22929 results for "ega"

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• Genomic_Advances_in_Sepsis__GAinS__genotyping

  This cohort comprises a subset of pateints enrolled in the Genomic Advances in Sepsis (GAinS) study, an established biobank of adult sepsis patients. Patients with sepsis due to community acquired pneumonia or faecal peritonitis were recruited from 35 hospitals across the UK from 2005-2018, with samples for functional genomics and detailed clinical information collected over the first five days of ICU admission. DNA was extracted from buffy coat or whole blood samples using the Qiagen DNA extraction protocol, the automated Maxwell Blood purification kit (Promega), or the QIAamp Blood Midi kit protocol (Qiagen). Genotyping data were generated using the Illumina HumanOmniExpress BeadChip (295 patients), the Infinium CoreExome BeadChip (655 patients), and the Infinium Global Screening Array BeadChip (307 patients). Genotyping QC and imputation into the Haplotype Reference Consortium was perfomed within each batch. The datasets were combined and following post-imputation filtering data were available on 1168 samples. 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 EGAS00001007786

• Germline biallelic mutation affecting the transcription factor Helios causes pleiotropic defects of immunity

  We studied a patient with recurrent respiratory infections and hypogammaglobulinemia and identified a germline homozygous missense mutation in IKZF2 encoding Helios (p.Ile325Val). We show that HeliosI325V retains DNA-binding and dimerization properties, but loses interaction with several partners, including epigenetic remodelers HDAC1, HDAC3 and the ATAC complex. Single-cell RNA-sequencing of peripheral blood mononuclear cells revealed gene expression signatures indicative of a shift towards pro-inflammatory, effector-like status in the patient’s T cells. We observed an upregulation of the anti-inflammatory gene, TSC22D3, encoding the glucocorticoid-induced leucine zipper (GILZ), whose expression was reported to increase upon IL-2 deprivation. We validated this finding in primary T cells, where we observed a pronounced reduction in IL-2 production upon their stimulation, together with a defect in their ability to proliferate, rendering them more anergic. Collectively, we identify a novel germline-encoded inborn error of immunity and define a role for Helios in conventional T cells, whereby interactions with specific binding partners is necessary to mediate the transcriptional programs that enable T-cell homeostasis in health and disease.

  Study EGAS00001005675

• The identification of genetic vulnerabilities in head and neck cancers for the development of novel treatments.

  The global cancer burden is expected to double by the year 2030 and this will disproportionately affect the Asian continent. Head and neck squamous cell carcinoma (HNSCC) is endemic in Asia. It is the sixth most common cancer worldwide and the second most common in Malaysia, with approximately 600,000 new cases per year globally. Longterm survival remains less than 50%, underscoring the need for better therapeutic strategies. In this proposal we aim to bring new technological advancements in the identification of critical genes in HNSCC and the identification of compounds for the development of new therapies in HNSCC. We propose to genomically characterise 16 unique Asian H&N cancer cell lines, to classify sub groups where identification of essential genes that can be targeted for tumor control can be applied in a more rational way. Further, we propose to use zebrafish, an established drug discovery platform to identify promising pathway-specific compounds that could be further studied using CRISPR/Cas9 lethality screens and biological models to better identify novel therapeutic strategies to combat this major health problem in Asia.

  Study EGAS00001002682

• Case Report: Rare IKZF1 gene fusions identified in neonate with congenital KMT2A-rearranged Acute Lymphoblastic Leukemia

  Here we describe a rare case of congenital KMT2Ar ALL presenting with co-occurring IKZF1 gene fusions and a predictably aggressive disease trajectory. We report for the first time, the novel IKZF1::TUT1 and KDM2A::IKZF1 gene fusions. Rearrangements in-volving KMT2A are commonly retained in relapsed infant ALL, however, in this case the KMT2A::AFF1 gene fusion did not appear to be the lesion driving leukemic relapse. Instead, our data suggest that relapse was driven by IKZF1::TUT1. This gene fusion remained in all samples investigated, including the on-blinatumomab therapy sample taken immediately prior to relapse. Conversely, the KMT2A::AFF1 gene fusion was only detected in the diagnosis and refractory post-induction samples highlighting a key role for IKZF1::TUT1 in disease pathogenesis. Intriguingly, both IKZF1 gene fusions are predicted to be out-of-frame, however, our data demonstrate the IKZF1 gene is still expressed. This is not unprecedented and it has previously been observed that out-of-frame fusions can cause transcriptional activation/repression of genes involved in the fusions leading to increases or decreases of their expression and the as-sociated functional outcomes.

  Study EGAS00001006947

• The DNA methylation landscape of glioblastoma disease progression shows extensive heterogeneity in time and space

  Glioblastoma is characterized by widespread genetic and transcriptional heterogeneity, yet little is known about the role of the epigenome in glioblastoma disease progression. Here, we present genome-scale maps of DNA methylation in matched primary and recurring glioblastoma tumors, using data from a highly annotated clinical cohort that was selected through a national patient registry. We demonstrate the feasibility of DNA methylation mapping in a large set of routinely collected FFPE samples, and we validate bisulfite sequencing as a multipurpose assay that allowed us to infer a range of different genetic, epigenetic, and transcriptional characteristics of the profiled tumor samples. On the basis of these data, we identified subtle differences between primary and recurring tumors, links between DNA methylation and the tumor microenvironment, and an association of epigenetic tumor heterogeneity with patient survival. In summary, this study establishes an open resource for dissecting DNA methylation heterogeneity in a genetically diverse and heterogeneous cancer, and it demonstrates the feasibility of integrating epigenomics, radiology, and digital pathology for a national cohort, thereby leveraging existing samples and data collected as part of routine clinical practice.

