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• Population Architecture using Genomics and Epidemiology (PAGE): Causal Variants Across the Life Course (CALiCo): Strong Heart Study (SHS) and Strong Heart Family Study (SHFS)

  The SHS is a study of cardiovascular disease and its risk factors among American Indian men and women. Using standardized methodology, it was designed to estimate cardiovascular disease mortality and morbidity and the prevalence of known and suspected cardiovascular disease risk factors and to assess the significance of these risk factors in a longitudinal analysis. The study included 13 American Indian tribes and communities in three geographic areas: an area near Phoenix, Arizona, the southwestern area of Oklahoma, and western and central North and South Dakota. The SHS included three components: The first was a survey to determine cardiovascular disease mortality rates from 1984 to 1994 among tribal members aged 35-74 years of age residing in the 3 study areas (the community mortality study). The second was the clinical examination of 4,500 tribal members aged 45-74. The SHS has completed three clinical examinations of the original Cohort (Phase I: 1989-1991; Phase II: 1993-1995; Phase III: 1998- 1999, respectively). The third component is the morbidity and mortality (M&M) surveillance of these 4,500 participants. Yearly SHS surveillance has only 0.2% loss to follow-up. All deaths and all nonfatal CVD events are classified by standardized criteria, including details of stroke and HF. The Strong Heart Family Study (SHFS) is a genetic epidemiological study designed to investigate the heritability of CVD and its risk factors and to localize genes that contribute to CVD risk in American Indians. SHFS participants include 3,838 family members that were >/=15 years old, and ascertained through sibships of the original SHS, from 94 extended (large, multigenerational) families. Exams have occurred in a pilot Phase III (1998-1999, 900 SHFS participants), in Phase IV (2001-2003), and Phase V (2006-2009). SHFS morbidity and mortality surveillance has occurred throughout the study phases, with 0.3% lost to follow-up. Genetic data includes complete pedigree information, DNA samples from all family members, a 10cM-spaced microsatellite map used for IBD estimation and to perform linkage analysis, genotypes for more than 12,000 SNPs in candidate regions, and genotypes from commercially available SNP assays.

  Study phs000580

• ALS Compute

  Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the progressive loss of brain and spinal cord motor neurons. Half of ALS patients display cognitive symptoms of frontotemporal dementia (FTD); reciprocally, about 40% of FTD patients show motor neuron deficits, and approximately 15% develop overt ALS. The clinical overlap between ALS and FTD means that the two conditions are thought to represent a disease spectrum (ALS/FTD). In recent years, the identification of several genetic causes of ALS/FTD has contributed significantly to our understanding of disease pathogenesis. Unfortunately, one-third of the underlying genetic causes of familial cases and ~90% of sporadic cases of ALS/FTD remain unexplained. As such, there is a dire need to identify additional genetic factors contributing to ALS/FTD. Such studies require huge cohorts of harmonized whole genome sequencing (WGS) data sets from cases and controls. Currently, there are several major ongoing sequencing efforts for ALS patients. Numerous centers lead to inefficiency, especially in terms of overall costs. At a minimum, this includes the cost of high-performance computing, the storage of large data files, and the duplication of effort between groups. This lack of data harmonization between groups precludes sharing of genetic information and weakens collaborative efforts. The cost and logistics are also a barrier to attracting talented investigators to the ALS/FTD field. To overcome this unmet need, we have founded the ALS Compute project. We are centralizing the storage of ALS/FTD WGS data from every significant sequencing effort in the United States and beyond within a single Cloud environment. This approach will facilitate data harmonization and improve accessibility to the data. To accomplish this, we have made the data and the computational infrastructure available via the Terra platform hosted by NHGRI's Genomic Data Science Analysis, Visualization, and Informatics Lab-Space (AnVIL). This will allow researchers worldwide to access this wealth of data, develop new theories of the disease, and yield breakthroughs in our understanding of ALS/FTD.

  Study phs003184

• Metagenomics-Guided Pathogen Discovery in Travelers' Diarrhea

  Traveler's diarrhea (TD) is caused by enterotoxigenic Escherichia coli (ETEC), other pathogenic gram-negative pathogens, norovirus and some parasites. Nevertheless, standard diagnostic methods fail to identify pathogens in more than 30% of TD patients, so it is predicted that new pathogens or groups of pathogens may be causative agents of disease. A comprehensive metagenomic study of the fecal microbiomes from 23 TD patients and seven healthy travelers was performed, all of which tested negative for the known etiologic agents of TD in standard tests. Metagenomic reads were assembled and the resulting contigs were subjected to semi-manual binning to assemble independent genomes from metagenomic pools. Taxonomic and functional annotations were conducted to assist identification of putative pathogens. We extracted 560 draft genomes, 320 of which were complete enough to be enough characterized as cellular genomes and 160 of which were bacteriophage genomes. We made predictions of the etiology of disease in individual subjects based on the properties and features of the recovered cellular genomes. Three subtypes of samples were observed. First were four patients with low diversity metagenomes that were predominated by one or more pathogenic E. coli strains. Annotation allowed prediction of pathogenic type in most cases. Second, five patients were co-infected with E. coli and other members of the Enterobacteriaceae, including antibiotic resistant Enterobacter, Klebsiella, and Citrobacter. Finally, several samples contained genomes that represented dark matter. In one of these samples we identified a TM7 genome that phylogenetically clustered with a strain isolated from wastewater and carries genes encoding potential virulence factors. We also observed a very high proportion of bacteriophage reads in some samples. The relative abundance of phage was significantly higher in healthy travelers when compared to TD patients. Our results highlight that assembly-based analysis revealed that diarrhea is often polymicrobial and includes members of the Enterobacteriaceae not normally associated with TD and have implicated a new member of the TM7 phylum as a potential player in diarrheal disease.

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• Drug Signatures for Prediction and Mitigation of Toxicity

  The overall goal of the drug signatures for prediction and mitigation of toxicity study is to use genomic and proteomic high-throughput measurements as the basis for computational analysis that integrates network analyses with structural constraints and dynamical models in multiple cell types to identify signatures that predict toxicity induced by 55 FDA-approved chemotherapeutic drugs and potential mitigation of this toxicity. Specifically, we have recruited and pre-screened healthy individuals to be included in our study. Ninety six male and female individuals that satisfied the initial pre-screening for the study and their sex, age, race/ethnicity recorded through an enrollment questionnaire were consented. Eighty five underwent a formal and thorough medical health screening. The evaluation involved assessment of all inclusion and exclusion criteria, a full medical history, measurement of weight, height, waist and hip circumference, heart rate, blood pressure, respiratory rate, and oxygen saturation, a physical exam and an electrocardiograph (ECG). Blood was drawn for analysis of clinical relevant parameters. A pregnancy test was included for female participants. All blood draws and pregnancy tests were sent to a certified clinical laboratory for clinical analysis. Of the 85 subjects that were screened 42 (48.3%) were deemed eligible for final inclusion. Of these 42, one declined to undergo skin biopsy and/or venesection and one subject didn’t show up for the scheduled biopsy, leaving 40 clinically healthy subjects on which a skin biopsy and venesection was performed. Fibroblast lines were established from the skin biopsies. Induced pluripotent cells (iPSC) were generated from the established fibroblast lines of these 40 clinically healthy racially diverse male and female study participants who ranged in age from 22 and 61 years through reprogramming using either the mRNA or Sendai-virus methods. One clone from each of the 40 iPSC line was fully characterized for normal karyotype, short-tandem repeat matching to the original fibroblast line (authentication), pluripotency by select pluripotent marker expression via immunocytochemistry and by mRNAseq-based PluriTest analysis, and whole-genome sequencing.

