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• Genetic landscape of SMM

  Identifying high risk smoldering myeloma patients and progression mechanism is a prerequisite to implement effective inception strategies and curve myeloma related morbidity and mortality. We hypothesize that genomics may help identify determinants of progression that may help predict outcome and offer effective chemo preventive targets. Eighty-two patients underwent a custom targeted panel sequencing with an additional capture for the translocation loci. These results were compared to 223 newly diagnosed patients (EGAS00001003223 and EGAD00001004117) and 17 MGUS and 10 early myeloma patients. DNA was obtained from either CD138+ cells from the bone marrow of multiple myeloma patients (tumor) or from stem cell harvests or peripheral blood cells from the same patient (control). 100 ng of DNA was fragmented, end-repaired, and adapters ligated using the HyperPlus kit (KAPA Biosystems). After PCR amplification the libraries were hybridized with probes against either a targeted panel consisting of 140 genes and chromosomal regions (Nimblegen) using SeqCap reagents (Nimblegen). Hybridized libraries underwent further amplification before being sequenced on a NextSeq500 (Illumina) using 75 bp paired end reads. Overall, these data highlight the importance of dysregulation of the MAPK pathway in the progression to MM.

  Dataset EGAD00001005285

• cfDNA analysis reveals relation of POLR1D amplification to bevacizumab resistance in colorectal cancer patients

  Bevacizumab is an approved anti-angiogenic drug for patients with metastasized colorectal cancer (mCRC) targeting VEGF. The survival benefit of anti-VEGF therapy in mCRC patients is limited to a few months and acquired resistance mechanisms are greatly unknown. Using plasma DNA, we studied the evolution of tumor genomes in a cohort of patients with mCRC (n=150) and observed a recurrent focal amplification (8.7% of cases) on chromosome 13q12.2. Analysis of TCGA data (n=619) suggested an association with later stages, which we confirmed by longitudinal plasma analyses. We defined the minimally amplified region and studied the mechanistic consequences of copy number gain of the involved genes. The amplification of one gene, POLR1D, impacted cell proliferation, resulting in upregulation of VEGFA, an important regulator of angiogenesis which has been implicated in the resistance to bevacizumab. In several patients, we observed the emergence of this 13q12.2 amplicon under bevacizumab treatment, which was invariably associated with evolution of therapy resistance. Hence, we describe a novel resistance mechanism against a widely applied treatment in mCRC patients which will impact clinical management .

  Dataset EGAD00001005761

• MutWP5: CRUK Mutographs of Cancer: Breast: Cancer Mastectomy (WG) (2020-01-29)

  Sequencing of LCM-derived microbiopsies from 40 women who underwent mastectomies due to breast cancer. LCM and sequencing will be conducted on both normal, unaffected breast, and, where possible, tumour tissue. Goal to assess the mutational burden, spectrum, and clonal dynamics within the tissue, and compare findings between the normal and associated cancer tissues. Whole-genome sequencing will be conducted on samples identified as promising from the initial targeted data. Results from this portion of the study will be compared to women who had cosmetic breast reduction surgeries and those who are BRCA carriers. . This dataset contains all the data available for this study on 2020-01-29.

  Dataset EGAD00001005922

• Cryopreservation of human cancers conserves tumour heterogeneity for single-cell multi-omics analysis

  Single-cell RNA-Sequencing of three primary breast cancers, two primary prostate cancers, and a metastatic melanoma sample from Wu et al. (2021) Genome Medicine study. Each tumour was sequenced across different cryopreservation conditions including Fresh Tissue (FT), cryopreserved single-cell suspensions (CCS), cryopreserved solid tissue fragments (CT) and a cryopreserved after overnight cold storage (CO). Data was generated using the Chromium controller (10X Genomics) and sequenced on the NextSeq platform.

  Dataset EGAD00001007030

• CBD-RAW-SC-GEX: 10X Single-Cell Gene Expression

  Raw Illumina sequencing data and CellRanger BAM output files. For further information regarding this dataset, please contact Stephen Sansom at contact@combat.ox.ac.uk.

  Dataset EGAD00001008007

• Alloreactive T-cell receptor (TCR) repertoire in kidney transplantation

  We analyzed the T-cell receptor (TCR) repertoires from twelve kidney transplant recipients. Six out of the twelve kidney transplant recipients experienced a cellular rejection after kidney transplantation. TCR repertoires of CD4+ and CD8+ positive T-cells were assessed prior to transplantation and after transplantation at time of allograft biopsy using RNA based T-cell receptor beta next generation sequencing (NGS). In addition, the pre-formed alloreactive TCR repertoire for each kidney transplant recipient was identified using mixed lymphocyte reaction and donor reactive T-cells were subjected to TCR beta sequencing. In two out of the six patients with cellular rejection the TCR repertoire of graft infiltrating T-cells was additionally captured. This dataset comprises a total of 98 samples. NGS TCR beta libraries of all samples were sequenced on an Illumina NextSeq 500 and raw sequencing data (in the form of fastq files) as well assembled clonotypes and their counts (in the form of clonotype tables) are provided.

  Dataset EGAD00001007695

• The mutational landscape of human somatic and germline cells

  During the course of a lifetime normal human cells accumulate mutations. Here, using multiple samples from the same individuals we compared the mutational landscape in 29 anatomical structures from soma and the germline. Two ubiquitous mutational signatures, SBS1 and SBS5/40, accounted for the majority of acquired mutations in most cell types but their absolute and relative contributions varied substantially. SBS18, potentially reflecting oxidative damage, and several additional signatures attributed to exogenous and endogenous exposures contributed mutations to subsets of cell types. The mutation rate was lowest in spermatogonia, the stem cell from which sperm are generated and from which most genetic variation in the human population is thought to originate. This was due to low rates of ubiquitous mutation processes and may be partially attributable to a low cell division rate of basal spermatogonia. The results provide important insights into how mutational processes affect the soma and germline.

  Dataset EGAD00001006642

• The mutational landscape of human somatic and germline cells

  During the course of a lifetime normal human cells accumulate mutations. Here, using multiple samples from the same individuals we compared the mutational landscape in 29 anatomical structures from soma and the germline. Two ubiquitous mutational signatures, SBS1 and SBS5/40, accounted for the majority of acquired mutations in most cell types but their absolute and relative contributions varied substantially. SBS18, potentially reflecting oxidative damage, and several additional signatures attributed to exogenous and endogenous exposures contributed mutations to subsets of cell types. The mutation rate was lowest in spermatogonia, the stem cell from which sperm are generated and from which most genetic variation in the human population is thought to originate. This was due to low rates of ubiquitous mutation processes and may be partially attributable to a low cell division rate of basal spermatogonia. The results provide important insights into how mutational processes affect the soma and germline.

  Dataset EGAD00001006643

• The Precision Medicine in Liver Cancer across an Asia-pacific NETwork (PLANET) - Intratumoural immune heterogeneity

  The clinical relevance of immune landscape intratumoural heterogeneity (immune-ITH) and its role in tumour evolution remain largely unexplored. Here, we uncover significant spatial and phenotypic immune–ITH from multiple tumour sectors and decipher its relationship with tumour evolution and disease progression in hepatocellular carcinomas (HCC). Immune–ITH is associated with tumour transcriptomic-ITH, mutational burden, and distinct immune microenvironments. Tumours with low immune–ITH experience higher immunoselective pressure and escape via loss of heterozygosity in human leukocyte antigens and immunoediting. Instead, the tumours with high immune-ITH evolve to a more immunosuppressive/exhausted microenvironment. This gradient of immune pressure along with immune-ITH represents a hallmark of tumour evolution, which is closely linked to the transcriptome-immune networks that contributes to disease progression and immune inactivation. Remarkably, high immune-ITH and its transcriptomic signature are predictive for worse clinical outcome in HCC patients. This in-depth investigation of ITH provides evidence on tumour-immune co-evolution along HCC progression.

