1010 results for "1993+article+mouse+line+professor+BA+psychology"

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• Study on the consequences of prenatal famine exposure on DNA methylation.

  The potential of the epigenome to undergo environmentally-induced changes during early development is central to its postulated role in human disease. Here, we report a genome-scale view of DNA methylation differences after early gestational malnutrition that was caused by the Dutch Hunger Winter, a severe famine at the end of World War II. We adopted an annotation guided analysis of reduced-representation bisulfite sequencing data generated in 24 middle-aged individuals who had been exposed to famine in utero and 24 unexposed sibling controls. We observed differential DNA methylation primarily at genomic regions with regulatory potential, including enhancers active during early development. In-depth annotation of 181 prenatally-induced differentially methylated regions (P-DMRs) showed that they were predominantly located in gene bodies and co-occurred with histone marks denoting an active state. In line with the early gestational timing of the exposure, the P-DMRs mapped to genes that were enriched for differential expression patterns during blastocyst development and early organogenesis. Validation and further explorative analyses of 6 P-DMRs mapping to SMAD7, CDH23, INSR, RFTN1, CPT1A and KLF13 highlighted the critical role of gestational timing, indicated that differential methylation extended along pathways involved in growth, development and metabolism, and suggested associations with birth weight and serum LDL cholesterol in adulthood and confirmed an enhancer function for the INSR and CPT1A P-DMRs. Our results point toward a link between prenatal malnutrition and epigenetic modulation of growth and lipid metabolism that may underlie the adverse metabolic phenotype of exposed individuals in later life.

  Study EGAS00001000668

• Sporadic and endemic Burkitt lymphoma have frequent FOXO1 mutations but distinct hotspots in the AKT recognition motif

  FOXO1 has an oncogenic role in adult germinal center derived lymphomas, in which mutations, predominately within the AKT recognition motif, cause nuclear retention of FOXO1 resulting in increased cell proliferation. To determine the prevalence and distribution of FOXO1 mutations in pediatric Burkitt lymphoma (BL), we sequenced a large number of sporadic and endemic BL patient samples. We report a high frequency of FOXO1 mutations in both sporadic and endemic BL at diagnosis, occurring in 23/78 (29%) and 48/89 (54%) samples respectively, as well as 8/16 (50%) cases at relapse. Mutations of T24 were the most common in sporadic BL but were rare in endemic cases, in which mutations of residue S22, also within the AKT recognition motif, were the most frequent. FOXO1 mutations were almost always present in the major tumor cell clone but were not associated with outcome. Analysis of other recurrent mutations reported in BL revealed that FOXO1 mutations were associated with mutations of DDX3X and ARID1A, but not MYC, TCF3/ID3 or members of the phosphatidylinositol 3’ OH kinase (PI3K) signaling pathway. We further show common nuclear retention of the FOXO1 protein, irrespective of mutation status, suggesting alternative unknown mechanisms for maintaining FOXO1 transcriptional activity in BL. CRISPR/Cas9 knockout of FOXO1 in an endemic cell line produced a significant decrease in cell proliferation, supporting an oncogenic role for FOXO1 in endemic BL. Thus, FOXO1 is frequently mutated in both sporadic and endemic BL and may offer a potential therapeutic target for pediatric BL patients worldwide.

  Study EGAS00001003719

• MYO5B mutations in Pheochromocytoma/Paraganglioma promote cancer progression

  Identification of additional cancer-associated genes and secondary mutations driving the metastatic progression in pheochromocytoma and paraganglioma (PPGL) is important. We recently reported novel recurrent nonsynonymous mutations in the MYO5B gene in metastatic PPGL. Here, we explored the functional impact of these MYO5B mutations, and analyzed MYO5B expression in primary PPGL tumor cases in relation to mutation status.Immunohistochemistry and mRNA expression analysis in 30 primary PPGL tumors revealed an increased MYO5B expression in metastatic compared to non-metastatic tumor cases. In addition, subcellular localization of MYO5B protein was altered from cytoplasmatic to membranous in some metastatic tumors, and the strongest and most abnormal expression pattern was observed in a PGL case harboring a MYO5B:p.G1611S mutation. Mutation analysis of additional PPGL tumors revealed additional, and hence recurrent, mutations in the paralog MYO5A. Three missense mutations in MYO5B were functionally studied using site directed mutagenesis and stable transfection into human neuroblastoma cells (SK-N-AS) and embryonic kidney cells (HEK293 cell line). Functional analysis of the mutants verified a significant increased proliferation rate in all MYO5B mutated clones (p.L587P, p.G1611S, and p.R1641C). The two somatically derived mutations p.L587P and p.G1611S were also found to increase the migration rate, and all three mutants affected intracellular transport by increased transferrin uptake. Downstream expression analysis of MYO5B mutants demonstrated a transcriptional activation of genes involved in glucose metabolism, proliferation, homeostasis, and migration. Our study validates the functional role of novel MYO5B mutations in proliferation and migration, and suggest the MYO5-pathway to be involved the malignant progression of PPGL tumors.

  Study EGAS00001003991

• Open_Targets_020_Epigenomes_of_Cell_Lines

  There is currently a drive to establish cell based assay systems of greater human biological and disease relevance through the use of well characterised transformed cell lines, primary cells and complex cellular models (e.g. co-culture, 3D models). However, although the field is gaining valuable experience in running more non-standard & complex cell assays for target validation and compound pharmacology studies, there is the lack of a systematic approach to determine if this expansion in cell assay models is reflected in increased human biological and disease relevance. The increasing wealth of publically available transcriptomic, and epigenome (ENCODE and Epigenome Roadmap) data represents an ideal reference mechanism for determining the relationship between cell types used for target & compound studies to primary human cells and tissues from both healthy volunteers & patients. The CTTV020 epigenomes of cell line project aims to generate epigenetic and transcriptomic profiles of cell lines and compare these with existing and newly generated reference data sets from human tissue and cell types. The aim is to identify assay systems which will provide greater confidence in translating target biology and compound pharmacology to patients. Multiple cell types commonly used within research have been grouped according to biology. Examples include erythroid, lung epithelial, hepatocyte cell types and immortalised models of monocyte / macrophage biology. 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 EGAS00001003137

• Open_Targets_020_Epigenomes_of_Cell_Lines

  There is currently a drive to establish cell based assay systems of greater human biological and disease relevance through the use of well characterised transformed cell lines, primary cells and complex cellular models (e.g. co-culture, 3D models). However, although the field is gaining valuable experience in running more non-standard & complex cell assays for target validation and compound pharmacology studies, there is the lack of a systematic approach to determine if this expansion in cell assay models is reflected in increased human biological and disease relevance. The increasing wealth of publically available transcriptomic, and epigenome (ENCODE and Epigenome Roadmap) data represents an ideal reference mechanism for determining the relationship between cell types used for target & compound studies to primary human cells and tissues from both healthy volunteers & patients. The CTTV020 epigenomes of cell line project aims to generate epigenetic and transcriptomic profiles of cell lines and compare these with existing and newly generated reference data sets from human tissue and cell types. The aim is to identify assay systems which will provide greater confidence in translating target biology and compound pharmacology to patients. Multiple cell types commonly used within research have been grouped according to biology. Examples include erythroid, lung epithelial, hepatocyte cell types and immortalised models of monocyte / macrophage biology. 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 EGAS00001003139

• The University of Hong Kong Intestinal Metaplasia Organoids Study

  Background: Gastric intestinal metaplasia (IM) is a pre-cancerous stage spanning a morphological spectrum that is poorly represented by human cell line models. Objective: We aim to establish and characterize human IM cell models to better understand IM progression along the cancer spectrum. Design: A large human gastric IM organoid (IMO) cohort (n=28), their clonal derivatives and normal gastric organoids (n=42) for comparison were established. Comprehensive multi-omics profiling and functional characterization were performed. Results: Single-cell transcriptomes revealed IMO cells spanning a spectrum from hybrid gastric/intestinal to advanced intestinal differentiation. Their lineage trajectories connected different cycling and quiescent stem and progenitors, highlighting differences in gastric to IM transition and the potential origin of IM from STMN1 cycling isthmus stem cells. Hybrid IMOs showed impaired differentiation potential, high lineage plasticity beyond gastric or intestinal fates, and reactivation of a fetal gene program. Cell populations in gastric IM and cancer tissues were highly similar to those derived from IMOs and exhibited a fetal signature. Genomically, IMOs showed elevated mutation burden, frequent chromosome 20 gain, and epigenetic de-regulation of many intestinal and gastric genes. Functionally, IMOs were FGF10 independent and showed downregulated FGFR2. Several IMOs exhibited a cell-matrix adhesion independent (CMi) subpopulation that displayed chromosome 20 gain but lacked key cancer driver mutations, potentially representing the earliest neoplastic precursor of IM-induced gastric cancer. Conclusions: Overall, our IM organoid biobank captured the heterogeneous nature of IM, revealing mechanistic insights on IM pathogenesis and progression, offering an ideal platform for studying early gastric neoplastic transformation and chemoprevention.

  Study EGAS00001007899

• Duplexseq of the interstrand crosslinks_WGS

  One of the most dangerous forms of DNA damage are interstrand crosslinks (ICLs), which covalently crosslink the two strands of the DNA double helix. The repair of these lesions is crucial for cellular survival due to their ability to block transcription and DNA replication. Initially, the major pathway that has been described in ICL repair involves a network of 22 genes that are mutated in a severe human genetic disease known as Fanconi Anemia (FA). Using synthetic lethality screens in the near-haploid human HAP1 cell line, we recently identified two potentially novel regulators of ICL repair, C1orf112 and THAP12. Loss of C1orf112 and THAP12 causes hypersensitivity to ICL-inducing DNA damaging agents, such as Mitomycin C (MMC). Additionally, C1orf112-depleted cells show elevated levels of micronuclei and accumulation of DNA damage in S-phase. To better understand how C1orf112 and THAP12 mediate the repair of ICLs, we want to perform mutational signature analysis, using the BotSeq method. Therefore, WT, C1orf112 and THAP12 knockout cells were cultured in vehicle or MMC treated conditions for 10 days and the genomic DNA was isolated. FANCA and FANCD2 knockout cells are taken along as controls in this experimental setting. . This dataset contains all the data available for this study on 2023-04-20.

  Dataset EGAD00001010298

• Characterization of mutational signatures in human cancer cell lines reveals episodic APOBEC mutagenesis

  Multiple signatures of somatic mutations have been identified in human cancer genomes. To investigate whether mutational signatures continue to be generated, and if so their temporal patterns of activity, subsets of cell lines were cultured in vitro for extended periods and subjected to single cell cloning and whole genome or exome sequencing or directly to single cell whole genome sequencing. As expected, signatures of past exogenous exposures, such as tobacco smoke and ultraviolet light, were not generated in vitro. In contrast, signatures of normal and defective DNA repair and replication continued to be generated at essentially constant mutation rates. Signatures of APOBEC cytidine deaminase DNA-editing activity exhibited a distinctive pattern with substantial fluctuations in mutation rate over time and episodic bursts of mutations. The initiating factors for these bursts are unclear although retrotransposon mobilisation may play a role. This cell line set now constitutes a comprehensive resource of live experimental models of mutational processes of both known and unknown aetiologies potentially retaining the patterns of activity and regulatory influences operative in human cells in vivo.