  Study phs002088

• Age related Macular Degeneration (AMD) - Michigan, Mayo, AREDS, Pennsylvania (MMAP) Cohort Study: Association and Sequencing Studies

  Age-related Macular Degeneration (AMD) is a leading cause of incurable blindness in people over the age of 65. AMD is a late-onset multi-factorial neurodegenerative disease and its pathogenesis involves interaction of genetic and environmental factors. Several chromosomal regions have been associated with AMD susceptibility through linkage analysis (Swaroop et al., 2009). More recent studies provide strong evidence that variants within the CFH gene cluster on chromosome 1 and at/near LOC387715/ARMS2 on chromosome 10 are strongly associated with the disease. Variants at other genes including C2/BF, C3, CFI and APOE4, also contribute to AMD susceptibility. Our primary goals are to identify genetic variants and haplotypes that are associated with AMD. The underlying hypothesis is that DNA variation(s) in multiple genetic susceptibility loci will predispose individuals to AMD pathogenesis, and comparison of DNA of cases and controls should identify these susceptibility variants. Our studies are focused on the genetic analysis of advanced AMD and should provide novel insights into disease diagnosis, progression and pathology. We have assembled a collaborative group of researchers from the University of Michigan, Mayo Clinic, University of Pennsylvania, and the AREDS group including National Eye Institute intramural investigators, who collected clinical data and DNA from a large number of patients affected with AMD and from unaffected controls. The primary source of funding was National Eye Institute. Study 1: To identify genetic variants and haplotypes that are associated with AMD, we submitted and obtained usable genotyping data on 2185 patients and 1155 controls from the Center for Inherited Disease Research (CIDR). Study 2: To identify rare coding variants associated with a large increase in risk of AMD, 10 candidate loci spanning 57 genes were sequenced in 2,335 cases and 789 controls. Probes were designed to capture 96.5% of the coding sequence and 35% of total locus sequence, generating an average 123Mb of on-target sequence per individual at 127x average depth. Substudies: phs000182 AMD-MMAP Cohort Study: A Joint Genome-Wide Asscociation Study phs000246 Fuchs' Corneal Dystrophy GWAS phs000457 MMAP Methylation in AMD phs000685 Age-Related Macular Degeneration Targeted Sequencing Study

  Study phs000684

• Longitudinal Studies of Patients with Familial Platelet Disorder with Associated Myeloid Malignancy (FPDMM)

  FPDMM (OMIM #601399) is a rare autosomal dominant disease characterized by defective megakaryocytic development, thrombocytopenia, platelet functional defects, and a life-long risk of developing hematological malignancies. FPDMM is caused by germline mutations in the RUNX1 gene, which encodes a master regulator of hematopoiesis. The overall lifetime risk of hematological malignancies in FPDMM patients is 35 - 45%. It is hypothesized that mutations in other genes cooperate with the germline RUNX1 mutations for leukemia development. However, it is not clear which genes need to be mutated for leukemia to develop and which biomarkers or clinical tests can be used to predict which patients will progress to leukemia. Likewise, there are no treatments and/or preventative measures that can reduce or stop disease progression except for stem cell transplantation, which is not available for all patients and may be associated with significant complications. To address these questions, we launched a longitudinal natural history study of patients associated with RUNX1 germline mutations at the NIH Clinical Center in early 2019 (Trial Registration ID: NCT03854318). The goals of this study are to: (1) perform comprehensive longitudinal phenotyping of FPDMM patients to document the full spectrum of both hematopoietic and non-hematopoietic manifestations of the disease; and (2) perform extensive genomic analyses of FPDMM patient samples to identify cooperating genomic variants, which may confer growth advantage that leads to clonal expansion of affected hematopoietic cells that may serve as the precursor to malignancies. The data available through dbGaP include exome sequencing and RNA-seq data from these patients. Diverse germline mutation types were identified in the RUNX1 gene, including splice site mutations and large deletions, in addition to nucleotide substitutions and small indels. Somatic mutations, especially in genes associated with clonal hematopoiesis of indeterminate potential (CHIP), were found in a substantial proportion of FPDMM patients who were leukemia-free. The findings suggest that the genomes in these patients are prone to somatic mutations and monitoring the dynamic changes of these somatic mutations prospectively may provide a way to predict progression to malignancies.

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• LCCC1122: Defining the Triple Negative Breast Cancer Kinome Response to GSK1120212

  In this study the investigators looked at adaptive reprogramming impact on the kinome when a MEK inhibitor called GSK1120212 (trametinib) was administered in a "window of opportunity" trial. GSK1120212 is not yet approved by the FDA for use in breast cancer patients. The investigators gave GSK1120212 for a short period of time (one week) to examine MEK and the other kinase expression in cancer cells both before and after the study drug is given. The investigators gave this drug for research purposes only. The length of time it was given is not intended to treat cancer. Recently researchers at UNC developed a process that can comprehensively profile the majority of the individual kinases in the kinome and examine the impact on kinase expression of kinase inhibitors (Duncan et al, Cell 2012, PMID: 22500798). This can tell us which kinases need to be concurrently blocked to augment responsiveness and prevent acquired resistance so that the investigators can design the best combinations of kinase blocking drugs for triple negative breast cancer. This is especially important for individuals with triple negative breast cancer (TNBC) because there are no targeted drugs available that can block molecules that affect tumor growth. The investigators believe that kinase-blocking drugs have the potential to be a more effective treatment for people with TNBC. In this recently published study (Zawistowski et al, Cancer Discovery 2017, PMID: 28108460), TNBC patients treated with trametinib for 7 days resulted in a transcriptional response characterized by significant reprogramming of the tyrosine kinome, and this adaptive bypass response in human tumors was found to be similar to that seen in preclinical models including TNBC cell lines and mouse xenografts. In this study we also examined whether reprogramming differed between TNBC molecular subtypes, finding that basal-like and claudin-low human TNBC cells and mouse tumor subtypes had different adaptive transcriptional responses to MEK-ERK inhibition. Mechanistically we found that genome-wide enhancer remodeling drove the adaptive transcriptional response, suggesting that epigenetic approaches to reprogramming may be more durable than kinase inhibitor polypharmacology.

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• CTN - 0051: Extended-Release Naltrexone vs. Buprenorphine for Opioid Treatment (X:BOT)

  The overarching goal of CTN-0051 is to foster adoption of new relapse-prevention pharmacotherapies in community-based treatment programs (CTPs) where these could have a substantial public health impact. To this end CTN-0051 will assess the comparative effectiveness of extended-release injectable naltrexone (XR-NTX, Vivitrol®), an opioid antagonist recently approved and indicated for the prevention of relapse to opioid dependence, versus buprenorphine-naloxone (BUP-NX, Suboxone®), a high affinity partial agonist indicated for maintenance treatment of opioid dependence, as pharmacotherapeutic aids to recovery. The study is conducted in 8 CTN-affiliated CTPs that provide or partner with detoxification services (inpatient/residential) which have the capacity to maintain participants opioid-free for approximately 3-7 days, have the capacity to provide medication-assisted therapy, and can provide a minimum of one group or individual counseling session per week during the 24-week treatment period. Up to 600 eligible participants will be randomized to treatment with XR-NTX or BUP-NX for 24 weeks (sufficient to include 350 participants who are randomized more than 72 hours after their last opioid). To maximize generalizability, the point of randomization is flexible, from shortly after program admission until just prior to program discharge. The primary goal of the study is to estimate the difference, if one exists, between XR-NTX and BUP-NX in the distribution of the time to relapse (i.e., loss of persistent abstinence) during the 6-month trial. The primary outcome measure is the time to the event, with the event called relapse. Secondary objectives are to: (1) compare outcome on XR-NTX versus BUP-NX across a range of clinical safety and secondary efficacy domains, (2) explore demographic, clinical, and genetic predictors of successful treatment and moderators of differential effectiveness (i.e., what variables may help clinicians choose which of these treatments is best for a given patient), and (3) collect a limited dataset to permit analyses of economic costs and benefits of the two treatments.

