Search Results - EGA European Genome-Phenome Archive

A novel TP53-KPNA3 translocation defines a de novo treatment-resistant clone in osteosarcoma

Osteosarcoma is the most common primary bone cancer. It can be cured by aggressive surgery and chemotherapy, but outcomes for metastatic or chemoresistant disease remain dismal. Cancer sequencing studies have shown that the p53 pathway is dysregulated in nearly every case, often by translocation; however, no studies of osteosarcoma evolution or intratumor heterogeneity have been done to date. We studied a patient with chemoresistant, metastatic disease over the course of three years. We performed exome sequencing on germline DNA and DNA collected from tumor at three separate timepoints. We compared variant calls and variant allele frequencies between different samples. We identified subclonal driver mutations in several different genes in the primary tumor sample and found that one particular subclone dominated subsequent tumor samples at relapse. This clone was marked by a novel TP53-KPNA3 translocation and loss of the opposite-strand wild-type TP53 allele. Future research must focus on functional significance of such clones and strategies to eliminate them.

Study EGAS00001001805

Physiological and genetic adaptations to diving in Sea Nomads

Understanding the physiology and genetics of human hypoxia tolerance has important medical implications, but this phenomenon has thus far only been investigated in high-altitude human populations. Another system, yet to be explored, is humans who engage in breath-hold diving. The indigenous Bajau people (“Sea Nomads”) of Southeast Asia live a subsistence lifestyle based on breath-hold diving and are renowned for their extraordinary breath holding abilities. However, it is unknown whether this has a genetic basis. Using a comparative genomic study, we show that natural selection on genetic variants in the PDE10A gene have increased spleen size in the Bajau, providing them with a larger reservoir of oxygenated red blood cells. We also find evidence of strong natural selection specific to the Bajau on BDKRB2, a gene affecting the human diving reflex. Thus, the Bajau, and possibly other natural diving populations, provide a new opportunity to study human adaptation to hypoxia tolerance.

Study EGAS00001002823

Single_Cell_RNAseq_at_various_stages_of_HiPSCs_differentiating_toward_definitive_endoderm_and_endoderm_derived_lineages

When comparing the differentiation capacities of pluripotent stem cell lines that have different genetic backgrounds, batch to batch experimental variablility poses a significant challenge, especially when trying to identify smaller effects. One way to address this issue is to differentiate several different lines in the same culture dish, thereby elimating experimental variation. In addition, it allows researchers to analyze many more lines with less experiments. Parallel single cell RNA-Seq exploits that individual cells are tagged and hence each cell can be reliably assigned to the donor of origin based on the genetic variants it contains. In addition, analyzing the genetic signature of single cells within a differentiating population can reveal differentation stages that are not easily detected in bulk RNAseq data. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/

Study EGAS00001002278

Robust detection of translocations in lymphoma FFPE samples using Targeted Locus Capture-based sequencing - NGS targeted capture control cohort

In routine diagnostic pathology, cancer biopsies are preserved by Formalin-Fixed, Paraffin-Embedding (FFPE) procedures for examination of (intra-) cellular morphology. Such procedures inadvertently induce DNA fragmentation, which compromises sequencing-based analyses of chromosomal rearrangements. Yet, rearrangements drive many types of hematolymphoid malignancies and solid tumors, and their manifestation is instructive for diagnosis, prognosis and treatment. Here, we present FFPE-Targeted Locus Capture (FFPE-TLC) for targeted sequencing of proximity-ligation products formed in FFPE tissue blocks, and PLIER, a computational framework that allows automated identification and characterization of rearrangements involving selected, clinically-relevant, loci. FFPE-TLC, blindly applied to 149 lymphoma and control FFPE samples, identifies the known and novel rearrangement partners. It outperforms fluorescence in situ hybridization (FISH) in sensitivity and specificity, and shows clear advantages over standard capture-NGS methods, finding rearrangements involving repetitive sequences which they typically miss. FFPE-TLC is therefore a powerful clinical diagnostics tool for accurate targeted rearrangement detection in FFPE specimens.

Study EGAS00001005325

Single_cell_analysis_of_cytokine_induced_T_cell_states

We isolated T cells from healthy platelet donors and cultured them in resting and stimulated conditions with addition of a range of cytokines. We performed scRNA sequencing to assess the variability of polarization in different cytokines treated cells. Cytokines affect T cell responses by polarising them to different phenotypes. Given that the inflammation and and autoimmune tissue distruction is driven by the local production of cytokines, we investigated cytokine induced changes in T cells by using a combination of immunological and genomics approaches. To characterize, the efficacy of cytokine induced porization, here we stimulated naive and memory CD4+ T cells in the presence of cytokine polarizing coctails and profiled single cell transcriptome five days following polarization. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/

Study EGAS00001003215

Modulation of macrophage inflammatory function through selective inhibition of the epigenetic reader protein SP140

SP140 is a bromodomain-containing protein expressed predominantly in immune cells. Genetic polymorphisms and epigenetic modifications in the SP140 locus have been linked to Crohn’s disease (CD), suggesting a role in inflammation. We report the development of the first small molecule SP140 inhibitor (GSK761) and utilize this to elucidate SP140 function in macrophages. We show that SP140 is highly expressed in CD68+ CD mucosal macrophages and in in vitro-generated inflammatory macrophages. SP140 inhibition through GSK761 reduced monocyte differentiation into inflammatory macrophages and lipopolysaccharide (LPS)-induced inflammatory activation. SP140 preferentially occupies transcriptional start sites (TSS) in inflammatory macrophages, with enrichment at gene loci encoding pro-inflammatory cytokines/chemokines and inflammatory pathways. GSK761 specifically reduced SP140 binding and thereby expression of SP140-bound genes. GSK761 inhibited the spontaneous expression of cytokines, including TNF, by CD14+ macrophages isolated from CD intestinal-mucosa. This study identifies SP140 as a druggable epigenetic therapeutic target for CD.