  Study EGAS00001002538

• Cross-tissue transcriptomic analysis of human secondary lymphoid organ residing ILC3 reveals a default quiescent state in the absence of inflammation

  A cross tissue transcriptional comparison of human ILC3 isolated from non-inflamed lymph nodes (LN) and spleens, inflamed tonsils and the peripheral blood (PB). Using uniform gating and high-purity cell-sorting, ILC3 were sorted as Lineage(CD3,CD19,CD34,CD14,CD94)-CD45intCD127+CRTH2-CD117+. From LN, spleen, tonsils and PB, NKp44- ILC3 were isolated and, in addition, NKp44+ ILC3 were sorted from spleen and tonsils. RNA quality was measured on a RNA 6000 Pico Kit (Agilent Technologies) using a 2100 Bioanalyzer (Agilent Technologies). 100 pg high-quality RNA was isolated and cDNA was generated using the SMART-Seq (v3) Ultra Low Input RNA Kit (Clontech) with 15 cycles of amplification. Amplified and Covaris-fragmented cDNA was quality-checked on a High Sensitivity DNA chip (Agilent Technologies) and further processed according the TruSeq Nano DNA Library Preparation Kit (Illumina). Data was processed as follows: SMARTer adapters were trimmed with cutadapt and the resulting sequences were aligned to the human RefSeq transcriptome using 14 TopHat2 (Kim et al., 2013). Normalization and quantification was performed using Cufflinks (Trapnell et al., 2012). FPKM counts were determined per gene with HTSeq-count.

  Study EGAS00001002636

• Ewings Sarcoma RNA-Seq

  Sarcomas are cancers of the bone and soft tissue often defined by their gene fusions. However, the timing, context, and processes by which these pathogenic fusions arise are unknown. We explored this in Ewing sarcoma, a cancer driven by EWSR1-ETS fusions, with very few cooperating mutations. Combining whole-genome sequencing with enhanced informatics, we found that the EWSR1-ETS fusion arose from striking rearrangement clusters in 42% of cases (52/124). Notably, these were organized in loops that universally contained the fusion at their center, while also weaving up to 18 genes together with it. We found the same pattern of rearrangements in three additional types of sarcoma. From these data, we define a new signature for sarcoma fusions that precedes other somatic changes, in the earliest replicating DNA of the genome. This dramatic, sudden process impinges on many genes – generating multiple coding changes that profoundly affect the transcriptome, with the disease-defining gene fusion at its core. These rearrangement loops emerge in an early ES clone from which both the primary tumor and the lethal relapse emerged, and then evolved in parallel until clinically detected.

  Study EGAS00001003062

• Gut microbiome modulates response to anti PD1 immunotherapy in metastatic melanoma patients

  There is a growing appreciation of the role of the microbiome in cancer, and evidence in pre-clinical models that the gut microbiome may modulate responses to immune checkpoint blockade though this has not been well-characterized in patients. We analyzed the oral (n=86)and gut (n=43) 16S microbiome in melanoma patients on PD-1 blockade. Significant differences were noted in the diversity and composition of the gut microbiome between responders and non-responders in patients with a fecal microbiome sample, with significantly higher alpha diversity and relative abundance of Ruminococcaceae bacteria) in R. Metagenomic studies (n=25) revealed functional differences in gut bacteria in R including enrichment of anabolic pathways. Immune profiling demonstrated enhanced systemic and anti-tumor immunity in patients with a favorable gut microbiome as well as in germ-free mice receiving fecal transplant from responding patients. Together, these data have important implications for treatment with immune checkpoint blockade in cancer. The fastq files associated with this dataset are stored at ENA under the following accession numbers: PRJEB22894 (Fecal 16S), PRJEB22874 (Oral 16S), PRJEB22895 (Murine 16S), PRJEB22893 (Fecal WGS).

  Study EGAS00001002698

• PATL2 is a key actor of oocyte maturation whose invalidation causes infertility in women and mice

  The genetic causes of oocyte meiotic deficiency (OMD), a form of primary infertility characterised by the production of immature oocytes, remain largely unexplored. Using whole exome sequencing, we found that 26% of a cohort of 23 subjects with OMD harboured the same homozygous nonsense pathogenic mutation in PATL2, a gene encoding a putative RNA-binding protein. Using Patl2 knockout mice, we confirmed that PATL2 deficiency disturbs oocyte maturation, since oocytes and zygotes exhibit morphological and developmental defects respectively. PATL2's amphibian orthologue is involved in the regulation of oocyte mRNA as a partner of CPEB. However, Patl2's expression profile throughout oocyte development in mice, alongside colocalisation experiments with Cpeb1, Msy2 and Ddx6 (three oocyte RNA-regulators) suggest an original role for Patl2 in Mammals. Accordingly, transcriptomic analysis of oocytes from WT and Patl2-/- animals demonstrated that in the absence of Patl2, expression levels of a select number of highly relevant genes involved in oocyte maturation and early embryonic development are deregulated. In conclusion, PATL2 is a novel actor of mammalian oocyte maturation whose invalidation causes OMD in humans.