  Study phs002876

• NHLBI GO-ESP: Lung Cohorts Exome Sequencing Project (Pulmonary Arterial Hypertension)

  The NHLBI "Grand Opportunity" Exome Sequencing Project (GO-ESP), a signature project of the NHLBI Recovery Act investment, was designed to identify genetic variants in coding regions (exons) of the human genome (the "exome") that are associated with heart, lung and blood diseases. These and related diseases that are of high impact to public health and individuals from diverse racial and ethnic groups will be studied. These data may help researchers understand the causes of disease, contributing to better ways to prevent, diagnose, and treat diseases, as well as determine whether to tailor prevention and treatments to specific populations. This could lead to more effective treatments and reduce the likelihood of side effects. GO-ESP is comprised of five collaborative components: 3 cohort consortia - HeartGO, LungGO, and WHISP - and 2 sequencing centers - BroadGO and SeattleGO. The syndrome of pulmonary hypertension (PH) is a pulmonary disease that carries very high morbidity and mortality. Pulmonary arterial hypertension (PAH) is a category of PH (WHO Group 1) that includes several entities (idiopathic or heritable PAH, and PAH associated with other diseases such as connective tissue diseases including scleroderma-associated PAH) and carries a dismal prognosis, in particular when it relates to scleroderma-associated PAH (median survival of about 4 years). It is believed that the severity of structural changes involving the pulmonary vasculature and right ventricular failure are genetically determined. The 'Genomics and Genetics of Pulmonary Arterial Hypertension' study at Johns Hopkins University aims to identify genetic determinants associated with risk of PAH in a cohort of European American and African American participants with and without PAH. The study also focuses on patients with scleroderma, who are further stratified according to those who have or do not have PAH. The broad goals of the Lung GO/ESP-GO falls into two general categories: (i) discovery of all variants (i.e., common and rare) in all protein-coding regions of the human genome (i.e., the exome) conferring risk to complex pulmonary diseases including PAH. The Johns Hopkins University PAH cohort offers a unique opportunity to elucidate genetic variants that cause PAH.

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• National Eye Institute (NEI) Ocular Hypertension Treatment Study (OHTS)

  The Ocular Hypertension Treatment Study (OHTS) is an National Eye Institute-sponsored multi-center, randomized, prospective treatment trial designed to determine whether lowering intraocular pressure (IOP) in individuals with ocular hypertension delays or prevents the development of primary open angle glaucoma (POAG). A total of 1,636 individuals with ocular hypertension between 40 and 80 years old were enrolled in the study. In addition to ocular hypertension, subjects in the OHTS were required to have normal optic nerve appearance as determined by the OHTS Optic Disc Reading Center and normal and reliable visual field tests at the time of enrollment by the OHTS Visual Field Reading Center. OHTS subjects were randomly assigned to either an observational group which received close observation or a topical medication group which received medication as needed to achieve a 20% reduction in IOP from their baseline levels. Subjects then were examined at regular intervals for optic disc cupping or visual field defects. Other clinical measures were also obtained from OHTS subjects including central corneal thickness (CCT) and intraocular pressure. The primary outcome monitored for the OHTS was the development of glaucoma in one or both eyes as defined by reproducible visual field abnormality or reproducible optic disc deterioration attributed to POAG by the masked OHTS Endpoint Committee. The OHTS study design has been reported in detail (Gordon, 1999; PMID: 10326953). The OHTS confirmed that ocular hypertension is a risk factor for developing POAG and showed that lowering IOP reduced the risk for developing POAG (Kass, 2002; PMID: 12049574). The OHTS also demonstrated that thin CCT is a significant risk factor for the development of POAG (Gordon, 2002; PMID: 12049575). Blood samples were also collected from 1,077 OHTS participants for an ancillary genetics study. DNA was prepared from these samples and used in a genome-wide association scan (GWAS) designed to identify genetic factors that control the magnitude of quantitative features of glaucoma (baseline IOP, baseline cup-to-disc ratio, and CCT). Genotypes from the GWAS and these clinical data (IOP, cup-to-disc ratio, and CCT) have been provided to dbGaP.

  Study phs000240

• Exploring the Genetic Variants Associated with Brain Growth in Children

  Understanding the genetic factors that control brain growth during childhood and adolescence is a priority for neuroscience. Its importance lies partly in the finding that disturbances in the course of brain development characterizes many common childhood behavioral problems. Recent advances in neuroimaging such as the use of non-invasive magnetic resonance imaging have provided a chance to define these trajectories in the living child. The intramural programs of the NIMH and NHGRI have conducted repeated neuroanatomic imaging on children along with the banking of DNA, providing a cohort which is studied for defining developmental trajectories of the brain. A central goal of this cohort is to define the disturbances in brain development that are linked with common behavioral problems, such as excessive impulsivity, hyperactivity and inattention. Studies have demonstrated that problems in these domains are distributed across the entire population and that children with Attention Deficit Hyperactivity Disorder (ADHD) lie at the extreme end of these behavioral dimensions. We found a similar dimensionality in the development of brain regions that are associated with these behavioral problems. Specifically, we showed that the rate of change in both the lateral prefrontal cortex and interconnected striatum during childhood and adolescence was linked to the severity of symptoms of hyperactivity and impulsivity. As symptoms increased, the rate of growth of both structures decreased. The lateral prefrontal cortex and striatum are core components of richly interconnected 'loops' that support a host of cognitive processes, including attention, cognitive control and motor planning. Anomalies in these structures and their connections have been implicated in many childhood behavioral problems, including ADHD. In summary, we aim to identify the common and rare genetic variants that are associated with the developmental trajectories of the lateral-prefrontal cortex and striatum. Identifying genetic variants influencing the growth of these structures may reveal the biological mechanisms underlying dysregulated control of attention, impulse and activity level, which in the extreme constitute ADHD. The study thus could inform our understanding of neurobiology underpinning one of the most common neuropsychiatric disorders of childhood.

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• NSIGHT North Carolina Newborn Exome Sequencing for Universal Screening (NC NEXUS)

  Since newborn screening (NBS) began in the 1960s, technological advances have resulted in its use in an increasing number of disorders. Recent developments in whole-genome sequencing and its simpler corollary, whole-exome sequencing (WES), now afford the opportunity to comprehensively define the variation within an individual's genome in a rapid and affordable manner. Many challenges arise with the clinical application of genome-scale sequencing and in deriving practical benefits to infants and children. Its utility in NBS has yet to be demonstrated and its application in the pediatric population requires special examination, not only for potential clinical benefits but also for the unique ethical challenges it presents. In this proposal, we outline a highly interdisciplinary approach to identify, confront, and overcome the major challenges that must be met in order to implement deep sequencing technology to enhance current newborn screening in a diverse pediatric population. Overarching Aim 1 will evaluate the utility of WES as a diagnostic tool to extend the utility of current NBS. Using diverse cohorts of infants and young children with known conditions identified through NBS, we will examine the sensitivity and specificity of WES. We will also utilize WES in cohorts of children with known conditions not currently screened as potential candidates for NBS in the future. Overarching Aim 2 will develop and assess a framework for analyzing WES in a clinically oriented framework based on principles of ethics and evidence-based medicine. We will develop strategies to guide clinicians, clinical laboratories, and patients/families in their decisions regarding the inevitable incidental findings that will be detected, in ways that respect the child and protect his/her future autonomy, while also respecting parental interests and rights. Overarching Aim 3 will explore ethical, legal, and social issues (ELSI) involved in informed decision making and develop best practices regarding the return of results after testing. We will develop novel decision support tools and evaluate their usefulness in parental decision making, and examine the burdens placed on clinicians as this new technology is deployed in the vulnerable and special population that are newborns and their families.

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• Neoadjuvant atezolizumab plus chemotherapy in gastric and gastroesophageal junction adenocarcinoma: the phase 2 PANDA trial

  Gastric and gastroesophageal junction (G/GEJ) cancers carry a poor prognosis, and despite recent advancements most patients die of their disease. While immune checkpoint blockade has become part of the standard of care for patients with metastatic G/GEJ cancers, its efficacy and impact on the tumor microenvironment in the early disease setting remain largely unknown. We hypothesized a higher efficacy of neoadjuvant immunotherapy plus chemotherapy in patients with non-metastatic G/GEJ cancer. In the phase 2 PANDA trial, patients with previously untreated resectable G/GEJ tumors (n=21) received neoadjuvant treatment with one cycle of atezolizumab monotherapy followed by 4 cycles of atezolizumab plus docetaxel, oxaliplatin and capecitabine. Treatment was well-tolerated, and all patients underwent resection without treatment-related delays, meeting the primary endpoint of safety and feasibility. Tissue was obtained at multiple time points, allowing analysis of the effects of single-agent PD-L1 blockade and the subsequent combination with chemotherapy on the tumor microenvironment. A total of 21 patients were included, of whom 20 patients underwent surgery and were evaluable for the secondary pathologic response and survival endpoints, while 19 were evaluable for exploratory translational analyses. A major pathologic response (MPR, ≤10% residual viable tumor) was observed in 14/20 (70%; 95% CI 46-88%) patients, including 9 (45%; 95% CI 23-68%) pathologic complete responses. At a median follow-up of 47 months, 13/14 responders were alive and disease-free, while 5/6 nonresponders had died due to disease recurrence. Notably, baseline PD-1+CD8+ T cell infiltration was significantly higher in responders than in nonresponders, and comparison of the tumor microenvironment alterations following anti-PD-L1 monotherapy versus the subsequent combination with chemotherapy revealed that increased immune activity was achieved upon single-agent PD-1/L1 axis blockade. Based on these data, anti-PD-L1 plus chemotherapy warrants further exploration in patients with non-metastatic G/GEJ cancer, and these results call for validation in a larger cohort.