  Dataset EGAD00001004201

• Successful Clinical Response in Pneumonia Therapy (SCRIPT)

  The goal of this study is to iteratively identify, validate and refine biomarkers to predict clinical failure, and identify novel targets for therapy, beyond traditional antibiotics, for patients with severe pneumonia requiring intubation, including severe community-acquired pneumonia (CAP), hospital-acquired pneumonia, and ventilator-associated pneumonia (HAP/VAP). We hypothesize that factors within the alveolar microenvironment in many patients with pneumonia prevent the active process of pneumonia resolution.The major emphasis will be on HAP. Mechanically ventilated CAP patients are a very high-risk group for subsequent VAP, and therefore CAP samples will be included to determine baseline bacteria, microbiome, and host defense patterns for those that do or do not develop HAP/VAP. Hospital-acquired pneumonia is the leading cause of death from nosocomial infections. Even in patients receiving pathogen-appropriate therapy, reported clinical failure rates are increasing, approaching 50% in recent clinical trials. Current approaches to improve outcomes in patients with pneumonia almost exclusively focus on the development of antibiotics to address the problem of resistance. However, the prevalence of antibiotic-resistant pathogens is insufficient to explain the high rates of clinical failure. Instead, we hypothesize that factors within the alveolar microenvironment in many patients with pneumonia prevent the active process of pneumonia resolution. These factors include pathogen-specific adaptations to the injured lung that modulate the innate and adaptive immune response, as well as changes to the alveolar microbiome, driven by selective pressure within our modern intensive care units. Understanding this complex adaptive system requires the integration of clinical information with unbiased measures of the host, pathogen, and microbiome changes within the alveolar microenvironment. These measures must be repeated over time to identify factors that distinguish successful from failed therapy for pneumonia sufficiently early during the clinical course to allow for effective interventions. To address this challenge, we have assembled a talented group of investigators in the Successful Clinical Response In Pneumonia Therapy (SCRIPT) Systems Biology Center. We will leverage our routine clinical practice of safe alveolar sampling in mechanically ventilated patients with pneumonia with repeated non-bronchoscopic bronchoalveolar lavage (NBBAL) sampling over the course of pneumonia. From this fluid, we will combine flow cytometry with multi-omic technologies (cell population-specific transcriptomics, epigenomics, shotgun microbiome sequencing, and pathogen-specific sequencing). Our systems scientists will integrate these genomic data with robust clinical -omics with the goal of identifying biomarkers that can be prospectively tested in patients and causally evaluated in humanized mouse models.The addition of a pooled sample artificially increases the number of automatically computed subjects by one.

  Study phs002300

• Development of cell lines and mouse models of bone and soft tissue sarcoma to establish novel treatment

  Malignant giant-cell tumors are extremely rare bone sarcomas that transform from conventional giant-cell tumors during long periods of treatment. We have applied the culture methods and succeeded in establishing patient-derived organoids (PDO) of rare sarcomas. This study aimed to investigate the genomic characteristics of our established novel organoids from human malignant giant-cell tumors. The developed tumors with xenograft were genomically analyzed to compare their characteristics with those of the original tumors. Genetic changes over time throughout treatment were analyzed, and the genomic status of the established organoid was confirmed. Next generation sequencing revealed that the established organoids lacked the H3-3A G34W mutation. The xenografted organoids of the malignant giant-cell tumor had phenotypes genetically similar to those of the original tumor. The established organoids were confirmed to be derived from human malignant giant-cell tumors. In summary, the present study demonstrated a novel organoid model of a malignant giant-cell tumor that was genetically confirmed to be a malignant transformed tumor. Our organoid model could be used to elucidate the molecular pathogenesis of a malignant giant-cell tumor and develop novel treatment modalities.

  Study JGAS000618

• RNA-seq of high grade serous ovarian tumours

  Somatic RNA for 37 samples was extracted using the Qiagen Qiasymphony RNA protcol (cat no 931636). The tissue was initially homogenised using a Qiagen Bioruptor, followed by the manufacturers recommended protocol (including DNase digestion). The resulting RNA the underwent quality control as follows: firstly, A260 and A280nm were measured on a Denovix DS-11 Fx to qualitatively illustrate A260/280nm and A260/230nm ratios as measures of RNA purity. A260/280 had to be 2.0 and A260/230 had to be 2.0-2.2. Then RNA was quantified using LifeTechnologies Qubit RNA BR kit (cat no Q10210). RNAseq was carried out by the Edinburgh Clinical Research Facility on an Illumina NExtSeq500. Total RNA samples were assessed on the Agilent Bioanalyser (Agilent Technologies, #G2939AA) with the RNA 6000 Nano Kit (#5067-1512) for quality and integrity of total RNA, and then quantified using the Qubit 2.0 Fluorometer (Thermo Fisher Scientific Inc, #Q32866) and the Qubit RNA HS assay kit (#Q32855). Libraries were prepared from total-RNA sample using the NEBNext Ultra 2 Directional RNA library prep kit for Illumina (#E7760S) with the NEBNext rRNA Depletion kit (#E6310) according to the provided protocol. 400ng of totalRNA was then added to the ribosomal RNA (rRNA) depletion reaction using the NEBNext rRNA depletion kit (Human/mouse/rat) (#E6310). This step uses specific probes that bind to the rRNA in order to cleave it. rRNA-depleted RNA was then DNase treated and purified using Agencourt RNAClean XP beads (Beckman Coulter Inc, #66514). RNA was then fragmented using random primers before undergoing first strand and second strand synthesis to create cDNA. cDNA was end repaired before ligation of sequencing adapters, and libraries were enriched by PCR using the NEBNext Multiplex oligos for Illumina set 1 and 2 (#E7500). Final libraries had an average peak size of 271bp. Libraries were quantified by fluorometry using the Qubit dsDNA HS assay and assessed for quality and fragment size using the Agilent Bioanalyser with the DNA HS Kit (#5067-4626). Sequencing was performed using the NextSeq 500/550 High-Output v2 (150 cycle) Kit (# FC- 404-2002) on the NextSeq 550 platform (Illumina Inc, #SY-415-1002). Libraries were combined in an equimolar pool based on the library quantification results and run across 5 High-Output Flow Cell v2.5.

  Dataset EGAD00001010139

• RNA-seq of high grade serous ovarian tumours

  Somatic RNA for 40 samples matched to the WGS was extracted using the Qiagen Qiasymphony RNA protcol (cat no 931636). The tissue was initially homogenised using a Qiagen Bioruptor, followed by the manufacturers recommended protocol (including DNase digestion). The resulting RNA the underwent quality control as follows: firstly, A260 and A280nm were measured on a Denovix DS-11 Fx to qualitatively illustrate A260/280nm and A260/230nm ratios as measures of RNA purity. A260/280 had to be 2.0 and A260/230 had to be 2.0-2.2. Then RNA was quantified using LifeTechnologies Qubit RNA BR kit (cat no Q10210). RNAseq was carried out by the Edinburgh Clinical Research Facility on an Illumina NExtSeq500. Total RNA samples were assessed on the Agilent Bioanalyser (Agilent Technologies, #G2939AA) with the RNA 6000 Nano Kit (#5067-1512) for quality and integrity of total RNA, and then quantified using the Qubit 2.0 Fluorometer (Thermo Fisher Scientific Inc, #Q32866) and the Qubit RNA HS assay kit (#Q32855). Libraries were prepared from total-RNA sample using the NEBNext Ultra 2 Directional RNA library prep kit for Illumina (#E7760S) with the NEBNext rRNA Depletion kit (#E6310) according to the provided protocol. 400ng of totalRNA was then added to the ribosomal RNA (rRNA) depletion reaction using the NEBNext rRNA depletion kit (Human/mouse/rat) (#E6310). This step uses specific probes that bind to the rRNA in order to cleave it. rRNA-depleted RNA was then DNase treated and purified using Agencourt RNAClean XP beads (Beckman Coulter Inc, #66514). RNA was then fragmented using random primers before undergoing first strand and second strand synthesis to create cDNA. cDNA was end repaired before ligation of sequencing adapters, and libraries were enriched by PCR using the NEBNext Multiplex oligos for Illumina set 1 and 2 (#E7500). Final libraries had an average peak size of 271bp. Libraries were quantified by fluorometry using the Qubit dsDNA HS assay and assessed for quality and fragment size using the Agilent Bioanalyser with the DNA HS Kit (#5067-4626). Sequencing was performed using the NextSeq 500/550 High-Output v2 (150 cycle) Kit (# FC- 404-2002) on the NextSeq 550 platform (Illumina Inc, #SY-415-1002). Libraries were combined in an equimolar pool based on the library quantification results and run across 5 High-Output Flow Cell v2.5.

  Dataset EGAD00001009048

• FASTQ files of total RNA-Seq data of POPS control samples

  Placental biopsies (n = 64 female placentas, n = 67 male placentas) were selected from healthy pregnancies from the POPs cohort. These patients had no evidence of hypertension at booking and during pregnancy, did not experience pre-eclampsia, Hemolysis, Elevated Liver enzymes, and Low Platelets (HELLP) syndrome, gestational diabetes, or diabetes mellitus type I or type II and other obstetric complications. They delivered live babies with a birth weight percentile in the normal range (20-80th percentile), with no evidence of slowing in fetal growth trajectory. Chorionic villi from the corresponding placentas (free from decidua, visible infarction, calcification, hematoma, or damage) were collected and processed within 30 minutes of separation from the uterus. After repeated washes in chilled phosphate buffered saline, the samples were placed in RNA later (Applied Biosystems) and stored at -80°C. Total placental RNA was extracted using mirVana Isolation Kit (Ambion). For each placenta, approximately 5 mg of tissue were homogenized in the Lysis/Binding solution for 20 sec at 6 m/s using a bead beater (FastPrep24) and Lysing Matrix D Tubes (MP Biomedicals). The samples were then spun at 13,000 rpm for 5 min at 4°C and the supernatants recovered. Afterwards, the manufacturer's instructions were followed. Immediately after the RNA extraction, placental RNA samples were DNase-treated using DNA-free DNA Removal Kit (Ambion), aliquoted, and stored in -80°C. Quantity and quality of the RNA samples were assessed using the Agilent 2100 Bioanalyzer, the Agilent RNA 6000 Nano Kit (Agilent Technologies), and Qubit fluorometer. Libraries were prepared starting with 300-500 ng of good quality total RNA (RIN ≥7.5) using the TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Human/Mouse/Rat (Illumina), according to the manufacturer's instructions. The kit contains 96 uniquely indexed adapter combinations in order to allow pooling of multiple samples prior to sequencing. After determining their size (with the Agilent 2100 Bioanalyzer and the Agilent High Sensitivity DNA Kit by Agilent Technologies) and concentration (by qPCR with the KAPA Illumina ABI Prism Library Quantification Kit, Kapa Biosystems), libraries have been pooled and sequenced (single-end, 125 bp) using a Single End V4 Cluster Kit and an Illumina HiSeq2500 or HiSeq4000 instrument.

