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Advances in protein structure determination and modeling allow us to study the structural context of human genetic variants on an unprecedented scale. Here, we analyze millions of cancer-associated missense mutations based on their structural locations and predicted perturbative effects. By considering the collective properties of mutations at the level of individual proteins, we identify distinct patterns associated with tumor suppressors and oncogenes. Tumor suppressors are enriched in structurally damaging mutations, consistent with loss-of-function mechanisms, while oncogene mutations tend to be structurally mild, reflecting selection for gain-of-function driver mutations and against loss-of-function mutations. Although oncogenes are difficult to distinguish from genes with no role in cancer using only structural damage, we find that the three-dimensional clustering of mutations is highly predictive. These observations allow us to identify candidate driver genes and speculate about their molecular roles, which we expect will have general utility in the analysis of cancer sequencing data., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

It is unclear how patterns of regional genetic differentiation in the UK and Ireland might impact the protein-coding fraction of the genome. We exploit UK Biobank (UKB) and Viking Genes whole exome sequencing data to study regional genetic differentiation across the UK and Ireland in protein coding genes, encompassing 44,696 unrelated individuals from 20 regions of origin. We demonstrate substantial exonic differentiation among Shetlanders, Orcadians, individuals with full or partial Ashkenazi Jewish ancestry and in several mainland regions (particularly north and south Wales, southeast Scotland and Ireland). With stringent filtering criteria, we find 67 regionally enriched (≥5-fold) variants likely to have adverse biomedical consequences in homozygous individuals. Here, we show that regional genetic variation across the UK and Ireland should be considered in the design of genetic studies and may inform effective genetic screening and counselling., (© 2024. The Author(s).)

DNA base damage is a major source of oncogenic mutations 1 . Such damage can produce strand-phased mutation patterns and multiallelic variation through the process of lesion segregation 2 . Here we exploited these properties to reveal how strand-asymmetric processes, such as replication and transcription, shape DNA damage and repair. Despite distinct mechanisms of leading and lagging strand replication 3,4 , we observe identical fidelity and damage tolerance for both strands. For small alkylation adducts of DNA, our results support a model in which the same translesion polymerase is recruited on-the-fly to both replication strands, starkly contrasting the strand asymmetric tolerance of bulky UV-induced adducts 5 . The accumulation of multiple distinct mutations at the site of persistent lesions provides the means to quantify the relative efficiency of repair processes genome wide and at single-base resolution. At multiple scales, we show DNA damage-induced mutations are largely shaped by the influence of DNA accessibility on repair efficiency, rather than gradients of DNA damage. Finally, we reveal specific genomic conditions that can actively drive oncogenic mutagenesis by corrupting the fidelity of nucleotide excision repair. These results provide insight into how strand-asymmetric mechanisms underlie the formation, tolerance and repair of DNA damage, thereby shaping cancer genome evolution., (© 2024. The Author(s).)

Diagnosing rare developmental disorders using genome-wide sequencing data commonly necessitates review of multiple plausible candidate variants, often using ontologies of categorical clinical terms. We show that Integrating Multiple Phenotype Resources Optimizes Variant Evaluation in Developmental Disorders (IMPROVE-DD) by incorporating additional classes of data commonly available to clinicians and recorded in health records. In doing so, we quantify the distinct contributions of sex, growth, and development in addition to Human Phenotype Ontology (HPO) terms and demonstrate added value from these readily available information sources. We use likelihood ratios for nominal and quantitative data and propose a classifier for HPO terms in this framework. This Bayesian framework results in more robust diagnoses. Using data systematically collected in the Deciphering Developmental Disorders study, we considered 77 genes with pathogenic/likely pathogenic variants in ≥10 individuals. All genes showed at least a satisfactory prediction by receiver operating characteristic when testing on training data (AUC ≥ 0.6), and HPO terms were the best predictor for the majority of genes, though a minority (13/77) of genes were better predicted by other phenotypic data types. Overall, classifiers based upon multiple integrated phenotypic data sources performed better than those based upon any individual source, and importantly, integrated models produced notably fewer false positives. Finally, we show that IMPROVE-DD models with good predictive performance on cross-validation can be constructed from relatively few individuals. This suggests new strategies for candidate gene prioritization and highlights the value of systematic clinical data collection to support diagnostic programs., Competing Interests: M.E.H. is a co-founder, consultant, and non-executive director of Congenica Ltd., (© 2022 The Authors.)

Purpose: High-grade serous ovarian carcinoma (HGSOC) is the most common ovarian cancer type; most patients experience disease recurrence that accumulates chemoresistance, leading to treatment failure. Genomic and transcriptomic features have been associated with differential outcome and treatment response. However, the relationship between events at the gene sequence, copy number, and gene-expression levels remains poorly defined., Experimental Design: We perform multiomic characterization of a large HGSOC cohort (n = 362) with detailed clinical annotation to interrogate the relationship between patient subgroups defined by specific molecular events., Results: BRCA2-mutant (BRCA2m) and EMSY-overexpressing cases demonstrated prolonged survival [multivariable hazard ratios (HR) 0.40 and 0.51] and significantly higher first- and second-line chemotherapy response rate. CCNE1-gained (CCNE1g) cases demonstrated underrepresentation of FIGO stage IV cases, with shorter survival but no significant difference in treatment response. We demonstrate marked overlap between the TCGA- and Tothill-derived subtypes. IMR/C2 cases displayed higher BRCA1/2m frequency (25.5%, 32.5%) and significantly greater immune cell infiltration, whereas PRO/C5 cases had the highest CCNE1g rate (23.9%, 22.2%) and were uniformly low in immune cell infiltration. The survival benefit for cases with aberrations in homologous recombination repair (HRR) genes was apparent across all transcriptomic subtypes (HR range, 0.48-0.68). There was significant co-occurrence of RB loss and HRR gene aberrations; RB loss was further associated with favorable survival within HRR-aberrant cases (multivariable HR, 0.50)., Conclusions: These data paint a high-resolution picture of the molecular landscape in HGSOC, better defining patients who may benefit most from specific molecular therapeutics and highlighting those for whom novel treatment strategies are needed to improve outcomes., (©2022 The Authors; Published by the American Association for Cancer Research.)

Colorectal cancer (CRC) is a common, multifactorial disease. While observational studies have identified an association between lower vitamin D and higher CRC risk, supplementation trials have been inconclusive and the mechanisms by which vitamin D may modulate CRC risk are not well understood. We sought to perform a weighted gene co-expression network analysis (WGCNA) to identify modules present after vitamin D supplementation (when plasma vitamin D level was sufficient) which were absent before supplementation, and then to identify influential genes in those modules. The transcriptome from normal rectal mucosa biopsies of 49 individuals free from CRC were assessed before and after 12 weeks of 3200IU/day vitamin D (Fultium-D3) supplementation using paired-end total RNAseq. While the effects on expression patterns following vitamin D supplementation were subtle, WGCNA identified highly correlated genes forming gene modules. Four of the 17 modules identified in the post-vitamin D network were not preserved in the pre-vitamin D network, shedding new light on the biochemical impact of supplementation. These modules were enriched for GO terms related to the immune system, hormone metabolism, cell growth and RNA metabolism. Across the four treatment-associated modules, 51 hub genes were identified, with enrichment of 40 different transcription factor motifs in promoter regions of those genes, including VDR:RXR. Six of the hub genes were nominally differentially expressed in studies of vitamin D effects on adult normal mucosa organoids: LCN2, HLA-C, AIF1L, PTPRU, PDE4B and IFI6. By taking a gene-correlation network approach, we have described vitamin D induced changes to gene modules in normal human rectal epithelium in vivo , the target tissue from which CRC develops., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Blackmur, Vaughan-Shaw, Donnelly, Harris, Svinti, Ochocka-Fox, Freile, Walker, Gurran, Reid, Semple, Din, Timofeeva, Dunlop and Farrington.)