  Study EGAS00001002903

• Japan PBC-GWAS Haplotype Study

  Primary biliary cholangitis (PBC) susceptibility loci have largely been discovered through single SNP association testing. In this study, we report genic haplotype patterns associated with PBC risk genome-wide in two Japanese cohorts. Among the 74 genic PBC risk haplotype candidates we detected with a novel methodological approach in a discovery cohort of 1,937 Japanese, nearly two-thirds were replicated (49 haplotypes, Bonferroni-corrected P<6.8x10-4) in an independent Japanese cohort (N=949). Along with corroborating known PBC-associated loci (TNFSF15, HLA-DRA), risk haplotypes may potentially model cis-interactions that regulate gene expression. For example, one replicated haplotype association (9q32-9q33.1, OR=1.7, P=3.0x10-21) consists of intergenic SNPs outside of the human leukocyte antigen (HLA) region that overlap regulatory histone mark peaks in liver and blood cells, and are significantly associated with TNFSF8 expression in whole blood. We also replicated a novel haplotype association involving non-HLA SNPs mapped to UMAD1 (7p21.3; OR=15.2, P=3.9x10-9) that overlap enhancer peaks in liver and memory Th cells. Our analysis demonstrates the utility of haplotype association analyses in discovering and characterizing PBC susceptibility loci.

  Study EGAS00001002915

• Low and variable tumor reactivity of the intratumoral TCR repertoire in human cancers

  Infiltration of human cancers by T cells is generally interpreted as a sign of immune recognition, and there is a growing effort to reactivate dysfunctional T cells at such tumor sites. However, these efforts only have value if the intratumoral T cell receptor (TCR) repertoire of such cells is intrinsically tumor-reactive, and this has not been established in an unbiased manner for most human cancers. To address this issue, we analyzed the intrinsic tumor-reactivity of the intratumoral TCR repertoire of CD8+ T cells in ovarian and colorectal cancer – two tumor types for which T cell infiltrates form a positive prognostic marker. Data obtained demonstrate that a capacity to recognize autologous tumor is limited to approximately 10% of intratumoral CD8+ T cells. Furthermore, in two out of four patient samples tested, no tumor-reactive TCRs were identified, despite infiltration of their tumors by T cells. These data indicate that the intrinsic capacity of intratumoral T cells to recognize adjacent tumor tissue can be rare and variable, and suggest that clinical efforts to reactivate intratumoral T cells will benefit from approaches that simultaneously increase the quality of the intratumoral TCR repertoire.

  Study EGAS00001003119

• Genomic Profiling of Thyroid Cancer Reveals a Role for Thyroglobulin in Metastasis -Part II

  Papillary thyroid carcinoma (PTC) has a wide geographic variation in incidence, being highest in Saudi Arabia where it is only second to breast cancer as the most common cancer among females. Genomic profiling of PTC from Saudi Arabia has not been attempted previously. We performed whole-exome sequencing of 101 PTC samples and the corresponding genomic DNA to identify genes with recurrent somatic mutations, followed by sequencing of these genes using a next-generation gene panel approach in further 785 samples. In addition to BRAF, N-RAS and H-RAS, which have previously been shown to be recurrently mutated in PTC, our analysis highlights additional genes, including thyroglobulin (TG), which harbored somatic mutations in 3% of the entire cohort. Surprisingly, although TG mutations were not exclusive to mutations in the RAS-MAP kinase pathway, their presence was associated with a significantly worse clinical outcome, which suggests a pathogenic role beyond driving initial oncogenesis. Analysis of metastatic PTC tissue revealed significant enrichment for TG mutations (p<0.001), including events of apparent clonal expansion. Our results suggest a previously unknown role of TG somatic mutations in the pathogenesis of PTC and its malignant evolution.

  Study EGAS00001003357

• Increased mutation accumulation during fetal development in Down syndrome

  Children with Down syndrome (DS) show a high incidence of leukemia, which is preceded by transient myeloproliferative disorder (TMD) in neonates. TMD is characterized by mutations in GATA1 that accumulate during fetal development, indicating that additional mutagenesis is required for leukemogenesis. Abnormalities in chromosome numbers can result in a mutator phenotype. However, it is unknown whether a trisomy of chromosome 21 (T21) results in enhanced mutation accumulation during human development. Here, we determined the mutational consequences of a constitutive T21 in DS fetuses compared to disomy 21 cells (D21) of D21 fetuses. We found that D21 fetal HPSCs had a higher somatic mutation rate compared to healthy adults HSPCs. Moreover, we observed an increase in somatic mutation load in T21 cells of DS fetuses, which was already apparent during the first cell divisions of embryogenesis. The mutation load could be explained by processes that are active during normal fetal development and were similar to those observed in TMD blast cells. Our study shows that T21 causes an increase in somatic load in fetal cells of DS fetuses, which may contribute to leukemia development early in life.