  Dataset EGAD00001003457

• Mucociliary Clearance Consortium (MCC) Longitudinal Study of Primary Ciliary Dyskinesia: Participants 5-18 Years of Age

  The purpose of this longitudinal study protocol is to define age-related prevalence of phenotypic characteristics and the progression of key features of lung disease in participants with one of these disorders, Primary Ciliary Dyskinesia (PCD). In PCD, the abnormal structure and function of cilia results in impaired clearance of secretions and consequent obstruction and chronic recurrent infection in the airways, sinuses and middle ears. Although it seems likely that the onset of PCD airway disease occurs early in childhood, as has been reported for cystic fibrosis (CF), the clinical course of PCD lung disease is not well defined, nor, is the time course of emergence of specific microbial pathogens, or the age of onset and rate of progression of airway disease and bronchiectasis. This longitudinal study is designed to define the rate of progression of PCD lung function in participants between 5-18 years of age using spirometry which tracks well with lung impairment and prognosis in other disorders of the airways such as cystic fibrosis. This longitudinal protocol will also systematically track other specific outcomes, including pathogens infecting the airways (assessed by respiratory cultures), and age at onset and progression of airway damage and bronchiectasis (assessed by high-resolution computerized tomography, HRCT, of the chest). Blood will be collected for DNA to test for genetic mutations. The blood samples will be processed to establish a cell line (lymphocyte transformation) for a source of DNA for future genetic studies, in participants with disease. For participants unable to provide a blood sample, a buccal sample will be obtained for DNA.

  Study phs000596

• Genomic Analysis of Mycosis Fungoides and Sézary Syndrome Identifies Recurrent Alterations in TNFR2

  Mycosis Fungoides (MF) and Sézary Syndrome (Sz) comprise the majority of Cutaneous T-Cell Lymphoma (CTCL) cases and are characterized by clinical heterogeneity. This array of symptoms includes skin patches, plaques and tumors as well as blood involvement and erythroderma. Because the genetic basis of CTCL is still poorly understood, we performed whole-exome sequencing on 11 MF/Sz samples and their matched, normal control DNA. Upon analyzing this data, we distilled a list of an additional 494 genes to be sequenced at depth. The majority of these 494 genes were sequenced in the exons alone, however, for a small subset of these genes we sequenced the entire genomic locus in search of structural variation events. This 494 gene targeted resequencing effort was performed upon 72 patients with MF/Sz and their matched, normal control DNA. An additional 5 MF/Sz and matched, normal DNA samples underwent whole genome amplification (WGA) prior to library preparation, which appears to have introduced mutations into these samples, however we also include them here. We additionally include 10 MF/Sz samples without a matched, normal control samples, 1 of which underwent WGA. In addition to primary disease samples, we also performed targeted resequencing on 4 CTCL cell lines (HH, MyLa, SeAx and Hut78) as well as on H9 cells (a subclone of Hut78 cells) and Jurkat cells, a T-ALL derived cell line that was used as an experimental system for our work. In sum, this data deposition includes exome and/or targeted resequencing from 92 individuals with 192 samples submitted.

  Study phs000913

• Non-coding RNAs Activated by the Wnt/Beta-catenin Signaling Pathway in Hepatoblastoma

  Hepatoblastoma (HB) is the most common pediatric liver tumor, affecting mostly children under 3 years of age. This rare tumor represents 1% of all pediatric cancers. Genetic studies have shown that HB is characterized by high frequency mutations of the CTNNB1 gene encoding beta-catenin (around 75%) and relative genomic stability. Here we have analyzed the transcriptional profile of 21 HBs compared to matched non-tumor livers by Cap Analysis of Gene Expression (CAGE), which provides accurate and quantitative profiling of all transcripts. CAGE analysis revealed strong upregulation of known Wnt target coding genes in most tumors analyzed, consistent with previous transcriptomic studies. To better define the Wnt-dependent transcriptional landscape of HB, we integrated CAGE data with TCF4 ChIP-seq data from a CTNNB1-mutated cancer cell line and with the FANTOM5 genomic coordinates of TCF/LEF binding motifs. Both TCF/LEF binding motifs and ChIP-seq peaks were strongly enriched in the immediate upstream region, not only for protein-coding genes, but also for non-coding transcripts. Among the selected 112 top Wnt target genes at FDR<1.0E-6 and fold change>8, we found clear over-representation (66%) of distant transcription start sites (TSSs) representing lncRNAs and enhancer RNAs, which raises the question of their role in HB pathogenesis. Analysis of the 112 promoters using CAGEd-oPOSSUM confirmed the predominant involvement of Tcf/Lef transcription factors, together with HNF4G, GATA2, Sox3 and Ets-related genes. Finally, the 112 Wnt target signature defined 3 tumor subclasses, T1, T2 and T3, characterized by progressive alteration of the non-coding part of the transcriptome and significant differences in clinical behavior.

  Study phs001433

• Investigating the Role of Neddylation in the Repair of Topoisomerase I-Mediated DNA Damage in Colorectal Cancer

  Colorectal cancer is one of the most lethal cancers worldwide. First-line chemotherapy for metastatic CRC (mCRC) entails a combination of irinotecan with 5-fluorouracil and leucovorin (FOLFIRI). Irinotecan is a prodrug that is converted within the cell to its active metabolite SN38, a potent TOP1 inhibitor. TOP1 relieves DNA torsional strain arising from DNA metabolism by cleaving one strand of the DNA duplex and forming an enzyme-DNA covalent intermediate that can be trapped by inhibitors. The resulting TOP1 DNA-protein crosslinks (TOP1-DPCs) interfere with replication and transcription and induce cell death if left unrepaired. I have established the role of the ubiquitin-proteasome pathway in TOP1-DPC repair, but it remains unknown how ubiquitylation is activated. One possibility is that collision between advancing replication forks and the DPCs triggers a specific ubiquitin ligase to target DPCs for degradation. The DDB1-CUL4 (Cullin 4)-RBX1 (CRL4) ubiquitin ligase complex performs a salient role in both replication and DNA repair upon its activation by auto-NEDD8 modification (neddylation), but it is unknown whether it plays a role for TOP1-DPC repair. We plan to define the role of neddylation for TOP1-DPC repair during replication and examine irinotecan plus pevonedistat (PEV), a NEDD8-activating enzyme inhibitor in multiple clinical trials, in CRC patient-derived preclinical models including 3D organoids. The three in-house organoids, from which samples have been submitted to dbGaP, were sequenced as a means of verifying their identity as colon cancer organoids, to allow us to investigate the combination of TOP1 inhibitors and PEV and the molecular mechanisms underlying this drug combination in these organoids.

  Study phs003257

• National Cancer Institute Clinical and Laboratory Analysis of Familial Cancer

  The purpose of this study is to continue the analysis begun on 09C0079 which was focused on identification of the genetic mutation associated with a new gastric polyposis syndrome, Gastric Adenocarcinoma and Proximal Polyposis of the Stomach (GAPPS). GAPPS, first described in 2012, is an autosomal dominant gastric polyposis syndrome that confers a substantial risk for gastric adenocarcinoma. The GAPPS phenotype consists of a carpeting of greater than 100 fundic gland polyps (FGPs) in the oxyntic mucosa of the gastric body and fundus, with antral sparing with some FGPs displaying high-grade dysplasia. This is in contrast to sporadic FGPs that are benign, most often are not associated with high-grade dysplasia though low-grade dysplasia has been reported, and are fewer in number. Gastric FGPs have been found to be associated with Familial Adenomatous Polyposis (FAP) and attenuated FAP, which is an autosomal dominant condition associated with germ line mutations in APC which confers a phenotype consisting of multiple (>100) adenomatous polyps in the colon and rectum developing after the first decade of life. FGPs are reported to occur in 12-84% of patients with FAP, whereas sporadic FGPs are identified in ~5% of patients undergoing upper gastrointestinal endoscopy. Gastric adenocarcinoma arising from FGPs in individuals with FAP have been reported. Other manifestations of FAP and AFAP include polyps in the upper gastrointestinal tract, congenital hypertrophy of retinal pigment epithelium, osteomas and epidermoid cysts, supernumerary teeth, desmoid formation, and other malignancies such as thyroid, small bowel cancer, hepatoblastoma, and medulloblastoma. Thus far there are no reports in GAPPS of polyposis arising in the esophagus, gastric antrum, pylorus, small intestine, or colon.

  Study phs001935

• Genetic Analysis of Parkinson's Disease

  A portion of the sample includes multiplex PD families recruited as part of a previously funded study (R01NS037167; PI: Tatiana Foroud). Families were ascertained initially through a living affected sibling pair. Subsequently, ascertainment was loosened to include PD probands having a positive family history of PD in a first degree relative, who was not required to be part of the study. All individuals completed an in-person evaluation at one of the > 65 Parkinson Study Group sites participating in this study. Individuals completed a detailed evaluation that included the Unified Parkinson Disease Rating Scale (UPDRS), Mini-Mental State Examination (MMSE), Geriatric Depression Scale (GDS) and a diagnostic checklist that implemented the UK PD Brain Bank inclusion and exclusion criteria. An autopsy program was established in this study and brain tissue for confirmation of diagnosis and further experimental study are available from over 100 participants. Autopsy confirmation of the PD diagnosis was nearly 100% for those who met UK PD Brain Bank criteria. In addition, because this was a cross sectional study and family members were often seen very early in disease course, among those who did not meet UK PD Brain Bank criteria but had a subsequent autopsy, a significant subset (>1/3) had autopsy confirmation of PD. The second source of samples is from the NINDS Human Genetics DNA and Cell Line Repository located at the Coriell Institute. Currently DNA from 4,509 PD patients is available from the Coriell Institute. We restricted the selected sample to include only Caucasians and ensured there was no overlap with samples contributed by the PROGENI Study.