Mutation in the germline is the ultimate source of genetic variation, but little is known about the influence of germline chromatin structure on mutational processes. Using ATAC-seq, we profile the open chromatin landscape of human spermatogonia, the most proliferative cell type of the germline, identifying transcription factor binding sites (TFBSs) and PRDM9 binding sites, a subset of which will initiate meiotic recombination. We observe an increase in rare structural variant (SV) breakpoints at PRDM9-bound sites, implicating meiotic recombination in the generation of structural variation. Many germline TFBSs, such as NRF1, are also associated with increased rates of SV breakpoints, apparently independent of recombination. Singleton short insertions (≥5 bp) are highly enriched at TFBSs, particularly at sites bound by testis active TFs, and their rates correlate with those of structural variant breakpoints. Short insertions often duplicate the TFBS motif, leading to clustering of motif sites near regulatory regions in this male-driven evolutionary process. Increased mutation loads at germline TFBSs disproportionately affect neural enhancers with activity in spermatogonia, potentially altering neurodevelopmental regulatory architecture. Local chromatin structure in spermatogonia is thus pervasive in shaping both evolution and disease., (© 2021 Kaiser et al.; Published by Cold Spring Harbor Laboratory Press.)

ISG15 is an ubiquitin-like modifier that is associated with reduced survival rates in breast cancer patients. The mechanism by which ISG15 achieves this however remains elusive. We demonstrate that modification of Rab GDP-Dissociation Inhibitor Beta (GDI2) by ISG15 (ISGylation) alters endocytic recycling of the EGF receptor (EGFR) in non-interferon stimulated cells using CRISPR-knock out models for ISGylation. By regulating EGFR trafficking, ISGylation enhances EGFR recycling and sustains Akt-signalling. We further show that Akt signalling positively correlates with levels of ISG15 and its E2-ligase in basal breast cancer cohorts, confirming the link between ISGylation and Akt signalling in human tumours. Persistent and enhanced Akt activation explains the more aggressive tumour behaviour observed in human breast cancers. We show that ISGylation can act as a driver of tumour progression rather than merely being a bystander., (© 2021. The Author(s).)

Endometrioid ovarian carcinoma (EnOC) is an under-investigated ovarian cancer type. Recent studies have described disease subtypes defined by genomics and hormone receptor expression patterns; here, we determine the relationship between these subtyping layers to define the molecular landscape of EnOC with high granularity and identify therapeutic vulnerabilities in high-risk cases. Whole exome sequencing data were integrated with progesterone and oestrogen receptor (PR and ER) expression-defined subtypes in 90 EnOC cases following robust pathological assessment, revealing dominant clinical and molecular features in the resulting integrated subtypes. We demonstrate significant correlation between subtyping approaches: PR-high (PR + /ER + , PR + /ER-) cases were predominantly CTNNB1-mutant (73.2% vs 18.4%, P < 0.001), while PR-low (PR-/ER + , PR-/ER-) cases displayed higher TP53 mutation frequency (38.8% vs 7.3%, P = 0.001), greater genomic complexity (P = 0.007) and more frequent copy number alterations (P = 0.001). PR-high EnOC patients experience favourable disease-specific survival independent of clinicopathological and genomic features (HR = 0.16, 95% CI 0.04-0.71). TP53 mutation further delineates the outcome of patients with PR-low tumours (HR = 2.56, 95% CI 1.14-5.75). A simple, routinely applicable, classification algorithm utilising immunohistochemistry for PR and p53 recapitulated these subtypes and their survival profiles. The genomic profile of high-risk EnOC subtypes suggests that inhibitors of the MAPK and PI3K-AKT pathways, alongside PARP inhibitors, represent promising candidate agents for improving patient survival. Patients with PR-low TP53-mutant EnOC have the greatest unmet clinical need, while PR-high tumours-which are typically CTNNB1-mutant and TP53 wild-type-experience excellent survival and may represent candidates for trials investigating de-escalation of adjuvant chemotherapy to agents such as endocrine therapy.

Purpose: The abundance and effects of structural variation at BRCA1/2 in tumors are not well understood. In particular, the impact of these events on homologous recombination repair deficiency (HRD) has yet to be demonstrated., Experimental Design: Exploiting a large collection of whole-genome sequencing data from high-grade serous ovarian carcinoma ( N = 205) together with matched RNA sequencing for the majority of tumors ( N = 150), we have comprehensively characterized mutation and expression at BRCA1/2 ., Results: In addition to the known spectrum of short somatic mutations (SSM), we discovered that multi-megabase structural variants (SV) were a frequent, unappreciated source of BRCA1/2 disruption in these tumors, and we found a genome-wide enrichment for large deletions at the BRCA1/2 loci across the cohort. These SVs independently affected a substantial proportion of patients (16%) in addition to those affected by SSMs (24%), conferring HRD and impacting patient survival. We also detail compound deficiencies involving SSMs and SVs at both loci, demonstrating that the strongest risk of HRD emerges from combined SVs at both BRCA1 and BRCA2 in the absence of SSMs. Furthermore, these SVs are abundant and disruptive in other cancer types., Conclusions: These results extend our understanding of the mutational landscape underlying HRD, increase the number of patients predicted to benefit from therapies exploiting HRD, and suggest there is currently untapped potential in SV detection for patient stratification., (©2021 American Association for Cancer Research.)

A balanced t(1;11) translocation that directly disrupts DISC1 is linked to schizophrenia and affective disorders. We previously showed that a mutant mouse, named Der1, recapitulates the effect of the translocation upon DISC1 expression. Here, RNAseq analysis of Der1 mouse brain tissue found enrichment for dysregulation of the same genes and molecular pathways as in neuron cultures generated previously from human t(1;11) translocation carriers via the induced pluripotent stem cell route. DISC1 disruption therefore apparently accounts for a substantial proportion of the effects of the t(1;11) translocation. RNAseq and pathway analysis of the mutant mouse predicts multiple Der1-induced alterations converging upon synapse function and plasticity. Synaptosome proteomics confirmed that the Der1 mutation impacts synapse composition, and electrophysiology found reduced AMPA:NMDA ratio in hippocampal neurons, indicating changed excitatory signalling. Moreover, hippocampal parvalbumin-positive interneuron density is increased, suggesting that the Der1 mutation affects inhibitory control of neuronal circuits. These phenotypes predict that neurotransmission is impacted at many levels by DISC1 disruption in human t(1;11) translocation carriers. Notably, genes implicated in schizophrenia, depression and bipolar disorder by large-scale genetic studies are enriched among the Der1-dysregulated genes, just as we previously observed for the t(1;11) translocation carrier-derived neurons. Furthermore, RNAseq analysis predicts that the Der1 mutation primarily targets a subset of cell types, pyramidal neurons and interneurons, previously shown to be vulnerable to the effects of common schizophrenia-associated genetic variants. In conclusion, DISC1 disruption by the t(1;11) translocation may contribute to the psychiatric disorders of translocation carriers through commonly affected pathways and processes in neurotransmission.

