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• Whole genome, whole exome, and targeted sequencing of high-grade meningioma tumor samples.

  High-grade meningiomas frequently recur and are associated with high rates of morbidity and mortality. To determine the factors that promote the development and evolution of these tumors, we analyzed the genomes of 274 high-grade meningiomas and compared this information with data from 456 low-grade meningiomas. High-grade meningiomas had a higher mutation burden than low-grade meningiomas but did not harbor any statistically significant mutated genes aside from NF2. High-grade meningiomas also possessed significantly elevated rates of chromosomal gains and losses, especially among tumors with monosomy 22. Meningiomas previously treated with adjuvant radiation had significantly more copy number alterations than radiation-induced or radiation-naïve meningiomas. Across serial recurrences, genomic disruption preceded the emergence of nearly all mutations, remained largely uniform across time, and when present in low-grade meningiomas, correlated with subsequent progression to a higher grade. In contrast to the largely stable copy number alterations, mutations were strikingly heterogeneous across tumor recurrences, likely due to extensive geographic heterogeneity in the primary tumor. While high-grade meningiomas harbored significantly fewer overtly targetable alterations than low-grade meningiomas, they contained numerous mutations that are predicted to be neoantigens, suggesting that immunologic targeting may be of therapeutic value.

  Study EGAS00001002294

• Clonal_haematopoiesis_in_patients_with_AAA

  Recent advances in genomics have demonstrated that clonal haemopoiesis driven by leukaemia associated somatic mutations is a relatively common phenomenon that increases in frequency with advancing age. Whilst individuals with clonal haemopoiesis have an increased risk of developing haematological malignancies, they also have an increased mortality from other causes. Additionally, certain mutations are almost exclusively seen in individuals aged 70 years or older, whilst others are seen in individuals with non-haematological cancers including breast and ovarian. Recently, clonal haemopoiesis was found to be associated with a significantly increased risk of atherosclerotic cardiovascular disease. This association is thought to be causative with clonally-derived macrophages showing elevated expression of several chemokine and cytokine genes that contribute to atherosclerosis. Another vascular pathology, abdominal aortic aneurysm (AAA), increases with age and shares risk factors with atherosclerosis (including smoking, male sex, high cholesterol). However, the impact of these risk factors and the overlap between AAA and atherosclerosis is poorly understood. To investigate a possible link between clonal haemopoiesis and AAA, we will study DNA samples from 300 patients with AAA and up to 200 controls for evidence of clonal haemopoiesis. This will be done using target DNA enrichment with biotinylated RNA baits followed by high throughput sequencing.

  Study EGAS00001002873

• UK10K OBESITY TWINSUK

  In the UK10K project we propose a series of complementary genetic approaches to find new low frequency/rare variants contributing to disease phenotypes. These will be based on obtaining the genome wide sequence of 4000 samples from the TwinsUK and ALSPAC cohorts (at 6x sequence coverage), and the exome sequence (protein coding regions and related conserved sequence) of 6000 samples selected for extreme phenotypes. Our studies will focus primarily on cardiovascular-related quantitative traits, obesity and related metabolic traits, neurodevelopmental disorders and a limited number of extreme clinical phenotypes that will provide proof-of-concept for future familial trait sequencing. We will analyse directly quantitative traits in the cohorts and the selected traits in the extreme samples, and also use imputation down to 0.1% allele frequency to extend the analyses to further sample sets with genome wide genotype data. In each case we will investigate indels and larger structural variants as well as SNPs, and use statistical methods that combine rare variants in a locus or pathway as well as single-variant approaches. These TwinsUK samples are a subset of the TwinsUK cohort samples with a BMI>=40 that will be exome sequenced by the Obesity group.

  Study EGAS00001000306

• Bisulfite-converted duplexes for the strand-specific detection and quantification of rare mutations

  The identification of mutations that are present at low frequencies in clinical samples is an essential component of precision medicine. The development of molecular barcoding for next generation sequencing has greatly enhanced the sensitivity of detecting such mutations by massively parallel sequencing. However, further improvements in specificity would be useful for a variety of applications. We herein describe a technology (BiSeqS) that can increase the specificity of sequencing by at least two orders of magnitude over and above that achieved with molecular barcoding and can be applied to any massively parallel sequencing instrument. BiSeqS employs bisulfite treatment to distinguish the two strands of molecularly barcoded DNA; its specificity arises from the requirement for the same mutation to be identified in both strands. Because no library preparation is required, the technology permits very efficient use of the template DNA as well as sequence reads, which are nearly all confined to the amplicons of interest. Such efficiency is critical for clinical samples, such as plasma, in which only tiny amounts of DNA are often available. We show here that BiSeqS can be applied to evaluate transversions, as well as small insertions or deletions, and can reliably detect one mutation among >10,000 wild type molecules.

  Study EGAS00001002406

• The UVA-induced single-base mutational signature of CX5461 in human cells

  Drug-related UVA-induced photoreactions have been reported for several therapeutic compounds, including fluoroquinolones. CX5461 is a clinically relevant quinolone-derived anti-cancer small molecule with documented UVA-sensitising activity. Here we compared, by bulk and clonal whole genome sequencing (WGS) under light-protected conditions, the mutational signatures in human retinal pigment epithelial cells (RPE1) exposed to UVA, CX5461, or co-exposed to UVA and CX5461. Treatment with CX5461 or UVA alone resulted in a low SNV burden and background-like mutational profiles. In contrast, bulk sequencing of human cells co-exposed to UVA and CX5461 had a markedly higher SNV burden characterized by T>A and T>C substitutions. Furthermore, single-cell clonal expansion and sequencing of CX5461 alone, UVA alone or CX5461+UVA treatments confirmed that the pattern was only observed when cells were exposed to both UVA and CX5461. The CX5461+UVA-associated SNV signature we report arises only when CX5461-treated cells are exposed to UVA, and is not observed when CX5461-treated cells are shielded from light. We do not observe strong single base mutagenic activity of CX5461 alone, under light protected conditions. Our data emphasise the need for appropriate controls and light-exposure precautions when studying base mutagenesis activity of known photosensitiser molecules.

  Study EGAS50000001145

• Unifying recovery dynamics in heterogeneous diseases exemplified by COVID-19

  Studying the recovery dynamics in different diseases poses many difficulties, as patients often display high heterogeneity in their recovery courses. Moreover, most attempts to study disease dynamics focus on disease progression rather than disease recovery mechanisms. To model recovery dynamics, using severe COVID-19 as the example, we align heterogeneous recovery trajectories via a novel computational scheme applied to longitudinally sampled blood transcriptomes. We thus generate pseudotime trajectories, which we then link to cellular and molecular mechanisms based on cell deconvolution analysis and molecular pathway prediction, thus presenting a unique framework for studying recovery processes over time. Specifically, mature neutrophils displayed a gradual decrease during recovery, allowing superior useability for outcome prediction compared to currently used clinical markers. Further, we discovered a recovery-related regulatory change in gene programs resulting in immune rebalancing between interferon and NFkB activity and the restoration of cell homeostasis. We thus propose regulatory mechanisms governing COVID-19 recovery and suggest mature neutrophils and additional gene markers, as novel clinical biomarkers for disease outcome. Overall, we present a novel clinically relevant computational framework for modeling disease recovery, paving the way for future studies of the recovery dynamics in other diseases and tissues.

  Study EGAS00001005735

• Sclerosing epithelioid fibrosarcoma case report

  Sclerosing epithelioid fibrosarcoma (SEF) is a rare and aggressive soft tissue sarcomathought to originate in fibroblasts of the tissues surrounding tendons, ligaments and muscles. Minimally responsive to conventional cytotoxic chemotherapies, greater than 50% of SEF patients experience relapse and/or metastatic disease. SEF is most commonly discovered in middle-aged and elderly adults, but also rarely in children. A common gene fusion occurring between the EWSR1 and CREB3L1 genes has been observed in 80-90% of SEF cases. We describe here the youngest SEF patient reported to date (a 3-year-old Caucasian male) who presented with numerous bony and lung metastases. Additionally, we perform a comprehensive literature review of all SEF-related articles published since the disease was first characterized. The patient described in this report experienced persistent disease progression despite being heavily treated with multiple surgeries, radiation, numerous chemotherapies and targeted therapeutics. Primary tumor, metastatic and relapse sites were sequenced by whole genome, whole exome, and deep transcriptome next generation sequencing with comparison to a patient-matched normal blood sample. Consistent across all sequencing analyses was the disease-defining EWSR1-CREB3L1 fusion as a single feature consensus. We provide an analysis of our genomic findings and a discussion as to potential therapeutic strategies for SEF.