  Study phs001004

• Genetic Aberrations and Subclonal Structure Impact Chronic Lymphocytic Leukemia

  Large-scale whole-exome sequencing (WES) of primary tumor samples enables the unbiased discovery of recurrent putative driver events and patterns of clonal evolution. We report the identification of 44 recurrently mutated genes and 11 recurrent CNVs through the WES of 538 chronic lymphocytic leukemia (CLL) and matched germline DNAs. These include previously unrecognized cancer drivers (e.g., RPS15, IKZF3), and collectively identify nuclear export, MYC activity and MAPK signaling as central pathways affected by somatic mutation in CLL. A clonality analysis of this large dataset further enabled the reconstruction of temporal relationships between these driver events. Several drivers were associated with shorter progression-free survival (PFS) in 280 samples that were collected prior to uniform treatment with front line chemo-immunotherapy, with mature follow up of greater than 10 years. Direct comparison between matched pretreatment and relapse CLL from 59 samples demonstrated marked clonal evolution occurring in more than 95% of these patients. Distinct patterns of clonal evolution in relationship to specific gene alteration were observed, suggesting a hierarchy of fitness amongst mutations. Thus, large WES datasets of clinically informative samples enable the discovery of novel driver genes as well as the network of relationships between the drivers and their impact on disease relapse and clinical outcome.Additionally, we performed RNA-seq for 268 CLL samples (including 26 follow-up samples) and used them to identify expression subtypes of CLL. RRBS for 30 of these samples was also generated. In an integrative analysis of genetic, transcriptomic, and epigenetic data, incorporating known and newly identified subtypes of CLL, we built new models to improve patient prognostication.

  Study phs000922

• Binding of Epstein Barr Virus EBNA2 Unifies Multiple Sclerosis Genetic Mechanisms

  This study is focused on multiple sclerosis (MS). MS is likely caused by a combination of genetic and environmental factors; however, the mechanisms contributing to these factors remain poorly understood. Epstein-Barr virus (EBV) in particular is a well-established environmental risk factor for MS. We have created a computational algorithm that systematically searches for common molecular mechanisms that might be impacted at multiple MS-associated loci. Using this algorithm, we have discovered that over 40% of MS-associated loci contain MS genetic variants that fall within regions of the human genome occupied by the EBV-encoded EBNA2 protein. The same MS-associated variants also impact gene expression levels of MS-associated genes in EBV-infected B cell lines. Our hypothesis is that allele-dependent binding of EBNA2 and its co-factors explains the allele-dependent risk at many MS genetic loci. Importantly, this hypothesis links the genetic associations of MS to the known molecular roles played by EBV and Notch signaling in disease processes.Subjects with and without MS were recruited into an Institutional Review Board (IRB)-approved protocol through the Cincinnati Autoimmune Registry and Repository. From each subject, DNA from peripheral blood was isolated for whole genome sequencing (WGS). These data will be used to identify genotype-dependent transcriptional regulation in cells assessed from patients and controls. This WGS data was deposited before the full integration of sequence data with other functional genomic data types.From each subject, 90 mL of blood is drawn for isolation of peripheral blood mononuclear cells from which primary B and T cells will be isolated. For each B cell isolation, an EBV-transformed B cell line will be derived.

  Study phs003240

• Genetic Neuroscience: How Human Genes and Alleles Shape Neuronal Phenotypes

  The goal of this collaborative, interdisciplinary project is to develop powerful, generalizable approaches for discovering how risk variants for psychiatric disorders shape neurobiological processes at multiple levels of analysis, and to identify the processes whose dysregulation underlies disease.Induced pluripotent stem cells (iPSCs) were used towards the development of these new experimental and inferential systems bridging gaps between human genetics and experimental biology. The largest publicly available collection of iPSCs (2607 lines) has been generated from 2184 donors by the California Institute for Regenerative Medicine (CIRM). To expand our donor collection, an additional 44 iPSC donors from the McLean_Levy cohort were identified. We wish to share the available SNP data for 2167 CIRM lines and whole genome sequence data generated at the Broad Institute for 473 of the CIRM and 44 McLean_Levy iPSC donors. These data can be used to identify (for experiments) lines with specific genotypes of interest and lines from donors with high or low polygenic risk scores for phenotypes of interest. The data can also be used to identify acquired mutations in the iPSC lines. The CIRM iPSC lines are available through Fujifilm Cellular Dynamics iPSC Repository (https://www.fujifilmcdi.com/cirm-ipsc-products/). The McLean_Levy lines are available through the NIMH Repository & Genomics Resource (https://www.nimhgenetics.org/).Additional project data registered with the study includes data from an iPSC line derived from an SMA patient (n=1), as well as single-cell RNA sequence data and supplemental processed genomic datasets in support of project publications.Molecular DatasetsSingle-cell RNA-seq: 10X Genomics, Illumina NovaSeqSupplemental "cell village" pooled genomic sequence data: Illumina NextSeqWhole Genome Genotyping: Infinium Global Screening Array-24 Kit, Illumina HumanCore chipWhole Genome Sequencing: Illumina NovaSeq

  Study phs002032

• Response and Resistance to ER-Directed Therapy in Metastatic Breast Cancer

  Despite the reduction in cancer recurrence and mortality provided by hormonal therapy, estrogen receptor positive (ER+) metastatic breast cancer (MBC) remains the most common cause of breast cancer death, resulting in more than 20,000 deaths in the U.S each year. Although several therapeutic options exist to target the estrogen receptor, resistance to these therapies invariably occurs. Genetic mechanisms of acquired resistance to targeted therapies in ER+ MBC are not well understood. To address this, we developed a prospective biopsy protocol where multiple research biopsies are collected from metastatic samples and used for pathology, ER/PR/HER2, OncoPanel (a CLIA 300 gene sequencing panel), whole exome sequencing (WES), transcriptome sequencing (RNA-Seq), and single-cell RNA-seq (scRNA-seq). In some cases, tissue is used for cell line and xenograft generation. Archival primary biopsies are also obtained when possible for parallel genomic studies. Patients are followed and serial metastatic biopsies are obtained. This data will be used to determine the landscape of genomic alterations in treatment-resistant ER+ MBC, and compared to data generated by TCGA for treatment-naive ER+ primary breast cancer. Comparison of genomic studies from resistant, metastatic specimens with corresponding pre-treatment primary samples will allow us to identify genomic association with resistance to specific therapies. Analysis of the serial metastatic biopsies collected over the course of multiple treatments will also shed light on tumor evolution under selective therapeutic pressure.NoteA subset of the MBC blood biopsies that are included in our study can be accessed and downloaded from the phs001977 accession.Another subset of patients in this study participated in a clinical trial (NCT02871791) and have their data deposited in the current study as well.

  Study phs001285

• Efficacy and Immune Effects of Anakinra Prophylaxis for Neurologic Toxicity and Cytokine Release Syndrome in Patients with Lymphoma Receiving Axicabtagene Ciloleucel

  Chimeric antigen receptor T cells have transformed the treatment of multiple hematologic malignancies, but are limited in application due to treatment-related toxicities. Cytokine release syndrome (CRS) and neurotoxicity (NT) are the primary toxicities associated with these therapies and are thought to be mediated by broad immune activation due to robust T-cell expansion. Current treatment of these toxicities utilize anti-IL-6 directed therapy and/or systemic glucocorticoids to mitigate the consequences of this complex inflammatory cascade. We sought to use anakinra, an IL-1R antagonist, as a prophylactic strategy to prevent clinically meaningful CRS/NT, defined as grade 2 or higher toxicity necessitating clinical intervention. Although our study, in line with others, demonstrated that IL-1R antagonism is insufficient to prevent such toxicities entirely, we were able to gain insight into the molecular immune signaling associated with breakthrough CRS & NT in the presence of anakinra prophylaxis using our scRNA dataset of patients treated with and without IL-1R antagonism. We demonstrate that IL-4 and IL-10 anti-inflammatory pathways in infused CAR-T products of both anakinra and non-anakinra cohorts were negatively associated with grade 2+ toxicities. We also found that expression of IFN-gamma pathways and ligand-receptor activities, as well as cytokine levels of IFN-gamma and CXCL10 in CD14+ monocytes, were significantly enriched in patients with breakthrough toxicity in the anakinra cohort. This correlated with an increase in IFN-gamma in the peripheral blood of patients with breakthrough toxicities, among other cytokines. These data identify IFN-gamma as a potential key mechanism in the development of CAR-T cell-associated toxcities and suggest that this pathway is also targetable and not inhibited by anakinra alone.

  Study phs003655

• CCR8-targeted specific depletion of clonally expanded Treg cells in tumor tissues evokes potent tumor immunity with long-lasting memory

  Foxp3-expressing CD25+CD4+ regulatory T cells (Tregs) are abundantly infiltrating into tumor tissues, hindering effective anti-tumor immunity. Here, assuming that such tumor Tregs were clonally expanding by recognizing tumor-associated antigens, we attempted to characterize them by their gene expression profiling and T-cell receptor (TCR) clonotyping at the single-cell level. We found that they predominantly expressed the chemokine receptor CCR8: ~40% of tumor Tregs, less than 10% of Tregs in other tissues, and few tumor-infiltrating conventional T cells expressed CCR8 in mice and humans. CCR8+ tumor Tregs were phenotypically highly differentiated and functionally stable. One injection of anti-CCR8 mAb with high antibody-dependent cellular cytotoxic activity was indeed able to specifically deplete clonally expanded tumor Tregs and thereby eradicate established tumors in mice. The depletion predominantly expanded tumor-infiltrating CD8+ effector T cells, which were different in activated or exhausted patterns from those seen after immune checkpoint blockade by anti-PD-1 mAb. The treatment also evoked potent secondary immune responses against the same tumor cell line inoculated several months after tumor eradication, indicating that the anti-CCR8-induced memory-type tumor-reactive effector T cells were, once induced, resistant to recovered Treg suppression. In contrast to such evocation of potent tumor immunity, anti-CCR8 treatment scarcely elicited discernable autoimmunity in mice, contrasting with a similar treatment for systemic whole Treg depletion, which induced histologically and serologically evident autoimmunity. Thus, temporary and specific depletion of clonally expanded Tregs in tumor tissues by cell-depleting anti-CCR8 mAb is sufficient to evoke potent tumor immunity with long-lasting memory and without serious autoimmunity.

  Study JGAS000312

• Stromal-induced epithelial-mesenchymal transition induces drug resistance in acute lymphoblastic leukemia

  Bone marrow (BM) microenvironment-induced drug resistance in acute lymphoblastic leukemia (ALL) is thought to be mediated by intercellular interaction of leukemic cells with BM niches, but the mechanistic basis of resistance is poorly understood. Here, we genomic and functional analyses, we show that ALL-BM mesenchymal stromal cells (MSC) interactions encompass direct cell-cell adhesion via integrin α4β1, uni-directional secretion of soluble factors from MSC to ALL, and bidirectional transfer of cellular vesicles through tunneling nanotubes. We show that this crosstalk drives an epithelial-mesenchymal transition (EMT)-like program in ALL cells adhering to MSC, thereby triggering stem cell-like features including drug resistance and survival advantage. Moreover, single-cell RNA sequencing of cell line and primary ALL samples identified a hybrid cell state derived from B-ALL adhered to MSC, that exhibits both B-ALL and MSC gene signatures that are orchestrated by an EMT-like program. Multiple EMT-activating pathways including WNT/β-catenin, cytokine/STAT3, TGFβ, NOTCH1, and NF-κB-dependent signaling pathways are activated in MSC-adherent ALL cells; however, only the inhibition of either WNT/β-catenin or cytokine/STAT3 pathway could attenuate the survival and drug resistance of ALL cells adherent to MSC. Notably, we identified that this hybrid cell state exhibited elevated WNT/β-catenin-mediated transcriptional activity. Finally, we show that blocking of the interaction of β-catenin and CBP interaction abrogates the survival and drug resistance of ALL cells adhered to MSC, indicating that targeting of an EMT-like program is potential therapeutic regimen in targeting cell extrinsically acquired drug resistance in ALL.

