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Childhood apraxia of speech (CAS), the prototypic severe childhood speech disorder, is characterized by motor programming and planning deficits. Genetic factors make substantive contributions to CAS aetiology, with a monogenic pathogenic variant identified in a third of cases, implicating around 20 single genes to date. Here we ascertained 70 unrelated probands with a clinical diagnosis of CAS and performed trio genome sequencing. Our bioinformatic analysis examined single nucleotide, indel, copy number, structural and short tandem repeat variants. We prioritised appropriate variants arising de novo or inherited that were expected to be damaging based on in silico predictions. We identified high confidence variants in 18/70 (26%) probands, almost doubling the current number of candidate genes for CAS. Three of the 18 variants affected SETBP1, SETD1A and DDX3X, thus confirming their roles in CAS, while the remaining 15 occurred in genes not previously associated with this disorder. Fifteen variants arose de novo and three were inherited. We provide further novel insights into the biology of child speech disorder, highlighting the roles of chromatin organization and gene regulation in CAS, and confirm that genes involved in CAS are co-expressed during brain development. Our findings confirm a diagnostic yield comparable to, or even higher, than other neurodevelopmental disorders with substantial de novo variant burden. Data also support the increasingly recognised overlaps between genes conferring risk for a range of neurodevelopmental disorders. Understanding the aetiological basis of CAS is critical to end the diagnostic odyssey and ensure affected individuals are poised for precision medicine trials.

Aims In 2003, an Australian woman was convicted by a jury of smothering and killing her four children over a 10-year period. Each child died suddenly and unexpectedly during a sleep period, at ages ranging from 19 days to 18 months. In 2019 we were asked to investigate if a genetic cause could explain the children’s deaths as part of an inquiry into the mother’s convictions. Methods and results Whole genomes or exomes of the mother and her four children were sequenced. Functional analysis of a novel CALM2 variant was performed by measuring Ca2+-binding affinity, interaction with calcium channels and channel function. We found two children had a novel calmodulin variant (CALM2 G114R) that was inherited maternally. Three genes (CALM1-3) encode identical calmodulin proteins. A variant in the corresponding residue of CALM3 (G114W) was recently reported in a child who died suddenly at age 4 and a sibling who suffered a cardiac arrest at age 5. We show that CALM2 G114R impairs calmodulin's ability to bind calcium and regulate two pivotal calcium channels (CaV1.2 and RyR2) involved in cardiac excitation contraction coupling. The deleterious effects of G114R are similar to those produced by G114W and N98S, which are considered arrhythmogenic and cause sudden cardiac death in children. Conclusion A novel functional calmodulin variant (G114R) predicted to cause idiopathic ventricular fibrillation, catecholaminergic polymorphic ventricular tachycardia, or mild long QT syndrome was present in two children. A fatal arrhythmic event may have been triggered by their intercurrent infections. Thus, calmodulinopathy emerges as a reasonable explanation for a natural cause of their deaths., Graphical Abstract

Aims Natriuretic peptides are useful for diagnosis and prognostication of heart failure of any cause. Now, research aims to discover novel biomarkers that will more specifically define the heart failure phenotype. DNA methylation plays a critical role in the development of cardiovascular disease with the potential to predict fundamental pathogenic processes. There is a lack of data relating DNA methylation in heart failure that specifically focuses on patients with severe multi‐vessel coronary artery disease. To begin to address this, we conducted a pilot study uniquely exploring the utility of powerful whole‐genome methyl‐binding domain‐capture sequencing in a cohort of cardiac surgery patients, matched for the severity of their coronary artery disease, aiming to identify candidate peripheral blood DNA methylation markers of ischaemic cardiomyopathy and heart failure. Methods and results We recruited a cohort of 20 male patients presenting for coronary artery bypass graft surgery with phenotypic extremes of heart failure but who otherwise share a similar coronary ischaemic burden, age, sex, and ethnicity. Methylation profiling in patient blood samples was performed using methyl‐binding domain‐capture sequencing. Differentially methylated regions were validated using targeted bisulfite sequencing. Gene set enrichment analysis was performed to identify differences in methylation at or near gene promoters in certain known Reactome pathways. We detected 567 188 methylation peaks of which our general linear model identified 68 significantly differentially methylated regions in heart failure with a false discovery rate

Background Inference of biological pathway activity via gene set enrichment analysis is frequently used in the interpretation of clinical and other omics data. With the proliferation of new omics profiling approaches and ever-growing size of data sets generated, there is a lack of tools available to perform and visualise gene set enrichments in analyses involving multiple contrasts. Results To address this, we developed mitch, an R package for multi-contrast gene set enrichment analysis. It uses a rank-MANOVA statistical approach to identify sets of genes that exhibit joint enrichment across multiple contrasts. Its unique visualisation features enable the exploration of enrichments in up to 20 contrasts. We demonstrate the utility of mitch with case studies spanning multi-contrast RNA expression profiling, integrative multi-omics, tool benchmarking and single-cell RNA sequencing. Using simulated data we show that mitch has similar accuracy to state of the art tools for single-contrast enrichment analysis, and superior accuracy in identifying multi-contrast enrichments. Conclusion mitch is a versatile tool for rapidly and accurately identifying and visualising gene set enrichments in multi-contrast omics data. Mitch is available from Bioconductor (https://bioconductor.org/packages/mitch).