  Study EGAS50000000168

• Columbia-Yale-Bilkent Study: Genetics Study of Essential Tremor

  This is a research development/planning grant application to initiate collaborative studies with investigators at Bilkent University in Turkey to study the genetics of Essential Tremor (ET). ET is one of the most common neurological diseases, with an estimated 7 million affected individuals in the United States. The most characteristic clinical feature of ET is a kinetic tremor in the hands or arms, which is mild early in the disease process. As the disease progresses, tremor becomes more severe and more anatomically widespread (e.g., head, trunk). Aside from tremor, patients with ET may also present with other motor features including gait ataxia. Non-motor features can include psychiatric manifestations, cognitive decline and dementia. Despite its extraordinarily high prevalence, the genetic causes of ET are largely unknown. We propose to develop a genetic study of ET focusing on consanguineous families in Turkey. Turkey with a population of about 70 million has a high rate of consanguineous marriages. The rate of consanguinity is estimated to be as much as 20-25% with approximately 70% of all consanguineous marriages involving first cousins. Studying the genetics of ET in the Turkish population, particularly in large consanguineous families, may identify novel genetic causes of ET that are also relevant to ET populations in the United States and worldwide. The proposed research has a high probability of success with our collaborators Drs Ozcelik and Tekinay's expertise in human genetics and functional studies together with an established resource of large consanguineous ET families. The specific aims of this planning grant are to: (1) further develop and solidify collaborations with the team in Bilkent and define the scope of the research that will be the focus of an R01 application, (2) assess the Bilkent team's resources and needs in order to successfully conduct the research, (3) implement cross-training between the United States and Turkish groups in the areas of human genetics, genetic epidemiology and risk factor assessment, genomics, clinical diagnosis, functional studies and personalized genomic medicine, and (4) conduct pilot studies to generate preliminary data necessary for an R01 application.

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• Foundation Medicine Genomic Data Used to Identify Prognostic Markers and Fusion Genes in Multiple Myeloma

  The purpose of this study is to identify prognostic markers and treatment targets using a clinically certified sequencing panel in multiple myeloma. Mutational burden was associated with maf and proliferation gene expression groups, and a high-mutational burden was associated with a poor prognosis. We identified homozygous deletions that were present in multiple myeloma within key genes, including CDKN2C, RB1, TRAF3, BIRC3 and TP53, and that bi-allelic inactivation was significantly enriched at relapse. Alterations in CDKN2C, TP53, RB1 and the t(4;14) were associated with poor prognosis. Alterations in RB1 were predominantly homozygous deletions and were associated with relapse and a poor prognosis which was independent of other genetic markers, including t(4;14), after multivariate analysis. Bi-allelic inactivation of key tumor suppressor genes in myeloma was enriched at relapse, especially in RB1, CDKN2C and TP53 where they have prognostic significance. In addition, chromosomal rearrangements that result in oncogenic kinase activation are present in many solid and hematological malignancies, but none have been reported in multiple myeloma (MM). Here we detected fusion genes in 1.5% of patients. These fusion genes were in-frame and the majority of them contained kinase domains from either receptor tyrosine kinases (ALK, ROS1, NTRK3, and FGFR1) or cytoplasmic kinases (BRAF, MAP3K14, and MAPK14) which would result in the activation of MEK/ERK, NF-κB or inflammatory signaling pathways. Fusion genes were present in smoldering MM, newly diagnosed MM and relapse patient samples indicating they are not solely late events. Most fusion genes were subclonal in nature, but one EML4-ALK fusion was clonal indicating it is a driver of disease pathogenesis. Samples with fusions of receptor tyrosine kinases were not found in conjunction with clonal Ras/Raf mutations indicating a parallel mechanism of MEK/ERK pathway activation. Fusion genes involving MAP3K14 (NIK), which regulates the NF-κB pathway, were detected as were t(14;17) rearrangements involving NIK in 2% of MM samples. Activation of kinases in myeloma through rearrangements presents an opportunity to use treatments existing in other cancers.

  Study EGAS00001002874

• Scottish High Grade Serous Ovarian Cancer

  Scottish HGSOC samples were collected via local Bioresource facilities at Edinburgh, Glasgow, Dundee and Aberdeen and stored in liquid Nitrogen until required. HGSOC patients were determined from pathology records and were included in the study where there was matched tumour and whole blood samples. On receipt of tumour material the tumour was processed as follows: firstly, the tumour sample was divided into two for DNA and RNA extraction. Slivers of tissue were cut from the front and rear faces of the DNA sample, then fixed in formalin and embedded in paraffin wax. Sections from the front and rear tissues from all samples were examined by H&E staining supplemented by WT1/p53 immunohistochemistry if required. Following pathology review, samples were only included if they met the following criteria: they were confirmed as HGSOC and there was greater than 40% tumour cellularity throughout the tumour, determined using the H&E sections. Somatic DNA was extracted using the Qiagen DNeasy Blood and tissue kit (cat no 69504). The tissue was initially homogenised using a Qiagen Bioruptor, followed by the manufacturers recommended protocol (including RNase digestion step). Germline DNA was extracted from 1-3ml whole blood using the Qiagen FlexiGene kit (cat no 51206) following the manufacturers recommended protocol. The resulting DNA 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 surrogate measures of DNA purity. A260/280 had to be 1.8 or greater and A260/230 had to be 2.0 or greater. Then, DNA was quantified using LifeTechnologies Qubit dsDNA BR kit (cat no Q32850) and we required a minimum of 50ul at 25ng/ul for WGS. Thirdly, DNA was diluted to 25ng/ul and a representative sample was loaded onto a 0.8% TAE gel, ran at 100v for 60mins and then imaged using a BioRad ChemiDoc imaging system to visualise the DNA quality. Only when all 4 quality control requirements were satisfied was the DNA sequenced. The DNA was sequenced at the Glasgow Precision Oncology Laboratories.

  Study EGAS00001004410

• Gene expression-based prediction of pazopanib efficacy in sarcoma (HIPO, H021)

  The multi-receptor tyrosine kinase (RTK) inhibitor pazopanib is approved for the treatment of advanced non-adipocytic soft-tissue sarcoma and has also shown activity in other sarcoma subtypes. However, its clinical efficacy is highly variable, and no reliable predictors exist to date to select patients who are most likely to benefit from this drug. We analyzed the molecular profiles and clinical outcomes of pazopanib-treated sarcoma patients enrolled in a prospective observational study by the German Cancer Consortium, DKTK MASTER, that employs whole-genome/exome sequencing (WGS/WES) and transcriptome sequencing to inform the care of adults with advanced cancer across histologies who are younger than 51 and patients with rare tumors, including rare subtypes of more common entities, regardless of age.​ Among a total of 109 patients with available WGS/WES data, there was no correlation between clinical parameters, specific genetic alterations, or mutational signatures and clinical outcome. In contrast, analysis of a subcohort of 62 patients who underwent molecular analysis before pazopanib treatment and had transcriptome sequencing data available showed that mRNA levels of NTRK3 (hazard ratio [HR]=0.53, p=0.021), IGF1R (HR=1.82, p=0.027), and KDR (HR=0.50, p=0.011) were independently associated with progression-free survival (PFS). Based on the expression of these RTK genes, i.e., the features NTRK3-high, IGF1R-low, and KDR-high, we developed a pazopanib efficacy predictor (PEP) that stratified patients into three groups with significantly different PFS (p<0.0001). Application of the PEP to an independent cohort of pazopanib-treated sarcoma patients from DKTK MASTER (n=43) confirmed its potential to separate patient groups with significantly different PFS (p=0.02), whereas no such association was observed in sarcoma patients from DKTK MASTER (n=77) or The Cancer Genome Atlas sarcoma cohort (n=256) who were not treated with pazopanib. A score based on the combined expression of NTRK3, IGF1R, and KDR allows identification of sarcoma patients with good, intermediate, and poor outcome following pazopanib therapy and warrants investigation as a predictive tool in prospective studies to optimize the use of this drug in the clinic.

