obtained signatures with those published in the COSMIC database and other sources. MutSpec offers an accessible framework for building reproducible analysis pipelines, integrating existing methods and scripts developed in-house with publicly available R packages. MutSpec may be used to analyse data from whole-exome, whole-genome or targeted sequencing experiments performed on human or mouse genomes. Results are provided in various formats including rich graphical outputs. An example is presented to illustrate the package functionalities, the straightforward workflow analysis and the richness of the statistics and publication-grade graphics produced by the tool. CONCLUSIONS: MutSpec offers an easy-to-use graphical interface embedded in the popular Galaxy platform that can be used by researchers with limited programming or bioinformatics expertise to analyse mutation signatures present in cancer genomes. MutSpec can thus effectively assist in the discovery of complex mutational processes resulting from exogenous and endogenous carcinogenic insults.

Reference-free population genomics from next-generation transcriptome data and the vertebrate-invertebrate gap.

In animals, the population genomic literature is dominated by two taxa, namely mammals and drosophilids, in which fully sequenced, well-annotated genomes have been available for years. Data from other metazoan phyla are scarce, probably because the vast majority of living species still lack a closely related reference genome. Here we achieve de novo, reference-free population genomic analysis from wild samples in five non-model animal species, based on next-generation sequencing transcriptome data. We introduce a pipe-line for cDNA assembly, read mapping, SNP/genotype calling, and data cleaning, with specific focus on the issue of hidden paralogy detection. In two species for which a reference genome is available, similar results were obtained whether the reference was used or not, demonstrating the robustness of our de novo inferences. The population genomic profile of a hare, a turtle, an oyster, a tunicate, and a termite were found to be intermediate between those of human and Drosophila, indicating that the discordant genomic diversity patterns that have been reported between these two species do not reflect a generalized vertebrate versus invertebrate gap. The genomic average diversity was generally higher in invertebrates than in vertebrates (with the notable exception of termite), in agreement with the notion that population size tends to be larger in the former than in the latter. The non-synonymous to synonymous ratio, however, did not differ significantly between vertebrates and invertebrates, even though it was negatively correlated with genetic diversity within each of the two groups. This study opens promising perspective regarding genome-wide population analyses of non-model organisms and the influence of population size on non-synonymous versus synonymous diversity.

Bio++: efficient extensible libraries and tools for computational molecular evolution.

Efficient algorithms and programs for the analysis of the ever-growing amount of biological sequence data are strongly needed in the genomics era. The pace at which new data and methodologies are generated calls for the use of pre-existing, optimized-yet extensible-code, typically distributed as libraries or packages. This motivated the Bio++ project, aiming at developing a set of C++ libraries for sequence analysis, phylogenetics, population genetics, and molecular evolution. The main attractiveness of Bio++ is the extensibility and reusability of its components through its object-oriented design, without compromising the computer-efficiency of the underlying methods. We present here the second major release of the libraries, which provides an extended set of classes and methods. These extensions notably provide built-in access to sequence databases and new data structures for handling and manipulating sequences from the omics era, such as multiple genome alignments and sequencing reads libraries. More complex models of sequence evolution, such as mixture models and generic n-tuples alphabets, are also included.

Phylogenomic analyses support the position of turtles as the sister group of birds and crocodiles (Archosauria).

BACKGROUND: The morphological peculiarities of turtles have, for a long time, impeded their accurate placement in the phylogeny of amniotes. Molecular data used to address this major evolutionary question have so far been limited to a handful of markers and/or taxa. These studies have supported conflicting topologies, positioning turtles as either the sister group to all other reptiles, to lepidosaurs (tuatara, lizards and snakes), to archosaurs (birds and crocodiles), or to crocodilians. Genome-scale data have been shown to be useful in resolving other debated phylogenies, but no such adequate dataset is yet available for amniotes. RESULTS: In this study, we used next-generation sequencing to obtain seven new transcriptomes from the blood, liver, or jaws of four turtles, a caiman, a lizard, and a lungfish. We used a phylogenomic dataset based on 248 nuclear genes (187,026 nucleotide sites) for 16 vertebrate taxa to resolve the origins of turtles. Maximum likelihood and Bayesian concatenation analyses and species tree approaches performed under the most realistic models of the nucleotide and amino acid substitution processes unambiguously support turtles as a sister group to birds and crocodiles. The use of more simplistic models of nucleotide substitution for both concatenation and species tree reconstruction methods leads to the artefactual grouping of turtles and crocodiles, most likely because of substitution saturation at third codon positions. Relaxed molecular clock methods estimate the divergence between turtles and archosaurs around 255 million years ago. The most recent common ancestor of living turtles, corresponding to the split between Pleurodira and Cryptodira, is estimated to have occurred around 157 million years ago, in the Upper Jurassic period. This is a more recent estimate than previously reported, and questions the interpretation of controversial Lower Jurassic fossils as being part of the extant turtles radiation. CONCLUSIONS: These results provide a phylogenetic framework and timescale with which to interpret the evolution of the peculiar morphological, developmental, and molecular features of turtles within the amniotes.