  Study EGAS00001003982

• Genome-wide cell-free DNA fragmentation in patients with cancer

  Cell-free DNA (cfDNA) in the blood provides a noninvasive diagnostic avenue for patients with cancer. However, characteristics of the origins and molecular features of cfDNA are poorly understood. We developed an approach to evaluate fragmentation patterns of cfDNA across the genome and found that cfDNA profiles of healthy individuals reflected nucleosomal patterns of white blood cells, while patients with cancer had altered fragmentation profiles. We applied this method to analyze fragmentation profiles of 236 patients with breast, colorectal, lung, ovarian, pancreatic, gastric, or bile duct cancers and 245 healthy individuals. A machine learning model incorporating genome-wide fragmentation features had sensitivities of detection ranging from 57% to >99% among the seven cancer types at 98% specificity, with an overall AUC of 0.94. Fragmentation profiles could be used to identify the tissue of origin of the cancers to a limited number of sites in 75% of cases. Combining our approach with mutation based cfDNA analyses detected 91% of cancer patients. The results of these analyses highlight important properties of cfDNA and provide a proof of principle approach for screening, early detection, and monitoring of human cancer.

  Study EGAS00001003611

• The Ovarian Cancer Association Consortium OncoArray genome-wide association study

  A custom Illumina genotyping array, the OncoArray. The array includes a genome-wide backbone, comprising 230,000 SNPs tagging most common genetic variants, together with dense mapping of known susceptibility regions, rare variants from sequencing experiments, pharmacogenetic markers, and cancer-related traits. A total of 494,763 SNPs passed quality control steps with a sample success rate of 97% of the samples. Participating sites performed ancestry analysis using a common set of markers and a scoring algorithm based on principal components analysis as described in PMID:27697780.The array was used to genotype ovarian cancer cases and controls from multiple studies participating in the Ovarian Cancer Association Consortium, of which 54,747 passes QC.An initial principal components analysis was carried out using a method described in PMID:27697780 to assign subject to intercontinental ancestry groups: “European”, “African”, “Asian” and "Other". Within ancestry principal components analysis was carried using a set of ~33k unlinked markers. Subsequent analyses were adjusted for the ancestry specific PCs: 9 Eur, 10 Asian, 1 African and 6 Mixed. These PCs are included in the imputed dataset phenotypes file

  Study EGAS00001002305

• Defective T-cell expansion in RASGRP1 deficiency

  Inherited CTPS1- CD27- and CD70-deficiencies in humans have revealed key factors of T-lymphocyte expansion, that is a prerequisite for an efficient immunity to Epstein-Barr virus (EBV) infection. RASGRP1 is a T-lymphocyte specific nucleotide exchange factor known to activate the MAP kinases pathway. A deleterious homozygous mutation in RASGRP1 leading to the loss RASGRP1 expression was identified in two siblings who both developed a persistent EBV infection leading to Hodgkin lymphoma. RASGRP1-deficient T cells exhibited defective MAPK activation and impaired proliferation that was restored by expression of wild-type RASGRP1. Similar defects were observed in T cells from healthy individuals when RASGRP1 was downregulated. RASGRP1-deficient T cells also exhibited decreased CD27-dependent proliferation toward CD70-expressing EBV-transformed B cells, a crucial pathway required for expansion of antigen-specific T cells in anti-EBV immunity. Furthermore, RASGRP1-deficient T cells failed to upregulate CTPS1, an important enzyme required for DNA synthesis. These results show that RASGRP1 deficiency leads to susceptibility to EBV infection, and demonstrate the key role of RASGRP1 at the crossroad of pathways required for the expansion of activated T lymphocytes.

  Study EGAS00001002753

• HCA_Thymus_Disease

  Single cell RNA sequencing on thymic cells from children with syndromic diseases such as Trisomy 21 and inflammatory/autoimmune diseases such as myasthenia gravis. Our group has previously performed atlasing of the healthy human thymus using single cell transcriptomics and obtained an unprecedented understanding of human thymus throughout development. In this study, we intend to survey diseased human thymus and compare this to healthy to obtain a better understanding of thymic dysfunction in human diseases. The diseases we will survey include congenital conditions such as Down's syndrome, CHARGE syndrome, DiGeorge syndrome as well as acquired diseases such as myasthenia gravis and thymic hyperplasia. We intend to perform genetic and genomic study on the samples, including expression analysis. This will not only help us to understand the cells and molecular pathways affected in dysfunctional thymus; but may also lead to generalisable understandings in human genetic diseases and inflammatory/autoimmune diseases. 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 EGAS00001004310

• ARAF Mutations Confer Resistance to RAF Dimer Inhibitor Belvarafenib in NRAS- and BRAF- Mutant Melanoma