  Study EGAS00001006120

• Whole-exome Sequencing Combined with Functional Genomics Reveals Novel Candidate Driver Cancer Genes in Endometrial Cancer

  Endometrial cancer is the most common gynecological malignancy, with more than 280,000 cases occurring annually worldwide. Although previous studies have identified important common somatic mutations in endometrial cancer, they have primarily focused on a small set of known cancer genes and have thus provided a limited view of the molecular basis underlying this disease. Here we developed an integrated systems biology approach to identifying novel cancer genes contributing to endometrial tumorigenesis. We first performed whole-exome sequencing on 13 endometrial cancers and matched normal samples, systematically identifying somatic alterations with high precision and sensitivity. We then combined bioinformatics prioritization with high-throughput screening (including both shRNA-mediated knockdown and expression of wild type and mutant constructs) in a highly sensitive cell viability assay. Our results revealed 12 potential driver cancer genes including 10 tumor suppressor candidates (ARID1A, INHBA, KMO, TTLL5, GRM8, IGFBP3, AKTIP, PHKA2, TRPS1 and WNT11) and two oncogene candidates (ERBB3 and RPS6KC1). The results in the “sensor” cell line were recapitulated by siRNA-mediated knockdown in endometrial cancer cell lines. Focusing on ARID1A, we integrated mutation profiles with functional proteomics in 222 endometrial cancer samples, demonstrating that ARID1A mutations frequently co-occur with mutations in the phosphatidylinositol 3-kinase (PI3K) pathway and are associated with PI3K pathway activation. siRNA knockdown in endometrial cancer cell lines increased AKT phosphorylation supporting ARID1A as a novel regulator of PI3K pathway activity. Our study presents the first unbiased view of somatic coding mutations in endometrial cancer and provides functional evidence for diverse driver genes and mutations in this disease.

  Study EGAS00001000318

• Intra-tumor heterogeneity and clonal evolution patterns towards platinum-resistant high-grade serous ovarian cancer

  Although many high-grade serous ovarian carcinoma (HGSOC) patients are initially sensitive to the standard-of-care platinum-based therapy, intra-tumor heterogeneity may fuel selection towards platinum-resistant clones over subsequent lines of therapy. We performed whole-exome coupled to ultra-deep re-sequencing and copy number alteration (CNA) profiling on 31 matched diagnosis and relapse tumors from HGSOC patients treated with platinum. Genetic heterogeneity within tumor pairs varied extremely, with some recurrent tumors being identical and others completely different from the primary tumor. All primary tumors showed evidence of homologous recombination (HR)-deficiency, but none of them carried reactivating mutations in the HR pathway at relapse, also not when becoming resistant to subsequent lines of platinum. We did also not identify other mutations, CNAs or pathways that were enriched in platinum-resistant clones. However, a platinum score consisting of 13 CNAs correlated significantly with platinum-free interval (PFI) after first-line therapy in multiple cohorts of >450 primary HGSOCs. A relative increase of this score in the relapsed tumor correlated with a change in PFI during subsequent therapy. Clonal evolution under platinum therapy contributes to genetic heterogeneity within tumor pairs, implicating that genetic tests linked to targeted treatment options for recurrent HGSOC need to be performed on biopsies collected at relapse. Platinum resistance of an initially platinum-sensitive HR-deficient HGSOC at relapse is not caused by reactivation of the HR pathway, suggesting that these tumors are still responsive to PARP inhibitors, instead, several chromosomal aberrations contribute to a score predictive of disease outcome under platinum therapy, both in first and subsequent lines of platinum therapy.

  Study EGAS00001001244

• Targeted Gene Panel for 171 PTCLs

  Mature T-cell lymphomas, including peripheral T-cell lymphoma (PTCL) and extranodal NK/T-cell lymphoma (NKTL), represent a heterogeneous group of non-Hodgkin lymphomas with dismal outcomes and limited treatment options. To determine the extent of involvement of the JAK/STAT pathway in this malignancy, we performed targeted capture sequencing of 188 genes in this pathway in 171 PTCL and NKTL cases. A total of 272 nonsynonymous somatic mutations in 101 genes were identified in 73% of the samples, including 258 single-nucleotide variants and 14 insertions or deletions. Recurrent mutations were most frequently located in STAT3 and TP53 (15%), followed by JAK3 and JAK1 (6%) and SOCS1 (4%). A high prevalence of STAT3 mutation (21%) was observed specifically in NKTL. Novel STAT3 mutations (p.D427H, E616G, p.E616K, and p.E696K) were shown to increase STAT3 phosphorylation and transcriptional activity of STAT3 in the absence of cytokine, in which p.E616K induced programmed cell death-ligand 1 (PD-L1) expression by robust binding of activated STAT3 to the PD-L1 gene promoter. Consistent with these findings, PD-L1 was overexpressed in NKTL cell lines harboring hotspot STAT3 mutations, and similar findings were observed by the overexpression of p.E616K and p.E616G in the STAT3 wild-type NKTL cell line. Conversely, STAT3 silencing and inhibition decreased PD-L1 expression in STAT3 mutant NKTL cell lines. In NKTL tumors, STAT3 activation correlated significantly with PD-L1 expression. We demonstrated that STAT3 activation confers high PD-L1 expression, which may promote tumor immune evasion. The combination of PD-1/PD-L1 antibodies and STAT3 inhibitors might be a promising therapeutic approach for NKTL, and possibly PTCL.

  Study EGAS00001002740

• Pharmacological and genomic profiling identifies NFκB-targeted treatment strategies for mantle cell lymphoma

  Mantle cell lymphoma (MCL) is an aggressive malignancy characterized by poor prognosis, underscoring the need for novel therapeutic strategies1. In this study, we employed large-scale pharmacological profiling of 16 pathway inhibitors across more than 100 hematological cell line models to identify novel therapeutic approaches. This screen identified a subset of MCL cell lines that were highly sensitive to the B-cell receptor (BCR) signaling inhibitors ibrutinib and sotrastaurin (STN), which are currently in clinical trials2, 3. Sensitive MCL models exhibited chronic activation of the CARD11-BCL10-MALT1 (CBM) complex and dependency on downstream classical NFκB signaling. In contrast, insensitive cell lines displayed activation of the alternative NFκB pathway. Massively parallel transcriptome sequencing identified mutations in negative regulators of the alternative NFκB pathway in ibrutinib-insensitive cell lines. Notably, sequencing of 165 MCL patient samples revealed that components of the alternative NFκB pathway, particularly TRAF2 and BIRC3, are mutated in roughly 15% of MCL patients. Functional analyses of patient-derived BIRC3 mutant alleles indicated that these are loss-of-function mutants that constitutively activate alternative NFκB signaling. While being associated with insensitivity to ibrutinib, genetic lesions in the alternative pathway conferred dependence on NIK, a kinase that mediates signaling downstream of the TRAF2/3-BIRC2/3 complex. Thus, NIK represents a novel therapeutic target for MCL treatment, particularly for tumors refractory to BCR pathway inhibitors. Collectively, these findings reveal a pattern of mutually exclusive activation of the CBM-NFκB or alternative NIK-NFκB pathways in MCL and provide critical insights into patient stratification strategies for NFκB-pathway targeted agents.

  Study EGAS00001000622

• Transcriptional_profiling_of_tauopathies_in_human_IPS_derived_neurons

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

  Study EGAS00001000382

• DSRCT RNA genomic sequencing

  Purpose: To further understand the molecular pathogenesis of desmoplastic small round cell tumor (DSRCT), a rare but often fatal malignancy occurring primarily in young males, we used next-generation RNA sequencing to investigate the gene expression profiles intrinsic to this disease.Experimental Design: RNA from DSRCT tumor samples were obtained from the Cooperative Human Tissue Network biorepository. RNA sequencing was completed using the Illumina HiSeq 2000 system. Presence of the EWSR1-WT1 fusion was verified using both RNA-sequencing and PCR. Validation studies, including WT1 ChIP-seq, RNA sequencing, and EWS-WT1 knockdown experiments, were performed on an established DSRCT cell line, JN-DSRCT-1. Protein expression of clinically relevant genes determined by RNA sequencing was confirmed using immunohistochemistry. A panel of immune signature genes was also evaluated to identify possible immune therapeutic targets. Results: We analyzed the RNA from 14 tumor samples, of which two were confirmed negative for the diagnostic EWSR1-WT1 translocation. Principal component analysis of the sequencing data indicated that fusion-negative and fusion-positive tumors are transcriptionally distinct entities. Overlapping the significant ChIP-seq peaks with genes found to be highly expressed in the RNA sequencing data revealed that IGF2 and FGFR4 were both highly expressed, and targets of the EWS-WT1 fusion gene. This finding has potential clinical relevance for targeted therapeutic intervention. In addition, we identified the immune checkpoints CD200 and CD276 as potentially targetable genes whose expression is independent of the EWS-WT1 fusion gene in vitro.Conclusions: Despite the transcriptional complexity of this disease, we were able to identify IGF2, FGFR4, CD200, and CD276 as potential therapeutic targets for patients suffering from DSRCT.

  Study EGAS00001002770

• Regenerative effects of MSC treatment on busulfan-induced small intestine damaged organoids.

  Allogenic hematopoietic stem cell transplantation (HSCT) is a curative treatment for leukemia and a range of non-malignant disorders. The success of the therapy is hampered by occurrence of acute graft-versus-host disease (aGvHD); an inflammatory response damaging recipient organs, with gut, liver, and skin being the most susceptible. Intestinal GvHD injury is often a life-threatening complication in patients unresponsive to steroid treatment. Second-line available therapies are immunosuppressants or mesenchymal stromal/stem cell (MSC) infusions. Data from our institution and others demonstrate rescue of approximately 40-50% of patients suffering from aGvHD with mesenchymal stromal/stem cells (MSCs) and minor side effects. Although promising, the better understanding of MSC mode of action and patient response to MSC-based therapy is essential to improve this lifesaving treatment. Here, we developed a novel 3D co-culture model of human small intestinal organoids and MSCs, which allows to study the regenerative effects of MSCs on intestinal epithelium in a more physiologically relevant setting than existing in vitro systems. Using this model we mimicked chemotherapy-mediated damage of the intestinal epithelium. The treatment with busulfan, the chemotherapeutic commonly used as conditioning regiment before the HSCT, affected pathways regulating EMT, proliferation, and apoptosis in small intestinal organoids, as shown by transcriptomic and proteomic analysis. The co-culture of busulfan treated intestinal organoids with MSCs reversed the effects of busulfan on the transcriptome and proteome of intestinal epithelium, which we also confirmed by functional evaluation of proliferation and apoptosis. Collectively, we demonstrate that our novel in vitro co-culture system is a new valuable tool to facilitate the investigation of the molecular mechanisms behind the therapeutic effects of MSCs on damaged intestinal epithelium.