Waterpipe and cigarette epigenome analysis reveals markers implicated in addiction and smoking type inference.

Waterpipe smoking is frequent in the Middle East and Africa with emerging prevalence worldwide. The epigenome acts as a molecular sensor to exposures and a crucial driver in several diseases. With the widespread use of waterpipe smoking, it is timely to investigate its epigenomic markers and their role in addiction, as a central player in disease prevention and therapeutic strategies. DNA methylome-wide profiling was performed on an exposure-rich population from the Middle East, constituting of 216 blood samples split equally between never, cigarette-only and waterpipe-only smokers. Waterpipe smokers showed predominantly distinct epigenetic markers from cigarette smokers, even though both smoking forms are tobacco-based. Moreover, each smoking form could be accurately (∼90 %) inferred from the DNA methylome using machine learning. Top markers showed dose-response relationship with extent of smoking and were validated using independent technologies and additional samples (total N = 284). Smoking markers were enriched in regulatory regions and several biological pathways, primarily addiction. The epigenetically altered genes were not associated with genetic etiology of tobacco use, and the methylation levels of addiction genes, in particular, were more likely to reverse after smoking cessation. In contrast, other epigenetic markers continued to feature smoking exposure after cessation, which may explain long-term health effects observed in former smokers. This study reports, for the first time, blood epigenome-wide markers of waterpipe smokers and reveals new markers of cigarette smoking, with implications in mechanisms of addiction and the capacity to discriminate between different smoking types. These markers may offer actionable targets to reverse the epigenetic memory of addiction and can guide future prevention strategies for tobacco smoking as the most preventable cause of illnesses worldwide.

Novel Probiotic Bacterium Rouxiella badensis subsp. acadiensis (Canan SV-53) Modulates Gut Immunity through Epigenetic Mechanisms.

Gut immune system homeostasis is crucial to overall host health. Immune disturbance at the gut level may lead to systemic and distant sites' immune dysfunction. Probiotics and prebiotics consumption have been shown to improve gut microbiota composition and function and enhance gut immunity. In the current study, the immunomodulatory and anti-inflammatory effects of viable and heat-inactivated forms of the novel probiotic bacterium Rouxiella badensis subsp. acadiensis (Canan SV-53), as well as the prebiotic protocatechuic acid (PCA) derived from the fermentation of blueberry juice by SV-53, were examined. To this end, female Balb/c mice received probiotic (viable or heat-inactivated), prebiotic, or a mixture of viable probiotic and prebiotic in drinking water for three weeks. To better decipher the immunomodulatory effects of biotics intake, gut microbiota, gut mucosal immunity, T helper-17 (Th17) cell-related cytokines, and epigenetic modulation of Th17 cells were studied. In mice receiving viable SV-53 and PCA, a significant increase was noted in serum IgA levels and the number of IgA-producing B cells in the ileum. A significant reduction was observed in the concentrations of proinflammatory cytokines, including interleukin (IL)-17A, IL-6, and IL-23, and expression of two proinflammatory miRNAs, miR-223 and miR425, in treated groups. In addition, heat-inactivated SV-53 exerted immunomodulatory properties by elevating the IgA concentration in the serum and reducing IL-6 and IL-23 levels in the ileum. DNA methylation analysis revealed the role of heat-inactivated SV-53 in the epigenetic regulation of genes related to Th17 and IL-17 production and function, including Il6, Il17rc, Il9, Il11, Akt1, Ikbkg, Sgk1, Cblb, and Smad4. Taken together, these findings may reflect the potential role of the novel probiotic bacterium SV-53 and prebiotic PCA in improving gut immunity and homeostasis. Further studies are required to ascertain the beneficial effects of this novel bacterium in the inflammatory state.