  Dataset EGAD00001006570

• Hypertension delays viral clearance and exacerbates airway hyperinflammation in patients with COVID-19

  In Coronavirus Disease 2019 (COVID-19), hypertension and cardiovascular diseases are major risk factors for critical disease progression. However, the underlying reasons and the effect of the main anti-hypertensive therapies—angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs)—remain unclear. Combining clinical data (n = 144) and single-cell sequencing data of airway samples (n = 48) with in vitro experiments, we observed a distinct inflammatory predisposition of immune cells in patients with hypertension that correlated with critical disease progression. ACEI treatment associated with dampened COVID-19-related hyperinflammation and with increased cell intrinsic anti-viral responses, whereas ARB treatment related to enhanced epithelial–immune cell interactions. Macrophages and neutrophils of patients with hypertension, in particular under ARB treatment, exhibited higher expression of the pro-inflammatory cytokines CCL3 and CCL4 and the chemokine receptor CCR1. Although the limited size of our cohort does not allow us to establish clinical efficacy, our data suggest that the clinical benefits of ACEI treatment in patients with COVID-19 who have hypertension warrant further investigation.

  Dataset EGAD00001006828

• T-cell reconstitution after reduced dose ATLG induction in kidney transplant patients

  We analyzed the T-cell receptor (TCR) repertoires from ten kidney transplant recipients. Five out of the ten kidney transplant recipients received ATLG while the other five recipients received basiliximab as induction therapy. TCR repertoires of CD4+ and CD8+ positive T-cells were assessed prior to transplantation and within the first month after transplantation as well as at three- and 12-months post-transplant. In addition, the pre-formed alloreactive TCR repertoire for each kidney transplant recipient was identified using mixed lymphocyte reaction and donor reactive T-cells were subjected to TCR beta sequencing. This dataset comprises a total of 106 samples. NGS TCR beta libraries of all samples were sequenced on an Illumina NextSeq 500 and raw sequencing data (in the form of fastq files) as well assembled clonotypes and their counts (in the form of clonotype tables) are provided.

  Dataset EGAD00001008478

• Myeloid cell programming in patients with non-medullary thyroid carcinoma

  This dataset contains single-cell RNA sequencing data from patients with thyroid cancer (n=7), multinodal Goiter (n=3) and healthy individuals (n=5). Mononuclear cells were taken from both the peripheral blood and the bone marrow compartments. We used a pooled single-cell design where multiple individuals were pooled in a single sample for sequencing (NextSeq 500-V2) and later demultiplexed using their genotype data. Associated metadata contains information on the phenotypes per individual, the pooling design and the linkage between the supplied files and sequenced pools. Due to limitations from EGA in uploading single-cell data, the raw fastq files were processed as follows: (i) I1/I2/R1/R2 fast files were concatenated over the different lanes. (ii) Concatenated I1 and I2 files were interleaved, as were the concatenated R1 and R2 files to generate two fastq files per pool containing all the information. To interleave the fastq files, the BBmap tool bbmap/reformat.sh was used, which can also be used to de-interleave the files.

  Dataset EGAD00001008108

• Dataset for head_and_neck_cancer-EXON

  Rare cancer sequencing data of 86 runs in tumor/control pairs, which were uploaded to umbrella studies. The sequencing was always paired

  Dataset EGAD00001008880

• Dataset for hepatopancreaticobiliary_malignancy-EXON

  Rare cancer sequencing data of 162 runs in tumor/control pairs, which were uploaded to umbrella studies. The sequencing was always paired

  Dataset EGAD00001008883

• Dataset for hepatopancreaticobiliary_malignancy-WHOLE_GENOME

  Rare cancer sequencing data of 83 runs in tumor/control pairs, which were uploaded to umbrella studies. The sequencing was always paired

  Dataset EGAD00001008884

• Dataset for neuroendocrine_adrenal_tumor-WHOLE_GENOME

  Rare cancer sequencing data of 112 runs in tumor/control pairs, which were uploaded to umbrella studies. The sequencing was always paired

  Dataset EGAD00001008893

• Transcriptome analysis for intrahepatic cholangiocarcinoma

  Intrahepatic cholangiocarcinomas (iCCs) are characterized by their rarity, difficulty in diagnosis, and overall poor prognosis. We performed comprehensive transcriptomic characterization of treatment-naive iCC. Whole transcriptome analyses identified two prognostic subtypes, concordant with previous reports.The findings could assist in patient stratification with iCCs and in developing rational therapeutic strategies.

  Dataset EGAD00001008544

• WES and RNA-seq of triple-negative breast cancers from the MyBrCa cohort

  The dataset contains a genomics characterization of 35 triple-negative Asian breast tumours from the Malaysian Breast Cancer cohort. This includes whole-exome sequencing of tumour tissue at 80X, whole-exome sequencing of matched normal (blood) tissue at 40X, and RNA-seq of tumour tissue at 40X coverage (>15 million reads). Whole-exome libraries were prepared using the Nextera Rapid Capture Exome Kit; exome capture was performed in pools of 3 and subjected to paired end 75 sequencing on a NovaSeq 6000 platform. RNA libraries were prepared using the TruSeq Stranded Total RNA HT kit with Ribo-Zero Gold as per manufacturer’s instructions and also subjected to paired end 75 sequencing on a NovaSeq 6000 platform. Uploaded bam files have been mapped to the hs37d5 human genome and processed using the standard GATK pipelines. Paired clinical, demographic, genotyping, and overall survival data for these patients are available from the associated publications or by request.

  Dataset EGAD00001009311

• Southern African Prostate Cancer Study (SAPCS) Ethnic Disparity

  The cohort (n=130) consists of prostate cancer patients from South Africa (n=123) and Brazil (n=7). For each patient, a pair of blood and tumour samples were collected. The sequencing data was mapped to hg38 reference. Blood BAMs are named with “-B” as suffix and tumour BAMs are named with “-T” as suffix.

  Dataset EGAD00001009067

• MutWP5: CRUK Mutographs of Cancer: Breast: Reduction Mammoplasty (WG)

  Sequencing of LCM-derived microbiopsies from 10 women who underwent reduction mammoplasty. Goal to assess the mutational burden, spectrum, and clonal dynamics within the tissue. Whole-genome sequencing will be conducted on samples identified as promising from the initial targeted data. Results from this portion of the study will be compared to women who had breast cancer and those who are BRCA 1/2 carriers. This dataset contains all the data available for this study on 2023-03-08.

  Dataset EGAD00001010109

• A next-generation dual guide CRISPR system for genetic interaction library screening

  Raw sgRNA sequencing data from a pilot CRISPR screening library to test the dual guide vector system (8,914 guide pairs), and from a large scale genetic interaction screen (~100,136 guide pairs) in HT-29 cells. This data refers to Burgold et al. Nature Communications 2025.