  Study EGAS00001005836

• METABRIC

  The elucidation of breast cancer subgroups and their molecular drivers requires integrated views of the genome and transcriptome from representative numbers of patients. We present an integrated analysis of copy number and gene expression in a discovery and validation set of 997 and 995 primary breast tumours, respectively, with long-term clinical follow-up. Inherited variants (CNVs, SNPs) and acquired somatic copy number aberrations (CNAs) were associated with expression in 40% of genes, although the landscape was dominated by cis and trans-acting CNAs. By delineating expression outlier genes driven in cis by CNAs, we identified putative cancer genes, including deletions in PPP2R2A, MTAP, and MAP2K4. Unsupervised analysis of paired DNA/RNA profiles revealed novel subgroups with distinct clinical outcomes, which reproduced in the validation cohort. These include a high-risk, ER-positive 11q13/14 cis-acting subgroup and a favourable prognosis subgroup devoid of CNAs. Trans-acting aberration 0152hotspots were found to modulate subgroup-specific gene networks, including a TCR deletion-mediated adaptive immune response in the 0152CNA-devoid sub-group and a Basal-specific chromosome 5 deletion-driven mitotic network. Our results provide a novel molecular stratification of the breast cancer population, derived from the impact of somatic copy number aberrations on the transcriptome.

  Study EGAS00000000083

• Multi-omic and functional analysis for classification and treatment of sarcomas with FUS-TFCP2 or EWSR1-TFCP2 fusions(H021/INF)

  Many rare cancers are not well understood, and pathogenesis-directed therapies are often lacking, resulting in poor patient outcomes. Leveraging two (inter)national precision oncology trials that enroll large numbers of rare cancers, we investigated the clinical, histopathologic, molecular, and functional characteristics of rhabdomyosarcoma (RMS) with fusions of FUS or EWSR1 to the TFCP2 transcription factor, a recently discovered ultra-rare entity whose classification, pathogenesis, and optimal treatment are unclear. Unusually for fusion-driven sarcomas, most cases had highly rearranged genomes, including chromothripsis, and signs of defective homologous recombination DNA repair. All tumors showed extreme expression of a truncated TERT variant and the ALK receptor tyrosine kinase, which was additionally affected by intragenic deletions and aberrant splicing, resulting in the expression of shortened variants in 58% of cases. Three ALK variants were oncogenic in immortalized cells, and patient-derived tumor cells expressing two variants responded to certain ALK inhibitors. Other recurrent alterations included CDKN2A/MTAP co-deletions and mutations in PAPPA2, encoding an IGFBP5-specific proteinase, in 67% and 25% of cases, respectively. DNA methylation analysis, along with 19 other soft-tissue sarcoma classes, revealed a close relationship with undifferentiated sarcoma but not other RMS subtypes, suggesting that TFCP2-rearranged RMS is a separate entity, possibly arising from a distinct cell of origin. Expression of TFCP2 fusions in immortalized human cells blocked late myogenic differentiation and significantly induced genes that were also highly expressed in patient tumors, including ALK, TERT, and two known regulators of skeletal muscle cells, IGFBP5 and PTH1R. Genome-wide chromatin profiling demonstrated direct binding of FUS-TFCP2 to the ALK and TERT loci outside their regular promoters, which correlated with the expression of alternative transcript variants. Finally, FUS-TFCP2 inhibited the repair of DNA double-strand breaks in immortalized cells, rendering them sensitive to treatment with cisplatin. This study provides insight into the pathogenesis and therapeutic vulnerabilities of a new RMS subtype and illustrates the value of linking comprehensive molecular diagnostics and functional annotation within multicenter consortia to improve the understanding and clinical management of rare cancers.

  Study EGAS00001006939

• DAC for A Karadimitris Lab

  Dac EGAC00001001003

• HuBMAP: A 3-D Tissue Map of the Human Lymphatic System

  The major goal of the 3-D Tissue Map of the Human Lymphatic System is to use microscopic and biomolecular procedures to facilitate co-registration pipelines and common 3D reconstruction algorithms. Tissue collected from human spleen, thymus and lymph node will be spatially resolved at the single cell level both within and across individuals. The approach employed involves sequencing of transcriptomes of dissociated cells and mapping to histological sections using CO-Detection by indEXing (CODEX) and /or Imaging mass spectroscopy, two highly multiplexed methods employing antibody-tagged target epitopes. Additionally, light sheet fluorescent microscopy is used to provide a higher level context for structural localization on a larger volume. The molecular data provided by this project is obtained through single-cell RNA-seq.

  Study phs002268

• Combined single-cell transcriptomics and T-cell receptor sequencing reveal heterogeneity of mycosis fungoides between and within patients and identify a CD4+ cytotoxic subtype

  Comprehensive analysis of the malignant cells present in the skin lesions of Mycosis fungoides, the most common subtype of cutaneous T-cell lymphoma, is complicated by the fact that malignant from reactive T cells are challenging to distinguish with certainty. To this end, combined single-cell RNA and TCR sequencing allows unambiguous identification of MF cells, thereby revealing a marked heterogeneity between and within patients with unexpected functional phenotypes. It should be further noted, that mRNA expression of established CTCL protein markers in single-cell transcriptomics to identify MF cells should be used with caution; however, our study uncovered an exciting gene expression pattern that serves as a beacon separating malignant from reactive T cells.

  Study EGAS50000000226

• RNAseq samples of patients with anti-PD-1 resistant HNSCC from a non-randomized, open-label phase 1b clinical trial

  Immune checkpoint blockade (ICB) is an effective treatment for a subset of patients with head and neck squamous cell carcinoma (HNSCC); however, the majority remain refractory. In a nonrandomized, open-label Phase 1b clinical trial, participants with recurrent and/or metastatic (R/M) HNSCC who had progressed on ICB were treated with low-dose 5-azacytidine (5-aza) daily for either 5 or 10 days, in combination with durvalumab and tremelimumab. The primary objective was to determine the biologically effective dose of 5-aza. In this study, RNA sequencing (RNA-seq) was performed on baseline and on-treatment samples from eight participants, and transcriptomic changes were analyzed to assess the impact of low-dose 5-aza treatment.

  Study EGAS50000000728

• WES samples of patients with anti-PD-1 resistant HNSCC from a non-randomized, open-label phase 1b clinical trial

  Immune checkpoint blockade (ICB) is an effective treatment for a subset of patients with head and neck squamous cell carcinoma (HNSCC); however, the majority remain refractory. In a nonrandomized, open-label Phase 1b clinical trial, participants with recurrent and/or metastatic (R/M) HNSCC who had progressed on ICB were treated with low-dose 5-azacytidine (5-aza) daily for either 5 or 10 days, in combination with durvalumab and tremelimumab. The primary objective was to determine the biologically effective dose of 5-aza. In this study, whole exome sequencing (WES) was performed on baseline samples from 12 participants, and genomic alterations were analyzed to access the genetic background of these participants.

  Study EGAS50000000729

• Multi-omics profiling identifies two epithelioid sarcoma molecular subtypes with distinct signaling and immune characteristics

  Epithelioid sarcoma (EpS) is an aggressive sarcoma, characterized by the loss of SMARCB1 expression. EpS is traditionally classified as distal or proximal according to clinicopathological features, but its molecular characteristics remain largely unknown. To establish an EpS molecular classification and uncover determinants of inter- and intra-patient heterogeneity in EpS, we used multi-omics profiling and integrated the genomic, transcriptional and methylome landscapes with single-cell RNA sequencing on fresh samples as well as spatial transcriptomics. We identified two molecular subtypes of EpS: “distal-like” and “proximal-like”, which were distinct from the histological subtypes. Distal-like tumors include all morphological distal subtypes and a subset of proximal and hybrid EpS. They express a specific molecular single-cell derived epithelial-to-mesenchymal transition signature, which associates with improved patient survival and includes Desmoglein 2 (DSG2), which we identify as a potential routine immunohistochemical biomarker to improve diagnosis. Distal-like EpS also display increased peri-tumoral CD8+ T cell infiltrates and specific tumor-immune cell interactions. Conversely, proximal-like EpS harbor a higher inter-tumoral molecular heterogeneity with some cases resembling other SMARCB1-deficient tumors by DNA methylation profiling, and increased intra-tumoral pro-tumoral macrophage infiltrates. Our study introduces a novel molecular classification of EpS with prognostic value, associated diagnostic biomarkers and distinct tumor immune microenvironments, which extends beyond traditional clinicopathological classification. This paves the way for precision medicine-based therapeutic strategies for patients with EpS.