  Study EGAS00001005214

• Variation and transmission of the human gut microbiota across generations - shotgun data

  Although composition and functional potential of the human gut microbiota evolve over lifespan, kinship has been identified as a key covariate of microbial community diversification. To date, sharing of microbiota features within families has however mostly been assessed between parents and their direct offspring. Here, we investigate potential transmission and persistence of familial microbiome patterns and microbial genotypes in a family cohort (N=102) spanning three to five generations over the same female bloodline. We observe microbiome community composition to be associated with kinship, with seven (low-abundant) genera displaying familial distribution patterns. While kinship and current cohabitation emerged as closely entangled variables, our explorative analyses of microbial genotype distribution and transmission estimates point at the latter as a key covariate of strain dissemination. Highest potential transmission rates are estimated between sisters and mother-daughter pairs, decreasing with increasing daughter’s age, and being higher among cohabiting pairs than those living apart. Although rare, we do detect potential transmission events spanning three and four generations, primarily involving species of the genera Alistipes and Bacteroides. Overall, while our analyses confirm the existence of family-bound microbiome community profiles, transmission or co-acquisition of bacterial strains appears to be strongly linked to cohabitation.

  Study EGAS00001005649

• Variation and transmission of the human gut microbiota across generations - 16S data

  Although composition and functional potential of the human gut microbiota evolve over lifespan, kinship has been identified as a key covariate of microbial community diversification. To date, sharing of microbiota features within families has however mostly been assessed between parents and their direct offspring. Here, we investigate potential transmission and persistence of familial microbiome patterns and microbial genotypes in a family cohort (N=102) spanning three to five generations over the same female bloodline. We observe microbiome community composition to be associated with kinship, with seven (low-abundant) genera displaying familial distribution patterns. While kinship and current cohabitation emerged as closely entangled variables, our explorative analyses of microbial genotype distribution and transmission estimates point at the latter as a key covariate of strain dissemination. Highest potential transmission rates are estimated between sisters and mother-daughter pairs, decreasing with increasing daughter’s age, and being higher among cohabiting pairs than those living apart. Although rare, we do detect potential transmission events spanning three and four generations, primarily involving species of the genera Alistipes and Bacteroides. Overall, while our analyses confirm the existence of family-bound microbiome community profiles, transmission or co-acquisition of bacterial strains appears to be strongly linked to cohabitation.

  Study EGAS00001005651

• Nanopore_and_Illumina_sequencing_of_human_glioblastomas

  Liquid biopsy analysis refers to methods designed to detect tumour-specific material (e.g., circulating tumour cells or tumour DNA) in body fluids, such as urine or blood samples. A widely-used liquid biopsy approach consists of genotyping the circulating tumour DNA (ctDNA) through sequencing of plasma/serum DNA. Although useful in the context of metastatic disease (where the concentration of ctDNA is high), current liquid biopsy technologies show limited sensitivity of detection for the early detection of cancer, and low specificity, as cancer-related mutations accumulate in healthy tissues as part of the ageing process, thus making it difficult to distinguish these from tumour mutations, and because sequencing errors and true mutations show overlapping profiles. Therefore, developing liquid biopsy protocols with increased sensitivity and specificity represents an urgent clinical need. Here we harness extrachromosomal circular DNA (eccDNA) elements, which are circular DNA structures physically separated from the chromosomes of up to several Mbp long pervasive in human cancers, for liquid biopsy analysis. In this pilot study we will focus on the analysis of glioblastomas, because there is strong evidence for the presence of eccDNA in these tumour types, and because developing liquid biopsy approaches for brain tumours to reduce the invasiveness of brain tumour biopsies remains an unmet clinical need.

  Study EGAS00001005812

• Sclerosing epithelioid fibrosarcoma case report

  Sclerosing epithelioid fibrosarcoma (SEF) is a rare and aggressive soft tissue sarcomathought to originate in fibroblasts of the tissues surrounding tendons, ligaments and muscles. Minimally responsive to conventional cytotoxic chemotherapies, greater than 50% of SEF patients experience relapse and/or metastatic disease. SEF is most commonly discovered in middle-aged and elderly adults, but also rarely in children. A common gene fusion occurring between the EWSR1 and CREB3L1 genes has been observed in 80-90% of SEF cases. We describe here the youngest SEF patient reported to date (a 3-year-old Caucasian male) who presented with numerous bony and lung metastases. Additionally, we perform a comprehensive literature review of all SEF-related articles published since the disease was first characterized. The patient described in this report experienced persistent disease progression despite being heavily treated with multiple surgeries, radiation, numerous chemotherapies and targeted therapeutics. Primary tumor, metastatic and relapse sites were sequenced by whole genome, whole exome, and deep transcriptome next generation sequencing with comparison to a patient-matched normal blood sample. Consistent across all sequencing analyses was the disease-defining EWSR1-CREB3L1 fusion as a single feature consensus. We provide an analysis of our genomic findings and a discussion as to potential therapeutic strategies for SEF.

  Study EGAS00001005215

• T cell responses of ALS patients

  In this project, we aimed to understand how T cell responses contribute to the disease progression of amyotrophic lateral sclerosis (ALS). The present data is on single-cell sequencing isolated from human cerebrospinal fluid (CSF) cells from both ALS patients (n=5) and controls (n=4). This analysis was conducted as part of a bigger project which is summarized in the section below. We used flow cytometry to define T cell subsets and phenotypes in blood and CSF samples collected at the time of diagnosis on a cohort of 89 newly diagnosed ALS patients in Stockholm, Sweden. High frequency of CD4+FOXP3- effector T cells in blood and CSF was associated with poor survival whereas high frequency of activated regulatory T (Treg) cells and high ratio between activated and resting Treg cells in blood was associated with better survival. T cell profiles also predicted disease progression rate. On an independent cohort of cases and controls, we used single cell transcriptomics data to demonstrate that ALS patients had altered T cell gene expression patterns and clonally expanded CD4+ and CD8+ T cells in CSF. In summary, T cell responses contribute to disease progression of ALS, supporting modulation of adaptive immunity as a viable therapeutic option.

  Study EGAS00001006675

• Longitudinal evaluation of serum microRNAs as biomarkers for neuroblastoma burden and therapeutic p53 reactivation

  Accurate assessment of treatment response and residual disease is indispensable for the evaluation of cancer treatment efficacy. However, performing tissue biopsies for longitudinal follow-up poses a major challenge in the management of solid tumours like neuroblastoma. In the present study, we evaluated whether circulating miRNAs are suitable to monitor neuroblastoma tumour burden and whether treatment-induced changes of miRNA abundance in the tumour are detectable in serum. We performed small RNA sequencing on longitudinally collected serum samples from mice carrying orthotopic neuroblastoma xenografts that were exposed to treatment with idasanutlin or temsirolimus. We identified 57 serum miRNAs to be differentially expressed upon xenograft tumour manifestation, out of which 21 were also found specifically expressed in the serum of human high-risk neuroblastoma patients. The murine serum levels of these 57 miRNAs correlated with tumour tissue expression and tumour volume, suggesting potential utility for monitoring tumour burden. In addition, we describe serum miRNAs that dynamically respond to p53 activation following treatment of engrafted mice with idasanutlin. We identified idasanutlin-induced serum miRNA expression changes upon one day and 11 days of treatment. By limiting to miRNAs with a tumour-related induction, we put forward hsa-miR-34a-5p as a potential pharmacodynamic biomarker of p53 activation in serum.

  Study EGAS00001006678

• Human leukocyte antigen alleles associate with COVID-19 vaccine immunogenicity and risk of breakthrough infection

  SARS-CoV-2 vaccine immunogenicity varies between individuals, and immune responses correlate with vaccine efficacy. Using data from 1,076 participants enrolled in ChAdOx1 nCov-19 vaccine efficacy trials in the United Kingdom, we find that inter-individual variation in normalised antibody responses against SARS-CoV-2 spike (S) and its receptor binding domain (RBD) at 28 days following first vaccination shows genome-wide significant association with major histocompatibility complex (MHC) class II alleles. The most statistically significant association with higher levels of anti-RBD antibody was HLA-DQB1*06 (P=3.2 x 10-9), which we replicate in 1,677 additional vaccinees. Individuals carrying HLA-DQB1*06 alleles were less likely to experience PCR-confirmed breakthrough infection during the Wuhan and Alpha-variant waves compared to non-carriers (HR 0.63, 0.42-0.93, P=0.02). We identify a distinct S-derived peptide that is predicted to bind effectively to HLA-DQB1*06 compared to other equivalent alleles, and find evidence of increased spike-specific memory B-cell responses in HLA-DQB1*06 carriers at 84 days post first vaccination. Our results demonstrate association of HLA type with COVID-19 vaccine antibody response and risk of breakthrough infection, with implications for future vaccine design and implementation.