  Study EGAS00001007432

• Multi-Layered Molecular Profiling Informs the Diagnosis and Targeted Therapy of Desmoplastic Small Round Cell Tumor

  Desmoplastic small round cell tumor (DSRCT) is an ultra-rare soft tissue sarcoma with dismal prognosis and limited therapeutic options. Here, we identified individual therapeutic targets in a cohort of 30 DSRCT patients by use of multilayered molecular profiling (genomic, transcriptomic, and (phospho-)proteomic analyses) within the DKFZ NCT DKTK MASTER program. 28 of the 30 patients received at least one personalized treatment recommendation. In line with the observation that DSRCTs are an entity with a low mutational load, most of the treatment recommendations were based on target gene or -protein expression. 13 patients received a recommended therapy, resulting in four partial responses, four stable diseases, and five progressive diseases. Eight of 13 patients received the multi tyrosine kinase inhibitor pazopanib, with mixed response. Based on high ERBB2 expression levels, two patients received the antibody drug conjugate trastuzumab deruxtecan (T-DXd), showed exceptional, long-lasting responses and are currently (8/2024) still on treatment and progression-free. We detected high ERBB2 protein expression in all DSRCT samples with proteome data (n = 9), which suggests that ERBB2 may be a particularly useful therapeutic target for DSRCT patients. For one of the two exceptional responders we did not detect a substantial ERBB2/HER2 overexpression, which may indicate that T-DXd could also be efficacious in DSRCT patients with low levels of HER2, as also described for breast cancer patients. In conclusion, multi-omic molecular profiling revealed personalized therapeutic targets in patients with DSRCT with HER2 targeting, with T-DXd as a promising therapeutic option for this ultra-rare and difficult-to-treat tumor entity.

  Study EGAS00001007934

• The transition from quiescent to activated states in human hematopoietic stem cells is governed by dynamic 3D genome reorganization

  To elucidate the epigenetic changes which occur when human long-term hematopoietic stem cells (LT-HSC) become activated we performed Bulk ATAC-Seq on 13 sorted bulk hematopoietic populations from cord bloodas well as single-cell ATAC-Seq upon CD34+CD38-CD45RA- cells enriched for HSC as well as CD34+/CD38+ progenitor cells both from cord blood. These studies revealed gains of chromatin accessibility around CTCF binding sites during HSPC activation, as such we additionally performed Low-C to directly profile the 3D conformation of human cord-blood derived LT-HSC and Short-term hematopoietic stem cells (ST-HSC), as well as Hi-C , ATAC-Seq and CTCF ChIP-Seq upon the OCIAML-2 cell line in which CTCF sites gained during LT-HSC activation are enriched. Finally we transduced human cord-blood LT-HSC with an shCTCF vector; in-vitro cultured LT-HSC cells harbouring shCTCF were used to perform RNA-Seq, and scATAC-Seq was performed on CD34+/CD38- human CB cells transduced with shCTCF, four weeks post xeno-transplantation into mice. Collectively these studies have helped us demonstrate the role of 3D chromatin conformation changes during human LT-HSC activation.

  Dataset EGAD00001006447

• Bioinformatic Methods and Bridging of Assay Results for Reliable Tumor Mutational Burden Assessment in Non-Small Cell Lung Cancer

  Tumor mutational burden (TMB) has emerged as a predictive biomarker of response to immune checkpoint inhibitors. Standardization of TMB measurement is essential for implementing diagnostic tools to guide treatment. Here we evaluate bioinformatic TMB analysis by whole exome sequencing (WES) in formalin-fixed, paraffin-embedded samples. In CheckMate 026, TMB was retrospectively assessed in 312 patients with non-small cell lung cancer (58% of the intent-to-treat population) who received first-line nivolumab treatment or chemotherapy. We examined the sensitivity of TMB assessment to bioinformatic filtering methods and assessed concordance between TMB data derived by WES and the FoundationOne CDx™ assay. TMB scores comprising synonymous, indel, frameshift, and nonsense mutations (all mutations) were 3.1-fold higher than data including missense mutations only, but values were highly correlated (Spearman’s r = 0.99). Scores from CheckMate 026 samples including missense mutations only were similar to those generated from data in The Cancer Genome Atlas, but those including all mutations were generally higher. Using databases for germline subtraction (instead of matched controls) showed a trend for race-dependent increases in TMB scores. Parameter variation can therefore impact TMB calculations, highlighting the need for standardization. Encouragingly, WES and FoundationOne CDx outputs were highly correlated (Spearman’s r = 0.90) and differences could be accounted for by empirical calibration, suggesting that reliable TMB assessment across assays, platforms and centers is achievable.

  Dataset EGAD00001005035

• scRNA-seq of total bone marrow and T cells from multiple myeloma long-term survivors

  scRNA This dataset contains 50 scRNA-seq samples from bone marrow aspirates of 11 multiple myeloma patients experiencing long-term survival and 3 healthy donors. For each donor, total bone marrow and CD3+ T cells were sequenced. For multiple myeloma patients, paired samples were collected at initial diagnosis and between 7-17 years after first-line therapy. Bone marrow mononuclear cells were isolated by Ficoll density gradient centrifugation. For sorting of total bone marrow cells singlet, live cells were gated and sorted, for sorting of T cells CD45+, CD3+ cells were gated and sorted on either FACSAria Fusion or FACSAria II. Single-cell RNA sequencing were generated using 10x Genomics single-cell RNAseq technology (Chromium Single Cell 3’ Solution v2) according to the manufacturer’s protocol and sequenced on an Illumina HiSeq4000 (paired end, 26 and 74 bp). Bulk RNA Singlet, live CD3+CD4- CXCR3+CD8+ and CD3+CD4- CXCR3-CD8+ cells were sorted from 7 bone marrow and 3 peripheral blood samples of 7 multiple myeloma patients using a FACSAria Fusion machine. Bulk-RNA sequencing libraries were generated using the SMART Seq Stranded Total RNA-Seq kit (Takara) and sequenced using the Illumina NovaSeq 6000 platform (2 x 100 bp).

  Dataset EGAD00001010025

• Transcriptional and DNA binding profiling of CEBPE in REH acute lymphoblastic leukaemia cells using shRNA RNA-Seq and ChIP-Seq

  ChIP-Seq - CEBPE - REH. The ETV6/RUNX1 translocated acute lymphoblastic leukaemia cell line. REH was used to perform ChIP-Seq using a CEBPE antibody. Cells were fixed in 1% formaldehyde for 10mins, prior to preparation of chromatin using Active Motif Express ChIP-IT. 2ug of antibody (anti CEBPE Atlas Antibodies HPA002928)was added to 25ug of chromatin O/N at 4C with rotation. Duplicate reactions were pooled and purified. 10ng of ChIP’d and input DNA used for Illumina NGS preparation (NEBNext ChIP-Seq Library kit; New England Biolabs), CEBPE and Input DNA ChIP samples were sequenced on a MiSeq using 150bp Kit v3 paired end and a HiSeq 2500 using 2x101 version 4 paired end (Illumina) respectively. Reactions performed in duplicate. shCEBPE RNA-Seq - REH. REH cells were lentivirally transduced with a pTRIPZ shRNA vector for transcriptional profiling of CEBPE. Two controls (empty and non-targeting) and two CEBPE shRNAs (V3THS_150517(A13), V3THS_404312(G3) Dharmacon, GE) were transduced into REH cells. Cells were treated with 1ug/ml doxycyclin for 144hrs and total RNA purified using Qiagen RNeasy. Knock down of CEBPE was validated by qRT. RNA integrity >7.7 for all samples. Libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit and sequenced on an Illuimna HiSeq 2500 using 2x101 version 4 paired end chemistry. 3 biological replicates of each samples were prepared.

  Dataset EGAD00001004084

• Open Targets 020 Epigenomes of Cell Lines (2018-10-23)

  There is currently a drive to establish cell based assay systems of greater human biological and disease relevance through the use of well characterised transformed cell lines, primary cells and complex cellular models (e.g. co-culture, 3D models). However, although the field is gaining valuable experience in running more non-standard & complex cell assays for target validation and compound pharmacology studies, there is the lack of a systematic approach to determine if this expansion in cell assay models is reflected in increased human biological and disease relevance. The increasing wealth of publically available transcriptomic, and epigenome (ENCODE and Epigenome Roadmap) data represents an ideal reference mechanism for determining the relationship between cell types used for target & compound studies to primary human cells and tissues from both healthy volunteers & patients. The CTTV020 epigenomes of cell line project aims to generate epigenetic and transcriptomic profiles of cell lines and compare these with existing and newly generated reference data sets from human tissue and cell types. The aim is to identify assay systems which will provide greater confidence in translating target biology and compound pharmacology to patients. Multiple cell types commonly used within research have been grouped according to biology. Examples include erythroid, lung epithelial, hepatocyte cell types and immortalised models of monocyte / macrophage biology. 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 please see http://www.sanger.ac.uk/datasharing/ . This dataset contains all the data available for this study on 2018-10-23.

  Dataset EGAD00001004415

• The African American Breast Cancer Epidemiology and Risk (AMBER) Consortium Study