  Dataset EGAD00001015754

• Public Access Defibrillation Community Trial (PAD)(PAD-BioLINCC)

  Data Access NOTE: Please refer to the “Authorized Access” section below for information about how access to the data from this accession differs from many other dbGaP accessions. Objectives The Public Access Defibrillation (PAD) Community Trial sought to evaluate broad implementation of Public Access Defibrillators (PAD) in urban community units. Survival to hospital discharge of participants with out-of-hospital cardiac arrest was the main outcome measure. Survival was compared in community units (e.g., apartment or office buildings, gated communities, sports venues, senior centers, shopping malls) served by non-medical responders trained in CPR and use of automated external defibrillators (AEDs), to units receiving the traditional optimum community standard of care (i.e., rescuers trained to recognize a cardiac emergency, call 911, and initiate CPR). Background Sudden out-of-hospital cardiac arrest (OOH-CA) remains a significant cause of death, in spite of recent declines in overall mortality from cardiovascular disease. Existing methods of emergency resuscitation are inadequate due to time delays inherent in transporting trained responders with defibrillation capabilities to the side of the OOH-CA victim. Existing Emergency Medical Services (EMS) systems typically combine paramedic Emergency Medical Technician (EMT) services with some level of community involvement, such as bystander cardiopulmonary resuscitation (CPR) training. Some communities include automated external defibrillators (AEDs) at isolated sites or in mobile police or fire vehicles. Such an approach typically varies in effectiveness, with an incremental improvement in effectiveness seen in communities that organize and integrate services with the existing EMS system. However, optimal improvement in survival from sudden OOH-CA may require a program that utilizes volunteer non-medical responders (who may not have a traditional duty to respond to an emergency) trained to use AEDs. Participants The PAD trial was a prospective, randomized community based trial. More than 19,000 volunteer responders from 993 community units in 24 North American regions participated. The two study arms had similar unit and volunteer characteristics. Participants with treated out-of-hospital cardiac arrest in the two groups were similar in age (mean: 69.8 years), proportion of men (67 perecnt), rate of cardiac arrest in a public location (70 percent), and rate of witnessed cardiac arrest (72 percent). Conclusions Community units with volunteers trained in CPR and AEDs had significantly more participants surviving to hospital discharge than units with volunteers trained to use CPR only. There were 30 survivors among 128 definite cardiac arrests in the CPR+AED units and 15 survivors among 107 definite cardiac arrests in the CPR only units (p = 0.03). Serious adverse effects were rarely reported. No volunteers received inadvertent shocks, and no participants were shocked unnecessarily. AED maintenance problems were infrequent. A few participating volunteers reported severe stress related to responding to emergency situations. Although residential complexes represented 16% of the units and 29% of the treatable cardiac arrests, only 5% of the survivors were from residential complexes. Such information should be helpful for individual facilities that are considering implementing PAD programs. (NEJM 2004; 351:637-46).

  Study phs003858

• Acute Respiratory Distress Network (ARDSNet) Studies 10 and 12 Statins for Acutely Injured Lungs from Sepsis (SAILS) (ARDSNet-SAILS-BioLINCC)

  Data Access NOTE: Please refer to the “Authorized Access” section below for information about how access to the data from this accession differs from many other dbGaP accessions. Biospecimens Access to Biospecimens is through the NHLBI Biologic Specimen and Data Repository Information Coordinating Center (BioLINCC). Biospecimens from ARDSNet-SAILS include Plasma, DNA, and Urine. Please note that use of biospecimens in genetic research is subject to a tiered consent. Objectives The SAILS trial was intended to assess the efficacy and safety of oral rosuvastatin in participants with sepsis-induced Acute Lung Injury (ALI) and test the hypothesis that rosuvastatin therapy would improve the clinical outcomes of critically ill participants with sepsis-associated acute respiratory distress syndrome (ARDS). Background In ARDS, inflammation in the lungs and other organs can cause life-threatening organ failure. Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins) can modulate inflammatory responses. Previous observational studies suggested that statins improved clinical outcomes in participants with sepsis. Participants There were 745 participants. Design Participants were randomly assigned in permuted blocks to receive either enteral rosuvastatin or placebo. A 40 mg loading dose of the study drug was administered within four hours after randomization. Subsequently, maintenance doses of 20 mg were administered daily until the third day after discharge from the intensive care unit, study day 28, hospital discharge, or death, whichever came first. The primary outcome measure was mortality before hospital discharge home or until study day 60 if the participant was still in a health care facility. Secondary outcome measures included the number of ventilator-free days to day 28, organ-failure-free days to day 14, and ICU-free days to day 28. Conclusions The study was stopped because of futility after 745 of an estimated 1000 participants had been enrolled. There was no significant difference between study groups in 60 day in-hospital mortality or in mean ventilator-free days. The rosuvastatin group had fewer days free of hepatic or renal failure. Thus, rosuvastatin therapy did not improve clinical outcomes in participants with sepsis-associated ARDS and may have contributed to hepatic and renal organ dysfunction.

  Study phs003736

• Whole Genome Sequencing and Variant Calling for Autism Families

  We extracted DNA from whole blood or lymphoblast-derived cell lines and assessed the DNA quality with PicoGreenTM and gel electrophoresis. Whole genome sequencing was performed (Illumina HiSeq2000 and Illumina HiSeq X). WGS reads were mapped to the human reference genome assembly hg19 (GRCh37) using Burrows-Wheeler Aligner v.0.7.12 (TCAG) or Isaac v.2.0.13 (Macrogen). For each genome, we performed local realignment and quality recalibration and detected SNVs and small indels using GATK Haplotype Caller v.3.4.6 without genotype refinement. We detected CNVs using ERDS (estimation by read depth with single nucleotide variants) and CNVnator. We detected structural variants using Manta v.0.29.6. When available by the variant caller (i.e. GATK and Manta), trio-based joint variant calling was conducted for each family.

  Dataset EGAD00001008452

• Characterizing the Role of the Immune Microenvironment in Multiple Myeloma Progression at a Single Cell Level

  The plasma cell disease multiple myeloma (MM) is highly dependent on its bone marrow microenvironment (BMME). Early alterations within the bone marrow microenvironment that contribute to the progression of multiple myeloma (MM) from its precursor stages could hold the key to identifying novel therapeutic approaches, yet the intrinsic variability in cellular populations between patients together with differences in sample processing and analysis methods have made it difficult to identify consistent changes between datasets. Here, we used single-cell RNA sequencing of BM cells from precursor stages, monoclonal gammopathy of unknown significance (MGUS), smoldering MM (SMM) and newly diagnosed MM and analyzed our data in combination with a previously published dataset that used a similar patient population and sample processing. Despite vast interpatient heterogeneity, some alterations were seen consistently in both datasets. We identified changes in immune cell populations as disease progressed that were characterized by a substantial decrease in memory and naïve CD4 T cells and an increase in CD8+ effector T cells and T-regulatory cells. These alterations were further accompanied by an enrichment of non-clonal memory B cells and an increase of CD14 and CD16 monocytes in MM compared to its precursor stages. These results provide crucial information of the immune changes associated with the progression to clinical MM and can help to develop immune based strategies for patient stratification and early therapeutic intervention. 