  Study EGAS00001006909

• Targeting TRIP13 in Wilms Tumor with Nuclear Export Inhibitors

  Wilms tumor (WT) is the most common renal malignancy of childhood. Despite improvements in the overall survival, relapse occurs in ~15% of patients with favorable histology WT (FHWT). Half of these patients will succumb to their disease. Identifying novel targeted therapies in a systematic manner remains challenging in part due to the lack of faithful preclinical in vitro models. We established ten short-term patient-derived WT cell lines and characterized these models using low-coverage whole genome sequencing, whole exome sequencing and RNA-sequencing, which demonstrated that these ex-vivo models faithfully recapitulate WT biology. We then performed targeted RNAi and CRISPR-Cas9 loss-of-function screens and identified the nuclear export genes (XPO1 and KPNB1) as strong vulnerabilities. We observed that these models are sensitive to nuclear export inhibition using the FDA approved therapeutic agent, selinexor (KPT-330). Selinexor treatment of FHWT suppressed TRIP13 expression, which was required for survival. We further identified in vitro and in vivo synergy between selinexor and doxorubicin, a chemotherapy used in high risk FHWT. Taken together, we identified XPO1 inhibition with selinexor as a potential therapeutic option to treat FHWTs and in combination with doxorubicin, leads to durable remissions in vivo.

  Study EGAS00001007389

• Integrative Multi-Omics and Drug Sensitivity Profiling Reveals Potential Predictive Biomarkers in Pediatric Solid Tumors from the INFORM Registry

  Cure rates for childhood malignancies using established therapy protocols have increased to an average of 80% but have reached a plateau. Moreover, survival rates are particularly low for some pediatric tumors (such as high-risk Group3 medulloblastomas, osteosarcomas, Ewing sarcomas, high-risk neuroblastomas and high-grade gliomas) and dismal for patients with relapsed malignancies. A functional drug response profiling platform for pediatric solid and brain tumors has been established within the INFORM program to identify patient-specific vulnerabilities, biomarkers and unravel molecular mechanisms associated with drug response profiles for clinical translation. Here, we perform a multi-omics analysis using drug sensitivity profiles, genomic, and transcriptomic data of 81 pediatric solid tumor samples. The integrative analysis revealed two multi-omics signatures with high drug sensitivity predictivity. One signature highlighted neuroblastoma samples with sensitivity to navitoclax (a BCL-2 family inhibitor). A second signature was significantly specific to a subset of Wilms tumors harboring the SIX1 (Q177R) hotspot mutation, which had high expression of MGAM, PTPN14, STAT4 and KDM2B and high sensitivity to MEK inhibitors. A patient-specific causal interaction network analysis elucidated the potential biological mechanism of action between MEK inhibitors and the SIX1 mutation in n = 6 Wilms tumor samples.

  Study EGAS00001008249

• Single-cell level characterization of B cell depletion and repopulation following rituximab in systemic lupus erythematosus

  Rituximab, a CD20+ B cell depletion therapy, is frequently used in the treatment of systemic lupus erythematosus (SLE). However, variability in patient response highlights the need for a deeper understanding of the underlying immune cell dynamics of B cell depletion and repopulation. In this study, we conducted longitudinal single-cell profiling of nine SLE patients treated with rituximab from pretreatment to up to 15 months post-treatment. These were compared to eight healthy controls. We profiled PBMCs via 10X Genomics single-cell RNA, surface protein (CITE-seq), B cell receptor (BCR), and T cell receptor (TCR) sequencing and sequenced bulk BCR repertoires in parallel. For single cell sequencing, 10 pools were created with an equal number of cells from 4 samples each. Samples collected at different timepoints from the same patient were distributed across different pools to allow demultiplexing of individuals using genotypes captured from the scRNA-seq data. Libraries were pooled using a ratio of GEX:CITE:TCR:BCR=9:2:1:1 and were sequenced across two lanes of the NovaSeq 6000. For bulk BCR sequencing, individual samples were demultiplexed using primer barcodes and paired-end BCR amplicon reads were merged prior to submission; data are provided as unmapped single-end reads.

  Study EGAS00001006798

• Multiple_myeloma_precursor_genomics

  Whole genome sequencing of up to 45 multiple myeloma precursor samples and matched normals pairs.

  Study EGAS00001006312

• OAC RNASeq

  Please see our related publication, 'Molecular profiling to predict immunochemotherapy outcomes in esophageal adenocarcinoma'

  Study EGAS00001006468

• Reference single cell SNP array dataset from Coriell for training and validation of method for accurate single cell genotyping

  This dataset contains DNA from B-lymphocytes from 2 Coriell families and 4 individuals hybridized to HumanKaryomap BeadChip Array. Single cells from subjects GM12878 and GM7228 were amplified using multiple displacement amplification (SureMDA) according to Infium Karyomapping Assay Guide. Bulk DNA was processed and hybridized to an array for subject GM12878, GM07224 and GM07225.

  Dataset EGAD00001006376

• A Single Cell Transcriptomic Analysis of Human Neocortical Development

  Defining the number, proportion, or lineage of distinct cell types in the developing human brain is an important goal of modern brain research. We produced single cell transcriptomic profiles for 40,000 cells at mid-gestation to define deep expression profiles corresponding to all known major cell types at this developmental period and compare this with bulk tissue profiles. We identified multiple transcription factors (TFs) and co-factors expressed in specific cell types, including multiple new cell-type-specific relationships, providing an unprecedented resource for understanding human neocortical development and evolution. This includes the first single-cell characterization of human subplate neurons and subtypes of developing glutamatergic and GABAergic neurons. We also used these data to deconvolute single cell regulatory networks that connect regulatory elements and transcriptional drivers to single cell gene expression programs in the developing CNS. We characterized major developmental trajectories that tie cell cycle progression with early cell fate decisions during early neurogenesis. Remarkably, we found that differentiation occurs on a transcriptomic continuum, so that differentiating cells not only express the few key TFs that drive cell fates, but express broad, mixed cell-type transcriptomes prior to telophase. Finally, we mapped neuropsychiatric disease genes to specific cell types, implicating dysregulation of specific cell types in ASD, ID, and epilepsy, as the mechanistic underpinnings of several neurodevelopmental disorders. Together these results provide an extensive catalog of cell types in human neocortex and extend our understanding of early cortical development, human brain evolution and the cellular basis of neuropsychiatric disease.

  Study phs001836

• Somatic Genome Dynamics of Barrett's Esophagus Patients with Non-Cancer and Cancer Outcomes

  A case-control study was designed with 80 participants diagnosed with Barrett's Esophagus (BE) selected from a larger case-cohort study (Li et al., 2014, PMID: 24253313) within the Seattle Barrett's Esophagus Program (SBEP) at the Fred Hutchinson Cancer Research Center. The study included 40 cases with BE with non-cancer outcomes, "NCO", who did not progress to esophageal adenocarcinoma (EA), and 40 controls who progressed to an endoscopically detected, incident EA (cancer outcome, "CO"). For each patient, two timepoints were evaluated (T1 and T2). NCO were matched to CO based on age at T1 (T1=first endoscopy with sufficient sample availability), and time between T1 and T2 (T2 in CO=time of diagnosis of incident EA). In 10 NCO, a third time point (T3), a mean of 13.2 years after T1, was also sequenced. Mapped endoscopic biopsies, at 1/3 and 2/3 annotated distances from the gastroesophageal junction within the Barrett's segment, were sampled per patient at each timepoint. Each biopsy was purified to separate BE epithelium from stroma, and DNA was extracted from purified epithelium for 60X WGS and 2.5M SNP array; normal control blood (N=62) or normal gastric sample (N=18) were analyzed by 30X WGS and 2.5M SNP array. An additional seven normal gastric biopsy samples in seven patients with normal control blood were also sequenced at 60X as controls. All research participants contributing clinical data and esophageal samples to this study provided written informed consent, subject to oversight by the Fred Hutchinson Cancer Research Center IRB Committee D (Reg ID 5619).