Neurodevelopmental disorders are highly heterogenous conditions resulting from abnormalities of brain architecture and/or function. FBXW7 (F-box and WD-repeat-domain-containing 7), a recognized developmental regulator and tumor suppressor, has been shown to regulate cell-cycle progression and cell growth and survival by targeting substrates including CYCLIN E1/2 and NOTCH for degradation via the ubiquitin proteasome system. We used a genotype-first approach and global data-sharing platforms to identify 35 individuals harboring de novo and inherited FBXW7 germline monoallelic chromosomal deletions and nonsense, frameshift, splice-site, and missense variants associated with a neurodevelopmental syndrome. The FBXW7 neurodevelopmental syndrome is distinguished by global developmental delay, borderline to severe intellectual disability, hypotonia, and gastrointestinal issues. Brain imaging detailed variable underlying structural abnormalities affecting the cerebellum, corpus collosum, and white matter. A crystal-structure model of FBXW7 predicted that missense variants were clustered at the substrate-binding surface of the WD40 domain and that these might reduce FBXW7 substrate binding affinity. Expression of recombinant FBXW7 missense variants in cultured cells demonstrated impaired CYCLIN E1 and CYCLIN E2 turnover. Pan-neuronal knockdown of the Drosophila ortholog, archipelago, impaired learning and neuronal function. Collectively, the data presented herein provide compelling evidence of an F-Box protein-related, phenotypically variable neurodevelopmental disorder associated with monoallelic variants in FBXW7.

Aggregation of islet amyloid polypeptide (IAPP) into islet amyloid results in β-cell toxicity in human type 2 diabetes. To determine the effect of islet amyloid formation on gene expression, we performed ribonucleic acid (RNA) sequencing (RNA-seq) analysis using cultured islets from either wild-type mice (mIAPP), which are not amyloid prone, or mice that express human IAPP (hIAPP), which develop amyloid. Comparing mIAPP and hIAPP islets, 5025 genes were differentially regulated (2439 upregulated and 2586 downregulated). When considering gene sets (reactomes), 248 and 52 pathways were up- and downregulated, respectively. Of the top 100 genes upregulated under two conditions of amyloid formation, seven were common. Of these seven genes, only steroidogenic acute regulatory protein (Star) demonstrated no effect of glucose per se to modify its expression. We confirmed this differential gene expression using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and also demonstrated the presence of STAR protein in islets containing amyloid. Furthermore, Star is a part of reactomes representing metabolism, metabolism of lipids, metabolism of steroid hormones, metabolism of steroids and pregnenolone biosynthesis. Thus, examining gene expression that is differentially regulated by islet amyloid has the ability to identify new molecules involved in islet physiology and pathology applicable to type 2 diabetes.

Hepatitis C virus (HCV) infection is the leading cause of chronic hepatitis, which often results in liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). HCV possesses an RNA genome and its replication is confined to the cytoplasm. Yet, infection with HCV leads to global changes in gene expression, and chromosomal instability (CIN) in the host cell. The mechanisms by which the cytoplasmic virus affects these nuclear processes are elusive. Here, we show that HCV modulates the function of the Structural Maintenance of Chromosome (SMC) protein complex, cohesin, which tethers remote regions of chromatin. We demonstrate that infection of hepatoma cells with HCV leads to up regulation of the expression of the RAD21 cohesin subunit and changes cohesin residency on the chromatin. These changes regulate the expression of genes associated with virus-induced pathways. Furthermore, siRNA downregulation of viral-induced RAD21 reduces HCV infection. During mitosis, HCV infection induces hypercondensation of chromosomes and the appearance of multi-centrosomes. We provide evidence that the underlying mechanism involves the viral NS3/4 protease and the cohesin regulator, WAPL. Altogether, our results provide the first evidence that HCV induces changes in gene expression and chromosome structure of infected cells by modulating cohesin.

Objectives Focal cortical dysplasia (FCD) is a major cause of drug‐resistant focal epilepsy in children, and the clinicopathological classification remains a challenging issue in daily practice. With the recent progress in DNA methylation–based classification of human brain tumors we examined whether genomic DNA methylation and gene expression analysis can be used to also distinguish human FCD subtypes. Methods DNA methylomes and transcriptomes were generated from massive parallel sequencing in 15 surgical FCD specimens, matched with 5 epilepsy and 6 nonepilepsy controls. Results Differential hierarchical cluster analysis of DNA methylation distinguished major FCD subtypes (ie, Ia, IIa, and IIb) from patients with temporal lobe epilepsy patients and nonepileptic controls. Targeted panel sequencing identified a novel likely pathogenic variant in DEPDC5 in a patient with FCD type IIa. However, no enrichment of differential DNA methylation or gene expression was observed in mechanistic target of rapamycin (mTOR) pathway–related genes. Significance Our studies extend the evidence for disease‐specific methylation signatures toward focal epilepsies in favor of an integrated clinicopathologic and molecular classification system of FCD subtypes incorporating genomic methylation.