Endometrioid ovarian carcinoma (EnOC) demonstrates substantial clinical and molecular heterogeneity. Here, we report whole exome sequencing of 112 EnOC cases following rigorous pathological assessment. We detect a high frequency of mutation in CTNNB1 (43%), PIK3CA (43%), ARID1A (36%), PTEN (29%), KRAS (26%), TP53 (26%) and SOX8 (19%), a recurrently-mutated gene previously unreported in EnOC. POLE and mismatch repair protein-encoding genes were mutated at lower frequency (6%, 18%) with significant co-occurrence. A molecular taxonomy is constructed, identifying clinically distinct EnOC subtypes: cases with TP53 mutation demonstrate greater genomic complexity, are commonly FIGO stage III/IV at diagnosis (48%), are frequently incompletely debulked (44%) and demonstrate inferior survival; conversely, cases with CTNNB1 mutation, which is mutually exclusive with TP53 mutation, demonstrate low genomic complexity and excellent clinical outcome, and are predominantly stage I/II at diagnosis (89%) and completely resected (87%). Moreover, we identify the WNT, MAPK/RAS and PI3K pathways as good candidate targets for molecular therapeutics in EnOC.

Cancers arise through the acquisition of oncogenic mutations and grow by clonal expansion 1,2 . Here we reveal that most mutagenic DNA lesions are not resolved into a mutated DNA base pair within a single cell cycle. Instead, DNA lesions segregate, unrepaired, into daughter cells for multiple cell generations, resulting in the chromosome-scale phasing of subsequent mutations. We characterize this process in mutagen-induced mouse liver tumours and show that DNA replication across persisting lesions can produce multiple alternative alleles in successive cell divisions, thereby generating both multiallelic and combinatorial genetic diversity. The phasing of lesions enables accurate measurement of strand-biased repair processes, quantification of oncogenic selection and fine mapping of sister-chromatid-exchange events. Finally, we demonstrate that lesion segregation is a unifying property of exogenous mutagens, including UV light and chemotherapy agents in human cells and tumours, which has profound implications for the evolution and adaptation of cancer genomes.

Human population isolates provide a snapshot of the impact of historical demographic processes on population genetics. Such data facilitate studies of the functional impact of rare sequence variants on biomedical phenotypes, as strong genetic drift can result in higher frequencies of variants that are otherwise rare. We present the first whole genome sequencing (WGS) study of the VIKING cohort, a representative collection of samples from the isolated Shetland population in northern Scotland, and explore how its genetic characteristics compare to a mainland Scottish population. Our analyses reveal the strong contributions played by the founder effect and genetic drift in shaping genomic variation in the VIKING cohort. About one tenth of all high-quality variants discovered are unique to the VIKING cohort or are seen at frequencies at least ten fold higher than in more cosmopolitan control populations. Multiple lines of evidence also suggest relaxation of purifying selection during the evolutionary history of the Shetland isolate. We demonstrate enrichment of ultra-rare VIKING variants in exonic regions and for the first time we also show that ultra-rare variants are enriched within regulatory regions, particularly promoters, suggesting that gene expression patterns may diverge relatively rapidly in human isolates., Competing Interests: The authors have declared that no competing interests exist.

The melanocyte-inducing transcription factor (MITF)-low melanoma transcriptional signature is predictive of poor outcomes for patients, but little is known about its biological significance, and animal models are lacking. Here, we used zebrafish genetic models with low activity of Mitfa (MITF-low) and established that the MITF-low state is causal of melanoma progression and a predictor of melanoma biological subtype. MITF-low zebrafish melanomas resembled human MITF-low melanomas and were enriched for stem and invasive (mesenchymal) gene signatures. MITF-low activity coupled with a p53 mutation was sufficient to promote superficial growth melanomas, whereas BRAF V600E accelerated MITF-low melanoma onset and further promoted the development of MITF-high nodular growth melanomas. Genetic inhibition of MITF activity led to rapid regression; recurrence occurred following reactivation of MITF. At the regression site, there was minimal residual disease that was resistant to loss of MITF activity (termed MITF-independent cells) with very low-to-no MITF activity or protein. Transcriptomic analysis of MITF-independent residual disease showed enrichment of mesenchymal and neural crest stem cell signatures similar to human therapy-resistant melanomas. Single-cell RNA sequencing revealed MITF-independent residual disease was heterogeneous depending on melanoma subtype. Further, there was a shared subpopulation of residual disease cells that was enriched for a neural crest G 0 -like state that preexisted in the primary tumor and remained present in recurring melanomas. These findings suggest that invasive and stem-like programs coupled with cellular heterogeneity contribute to poor outcomes for MITF-low melanoma patients and that MITF-independent subpopulations are an important therapeutic target to achieve long-term survival outcomes. SIGNIFICANCE: This study provides a useful model for MITF-low melanomas and MITF-independent cell populations that can be used to study the mechanisms that drive these tumors as well as identify potential therapeutic options. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/22/5769/F1.large.jpg., (©2019 American Association for Cancer Research.)

Background: Disease relapse is the primary cause of death from ovarian carcinoma. Isolated lymph node relapse is a rare pattern of ovarian carcinoma recurrence, with a reported median postrelapse survival of 2.5 to 4 years. To date, investigations have not compared isolated lymph node relapse ovarian carcinoma directly to a matched extranodal relapse cohort or performed molecular characterization of cases that subsequently experience isolated lymph node relapse., Objective: Here we seek to compare the clinical outcome, tumor-infiltrating lymphocyte burden, and frequency of known prognostic genomic events in isolated lymph node relapse ovarian carcinoma vs extranodal relapse ovarian carcinoma., Study Design: Forty-nine isolated lymph node relapse ovarian carcinoma patients were identified and matched to 49 extranodal relapse cases using the Edinburgh Ovarian Cancer Database, from which the clinical data for identified patients were retrieved. Matching criteria were disease stage, histologic subtype and grade, extent of residual disease following surgical debulking, and age at diagnosis. Clinicopathologic factors and survival data were compared between the isolated lymph node relapse and extranodal relapse cohorts. Genomic characterization of tumor material from diagnosis was performed using panel-based high-throughput sequencing and tumor-infiltrating T cell burden was assessed using immunohistochemistry for CD3+ and CD8+ cells., Results: Isolated lymph node relapse cases demonstrated significantly prolonged postrelapse survival and overall survival vs extranodal relapse upon multivariable analysis (HR multi  = 0.52 [0.33-0.84] and 0.51 [0.31-0.84]). Diagnostic specimens from high-grade serous ovarian carcinomas that subsequently displayed isolated lymph node relapse harbored significantly greater CD3+ and CD8+ cell infiltration compared to extranodal relapse cases (P = .001 and P = .009, Bonferroni-adjusted P = .003 and P = .019). Isolated lymph node relapse high-grade serous ovarian carcinoma cases did not show marked enrichment or depletion of cases with BRCA1/2 mutation or CCNE1 copy number gain when compared to their extranodal relapse counterparts (24.4% vs 19.4% and 18.2% vs 22.6%, P = .865 and P = .900)., Conclusion: Isolated lymph node relapse ovarian carcinoma represents a distinct clinical entity with favorable outcome compared to extranodal relapse. There was no clear enrichment or depletion of BRCA1/2 mutation or CCNE1 gain in the isolated lymph node relapse ovarian carcinoma cohort compared with extranodal relapse cases, suggesting that these known prognostic genomically defined subtypes of disease do not display markedly altered propensity for isolated lymph node relapse. Diagnostic tumor material from isolated lymph node relapse patients demonstrated greater CD3+ and CD8+ cell infiltration, indicating stronger tumor engagement by T cell populations, which may contribute to the more indolent disease course of isolated lymph node relapse., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The Viking Health Study Shetland is a population-based research cohort of 2,122 volunteer participants with ancestry from the Shetland Isles in northern Scotland. The high kinship and detailed phenotype data support a range of approaches for associating rare genetic variants, enriched in this isolate population, with quantitative traits and diseases. As an exemplar, the c.1750G > A; p.Gly584Ser variant within the coding sequence of the KCNH2 gene implicated in Long QT Syndrome (LQTS), which occurred once in 500 whole genome sequences from this population, was investigated. Targeted sequencing of the KCNH2 gene in family members of the initial participant confirmed the presence of the sequence variant and identified two further members of the same family pedigree who shared the variant. Investigation of these three related participants for whom single nucleotide polymorphism (SNP) array genotypes were available allowed a unique shared haplotype of 1.22 Mb to be defined around this locus. Searching across the full cohort for this haplotype uncovered two additional apparently unrelated individuals with no known genealogical connection to the original kindred. All five participants with the defined haplotype were shown to share the rare variant by targeted Sanger sequencing. If this result were verified in a healthcare setting, it would be considered clinically actionable, and has been actioned in relatives ascertained independently through clinical presentation. The General Practitioners of four study participants with the rare variant were alerted to the research findings by letters outlining the phenotype (prolonged electrocardiographic QTc interval). A lack of detectable haplotype sharing between c.1750G > A; p.Gly584Ser chromosomes from previously reported individuals from Finland and those in this study from Shetland suggests that this mutation has arisen more than once in human history. This study showcases the potential value of isolate population-based research resources for genomic medicine. It also illustrates some challenges around communication of actionable findings in research participants in this context.