  Study phs002756

• Multimodal Immune Profiling to Determine Mechanisms of COVID-19 Clinical Trajectory in Uganda

  We conducted a prospective cohort study of adults (RESERVE-U-C19) admitted to Entebbe Regional Referral Hospital (ERRH), a national COVID-19 referral center, across three viral variant-driven phases of the pandemic in Uganda. In this cohort, which reflects the entire spectrum of SARS-CoV-2 infection, we integrated clinical data with serum proteomics (N=306) and whole-blood transcriptomics (N=100) to determine a range of host responses associated with COVID-19 severity, SARS-CoV-2/HIV co-infection, and circulating viral variants. Beyond the prognostic importance of myeloid cell-driven immune activation and lymphopenia, we show that multifaceted impairment of host protein synthesis and extensive redox imbalance define core metabolic signatures of severe COVID-19, with central roles for IL-7, IL-15, and lymphotoxin-α in COVID-19 respiratory failure. While prognostic signatures were generally consistent in SARS-CoV-2/HIV-coinfection, type I interferon responses uniquely scaled with COVID-19 severity in persons living with HIV. Throughout the pandemic, COVID-19 severity peaked during phases dominated by A.23/A.23.1 and Delta B.1.617.2/AY variants, with Delta phase COVID-19 distinguished by exaggerated pro-inflammatory myeloid cell and inflammasome activation, NK and CD8+ T-cell depletion, impaired host protein synthesis, and upregulation of viral cell entry and trafficking pathways. Data from this study available through dbGaP include raw RNA-Seq data from whole-blood of adult participants (N=100).

  Study phs003246

• NIMH (National Institute of Mental Health) De Novo Mutation Identification in Taiwanese Schizophrenia Trios

  The substantial reproductive impact of schizophrenia, for which affected individuals have fewer than half as many offspring as unaffected individuals do, implies that mutations of largest effect will frequently be de novo mutations. Ascertaining exome sequence variation in father-mother-offspring trios allows such mutations to be identified and distinguished from the far-larger amount of rare variation that is inherited by each individual. The pursuit of this approach in a large, well-powered cohort of trios can also provide lessons that inform the development of such gene discovery strategies more generally in human genetics. Schizophrenia trios from the Taiwanese population are being collected by Dr. Ming Tsuang (PI, UC San Diego, California) and investigators in Taiwan (PI, Dr. Hai Gwo Hwu; both funded by NIMH grant 1R01MH085560; Expanding Rapid Ascertainment Networks of Schizophrenia Families in Taiwan). A total of 3800 trios are anticipated to be collected by May 2013. This represents a highly homogenous national sample from the same ancestral population. DNA samples will be obtained from the NIMH Repository, Rutgers University Cell and DNA Repository (described below) and stored at the Broad Institute. Genetic and data analyses will be performed at the Broad Institute. We propose to sequence the whole exome of trios by hybrid capture and Illumina next generation sequencing and perform targeted genotyping and validation of variants (SNPs, indels and CNVs) using several molecular methods, to include emulsion-based PCR and Sanger sequencing.

  Study phs001196

• Genome and Transcriptome Assembly Reveals SVA-Mediated Aberrant Splicing in X-Linked Dystonia Parkinsonism

  X-linked Dystonia-Parkinsonism (XDP, DYT3) is a long-standing quandary in human disease genetics. XDP is predominantly observed on Panay island in the Philippines. This study is one of the first of its kind to interrogate an unsolved Mendelian disorder by integrating genome and transcriptome assembly methods using Illumina, 10X Genomics, Pacific Biosciences, and Agilent genome targeting technologies. These data provide strong evidence for a pathogenic link between a noncoding SVA retrotransposon and XDP. We demonstrate that this Mendelian disorder is associated with a sine-VNTR-Alu (SVA) retrotransposon that inserted into the TAF1 gene and is shared by all XDP probands, yet never observed in controls from worldwide populations. Transcriptome assembly in iPSC-derived neural stem cells (NSCs) and neurons revealed that this SVA caused aberrant splicing and significant intron retention, which was negatively correlated with TAF1 expression. Remarkably, CRISPR/Cas9 excision of the SVA rescued the aberrant transcriptional signature and normalized expression of TAF1 in patient-derived NSCs. We have also interrogated iPSC-derived microglia to investigate the contribution of glial components to the cellular and molecular deficit, effect of TNF treatment on XDP transcriptional signatures. To further understand what XDP may have in common with other hereditary dystonias (such as DYT6), we are further comparing these signatures to ones associated with dystonia-specific variants in proteins that, like TAF1, are also involved in regulating transcription, i.e. THAP1. 

  Study phs001525

• National Institute on Alcohol Abuse and Alcoholism (NIAAA) Postmortem Prefrontal Cortex eQTL and mQTL Study

  Mapping expression and methylation quantitative trait loci (eQTLs and mQTLs) using genome-wide genotype, transcriptome, and DNA methylome data can provide an unbiased approach to identify novel functional variants. One critical issue of the eQTL/mQTL analysis is that the role of genetic variants in regulating gene expression (or DNA methylation) can be tissue- or cell-specific. To unravel causal variants from disease-associated loci, it would be necessary to map eQTLs and mQTLs in both patients and controls. Those eQTLs and mQTLs in patients are more likely to be causal variants for the disease. Identification of disease- and tissue-specific eQTLs and mQTLs will help disentangle the role of the specific tissue in disease development as well. In the present study, we mapped eQTLs and mQTLs in postmortem prefrontal cortex of patients with alcoholism or alcohol use disorder (AUD) and matched control subjects by integrative analyses of genome-wide SNP genotype, mRNA transcriptome, and DNA methylome data. As AUD is comorbid with schizophrenia (SCZ), bipolar disorder (BIP), major depressive disorder (MDD), and attention deficit/hyperactivity disorder (ADHD), we further examined the co-localization of PFC eQTLs/mQTLs and GWAS-identified SNPs (mainly noncoding SNPs) that were associated with AUD as well as comorbid SCZ, BIP, MDD, and ADHD. Our study demonstrated the enrichment of PFC eQTLs/mQTLs in the above psychiatric disorder-associated genetic variants identified by genome-wide association studies (GWAS).

  Study phs001981

• Gabriella Miller Kids First Pediatric Research Program in Craniofacial Microsomia

  The Gabriella Miller Kids First Pediatric Research Program (Kids First) is a trans-NIH effort initiated in response to the 2014 Gabriella Miller Kids First Research Act and supported by the NIH Common Fund. This program focuses on gene discovery in pediatric cancers and structural birth defects and the development of the Gabriella Miller Kids First Pediatric Data Resource (Kids First Data Resource). All of the WGS and phenotypic data from this study are accessible through dbGaP and kidsfirstdrc.org, where other Kids First datasets can also be accessed. Craniofacial microsomia (CFM), also termed hemifacial microsomia or oculo-auricular-vertebral spectrum, is the third most common congenital craniofacial condition. CFM comprises a variable phenotype and the most common features include malformations of the ear (i.e. microtia) and lower jaw (i.e. mandibular hypoplasia) on one or both sides. Microtia in the absence of other anomalies is believed to represent the mildest form of CFM. The cause of CFM is unknown for most affected individuals. We have established a cohort through previous studies and collected DNA to identify the genetic contributions to CFM, which could facilitate diagnosis, tailored treatments and guide prevention strategies. Successful completion of this proposal will advance knowledge in the genetic architecture of susceptibility to CFM and will provide insight about the biological mechanisms underlying craniofacial development. The results from this study have the potential to further research on the etiology of other craniofacial conditions, and the pathogenesis of typical and atypical craniofacial development.