  Dac EGAC50000000552

• Investigation of a method for generating cells for regenerative medicine using comprehensive nucleic acid analysis of iPS cell-derived cardiomyocytes.

  Regenerative medicine for heart failure is expected to restore cardiac function by using human iPS cells (hiPSCs). In our laboratory, it was shown that transplantation of cardiomyocytes (CMs) derived from hiPSCs into a pig model of myocardial infarction improves the disease condition. However, the instability of differentiation efficiency and tumorigenicity after transplantation are still issues to be resolved. We attempt to identify and analyze the genes and DNA methylation associated with the induction of differentiation of hiPSCs into CMs with the aim of achieving stable induction of CMs differentiation. Furthermore, we aim to analyze genes involved in tumorigenesis in CMs, and to develop a predictive marker for tumorigenesis to ensure the safety of cells before transplantation.

  Study JGAS000665

• A study of resistance to novel coronavirus infection in health care workers

  There have been cases of what appear to be nosocomial infections among healthcare workers at several hospitals. The infected healthcare workers were assumed to have contracted the disease through close contact with infected patients. However, we confirmed at each medical institution that there were many healthcare workers who had apparently had closer contact with infected patients than with infected patients, but had not contracted the disease. Thus, the purpose of this study was to clarify the internal factors that make individuals less susceptible to novel coronavirus infection and their stress tolerance, and to establish a system that contributes to the appropriate allocation of healthcare workers and the creation of a system that can more safely deal with this disease in the future.

  Study JGAS000562

• Fixation effects on variant-calling in a clinical resequencing panel

  Formalin fixation is the standard method for preservation of tissue for diagnostic purposes, including pathological review and molecular assays. However, this method is known to cause artifacts that can affect the accuracy of molecular genetic tests. We assessed the applicability of alternative fixatives to determine whether these perform significantly better on next-generation sequencing assays, and whether adequate morphology is retained for primary diagnosis, in a prospective study using a clinical-grade laboratory-developed targeted resequencing assay. We examined and quantified several parameters relating to sequencing quality and variant-calling from tumor and normal colon epithelial tissues. We identify one alterative fixative, Sakura Universal Molecular Fixative that suppresses many formalin-related artifacts while retaining adequate morphology for pathological review.

  Study EGAS00001003507

• Aberrant ERBB4-SRC Signaling as a Hallmark of Group 4 Medulloblastoma Revealed by Integrative Phosphoproteomic Profiling

  The four main medulloblastoma subgroups are characterized by distinct biology, clinicopathological features and clinical outcomes. While the WNT and SHH subgroups are characterized by clearly defined aberrant signaling of developmental pathways, the underlying biology of Group 3 and 4 remains poorly understood. Here, we delineate distinct post-translational regulation orchestrating active oncogenic signaling networks in Groups 3 and 4. Specifically, aberrant ERBB4-SRC signaling constitutes a novel hallmark feature of Group 4, with great promise as a novel oncogenic mechanism and potential vulnerability for therapeutic intervention in the most common subgroup. Altogether, this novel integrative proteogenomic landscape demonstrates the added value of proteomics and phosphoproteomics analysis to discover and exploit unexpected oncogenic mechanisms in medulloblastoma and beyond.

  Study EGAS00001002757

• NOTCH1 fusion genes in pediatric T-cell lymphoblastic lymphoma hallmark a common high-risk subgroup with blood TARC levels as possible biomarker

  T-cell lymphoblastic lymphoma (T-LBL) is a common pediatric malignancy accounting for approximately 20% of the non-Hodgkin lymphomas during childhood. Survival rates of T-LBL are ~80%, but outcome after relapse is dismal, with salvage rates reaching only ~15. Considering the extremely poor prognosis after relapse and absence of clinically relevant high-risk genetics, there is an urgent need for the identification of molecular risk factors and new prognostic biomarkers in T-LBL, as well as identification of new therapeutic strategies. In this study we present a novel entity of high-risk pediatric T-LBL patients characterized by previously unknown NOTCH1 gene fusions and highly elevated blood TARC levels

  Study EGAS00001007703

• minION fastq files of 10 different tumor samples from the Master program (H021)

  Nanopore RNA Sequencing was done for 10 tumor samples. Direct cDNA sequencing was performed using the SQK-DCS109 kit (Oxford Nanopore Technologies). For analysis of a single sample on a MinION flow cell (version R9.4.1), 5 μg RNA was used as input. For multiplexing on a MinION flow cell, 2.5 μg RNA per sample was used as input, and the native barcoding expansion kit EXP-NBD104 was employed in conjunction with SQK-DCS109. After reverse transcription with Maxima H Minus Reverse Transcriptase (Thermo Scientific), second-strand synthesis was performed using the 2x LongAmp Taq Master Mix (New England Biolabs). The resulting double-stranded cDNA was subjected to end-repair and dA-tailing using the NEBNext Ultra End Repair/dA-Tailing Module (New England Biolabs). For multiplexed libraries, this step was followed by barcode ligation and library pooling. Next, libraries were quantified with a Qubit Fluorometer 3.0 (Life Technologies). Finally, sequencing adapters were added to the library preparations and ligated with Blunt/TA Ligase Master Mix (New England Biolabs), followed by further quality control using a Qubit. Samples ACC1 and ACC2 were analyzed on individual MinION flow cells, while the remaining eight samples were sequenced as multiplexed libraries on two MinION flow cells by pooling four samples for each run. Five ACC samples were also analyzed individually on Flongle flow cells

  Dataset EGAD00001008970

• A Genome-Wide Association Study of Heroin Dependence

  This collaboration of Australian and American investigators aims to identify genes associated with liability for heroin dependence. The project uses a case-control design in which cases met lifetime DSM-IV criteria for heroin dependence. Controls included assessed individuals who did not meet DSM-IV heroin dependence criteria and unassessed general population controls. Cases and controls were obtained from the several large investigations including: The Comorbidity and Trauma Study, Heroin Dependence in Western Australia, the OZ-ALC Study, a Twin Study of Mole Development in Adolescence, and ongoing genetic studies of substance dependence conducted by investigators at Yale and collaborating institutions. These projects are briefly described below. The Comorbidity and Trauma Study (PI: Elliot Nelson), a retrospective case-control study examining genetic and environmental factors contributing to heroin dependence liability. The study was funded by the National Institute on Drug Abuse (NIDA), and was run in collaboration with Washington University, the Queensland Institute of Medical Research (QIMR), and the National Drug and Alcohol Research Centre (NDARC), University of New South Wales. Case participants were recruited from maintenance clinics in the greater Sydney area. Control participants were recruited from employment centres and community centres, open street malls, and local press servicing the same geographical area as the opioid maintenance treatment clinics and either denied recreational use of opioids or had used these drugs recreationally fewer than 11 times lifetime. The prevalence in these individuals of non-opioid licit drug dependence and illicit drug dependence as well as childhood trauma exposure and other psychiatric disorders is elevated considerably versus estimates of similar measures in Australian general population samples. Participants provided blood samples as a source of DNA and completed a comprehensive psychiatric diagnostic interview based on the Semi-Structured Assessment of the Genetics of Alcoholism - Australia (SSAGA-OZ) augmented with sections drawn from other instruments assessing childhood trauma exposure, family history, and screening for borderline personality disorder. Heroin Dependence in Western Australia (PI: Sybille Schwab) is a study focusing both on genetic contributions to heroin dependence and response to naltrexone treatment of the disorder. Participants completed a clinical assessment and provided blood samples during their treatment at the Perth Naltrexone Clinic now name as the Fresh Start Recovery Programme. Funding for the project was provided by the Australia Government's National Health and Medical Research Council (Grant # 513862; PI: Sybille Schwab) Affected subjects from ongoing genetic studies of substance dependence conducted by investigators at Yale (PI: Joel Gelernter) and collaborating institutions were collected in the course of several NIDA-funded studies. Those included in the current set were assessed by means of the SSADDA (Semi-Structured Assessment for Drug Dependence and Alcoholism). All are opioid dependent European-Americans, and all list heroin as the opioid must used. Most were collected at Yale University School of Medicine or University of CT School of Medicine under the supervision of Drs Joel Gelernter and Henry Kranzler. Control subjects were also collected in the course of several NIDA- and NIAAA-funded studies. Those included in the current set were assessed by means of the SSADDA (Semi-structured Assessment for Drug Dependence and Alcoholism). Most were collected at Yale University School of Medicine or University of Connecticut School of Medicine under the supervision of Drs Joel Gelernter and Henry Kranzler. The OZ-ALC Study (PI: Andrew Heath) consists of a large group of twins and their family members ascertained from the general population Australian Twin Registry who have participated in ongoing research projects. For the control investigation, we have selected individuals who do meet criteria for illicit drug dependence who have had GWAS genotyping with the Illumina Human CNV370-Quad. Inclusion of individuals with alcohol dependence or nicotine dependence was minimized. For a more detailed description of the study, please see: http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?study_id=phs000181.v1.p1 The Twin Study of Mole Development in Adolescence (PI: Nick Martin) is an ongoing investigation of melanocytic naevi funded by the Australian Government's National Health and Medical Research Council (Grant # 389891; PI: Nick Martin). For the current project, unassessed parents of these twins will serve as a control group. These individuals will either have been previously genotyped with the Illumina Human 610-Quad BeadChip or will be genotyped as part of the current project. Parents have largely survived the period of risk for heroin dependence and, by virtue of their participation in this research, are very likely to have a prevalence of heroin dependence lower than that in the general population (i.e., <0.7%). In order to understand the immune modulatory effect of opioids across peripheral blood immune populations, a second study (PI: Christine S. Cheng) analyzed single cell RNA-seq profiles from the PBMCs of a subset of case participants and control participants from the Comorbidity and Trauma Study (PI: Elliot Nelson). The study assessed and identified transcriptional changes between opioid dependent and control samples across naive and LPS stimulated immune populations.