Background: Structural variants (SVs) are known to play important roles in a variety of cancers, but their origins and functional consequences are still poorly understood. Many SVs are thought to emerge from errors in the repair processes following DNA double strand breaks (DSBs)., Results: We used experimentally quantified DSB frequencies in cell lines with matched chromatin and sequence features to derive the first quantitative genome-wide models of DSB susceptibility. These models are accurate and provide novel insights into the mutational mechanisms generating DSBs. Models trained in one cell type can be successfully applied to others, but a substantial proportion of DSBs appear to reflect cell type-specific processes. Using model predictions as a proxy for susceptibility to DSBs in tumors, many SV-enriched regions appear to be poorly explained by selectively neutral mutational bias alone. A substantial number of these regions show unexpectedly high SV breakpoint frequencies given their predicted susceptibility to mutation and are therefore credible targets of positive selection in tumors. These putatively positively selected SV hotspots are enriched for genes previously shown to be oncogenic. In contrast, several hundred regions across the genome show unexpectedly low levels of SVs, given their relatively high susceptibility to mutation. These novel coldspot regions appear to be subject to purifying selection in tumors and are enriched for active promoters and enhancers., Conclusions: We conclude that models of DSB susceptibility offer a rigorous approach to the inference of SVs putatively subject to selection in tumors.

The neuromodulatory gene DISC1 is disrupted by a t(1;11) translocation that is highly penetrant for schizophrenia and affective disorders, but how this translocation affects DISC1 function is incompletely understood. N-methyl-D-aspartate receptors (NMDAR) play a central role in synaptic plasticity and cognition, and are implicated in the pathophysiology of schizophrenia through genetic and functional studies. We show that the NMDAR subunit GluN2B complexes with DISC1-associated trafficking factor TRAK1, while DISC1 interacts with the GluN1 subunit and regulates dendritic NMDAR motility in cultured mouse neurons. Moreover, in the first mutant mouse that models DISC1 disruption by the translocation, the pool of NMDAR transport vesicles and surface/synaptic NMDAR expression are increased. Since NMDAR cell surface/synaptic expression is tightly regulated to ensure correct function, these changes in the mutant mouse are likely to affect NMDAR signalling and synaptic plasticity. Consistent with these observations, RNASeq analysis of the translocation carrier-derived human neurons indicates abnormalities of excitatory synapses and vesicle dynamics. RNASeq analysis of the human neurons also identifies many differentially expressed genes previously highlighted as putative schizophrenia and/or depression risk factors through large-scale genome-wide association and copy number variant studies, indicating that the translocation triggers common disease pathways that are shared with unrelated psychiatric patients. Altogether, our findings suggest that translocation-induced disease mechanisms are likely to be relevant to mental illness in general, and that such disease mechanisms include altered NMDAR dynamics and excitatory synapse function. This could contribute to the cognitive disorders displayed by translocation carriers.

The promoters of immediate early genes (IEGs) are rapidly activated in response to an external stimulus. These genes, also known as primary response genes, have been identified in a range of cell types, under diverse extracellular signals and using varying experimental protocols. Whereas genomic dissection on a case-by-case basis has not resulted in a comprehensive catalogue of IEGs, a rigorous meta-analysis of eight genome-wide FANTOM5 CAGE (cap analysis of gene expression) time course datasets reveals successive waves of promoter activation in IEGs, recapitulating known relationships between cell types and stimuli: we obtain a set of 57 (42 protein-coding) candidate IEGs possessing promoters that consistently drive a rapid but transient increase in expression over time. These genes show significant enrichment for known IEGs reported previously, pathways associated with the immediate early response, and include a number of non-coding RNAs with roles in proliferation and differentiation. Surprisingly, we also find strong conservation of the ordering of activation for these genes, such that 77 pairwise promoter activation orderings are conserved. Using the leverage of comprehensive CAGE time series data across cell types, we also document the extensive alternative promoter usage by such genes, which is likely to have been a barrier to their discovery until now. The common activation ordering of the core set of early-responding genes we identify may indicate conserved underlying regulatory mechanisms. By contrast, the considerably larger number of transiently activated genes that are specific to each cell type and stimulus illustrates the breadth of the primary response., (© 2018 The Authors.)

Background: Chromatin loops form a basic unit of interphase nuclear organization, with chromatin loop anchor points providing contacts between regulatory regions and promoters. However, the mutational landscape at these anchor points remains under-studied. Here, we describe the unusual patterns of somatic mutations and germline variation associated with loop anchor points and explore the underlying features influencing these patterns., Results: Analyses of whole genome sequencing datasets reveal that anchor points are strongly depleted for single nucleotide variants (SNVs) in tumours. Despite low SNV rates in their genomic neighbourhood, anchor points emerge as sites of evolutionary innovation, showing enrichment for structural variant (SV) breakpoints and a peak of SNVs at focal CTCF sites within the anchor points. Both CTCF-bound and non-CTCF anchor points harbour an excess of SV breakpoints in multiple tumour types and are prone to double-strand breaks in cell lines. Common fragile sites, which are hotspots for genome instability, also show elevated numbers of intersecting loop anchor points. Recurrently disrupted anchor points are enriched for genes with functions in cell cycle transitions and regions associated with predisposition to cancer. We also discover a novel class of CTCF-bound anchor points which overlap meiotic recombination hotspots and are enriched for the core PRDM9 binding motif, suggesting that the anchor points have been foci for diversity generated during recent human evolution., Conclusions: We suggest that the unusual chromatin environment at loop anchor points underlies the elevated rates of variation observed, marking them as sites of regulatory importance but also genomic fragility.