  Study phs002130

• Genomic Analysis of Pediatric Low Grade Gliomas

  Pediatric low-grade gliomas (PLGGs) are among the most common solid tumors in children but, apart from mutations or duplications in the BRAF kinase in specific subclasses, few genetic driver events are known. Diffuse PLGGs compose a set of uncommon subtypes that exhibit invasive growth and are therefore especially challenging clinically. These tumors are particularly poorly understood. We performed high-resolution copy-number analysis of 44 diffuse PLGGs to identify recurrent alterations. Diffuse PLGGs exhibited fewer such alterations than adult low-grade gliomas, but we identified several significantly recurrent events. The most significant event, 8q13.1 gains, was observed in 28% of diffuse astrocytoma WHO grade II (DA2) and resulted in partial duplication of the transcription factor MYBL1 with truncation of its C-terminal negative-regulatory domain. A similar recurrent deletion-truncation breakpoint was identified in two angiocentric gliomas in the related gene MYB on 6q23.3. Whole genome sequencing of a MYBL1-rearranged diffuse astrocytoma grade II demonstrated MYBL1 tandem duplication and few other events. Two truncated MYBL1 transcripts identified in this tumor induced anchorage-independent growth when expressed in 3T3 cells and tumor formation in nude mice. Truncated transcripts were also expressed in two additional tumors with MYBL1 partial duplication. Our results define clinically relevant molecular subclasses of diffuse PLGGs and highlight a potential role for the MYB family in the biology of low-grade gliomas. "Reprinted from www.pnas.org/cgi/doi/10.1073/pnas.1300252110 with permission from PNAS."

  Study phs000614

• A New Pipeline to Predict and Confirm Tumor Neoantigens Predicts Better Response to Checkpoint Blockade

  Mutations that drive oncogenesis in cancer can generate neoantigens that may be recognized by the immune system. Identification of these neoantigens remains challenging due to the complexity of the major histocompatibility complex (MHC) antigen and T-cell receptor interaction. Here we describe the development of a systematic approach to efficiently identify and validate immunogenic neoantigens. Whole exome sequencing of tissue from a melanoma patient was used to identify nonsynonymous mutations, followed by MHC binding prediction and identification of tumor clonal architecture. The top 18 putative class I neoantigens were selected for immunogenicity testing via a novel in vitro pipeline in HLA-A201 healthy donor blood. Naive CD8 T cells from donors were stimulated with allogeneic dendritic cells pulsed with peptide pools and then with individual peptides. The presence of antigen-specific T cells was determined via functional assays. We identified one putative neoantigen that expanded T cells specific to the mutant form of the peptide and validated this pipeline in a subset of patients with bladder tumors treated with durvalumab (N = 5). Within this cohort, the top predicted neoantigens from all patients were immunogenic in vitro. Finally, we looked at overall survival in the whole durvalumab-treated bladder cohort (N = 37) by stratifying patients by tertile measure of tumor mutation burden (TMB) or neoantigen load. Patients with higher neoantigen and TMB load tended to show better overall survival.

  Study phs002269

• Tumor Mutation Burden, Expressed Neoantigen and Immune Microenvironment in Diffuse Gliomas

  A consistent correlation between tumor mutation burden (TMB) and tumor immune microenvironment has not been observed in gliomas as in other cancers. Driver germline and somatic mutations, TMB, neoantigen, and immune cell signatures were analyzed using whole exome sequencing (WES) and transcriptome sequencing of tumor and WES of matched germline DNA in a cohort of 66 glioma samples (44 IDH-mutant and 22 IDH-wildtype). Fourteen samples revealed a hypermutator phenotype (HMP). Eight pathogenic (P) or likely pathogenic (LP) germline variants were detected in 9 (19%) patients. Six of these 8 genes were DNA damage repair genes. P/LP germline variants were found in 22% of IDH-mutant gliomas and 12.5%of IDH-wildtype gliomas (p= 0.7). TMB was correlated with expressed neoantigen but showed an inverse correlation with immune score (R =−0.46, p= 0.03) in IDH-wildtype tumors and no correlation in IDH-mutant tumors. Antigen Processing and Presentation (APP) score correlates with immune score. The APP is higher in non-hypermutator phenotype (NHMP) samples versus HMP samples in the IDH-wildtype gliomas, but higher in HMP samples versus NHMP samples in the IDH-mutant gliomas. Conclusion: TMB was inversely correlated with the immune score in the IDH-wildtype gliomas, and showed no correlation in the IDH-mutant tumors. The APP was correlated with the immune score, and may be further investigated as a biomarker for response to immunotherapy in gliomas. Studies of germline variants in a large glioma cohort are warranted.

  Study phs002653

• Extent and Significance of Bacterial DNA Integrations in the Human Cancer Genome

  The integration of exogenous DNA into the human genome can cause somatic mutations associated with oncogenesis. For example, the insertion of HPV DNA into human chromosomes is the single most important event leading to tumorigenesis in cervical cancer. It is also now preventable with vaccines against HPV. In contrast to viral DNA integrations, the instances and repercussions of bacterial DNA integration into the somatic human genome are less clear. Yet bacterial DNA integrations (BDI) found to be associated with tumorigenesis could also be prevented using therapeutics like vaccines that limit exposure to the bacteria. Previous analysis of publicly available sequencing data from the Cancer Genome Atlas (TCGA) showed BDIs from Pseudomonas spp. rRNA into the 5'-UTR of four proto-oncogenes as well as in Ig (the immunoglobulin gene) of gastric cancer samples. The original samples from TCGA were not available to validate these findings. Therefore, a different cohort of gastric cancer patients was identified in order to establish the presence BDIs. Whole exome sequencing was completed on seven tumor samples and six adjacent gastric samples, as well as one intestinal metaplasia sample and one non-atrophic gastritis sample. Four of those samples were also investigated with transcriptomics and whole genome sequencing. Given that 15-20% of cancers worldwide are linked to infections, more work is needed to understand the role of infectious agents in oncogenesis and the mechanisms underlying that role.

  Study phs001936

• Medulloblastoma exome sequence analysis

  Medulloblastomas are the most common malignant brain tumors in children. Identifying and understanding the genetic events that drive these tumors is critical for the development of more effective diagnostic, prognostic and therapeutic strategies. Recently, our group and others described distinct molecular subtypes of medulloblastoma based on transcriptional and copy number profiles. Here, we utilized whole exome hybrid capture and Illumina sequencing to identify somatic mutations across the coding regions of 92 primary medulloblastoma/normal pairs. Overall, medulloblastomas exhibit low mutation rates consistent with other pediatric tumors, with a median of 0.35 non-silent mutations per megabase. We identified twelve genes mutated at statistically significant frequencies, including previously known mutated genes in medulloblastoma such as CTNNB1, PTCH1, MLL2, SMARCA4 and TP53. Recurrent somatic mutations were identified in an RNA helicase gene, DDX3X, often concurrent with CTNNB1 mutations, and in the nuclear co-repressor (N-CoR) complex genes GPS2, BCOR, and LDB1, to our knowledge novel findings in medulloblastoma and all cancer. We show that mutant DDX3X potentiates transactivation of a TCF promoter and enhances cell viability in combination with mutant but not wild type beta-catenin. Together, our study reveals the alteration of Wnt, Hedgehog, histone methyltransferase and now N-CoR pathways across medulloblastomas and nominates the RNA helicase DDX3X as a component of pathogenic beta-catenin signaling in medulloblastoma. "Reprinted from 'MEDULLOBLASTOMA EXOME SEQUENCING UNCOVERS SUBTYPE-SPECIFIC SOMATIC MUTATION', with permission from Nature"