  Study phs000277

• Diet, Genetic Factors, and the Gut Microbiome in Crohn's Disease

  We investigate the hypothesis that consistent changes in the human gut microbiome are associated with Crohn's disease, a form of inflammatory bowel disease, and that altered microbiota contributes to pathogenesis. Analysis of this problem is greatly complicated by the fact that multiple factors influence the composition of the gut microbiota, including diet, host genotype, and disease state. For example, data from us and others document a drastic impact of diet on the composition of the gut microbiome. No amount of sequencing will yield a useful picture of the role of the microbiota in disease if samples are confounded with uncontrolled variables. We aim to characterize the composition of the gut microbiome while controlling for diet, host genotype, and disease state. Diet is controlled by analyzing children treated for Crohn's disease by placing them on a standardized elemental diet, and by testing effects of different diets on the gut microbiome composition in adult volunteers. Genotype is analyzed by large scale SNP genotyping, which is already underway and separately funded--team member Hakon Hakonarson is currently genotyping 50 children a week at ~half a million loci each and investigating connections with inflammatory bowel disease. Clinical status is ascertained in the very large IBD practice in the UPenn/CHOP hospital system. Effects of diet, host genotype, and disease state on the gut microbiome are summarized in a multivariate model, allowing connections between microbiome and disease to be assessed free of confounding factors. This project is divided into four sub-studies. In the Fecal Storage Methods (FSM) study, methods of stool storage and DNA extraction are compared to examine their impact on DNA sequence analysis results. The Controlled Feeding Experiment (CaFE) addresses the effects of controlled diets on the gut microbiome. In the Cross-sectional Study of Diet and Stool Microbiome Composition (COMBO), the effects of diet analyzed using surveys and deep sequencing of stool specimens. The fourth study, Pediatric Longitudinal Study of Elemental Diet and Stool Microbiome Composition (PLEASE), examines the effects of an elemental diet treatment on pediatric patients diagnosed with inflammatory bowel disease (IBD), particularly Crohn's disease. Fecal Storage Methods (FSM): Cross-sectional study Controlled Feeding Experiment (CaFE): Controlled trial Cross-sectional Study of Diet and Stool Microbiome Composition (COMBO): Cross-sectional study Pediatric Longitudinal Study of Elemental Diet and Stool Microbiome Composition (PLEASE): Longitudinal cohort study

  Study phs000252

• Cystic Fibrosis Nasal Epithelium Gene Expression by RNAseq

  Cystic fibrosis (CF) is a monogenic disorder leading to multi-organ system dysfunction, with the primary cause of morbidity and mortality being lung disease. Mutations in CFTR result in defective ion channel transport leading to hyperviscous mucus, airway infection, inflammation and lung function decline. While the Phe508del mutation is the most common CFTR mutation in North American people with CF, it is well known that there is a broad range of lung disease severity even amongst Phe508del homozygotes, including twins/siblings. Heritability analyses showing the relative contribution of non-CFTR modifier genes in such differences in phenotype, have led to our genomic and transcriptomic studies of gene modifiers in CF. Complementary to two international CF GWAS studies, this study was performed to a) determine differential expression of genes relative to a quantitative lung disease severity phenotype score (KNoRMA; as used in CF GWAS studies) and b) to assess expression quantitative trait loci. To better understand genetic modification of CF lung disease severity, we analyzed gene expression (transcriptomic) profiles by mRNA sequencing from nasal mucosal biopsy specimens of 134 people with CF. Each study participants was characterized for CF lung disease severity phenotype by averaging three years of lung function data (forced expiratory volume in 1 second; FEV1), normalized to a CF-specific reference population matching for age, sex, and height. Participants completed nasal lavage and nasal curettage mucosal sampling, with directed sampling to the inferior nasal turbinate, a well validated surrogate for cellular characteristics and CFTR function of the lower CF airways. Participants were further invited to complete a second set of these sampling procedures for validation analysis. Specimen and concurrent clinical data collection occurred across four North American study sites and nasal mucosal specimens were subjected to RNASeq analysis. These data were to be made available in the dbGaP. Our association analyses of gene expression relative to quantitative CF lung disease phenotype, taken together with previously identified GWAS signals, identified biological pathways correlated with CF lung disease pathogenesis. Our further association analyses of gene expression relative to single nucleotide polymorphism variation at significant CF GWAS loci further identified potential mechanisms of genetic variants associated with CF lung disease. This resource is intended to aid researchers in characterizing airway mucosal gene expression from the CF airways for the purpose of gene expression profiling in CF and in muco-obstructive airways disease.

  Study phs002254

• CCDG CVD: VIRGO - Variation in Recover-Role of Gender on Outcomes of Young Acute Myocardial Infarction (AMI) Patients

  The VIRGO study is a four year research project funded by the NHLBI to study young women and men with heart attacks. A total of 2,000 women who are 18 - 55 years of age and a smaller comparison group of 1,000 similarly aged men will be enrolled in this multi-site observational study from approximately 120 different hospitals across the country. Young women have a much greater risk of dying after an acute myocardial infarction (AMI) than similarly aged men. Their mortality risk is markedly higher than that of young men, and limited data on young minority women suggest that they may have the highest risk of any young subgroup. Findings from the small number of published studies of young women with ischemic heart disease suggest that the biology, epidemiology, care, and outcomes of this group are distinct from those of men. To date, there have been no large studies of this population, even as the death toll is comparable to that from breast cancer in this country. This study will help researchers identify key factors that influence recovery from a heart attack and potentially improve the care and outcomes of young women and men with AMI. Specific Aims: To investigate the excess risk in young women with AMI and to identify key demographic, clinical, metabolic, psychosocial, health care delivery, and biological determinants of prognosis, we propose a national, prospective, observational study of 2,000 women who are 55 years or younger with AMI and a smaller comparison group of 1,000 men. Our prior work and preliminary studies indicate that the sex differences are most prominent in this age group and suggest that by 1 year after discharge, there are substantial sex differences in outcomes. We will also develop risk stratification tools that will help clinicians to identify young women with the highest and lowest risk. Specific Aim 1: Determine sex differences in outcomes following AMI. Specific Aim 2: Determine sex differences in the prevalence and prognostic importance of demographic, clinical, and psychosocial risk factors. Specific Aim 3:Determine sex differences in quality of care. Specific Aim 4: Determine sex differences in the prevalence and prognostic importance of selected biochemical biomarkers following AMI. These samples have been whole genome sequenced as part of the NHGRI's Center for Common Disease Genetics at the Broad Institute.