Study EGAS00001004460

Genome-wide discovery of somatic coding and regulatory variants in Diffuse Large B-cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer originating from mature B-cells. Prognosis is strongly associated with molecular subgroup, although the driver mutations that distinguish the two main subgroups remain poorly defined. Through an integrative analysis of whole genomes, exomes, and transcriptomes, we have uncovered genes and non-coding loci that are commonly mutated in DLBCL. Our analysis has identified novel cis-regulatory sites, and implicates recurrent mutations in the 3'UTR of NFKBIZ as a novel mechanism of oncogene deregulation and NFκB pathway activation in the activated B-cell (ABC) subgroup. Small amplifications associated with over-expression of FCGR2B (the Fcγ receptor protein IIB), primarily in the germinal centre B-cell (GCB) subgroup, correlate with poor patient outcomes suggestive of a novel oncogene. These results expand the list of subgroup driver mutations that may facilitate implementation of improved diagnostic assays and could offer new avenues for the development of targeted therapeutics.

Study EGAS00001002936

Sequence data for study: Mobilization of tissue-resident memory CD4+ T lymphocytes from bone marrow and their contribution to a systemic secondary immune reaction

Resident memory T lymphocytes (TRM) of epithelial tissues and the bone marrow protect their host tissue. To what extent these cells are mobilized and contribute to systemic immune reactions is less clear. Here we show that in secondary immune reactions to the measles-mumps-rubella (MMR) vaccine, CD4+ TRM are mobilized into the blood within 16 to 48 hours after immunization. This mobilization of TRM is cognate: TRM recognizing other antigens are not mobilized, unless they cross-react with the vaccine. We also demonstrate through methylome analyses that TRM are mobilized from the bone marrow. These mobilized cells make significant contribution to the systemic immune reaction, as evidenced by their T-cell receptor Vβ clonotypes represented among the newly generated circulating memory T-cells, 14 days after vaccination. Thus, TRM of the bone marrow confer not only local, but also systemic immune memory.

Study EGAS00001005475

Exome sequencing of familial high-grade serous ovarian carcinoma reveals heterogeneity for rare candidate susceptibility genes

High-grade serous ovarian carcinoma (HGSOC) has a significant hereditary component, approximately half of which cannot be explained by known genes. To discover genes, we analyse germline exome sequencing data from 516 BRCA1/2-negative women with HGSOC, focusing on genes enriched with rare, protein-coding loss-of-function (LoF) variants. Overall, there is a significant enrichment of rare protein-coding LoF variants in the cases (p < 0.0001, chi-squared test). Only thirty-four (6.6%) have a pathogenic variant in a known or proposed predisposition gene. Few genes have LoF mutations in more than four individuals and the majority are detected in one individual only. Forty-three highly-ranked genes are identified with three or more LoF variants that are enriched by three-fold or more compared to GnomAD. These genes represent diverse functional pathways with relatively few involved in DNA repair, suggesting that much of the remaining heritability is explained by previously under-explored genes and pathways.

Study EGAS00001004235

Gut metagenome associations with extensive digital health data in a volunteer-based EstMB cohort

Microbiome research is starting to move beyond the exploratory phase towards interventional trials and therefore well-characterized cohorts will be instrumental for generating hypothesis and providing new knowledge. As part of the Estonian Biobank, we established the Estonian Microbiome Cohort which includes stool, oral and plasma samples from 2,509 participants and is supplemented with multi-omic measurements, questionnaires, and regular linkages to national electronic health records. In this study, we analyzed stool data from deep metagenomic sequencing together with rich phenotyping, including 71 diseases, 136 medications, 21 dietary questions, 5 procedures, and 19 other factors. The data revealed numerous relationships with different microbiome features. Additionally, the long-term antibiotic usage, independent from recent administration, has a significant impact on the microbiome composition, partly explaining the common associations between diseases. Overall, this study extends the understanding of microbiome–host interactions and facilitates the development of microbiome-related studies.

Study EGAS00001005900

Fibroblast heterogeneity drives metastatic spread in breast cancer through distinct mechanisms

The major cause of death among breast cancer patients is metastatic dissemination, suggesting that understanding the mechanisms driving this process remains an unmet medical need. Here, by concomitantly using 5 markers, we identify 4 Carcinoma-Associated Fibroblasts (CAF) subsets in primary tumors and corresponding metastatic lymph nodes (LN). Two CAF subsets, called CAF-S1 and CAF-S4, correlate with the proportion of cancer cells in invaded LN and exert distinct functions in tumor cell spread. CAF-S1 promote cancer cell migration and initiates an epithelial to mesenchymal transition through a CXCL12 / TGFb positive loop. For their part, the highly contractile CAF-S4 foster cancer cell motility and invasion in 3-dimensional matrix via Notch pathway activation. Finally, CAF-S1 and CAF-S4 accumulation in LN is a new prognostic factor at diagnostic, independent of breast cancer subtypes and LN status, strengthening the validity of our findings in human breast cancer pathophysiology.