Supplemental Digital Content is available in the text., Background: The inability of the adult mammalian heart to regenerate following injury represents a major barrier in cardiovascular medicine. In contrast, the neonatal mammalian heart retains a transient capacity for regeneration, which is lost shortly after birth. Defining the molecular mechanisms that govern regenerative capacity in the neonatal period remains a central goal in cardiac biology. Here, we assemble a transcriptomic framework of multiple cardiac cell populations during postnatal development and following injury, which enables comparative analyses of the regenerative (neonatal) versus nonregenerative (adult) state for the first time. Methods: Cardiomyocytes, fibroblasts, leukocytes, and endothelial cells from infarcted and noninfarcted neonatal (P1) and adult (P56) mouse hearts were isolated by enzymatic dissociation and fluorescence-activated cell sorting at day 3 following surgery. RNA sequencing was performed on these cell populations to generate the transcriptome of the major cardiac cell populations during cardiac development, repair, and regeneration. To complement our transcriptomic data, we also surveyed the epigenetic landscape of cardiomyocytes during postnatal maturation by performing deep sequencing of accessible chromatin regions by using the Assay for Transposase-Accessible Chromatin from purified mouse cardiomyocyte nuclei (P1, P14, and P56). Results: Profiling of cardiomyocyte and nonmyocyte transcriptional programs uncovered several injury-responsive genes across regenerative and nonregenerative time points. However, the majority of transcriptional changes in all cardiac cell types resulted from developmental maturation from neonatal stages to adulthood rather than activation of a distinct regeneration-specific gene program. Furthermore, adult leukocytes and fibroblasts were characterized by the expression of a proliferative gene expression network following infarction, which mirrored the neonatal state. In contrast, cardiomyocytes failed to reactivate the neonatal proliferative network following infarction, which was associated with loss of chromatin accessibility around cell cycle genes during postnatal maturation. Conclusions: This work provides a comprehensive framework and transcriptional resource of multiple cardiac cell populations during cardiac development, repair, and regeneration. Our findings define a regulatory program underpinning the neonatal regenerative state and identify alterations in the chromatin landscape that could limit reinduction of the regenerative program in adult cardiomyocytes.

Background RNA sequencing (RNA-seq) is an indispensable tool in the study of gene regulation. While the technology has brought with it better transcript coverage and quantification, there remain considerable barriers to entry for the computational biologist to analyse large data sets. There is a real need for a repository of uniformly processed RNA-seq data that is easy to use. Findings To address these obstacles, we developed Digital Expression Explorer 2 (DEE2), a web-based repository of RNA-seq data in the form of gene-level and transcript-level expression counts. DEE2 contains >5.3 trillion assigned reads from 580,000 RNA-seq data sets including species Escherichia coli, yeast, Arabidopsis, worm, fruit fly, zebrafish, rat, mouse, and human. Base-space sequence data downloaded from the National Center for Biotechnology Information Sequence Read Archive underwent quality control prior to transcriptome and genome mapping using open-source tools. Uniform data processing methods ensure consistency across experiments, facilitating fast and reproducible meta-analyses. Conclusions The web interface allows users to quickly identify data sets of interest using accession number and keyword searches. The data can also be accessed programmatically using a specifically designed R package. We demonstrate that DEE2 data are compatible with statistical packages such as edgeR or DESeq. Bulk data are also available for download. DEE2 can be found at http://dee2.io.

The increasing worldwide prevalence of Hepatocellular carcinoma (HCC), characterized by resistance to conventional chemotherapy, poor prognosis and eventually mortality, place it as a prime target for new modes of prevention and treatment. Hepatitis C Virus (HCV) is the predominant risk factor for HCC in the US and Europe. Multiple epidemiological studies showed that sustained virological responses (SVR) following treatment with the powerful direct acting antivirals (DAAs), which have replaced interferon-based regimes, do not eliminate tumor development. We aimed to identify an HCV-specific pathogenic mechanism that persists post SVR following DAAs treatment. We demonstrate that HCV infection induces genome-wide epigenetic changes by performing chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) for histone post-translational modifications that are epigenetic markers for active and repressed chromatin. The changes in histone modifications correlate with reprogramed host gene expression and alter signaling pathways known to be associated with HCV life cycle and HCC. These epigenetic alterations require the presence of HCV RNA or/and expression of the viral proteins in the cells. Importantly, the epigenetic changes induced following infection persist as an "epigenetic signature" after virus eradication by DAAs treatment, as detected using in vitro HCV infection models. These observations led to the identification of an 8 gene signature that is associated with HCC development and demonstrate persistent epigenetic alterations in HCV infected and post SVR liver biopsy samples. The epigenetic signature was reverted in vitro by drugs that inhibit epigenetic modifying enzyme and by the EGFR inhibitor, Erlotinib. This epigenetic “scarring” of the genome, persisting following HCV eradication, suggest a novel mechanism for the persistent pathogenesis of HCV after its eradication by DAAs. Our study offers new avenues for prevention of the persistent oncogenic effects of chronic hepatitis infections using specific drugs to revert the epigenetic changes to the genome., Author summary Hepatitis C virus (HCV) is a leading cause of hepatocellular carcinoma (HCC) in western countries. While direct acting antivirals (DAAs) therapy for HCV efficiently eradicates the infection, sustained virological response (SVR) following anti-HCV treatment does not eliminate the risk for HCC development. With the 3–4 million newly infected cases each year, it is estimated that HCV-associated disease burden remains high in the next decade and the population of post-DAA-based SVR patients is expected to grow exponentially in the near future. Therefore, Post-SVR HCC is an emerging problem with an urgent unmet need for the elucidation of its molecular mechanisms for therapeutic target and biomarker discovery. We demonstrate that HCV-induce epigenetic missregulation. These HCV-induced epigenetic changes reprogram host gene expression and persist as an "epigenetic signature" following virus eradication. Treatment of HCV-cured cells with specific inhibitors reverted the epigenetic signature. These results suggest a "hit and run" scenario that may explain why some chronic HCV infected patients do proceed to develop HCC after HCV eradication. Our discoveries provide an insight into the outcomes of HCV infection and HCC development, also following SVR, and novel approaches for its prevention.