Background: Approximately 10-15% of ovarian carcinomas (OC) are attributed to inherited susceptibility, the majority of which are due to mutations in BRCA1 or BRCA2 (BRCA1/2). These patients display superior clinical outcome, including enhanced sensitivity to platinum-based chemotherapy. Here, we seek to investigate whether BRCA1/2 status influences the response rate to single-agent pegylated liposomal doxorubicin (PLD) in high grade serous (HGS) OC., Methods: One hundred and forty-eight patients treated with single-agent PLD were identified retrospectively from the Edinburgh Ovarian Cancer Database. DNA was extracted from formalin-fixed paraffin-embedded (FFPE) archival tumour material and sequenced using the Ion Ampliseq BRCA1 and BRCA2 panel. A minimum variant allele frequency threshold was applied to correct for sequencing artefacts associated with formalin fixation., Results: A superior response rate to PLD was observed in patients with HGS OC who harboured variants likely to affect BRCA1 or BRCA2 function compared to the BRCA1/2 wild-type population (36%, 9 of 25 patients versus 12.1%, 7 of 58 patients; p = 0.016). An enhanced response rate was also seen in patients harbouring only the BRCA1 SNP rs1799950, predicted to be detrimental to BRCA1 function (50%, 3 of 6 patients versus 12.1%, 7 of 58 patients; p = 0.044)., Conclusions: These data demonstrate that HGS OC patients with BRCA1/2 variants predicted damaging to protein function experience superior sensitivity to PLD, consistent with impaired DNA repair. Further characterisation of rs1799950 is now warranted in relation to chemosensitivity and susceptibility to developing ovarian carcinoma.

The circadian regulation of gene expression allows plants and animals to anticipate predictable environmental changes. While the influence of the circadian clock has recently been shown to extend to ribosome biogenesis, the dynamics and regulation of the many small nucleolar RNA that are required in pre-ribosomal RNA folding and modification are unknown. Using a novel computational method, we show that 18S and 28S pre-rRNA are subject to circadian regulation in a nuclear RNA sequencing time course. A population of snoRNA with circadian expression is identified that is functionally associated with rRNA modification. More generally, we find the abundance of snoRNA known to modify 18S and 28S to be inversely correlated with the abundance of their target. Cyclic patterns in the expression of a number of snoRNA indicate a coordination with rRNA maturation, potentially through an upregulation in their biogenesis, or their release from mature rRNA at the end of the previous cycle of rRNA maturation, in antiphase with the diurnal peak in pre-rRNA. Few cyclic snoRNA have cyclic host genes, indicating the action of regulatory mechanisms in addition to transcriptional activation of the host gene. For highly expressed independently transcribed snoRNA, we find a characteristic RNA polymerase II and H3K4me3 signature that correlates with mean snoRNA expression over the day., (© 2017 The Author(s).)

Chromatin in the interphase nucleus is organised as a hierarchical series of structural domains, including self-interacting domains called topologically associating domains (TADs). This arrangement is thought to bring enhancers into closer physical proximity with their target genes, which often are located hundreds of kilobases away in linear genomic distance. TADs are demarcated by boundary regions bound by architectural proteins, such as CTCF and cohesin, although much remains to be discovered about the structure and function of these domains. Recent studies of TAD boundaries disrupted in engineered mouse models show that boundary mutations can recapitulate human developmental disorders as a result of aberrant promoter-enhancer interactions in the affected TADs. Similar boundary disruptions in certain cancers can result in oncogene overexpression, and CTCF binding sites at boundaries appear to be hyper-mutated across cancers. Further insights into chromatin organisation, in parallel with accumulating whole genome sequence data for disease cohorts, are likely to yield additional valuable insights into the roles of noncoding sequence variation in human disease., Competing Interests: Competing interests: The authors declare that they have no competing interests.No competing interests were disclosed.No competing interests were disclosed.

Disruption of gene regulation is known to play major roles in carcinogenesis and tumour progression. Here, we comprehensively characterize the mutational profiles of diverse transcription factor binding sites (TFBSs) across 1,574 completely sequenced cancer genomes encompassing 11 tumour types. We assess the relative rates and impact of the mutational burden at the binding sites of 81 transcription factors (TFs), by comparing the abundance and patterns of single base substitutions within putatively functional binding sites to control sites with matched sequence composition. There is a strong (1.43-fold) and significant excess of mutations at functional binding sites across TFs, and the mutations that accumulate in cancers are typically more disruptive than variants tolerated in extant human populations at the same sites. CTCF binding sites suffer an exceptionally high mutational load in cancer (3.31-fold excess) relative to control sites, and we demonstrate for the first time that this effect is seen in essentially all cancer types with sufficient data. The sub-set of CTCF sites involved in higher order chromatin structures has the highest mutational burden, suggesting a widespread breakdown of chromatin organization. However, we find no evidence for selection driving these distinctive patterns of mutation. The mutational load at CTCF-binding sites is substantially determined by replication timing and the mutational signature of the tumor in question, suggesting that selectively neutral processes underlie the unusual mutation patterns. Pervasive hyper-mutation within transcription factor binding sites rewires the regulatory landscape of the cancer genome, but it is dominated by mutational processes rather than selection.

Mammalian chromosomes fold into arrays of megabase-sized topologically associating domains (TADs), which are arranged into compartments spanning multiple megabases of genomic DNA. TADs have internal substructures that are often cell type specific, but their higher-order organization remains elusive. Here, we investigate TAD higher-order interactions with Hi-C through neuronal differentiation and show that they form a hierarchy of domains-within-domains (metaTADs) extending across genomic scales up to the range of entire chromosomes. We find that TAD interactions are well captured by tree-like, hierarchical structures irrespective of cell type. metaTAD tree structures correlate with genetic, epigenomic and expression features, and structural tree rearrangements during differentiation are linked to transcriptional state changes. Using polymer modelling, we demonstrate that hierarchical folding promotes efficient chromatin packaging without the loss of contact specificity, highlighting a role far beyond the simple need for packing efficiency., (© 2015 The Authors. Published under the terms of the CC BY 4.0 license.)

Homozygous loss of function (HLOF) variants provide a valuable window on gene function in humans, as well as an inventory of the human genes that are not essential for survival and reproduction. All humans carry at least a few HLOF variants, but the exact number of inactivated genes that can be tolerated is currently unknown—as are the phenotypic effects of losing function for most human genes. Here, we make use of 1432 whole exome sequences from five European populations to expand the catalogue of known human HLOF mutations; after stringent filtering of variants in our dataset, we identify a total of 173 HLOF mutations, 76 (44%) of which have not been observed previously. We find that population isolates are particularly well suited to surveys of novel HLOF genes because individuals in such populations carry extensive runs of homozygosity, which we show are enriched for novel, rare HLOF variants. Further, we make use of extensive phenotypic data to show that most HLOFs, ascertained in population-based samples, appear to have little detectable effect on the phenotype. On the contrary, we document several genes directly implicated in disease that seem to tolerate HLOF variants. Overall HLOF genes are enriched for olfactory receptor function and are expressed in testes more often than expected, consistent with reduced purifying selection and incipient pseudogenisation., (© The Author 2015. Published by Oxford University Press.)