Study EGAS00001003238

SARS‐CoV‐2 receptor ACE2 and TMPRSS2 are primarily expressed in bronchial transient secretory cells

The SARS‐CoV‐2 pandemic affecting the human respiratory system severely challenges public health and urgently demands for increasing our understanding of COVID‐19 pathogenesis, especially host factors facilitating virus infection and replication. SARS‐CoV‐2 was reported to enter cells via binding to ACE2, followed by its priming by TMPRSS2. Here, we investigate ACE2 and TMPRSS2 expression levels and their distribution across cell types in lung tissue (twelve donors, 39,778 cells) and in cells derived from subsegmental bronchial branches (four donors, 17,521 cells) by single nuclei and single cell RNA sequencing, respectively. While TMPRSS2 is strongly expressed in both tissues, in the subsegmental bronchial branches ACE2 is predominantly expressed in a transient secretory cell type. Interestingly, these transiently differentiating cells show an enrichment for pathways related to RHO GTPase function and viral processes suggesting increased vulnerability for SARS‐CoV‐2 infection. Our data provide a rich resource for future investigations of COVID‐19 infection and pathogenesis.

Study EGAS00001004419

Detection of cell-free DNA fragmentation and copy number alterations in cerebrospinal fluid from glioma patients

Glioma is difficult to detect or characterise using current liquid biopsy approaches. Detection of cell-free tumor DNA (cftDNA) in cerebrospinal fluid (CSF) has been proposed as an alternative to detection in plasma. We used shallow whole-genome sequencing (sWGS, at a coverage of < 0.4x) of cell-free DNA from the CSF of 13 patients with primary glioma to determine somatic copy number alterations and DNA fragmentation patterns. This allowed us to determine the presence of cftDNA in CSF without any prior knowledge of point mutations present in the tumor. We also showed that the fragmentation pattern of cell-free DNA in CSF is different from that in plasma. This low-cost screening method provides information on the tumor genome and can be used to target those patients with high levels of cftDNA for further larger-scale sequencing, such as by whole-exome and whole-genome sequencing.

Study EGAS00001003255

A Proteogenomic Analysis of Clear Cell Renal Cell Carcinoma in a Chinese Population

Clear cell renal cell carcinoma (ccRCC) is a common and aggressive subtype of renal cancer. Here we conduct a comprehensive proteogenomic analysis of 232 tumor and adjacent non-tumor tissue pairs from Chinese ccRCC patients. By comparing with tumor adjacent tissues, we find that ccRCC shows extensive metabolic dysregulation and an enhanced immune response. Molecular subtyping classifies ccRCC tumors into three subtypes (GP1–3), among which the most aggressive GP1 exhibits the strongest immune phenotype, increased metastasis, and metabolic imbalance, linking the multi-omics-derived phenotypes to clinical outcomes of ccRCC. Nicotinamide N-methyltransferase (NNMT), a one-carbon 38 metabolic enzyme, is identified as a potential marker of ccRCC and a drug target for GP1. We demonstrate that NNMT induces DNA-dependent protein kinase catalytic subunit (DNA-PKcs) homocysteinylation, increases DNA repair, and promotes ccRCC tumor growth. This study provides insights into the biological underpinnings and prognosis assessment of ccRCC, revealing targetable metabolic vulnerabilities.

Study EGAS00001006005

Molecular and functional profiling of plasmablastic lymphoma

Plasmablastic lymphoma (PBL) represents a rare and aggressive lymphoma subtype frequently associated with immunosuppression. Clinically, patients with PBL are characterized by poor outcome. The current understanding of the molecular pathogenesis is limited. A hallmark of PBL represents its plasmacytic differentiation with loss of B-cell markers and, in 60% of cases, its association with Epstein-Barr virus (EBV). Roughly 50% of PBLs harbor a MYC translocation. Here, we provide a comprehensive integrated genomic analysis using whole exome sequencing (WES) and genome-wide copy number determination in a large cohort of 96 primary PBL samples. We identify alterations activating the RAS-RAF, JAK-STAT, and NOTCH pathways as well as frequent high-level amplifications in MCL1 and IRF4. The functional impact of these alterations is assessed using an unbiased shRNA screen in a PBL model. These analyses identify the IRF4 and JAK-STAT pathways as promising molecular targets to improve outcome of PBL patients.

Study EGAS00001004659

Enhanced cortical neural stem cell identity through SMAD/WNT inhibition in human cerebral organoids facilitates emergence of outer radial glial cells

Cerebral organoids exhibit broad regional heterogeneity accompanied by limited cortical cellular diversity despite the tremendous upsurge in derivation methods, suggesting inadequate patterning of early neural stem cells (NSCs). Here we show that a short and early dual-SMAD/WNT inhibition course is necessary and sufficient for establishing robust and lasting cortical organoid NSC identity, efficiently suppressing of non-cortical NSC fates, while other widely used methods are inconsistent in their cortical NSC specification capacity. Accordingly, this method selectively enriches for outer radial glia (oRG) NSCs, which cytoarchitecturally demarcate well-defined outer sub-ventricular (oSVZ)-like regions propagating from superiorly radially organized, apical cortical rosette NSCs. Finally, this method culminates in the emergence of molecularly distinct deep and upper cortical layer neurons, and reliably uncovers cortex-specific microcephaly defects. Thus, a short SMAD/WNT inhibition is critical for establishing a rich cortical cell repertoire that enables mirroring fundamental molecular and cytoarchitectural features of cortical development and meaningful disease modeling.