Background The failure of spontaneous resolution underlies chronic inflammatory conditions, including microvascular complications of diabetes such as diabetic kidney disease. The identification of endogenously generated molecules that promote the physiologic resolution of inflammation suggests that these bioactions may have therapeutic potential in the context of chronic inflammation. Lipoxins (LXs) are lipid mediators that promote the resolution of inflammation. Methods We investigated the potential of LXA(4) and a synthetic LX analog (Benzo-LXA(4)) as therapeutics in a murine model of diabetic kidney disease, ApoE(−/−) mice treated with streptozotocin. Results Intraperitoneal injection of LXs attenuated the development of diabetes-induced albuminuria, mesangial expansion, and collagen deposition. Notably, LXs administered 10 weeks after disease onset also attenuated established kidney disease, with evidence of preserved kidney function. Kidney transcriptome profiling defined a diabetic signature (725 genes; false discovery rate P≤0.05). Comparison of this murine gene signature with that of human diabetic kidney disease identified shared renal proinflammatory/profibrotic signals (TNF-α, IL-1β, NF-κB). In diabetic mice, we identified 20 and 51 transcripts regulated by LXA(4) and Benzo-LXA(4), respectively, and pathway analysis identified established (TGF-β1, PDGF, TNF-α, NF-κB) and novel (early growth response–1 [EGR-1]) networks activated in diabetes and regulated by LXs. In cultured human renal epithelial cells, treatment with LXs attenuated TNF-α–driven Egr-1 activation, and Egr-1 depletion prevented cellular responses to TGF-β1 and TNF-α. Conclusions These data demonstrate that LXs can reverse established diabetic complications and support a therapeutic paradigm to promote the resolution of inflammation.

Traumatic brain injury (TBI) induces a wide variety of cellular and molecular changes that can continue for days to weeks to months, leading to functional impairments. Currently, there are no pharmacotherapies in clinical use that favorably modify the post-TBI outcome, due in part to limited understanding of the mechanisms of TBI-induced pathologies. Our system biology analysis tested the hypothesis that chronic transcriptomics changes induced by TBI are controlled by altered DNA-methylation in gene promoter areas or by transcription factors. We performed genome-wide methyl binding domain (MBD)-sequencing (seq) and RNA-seq in perilesional, thalamic, and hippocampal tissue sampled at 3 months after TBI induced by lateral fluid percussion in adult male Sprague-Dawley rats. We investigated the regulated molecular networks and mechanisms underlying the chronic regulation, particularly DNA methylation and transcription factors. Finally, we identified compounds that modulate the transcriptomics changes and could be repurposed to improve recovery. Unexpectedly, DNA methylation was not a major regulator of chronic post-TBI transcriptomics changes. On the other hand, the transcription factors Cebpd, Pax6, Spi1, and Tp73 were upregulated at 3 months after TBI (False discovery rate

Scope Early life nutrition has long-lasting influence in adults through key mediators that modulate epigenetic states, although the determinants involved that underlie this response remain controversial. Because of the similarities between metabolic, physiological, and endocrine changes and those occurring in human type 2 diabetes, we studied the interaction of diet during pregnancy regulating RNA adenosine methylation (N6-methyladenosine [m6A]) and the transcriptome in Psammomys obesus. Methods and results Breeding pairs were randomly allocated standard diet (total digestible energy 18 MJ kg-1 ) or low-fat diet (15 MJ kg-1 ). Offspring were weaned onto the low-fat diet at 4 weeks of age and given ad libitum access, resulting in two experimental groups: 1) male offspring of animals fed a low-fat diet and weaned onto the low-fat diet and 2) male offspring of animals fed a standard diet and weaned onto the low-fat diet. Hypothalamic RNA was used to assess m6A by immunoprecipitation. Parental low-fat diet alters the metabolic phenotype in offspring. An association between parental diet and hypothalamic m6A was observed in regulating the expression of FTO and METTL3 in the offspring. Conclusions We propose the regulatory capacity is now broadened for the first time to include m6A in developmental programming and obesity phenotype.