  Study phs001912

• Clonally selected lines after CRISPR/Cas editing are not isogenic

  The CRISPR-Cas9 system has enabled researchers to precisely modify/edit the sequence of a genome. A typical editing experiment consists of two steps: (i) editing cultured cells and (ii) selection of clones, which are presumed to be isogenic, with and without the intended edit. The application of the CRISPR-Cas9 system may result in off-target edits, while cloning would reveal culture-acquired mutations. We analyzed the extent of the former and of the latter by whole genome sequencing (WGS) involving separate genomic loci in three experiments conducted by three independent laboratories. In all experiments we hardly found any off-target edits, while we detected hundreds to thousands of single nucleotide mutations unique to each clone after relatively short culture of 10-20 passages. Notably, clones also differed in copy number alterations that were several kb to several mb in size, representing the largest source of genomic divergence among clones. This study in dbGaP includes data from experiments carried out by investigators at the Oklahoma Medical Research Foundation and Baylor College of Medicine (OMRF/BCM) involving an iPSC line derived from fibroblasts of a female patient (line c7) carrying a constitutional heterozygous variant (chr1:1,464,679 C>T; GRCh37) in exon 15 of the ATPase family, AAA domain containing 3A (ATAD3A) gene. The parental line was edited by introducing a double stranded break at the variant allele to correct the variant by homology directed repair. Two unedited control clones (clone7 and clone8) and one edited clone (SC20) were selected for WGS.

  Study phs003110

• Norepinephrine Transporter Blockade as a Pathophysiological Biomarker in Neurogenic Orthostatic Hypotension

  The clinical picture of autonomic failure is dominated by disabling orthostatic hypotension. Recent developments in the understanding of the underlying pathophysiology of the discrete clinical forms of autonomic failure have revealed that participants with multiple system atrophy (MSA) are characterized by impairment of central autonomic pathways crucial for autonomic cardiovascular control, but have intact peripheral postganglionic noradrenergic fibers. Participants with MSA have peripheral residual sympathetic tone that is no longer modulated by central autonomic centers and is not under baroreflex control and, therefore, cannot be harnessed to improve their orthostatic hypotension. On the other hand, participants with pure autonomic failure (PAF) and with Parkinson' s disease (PD) are characterized by neurodegeneration and loss of peripheral noradrenergic fibers, as evidenced by low levels of plasma norepinephrine and absent cardiac uptake of labeled catechols. Pharmacological inhibition of the norepinephrine transporter (NET) is an example of an approach that can take advantage of the participants' own residual sympathetic tone. NET inhibition will increase synaptic norepinephrine that is tonically released in MSA participants by preventing its reuptake. This should result in an increase in blood pressure. This is a longitudinal study of 50 participants, age 18-80 years with neurogenic orthostatic hypotension associated with impaired reflexes. The purpose of this study is to determine if the magnitude of the pressor response to atomoxetine at study entry, will be useful as a biomarker to differentiate those participants that will ultimately be shown to have pre-ganglionic (central) lesions (MSA) with those shown to have post ganglionic (peripheral) lesions (PAF and PD).

  Study phs001595

• Follow-up of Ovarian Cancer Genetic Association and Interaction Studies (FOCI)

  (Excerpted/paraphrased from original grant application): FOCI seeks to expand our understanding of epithelial ovarian cancer through a coordinated and comprehensive approach. Project 1 will focus on discovery, expansion, and replication. By pooling GWAS, we expect to identify new associations and achieve independent replication, explore whether there are risk variants specific for histologic subtypes, and evaluate structural polymorphisms - copy number variants - as risk factors. Finally, Project 1 will leverage the GWAS data to correlate DNA variants with a new endpoint - survival. Project 2 will focus on biological studies designed to help inform interpretation of findings from Project 1. This will include efforts to identify the functional consequences of variants and improve understanding of biological mechanisms. Project 3 will include epidemiologic studies of gene by gene interaction, gene by environment interaction, and development of risk prediction models. The collective effort builds upon the strengths and history of collaboration inherent in the Ovarian Cancer Association Consortium (OCAC), a multidisciplinary group comprised of epidemiologists, genetic epidemiologists, statistical geneticists, molecular and cell biologists and clinicians that was formed in 2005. The FOCI Cohort is utilized in the following dbGaP sub-studies. To view genotypes, other molecular data, and derived variables collected in these sub-studies, please click on the following sub-studies below or in the "Sub-studies" box located on the right hand side of this top-level study page phs001133 FOCI Cohort. phs001131 Affymetrix Exome Chip phs001132 GWAS Meta Analysis phs001142 Mayo Omni Express phs001150 Mayo 2 5M

  Study phs001133

• Decoding Human Heart Morphogenesis through Single-cell Multi-modal Analyses

  The heart, which is the first organ to develop, is highly dependent on its form to function. However, how diverse cardiac cell types spatially coordinate to create complex morphological structures critical for heart function remains to be elucidated. Here, we show that integration of single cell RNA-sequencing with high-resolution multiplexed error-robust fluorescent in situ hybridization (MERFISH) not only resolves the identity of cardiac cell types developing the human heart but also provides a spatial mapping of individual cells that enables illumination of their organization into cellular communities forming distinct cardiac structures. We discovered that many of these cardiac cell types further specified into subpopulations exclusive to specific communities, supporting their specialization according to cellular ecosystem and anatomic region. In particular, ventricular cardiomyocyte subpopulations displayed an unexpected complex laminar organization across the ventricular wall and formed, with other cell subpopulations, several cellular communities. Interrogating cell-cell interactions within these communities revealed signaling pathways orchestrating the spatial organization of cardiac cell subpopulations during ventricular wall morphogenesis. In vivo conditional genetic mouse models and in vitro human pluripotent stem cell studies confirmed an intricate multicellular PLXN-SEMA crosstalk among specific ventricular cardiomyocyte, fibroblast and endothelial cell subpopulations that directs the compaction of the ventricular wall layers. Thus, these detailed findings into the cellular social interactions and specialization of cardiac cell types constructing and remodeling the human heart offer new insights into structural heart diseases as well as engineering complex multi-cellular tissues for human heart repair. Reprinted from Farah et al. (In press), with permission from Nature.

  Study phs002031

• NCI Cancer Model Development for the Human Cancer Model Initiative (HCMI)

  The Cancer Model Development Center (CMDC) project is part of the Human Cancer Model Initiative (HCMI) and is managed by the Office of Cancer Genomics within the Center for Cancer Genomics at the National Cancer Institute (NCI). The goal of the HCMI is to generate approximately 1000 novel human "next generation" tumor-derived models as a community resource. The models aim to faithfully recapitulate the cellular complexity and heterogeneity of human tumors. The CMDCs are tasked by the NCI with producing a proportion of the cancer models. Wellcome Sanger Institute (WSI) is a contributing member of the HCMI. Models developed by HCMI are being derived from a number of tumor types and subtypes (see below), including rare adult, pediatric and understudied cancers. The number of models and the clinical sites from which patient samples are acquired is increasing. Normal tissue, originating tumor, and tumor-derived models are sequenced. The next generation cancer models are associated with rich clinical and genomic data through the NCI Genomic Data Commons for all researchers to advance cancer research. The CMDC and HCMI models will serve as excellent tools in research to provide insight into the pathways that influence tumor growth and progression, how tumors respond to therapy, etc. Ultimately, the data generated using these models will support the development of personalized oncology. There is a delay between registered IDs and model availability. The current model count is found in The HCMI Searchable Catalog which is updated when new models are available for distribution to the research community.

  Study phs001486

• Epigenetic Control of Topoisomerase 1 by MacroH2A1.1

  DNA transactions introduce torsional constraints that pose an inherent risk to genome integrity. While topoisomerase 1 (TOP1) activity is essential for removing DNA supercoiling, aberrant stabilization of TOP1:DNA cleavage complexes (TOP1ccs) can result in cytotoxic single-stranded DNA lesions (SSLs). What protects the genome from aberrant TOP1 activity remains unknown. Using a combination of CUT&RUN and TOP1 Covalent Adduct Detection (CAD) Seq in MDA-MB-231 breast cancer cells (American Type Culture Collection, ATCC), we identified chromatin context as an essential means to ensure TOP1cc resolution at TOP1 hot spots. Through its ability to bind poly(ADP-ribose) (PAR), a protein modification required for TOP1cc repair, the histone variant macroH2A1.1 establishes a TOP1-permissive chromatin environment, while the alternatively spliced macroH2A1.2 isoform is unable to bind PAR or protect from TOP1ccs. MacroH2A1 isoform-specific analyses were based on reconstitution of macroH2A1.1 knockout cells with wild-type or PAR binding-deficient FLAG-tagged macroH2A1.1 in human breast cancer cells. Mechanistically, we find that macroH2A1.1 facilitates the recruitment of the TOP1cc repair factor XRCC1 in response to both endogenous and drug-induced topological stress. Impaired macroH2A1.1 splicing, a frequent cancer feature, was predictive of increased sensitivity to TOP1 poisons in a pharmaco-genomic screen in breast cancer cells, and macroH2A1.1 inactivation mirrored this effect in breast and ovarian cancer cells. Consistent with this, low macroH2A1.1 expression correlated with improved survival in cancer patients treated with TOP1 inhibitors (TOP1i). We propose that macroH2A1 alternative splicing serves as an epigenetic modulator of TOP1-associated genome maintenance and a potential cancer vulnerability.