  Study phs000504

• Molecular Characterization of Hemimegalencephaly

  Somatic mutations, in which a fraction of the cells in the body have a deleterious mutation, are well recognized in cancer but only recently appreciated in neurological disease. Given a somatic mutation in a population of progenitor cells, all daughter cells inherit the mutation and are able to express the resultant phenotype as a function of the differentiation program. We recently identified the first de novo somatic mutations in the developing brain in the condition hemimegalencephaly (HME), a catastrophic focal epilepsy condition associated with a malformation of cerebral cortical development (MCD). HME is one of the most severe MCD syndromes, characterized by massive hamartomatous overgrowth of either of the two cerebral hemispheres. Cerebral hemispherectomy is a frequent treatment for refractory epilepsy, allowing the sampling of diseased tissue. By comparing DNA from a diseased brain with DNA from blood/saliva, we identified de novo somatic mutations in PIK3CA, AKT3 or MTOR, part of the mTOR pathway. Mutations were present in 8-40% of sequenced alleles in various brain regions sampled during surgery and in some codons known to activate proteins.• Population informationPatient samples were collected from multiple centers. The population is Caucasian and Hispanic• Molecular technologies employedWhole-exome sequencing• Principal findings of the studyWe were the first to describe mTOR-related and non-mTOR mutations causing hemimegalencephaly, and we described two-hit mutational models to explain the genetic pathogenesis of hemimegalencephaly

  Study phs002156

• Single Cell Genotypic and Phenotypic Analysis of Measurable Residual Disease in Acute Myeloid Leukemia

  Acute myeloid leukemia (AML) is a group of blood cancers with high mortality. Even though complete remission is often achieved, majority of the AML patients still relapse. Measurable residual disease (MRD), defined as the population of cancer cells which persists following chemotherapy, is responsible for AML relapse. Understanding the biology enabling MRD clones to resist therapy is necessary to guide the development of curative treatments more effectively. Discriminating residual leukemic clones, preleukemic clones, and normal precursors remains a challenge with current MRD tools. We developed a single cell MRD (scMRD) assay by combining flow cytometric enrichment of the targeted precursor/blast population with integrated single cell DNA sequencing (scDNA-seq) and immunophenotyping (Tapestri, Missionbio). The platform utilizes a custom amplicon panel covering 109 amplicons across 31 genes frequently mutated in AML and a panel of 42 oligo-conjugated antibodies against surface makers. We applied our scMRD assay to sequence 30 MRD samples (multiplexed into 6) from 29 AML patients to study the clonal architecture and protein correlation. In addition, 2 MRD samples were sequenced with scDNA+protein sequencing technology without enrichment or multiplexing. Five patients with AML at diagnosis were also sequenced with scDNA+protein sequencing technology without enrichment or multiplexing. In order to establish the sensitivity of our scMRD assay, we spiked AML cells into normal bone marrow cells. We sequenced 12 such spiked samples after multiplexing.

  Study phs003233

• MILK-Omics: Systems Biology of Human Milk and Its Links to Maternal and Infant Health

  The primary objective of the project is to generate a systems-level view of human milk in the context of healthy mothers and their term infants. The study uses biospecimens and clinical data from the Mothers and Infants LinKed for Healthy Growth Study (MILk Study), a prospective observational cohort of approximately 500 exclusively breastfeeding women and their infants who are followed to 6 months of age. The project focuses on mature milk samples collected at 1 month postpartum and infant gut microbiome samples collected at 1 and 6 months of age. Specific Aim 1 is to identify maternal genetic and clinical factors that shape human milk gene expression. Using bulk and single-cell RNA sequencing of the milk cell pellet and genotypes from maternal DNA, we will identify novel genetic determinants of the milk transcriptome and assess potential modification of these genetic associations by gestational weight gain, diet, and other clinical factors. Specific Aim 2 is to describe key features of the normative human milk biosystem and their interactions with one another. Specifically, machine learning techniques will be used to characterize interaction networks and correlational structures among the following features of human milk: transcriptomics, microbiomes, oligosaccharides, metabolomics, lipidomics, and milk macronutrient composition. Specific Aim 3 is to establish how the milk biosystem is related to variation in infant gut microbiomes and health. dbGaP will house the maternal genotype and milk transcriptomics data.The infant microbiome sequence data are associated with BioProject PRJNA1019702.

  Study phs003408

• Longitudinal Multi-Omic Immune Resource Reveals Dynamic Responses in Healthy Human Aging

  The generation and maintenance of protective immunity is a dynamic interplay between host and environment that is impacted by age. Understanding fundamental changes in the healthy immune system that occur over a lifespan is critical in developing interventions for age-related susceptibility to infections and diseases. Here, we use multi-omic profiling (scRNA-seq, proteomics, flow cytometry) to examine human peripheral immunity in over 300 healthy adults, with 96 young and older adults followed over two years with yearly vaccination. The resulting resource includes scRNA-seq datasets of >16 million PBMCs, interrogating 71 immune cell subsets from our new Immune Health Atlas. This study allows unique insights into the composition and transcriptional state of immune cells at homeostasis, with vaccine perturbation, under Cytomegalovirus (CMV) infection and across age. We find that T cells specifically accumulate age-related transcriptional changes more than other immune cells, independent from inflammation and chronic perturbation. Moreover, impaired memory B cell responses to vaccination are linked to a Th2-like state shift in older adults' memory CD4 T cells, revealing possible mechanisms of immune dysregulation during healthy human aging. This extensive resource is provided with a suite of exploration tools at https://apps.allenimmunology.org/aifi/insights/dynamics-imm-health-age/ to enhance data accessibility and further the understanding of immune health across age. Please note that the Sound Life Cohort raw data are spread across three dbGap submissions: phs003841.v1 (this study), phs003944.v1, and phs003400.v1.

  Study phs003841

• Space Associated Stem Cell Hallmarks of Aging in Astronauts

  This study examined the effects of the stress of space, specifically low earth orbit, on hematopoietic stem and progenitor cells (HSPCs) that maintain lifelong hematopoiesis and immune responses. Enriched CD34+ HSPCs were derived from 7 astronauts before, during and after 10 and 21-day ISS missions on the International Space Station. The samples were used as input to whole transcriptome sequencing by bulk RNA-Seq performed at The Scripps Research Institute Next Generation Sequencing Core on Illumina NextSeq 2000 sequencer and single cell RNA-Seq performed at The Salk Institute for Biological Studies on Illumina NovaSeq X Plus sequencer. Subsequent expression, RNA editing, and retrotransposon analyses were performed to assess HSPC and immune subpopulation survival dynamics. Overall, these analyses revealed space-associated stem cell hallmarks of aging (SASHA), including spaceflight dependent alterations in HSPC survival and self-renewal, ADAR1, telomere maintenance, mobilization and cell cycle gene expression, combined with space-induced clonal hematopoietic mutations, APOBEC3C activation and retrotransposon deregulation to warrant countermeasure development to enable long-duration spaceflight. Post-spaceflight analysis revealed increased C-to-T and clonal hematopoiesis-associated mutations, repetitive element and base deaminase deregulation, decreased telomere maintenance gene expression, and decreased self-renewal capacity compared to ground controls. These results demonstrate the space-associated aging effects in HSPCs after month-long exposure to LEO, providing insights into utilizing the LEO environment to model space-accelerated aging and other age-related malignancies.