Given the skyrocketing costs to develop new drugs, repositioning of approved drugs, such as histone deacetylase (HDAC) inhibitors, may be a promising strategy to develop novel therapies. However, a gap exists in the understanding and advancement of these agents to meaningful translation for which new indications may emerge. To address this, we performed systems-level analyses of 33 independent HDAC inhibitor microarray studies. Based on network analysis, we identified enrichment for pathways implicated in metabolic syndrome and diabetes (insulin receptor signaling, lipid metabolism, immunity and trafficking). Integration with ENCODE ChIP-seq datasets identified suppression of EP300 target genes implicated in diabetes. Experimental validation indicates reversal of diabetes-associated EP300 target genes in primary vascular endothelial cells derived from a diabetic individual following inhibition of HDACs (by SAHA), EP300, or EP300 knockdown. Our computational systems biology approach provides an adaptable framework for the prediction of novel therapeutics for existing disease.

Relatively little is known about the epigenetic control mechanisms that guide postnatal organ maturation. The goal of this study was to determine whether DNA methylation plays an important role in guiding transcriptional changes during the first 2 wk of mouse heart development, which is an important period for cardiomyocyte maturation, loss of proliferative capacity and loss of regenerative potential. Gene expression profiling (RNA-seq) and genome-wide sequencing of methylated DNA (MBD-seq) identified dynamic changes in the cardiac methylome during postnatal development [2545 differentially methylated regions (DMRs) from P1 to P14 in the mouse]. The vast majority (~80%) of DMRs were hypermethylated between P1 and P14, and these hypermethylated regions were associated with transcriptional shut down of important developmental signaling pathways, including Hedgehog, bone morphogenetic protein, TGF-β, fibroblast growth factor, and Wnt/β-catenin signaling. Postnatal inhibition of DNA methylation with 5-aza-2'-deoxycytidine induced a marked increase (~3-fold) in cardiomyocyte proliferation and ~50% reduction in the percentage of binucleated cardiomyocytes compared with saline-treated controls. This study provides novel evidence for widespread alterations in DNA methylation during postnatal heart maturation and suggests that cardiomyocyte cell cycle arrest during the neonatal period is subject to regulation by DNA methylation.

Epilepsy is a frequent neurological disorder, although onset and progression of seizures remain difficult to predict in affected patients, irrespective of their epileptogenic condition. Previous studies in animal models as well as human epileptic brain tissue revealed a remarkably diverse pattern of gene expression implicating epigenetic changes to contribute to disease progression. Here we mapped for the first time global DNA methylation patterns in chronic epileptic rats and controls. Using methyl-CpG capture associated with massive parallel sequencing (Methyl-Seq) we report the genomic methylation signature of the chronic epileptic state. We observed a predominant increase, rather than loss of DNA methylation in chronic rat epilepsy. Aberrant methylation patterns were inversely correlated with gene expression changes using mRNA sequencing from same animals and tissue specimens. Administration of a ketogenic, high-fat, low-carbohydrate diet attenuated seizure progression and ameliorated DNA methylation mediated changes in gene expression. This is the first report of unsupervised clustering of an epigenetic mark being used in epilepsy research to separate epileptic from non-epileptic animals as well as from animals receiving anti-convulsive dietary treatment. We further discuss the potential impact of epigenetic changes as a pathogenic mechanism of epileptogenesis. Electronic supplementary material The online version of this article (doi:10.1007/s00401-013-1168-8) contains supplementary material, which is available to authorized users.

Reliable identification of cis regulatory elements influencing transcription remains a challenging problem in molecular bioinformatics. This is especially true for enhancer elements which are often located hundreds of kilobases from the gene promoter. High resolution DNase hypersensitivity and connectivity profiling by the ENCODE consortium provides evidence of millions of interacting cis-acting elements in the human genome. This prior knowledge can be incorporated into genome-wide expression analyses, in the form of gene sets sharing regulatory sequence motifs in known DNase hypersensitivity peak regions. High proportions of enrichment among the most extreme differentially transcribed genes from controlled biological experiments may suggest novel hypotheses about signalling pathways. The utility of this approach is demonstrated with the reanalysis of a microarray-derived gene expression data set through the Gene Set Enrichment Analysis pipeline, uncovering new putative distal cis elements in the context of innate immunity. The DNase Hypersensitivity Connectivity informed Motif Enrichment in Gene Expression (DHC-MEGE) method described here has the advantage of identifying distal elements such as enhancers, which are often overlooked with standard promoter motif analysis. Availability The DHC-MEGE shell script can be obtained from Sourceforge https://sourceforge.net/projects/dhcmege/ and the generated GMT file is attached as supplementary data.

This study tested the hypothesis that acquired epileptogenesis is accompanied by DNA methylation changes independent of etiology. We investigated DNA methylation and gene expression in the hippocampal CA3/dentate gyrus fields at 3 months following epileptogenic injury in three experimental models of epilepsy: focal amygdala stimulation, systemic pilocarpine injection, or lateral fluid-percussion induced traumatic brain injury (TBI) in rats. In the models studies, DNA methylation and gene expression profiles distinguished controls from injured animals. We observed consistent increased methylation in gene bodies and hypomethylation at non-genic regions. We did not find a common methylation signature in all three different models and few regions common to any two models. Our data provide evidence that genome-wide alteration of DNA methylation signatures is a general pathomechanism associated with epileptogenesis and epilepsy in experimental animal models, but the broad pathophysiological differences between models (i.e. pilocarpine, amygdala stimulation and post-TBI) are reflected in distinct etiology-dependent DNA methylation patterns.