Buffy coat signatures of breast cancer risk in a prospective cohort study.

BACKGROUND: Epigenetic alterations are a near-universal feature of human malignancy and have been detected in malignant cells as well as in easily accessible specimens such as blood and urine. These findings offer promising applications in cancer detection, subtyping, and treatment monitoring. However, much of the current evidence is based on findings in retrospective studies and may reflect epigenetic patterns that have already been influenced by the onset of the disease. METHODS: Studying breast cancer, we established genome-scale DNA methylation profiles of prospectively collected buffy coat samples (n = 702) from a case-control study nested within the EPIC-Heidelberg cohort using reduced representation bisulphite sequencing (RRBS). RESULTS: We observed cancer-specific DNA methylation events in buffy coat samples. Increased DNA methylation in genomic regions associated with SURF6 and REXO1/CTB31O20.3 was linked to the length of time to diagnosis in the prospectively collected buffy coat DNA from individuals who subsequently developed breast cancer. Using machine learning methods, we piloted a DNA methylation-based classifier that predicted case-control status in a held-out validation set with 76.5% accuracy, in some cases up to 15 years before clinical diagnosis of the disease. CONCLUSIONS: Taken together, our findings suggest a model of gradual accumulation of cancer-associated DNA methylation patterns in peripheral blood, which may be detected long before clinical manifestation of cancer. Such changes may provide useful markers for risk stratification and, ultimately, personalized cancer prevention.

Chromatin Remodeler Smarca5 Is Required for Cancer-Related Processes of Primary Cell Fitness and Immortalization.

Smarca5, an ATPase of the ISWI class of chromatin remodelers, is a key regulator of chromatin structure, cell cycle and DNA repair. Smarca5 is deregulated in leukemia and breast, lung and gastric cancers. However, its role in oncogenesis is not well understood. Chromatin remodelers often play dosage-dependent roles in cancer. We therefore investigated the epigenomic and phenotypic impact of controlled stepwise attenuation of Smarca5 function in the context of primary cell transformation, a process relevant to tumor formation. Upon conditional single- or double-allele Smarca5 deletion, the cells underwent both accelerated growth arrest and senescence entry and displayed gradually increased sensitivity to genotoxic insults. These phenotypic characteristics were explained by specific remodeling of the chromatin structure and the transcriptome in primary cells prior to the immortalization onset. These molecular programs implicated Smarca5 requirement in DNA damage repair, telomere maintenance, cell cycle progression and in restricting apoptosis and cellular senescence. Consistent with the molecular programs, we demonstrate for the first time that Smarca5-deficient primary cells exhibit dramatically decreased capacity to bypass senescence and immortalize, an indispensable step during cell transformation and cancer development. Thus, Smarca5 plays a crucial role in key homeostatic processes and sustains cancer-promoting molecular programs and cellular phenotypes.

DNA Methylome Changes of Muscle- and Neuronal-Related Processes Precede Bladder Cancer Invasiveness.

Bladder cancer (BC) is the ninth leading cause of cancer death with one of the highest recurrence rates among all cancers. One of the main risks for BC development is exposure to nitrosamines present in tobacco smoke or in other products. Aberrant epigenetic (DNA methylation) changes accompanied by deregulated gene expression are an important element of cancer pathogenesis. Therefore, we aimed to determine DNA methylation signatures and their impacts on gene expression in mice treated with N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN), a carcinogen similar to compounds found in tobacco smoke. Following BBN administration mice developed non-invasive or invasive bladder cancers. Surprisingly, muscle- and neuronal-related pathways emerged as the most affected in those tumors. Hypo- and hypermethylation changes were present within non-invasive BC, across CpGs mapping to the genes involved in muscle- and neuronal-related pathways, however, methylation differences were not sufficient to affect the expression of the majority of associated genes. Conversely, invasive tumors displayed hypermethylation changes that were linked with alterations in gene expression profiles. Together, these findings indicate that bladder cancer progression could be revealed through methylation profiling at the pre-invasive cancer stage that could assist monitoring of cancer patients and guide novel therapeutic approaches.