  The AMBER Consortium Study was formed to pool interview data, questionnaire data, and biological samples from epidemiological studies of breast cancer in African-American women to discover the potential causes of early-onset and aggressive breast cancer in African-American women. AMBER is funded through a Program Project grant from the National Cancer Institute. Genetic data submitted to dbGaP come from participants in the Carolina Breast Cancer Study, Women's Circle of Health Study, and Black Women's Health Study. The P01 consists of four scientific projects; the aims include follow-up on previous GWAS findings for breast cancer susceptibility in AA women as well as investigation of SNPs in candidate genes in biologically plausible pathways. These SNPs were genotyped using DNA from 3130 African-American women with breast cancer and 3700 controls. Descriptions of the original studies that provided the data and samples for this collaborative study are given below. The Carolina Breast Cancer Study (CBCS): a North Carolina population-based case-control study of breast cancer, conducted in three phases. The current study phase, phase 3 (years 2008-2014), includes women residents in 44 counties. CBCS phases 1 and 2 were conducted in 24 counties. Breast cancer cases are identified using Rapid Case Ascertainment in cooperation with the NC Central Cancer Registry. Controls were identified for phases 1 and 2 only (1993-1996 and 1996-2001), using Division of Motor Vehicles lists for women under age 65 and Health Care Financing Administration lists for women 65 and older. Randomized recruitment was used to oversample AA women and women under age 50. In-depth interviews are conducted by study nurses in participants' homes to obtain information on potential risk factors for breast cancer. DNA samples have been obtained from most participants. Overall response rates for Phases 1 and 2 were 74% for AA cases and 54% for AA controls. Phase 3, conducted in 44 counties from 2008-2014, includes cases only. The response rate for AA cases in Phase 3 was 70.5%. The Women's Circle of Health Study (WCHS): a multi-site case-control study in New York City (NYC) and New Jersey (NJ) aimed at evaluating risk factors for early and aggressive breast cancer in women of AA and EA ancestry. Recruitment in NYC took place between January 2002 and December 2008 and involved hospital-based ascertainment of cases, while controls were identified through random digit dialing (RDD). Recruitment at the NJ site started in March 2006 and is ongoing. Phase I of the study ended in April 2012 and covered seven counties in NJ. WCHS2 includes two additional counties. Cases in NJ were identified from 2006 to 2012 by the NJ State Cancer Registry using rapid case ascertainment. Controls were initially recruited though RDD (2006 to 2010) and later through community-based efforts (2009-2012). In-person interviews ascertained data on established and suspected risk factors for breast cancer. DNA samples were obtained. Among eligible AA women, 75% in NY and 54% in NJ completed an interview and provided a biologic specimen. Black Women's Health Study (BWHS): an ongoing prospective cohort study of health and illness among U.S. black women, with a focus on cancer. The study began in 1995 when 59,000 AA women 21-69 years of age from across the United States completed a 14-page postal health questionnaire. The median age at entry was 38, and participants were residents of 17 states in mainland U.S.: Northeast, 28%; South, 30%; Midwest, 23%; West, 19%. The baseline questionnaire elicited information on a wide range of variables, including demographic factors, use of medical care, family history of breast cancer, reproductive and medical history, cigarette and alcohol use, weight, height, waist and hip circumference, medication use, diet, and exercise. Biennial follow-up questionnaires ascertain new cases of breast cancer and other illnesses and update covariate information. Medical record and cancer registry data are sought for all participants who report a diagnosis of breast cancer. As of 2014, approximately 80% of the baseline cohort have completed follow-up. DNA samples were obtained from about 50% of participants. BWHS data for the AMBER consortium were prepared as a nested case-control study, with controls frequency-matched to cases on year of birth, geographic region, and most recent questionnaire completed prior to the end of the at-risk period.

  Study phs000669

• FASTQ files of total RNA-Seq data from the POPS PET (pre-eclamptic) samples

  All the samples were obtained from the Pregnancy Outcome Prediction–a prospective cohort study of nulliparous women attending the Rosie Hospital, Cambridge (UK) for their dating ultrasound scan between January 14, 2008, and July 31, 2012. Ethical approval for the study was given by the Cambridgeshire 2 Research Ethics Committee (reference number 07/H0308/163) and all participants provided written informed consent. Cases of preeclampsia (PET) were defined on the basis of the 2013 ACOG criteria and cases of small for gestational age (SGA)infants were confined to severe SGA, i.e. a customized birth weight <5th percentile. Chorionic villi from the corresponding placentas (free from decidua, visible infarction, calcification, hematoma, or damage) were collected and processed within 30 minutes of separation from the uterus. After repeated washes in chilled phosphate buffered saline, the samples were placed in RNA later (Applied Biosystems) and stored at -80°C. Total placental RNA was extracted using mirVana Isolation Kit (Ambion). For each placenta, approximately 5 mg of tissue were homogenized in the Lysis/Binding solution for 20 sec at 6 m/s using a bead beater (FastPrep24) and Lysing Matrix D Tubes (MP Biomedicals). The samples were then spun at 13,000 rpm for 5 min at 4°C and the supernatants recovered. Afterwards, the manufacturer's instructions were followed. Immediately after the RNA extraction, placental RNA samples were DNase-treated using DNA-free DNA Removal Kit (Ambion), aliquoted, and stored in -80°C. Quantity and quality of the RNA samples were assessed using the Agilent 2100 Bioanalyzer, the Agilent RNA 6000 Nano Kit (Agilent Technologies), and Qubit fluorometer. Libraries were prepared starting with 300-500 ng of good quality total RNA (RIN ≥7.5) using the TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Human/Mouse/Rat (Illumina), according to the manufacturer's instructions. The kit contains 96 uniquely indexed adapter combinations in order to allow pooling of multiple samples prior to sequencing. After determining their size (with the Agilent 2100 Bioanalyzer and the Agilent High Sensitivity DNA Kit by Agilent Technologies) and concentration (by qPCR with the KAPA Illumina ABI Prism Library Quantification Kit, Kapa Biosystems), libraries have been pooled and sequenced (single-end, 125 bp) using a Single End V4 Cluster Kit and an Illumina HiSeq2500 or HiSeq4000 instrument.

  Dataset EGAD00001003508

• FASTQ files of total RNA-Seq data from the POPS SGA (Small for Gestational Age) samples

  All the samples were obtained from the Pregnancy Outcome Prediction–a prospective cohort study of nulliparous women attending the Rosie Hospital, Cambridge (UK) for their dating ultrasound scan between January 14, 2008, and July 31, 2012. Ethical approval for the study was given by the Cambridgeshire 2 Research Ethics Committee (reference number 07/H0308/163) and all participants provided written informed consent. Cases of preeclampsia (PET) were defined on the basis of the 2013 ACOG criteria and cases of small for gestational age (SGA)infants were confined to severe SGA, i.e. a customized birth weight <5th percentile. Chorionic villi from the corresponding placentas (free from decidua, visible infarction, calcification, hematoma, or damage) were collected and processed within 30 minutes of separation from the uterus. After repeated washes in chilled phosphate buffered saline, the samples were placed in RNA later (Applied Biosystems) and stored at -80°C. Total placental RNA was extracted using mirVana Isolation Kit (Ambion). For each placenta, approximately 5 mg of tissue were homogenized in the Lysis/Binding solution for 20 sec at 6 m/s using a bead beater (FastPrep24) and Lysing Matrix D Tubes (MP Biomedicals). The samples were then spun at 13,000 rpm for 5 min at 4°C and the supernatants recovered. Afterwards, the manufacturer's instructions were followed. Immediately after the RNA extraction, placental RNA samples were DNase-treated using DNA-free DNA Removal Kit (Ambion), aliquoted, and stored in -80°C. Quantity and quality of the RNA samples were assessed using the Agilent 2100 Bioanalyzer, the Agilent RNA 6000 Nano Kit (Agilent Technologies), and Qubit fluorometer. Libraries were prepared starting with 300-500 ng of good quality total RNA (RIN ≥7.5) using the TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Human/Mouse/Rat (Illumina), according to the manufacturer's instructions. The kit contains 96 uniquely indexed adapter combinations in order to allow pooling of multiple samples prior to sequencing. After determining their size (with the Agilent 2100 Bioanalyzer and the Agilent High Sensitivity DNA Kit by Agilent Technologies) and concentration (by qPCR with the KAPA Illumina ABI Prism Library Quantification Kit, Kapa Biosystems), libraries have been pooled and sequenced (single-end, 125 bp) using a Single End V4 Cluster Kit and an Illumina HiSeq2500 or HiSeq4000 instrument.

  Dataset EGAD00001003507

• NINDS-Genome-Wide Genotyping in Parkinson's Disease: First Stage Analysis and Public Release of Data

  Epidemiological studies have estimated a cumulative prevalence of PD of greater than 1 per thousand. When prevalence is limited to senior populations, this proportion increases nearly 10-fold. The estimated genetic risk ratio for PD is approximately 1.7 (70% increased risk for PD if a sibling has PD) for all ages, and increases over 7-fold for those under age 66 years. The role for genes contributing to the risk of PD is therefore significant. This study utilized the well characterized collection of North American Caucasians with Parkinson's disease, and neurologically normal controls from the sample population which are banked in the National Institute of Neurological Disorders and Stroke (NINDS Repository) collection for a first stage whole genome analysis. Genome-wide, single nucleotide polymorphism (SNP) genotyping of these publicly available samples was originally done in 267 Parkinson's disease patients and 270 controls, and this has been extended to include genome wide genotyping in 939 Parkinson's disease cases and 802 controls. The NINDS repository was established in 10-2001 towards the goal of developing standardized, broadly useful diagnostic and other clinical data and a collection of DNA and cell line samples to further advances in gene discovery of neurological disorders. All samples, phenotypic, and genotypic data are available to the research community including to academics and industry scientists. In addition, well characterized neurologically normal control subjects are a part of the collection. This collection formed the basis of this first stage study by Fung et al., and the expanded study by Simon-Sanchez et al. The genotyping data was generated and provided by the laboratory of Dr. Andrew Singleton NIA, and Dr. John Hardy NIA (NIH Intramural, funding from NIA and NINDS). Important links to apply for individual-level data Data Use Certification Requirements (DUC) Apply here for controlled access to individual level data Participant Protection Policy FAQ

  Study phs000089

• Identifying novel DNA damage response genes in radiosensitive individuals

  Ionizing radiation is an effective therapeutic agent for cancer treatment as well as a potent carcinogen. Sensitivity to the cell-killing effects of radiation can vary across human population with a subset of individuals displaying extreme hypersensitivity. It is usually attributable to inherited defects in DNA damage response pathways. The present study was designed to elucidate the genetic basis of variation in hypersensitivity to radiation exposure through exome sequencing of radiosensitive individuals, with the ultimate goal of identifying genes with the most significant effects on cellular DNA damage responses. The study participants included subjects referred for clinical testing for Ataxia-telangiectasia (A-T), Nijmegen Breakage Syndrome (NBS) or Ligase IV Syndrome. These are rare, recessive genetic disorders and hypersensitivity to radiation exposure is a common phenotype among individuals affected by all the three disorders. The study participants exhibited phenotypic characteristics similar to individuals with A-T, NBS or Ligase IV Syndrome, but lacked the causative mutations in ATM (GeneID:472) or NBN (GeneID:4683) genes. For further validation of the radiation sensitivity phenotype among the enrolled subjects, B-lymphoblastoid cells lines were established for each subject from peripheral blood lymphocytes. Each cell line was evaluated for displaying impaired survival rates relative to normal controls after exposure to ionizing radiation. 53 subjects with validated phenotype were finally included in the study and DNA extracted from their B-lymphoblastoid cell lines was used for exome sequencing. This sequencing data for radiation sensitive subjects is being made available in the dbGaP. It is hoped that this resource will be beneficial for researchers who wish to further investigate components of human cellular DNA damage response pathways and/or genetic architecture underlying radiation hypersensitivity. This data may also aid in the rational design of new radiosensitizing or radioprotective agents.