The rising incidence of obesity is a major public health issue worldwide. Recent human and animal studies suggest that parental diet can influence fetal development and is implicated with risk of obesity and type 2 diabetes in offspring. The hypothalamus is central to body energy homoeostasis and appetite by controlling endocrine signals. We hypothesise that offspring susceptibility to obesity is programmed in the hypothalamus in utero and mediated by changes to DNA methylation, which persist to adulthood. We investigated hypothalamic genome-wide DNA methylation in Psammomys obesus diet during pregnancy to the offspring's risk of obesity.Using methyl-CpG binding domain capture and deep sequencing (MBD-seq), we examined the hypothalamus of offspring exposed to a low-fat diet and standard chow diet during the gestation and lactation period.Offspring exposed to a low-fat parental diet were more obese and had increased circulating insulin and glucose levels. Methylome profiling identified 1447 genomic regions of differential methylation between offspring of parents fed a low-fat diet compared with parents on standard chow diet. Pathway analysis shows novel DNA methylation changes of hypothalamic genes associated with neurological function, nutrient sensing, appetite and energy balance. Differential DNA methylation corresponded to changes in hypothalamic gene expression of Tas1r1 and Abcc8 in the offspring exposed to low-fat parental diet.Subject to parental low-fat diet, we observe DNA methylation changes of genes associated with obesity in offspring.

Genomic alignment of small RNA (smRNA) sequences such as microRNAs poses considerable challenges due to their short length (∼21 nucleotides [nt]) as well as the large size and complexity of plant and animal genomes. While several tools have been developed for high-throughput mapping of longer mRNA-seq reads (>30 nt), there are few that are specifically designed for mapping of smRNA reads including microRNAs. The accuracy of these mappers has not been systematically determined in the case of smRNA-seq. In addition, it is unknown whether these aligners accurately map smRNA reads containing sequence errors and polymorphisms. By using simulated read sets, we determine the alignment sensitivity and accuracy of 16 short-read mappers and quantify their robustness to mismatches, indels, and nontemplated nucleotide additions. These were explored in the context of a plant genome (Oryza sativa, ∼500 Mbp) and a mammalian genome (Homo sapiens, ∼3.1 Gbp). Analysis of simulated and real smRNA-seq data demonstrates that mapper selection impacts differential expression results and interpretation. These results will inform on best practice for smRNA mapping and enable more accurate smRNA detection and quantification of expression and RNA editing.

The evolutionary theory assumes that occurrence of mutations in cancer is random. However, recent studies suggest that passenger mutations are not randomly scattered in cancer genomes and that chromatin organization dictate mutations profiles. Hepatocellular carcinoma (HCC) serves as a model of diverse spectrum of cancers, since it is induced by a number of well-known etiological agents, mainly Hepatitis C virus (HCV) and Hepatitis B virus (HBV). This provides a unique opportunity to test whether different etiological agents dictate distinct mutational landscapes across the genome through a unique epigenetic signature. The HCC-specific genomic mutations were studied in 80 HCC biopsies from three etiology groups - HBV, HCV, or other. To explore the mutational signature we performed target high-resolution sequencing that enables the detection of low-frequency passenger mutations. By using this strategy we identified novel distinct etiology-dependent regional mutations signature in specific genes and pathways in HCC that was not detected previously by exome sequencing of liver tumors. These observations suggest that viral infection modulate somatic mutations causing HCC. To explore the link between genomic signature and genome wide chromatin organization we studied the epigenetic changes occur following HCV infection. Epigenetic analysis unraveled known and novel pathways that are controlled by the virus such as Hepatic lipid metabolism, cell motility, cell cycle and immunity. Invers correlation between high mutation rate and enrichment of chromatin modifications associated with active transcription provides a link between etiology and cancer genome. Our novel approach offers a perspective into the mechanisms that shape mutational signature development in cancer. Note: This abstract was not presented at the meeting. Citation Format: Izhak Haviv, Shira Perez, Anat Lavi-Itzkovitz, Antony Kaspi, Ana Tover, Assam El-Osta, Gur Yaari, Salomon Stemmer, Meital Gal-Tanamy. The interplay between etiology-dependent genomic and epigenetic signatures: The paradigm of liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5564. doi:10.1158/1538-7445.AM2017-5564

HDAC inhibitors can regulate gene expression by post-translational modification of histone as well as nonhistone proteins. Often studied at single loci, increased histone acetylation is the paradigmatic mechanism of action. However, little is known of the extent of genome-wide changes in cells stimulated by the hydroxamic acids, TSA and SAHA. In this article, we map vascular chromatin modifications including histone H3 acetylation of lysine 9 and 14 (H3K9/14ac) using chromatin immunoprecipitation (ChIP) coupled with massive parallel sequencing (ChIP-seq). Since acetylation-mediated gene expression is often associated with modification of other lysine residues, we also examined H3K4me3 and H3K9me3 as well as changes in CpG methylation (CpG-seq). RNA sequencing indicates the differential expression of ∼30% of genes, with almost equal numbers being up- and down-regulated. We observed broad deacetylation and gene expression changes conferred by TSA and SAHA mediated by the loss of EP300/CREBBP binding at multiple gene promoters. This study provides an important framework for HDAC inhibitor function in vascular biology and a comprehensive description of genome-wide deacetylation by pharmacological HDAC inhibition.