  Belvarafenib, a potent and selective RAF dimer (type II) inhibitor, exhibits clinical activity in BRAFV600E- and NRAS-mutant melanoma patients. Here, we report the first-in-human phase I study investigating maximum tolerated dose, assessing safety and preliminary efficacy of belvarafenib in BRAF- and RAS-mutated advanced solid tumors (NCT02405065, NCT03118817). Through generation of belvarafenib resistant NRAS- mutant melanoma cells and analysis of circulating tumor DNA from patients treated with belvarafenib, we identified novel recurrent mutations in ARAF within the kinase domain. ARAF mutants conferred resistance to belvarafenib in both a dimer- and kinase activity- dependent manner. Belvarafenib induced ARAF mutant dimers, and dimers containing mutant ARAF were active in the presence of inhibitor. ARAF mutations may serve as a general resistance mechanism for RAF dimer inhibitors as the mutants exhibit reduced sensitivity to a panel of type II RAF inhibitors. The combination of RAF plus MEK inhibition may be used to delay ARAF-driven resistance and suggests a rational combination for clinical use. Taken together, our findings reveal specific and compensatory functions for the ARAF isoform and implicate ARAF mutations as a driver of resistance to RAF dimer inhibitors.

  Study EGAS00001005086

• SmMIP-tools:a computational toolset for processing and analysis of single-molecule molecular inversion probes derived data

  Single-molecule molecular inversion probes (smMIPs) provides a modular and cost-effective platform for high-multiplex targeted next-generation sequencing (NGS). Nevertheless, translating the raw smMIP-derived sequencing data into accurate and meaningful information currently requires proficient computational skills and a large amount of computational work, prohibiting wide-scale adoption of smMIP-based technologies. To enable easy, efficient, and accurate interrogation of smMIP-derived data, we developed SmMIP-tools, a computational toolset that combines the critical analytic steps for smMIP data interpretation into a single computational pipeline. Here, we describe in detail two major components of the software. The first is a read processing tool that performs quality control steps, generates read-smMIP linkages and retrieves molecular tags. The second is an error-aware variant caller capable of detecting single nucleotide variants (SNVs) and short insertions and deletions (indels). Using a cell-line DNA dilution series and a cohort of blood cancer patients, we benchmarked SmMIP-tools and evaluated its performance against clinical sequencing reports. We anticipate that SmMIP-tools will increase accessibility to smMIP-technology, enabling cost-effective genetic research to push personalized medicine forward.

  Study EGAS00001005359

• Single-nucleus transcriptomic profiling of aging Down Syndrome brains

  Down syndrome (DS) – trisomy of human chromosome 21 (HSA21) – is characterized by lifelong cognitive impairments and development of neuropathological hallmarks of Alzheimer’s disease (AD). The cellular and molecular modifications responsible for these effects are not understood. Here we performed single-nucleus RNA-sequencing (snRNA-seq) employing both short (Illumina) and long-read (Pacific Biosciences) sequencing technologies on a total of 29 DS and non-DS control prefrontal cortex samples. In DS, the ratio of inhibitory-to-excitatory neurons was significantly increased, which was not observed in previous reports examining sporadic AD. DS microglial transcriptomes displayed AD-related aging and activation signatures in advance of AD neuropathology, with increased microglial expression of C1q complement genes (associated with dendritic pruning) and the HSA21 transcription factor gene RUNX1. Long-read sequencing detected vast RNA isoform diversity within and amongst specific cell types including numerous novel sequences that differed between DS and normal brains. Notably, over 8,000 genes produced RNAs containing intra-exonic junctions (IEJs), including APP that had previously been associated with somatic gene recombination. These and related results illuminate large-scale cellular and transcriptomic alterations as novel features of the aging DS brain.

  Study EGAS00001005691

• Molecular_risk_stratification_in_patients_with_T1_colorectal_cancer_WES

  The introduction of bowel cancer screening has led to a significant increase in the proportion of patients being diagnosed with asymptomatic, early-stage colorectal cancer (CRC). Although the majority of these patients are successfully treated with surgery alone, a small proportion of patients have ‘born-to-be-bad’ aggressive lesions with early dissemination leading to distant metastases. Current standard of care histological assessment is unable to distinguish between these aggressive versus non-aggressive early lesions which is essential to provide appropriate clinical management decisions. This study aims to carry out molecular and histological profiling of approximately 300 T1 CRCs in order to develop a molecular stratifier based on the risk of relapse in early-invasive CRC. This novel T1 cohort will represent the world’s largest molecularly characterised T1 cohort of samples, with digital pathology assessment alongside whole exome sequencing, copy number variation analysis and 3’ RNA-seq. This data will be used to generate a robust panel of molecular and/or histological markers applicable to formalin-fixed paraffin embedded (FFPE) archival tissue which discriminates between T1 lesions based on risk of relapse, which will ultimately be used to inform clinical management of CRC at the earliest stages of the disease.