  Study phs001911

• Genomic Basis of Phenotypic Variability of Complex Disorders

  The 520-kbp deletion on chromosome 16p12.1 is associated with multiple neurodevelopmental outcomes, including intellectual disability/developmental delay (ID/DD), schizophrenia, autism, and epilepsy. This variant is inherited in >95% of cases from carrier parents manifesting neuropsychiatric features, while affected children with the deletion were more likely to carry another large Copy Number Variation (CNV) or rare deleterious mutation elsewhere in the genome, or "second-hits", compared to carrier parents. Thus, while the 16p12.1 deletion confers significant risk for a range of disorders, the ultimate phenotypic trajectory is determined by "second-hit" variants in the genetic background. However, outstanding questions remain about the genetic background in these disorders, including the functional effects of "second-hit" variants towards gene expression or developmental phenotypes, the full spectrum of "second-hit" variants, and the mechanisms driving accumulation of these variants.In version 1, we performed deep clinical phenotyping, whole-genome sequencing (WGS), SNP microarray analysis, and RNA-sequencing of lymphoblastoid cell lines of 32 individuals in five large families with the 16p12.1 deletion. We found that specific classes of rare single-nucleotide and structural variants in coding and non-coding regions contributed towards expression changes in affected children based on their inheritance patterns, including a subset of variants that acted synergistically with the deletion to alter expression. We further found that rare variant-mediated expression changes contribute to specific developmental phenotypes in the carrier children, suggesting a potential genetic mechanism for variable expressivity of the deletion.In version 2, we additionally analyzed genetic and phenotypic data from individuals with 16p12.1 deletion and their spouses to assess the role of assortative mating modulating phenotypic outcomes of the 16p12.1 deletion. We identified significant within- and cross- disorder phenotypic correlations between spouses, but observed no significant genetic correlations between spouses, in line with expectations.

  Study phs002450

• Precision High Intensity Training Through Epigenetics (PHITE)

  Exercising regularly promotes health, but these benefits are complicated by acute inflammation induced by exercise. A potential source of exercise-induced inflammation is cell-free DNA (cfDNA), yet the cellular origins, molecular causes, and immune system interactions of exercise-induced cfDNA are unclear. To study these, 10 healthy individuals were randomized to a 12-week exercise program of either high-intensity tactical training (HITT) or traditional moderate-intensity training (TRAD). Blood plasma was collected pre- and post exercise at weeks 0 and 12 and after 4 week of detraining upon program completion. Whole-genome enzymatic methylation sequencing (EM-seq) with cell-type proportion deconvolution was applied to cfDNA obtained from the 50 plasma samples and paired to concentration measurements for 90 circulating cytokines. Acute exercise increased the release of cfDNA from neutrophils, dendritic cells (DCs), and macrophages proportional to exercise intensity (HITT > TRAD). Exercise training reduced cfDNA released in HITT participants but not TRAD and from DCs and macrophages but not neutrophils. For most participants, training lowered mitochondrial cfDNA at rest, even after detraining. Using a sequencing analysis approach we developed, we concluded that rapid extracellular traps (ETosis) was the likely source of cfDNA, demonstrated by enrichment of nuclear DNA. Further, several cytokines were induced by acute exercise, such as IL-6, IL-10, and IL-16, and training attenuated the induction of only IL-6 and IL-17F. Cytokine levels were not associated with cfDNA induction, suggesting that these cytokines are not the main cause of exercise-induced cfDNA. Overall, exercise intensity and training modulated cfDNA release and cytokine responses, contributing to the anti-inflammatory effects of regular exercise. One additional subject was also used in the methyl-seq data as a control for comparing bisulfite-sequencing to enzymatic methyl sequencing. DNA methylation for MPO gene was found to be significantly different as well, in line with interpretation that neutrophils are primarily undergoing ETosis.

  Study phs003873

• A prospective study identifies MisMatch Repair genes as candidate predisposing genes for uveal melanoma.

  Objective: The aim of this study is to identify new predisposing genes for uveal melanoma (UM), the most common intraocular malignancy in adults. Design: Prospective cohort study from Institut Curie. Participants: 381 unselected patients diagnosed with UM between July 2021 and February 2023 at Institut Curie. Methods: All patients received genetic counseling and consented to extended genetic testing. A panel of 122 genes was analyzed by targeted sequencing on germline DNA from these patients. Main outcomes measures: UM was diagnosed by ophthalmologists at Institut Curie, and a senior pathologist confirmed the diagnosis when tumor or biopsy was available. Classification of the germline genetic variants was performed according to the ACMG guidelines. Results: We identified at least one pathogenic variant (PV) in 79 patients. Among those variants, 20 were found in clinically relevant genes for the patient’s follow-up and genetic counseling. We found a significant enrichment of germline pathogenic variants in Mismatch Repair (MMR) MLH1, MSH2 and MSH6 genes in this UM cohort, suggesting that MMR germline alterations could predispose to UM. From this cohort, one tumor was available from a patient carrying a MLH1 germline mutation. The tumor was characterized by a monosomy 3, in line with a loss of heterozygosity (LOH) of MLH1, located on chromosome 3. Loss of expression of MLH1 and PMS2 was observed by immunohistochemistry, and MMR mutational signatures SBS6, ID1 and ID2 were identified from the whole genome sequencing of the tumor. These data suggest that MLH1 is implicated in the oncogenesis of this UM. Conclusions: This study is the largest prospective constitutional study performed on UM patients, and the first demonstrating that MMR germline alterations are significantly enriched among these patients. Further analyses will be needed to estimate the risk of developing UM for patients with Lynch syndrome

  Study EGAS50000000914

• Single-Gene vs. Panel Sequencing in Advanced HR+/HER2− Breast Cancer

  Background: Hormone receptor-positive (HR+)/HER2-negative (HER2−) breast cancer is the most common subtype of breast cancer, with biomarker-driven therapies improving outcomes. Liquid biopsies, which analyze circulating tumor DNA (ctDNA), are increasingly essential in clinical management, offering a minimally invasive approach to assess somatic alterations and guide therapy decisions. However, the choice of assay directly affects clinical utility and global access to validated testing platforms remains inconsistent. To address these challenges, this study aimed to compare the performance of single-gene and panel-based sequencing for detecting PIK3CA mutations in HR+/HER2− breast cancer and evaluate the added value of broader genomic profiling in guiding treatment decisions. Patients and methods: We conducted a prospective, multicenter study comparing our previously established SiMSen-Seq (SSS) assay, a single-gene method targeting PIK3CA hotspot mutations, with the AVENIO ctDNA Expanded assay, enriching for 77 clinically relevant cancer genes. Additionally, tumor fractions were estimated with mFAST-SeqS by assessing chromosomal aneuploidy. We analyzed 161 plasma samples collected from 146 patients with HR+/HER2− advanced breast cancer before starting a new line of palliative therapy. Results: High concordance (92.6%) was observed between SSS and AVENIO for PIK3CA mutation detection, with accurate variant allele frequency (VAF) quantification confirmed by Bland-Altman analysis and linear mixed-effects modeling. AVENIO identified additional PIK3CA mutations in 19% of samples as well as other actionable alterations, including ESR1 mutations (17.5%) and PI3K pathway alterations (40.6%). Panel sequencing improved the interpretation of negative liquid biopsy results by integrating tumor fraction and VAF metrics. Conclusions: Panel sequencing assays such as AVENIO provide broader genomic coverage, enhance the detection of actionable mutations and refine the interpretation of negative liquid biopsy findings. These findings support the integration of comprehensive ctDNA profiling into routine clinical practice for HR+/HER2− advanced breast cancer while highlighting the need for improved assay accessibility and expanded genomic targets.

  Study EGAS00001008200

• Genetic analysis of HLA and immune escape genes in Diffuse Large B-cell Lymphoma

  Fifty percent of diffuse large B-cell lymphoma (DLBCL) cases lack cell-surface expression of the class-I major histocompatibility complex (MHC-I), thereby escaping immune recognition by cytotoxic T cells. In order to comprehensively identify the mechanisms involved in MHC-I loss, we first performed immunophenotypic analysis of both MHC class-I and -II in 657 cases across the spectrum of B-NHL, revealing that loss of MHC-I, but not MHC-II, is preferentially restricted to DLBCL. We then used whole exome and targeted deep-sequencing to examine genes involved in MHC-I expression in 74 DLBCL samples representative of MHC-I positive and negative cases. We show here that somatic biallelic or monoallelic inactivation of B2M and/or HLA-I is present in 80.9% (34/42) of MHC-I negative tumors. Furthermore, 68.8% (22/32) of MHC-I positive DLBCLs also harbored monoallelic HLA-I genetic alterations (MHC-I positivemono) that lead to allelic imbalance, suggesting allele-specific inactivation. Both MHC-I negative and MHC-I positivemono cases showed a significantly higher mutational burden as well as a higher number of inferred neo-antigens, suggesting co-selection of HLA-I loss and sustained neo-antigen production. Interestingly, the analysis of > 500.000 individuals in two databases revealed homozygosity of germline HLA-I genes in 26-38% of DLBCL patients, a frequency significantly higher than that observed in any other cancer type. In mice, germinal-center B cells lacking HLA-I expression did not progress to lymphoma and were counterselected in the context of oncogene-driven lymphomagenesis, suggesting that additional events are needed to license immune evasion. These results suggest a multi-step process of HLA-I loss including both germ-line and somatic events in DLBCL development, and have direct implications for the pathogenesis and immunotherapeutic targeting of this disease.

  Study EGAS00001005054

• The University of Hong Kong Colon Cancer GCV Study

  Background: Ganciclovir (GCV) is widely used in solid organ and hematopoietic stem cell transplant patients for prophylaxis and treatment of cytomegalovirus. It has long been considered a mutagen and carcinogen. However, the contribution of GCV to cancer incidence and other factors that influence its mutagenicity remains unknown. Methods: This retrospective cohort study analysed genomics data for 121,771 patients who had undergone targeted sequencing compiled by the Genomics Evidence Neoplasia Information Exchange (GENIE) or Foundation Medicine (FM). A statistical approach was developed to identify patients with GCV-associated mutational signature (GCVsig) from targeted sequenced data of tumour samples. Cell line exposure models were further used to quantify mutation burden and DNA damage caused by GCV and other antiviral and immunosuppressive drugs. Results: Mutational profiles from 22 of 121,771 patient samples in the GENIE and FM cohorts showed evidence of GCVsig. A diverse range of cancers was represented. All patients with detailed clinical history available had previously undergone solid organ transplantation and received GCV and mycophenolate treatment. RAS hotspot mutations associated with GCVsig¬¬ were present in 9 of the 22 samples, with all samples harbouring multiple GCV-associated protein-altering mutations in cancer driver genes. In vitro testing in cell lines showed that elevated DNA damage response and GCV¬sig are uniquely associated with GCV but not acyclovir, a structurally similar antiviral. Combination treatment of GCV with the immunosuppressant, mycophenolate mofetil (MMF), increased the misincorporation of GCV in genomic DNA and mutations attributed to GCVsig in cell lines and organoids. Conclusions: In summary, GCV can cause a diverse range of cancers. Its mutagenicity may be potentiated by other therapies, such as mycophenolate, commonly co-prescribed with GCV for post-transplant patients. Further investigation of the optimal use of these drugs could help reduce GCV-associated mutagenesis in post-transplant patients.

  Study EGAS00001006707