Epigenetic modifications have emerged as central players in the coordination of gene expression networks during cardiac development. Much attention has focused on the role of histone modifications during embryonic heart development, but relatively little is known about the epigenetic control mechanisms that guide post-natal heart maturation. Furthermore, few studies have investigated the role of DNA methylation during cardiac development, despite the fundamental importance of this biological process for transcriptional regulation. The purpose of the current study was to determine whether DNA methylation plays an important role in guiding transcription during the neonatal period, which is an important developmental window for cardiac maturation, including cardiomyocyte cell cycle withdrawal and loss of endogenous regenerative capacity. Here, we interrogated genome-wide changes in transcription and CpG methylation during post-natal cardiac maturation in the mouse (P1 vs. P14). CpG sequencing (CpG-seq) identified 2251 differentially methylated regions (DMRs) between P1 and P14. Intersection of DMRs with different genomic features revealed that 1248 DMRs were associated with promoter regions and transcription start sites. Interestingly, increased methylation of genes associated with well-known signalling pathways for muscle development and differentiation, such as the bone morphogenetic protein, fibroblast growth factor, Wnt and Notch signalling pathways, was associated with transcriptional repression of these regulatory networks at P14. To determine the functional significance of these dynamic changes in the cardiac methylome, we inhibited DNA methylation in vivo by administration of the DNA hypomethylating agent 5-azacytidine from P2 to P12. Post-natal inhibition of DNA methylation caused a marked increase in heart size and was associated with increased cardiomyocyte proliferation. This study provides evidence for widespread alterations in DNA methylation during post-natal heart maturation and suggests that DNA methylation may play an important role in the transcriptional silencing of key regulatory networks for muscle development and cardiomyocyte proliferation during neonatal life.

SummaryEarly full-term pregnancy is one of the most effective natural protections against breast cancer. To investigate this effect, we have characterized the global gene expression and epigenetic profiles of multiple cell types from normal breast tissue of nulliparous and parous women and carriers of BRCA1 or BRCA2 mutations. We found significant differences in CD44+ progenitor cells, where the levels of many stem cell-related genes and pathways, including the cell-cycle regulator p27, are lower in parous women without BRCA1/BRCA2 mutations. We also noted a significant reduction in the frequency of CD44+p27+ cells in parous women and showed, using explant cultures, that parity-related signaling pathways play a role in regulating the number of p27+ cells and their proliferation. Our results suggest that pathways controlling p27+ mammary epithelial cells and the numbers of these cells relate to breast cancer risk and can be explored for cancer risk assessment and prevention.

The advent of massive parallel sequencing of immunopurified chromatin and its determinants has provided new avenues for researchers to map epigenome-wide changes and there is tremendous interest to uncover regulatory signatures to understand fundamental questions associated with chromatin structure and function. Indeed, the rapid development of large genome annotation projects has seen a resurgence in chromatin immunoprecipitation (ChIP) based protocols which are used to distinguish protein interactions coupled with large scale sequencing (Seq) to precisely map epigenome-wide interactions. Despite some of the great advances in our understanding of chromatin modifying complexes and their determinants, the development of ChIP-Seq technologies also pose specific demands on the integration of data for visualization, manipulation and analysis. In this article we discuss some of the considerations for experimental design planning, quality control, and bioinformatic analysis. The key aspects of post sequencing analysis are the identification of regions of interest, differentiation between biological conditions and the characterization of sequence differences for chromatin modifications. We provide an overview of best-practise approaches with background information and considerations of integrative analysis from ChIP-Seq experiments.

Emerging evidence suggests that poor glycemic control mediates post-translational modifications to the H3 histone tail. We are only beginning to understand the dynamic role of some of the diverse epigenetic changes mediated by hyperglycemia at single loci, yet elevated glucose levels are thought to regulate genome-wide changes, and this still remains poorly understood. In this article we describe genome-wide histone H3K9/K14 hyperacetylation and DNA methylation maps conferred by hyperglycemia in primary human vascular cells. Chromatin immunoprecipitation (ChIP) as well as CpG methylation (CpG) assays, followed by massive parallel sequencing (ChIP-seq and CpG-seq) identified unique hyperacetylation and CpG methylation signatures with proximal and distal patterns of regionalization associative with gene expression. Ingenuity knowledge-based pathway and gene ontology analyses indicate that hyperglycemia significantly affects human vascular chromatin with the transcriptional up-regulation of genes involved in metabolic and cardiovascular disease. We have generated the first installment of a reference collection of hyperglycemia-induced chromatin modifications using robust and reproducible platforms that allow parallel sequencing-by-synthesis of immunopurified content. We uncover that hyperglycemia-mediated induction of genes and pathways associated with endothelial dysfunction occur through modulation of acetylated H3K9/K14 inversely correlated with methyl-CpG content.