Study EGAS00001006063

The immunopeptidome landscape associated with T cell infiltration, inflammation and immune-editing in lung cancer

One key barrier for improving efficacy of cancer immunotherapy remains patient stratification. While patients with CD3+CD8+ T cell inflamed ‘hot’ tumors typically show better response to immune checkpoint inhibitors, it is still unknown if the repertoire of HLA bound peptides (HLAp) presented in ‘hot’ and ‘cold’ tumors is substantially different. We surveyed 61 tumor regions and adjacent non-malignant lung tissues from eight lung cancer patients and performed deep antigen discovery combining immunopeptidomics, genomics, bulk and spatial transcriptomics and explored the heterogenic expression and presentation of tumor (neo)antigens. We associated diverse immune cell populations with the immunopeptidome in CD3+CD8+ T cell excluded, infiltrated, hot and cold tumors and found evidence for lack of immune-editing and higher presentation efficiency of tumor antigens in cold and CD3+CD8+ T cell excluded tumors. This could have implications for the choice of combination therapies tailored to the patient’s mutanome and microenvironment.

Study EGAS00001006298

RNA-seq on patient-derived, stage II, CRC cell lines

The gut microbiome is a key player in the immunomodulatory and pro-tumorigenic microenvironment in colorectal cancer (CRC). It is well established that Fusobacterium’s virulence factors are responsible for its pro-tumorigenic properties, however the role of its metabolic cross-talk with the tumor remains unexplored. Here we use in vitro, in vivo, and in silico methods to describe a novel microbe-host interaction keystone: formate. Omics data reveals molecular signatures, linking a metabolically driven CRC phenotype with Fusobacteria. We observed a metabolic shift towards increased F. nucleatum formate secretion in gut-on-chip model co-cultures with patient-derived CRC cells, along with an accelerated cancer glutamine metabolism. We show that high formate levels trigger AhR signaling, increase cancer stemness and cellular invasion. Finally, we observed an expansion of Th17 cells accompanying F. nucleatum-induced tumorigenesis. Moving beyond observational studies, we applied new experimental approaches for gaining ecosystem-level mechanistic understanding of F. nucleatum’s role in cancer pathogenesis.

Study EGAS00001005948

GENOMIC MUTATION LANDSCAPE OF SKIN CANCERS FROM DNA REPAIR-DEFICIENT XERODERMA PIGMENTOSUM PATIENTS

Xeroderma pigmentosum (XP) is a genetic disorder caused by mutations in genes of the Nucleotide Excision Repair (NER) pathway (groups A-G) or in Translesion Synthesis (TLS) DNA polymerase η (group V). XP is associated with an increased skin cancer risk, reaching, for some groups, several thousand-fold compared to the general population. Here, we analyzed 38 skin cancer genomes from five XP groups. We found that the activity of NER determines heterogeneity of the mutation rates across skin cancer genomes and that transcription-coupled NER extends beyond the gene boundaries reducing the intergenic mutation rate. Mutational profile in XP-V tumors revealed the role of polymerase η in the error-free bypass of (i) rare TpG and TpA DNA lesions, (ii) 3’ nucleotides in pyrimidine dimers, and (iii) TpT photodimers. Our study unravels the genetic basis of skin cancer risk in XP and provides insights into the mechanisms reducing UV-induced mutagenesis in the general population.

Study EGAS00001006732

The landscape of chromothripsis across adult cancer types

Chromothripsis is a recently identified mutational phenomenon, by which a presumably single catastrophic event generates extensive genomic rearrangements of one or a few chromosome(s). Considered as an early event in tumour development, this form of genome instability plays a prominent role in tumour onset. Chromothripsis prevalence might have been underestimated when using low-resolution methods, and pan-cancer studies based on sequencing are rare. We analysed chromothripsis in 28 tumour types covering all major adult cancers (634 tumours, 316 whole-genome and 318 whole-exome sequences). We show that chromothripsis affects a substantial proportion of human cancers, with a prevalence of 49% across all cases. Chromothripsis generates entity-specific genomic alterations driving tumour development, including clinically relevant druggable fusions. Chromothripsis is linked with specific telomere patterns and univocal mutational signatures in distinct tumour entities. Longitudinal analysis of chromothriptic patterns in 24 matched tumour pairs reveals novel insights in the clonal evolution of tumours with chromothripsis. (H021)

Study EGAS00001004250

Multi-omics identify falling LRRC15 as a COVID-19 severity marker and persistent pro-thrombotic signals in convalescence

Patients with end-stage kidney disease (ESKD) are at high risk of severe COVID-19. Here, we perform longitudinal blood sampling of ESKD haemodialysis patients with COVID-19, collecting samples pre-infection, serially during infection, and after clinical recovery. Using plasma proteomics, and RNA-sequencing and flow cytometry of immune cells, we identify transcriptomic and proteomic signatures of COVID-19 severity, and find distinct temporal molecular profiles in patients with severe disease. Supervised learning reveals that the plasma proteome is a superior indicator of clinical severity than the PBMC transcriptome. We show that a decreasing trajectory of plasma LRRC15, a proposed co-receptor for SARS-CoV-2, is associated with a more severe clinical course. Strikingly, we observe that two months after the acute infection, patients still display dysregulated gene expression related to vascular, platelet and coagulation pathways, including PF4 (platelet factor 4), which may explain the prolonged thrombotic risk following COVID-19.