  Study phs004267

• CRISPR screening in human trophoblast stem cells reveals both shared and distinct aspects of human and mouse placental development

  The placenta serves as the interface between the mother and fetus, facilitating the exchange of gases and nutrients between their separate blood circulation systems. Trophoblasts in the placenta play a central role in this process. Our current understanding of mammalian trophoblast development relies largely on mouse models. However, given the diversification of mammalian placentas, findings from the mouse placenta cannot be readily extrapolated to other mammalian species, including humans. To fill this knowledge gap, we performed CRISPR knockout (KO) screening in human trophoblast stem cells (hTSCs). We targeted genes essential for mouse placental development and identified more than 100 genes as critical regulators in both human hTSCs and mouse placentas. Among them, we further characterized in detail two transcription factors, DLX3 and GCM1, and revealed their essential roles in hTSC differentiation. Moreover, a gene function-based comparison between human and mouse trophoblast subtypes suggests that their relationship may differ significantly from previous assumptions based on tissue localization or cellular function. Notably, our data reveal that hTSCs may not be analogous to mouse TSCs or the extraembryonic ectoderm (ExE) in which in vivo TSCs reside. Instead, hTSCs may be analogous to progenitor cells in the mouse ectoplacental cone and chorion. This finding is consistent with the absence of ExE-like structures during human placental development. Our data not only deepen our understanding of human trophoblast development but also facilitate cross-species comparison of mammalian placentas.

  Study JGAS000659

• Targeting the p53 pathway to treat Malignant Rhabdoid and High-Risk Atypical Teratoid Rhabdoid Tumors

  Rhabdoid tumors (RTs) predominantly affect young children and are among the deadliest pediatric solid tumors. Despite multimodal therapy consisting of surgery, radiation, and chemotherapy, children with these tumors have median survival of less than one year. RTs can arise throughout the body, including the central nervous system (CNS) where they are called atypical teratoid rhabdoid tumors (AT/RTs), and in extra-CNS locations such as the kidneys and other soft tissues where they are designated malignant RTs (MRTs). We previously identified MDM2 and MDM4 as therapeutic vulnerabilities in RTs and showed that treatment with the MDM2 inhibitor idasanutlin increased survival in mice bearing MRT xenografts. However, the therapeutic potential of idasanutlin in CNS RT tumors is unknown. Single agent therapies are prone to resistance and show limited clinical benefit on their own. Therefore, we sought to identify combination strategies incorporating idasanutlin that would be effective in both CNS and extra-CNS RTs. Here, we show that the XPO1 inhibitor selinexor increased nuclear retention of p53 and potentiated idasanutlin-induced p53 pathway activation and cytotoxicity in AT/RT and MRT cell lines in vitro. Importantly, combination therapy limited acquired resistance through TP53 mutation. In vivo, combination therapy was well-tolerated, reduced tumor burden, and increased survival in orthotopic models of both AT/RT and MRT. Our results demonstrate that combining idasanutlin with selinexor is a promising therapeutic strategy for children with rhabdoid tumors.

  Study EGAS00001007680

• Pseudodiastrophic dysplasia expands the known phenotypic spectrum of defects in proteoglycan biosynthesis

  Pseudodiastrophic dysplasia (PDD) is a severe skeletal dysplasia associated with prenatal manifestation and early lethality. Clinically, PDD is classified as a ‘dysplasia with multiple joint dislocations’ however, the molecular aetiology of the disorder is currently unknown. In order to identify the genetic defects underlying PDD, we performed whole exome sequencing (WES) on 3 patients from 2 unrelated families, clinically diagnosed with PDD. WES resulted in the identification of bi-allelic variants in the established skeletal dysplasia genes, B3GAT3 (Family 1) and CANT1 (Family 2). Mutations in these genes have previously been reported to cause ‘multiple joint dislocations, short stature, and craniofacial dysmorphism with or without congenital heart defects’ (B3GAT3) and Desbuquois dysplasia 1 (CANT1); disorders in the same nosological group as PDD. Follow-up of the B3GAT3 variants demonstrated significantly reduced B3GAT3/GlcAT-I expression. Downstream in vitro functional analysis revealed abolished biosynthesis of glycosaminoglycan side chains on proteoglycans. Functional evaluation of the CANT1 variant showed impaired nucleotidase activity, which results in inhibition of glycosaminoglycan synthesis through accumulation of uridine diphosphate. For the families described in this study, the PDD phenotype was caused by mutations in the known skeletal dysplasia genes B3GAT3 and CANT1, demonstrating the advantage of genomic analyses in delineating the molecular diagnosis of skeletal dysplasias. This finding expands the phenotypic spectrum of B3GAT3- and CANT1- related skeletal dysplasias to include PDD, and highlights the significant phenotypic overlap of conditions within the proteoglycan biosynthesis pathway.

  Study EGAS00001004145

• Genomic landscapes of endometrioid and mucinous ovarian cancers and morphologically similar tumor types

  While endometrioid and mucinous ovarian cancers represent nearly a quarter of ovarian cancers, their molecular characteristics and pathologic origins are poorly understood. To characterize the genomic and epigenomic alterations of these ovarian cancer subtypes and evaluate links to morphologically similar tumors from other sites, we performed sequence, copy number, mutation signature, rearrangement, and methylation analyses from tumor samples and matched normal tissues of 133 patients. These analyses included patients with ovarian endometrioid (n=44), ovarian mucinous (n=43), uterine endometrial (n=15), and gastrointestinal mucinous cancers (n=31). In addition to identifying genes previously known to be involved in these tumors, we identified alterations in RAD51C, NOTCH4, SMARCA1/4 and JAK1 in ovarian endometrioid, ESR1 in uterine endometrioid, and SMARCA4 in ovarian mucinous cancers. Whole genome sequencing revealed rearrangements involving PTEN, NF1, and NF2 in ovarian endometrioid tumors, and NF1 and MED1 in ovarian mucinous cancers. The number of alterations and affected genes in ovarian and uterine endometrioid cancers were similar as were genome-wide methylation profiles, suggesting that these tumors may have a similar tissue of origin. By contrast, methylation patterns in ovarian mucinous cancers were more similar to other gastrointestinal mucinous cancers, including mucinous cancers of the stomach, colon, and pancreas. These analyses provide insights into the genomic landscapes and origins of mucinous and endometrioid ovarian carcinomas, providing new avenues for early clinical intervention and management of patients with these cancers.

  Study EGAS50000000523

• Targets of MEK inhibition in DIPG

  The survival of children with diffuse intrinsic pontine glioma (DIPG) remains dismal, with new treatments desperately needed. In the era of precision medicine, targeted therapies represent an exciting treatment opportunity, yet resistance can rapidly emerge, playing an important role in treatment failure. In a prospective biopsy-stratified clinical trial, we combined detailed molecular profiling (methylation BeadArray, exome, RNAseq, phospho-proteomics) linked to drug screening in newly-established patient-derived models of DIPG in vitro and in vivo. We identified a high degree of in vitro sensitivity to the MEK inhibitor trametinib in samples which harboured genetic alterations targeting the MAPK pathway, including the non-canonical BRAF_G469V mutation, and those affecting PIK3R1 and NF1. However, treatment of PDX models and of a patient with trametinib at relapse failed to elicit a significant response. We generated trametinib-resistant clones in the BRAF_G469V model through continuous drug exposure, and identified acquired mutations in MEK1/2 (MEK1_K57N, MEK1_I141S and MEK2_I115N) with sustained pathway up-regulation. These cells showed the hallmarks of mesenchymal transition, and expression signatures overlapping with inherently trametinib-insensitive primary patient-derived cells that predicted an observed sensitivity to dasatinib. Combinations of trametinib with dasatinib and the downstream ERK inhibitor ulixertinib showed highly synergistic effects in vitro. These data highlight the MAPK pathway as a therapeutic target in DIPG, and show the importance of parallel resistance modelling and rational combinatorial treatments likely to be required for meaningful clinical translation.

  Study EGAS00001004495

• Assessing mitochondrial bioenergetics in coronary artery disease: A translational left ventricular tissue study in humans (The AMBITION study).