  Study phs003729

• Genomic Characterization of Duke Melanoma Brain Metastases

  Previous work has shown that melanoma brain metastases (MBM) have a unique molecular profile compared to extracranial metastases (ECM). Description of the biology of MBM and associations between biological features and clinical outcomes will facilitate the design of rational therapies for patients with MBM. To better characterize the molecular profile of MBM, we conducted whole exome sequencing (WES) and total RNA sequencing on MBM tissues obtained from an independent cohort of 14 patients from Duke University Medical Center. This cohort adds valuable patient samples to the field and enables strengthened investigation of somatic mutations and gene expression profiles within and between cohorts. The mutational landscape and gene expression of MBM from the Duke cohort resembled those previously reported in a previously published dataset of WES and RNA sequencing in MBM and matched ECM tissues from UT MD Anderson Cancer Center (Fischer et al., 2019 30787016). Duke MBM demonstrated similar rates of mutations in genes associated with melanoma, including BRAF, NRAS, and PTEN, as the MD Anderson cohort. Immune populations in MBM were similar between Duke and MD Anderson MBM, though Duke samples demonstrated more resting CD4+ memory T cells. The ratio of M2 to M1 macrophages in the Duke cohort was similar to that of the MD Anderson cohort. In MBM, a higher M2:M1 ratio may contribute to an immune-suppressive tumor microenvironment. Sequencing data and sample attributes from MBM tissues obtained from 14 patients from Duke University Medical Center will be available through dbGaP.

  Study phs003009

• Colon Cancer Family Registry (Colon CFR)

  The Colon Cancer Family Registry Cohort (CCFRC www.coloncfr.org) is a cohort of families recruited through six study sites located in the USA, Canada, Australia, and New Zealand. The CCFRC was formed as a resource to support studies on the etiology, prevention, and clinical management of colorectal cancer. Recruitment protocols fall broadly into two main categories: population-based and clinic-based ascertainment. Between 1998 and 2013, the CCFR recruited and interviewed probands who were either recently diagnosed with colorectal cancer that was reported to state or regional population cancer registries in the United States (Washington, California, Arizona, Minnesota, Colorado, New Hampshire, North Carolina, and Hawaii), Australia (Victoria), and Canada (Ontario) or were from multiple-case families referred to family-cancer clinics in the United States (Mayo Clinic in Rochester, Minnesota, and Cleveland Clinic in Cleveland, Ohio), Australia (Melbourne, Adelaide, Perth, Brisbane, and Sydney), New Zealand (Auckland), and Canada. Cases with known familial adenomatous polyposis were excluded. Once recruited, probands were asked for permission to contact their relatives for recruitment. Collectively, we have enrolled 42,49874 participants in 15,048 families who participated in standardized protocols used to collect information regarding family cancer history and colorectal cancer risk factors and biospecimens. All participants of population-based case families (excluding control-families) and clinic-based families are asked to provide updates on their personal and family history of cancer as well as their history of surgery, cancer screening, and some risk factors every 4-5 years either by telephone interview or by self-completed questionnaire via mail or online (PMID: 17982118).

  Study phs002733

• CAGE-seq analysis of osteoblast derived from cleidocranial dysplasia human induced pluripotent stem cells

  Non-coding RNAs (ncRNAs) comprise a major portion of transcripts and serve an essential role in biological processes. Although the importance of major transcriptomes in osteogenesis has been extensively studied, the function of ncRNAs in human osteogenesis remains unclear. Previously, we developed hiPSCs from patients with cleidocranial dysplasia (CCD) caused by runt-related transcription factor 2 (RUNX2) haploinsufficiency. To gain insight into ncRNAs in osteogenesis, we surveyed differential ncRNA expression profiling and promoter differences of RUNX2 using patient-specific iPSCs and cap analysis gene expression (CAGE) technology to define the promoter landscape. Revertant iPSCs (Rev1 iPSCs) edited by CRISPR/Cas9 system to harbor mutation-corrected RUNX2 exhibited increased proximal promoter expression of RUNX2, while CCD iPSCs did not. We identified 2271 ncRNA genes with altered expression levels before and after differentiation, 31 of which showed at least 20-fold higher expression in Rev1 iPSCs. Bioinformatic analysis also categorized AC007392.3, LINC00379, RP11-122D10.1, and RP11-90J7.2 as enhancer regulatory regions, and HOXA-AS2, MIR219-2, and RP11-834C11.3 as dyadic regulatory regions of these ncRNAs. In addition, two miRNAs, termed MIR199A2 and MIR152, were found to have high enrichment of osteogenic-related terms. Upon further examination of the role of MIR152 on osteoblast differentiation, we found that MIR152 knockdown induced upregulation of ALP and COL1A1 in Saos-2 cells. Thus, ncRNAs were found to regulate the osteogenic differentiation potentials of hiPSCs that are used for bone regeneration and repair owing to their differentiation potentials. These data allow understanding ncRNA profiles of hiPSCs during osteogenesis.

  Study JGAS000248

• DNA demethylation is associated with malignant progression of low-grade gliomas

  To elucidate the mechanisms of tumor progression is key to discovering strategies for treating patients with lower-grade glioma. Thus to characterize the changes in the molecular profile during malignant progression, we performed methylation array analysis (122 tumors), whole-exome sequencing (36 tumors and matched normal samples), and RNA sequencing (31 tumors). This cohort included 24 matched pairs of initial lower-grade gliomas and recurrent tumors, most of which showed malignant progression when they recurred. Nearly half of IDH-mutant glioblastomas that had progressed from lower-grade gliomas exhibited characteristic partial DNA demethylation in previously methylated genomic regions of their corresponding initial tumors, which had the glioma CpG island methylator phenotype (G-CIMP). In these glioblastomas, cell cycle-related genes were upregulated, RB and PI3K-AKT pathway genes were frequently altered. Notably, bioinformatics analysis revealed that late-replicating domain was significantly enriched in the demethylated regions that were mostly located in non-regulatory regions, suggesting that the loss of DNA methylation during malignant transformation may involve mainly passive demethylation due to a delay in maintenance of methylation during accelerated cell division. Nonetheless, a limited number of genes including IGF2BP3, which potentially drives cell proliferation, were presumed to be upregulated due to demethylation of their promoter. Thus, cell proliferation was further promoted by passive demethylation in the promoter region of these oncogenic genes. Our data indicated that demethylation of the G-CIMP profile found in a subset of recurrent gliomas reflects accelerated cell divisions accompanied by malignant transformation. Oncogenic genes activated by such epigenetic change represent potential therapeutic targets.

  Study JGAS000146

• Sequencing and analysis of a South Asian-Indian personal genome

  India with over 1.3 billion people is estimated to carry three times more genetic diversity compared to Europe. Next-generation sequencing technologies have facilitated the understanding of diversity by enabling whole genome sequencing at greater speed and lower cost. While multiple genomes from people of European and a few of Asian descent have been sequenced, only recently a single male genome from the Indian subcontinent at sufficient depth and coverage was published. We have in this study sequenced and analyzed the genome of a South Asian Indian female (SAIF) from the Indian state of Kerala. We identified over 3.4 million SNPs in this genome including over 89,873 private variations. Comparison of the SAIF genome with several published personal genomes revealed that this individual shared ~50% of the SNPs with each of these genomes. Analysis of the SAIF mitochondrial genome showed that it is closely related to the U1a3 haplogroup which has been previously observed in Kerala. We assessed the SAIF genome for SNPs with health and disease consequences and found that the individual was at a higher risk for multiple sclerosis and a few other diseases. In assessing SNPs that modulate drug response we found a variation that predicts a favorable response to metformin used for treating diabetes. SNPs predictive of adverse reaction to warfarin indicated that the SAIF individual is not at risk for bleeding if treated with typical doses of warfarin. In addition to these, we report the presence of several other SNPs of medical relevance.