Study EGAS00001006778

Early on-treatment changes in circulating tumor DNA fraction and response to enzalutamide or abiraterone in metastatic castration-resistant prostate cancer

On-treatment circulating tumor DNA (ctDNA) quantification is touted as a surrogate biomarker for understanding metastatic cancer response to systemic therapy, but there is relatively little supporting data from standardized patient cohorts. Here, in a prospective observational cohort, we demonstrate that 4-week on-treatment changes in plasma ctDNA levels can accurately identify metastatic castration-resistant prostate cancer that will progress within 6 months of initiating first-line abiraterone or enzalutamide treatment. On-treatment ctDNA measurements outperformed existing clinical variables for predicting disease response and were also closely associated with differential overall survival. Although optimal timing of blood collections and thresholds for ctDNA detection will require further testing, our data highlights the potential for sequential ctDNA measurements to provide an early and reliable read-out for therapy response. The high accuracy of this information suggests that ctDNA could be useful for guiding clinical trials of therapy changes in initially non-responsive metastatic cancers.

Study EGAS00001006856

Study of complex rearrangements and mutational signatures in neuroblastoma heterogeneous risk groups.

Neuroblastoma is a pediatric solid tumor characterized by strong clinical heterogeneity. Although certain complex genomic alterations, such as extrachromosomal DNA amplifications (ecDNA), are associated with adverse outcome and have been recurrently detected in neuroblastomas, the mutational processes involved in their generation remain largely unclear. By examining the topography of complex rearrangements along with mutational signatures derived from all variant classes, we identify previously unrecognized co-occurring mutational footprints, which we termed mutational scenarios. We demonstrate that clinical neuroblastoma heterogeneity is linked to differences in the processes driving these mutational scenarios. Whereas high-risk MYCN-amplified neuroblastoma genomes were characterized by signs of replication slippage and stress, homologous recombination-associated signatures defined high-risk non-MYCN-amplified patients. Non-high-risk neuroblastomas, on the other hand, were marked by footprints of chromosome missegregation and TOP1 mutational activity. This analysis provides a systematic perspective on the repertoire of mutational patterns that contribute to clinical neuroblastoma heterogeneity.

Study EGAS00001006983

Neuroblastoma heterogeneity

Neuroblastoma is a pediatric solid tumor characterized by strong clinical heterogeneity. Although certain complex genomic alterations, such as extrachromosomal DNA amplifications (ecDNA), are associated with adverse outcomes and have been recurrently detected in neuroblastomas, the mutational processes involved in their generation remain largely unclear. By examining the topography of complex rearrangements along with mutational signatures derived from all variant classes, we identify previously unrecognized co-occurring mutational footprints, which we termed mutational scenarios. We demonstrate that clinical neuroblastoma heterogeneity is linked to differences in the processes driving these mutational scenarios. Whereas high-risk MYCN-amplified neuroblastoma genomes were characterized by signs of replication slippage and stress, homologous recombination-associated signatures defined high-risk non-MYCN-amplified patients. Non-high-risk neuroblastomas, on the other hand, were marked by footprints of chromosome missegregation and TOP1 mutational activity. This analysis provides a systematic perspective on the repertoire of mutational patterns that contribute to clinical neuroblastoma heterogeneity.

Study EGAS00001007019

Integrative genomic analyses in adipocytes implicate DNA methylation in human obesity and diabetes

DNA methylation variations are prevalent in human obesity but evidence of a causative role in disease pathogenesis is limited. Here, we combine epigenome-wide association and integrative genomics to investigate the impact of adipocyte DNA methylation variations in human obesity. We discover extensive DNA methylation changes that are robustly associated with obesity (N=190 samples, 691 loci in subcutaneous and 173 loci in visceral adipocytes, P<1x10-7). We connect obesity-associated methylation variations to transcriptomic changes at >500 target genes, and identify putative methylation-transcription factor interactions. Through Mendelian Randomisation, we infer causal effects of methylation on obesity and obesity-induced metabolic disturbances at 59 independent loci. Targeted methylation sequencing, CRISPR-activation and gene silencing in adipocytes, further identifies regional methylation variations, underlying regulatory elements and novel cellular metabolic effects. Our results indicate DNA methylation is an important determinant of human obesity and its metabolic complications, and reveal mechanisms through which altered methylation may impact adipocyte functions.