  Severe or recurrent myocardial ischemia can lead to long-term left ventricular (LV) dysfunction and heart failure in patients with coronary artery disease (CAD). In this study, we assessed for chronic metabolomic and transcriptomic adaptations within LV myocardium of patients with evidence of preoperative inducible ischemia undergoing coronary artery bypass grafting (CABG). During surgery, paired transmural LV biopsies were acquired on the beating heart from a region of inducible ischemia (determined by stress perfusion cardiovascular magnetic resonance), and a remote LV segment. From 33 patients, 63 LV biopsies were acquired on the beating heart during CABG, comparing to analysis of LV samples from 11 donor hearts. The global myocardial ATP/ADP ratio was reduced in CAD patients as compared to donor LV tissue with increased expression of oxidative phosphorylation (OXPHOS) genes encoding the electron transport chain complexes across multiple cell types. Paired analyses of biopsies obtained during CABG from LV segments with or without inducible ischemia revealed no significant difference in the ATP/ADP ratio, broader metabolic profile or expression of ventricular cardiomyocyte genes implicated in OXPHOS. Finally, differential metabolite analysis suggested dysregulation of several intermediates in patients with impaired LVEF; this notably included the tricyclic acid cycle metabolite, succinate. Overall, our results suggest that viable human myocardium in patients with stable CAD has global alterations in bioenergetic and transcriptional profile without large regional differences between areas with or without inducible ischemia.

  Study EGAS00001007351

• Delineating pediatric brain tumor progression using single-nuclei sequencing

  Pediatric brain cancers are the most common solid tumors in children with varying survival rates depending on molecular features of cancer cells and associated microenvironment. Several studies have used single-cell RNA sequencing to define transcriptional programs active in cancer cells and highlighted supportive roles of non-cancerous cells embedded in these tumors. Although application of single-cell RNA sequencing technology has expanded our understanding of intratumoral heterogeneity and underlying transcriptional mechanisms in brain tumors at diagnosis, mechanisms of disease remains elusive. Therefore, the purpose of this proposal is to characterize cellular and transcriptional states of malignant and non-malignant compartments at primary diagnosis and at relapse. In this study, we performed single-nuclei sequencing of 42 patient samples taken at various disease stages (initial diagnosis, progressive disease, and relapsed disease) from 20 patients with low-grade glioma or Atypical Teratoid Rhabdoid Tumor (ATRT). Bulk whole genome and RNA-seq data for these samples are available from the Children’s Brain Tumor Network and the Gabriella Miller Kids First Data Resource Center. Findings from our study will help define the mechanistic shifts of tumor and non-cancerous cells over course of disease progression. Collectively with the single-cell Pediatric Cancer Atlas funded, this project lays the foundation of an important data set focused on lethal relapsed cancers that can continue to be expanded to identify cancer-intrinsic and extrinsic microenvironmental factors supporting recurrence of pediatric brain tumors.

  Study EGAS50000001288

• Whole exome sequencing of an invasive diffuse intrinsic pontine glioma sampled from different sites

  The lateral ventricle subventricular zone (SVZ) is a frequent site of high-grade glioma (HGG) spread and tumor invasion of the SVZ is a predictor of a worse clinical prognosis. A range of HGG types invade the stem cell niche of the SVZ, including both adult glioblastoma and pediatric high-grade gliomas such as diffuse intrinsic pontine glioma (DIPG). The cellular and molecular mechanisms mediating this frequent invasion of the SVZ are poorly understood. Here we demonstrate that neural precursor cell (NPC):glioma cell communication underpins the propensity of glioma to colonize the SVZ. SVZ NPCs secrete chemoattractant signals toward which glioma cells home. Biochemical and proteomic analyses of factors secreted by SVZ NPCs revealed a discrete list of candidate proteins. Necessity and sufficiency testing implicated the neurite outgrowth-promoting factor pleiotrophin, along with three required binding partners (secreted protein acidic and rich in cysteine (SPARC), SPARC-like protein 1, and heat shock protein 90B), as key mediators of this chemoattractant effect. Pleiotrophin protein expression is strongly enriched in the SVZ, and knockdown of pleiotrophin expression starkly reduced glioma invasion of the SVZ in the adult murine brain. Pleiotrophin, in complex with the three binding partners, activated the Rho/ROCK pathway in DIPG cells, and inhibition of ROCK resulted in decreased DIPG invasion toward SVZ neural precursor cell-secreted factors. These findings demonstrate a pathogenic role for neural precursor cell – glioma interactions and potential therapeutic targets to limit glioma invasion.

  Study EGAS00001002326

• Genetic variability in exon 1 of the glucocorticoid receptor gene NR3C1 is associated with postoperative complications

  Patients having major surgery experience inflammation post-operatively, which may contribute to post-operative morbidity. Endogenous glucocorticoids (GCs) are an essential part of the stress response, but this response varies between individuals, which may in turn affect clinical outcome in general, and post-operative inflammation, specifically. Exon 1 of the NR3C1 gene, encoding the glucocorticoid receptor, contains an established region of differential regulation. DNA methylation patterns in this region have been found to differ between individuals. We investigated the methylation status and genotype in the Cytosine-phosphate-Guanine (CpG) island in exon 1 of NR3C1 in 24 patients who underwent major abdominal surgery and correlated it with post-operative complications. DNA was extracted from peripheral blood leukocytes and underwent targeted bisulfite sequencing of the CpG island. Complications were graded according to the Clavien-Dindo classification and 14 out of 24 had post-operative complications. Multifactorial and partial least square methods were used to analyse the data. A homogenous demethylated pattern was observed in all patients and no single CpG methylation was associated with post-operative complications. Four single nucleotide polymorphisms (SNPs) were significantly correlated with higher Clavien-Dindo scores. Genetic variability in the chr5:143,402,505-143,405,805 region of exon 1 of the glucocorticoid receptor gene NR3C1, but not DNA methylation, was associated with more severe post-operative complications in patients having major abdominal surgery. This indicates that the response to GCs may be of clinical importance for inflammatory conditions.

  Study EGAS00001005737

• The patterns and dynamics of genomic instability in metastatic pancreatic cancer

  Pancreatic cancer is an aggressive malignancy with a five-year mortality of 97–98%, usually due to widespread metastatic disease. Previous studies indicate that this disease has a complex genomic landscape, with frequent copy number changes and point mutations, but genomic rearrangements have not been characterized in detail. Despite the clinical importance of metastasis, there remain fundamental questions about the clonal structures of metastatic tumours, including phylogenetic relationships among metastases, the scale of ongoing parallel evolution in metastatic and primary sites, and how the tumour disseminates. Here we harness advances in DNA sequencing to annotate genomic rearrangements in 13 patients with pancreatic cancer and explore clonal relationships among metastases. We find that pancreatic cancer acquires rearrangements indicative of telomere dysfunction and abnormal cell-cycle control, namely dysregulated G1-to-S-phase transition with intact G2–M checkpoint. These initiate amplification of cancer genes and occur predominantly in early cancer development rather than the later stages of the disease. Genomic instability frequently persists after cancer dissemination, resulting in ongoing, parallel and even convergent evolution among different metastases. We find evidence that there is genetic heterogeneity among metastasis-initiating cells, that seeding metastasis may require driver mutations beyond those required for primary tumours, and that phylogenetic trees across metastases show organ-specific branches. These data attest to the richness of genetic variation in cancer, brought about by the tandem forces of genomic instability and evolutionary selection.

  Study EGAS00000000064