Cutaneous and acral melanoma cross-OMICs reveals prognostic cancer drivers associated with pathobiology and ultraviolet exposure.

Ultraviolet radiation (UV) is causally linked to cutaneous melanoma, yet the underlying epigenetic mechanisms, known as molecular sensors of exposure, have not been characterized in clinical biospecimens. Here, we integrate clinical, epigenome (DNA methylome), genome and transcriptome profiling of 112 cutaneous melanoma from two multi-ethnic cohorts. We identify UV-related alterations in regulatory regions and immunological pathways, with multi-OMICs cancer driver potential affecting patient survival. TAPBP, the top gene, is critically involved in immune function and encompasses several UV-altered methylation sites that were validated by targeted sequencing, providing cost-effective opportunities for clinical application. The DNA methylome also reveals non UV-related aberrations underlying pathological differences between the cutaneous and 17 acral melanomas. Unsupervised epigenomic mapping demonstrated that non UV-mutant cutaneous melanoma more closely resembles acral rather than UV-exposed cutaneous melanoma, with the latter showing better patient prognosis than the other two forms. These gene-environment interactions reveal translationally impactful mechanisms in melanomagenesis.

A molecular map of lung neuroendocrine neoplasms.

BACKGROUND: Lung neuroendocrine neoplasms (LNENs) are rare solid cancers, with most genomic studies including a limited number of samples. Recently, generating the first multi-omic dataset for atypical pulmonary carcinoids and the first methylation dataset for large-cell neuroendocrine carcinomas led us to the discovery of clinically relevant molecular groups, as well as a new entity of pulmonary carcinoids (supra-carcinoids). RESULTS: To promote the integration of LNENs molecular data, we provide here detailed information on data generation and quality control for whole-genome/exome sequencing, RNA sequencing, and EPIC 850K methylation arrays for a total of 84 patients with LNENs. We integrate the transcriptomic data with other previously published data and generate the first comprehensive molecular map of LNENs using the Uniform Manifold Approximation and Projection (UMAP) dimension reduction technique. We show that this map captures the main biological findings of previous studies and can be used as reference to integrate datasets for which RNA sequencing is available. The generated map can be interactively explored and interrogated on the UCSC TumorMap portal (https://tumormap.ucsc.edu/?p=RCG_lungNENomics/LNEN). The data, source code, and compute environments used to generate and evaluate the map as well as the raw data are available, respectively, in a Nextjournal interactive notebook (https://nextjournal.com/rarecancersgenomics/a-molecular-map-of-lung-neuroendocrine-neoplasms/) and at the EMBL-EBI European Genome-phenome Archive and Gene Expression Omnibus data repositories. CONCLUSIONS: We provide data and all resources needed to integrate them with future LNENs transcriptomic studies, allowing meaningful conclusions to be drawn that will eventually lead to a better understanding of this rare understudied disease.

The methylome of the celiac intestinal epithelium harbours genotype-independent alterations in the HLA region.

The Human Leucocyte Antigen (HLA) locus and other DNA sequence variants identified in Genome-Wide Association (GWA) studies explain around 50% of the heritability of celiac disease (CD). However, the pathogenesis of CD could be driven by other layers of genomic information independent from sequence variation, such as DNA methylation, and it is possible that allele-specific methylation explains part of the SNP associations. Since the DNA methylation landscape is expected to be different among cell types, we analyzed the methylome of the epithelial and immune cell populations of duodenal biopsies in CD patients and controls separately. We found a cell type-specific methylation signature that includes genes mapping to the HLA region, namely TAP1 and HLA-B. We also performed Immunochip SNP genotyping of the same samples and interrogated the expression of some of the affected genes. Our analysis revealed that the epithelial methylome is characterized by the loss of CpG island (CGI) boundaries, often associated to altered gene expression, and by the increased variability of the methylation across the samples. The overlap between differentially methylated positions (DMPs) and CD-associated SNPs or variants contributing to methylation quantitative trait loci (mQTLs) is minimal. In contrast, there is a notable enrichment of mQTLs among the most significant CD-associated SNPs. Our results support the notion that DNA methylation alterations constitute a genotype-independent event and confirm its role in the HLA region (apart from the well-known, DQ allele-specific effect). Finally, we find that a fraction of the CD-associated variants could exert its phenotypic effect through DNA methylation.