  Study EGAS00001005734

• Field_effect_of_healthy_and_diseased_livers_WGS

  Recent work in the Campbell group has revealed somatic mutations present in normal, non-cancerous human skin. A subset of the mutations conferred selective advantages to the host cells, leading to clonal expansions and raising the risk for future cancer development. Capturing such somatic mutations in normal tissue is important to advance our understanding about carcinogenesis and could provide prospective medical insights. In this project, our goal is to detect somatic mutations in normal (pre-cancerous) liver tissue. Using Laser Microdissection technology, we will dissect individual liver lobules from patient samples and submit these to sequencing. For each patient sample, we aim to sequence multiple lobules to characterise the mutagenic burden. Samples will be taken from patients with different liver disease aetiologies, including alcoholism and obesity, with a view on distinguishing the prevalent mutation types occurring in each disease context. We will perform both whole genome and targeted sequencing, initially using the WTSI cancer panel. Later we aim to use a novel bait set that captures both cancer genes as well as genes relevant to the non-cancerous samples (ie. genes implicated in hereditary disorders, immune sequences).

  Study EGAS00001002413

• Whole Mitochondrial Sequencing of Gingivo-buccal Cancer: ICGC-India Project

  As a part of the ICGC, India has undertaken genomic studies on gingivobuccal cancer of the oral cavity, which is the most prevalent form of cancer among men in India. There are various known environmental (life-style) correlates of this cancer, the most important of which are tobacco chewing and HPV infection. Detailed clinical characterization of the patients, collection of data on demographic and environmental exposures, and isolation of DNA samples from blood and tumor tissues collected from each patient are being done at the Advanced Centre for Research, Treatment and Education on Cancer, Mumbai. Whole genome and other sequencing experiments are being performed at the National Institute of Biomedical Genomics, Kalyani. Whole mitochondria sequencing of 89 patients was performed in this study. The mean depth of sequencing was at least 2500X, but variable across platforms, 2689X and 9894X for amplicon sequencing in Ion Torrent PGM (Tumor-blood pairs=16) and Illumina HiSeq 2500 (Tumor-blood pairs =48), respectively; and, 14,190X for whole genome sequencing in Illumina HiSeq 2500 (Tumor-blood pairs =25). Associations of mutation data with important clinical aspects have been found.

  Study EGAS00001002425

• BARIA baseline first 100 individuals transcriptomes

  Prevalence of obesity and associated diseases, including type 2 diabetes mellitus, dyslipidaemia and non-alcoholic fatty liver disease (NAFLD), are increasing. Underlying mechanisms, especially in humans, are unclear. Bariatric surgery provides the unique opportunity to obtain biopsies and portal vein blood-samples. Methods The BARIA Study aims to assess how microbiota and their metabolites affect transcription in key tissues and clinical outcome in obese subjects and how baseline anthropometric and metabolic characteristics determine weight loss and glucose homeostasis after bariatric surgery. We phenotype patients undergoing bariatric surgery (predominantly laparoscopic Roux-en-Y gastric bypass), before weight loss, with biometrics, dietary and psychological questionnaires, mixed meal test (MMT) and collect fecal-samples and intra-operative biopsies from liver, adipose tissues and jejunum. We aim to include 1500 patients. A subset (approximately 25%) will undergo intra-operative portal vein blood-sampling. Fecal-samples are analyzed with shotgun metagenomics and targeted metabolomics, fasted and postprandial plasma-samples are subjected to metabolomics, and RNA is extracted from the tissues for RNAseq-analyses. Data will be integrated using state-of-the-art neuronal networks and metabolic modeling. Patient follow-up will be ten years.

  Study EGAS00001005704

• The demographic history and mutational load of African hunter-gatherers and farmers

  The distribution of deleterious genetic variation across human populations is a key issue in evolutionary biology and medical genetics. However, the impact of different modes of subsistence on recent changes in population size, patterns of gene flow, and deleterious mutational load remains unclear. Here, we report high-coverage exomes from various populations of rainforest hunter-gatherers and farmers from central Africa. We find that the recent demographic histories of hunter-gatherers and farmers differed considerably, with population collapses for hunter-gatherers and expansions for farmers, accompanied by increased gene flow. We show that purifying selection against newly arising deleterious alleles is of similar efficiency across African populations, in contrast with Europeans where we detect weaker purifying selection. Furthermore, the per-individual mutation load of rainforest hunter-gatherers is similar to that of farmers, under both additive and recessive models. Our results indicate that differences in the cultural practices and demographic regimes of African populations have not resulted in large differences in mutational burden, and highlight the beneficial role of gene flow in reshaping the distribution of deleterious genetic variation across human populations.

  Study EGAS00001002457

• Cryptic Relatedness in the Singapore Living Biobank Project

  The Singapore Living Biobank is a collection of healthy population-based Chinese and Malay individuals, for the purpose of phenotype recall study of high-impact variant carriers. These individuals are sampled from two studies: Multi-Ethnic Cohort (MEC), and the Singapore Health 2012 (SH2012). The MEC is a population-based cohort initiated in 2007 to investigate the genetic and lifestyle factors that affect the risk of developing chronic diseases such as diabetes and cardiovascular outcomes in the three ethnic groups (Chinese, Malays, and Indians). The SH2012 study is a population-based cross-sectional survey conducted in Singapore between 2012 and 2013, with over-sampling of Malays and Indians. Participants in MEC and SH2012 completed a similar set of questionnaire components, health examination, and biochemisty panels. Description of the MEC and SH2012 studies can be found at http://blog.nus.edu.sg/sphs/. We generated whole-exome sequencing data and Illumina OmniExpress array genotyping data for 1,299 Chinese and 1,229 Malays from the Singapore Living Biobank. This study includes a subset of 762 individuals that were found to be closely related (≤3rd degree), including 263 Chinese and 499 Malays.