  Study EGAS00001000328

• An autoinflammatory RIG-I variant causing Singleton-Merten Syndrome associates with small non-coding Y-RNAs

  The RNA sensor RIG-I performs a critical role in surveying the cytoplasm for the presence of viral nucleic acids and initiating the downstream anti-viral type I interferon pathway. Through recognition of specific features, such as the presence of a 5’ di/triphosphate motif or highly structured base-paired regions, RIG-I effectively differentiates between RNA of viral- and self-origin. In Singleton-Merten syndrome (SMS), gain-of-function variants in RIG-I lead to a breakdown in this surveillance system and results in aberrant sensing of self-RNAs and deleterious upregulation of type I interferons. The identity of the self-RNAs binding to gain-of-function RIG-I mutants has remained elusive and would provide a greater understanding of the aetiology of SMS. Here we used an infrared individual-nucleotide resolution UV-crosslinking and immunoprecipitation (irCLIP) approach to determine the RNA profile bound to a previously characterised ATPase-deficient SMS variant, RIG-IC268F. irCLIP identified a broad array of self-RNAs, primarily those transcribed by RNA polymerase III, that were bound to RIG-I. Subsequent native RNA immunoprecipitation confirmed a prominent and specific interaction between RIG-IC268F and Y-RNAs, a family of four structurally similar non-coding RNAs. Manipulation of Y-RNAs alone by targeting either Y-RNA transcripts or Y-RNA stabilising proteins was insufficient to negate RIG-I induce interferon, hinting at a broader profile of RNA polymerase III-derived RNAs being influential in driving the sterile activation of gain-of-function RIG-I variants.

  Study EGAS50000001660

• UK10K NEURO FSZNK

  In the UK10K project we propose a series of complementary genetic approaches to find new low frequency/rare variants contributing to disease phenotypes. These will be based on obtaining the genome wide sequence of 4000 samples from the TwinsUK and ALSPAC cohorts (at 6x sequence coverage), and the exome sequence (protein coding regions and related conserved sequence) of 6000 samples selected for extreme phenotypes. Our studies will focus primarily on cardiovascular-related quantitative traits, obesity and related metabolic traits, neurodevelopmental disorders and a limited number of extreme clinical phenotypes that will provide proof-of-concept for future familial trait sequencing. We will analyse directly quantitative traits in the cohorts and the selected traits in the extreme samples, and also use imputation down to 0.1% allele frequency to extend the analyses to further sample sets with genome wide genotype data. In each case we will investigate indels and larger structural variants as well as SNPs, and use statistical methods that combine rare variants in a locus or pathway as well as single-variant approaches. This Finnish schizophrenia sample set has been collected from a population cohort using national registers. The entire sample collection consists of 2756 individuals from 458 families of whom 931 are diagnosed with schizophrenia spectrum disorder. Families outside Kuusamo (n=288) all had at least two affected siblings. All diagnoses are based on DSM-IV and for a large fraction of cases there is cognitive data.For further details/descriptions with regard to this data set please contact Tiina Paunio (tiina.paunio@thl.fi)

  Study EGAS00001000119

• UK10K NEURO ASD SKUSE

  In the UK10K project we propose a series of complementary genetic approaches to find new low frequency/rare variants contributing to disease phenotypes. These will be based on obtaining the genome wide sequence of 4000 samples from the TwinsUK and ALSPAC cohorts (at 6x sequence coverage), and the exome sequence (protein coding regions and related conserved sequence) of 6000 samples selected for extreme phenotypes. Our studies will focus primarily on cardiovascular-related quantitative traits, obesity and related metabolic traits, neurodevelopmental disorders and a limited number of extreme clinical phenotypes that will provide proof-of-concept for future familial trait sequencing. We will analyse directly quantitative traits in the cohorts and the selected traits in the extreme samples, and also use imputation down to 0.1% allele frequency to extend the analyses to further sample sets with genome wide genotype data. In each case we will investigate indels and larger structural variants as well as SNPs, and use statistical methods that combine rare variants in a locus or pathway as well as single-variant approaches. This sample set of UK origin consists of clinically identified subjects with Autism Spectrum Disorders, mostly without intellectual disability (ie. Verbal IQs >70). The subjects represent children and adults with Autism, Asperger syndrome or Atypical Autism, identified according to standardized research criteria (ADI-algorithm, ADOS). A minority has identified comorbid neurodevelopmental disorders (e.g. ADHD). Family histories are available, with measures of broader phenotype in first-degree relatives.For further information on this cohort please contact David Skuse (d.skuse@ucl.ac.uk).

  Study EGAS00001000114

• Characterization of a human iPSC-derived islet differentiation model

  To explore the contribution of islet development to T2D pathogenesis, we characterised the transcriptomes of 3 human iPSC lines (from independent donors) differentiated along the islet development lineage. Whole transcriptome RNA-seq was performed at 7 developmental stages: definitive endoderm, primitive gut tube, posterior foregut (PF), pancreatic endoderm, endocrine progenitors, endocrine-like cells, and beta-like cells (BLC). Differentially-expressed (ΔExp) genes were assigned to the stage in which they were most upregulated (versus baseline iPSC profile), and used to assess the enrichment of T2D GWAS genes, as well as to predict the upstream transcription factors at each developmental stage.We found 9409 ΔExp genes across all stages, including known markers of islet development (NEUROG3, INS). Genes ΔExp in the most-differentiated (BLC) stage were significantly enriched for genes within the 99% credible sets of T2D GWAS loci. Despite this enrichment in BLC only, over 70% of genes mapping within the credible sets were ΔExp before this stage, highlighting the relevance of key T2D GWAS genes in islet development: e.g. expression of TCF7L2 peaked during the PF stage (log2FC=1.2; q=8.5×10-10). REST and LMNA are also highlighted by the analyses as having a potential function in islet development, considering their pattern of expression and those of the genes they regulate. Transcriptomic signatures derived from this iPSC differentiation model highlight T2D-associated GWAS loci, monogenic diabetes genes and potential master regulators of gene networks with plausible function in islet development. All these provide clues for developmental mechanisms contributing to T2D diabetes pathology.

  Study EGAS00001002721

• TRACERx 100: whole exome data of the first 100 TRACERx tumours

  BACKGROUNDTRACERx (TRAcking Cancer Evolution through therapy (Rx)) is a prospective cohort study designed to investigate intratumor heterogeneity (ITH) in relation to clinical outcome, and to determine the clonal nature of driver events and evolutionary processes in early stage non-small cell lung cancer (NSCLC). METHODSMultiregion high-depth whole-exome sequencing (M-seq) was performed on 100 early stage NSCLC tumors resected prior to systemic therapy. A total of 327 tumor regions were sequenced and analyzed to define evolutionary histories, obtain a census of clonal and subclonal events, and assess the relationship between ITH and recurrence-free survival (RFS). RESULTSWidespread ITH was observed for both somatic copy number alterations (median 48% [0.03-88%]) and mutations (median 30% [0.5-93%]). Driver mutations in EGFR, MET, BRAF and TP53 were almost always clonal. However, heterogeneous driver alterations occurring later in evolution were found in over 75% of tumors and were common in PIK3CA, NF1 and genes involved in chromatin modification and DNA response and repair. Genome doubling and ongoing dynamic chromosomal instability (CIN), illustrated by mirrored subclonal allelic imbalance, were identified as causes of ITH resulting in parallel evolution of driver copy number events, including amplifications of CDK4, FOXA1, and BCL11A. Elevated copy number heterogeneity was associated with shorter RFS (HR=4.9, P=0.00044), which remained significant in a multivariate analysis.CONCLUSIONSITH mediated through CIN, rather than point mutational heterogeneity, was associated with increased risk of relapse, supporting its value as a prognostic predictor, and the need to target this high-risk phenotype.

  Study EGAS00001002247

• MYCN Amplification and ATRX Mutations are Incompatible in Neuroblastoma

  Age and stage of disease at diagnosis is associated with outcome in neuroblastoma. A previous study identified ATRX point mutations and in-frame deletions in older patients with stage 4 disease. To validate the frequency of ATRX mutations and assess the relation of mutations and known prognostic variables, we collaborated with the Children’s Oncology Group and sequenced the entire ATRX genomic locus in 475 neuroblastoma tumor samples to validate the frequency of ATRX mutations and assess the relation of mutations and known prognostic variables. Older age at diagnosis, stage 4 disease, and unfavorable histology were significantly associated with mutations in the ATRX gene; MYCN amplification was mutually exclusive from ATRX mutations. To directly test if ATRX mutations and MYCN amplification are incompatible in neuroblastoma, we mutated the ATRX gene in MYCN amplified neuroblastoma cell lines and ectopically expressed MYCN in ATRX mutant neuroblastoma cell lines. Both approaches showed that ATRX and MYCN amplification are incompatible in neuroblastoma in culture and in vivo. To gain a better understanding of the underlying molecular and cellular mechanisms of this incompatibility, we performed a series of experiments including telomere analysis, epigenetic profiling, electron microscopic analysis and metabolic profiling. We discovered that induction of MYCN expression in ATRX mutant neuroblastomas cells leads to metabolic reprogramming, mitochondrial dysfunction, increased reactive oxygen species and replicative stress. We propose that this leads to synthetic lethality in ATRX mutant neuroblastomas because of the underlying replicative stress in those cells as a result of dysfunction of this essential histone chaperone.