Background: Interphase chromosomes adopt a hierarchical structure, and recent data have characterized their chromatin organization at very different scales, from sub-genic regions associated with DNA-binding proteins at the order of tens or hundreds of bases, through larger regions with active or repressed chromatin states, up to multi-megabase-scale domains associated with nuclear positioning, replication timing and other qualities. However, we have lacked detailed, quantitative models to understand the interactions between these different strata., Results: Here we collate large collections of matched locus-level chromatin features and Hi-C interaction data, representing higher-order organization, across three human cell types. We use quantitative modeling approaches to assess whether locus-level features are sufficient to explain higher-order structure, and identify the most influential underlying features. We identify structurally variable domains between cell types and examine the underlying features to discover a general association with cell-type-specific enhancer activity. We also identify the most prominent features marking the boundaries of two types of higher-order domains at different scales: topologically associating domains and nuclear compartments. We find parallel enrichments of particular chromatin features for both types, including features associated with active promoters and the architectural proteins CTCF and YY1., Conclusions: We show that integrative modeling of large chromatin dataset collections using random forests can generate useful insights into chromosome structure. The models produced recapitulate known biological features of the cell types involved, allow exploration of the antecedents of higher-order structures and generate testable hypotheses for further experimental studies.

The immediate-early response mediates cell fate in response to a variety of extracellular stimuli and is dysregulated in many cancers. However, the specificity of the response across stimuli and cell types, and the roles of non-coding RNAs are not well understood. Using a large collection of densely-sampled time series expression data we have examined the induction of the immediate-early response in unparalleled detail, across cell types and stimuli. We exploit cap analysis of gene expression (CAGE) time series datasets to directly measure promoter activities over time. Using a novel analysis method for time series data we identify transcripts with expression patterns that closely resemble the dynamics of known immediate-early genes (IEGs) and this enables a comprehensive comparative study of these genes and their chromatin state. Surprisingly, these data suggest that the earliest transcriptional responses often involve promoters generating non-coding RNAs, many of which are produced in advance of canonical protein-coding IEGs. IEGs are known to be capable of induction without de novo protein synthesis. Consistent with this, we find that the response of both protein-coding and non-coding RNA IEGs can be explained by their transcriptionally poised, permissive chromatin state prior to stimulation. We also explore the function of non-coding RNAs in the attenuation of the immediate early response in a small RNA sequencing dataset matched to the CAGE data: We identify a novel set of microRNAs responsible for the attenuation of the IEG response in an estrogen receptor positive cancer cell line. Our computational statistical method is well suited to meta-analyses as there is no requirement for transcripts to pass thresholds for significant differential expression between time points, and it is agnostic to the number of time points per dataset.

Human serum uric acid concentration (SUA) is a complex trait. A recent meta-analysis of multiple genome-wide association studies (GWAS) identified 28 loci associated with SUA jointly explaining only 7.7% of the SUA variance, with 3.4% explained by two major loci (SLC2A9 and ABCG2). Here we examined whether gene-gene interactions had any roles in regulating SUA using two large GWAS cohorts included in the meta-analysis [the Atherosclerosis Risk in Communities study cohort (ARIC) and the Framingham Heart Study cohort (FHS)]. We found abundant genome-wide significant local interactions in ARIC in the 4p16.1 region located mostly in an intergenic area near SLC2A9 that were not driven by linkage disequilibrium and were replicated in FHS. Taking the forward selection approach, we constructed a model of five SNPs with marginal effects and three epistatic SNP pairs in ARIC-three marginal SNPs were located within SLC2A9 and the remaining SNPs were all located in the nearby intergenic area. The full model explained 1.5% more SUA variance than that explained by the lead SNP alone, but only 0.3% was contributed by the marginal and epistatic effects of the SNPs in the intergenic area. Functional analysis revealed strong evidence that the epistatically interacting SNPs in the intergenic area were unusually enriched at enhancers active in ENCODE hepatic (HepG2, P = 4.7E-05) and precursor red blood (K562, P = 5.0E-06) cells, putatively regulating transcription of WDR1 and SLC2A9. These results suggest that exploring epistatic interactions is valuable in uncovering the complex functional mechanisms underlying the 4p16.1 region., (© The Author 2014. Published by Oxford University Press.)

DNA methylation and chromatin states play key roles in development and disease. However, the extent of recent evolutionary divergence in the human epigenome and the influential factors that have shaped it are poorly understood. To determine the links between genome sequence and human epigenome evolution, we examined the divergence of DNA methylation and chromatin states following segmental duplication events in the human lineage. Chromatin and DNA methylation states were found to have been generally well conserved following a duplication event, with the evolution of the epigenome largely uncoupled from the total number of genetic changes in the surrounding DNA sequence. However, the epigenome at tissue-specific, distal regulatory regions was observed to be unusually prone to diverge following duplication, with particular sequence differences, altering known sequence motifs, found to be associated with divergence in patterns of DNA methylation and chromatin. Alu elements were found to have played a particularly prominent role in shaping human epigenome evolution, and we show that human-specific AluY insertion events are strongly linked to the evolution of the DNA methylation landscape and gene expression levels, including at key neurological genes in the human brain. Studying paralogous regions within the same sample enables the study of the links between genome and epigenome evolution while controlling for biological and technical variation. We show DNA methylation and chromatin divergence between duplicated regions are linked to the divergence of particular genetic motifs, with Alu elements having played a disproportionate role in the evolution of the epigenome in the human lineage., (© The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: DNA methylation contributes to genomic integrity by suppressing repeat-associated transposition. In addition to the canonical DNA methyltransferases, several auxiliary chromatin factors are required to maintain DNA methylation at intergenic and satellite repeats. The interaction between Lsh, a chromatin helicase, and the de novo methyltransferase Dnmt3b facilitates deposition of DNA methylation at stem cell genes, which are hypomethylated in Lsh-/- embryos. We wished to determine if a similar targeting mechanism operates to maintain DNA methylation at repetitive sequences., Results: We mapped genome-wide DNA methylation patterns in Lsh-/- and Dnmt3b-/- somatic cells. DNA methylation is predominantly lost from specific genomic repeats in Lsh-/- cells: LTR -retrotransposons, LINE-1 repeats and mouse satellites. RNA-seq experiments demonstrate that specific IAP LTRs and satellites, but not LINE-1 elements, are aberrantly transcribed in Lsh-/- cells. LTR hypomethylation in Dnmt3b-/- cells is moderate, whereas IAP, LINE-1 and satellite elements are hypomethylated but silent. Repressed LINE-1 elements in Lsh-/- cells gain H3K4me3, but H3K9me3 levels are unaltered, indicating that DNA hypomethylation alone is not permissive for their transcriptional activation. Mis-expressed IAPs and satellites lose H3K9me3 and gain H3K4me3 in Lsh-/- cells., Conclusions: Our study emphasizes that regulation of repetitive elements by Lsh and DNA methylation is selective and context dependent. Silencing of repeats in somatic cells appears not to be critically dependent on Dnmt3b function. We propose a model where Lsh is specifically required at a precise developmental window to target de novo methylation to repeat sequences, which is subsequently maintained by Dnmt1 to enforce selective repeat silencing.