Study EGAS00001007118

Transcriptional profiling of tauopathies in human IPS-derived neurons (2019-08-21)

This project is a pilot study, in collaboration with Maria Grazia Spillantini and Mariangela Iovino (Cambridge Centre for Brain Repair), to investigate the utility of IPS-derived neurons for the study of neurodegenerative disorders. Our aim is to characterise the transcriptional consequences of tauopathies using neurons derived from differentiated IPSCs as a model system. We will use IPS cells derived from six individuals, four with known mutations in the tau protein, 2 without. RNA will be extracted at Day 0 and Day 65 of differentiation by which time the neuronal tauopathy is apparent. RNA will be extracted and the transcriptome of each line characterised using RNAseq. We will then search for genes that are differentially expressed between the transcriptomes of individuals with tau mutations versus those in controls. My lab will analyse the RNAseq data, comparing both affected and controls and both time-points, to establish candidate genes. Darren Logan’s lab, along with our collaborators, will experimentally verify and further investigate these genes in additional lines and animal models. From this analysis we will generate a list of candidate genes that are differentially expressed between cases and controls. This study will not only help us understand the molecular basis of tauopathies, but also identify gene candidates for biomarkers of neurodegenerative disease. It will serve as a proof of principle for future planned studies into generating and transcriptomically analysing an allelic series of Tau mutations in IPSCs with a controlled genetic background. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/ . This dataset contains all the data available for this study on 2019-08-21.

Dataset EGAD00001005277

Constitutional and somatic genomic rearrangements coherently restructure chromosome 21 in acute lymphoblastic leukaemia

Changes in gene dosage are a major driver of cancer1, engineered from a finite, but increasingly well annotated, repertoire of mutational mechanisms2-6. These processes operate over levels ranging from individual exons to whole chromosomes, often generating correlated copy number alterations across hundreds of linked genes. An example of the latter is the 2% of childhood acute lymphoblastic leukemia (ALL) characterized by recurrent intrachromosomal amplification of megabase regions of chromosome 21 (iAMP21)7,8 To dissect the interplay between mutational processes and selection on this scale, we used genomic, cytogenetic and transcriptional analysis, coupled with novel bioinformatic approaches, to reconstruct the evolution of iAMP21 ALL. We find that individuals born with the rare constitutional Robertsonian translocation between chromosomes 15 and 21, rob(15;21)(q10;q10)c, have ~2700-fold increased risk of developing iAMP21 ALL compared to the general population. In such cases, amplification is initiated by chromothripsis involving both sister chromatids of the dicentric Robertsonian chromosome. In contrast, sporadic iAMP21 is typically initiated by breakage-fusion-bridge (BFB) events, often followed by chromothripsis or other rearrangements. In both sporadic and iAMP21 in rob(15;21)c individuals, the final stages of amplification frequently involve large-scale duplications of the abnormal chromosome. The end-product is a derivative chromosome 21 or a derivative originating from the rob(15;21)c chromosome, der(15;21), respectively, with gene dosage optimised for leukemic potential, showing constrained copy number levels over multiple linked genes. In summary, the constitutional translocation, rob(15;21)c, predisposes to leukemia through a novel mechanism, namely a propensity to undergo chromothripsis, likely related to its dicentric nature. More generally, our data illustrate that several cancer-specific mutational processes, applied sequentially, can co-ordinate to fashion copy number profiles over large genomic scales, incrementally refining the fitness benefits of aggregated gene dosage changes.

Dataset EGAD00001000658

Immune variation leads to diverse outcomes in human malaria (2020-01-15)

Falciparum malaria is clinically heterogeneous and yet in most cases the risk of life-threatening disease dramatically declines after the first few infections of life because children rapidly acquire disease tolerance (resistance to severe malaria without improved control of parasite burden). Identifying the factors that determine clinical outcome in a malaria-naive host is therefore paramount to reduce malaria mortality. However, the relative contribution of disease-causing variants of the Plasmodium var gene family versus pathogenic inflammatory cytokine cascades remains fiercely debated - we sought to reconcile these conflicting arguments by studying their interaction in vivo. To this end, two human challenge models were used to reveal the parasite-host interactions that underpin variation in falciparum malaria. To capture the diversity of human immune responses, each individual was analysed independently by tracking dynamic changes in their whole blood transcriptome through time. And to uncover evidence of preferential expansion of disease-causing variants, var gene expression was tracked in vivo from the start to end of infection. In this way, we could show that group A var genes are always expressed upon liver egress but in a minority population that does not increase over 10-days of blood cycling; there is no selection of disease-causing variants in the naive host. In fact, parasites do not respond in any way to differences or changes in host environment. On the other hand, host-intrinsic variation determines the intensity of inflammation and progression to clinical malaria. And furthermore, regulation of the interferon signaling network controls host fate. These data emphasise the role of human immune decision-making in shaping course & outcome of infection. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/ . This dataset contains all the data available for this study on 2020-01-15.

Dataset EGAD00001005790

Identification of gene mutations and fusion genes in patients with Sézary Syndrome

Sezary syndrome (SS) is a leukemic form of cutaneous T-cell lymphoma (CTCL) with an aggressive clinical course. The goal of our study is to understand the genetic basis of the disease by looking for driver gene mutations and fusion genes in 15 erythrodermic patients with circulating Sezary cels, fourteen of them fulfilling the diagnostic criteria of SS. Peripheral blood samples were collected for each patient and CD4+ T-lymphocytes and Granulocytes were isolated. DNA and RNA was extracted for matched normal and tumor materials. Whole exome sequencing was performed on matched normal and tumor tissues of 12 patients and RNA sequencing was performed on 10 patients. For 7 of these patients, we have both exome as well as RNA sequencing data. In this study, we present a complex genomic landscape of Sezary syndrome with several point mutations, copy number variations and fusion events which could contribute to the pathogenesis of SS.