  Study EGAS00001003257

• Transcriptome_human_nasal_epithelium

  1. Odors are detected, firstly, by olfactory sensory neurons (OSNs) in the olfactory epithelium of the nose. This neurons then project directly to the olfactory bulb in the brain. Olfaction depends on cellular regeneration of the OE, olfactory bulb and hippocampus, and on their continual re-wiring. The olfactory neural pathway includes regions of the frontal, temporal and limbic brain, which in turn overlap with brain areas involved in brain disorders. OSNs are the only aspect of the human brain exposed to the external environment. This not only makes them vulnerable to environmental changes, but also accessible for biomedical studies.We have already sequenced and developed a protocol for analyzing the transcriptome of mouse main olfactory epithelium and single OSNs. We propose here to perform a similar study for samples from the human olfactory epithelium. We have developed a minimally invasive method for obtaining human OSNs, among other cells from the nasal epithelium. In this experiment, we have obtained cell samples from the olfactory epithelium, including OSN, from healthy volunteers. We would like to further characterize them by RNA sequencing. This will give us valuable insight into human olfaction. It will also provide a first step into a new avenue to study, and find biomarkers for, brain diseases though the analysis of these easily available neurons. 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 EGAS00001001294

• Phylogenetic analysis of treatment-naive metastases using whole exome and genome sequencing data

  Metastases are responsible for the majority of cancer related deaths and are often difficult to treat successfully. Even for metastases that occur subsequent to treatment, we do not know whether such relapsing metastases were originally heterogeneous or if the observed heterogeneity is a consequence of therapy. Given that treatment can influence evolutionary dynamics by selecting resistant clones, imposing bottlenecks on cancer cell populations, and even inducing novel somatic mutations, characterizing the prior standing variation among metastases remains an important goal to predict initial therapeutic response. To quantify the heterogeneity at clinical presentation of advanced disease, we surveyed the literature for patients in which at least two treatment-naïve metastases underwent genome/exome-wide sequencing. Across all cancer types surveyed, only 18 subjects were found to fulfill this requirement. Including previously unpublished data from two subjects, we analyzed data of 74 untreated metastases and inferred cancer phylogenies. Putative driver gene mutations were acquired at a 2-fold higher rate on the trunk of all metastases than on branches. Mutations in driver genes along the trunk were strongly enriched for predicted functional consequences. Using a stochastic mathematical model, we find that driver gene heterogeneity among metastases mostly occurs in slowly growing cancers for highly advantageous driver gene mutations. Seeding efficiency only weakly affects the heterogeneity among metastases. These results explain why functional driver gene heterogeneity is uncommon in advanced disease prior to treatment, thus providing optimism for future therapies that depend on mutations across metastases.

  Study EGAS00001002777

• Mutation-specific non-canonical pathway of PTEN as a distinct therapeutic target for glioblastoma

  PTEN is one of most frequently altered tumor suppressor genes in malignant tumors. The dominant negative effect of PTEN alteration suggests that aberrant function of PTEN mutation might be more disastrous than deletion, the most frequent genomic event in glioblastoma (GBM). This study aimed to understand the functional properties of various PTEN missense mutations and to investigate their clinical relevance. The genomic landscape of PTEN alteration was analyzed using the Samsung Medical Center GBM cohort and validated via The Cancer Genome Atlas dataset. Several hot-spot mutations were identified, and their subcellular distributions and phenotypes were evaluated. We established a library of cancer cell lines that over-express these mutant proteins using the U87MG and patient-derived cell models lacking functional PTEN. PTEN mutations were categorized into two major subsets: missense mutations in the phosphatase domain and truncal mutations in the C2 domain. We determined the subcellular compartmentalization of four mutant proteins (H93Y, C124S, R130Q, and R173C) from the former group and found that they had distinct localizations; those associated with invasive phenotypes (‘edge mutations’) localized to the cell periphery, while the R173C mutant localized to the nucleus. Invasive phenotypes derived from edge substitutions were unaffected by an anti-PI3K/Akt agent but were disrupted by microtubule inhibitors. PTEN mutations exhibit distinct functional properties with regard to their subcellular localization. Further, some missense mutations (‘edge mutations’) in the phosphatase domain caused enhanced invasiveness associated with dysfunctional cytoskeletal assembly, thus suggesting it to be a potent therapeutic target.

  Study EGAS00001004753

• Non-viral precision T cell receptor replacement for personalized cell therapy

  The T cell receptor (TCR) provides the fine specificity of T cells to recognize mutations in cancer cells 1-3. We developed a clinical-grade approach based on CRISPR/Cas9 non-viral precision genome editing to simultaneously knock-out the two endogenous TCR genes, TCRα (TRAC) and TCRβ (TRBC), and insert in the TRAC locus the two chains of a neoantigen-specific TCR (neoTCR), isolated from the patient’s own circulating T cells using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with refractory solid cancers received up to three distinct neoTCR-transgenic cell products, each expressing a patient-specific neoTCR, in a cell dose-escalation, first-in-human phase 1 clinical trial (NCT03970382). One patient had grade 1 cytokine release syndrome, and one grade 3 encephalitis. All had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease, and the other 11 had disease progression as best response on therapy. NeoTCR-transgenic T cells were detected in tumour biopsies post-infusion at frequencies higher than the native TCRs pre-infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs recognizing mutational neoantigens, the simultaneous knock-out of the endogenous TCR and knock-in of the neoTCRs using single-step, non-viral precision genome editing, the manufacturing of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene edited neoTCR T cell products, and the ability of the transgenic T cells to traffic to the patients’ tumours.

  Study EGAS00001006898

• Targeted and shallow whole genome sequencing identifies therapeutic opportunities in p53abn endometrial cancers

  Purpose: Shallow whole genome sequencing (sWGS) can detect copy number (CN) aberrations. In high-grade serous ovarian (HGSOC) sWGS identified CN signatures such as homologous recombination deficiency (HRD) to direct therapy. We applied sWGS with targeted sequencing to p53abn endometrial cancers (ECs) to identify additional prognostic stratification and therapeutic opportunities. Experimental Design: sWGS and targeted panel sequencing was performed on formalin-fixed paraffin-embedded p53abn ECs. CN alterations, mutational data and CN signatures were derived, and associations to clinicopathologic and outcomes data were assessed. Results: In 187 p53abn ECs, 5 distinct CN signatures were identified. Signature 5 was associated with BRCA1/2 CN loss with features similar to HGSOC HRD signature. 22% potential HRD cases were identified, 35 patients with signature 5, and 8 patients with BRCA1/2 somatic mutations. Signatures 3 and 4 were associated with whole genome duplication, and CCNE1, ERBB2 and MYC amplifications, with mutations in PIK3CA enriched in signature 3. We observed improved overall survival (OS) for patients with signature 2 and worse OS for signatures 1 and 3. 28% of patients had CCNE1 amplification and this subset was enriched with carcinosarcoma histotype. 34% of patients, across all histotypes, had ERBB2 amplification and/or HER2 overexpression on immunohistochemistry, which was associated with worse outcomes. Mutations in PPP2R1A (29%) and FBXW7 (16%) were among the top 5 most common mutations. Conclusions: sWGS and targeted sequencing identified therapeutic opportunities in 75% of p53abn EC patients. Further research is needed to determine the efficacy of treatments targeting these identified pathways within p53abn ECs.

  Study EGAS00001007661

• Benchmarking for alignment and variant calling

  Background: Lung cancer is a heterogeneous disease and the primary cause of cancer-related mortality worldwide. Somatic mutations, including large structural variants, are important biomarkers in lung cancer for selecting targeted therapy. Genomic studies in lung cancer have been conducted using short-read sequencing. Emerging long-read sequencing technologies are a promising alternative to study somatic structural variants, however there is no current consensus on how to process data and call somatic events. In this study, we preformed whole genome sequencing of lung cancer and matched non-tumour samples using long and short read sequencing to comprehensively benchmark three sequence aligners and six structural variant callers comprised of four generic callers (Sniffles2, cuteSV, SVIM and DELLY in generic mode) and two somatic callers (nanomonsv and DELLY in somatic modes). Results: Different combinations of aligners and variant callers influenced somatic structural variant detection. The choice of caller had a significant influence on somatic structural variant detection in terms of variant type, size, sensitivity, and accuracy. The performance of each variant caller was assessed by comparing to somatic structural variants identified by short read sequencing. When compared to somatic structural variants detected with short read sequencing, more events were detected with long read sequencing. The mean recall of somatic variant events identified by short read sequencing was higher for the somatic callers (64%) than generic callers (52%), with nanomonsv (69%) showing the best performance. Conclusion: Long read sequencing can identify somatic structural variants. The longer reads have the potential to improve our understanding of cancer development and inform personalized cancer treatment.