Local interactions between neighbouring SNPs are hypothesized to be able to capture variants missing from genome-wide association studies (GWAS) via haplotype effects but have not been thoroughly explored. We have used a new high-throughput analysis tool to probe this underexplored area through full pair-wise genome scans and conventional GWAS in diastolic and systolic blood pressure and six metabolic traits in the Northern Finland Birth Cohort 1966 (NFBC1966) and the Atherosclerosis Risk in Communities study cohort (ARIC). Genome-wide significant interactions were detected in ARIC for systolic blood pressure between PLEKHA7 (a known GWAS locus for blood pressure) and GPR180 (which plays a role in vascular remodelling), and also for triglycerides as local interactions within the 11q23.3 region (replicated significantly in NFBC1966), which notably harbours several loci (BUD13, ZNF259 and APOA5) contributing to triglyceride levels. Tests of the local interactions within the 11q23.3 region conditional on the top GWAS signal suggested the presence of two independent functional variants, each with supportive evidence for their roles in gene regulation. Local interactions captured 9 additional GWAS loci identified in this study (3 significantly replicated) and 73 from previous GWAS (24 in the eight traits and 49 in related traits). We conclude that the detection of local interactions requires adequate SNP coverage of the genome and that such interactions are only likely to be detectable between SNPs in low linkage disequilibrium. Analysing local interactions is a potentially valuable complement to GWAS and can provide new insights into the biology underlying variation in complex traits.

Competing Interests: The authors have declared that no competing interests exist.

Several recent studies have examined different aspects of mammalian higher order chromatin structure - replication timing, lamina association and Hi-C inter-locus interactions - and have suggested that most of these features of genome organisation are conserved over evolution. However, the extent of evolutionary divergence in higher order structure has not been rigorously measured across the mammalian genome, and until now little has been known about the characteristics of any divergent loci present. Here, we generate a dataset combining multiple measurements of chromatin structure and organisation over many embryonic cell types for both human and mouse that, for the first time, allows a comprehensive assessment of the extent of structural divergence between mammalian genomes. Comparison of orthologous regions confirms that all measurable facets of higher order structure are conserved between human and mouse, across the vast majority of the detectably orthologous genome. This broad similarity is observed in spite of many loci possessing cell type specific structures. However, we also identify hundreds of regions (from 100 Kb to 2.7 Mb in size) showing consistent evidence of divergence between these species, constituting at least 10% of the orthologous mammalian genome and encompassing many hundreds of human and mouse genes. These regions show unusual shifts in human GC content, are unevenly distributed across both genomes, and are enriched in human subtelomeric regions. Divergent regions are also relatively enriched for genes showing divergent expression patterns between human and mouse ES cells, implying these regions cause divergent regulation. Particular divergent loci are strikingly enriched in genes implicated in vertebrate development, suggesting important roles for structural divergence in the evolution of mammalian developmental programmes. These data suggest that, though relatively rare in the mammalian genome, divergence in higher order chromatin structure has played important roles during evolution.

Background: Genome-wide association studies have identified thousands of SNP variants associated with hundreds of phenotypes. For most associations the causal variants and the molecular mechanisms underlying pathogenesis remain unknown. Exploration of the underlying functional annotations of trait-associated loci has thrown some light on their potential roles in pathogenesis. However, there are some shortcomings of the methods used to date, which may undermine efforts to prioritize variants for further analyses. Here, we introduce and apply novel methods to rigorously identify annotation classes showing enrichment or depletion of trait-associated variants taking into account the underlying associations due to co-location of different functional annotations and linkage disequilibrium., Results: We assessed enrichment and depletion of variants in publicly available annotation classes such as genic regions, regulatory features, measures of conservation, and patterns of histone modifications. We used logistic regression to build a multivariate model that identified the most influential functional annotations for trait-association status of genome-wide significant variants. SNPs associated with all of the enriched annotations were 8 times more likely to be trait-associated variants than SNPs annotated with none of them. Annotations associated with chromatin state together with prior knowledge of the existence of a local expression QTL (eQTL) were the most important factors in the final logistic regression model. Surprisingly, despite the widespread use of evolutionary conservation to prioritize variants for study we find only modest enrichment of trait-associated SNPs in conserved regions., Conclusion: We established odds ratios of functional annotations that are more likely to contain significantly trait-associated SNPs, for the purpose of prioritizing GWAS hits for further studies. Additionally, we estimated the relative and combined influence of the different genomic annotations, which may facilitate future prioritization methods by adding substantial information.

Motivation: Gene-gene interactions (epistasis) are thought to be important in shaping complex traits, but they have been under-explored in genome-wide association studies (GWAS) due to the computational challenge of enumerating billions of single nucleotide polymorphism (SNP) combinations. Fast screening tools are needed to make epistasis analysis routinely available in GWAS., Results: We present BiForce to support high-throughput analysis of epistasis in GWAS for either quantitative or binary disease (case-control) traits. BiForce achieves great computational efficiency by using memory efficient data structures, Boolean bitwise operations and multithreaded parallelization. It performs a full pair-wise genome scan to detect interactions involving SNPs with or without significant marginal effects using appropriate Bonferroni-corrected significance thresholds. We show that BiForce is more powerful and significantly faster than published tools for both binary and quantitative traits in a series of performance tests on simulated and real datasets. We demonstrate BiForce in analysing eight metabolic traits in a GWAS cohort (323 697 SNPs, >4500 individuals) and two disease traits in another (>340 000 SNPs, >1750 cases and 1500 controls) on a 32-node computing cluster. BiForce completed analyses of the eight metabolic traits within 1 day, identified nine epistatic pairs of SNPs in five metabolic traits and 18 SNP pairs in two disease traits. BiForce can make the analysis of epistasis a routine exercise in GWAS and thus improve our understanding of the role of epistasis in the genetic regulation of complex traits., Availability and Implementation: The software is free and can be downloaded from http://bioinfo.utu.fi/BiForce/., Contact: wenhua.wei@igmm.ed.ac.uk, Supplementary Information: Supplementary data are available at Bioinformatics online.

Genome-wide association studies (GWAS) have discovered many loci associated with common disease and quantitative traits. However, most GWAS have not studied the gene-gene interactions (epistasis) that could be important in complex trait genetics. A major challenge in analysing epistasis in GWAS is the enormous computational demands of analysing billions of SNP combinations. Several methods have been developed recently to address this, some using computers equipped with particular graphical processing units, most restricted to binary disease traits and all poorly suited to general usage on the most widely used operating systems. We have developed the BiForce Toolbox to address the demand for high-throughput analysis of pairwise epistasis in GWAS of quantitative and disease traits across all commonly used computer systems. BiForce Toolbox is a stand-alone Java program that integrates bitwise computing with multithreaded parallelization and thus allows rapid full pairwise genome scans via a graphical user interface or the command line. Furthermore, BiForce Toolbox incorporates additional tests of interactions involving SNPs with significant marginal effects, potentially increasing the power of detection of epistasis. BiForce Toolbox is easy to use and has been applied in multiple studies of epistasis in large GWAS data sets, identifying interesting interaction signals and pathways.

Background: Chromatin structure at a given site can differ between chromosome copies in a cell, and such imbalances in chromatin structure have been shown to be important in understanding the molecular mechanisms controlling several disease loci. Human genetic variation, DNA methylation, and disease have been intensely studied, uncovering many sites of allele-specific DNA methylation (ASM). However, little is known about the genome-wide occurrence of sites of allele-specific histone modification (ASHM) and their relationship to human disease. The aim of this study was to investigate the extent and characteristics of sites of ASHM in human embryonic stem cells (hESCs)., Results: Using a statistically rigorous protocol, we investigated the genomic distribution of ASHM in hESCs, and their relationship to sites of allele-specific expression (ASE) and DNA methylation. We found that, although they were rare, sites of ASHM were substantially enriched at loci displaying ASE. Many were also found at known imprinted regions, hence sites of ASHM are likely to be better markers of imprinted regions than sites of ASM. We also found that sites of ASHM and ASE in hESCs colocalize at risk loci for developmental syndromes mediated by deletions, providing insights into the etiology of these disorders., Conclusion: These results demonstrate the potential importance of ASHM patterns in the interpretation of disease loci, and the protocol described provides a basis for similar studies of ASHM in other cell types to further our understanding of human disease susceptibility.