Background The primary goal of genetic linkage analysis is to identify genes affecting a phenotypic trait. After localisation of the linkage region, efficient genetic dissection of the disease linked loci requires that functional variants are identified across the loci. These functional variations are difficult to detect due to extent of genetic diversity and, to date, incomplete cataloguing of the large number of variants present both within and between populations. Massively parallel sequencing platforms offer unprecedented capacity for variant discovery, however the number of samples analysed are still limited by cost per sample. Some progress has been made in reducing the cost of resequencing using either multiplexing methodologies or through the utilisation of targeted enrichment technologies which provide the ability to resequence genomic areas of interest rather that full genome sequencing. Results We developed a method that combines current multiplexing methodologies with a solution-based target enrichment method to further reduce the cost of resequencing where region-specific sequencing is required. Our multiplex/enrichment strategy produced high quality data with nominal reduction of sequencing depth. We undertook a genotyping study and were successful in the discovery of novel SNP alleles in all samples at uniplex, duplex and pentaplex levels. Conclusion Our work describes the successful combination of a targeted enrichment method and index barcode multiplexing to reduce costs, time and labour associated with processing large sample sets. Furthermore, we have shown that the sequencing depth obtained is adequate for credible SNP genotyping analysis at uniplex, duplex and pentaplex levels.

Motivation: Galaxy is a software application supporting high-throughput biology analyses and work flows, available as a free on-line service or as source code for local deployment. New tools can be written to extend Galaxy, and these can be shared using public Galaxy Tool Shed (GTS) repositories, but converting even simple scripts into tools requires effort from a skilled developer. Results: The Tool Factory is a novel Galaxy tool that automates the generation of all code needed to execute user-supplied scripts, and wraps them into new Galaxy tools for upload to a GTS, ready for review and installation through the Galaxy administrative interface. Availability and implementation: The Galaxy administrative interface supports automated installation from the main GTS. Source code and support are available at the project website, https://bitbucket.org/fubar/galaxytoolfactory. The Tool Factory is implemented as an installable Galaxy tool. Contact: [email protected]

Differentiation is an epigenetic program that involves the gradual loss of pluripotency and acquisition of cell type–specific features. Understanding these processes requires genome-wide analysis of epigenetic and gene expression profiles, which have been challenging in primary tissue samples due to limited numbers of cells available. Here we describe the application of high-throughput sequencing technology for profiling histone and DNA methylation, as well as gene expression patterns of normal human mammary progenitor-enriched and luminal lineage-committed cells. We observed significant differences in histone H3 lysine 27 tri-methylation (H3K27me3) enrichment and DNA methylation of genes expressed in a cell type–specific manner, suggesting their regulation by epigenetic mechanisms and a dynamic interplay between the two processes that together define developmental potential. The technologies we developed and the epigenetically regulated genes we identified will accelerate the characterization of primary cell epigenomes and the dissection of human mammary epithelial lineage-commitment and luminal differentiation., Author Summary Cellular differentiation is a precisely controlled and largely irreversible process orchestrated by cell type–specific epigenetic programs. Abnormalities in these programs lead to developmental disorders and play a key role in tumorigenesis. To better understand the regulation of human mammary epithelial cell type specification, we analyzed the gene expression, DNA methylation, and histone H3 K4 and K27 trimethylation profiles of progenitor-enriched and more differentiated luminal epithelial cell populations from multiple individuals. Network analysis of these profiles and their comparison to that of human embryonic stem cells identified key regulators of mammary epithelial and luminal lineage commitment. The list of genes epigenetically regulated in a cell type–specific manner provides a rich resource for the further analysis of human breast development and the role of epigenetic mechanisms in breast tumorigenesis.

Background and Aims: A major impediment to establishing new treatments for non-alcoholic steatohepatitis is the lack of suitable animal models that accurately mimic the biochemical and metabolic characteristics of the disease. The aim of this study was to explore a unique polygenic animal model of metabolic disease as a model of non-alcoholic steatohepatitis by determining the effects of 2% dietary cholesterol supplementation on metabolic and liver endpoints in Psammomys obesus (Israeli sand rat). Methods: P. obesus were provided ad libitum access to either a standard rodent diet (20% kcal/fat) or a standard rodent diet supplemented with 2% cholesterol (w/w) for 4 weeks. Histological sections of liver from animals on both diets were examined for key features of non-alcoholic steatohepatitis. The expression levels of key genes involved in hepatic lipid metabolism were measured by real-time PCR. Results: P. obesus fed a cholesterol-supplemented diet exhibited profound hepatomegaly and steatosis, and higher plasma transaminase levels. Histological analysis identified extensive steatosis, inflammation, hepatocyte injury and fibrosis. Hepatic gene expression profiling revealed decreased expression of genes involved in delivery and uptake of lipids, and fatty acid and triglyceride synthesis, and increased expression of genes involved in very low density lipoprotein cholesterol synthesis, triglyceride and cholesterol export. Conclusions: P. obesus rapidly develop non-alcoholic steatohepatitis when fed a cholesterol-supplemented diet that appears to be histologically and mechanistically similar to patients. © 2014 Spolding et al.

Current computational methods used to analyze changes in DNA methylation and chromatin modification rely on sequenced genomes. Here we describe a pipeline for the detection of these changes from short-read sequence data that does not require a reference genome. Open source software packages were used for sequence assembly, alignment, and measurement of differential enrichment. The method was evaluated by comparing results with reference-based results showing a strong correlation between chromatin modification and gene expression. We then used our de novo sequence assembly to build the DNA methylation profile for the non-referenced Psammomys obesus genome. The pipeline described uses open source software for fast annotation and visualization of unreferenced genomic regions from short-read data.