  Study EGAS00001002619

• Evolutionary analysis of primary tumors and metastatic lesions from 20 breast cancer patients (99 samples in total) using exome sequencing data.

  Metastatic breast cancers are still incurable. Characterizing their evolutionary landscape including the role of metastatic axillary lymph nodes to seed distant organ metastasis can provide rational basis for effective treatments. Here, we describe the genomic analyses of the primary tumors and metastatic lesions from 20 breast cancer patients (99 samples). Our evolutionary analyses revealed diverse spreading and seeding patterns governing tumor progression. Although linear evolution to successive metastatic sites was common, parallel evolution from the primary tumor to multiple distant sites was also evident. Metastatic spreading was frequently coupled with polyclonal seeding, where multiple metastatic subclones originated from primary tumor and/or other distant metastases. Synchronous axillary lymph node metastasis, a well-established prognosticator of breast cancer, was not involved in seeding distant metastasis, suggesting haematogenous route for cancer dissemination. Clonal evolution coincided frequently with emerging driver alterations and evolving mutational processes, notably a significant increase in apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) associated mutagenesis. Our data provide the first genomic evidence regarding the role of axillary lymph node metastasis in seeding distant organ metastasis and elucidates the evolving mutational landscape during cancer progression.

  Study EGAS00001002737

• Germline HAVCR2 mutations altering TIM-3 characterize subcutaneous panniculitis-like T-cell lymphomas with hemophagocytic lymphohistiocytic syndrome

  Sub-cutaneous panniculitis-like T-cell lymphomas (SPTCL), a non-Hodgkin lymphoma, can be associated with hemophagocytic lymphohistiocytosis (HLH), a life-threatening immune activation which adversely impacts survival1,2. T-cell-immunoglobulin mucin-3 (TIM-3) is a modulator of immune responses expressed on subgroups of T- and innate immune cells. We identify in ~60% of SPTCL cases germline, loss-of-function, missense variants in highly conserved residues of TIM-3, c.245A>G(p.Y82C) and c.291A>G(p.I97M), each with specific geographic distribution. Y82C-TIM-3 occurs on a potential founder chromosome in patients with East Asian and Polynesian ancestry, while I97M-TIM-3 occurs in Caucasians. Both variants induce protein misfolding and abrogate TIM-3’s plasma-membrane expression leading to persistent immune activation, increased production of inflammatory cytokines, including TNF-α and IL-1β, promoting HLH and SPTCL. Our findings highlight HLH/SPTCL as a new genetic entity and showcase TIM-3 mutations as the first causative genetic defect in SPTCL. While TIM-3-mutant HLH/SPTCL benefit from immunomodulation, therapeutic repression of the TIM-3 checkpoint may have adverse consequences.

  Study EGAS00001002765

• Somatic IL4R Hotspot Mutations in Primary Mediastinal Large B-cell lymphoma lead to constitutive JAK-STAT activation

  Primary mediastinal large B cell lymphoma (PMBCL) is a distinct subtype of diffuse large B cell lymphoma thought to arise from thymic medullary B cells. Gene mutations underlying the molecular pathogenesis of the disease are incompletely characterized. Here, we describe novel somatic IL4R mutations in 15 out of 62 primary cases of PMBCL (24.2%) and in all PMBCL-derived cell lines tested. The majority of mutations (11/21; 52%) were hotspot single nucleotide variants in exon 8 leading to an I242N amino acid change in the transmembrane domain. Functional analyses establish this mutation as gain-of-function leading to constitutive activation of the JAK-STAT pathway and upregulation of downstream cytokine expression profiles and B cell specific antigens. Moreover, expression of I242N mutant IL4R in a mouse xenotransplantation model conferred growth advantage in vivo. The pattern of concurrent mutations within the JAK-STAT signaling pathway suggests additive/synergistic effects of these gene mutations contributing to lymphomagenesis. Our data establish IL4R mutations as novel driver alterations and provide a strong preclinical rationale for therapeutic targeting of JAK-STAT signaling in PMBCL.

  Study EGAS00001002796

• Hominin-specific NOTCH2 paralogs expand human cortical neurogenesis through regulation of Delta/Notch interactions.

  The human cerebral cortex has undergone rapid expansion and increased complexity during recent evolution. Hominid-specific gene duplications represent a major driving force of evolution, but their impact on human brain evolution remains unclear. Using tailored RNA sequencing (RNAseq), we profiled the spatial and temporal expression of Hominid-specific duplicated (HS) genes in the human fetal cortex, leading to the identification of a repertoire of 36 HS genes displaying robust and dynamic patterns during cortical neurogenesis. Among these we focused on NOTCH2NL, previously uncharacterized HS paralogs of NOTCH2. NOTCH2NL promote the clonal expansion of human cortical progenitors by increasing self-renewal, ultimately leading to higher neuronal output. NOTCH2NL function by activating the Notch pathway, through inhibition of Delta/Notch interactions. Our study uncovers a large repertoire of recently evolved genes linking genomic evolution to human brain development, and reveals how hominin-specific NOTCH paralogs may have contributed to the expansion of the human cortex. The dataset contains raw sequences (FASTQ files) from the Illumina 2x150bp paired-end RNA sequencing profiles of 11 fetal human brain samples at 7, 9, 12, 15 and 21 gestational weeks.