  Study EGAS00001007819

• Exome sequencing

  Human telomere biology disorders (TBD), a heterogeneous group of disorders characterized by telomere attrition and premature aging phenotypes, are caused by inherited loss-of-function mutations in telomere-associated genes. Here, we identify three germline heterozygous missense variants in RPA1 gene in four unrelated probands presenting with short telomeres and varying clinical features of TBD including bone marrow failure, myelodysplastic syndrome, T- and B-cell lymphopenia, pulmonary fibrosis, and skin manifestations. All variants cluster to DNA binding domain A of RPA1 protein. RPA1 is an essential single-strand DNA-binding protein essential for DNA replication and repair. RPA1 has been implicated in telomere maintenance but the mechanism remains elusive. Thus far, no genetic disorder had been linked to RPA1. Using FRET assays, we showed that RPA1E240K and RPA1V227A exhibit increased binding to single-strand and telomeric DNA, implying a gain in DNA-binding function while RPA1T270A has binding properties similar to wild type. To study the mutational effect, RPA1E240K, with the highest binding capacity, was knocked-in iPSCs using CRISPR/Cas9, which showed severe telomere shortening and impaired hematopoietic differentiation. Furthermore, in the RPA1E240K patient, stable blood counts over two decades due coincided with clonal rescue hematopoiesis with an RNA-degrading truncating RPA1 mutation and a uniparental isodisomy 17p with loss of RPA1E240K allele. Using single cell DNA sequencing, the two somatic genetic rescue events were proven to be independently acquired in hematopoietic stem cells. In summary, we describe the first human disease caused by germline RPA1 variants in individuals with TBD.

  Study EGAS00001005761

• scRNAseq for patients with immunodeficiency and HCs

  The NFAT family of transcription factors plays central roles in adaptive immunity in murine models, however, their contribution to human immune homeostasis remains poorly defined. In a multigenerational pedigree, we identified three patients carrying germline biallelic missense variants in NFATC1, presenting with recurrent infections, hypogammaglobulinemia and decreased antibody responses. The compound heterozygous NFATC1 variants identified in the patients caused decreased stability and reduced binding of DNA and interacting proteins. We observed defects in early activation and proliferation of T and B cells from these patients, amenable to reconstitution upon genetic rescue. Following stimulation, T-cell activation and proliferation were impaired, reaching that of healthy controls with delay indicative of an adaptive capacity of the cells. Assessment of the metabolic capacity of patient T cells, revealed that NFATc1-dysfunction rendered T cells unable to engage in glycolysis following stimulation, although oxidative metabolic processes were intact. We hypothesized that NFATc1-mutant T cells could compensate for the energy deficit due to defective glycolysis by enhanced lipid metabolism as an adaptation, leading to a delayed, but not lost activation responses. Indeed, we observed increased 13C-labelled palmitate incorporation into citrate indicating higher fatty acid oxidation and we demonstrated that metformin and rosiglitazone improved patient T-cell effector functions. Collectively, enabled by our molecular dissection of NFATC1 mutations and extending the role of NFATc1 in human immunity beyond receptor signaling, and reveal evidence of metabolic plasticity in the context of impaired glycolysis observed in patient T cells to remedy delayed effector responses.

  Study EGAS00001007271

• Efficacy of nivolumab in pediatric cancers with high mutation burden and mismatch-repair deficiency

  Purpose: Checkpoint-inhibitors have limited efficacy for children with unselected solid and brain tumors. We report the first prospective pediatric trial (NCT02992964) using nivolumab exclusively for refractory non-hematological cancers harboring tumor mutation burden (TMB) ≥5 mutations/megabase (mut/Mb) and/or mismatch-repair deficiency (MMRD). Patients and methods: Twenty patients were screened, and ten ultimately included in the response cohort of whom nine had TMB >10mut/Mb (three initially eligible based on MMRD) and one patient had TMB between 5-10 mut/Mb. Results: Delayed immune responses contributed to best overall response of 50%, improving on initial objective responses (20%) and leading to 2-year overall survival (OS) of 50% (95% CI; 27, 93). Four children, including three with refractory malignant gliomas are in complete remission at a median follow-up of 37-months (range: 32.4-60), culminating in 2-year OS of 43% (95% CI; 18.2, 100). Biomarker analyses confirmed benefit in children with germline MMRD, microsatellite instability, higher activated and lower regulatory circulating T-cells. Stochastic mutation accumulation driven by underlying germline MMRD impacted the tumor microenvironment, contributing to delayed responses. No benefit was observed in the single patient with a MMR-proficient tumour and TMB 7.4 mut/Mb. Conclusions: Nivolumab resulted in durable responses and prolonged survival for the first time in a pediatric trial of refractory hypermutated cancers including malignant gliomas. Novel biomarkers identified here need to be translated rapidly to clinical care to identify children who can benefit from checkpoint-inhibitors, including for upfront management of their cancers.

  Study EGAS00001007393

• Targeting vulnerabilities in IDH mutant tumours: The model matters WES study

  Introduction: Synthetic lethal interactions with IDH1 and IDH2 (IDH) mutations were identified in non-endogenous IDH mutant (IDHmut) AML and glioma models, but are absent in endogenous IDHmut chondrosarcoma cell lines. The translation into successful clinical applications has remained challenging, implying artificially created models do not fully recapitulate endogenous IDHmut tumour biology. The aim of this study was to elucidate if the model system is indeed an important factor to consider when studying therapeutic vulnerabilities in IDHmut tumours. Methods: Vector-based and CRISPR-Cas9 approaches were used to introduce or revert the IDH1 mutation in chondrosarcoma cell lines. These isogenic cell line pairs were used to examine the presence of known therapeutic vulnerabilities and their underlying biological mechanisms. Results: Vector-based IDHmut chondrosarcoma models showed the previously reported synthetic lethal interactions, but these treatment sensitivities were absent in the CRISPR-edited models. Interestingly, not all vector-based IDHmut cell lines displayed the same therapeutic vulnerabilities. Differences in treatment response were associated with multiple factors, including IDHmut protein expression and D-2-HG levels, in line with the fact that therapeutic vulnerabilities could be induced in the CRISPR-edited models by enhancing D-2-HG levels. Conclusion: Our findings demonstrate that synthetic lethal interactions observed in vector-based models are often a consequence of IDHmut protein overexpression and supra-physiological D-2-HG levels. These results highlight that relying on artificially created IDHmut models may lead to the identification of therapeutic vulnerabilities that are not present in IDHmut tumours, potentially explaining the poor translation of preclinical findings to clinical trials.

  Study EGAS50000001870

• Targeting vulnerabilities in IDH mutant tumours: The model matters RNAseq study

  Introduction: Synthetic lethal interactions with IDH1 and IDH2 (IDH) mutations were identified in non-endogenous IDH mutant (IDHmut) AML and glioma models, but are absent in endogenous IDHmut chondrosarcoma cell lines. The translation into successful clinical applications has remained challenging, implying artificially created models do not fully recapitulate endogenous IDHmut tumour biology. The aim of this study was to elucidate if the model system is indeed an important factor to consider when studying therapeutic vulnerabilities in IDHmut tumours. Methods: Vector-based and CRISPR-Cas9 approaches were used to introduce or revert the IDH1 mutation in chondrosarcoma cell lines. These isogenic cell line pairs were used to examine the presence of known therapeutic vulnerabilities and their underlying biological mechanisms. Results: Vector-based IDHmut chondrosarcoma models showed the previously reported synthetic lethal interactions, but these treatment sensitivities were absent in the CRISPR-edited models. Interestingly, not all vector-based IDHmut cell lines displayed the same therapeutic vulnerabilities. Differences in treatment response were associated with multiple factors, including IDHmut protein expression and D-2-HG levels, in line with the fact that therapeutic vulnerabilities could be induced in the CRISPR-edited models by enhancing D-2-HG levels. Conclusion: Our findings demonstrate that synthetic lethal interactions observed in vector-based models are often a consequence of IDHmut protein overexpression and supra-physiological D-2-HG levels. These results highlight that relying on artificially created IDHmut models may lead to the identification of therapeutic vulnerabilities that are not present in IDHmut tumours, potentially explaining the poor translation of preclinical findings to clinical trials.

  Study EGAS50000001871