Molecular features of premenopausal breast cancers in Latin American women: Pilot results from the PRECAMA study.

BACKGROUND: In Latin America (LA), there is a high incidence rate of breast cancer (BC) in premenopausal women, and the genomic features of these BC remain unknown. Here, we aim to characterize the molecular features of BC in young LA women within the framework of the PRECAMA study, a multicenter population-based case-control study of BC in premenopausal women. METHODS: Pathological tumor tissues were collected from incident cases from four LA countries. Immunohistochemistry (IHC) was performed centrally for ER, PR, HER2, Ki67, EGFR, CK5/6, and p53 protein markers. Targeted deep sequencing was done on genomic DNA extracted from formalin-fixed, paraffin-embedded tumor tissues and their paired blood samples to screen for somatic mutations in eight genes frequently mutated in BC. A subset of samples was analyzed by exome sequencing to identify somatic mutational signatures. RESULTS: The majority of cases were positive for ER or PR (168/233; 72%), and 21% were triple-negative (TN), mainly of basal type. Most tumors were positive for Ki67 (189/233; 81%). In 126 sequenced cases, TP53 and PIK3CA were the most frequently mutated genes (32.5% and 21.4%, respectively), followed by AKT1 (9.5%). TP53 mutations were more frequent in HER2-enriched and TN IHC subtypes, whereas PIK3CA/AKT1 mutations were more frequent in ER-positive tumors, as expected. Interestingly, a higher proportion of G:C>T:A mutations was observed in TP53 in PRECAMA cases compared with TCGA and METABRIC BC series (27% vs 14%). Exome-wide mutational patterns in 10 TN cases revealed alterations in signal transduction pathways and major contributions of mutational signatures caused by altered DNA repair pathways. CONCLUSIONS: These pilot results on PRECAMA tumors give a preview of the molecular features of premenopausal BC in LA. Although the overall mutation burden was as expected from data in other populations, mutational patterns observed in TP53 and exome-wide suggested possible differences in mutagenic processes giving rise to these tumors compared with other populations. Further -omics analyses of a larger number of cases in the near future will enable the investigation of relationships between these molecular features and risk factors.

Lowly methylated region analysis identifies EBF1 as a potential epigenetic modifier in breast cancer.

Breast cancer (BC) encompasses heterogeneous pathologies with different subtypes exhibiting distinct molecular changes, including those related to DNA methylation. However, the role of these changes in mediating BC heterogeneity is poorly understood. Lowly methylated regions (LMRs), non-CpG island loci that usually contain transcription factor (TF) binding sites, have been suggested to act as regulatory elements that define cellular identity. In this study, we aimed to identify the key subtype-specific TFs that may lead to LMR generation and shape the BC methylome and transcription program. We initially used whole-genome bisulfite sequencing (WGBS) data available at The Cancer Genome Atlas (TCGA) portal to identify subtype-specific LMRs. Differentially methylated regions (DMRs) within the BC PAM50 subtype-specific LMRs were selected by comparing tumors and normal tissues in a larger TCGA cohort assessed by HumanMethylation450 BeadChip (450K) arrays and TF enrichment analyses were performed. To assess the impact of LMRs on gene expression, TCGA RNA sequencing data were downloaded and Pearson correlations between methylation levels of loci presenting subtype-specific TF motifs and expression of the nearest genes were calculated. WGBS methylome data revealed a large number of LMRs for each of the BC subtypes. Analysis of these LMRs in the 450K datasets available for a larger sample set identified 7,765, 5,657, and 19 differentially methylated positions (DMPs) between normal adjacent tissues and tumor tissues from basal, luminal, and HER2-enriched subtypes, respectively. Unsupervised clustering showed that the discriminatory power of the top DMPs was remarkably strong for basal BC. Interestingly, in this particular subtype, we found 4,409 differentially hypomethylated positions grouped into 1,185 DMRs with a strong enrichment for the early B-cell factor 1 (EBF1) motifs. The methylation levels of the DMRs containing EBF1 motifs showed a strong negative correlation with the expression of 719 nearby genes, including BTS2 and CD74, two oncogenes known to be specific for basal BC subtype and for poor outcome. This study identifies LMRs specific to the three main BC subtypes and reveals EBF1 as a potentially important regulator of BC subtype-specific methylation and gene expression program.