Study EGAS00001001706

Sensitive and Frequent Identification of High Avidity Neo-epitope Specific CD8 + T-cells in Immunotherapy-naïve Ovarian Cancer

Immunotherapy directed against private tumor neo-antigens derived from non-synonymous somatic mutations is a promising strategy of personalized cancer immunotherapy. However, feasibility in low mutational load tumor types remains unknown. Comprehensive and deep analysis of circulating and tumor-infiltrating lymphocytes (TILs) for neo-epitope specific CD8 + T cells allowed prompt identification of oligoclonal and polyfunctional such cells from most immunotherapy-naïve patients with advanced epithelial ovarian cancer studied. Neo-epitope recognition was discordant between circulating T cells and TILs, and was more likely to be found among TILs, which displayed higher functionalavidity and unique TCRs with higher predicted affinity than their blood counterparts. Our results imply that identification of neo-epitope specific CD8 + T cells is achievable even in tumors with relatively low number of somatic mutations, and neo-epitope validation in TILs extends opportunities for mutanome-based personalized immunotherapies to such tumors.

Study EGAS00001002803

Disease specific alterations in the olfactory mucosa of patients with Alzheimer’s disease

OOlfactory dysfunction manifests early in several neurodegenerative disorders. Olfaction is orchestrated by olfactory mucosal cells located in the upper nasal cavity. However, it is unclear how this tissue reflects key neurodegenerative features in Alzheimer’s disease. Here we report that Alzheimer´s disease olfactory mucosal cells secrete toxic amyloid-beta. We detail cell-type-specific gene expression patterns, unveiling 147 differentially expressed disease-associated genes compared to the cognitively healthy controls, and 5 distinct populations in globose basal cell -, myofibroblast-, and fibroblast/ stromal – like cells in vitro. Overall, coordinated alteration of RNA and protein metabolism, inflammatory processes and signal transduction were observed in multiple cell populations, suggesting a key role in pathophysiology. Our results demonstrate the potential of olfactory cell cultures in modelling Alzheimer´s disease. Moreover, for the first time we provide single cell data on olfactory mucosa in Alzheimer´s disease for investigating molecular and cellular mechanisms associated with the disease.

Study EGAS00001006019

An IL-1β driven neutrophil-stromal cell axis fosters a BAFF-rich microenvironment in multiple myeloma

The bone marrow is continuously occupied by high numbers of neutrophils, and a tumor-supportive bias of these cells could significantly impact bone marrow-confined malignancies. In multiple myeloma, the bone marrow is characterized by inflammatory stromal cells with the potential to influence neutrophils. Here, we investigated myeloma-associated alterations in marrow neutrophils and the impact of stromal inflammation on neutrophil function. Mature neutrophils in myeloma marrow are activated and tumor-supportive, transcribing increased levels of pro-inflammatory cytokines, including IL-1β, and myeloma cell survival factors, such as the BCMA-ligand BAFF. Neutrophils were re-activated after first-line treatment, while this regimen reduced, but did not normalize, stromal inflammation. Interactions with inflammatory stroma induced neutrophil activation, including BAFF secretion, in a STAT3-dependent manner and once activated, neutrophils gained the ability to reciprocally induce stromal activation. Combined, our data define the presence of a neutrophil-stromal cell feed-forward loop driving tumor-supportive inflammation that could impact disease recurrence.

Study EGAS00001007038

SMARCB1 loss activates patient-specific distal oncogenic enhancers in malignant rhabdoid tumors

Malignant rhabdoid tumor (MRT) is a highly malignant and often lethal childhood cancer. MRTs are genetically defined by bi-allelic inactivating mutations in SMARCB1, a member of the BRG1/BRM-associated factors (BAF) chromatin remodeling complex. Mutations in BAF complex members are common in human cancer, yet their contribution to tumorigenesis remains in many cases poorly understood. Here, we studied derailed regulatory landscapes as a consequence of SMARCB1 loss in the context of MRT. Our multi-omics approach on patient-derived MRT organoids revealed a dramatic reshaping of the regulatory landscape upon SMARCB1 reconstitution. Chromosome conformation capture experiments subsequently revealed patient-specific looping of distal enhancer regions with the promoter of the MYC oncogene. This intertumoral heterogeneity in MYC enhancer utilization is also present in patient MRT tissues as shown by combined single-cell RNA-seq and ATAC-seq. We show that loss of SMARCB1 drives patient-specific epigenetic reprogramming underlying MRT tumorigenesis.

Study EGAS00001007590

TOPDECC-Trans-omics for Precision Dentistry and Early Childhood Caries: Genome-Wide Genotyping (CIDR) and Microbiome in the ZOE 2.0 Study