  Study EGAS00001002798

• Clonal evolution in myeloma: the impact of maintenance lenalidomide and depth of response on the genetics and sub-clonal structure of relapsed disease in uniformly treated newly diagnosed patients

  The emergence of treatment resistant sub-clones is a key feature of relapse in multiple myeloma. Therapeutic attempts to extend remission and prevent relapse include the maximisation of response and use of maintenance therapy. We used whole exome sequencing to study the genetics of paired presentation and relapse samples from 56 newly diagnosed patients, following induction therapy, randomised to receive either lenalidomide maintenance or observation as part of the Myeloma XI trial. Patients included were considered high risk, relapsing within 30 months of maintenance randomisation. Patients achieving a complete response had predominantly branching evolutionary patterns leading to relapse, characterised by a greater mutational burden, an altered mutational profile, bi-allelic inactivation of tumour suppressor genes, and acquired structural aberrations. Conversely, in patients achieving a partial response the evolutionary features were predominantly stable with a similar mutational and structural profile. There were no significant differences between patients relapsing after maintenance lenalidomide vs observation. This study shows that the depth of response is a key determinant of the evolutionary patterns seen at relapse.

  Study EGAS00001003539

• Reference Exome Data for a Northern Brazilian population

  Whole exome sequencing (WES) is a popular technology widely used in the diagnosis of rare genetic diseases and provides useful variant data for analysis of complex diseases. However, there is not always adequate population-specific reference data available to assist clinical geneticists in assigning a diagnostic variant to a specific clinical condition. Here we provide a catalogue of variants called after sequencing the exomes of 45 babies from the State of Rio Grande do Nord in Brazil. Sequence data were processed using an ‘intersect-then-combine’ (ITC) approach, using GATK and SAMtools to call variants. A total of 612,761 variants were identified in at least one individual in this Brazilian Cohort, including 559,448 single nucleotide variants (SNVs) and 53,313 insertion/deletions. Of these, 58,111 overlapped with nonsynonymous (nsSNVs) or splice site (ssSNVs) SNVs in dbNSFP. As an aid to clinical diagnosis of rare diseases, we used the American College of Medicine Genetics and Genomics (ACMG) guidelines to assign pathogenic/likely pathogenic status to 439 (0.755%) of the 58,111 nsSNVs and ssSNVs. Our data set provides a useful reference point for diagnosis of rare diseases in Brazil.

  Study EGAS00001004112

• SHANK2 mutations associated with Autism Spectrum Disorder cause hyperconnectivity of human neurons

  Heterozygous loss-of-function mutations in the synaptic scaffolding gene SHANK2 are strongly associated with autism spectrum disorder (ASD). To investigate their effect on synaptic connectivity, we generated cortical neurons from induced pluripotent stem cells (iPSC) derived from neurotypic and ASD-affected donors. We developed Sparse coculture for Connectivity (SparCon) assays where SHANK2 and control neurons were differentially labeled and sparsely seeded together on a lawn of unlabeled control neurons. We observed striking increases in dendrite length, dendrite complexity, total synapse number, and frequency of spontaneous excitatory postsynaptic currents. These findings were phenocopied in gene-edited homozygous SHANK2 knockout cells and rescued by gene correction of an ASD SHANK2 muation, supporting a role for SHANK2 as a regulator of connectivity in developing human neurons. Dendrite length increases were exacerbated by IGF1, TG003, or BDNF, and suppressed by DHPG treatment. The transcriptome in these isogenic SHANK2 neurons was deeply perturbed in synapse, plasticity, and neuronal morphogenesis gene sets and ASD gene modules, and activity-dependent dendrite extension was impaired. Our unexpected findings provide evidence for hyperconnectivity and profoundly altered transcriptome in SHANK2 neurons derived from ASD subjects.

  Study EGAS00001003436

• Spatial_transcriptome_analysis_of_Paediatric_Thymus

  Cell atlas of Paediatric Thymus Cell atlas of Paediatric Thymus - The Human Cell Atlas initiative is an ambitious global research effort which aims to describe every cell in the human body across all developmental stages to generate a reference map to accelerate progress in science and medicine particularly relating to human development and disease. The initiative is led by investigators based at the Wellcome Sanger Institute and the Broad Institute of MIT and Harvard and funding support from the Wellcome and NIH. We aim to interrogate the cellular composition and molecular regulators underpinning a cells identity through; genetic and genomic analysis, expression analysis, non-identifiable video imaging and cell line and organoid production, as well as, computational algorithms to predict cellular developmental trajectories and cell-cell interactions. We will subsequently validate these findings in situ using spatial genomic and imaging technologies e.g. immunohistochemistry, RNAscope, in situ sequencing and spatial transcriptomic analyses. 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 EGAS00001004281