Background: Germline variation in the 71 Crohn's disease (CD) loci implicated by genome-wide association studies (GWAS) only accounts for approximately 25% of estimated heritability. The contribution of epigenetic alterations to disease pathogenesis is emerging as a research priority., Materials and Methods: The methylation status of 27,578 CpG sites across the genome was analyzed using the Illumina Human Methylation27 assay in DNA extracted from whole blood samples from 40 adult females (21 ileal CD, 19 healthy controls) and 16 girls with childhood-onset CD, all nonsmokers. Our primary analysis compared methylation profiles in adult cases and controls., Results: Our data define a global methylation profile characteristic of ileal CD. In all, 1117 sites were differentially methylated (corrected P < 0.01); 50 showed significantly altered methylation in cases compared with controls (uncorrected P < 10(-6), corrected P < 0.0006), including genes altering immune activation: MAPK13, FASLG, PRF1, S100A13, RIPK3, and IL-21R. Gene ontology analyses implicated immunity-related pathways as targets of epigenetic modification (immune system processes [P = 1.3 × 10(-22)], immune response [P = 8.1 × 10(-16)], defense responses to bacteria [P = 1.8 × 10(-15)]). Ingenuity canonical pathway analyses implicated dendritic cell activity (P = 2.4 × 10(-8)) and differential regulation of cytokines by interleukin (IL)-17A and IL-17F (P = 5.8 × 10(-7)). We identified a significant enrichment of methylation changes within 50 kb of CD GWAS loci (8.6-fold [P = 0.021] in adults; 2.4-fold [P = 0.009] in adults and children combined), including IL-27, IL-19, TNF, MST1, and NOD2. Methylation status was predictive of disease status (sensitivity 0.71, specificity 0.83). Disease activity, drug therapy, NOD2 and DNMT3A genotypes were not associated with methylation changes., Conclusions: These data provide an important insight into the impact of epigenetic mechanisms in the pathogenesis of CD., (Copyright © 2011 Crohn's & Colitis Foundation of America, Inc.)

Evolutionary change in gene expression is generally considered to be a major driver of phenotypic differences between species. We investigated innate immune diversification by analyzing interspecies differences in the transcriptional responses of primary human and mouse macrophages to the Toll-like receptor (TLR)-4 agonist lipopolysaccharide (LPS). By using a custom platform permitting cross-species interrogation coupled with deep sequencing of mRNA 5' ends, we identified extensive divergence in LPS-regulated orthologous gene expression between humans and mice (24% of orthologues were identified as "divergently regulated"). We further demonstrate concordant regulation of human-specific LPS target genes in primary pig macrophages. Divergently regulated orthologues were enriched for genes encoding cellular "inputs" such as cell surface receptors (e.g., TLR6, IL-7Rα) and functional "outputs" such as inflammatory cytokines/chemokines (e.g., CCL20, CXCL13). Conversely, intracellular signaling components linking inputs to outputs were typically concordantly regulated. Functional consequences of divergent gene regulation were confirmed by showing LPS pretreatment boosts subsequent TLR6 responses in mouse but not human macrophages, in keeping with mouse-specific TLR6 induction. Divergently regulated genes were associated with a large dynamic range of gene expression, and specific promoter architectural features (TATA box enrichment, CpG island depletion). Surprisingly, regulatory divergence was also associated with enhanced interspecies promoter conservation. Thus, the genes controlled by complex, highly conserved promoters that facilitate dynamic regulation are also the most susceptible to evolutionary change.

β-Defensins are cationic host defense peptides that form an amphipathic structure stabilized by three intramolecular disulfide bonds. They are key players in innate and adaptive immunity and have recently been shown to limit the production of pro-inflammatory cytokines in TLR4-stimulated macrophages. In the present study, we investigate the mechanism underlying the anti-inflammatory effect of human β-defensin 3 (hBD3). We show that the canonical structure of hBD3 is required for this immunosuppressive effect and that hBD3 rapidly associates with and enters macrophages. Examination of the global effect of hBD3 on transcription in TLR4-stimulated macrophages shows that hBD3 inhibits the transcription of pro-inflammatory genes. Among the altered genes there is significant enrichment of groups involved in the positive regulation of NF-κB including components of Toll-like receptor signaling pathways. We confirm these observations by showing corresponding decreases in protein levels of pro-inflammatory cytokines and cell surface molecules. In addition, we show that hBD3 reduces NF-κB signaling in cells transfected with MyD88 or TRIF and that hBD3 inhibits the TLR4 response in both MyD88- and TRIF-deficient macrophages. Taken together these findings suggest that the mechanism of hBD3 anti-inflammatory activity involves specific targeting of TLR signaling pathways resulting in transcriptional repression of pro-inflammatory genes., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this study we investigated the strengths and modes of selection associated with nucleosome positioning in the human lineage through the comparison of interspecies and intraspecies rates of divergence. We identify significant evidence for both positive and negative selection linked to human nucleosome positioning for the first time, implicating a widespread and important role for DNA sequence in the location of well-positioned nucleosomes. Selection appears to be acting on particular base substitutions to maintain optimum GC compositions in core and linker regions, with, e.g., unexpectedly elevated rates of C→T substitutions during recent human evolution at linker regions 60-90 bp from the nucleosome dyad but significant depletion of the same substitutions within nucleosome core regions. These patterns are strikingly consistent with the known relationships between genomic sequence composition and nucleosome assembly. By stratifying nucleosomes according to the GC content of their genomic neighborhood, we also show that the strength and direction of selection detected is dictated by local GC content. Intriguingly these signatures of selection are not restricted to nucleosomes in close proximity to exons, suggesting the correct positioning of nucleosomes is not only important in and around coding regions. This analysis provides strong evidence that the genomic sequences associated with nucleosomes are not evolving neutrally, and suggests that underlying DNA sequence is an important factor in nucleosome positioning. Recent signatures of selection linked to genomic features as ubiquitous as the nucleosome have important implications for human genome evolution and disease.

Ascertaining when and where genes are expressed is of crucial importance to understanding or predicting the physiological role of genes and proteins and how they interact to form the complex networks that underlie organ development and function. It is, therefore, crucial to determine on a genome-wide level, the spatio-temporal gene expression profiles at cellular resolution. This information is provided by colorimetric RNA in situ hybridization that can elucidate expression of genes in their native context and does so at cellular resolution. We generated what is to our knowledge the first genome-wide transcriptome atlas by RNA in situ hybridization of an entire mammalian organism, the developing mouse at embryonic day 14.5. This digital transcriptome atlas, the Eurexpress atlas (http://www.eurexpress.org), consists of a searchable database of annotated images that can be interactively viewed. We generated anatomy-based expression profiles for over 18,000 coding genes and over 400 microRNAs. We identified 1,002 tissue-specific genes that are a source of novel tissue-specific markers for 37 different anatomical structures. The quality and the resolution of the data revealed novel molecular domains for several developing structures, such as the telencephalon, a novel organization for the hypothalamus, and insight on the Wnt network involved in renal epithelial differentiation during kidney development. The digital transcriptome atlas is a powerful resource to determine co-expression of genes, to identify cell populations and lineages, and to identify functional associations between genes relevant to development and disease., Competing Interests: The authors have declared that no competing interests exist.