The ZOE 2.0 study is a cross-sectional genetic epidemiologic investigation of early childhood oral health, conducted in a state-wide, community-based, multi-ethnic sample of 3-5-year-old preschool-age children in North Carolina, United States. Between 2016-2019, 8,059 children were enrolled and 6,404 of them underwent comprehensive oral-clinical examinations that provided usable data on dental caries (i.e., early childhood caries; ECC) phenotypes. Saliva samples and supragingival biofilm samples were collected by 10 trained and calibrated examiners during the clinical encounters that took place in enrolled children's preschool (Head Start) centers. Specifically, the study enrolled participants and conducted clinical examinations in 260 Head Start centers that belonged to 34 Head Start programs in NC. 6,262 participants provided clinical data and had extracted DNA that met quantity and quality criteria for high-density genotyping and 6,144 of them were retained in the genome-wide association study (GWAS). Additional information on oral health-related behaviors, risk factors and self-reported outcomes was collected prior to the conduct of the clinical exam using self-administered questionnaires completed by children's guardians. Height and weight measurements were used to derive measures of adiposity. A domestic water sample for fluoride concentration measurement was obtained after the clinical examination for approximately 25% of study participants, to provide a measure of environmental exposure to this known caries-preventive agent and this information was subsequently imputed to the entire cohort using a novel GIS-informed method. Supragingival biofilm samples for microbiome analyses were collected from all participating children. Biofilm samples from a pilot 5% (n=300) of the cohort were carried forward to WGS (metagenomics; MTG) and RNAseq (metatranscriptomics; MTX) analyses, and these sequence data are being released via the Sequence Read Archive (SRA) in this dbGaP accession: 300 participants with MTG, 297 of those with MTX. Of note, the 17 participants with MTG data did not have genotypes produced, therefore the study's released participant total count is 6161, including 6144 (with genotypes released) plus 17 (with no genotypes released but with MTG data in the SRA).The study's primary objective is to identify risk loci and gene sets/pathways for ECC and related traits (e.g., caries prevalence and severity) by conducting a trans-ethnic GWAS. Genotyping is based on the Infinium™ Global Diversity Array and the TOPMed panel is used for imputation via the University of Michigan Imputation server. Secondarily, the ZOE 2.0 seeks to determine prevalence and severity of dental caries, other oral disease outcomes, and putative risk factors for dental caries in this low-income population-based sample of European-, African-, and Hispanic/Latino-American preschool-age children.

Study phs002232

NIDA Genetic Epidemiology of Opioid Dependence in Bulgaria (GEODB)

The study was conducted in Bulgaria as a collaboration between the Molecular Medicine Center (Medical University, Sofia), the Bulgarian Addictions Institute (Sofia), the Initiative for Health Foundation (Sofia) and Washington University School of Medicine (Saint Louis, MO, USA). This is a case-population control study. Participants (N=2,499 interviews; N=1,995 with GWAS data) in our study are primarily active heroin users (78%) aged 18 and over, with a median 8.9 years of daily heroin use (minimum of 12 months). Virtually all participants (99.1%) qualified for a lifetime DSM-IV diagnosis of dependence, symptom endorsement probabilities ranging from 77% to 99.3%; and 61% endorsing all symptoms. 85% were heroin injectors and 97% reported using (or having used) heroin daily. Anonymized, population-representative control samples (no phenotypic data, N = 1,062 with GWAS data) were drawn from the MMC repository. Unlike other studies, where samples are clinically ascertained (e.g., from ORT programs), our participants are primarily untreated. PTSD, major depression, suicidality, mania, alcohol, nicotine and substance dependence (separating heroin and opioids) were assessed using the MAGIC (Todd et al., 2003, PMID: 14627881), a broad semi-structured psychiatric assessment tool with good reliability and prospective stability. The onset of (any) illicit drug use in this sample is not particularly remarkable (age~16), but the age of onset of heroin use is much lower than in the USA: 25% by age 16; 50% by age 19, with no differences between ethnicities or gender. This is consistent with findings from other studies in Bulgaria. In contrast, in 2008, SAMHSA estimated an average age of 23.4 for new heroin initiates in the USA. Further, progression from first use to regular use appears to be more rapid, with the majority of participants reporting onset of dependence problems within one year of initiation. Another unique aspect of this study population are the comparatively low levels of polysubstance use (outside of nicotine and alcohol). This early-use, fast-transition and monosubstance heroin pattern stands in sharp contrast to what is reported in many studies in the Western World. In our study, 47% of Ethnic Bulgarians and 66% of Roma used only opioids, even years after the onset of drug use. 15.4% of Ethnic Bulgarians and 66.2% of Roma reported heroin as the very first illicit drug used. Lifetime, 12.5% of Ethnic Bulgarians and 52.8% of Roma report never having used another illicit drug other than heroin more than five times. While both Roma and Ethnic Bulgarians certainly experiment with other drugs, abuse/dependence rates for these drugs are low in Ethnic Bulgarians (from 8.4% for inhalants to 28% for stimulants) and in the Roma population (from 1.3% for hallucinogens to 11.2% for stimulants).

Study phs001804

EGAD00000000049

RNA-SEQ data from oestrogen-receptor-alpha-positive metastatic lobular breast cancer sample

Dataset EGAD00000000049

EGAD00010000636

WTCCC2 Visceral Leishmaniasis samples from Brazil using Illumina 670k

Dataset EGAD00010000636

EGAD00010000638

WTCCC2 Visceral Leishmaniasis samples from Indial using Illumina 670k

Dataset EGAD00010000638

EGAD00010000640

WTCCC2 Visceral Leishmaniasis samples from Sudanl using Illumina 670k

Dataset EGAD00010000640

Non Tumour Renal Cell Line Sequencing

Dataset EGAD00001000091

Plasma DNA tissue mapping by genome-wide methylation sequencing for noninvasive prenatal, cancer, and transplantation assessments

Dataset EGAD00001001602

Second generation noninvasive fetal genome analysis reveals de novo mutations, single-base parental inheritance, and preferred DNA ends

Dataset EGAD00001003778

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