  Study phs001259

• Integrative Single-Cell Analysis of Transcriptome, Epigenome, and Lineage in HIV Latency and Activation

  With the development of triple combination antiretroviral therapy, routine HIV treatment eliminates nearly all actively infected cells. Nevertheless, the small reservoir of latently infected cells, which can remain dormant for long periods of time before becoming active and producing new virus particles, represents a crucial barrier to completely curing the disease. Identifying markers that identify latently infected cells or the biochemical factors that control latency activation could enable the effective use of a “shock and kill” strategy, where specific targeting or activation of latently infected cells eliminates the viral reservoir. Our recent work suggests that the global transcriptomic and epigenomic changes during hematopoietic differentiation affect viral latency and activation. Additionally, we recently found that global inhibition of histone deacetylase activity increases viral activation in these cells, further implicating epigenomic changes during activation. These results raise fundamental questions: What are the markers of latently infected cells? How do the transcriptomic and epigenomic states of a cell affect latency and activation? How does the differentiation state relate to viral latency? Here, we leverage our experimental platform for identifying latently and actively infected cells, single-cell transcriptome and epigenome sequencing, and our recently developed computational integration methods to investigate these questions. Our interdisciplinary team combines expertise in HIV basic science, HIV clinical treatment, and bioinformatics to develop an experimental and computational framework for integrated gene expression, chromatin accessibility, and lineage into a single picture of viral latency and activation. Specifically, this project will (1) use single-cell RNA-seq and single-cell ATAC-seq to map the diversity of infected cells, (2) investigate the relationship between hematopoietic differentiation state and viral activation, (3) determine viral integration sites through single-cell RNA-seq, (4) computationally integrate single-cell transcriptome and epigenome profiles, and (5) computationally infer cell lineage relationships among viral genomes and infected cells. To accomplish these goals, we will carry out the following aims: (1) Characterize lineage, transcriptomic and epigenomic diversity of single latently and actively infected primary cells. (2) Investigate latency and activation during in vitro differentiation. (3) Survey single cell diversity of re-activated and in vitro infected cells from cART-suppressed patients. Together, these aims will produce a comprehensive, integrated transcriptomic and epigenomic atlas of the HIV reservoir, identify DNA and RNA biomarkers of latency, and characterize clonal expansion patterns. Our work also develops a broadly applicable experimental and computational framework, laying a foundation for the discovery of novel insights into HIV latency and activation.

  Study phs002915

• DNA methylomes of monozygotic twins clinically discordant for multiple sclerosis

  Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Although genetic susceptibility is important, a modest concordance rate for MS in monozygotic (MZ) twins suggests that interaction with other risk factors is required to develop clinical symptoms. In this study, we examined whether DNA methylation differences contribute to the discordant clinical manifestation of MS in MZ twins, and studied the impact of MS treatments on the DNA methylome. Genome-wide DNA methylation profiles of peripheral blood mononuclear cells (PBMCs) of 45 MZ twins clinically discordant for MS were generated using the Illumina HumanMethylationEPIC array. Repetitive element methylation and selected differentially methylated positions (DMPs) were validated using targeted deep bisulfite sequencing (TDBS). In addition, we performed whole genome bisulfite sequencing (WGBS) to profile CD4+ memory T-cells of a subset of four MS discordant MZ twins. Our results show that interferon treatment causes robust DNA methylation changes and several epigenetic biomarkers for interferon treatment response were identified. However, overall the PBMC-based methylomes of the MS discordant MZ twins were highly similar, since large systematic methylation differences (>5%) were absent in the data. This suggests that previously reported large methylation changes are probably caused by genetic rather than epigenetic differences. In addition, our data does not support the hypothesis that the observed maternal parent-of-origin effect in MS is due to genomic imprinting errors, and no evidence was found that copy number variations explain the discordant phenotype in these MZ twins. Although not genome-wide significant, a couple of DMPs associated with the MS phenotype were identified and successfully technically replicated using TDBS, including a differentially methylated region (DMR) in the promoter of the transmembrane protein encoding gene TMEM232. Another MS-associated DMP, located in an enhancer in the gene body of the transcription factor ZBTB16, was also associated with medium-term glucocorticoid treatment history. WGBS analysis confirmed this DMP as a promising epigenetic biomarker for glucocorticoid treatment. In conclusion, this epigenome-wide association study (EWAS) in genetically identical twins identified a DMR in the TMEM232 promoter as a candidate loci associated with the clinical manifestation of MS. In addition, epigenetic biomarkers for MS treatments were identified, revealing that not only short-term, but also medium-term treatment effects are detectable in immune cells, which should be taken into account in future EWAS designs.

  Study EGAS00001003147

• HG_Retroduplications_in_Neurodevelopmental_Disorders

  Mobile genetic Elements (MEs) are pieces of DNA which, through an RNA intermediate, can generate new copies of themselves elsewhere in the host genome. Additionally, MEs can facilitate the duplication of non-ME transcripts, typically genes, through the mechanism of retroduplication. In humans, several disorders have been directly attributed to ME-derived mutagenesis but not to retroduplication events. We propose to whole genome sequence the 7 individuals with putative de novo gene retroduplications in order to identify the site of insertion. This sequencing will serve two primary purposes: 1.) Identify the first documented case of a de novo retrogene insertion event causing a disease in humans 2.) provide additional whole genome sequencing of undiagnosed probands recruited into the DDD 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 EGAS00001002907

• NOWAC blood-based breast cancer case-control study

  Tumor-host interactions extend beyond the local microenvironment and cancer development largely depends on the ability of malignant cells to hijack and exploit the normal physiological processes of the host. Although abnormalities in a host’s systemic immunity are associated with increased cancer susceptibility, the functional interplay between tumor cells and circulating immune cells in regulating tumorigenic responses is unclear. We employed the Norwegian Women and Cancer study, a large prospective population-based cohort study, to identify gene expression changes in blood cells that provide a robust and reproducible diagnostic signal specific to breast cancer patients. We further show that circulating blood cells in breast cancer patients are enriched in genes involved in systemic immunosuppression and the motility, metabolism, growth, and proliferation of immune cells. By mining of the cancer-associated blood transcriptome, we identified immune mediators or biomarkers that could permit early detection of breast cancer and open avenues to novel targeted immunotherapies.

  Study EGAS00000000134

• PETAL Network: Outcomes Related to COVID-19 Treated With Hydroxychloroquine Among Inpatients With Symptomatic Disease (ORCHID) Trial

  ORCHID was a multicenter, blinded, placebo-controlled randomized trial conducted at 34 hospitals in the US between April 2 and June 19, 2020. Adults hospitalized with respiratory symptoms from severe acute respiratory syndrome coronavirus 2 infection were enrolled, with the last outcome assessment on July 17, 2020. The planned sample size was 510 patients with five interim analyses; however, the trial was stopped at the fourth interim analysis for futility with a sample size of 479 patients.The distribution of the day 14 clinical status score (measured using a 7-category ordinal scale) was not significantly different for patients randomized to receive hydroxychloroquine compared with placebo.Instructions for requesting individual-level data are available on BioData Catalyst at https://biodatacatalyst.nhlbi.nih.gov/resources/data/. Apply for data access in dbGaP. Upon approval, users may begin accessing requested data in BioData Catalyst. For questions about availability, you may contact the BioData Catalyst team at https://biodatacatalyst.nhlbi.nih.gov/contact.

  Study phs002299

• Molecular and Clinical Analyses of PHF6 Mutant Myeloid Neoplasia Provide Clues as to Their Pathogenesis and Therapeutic Targeting

  PHF6 mutations (PHF6MT) are identified in various myeloid neoplasms (MN). However, little is known about the precise function and consequences of PHF6 in MN. Our comprehensive genomic analyses yielded three main findings. Firstly, we revealed a different pattern of genes correlating with PHF6MT in male and female cases. When analyzing male and female cases separately, in only male cases, RUNX1 and U2AF1 were co-mutated with PHF6. In contrast, female cases revealed co-occurrence of ASXL1 mutations and X-chromosome deletions with PHF6MT. Next, proteomics analysis revealed a direct interaction between PHF6 and the pioneer transcription factor RUNX1. Both proteins co-localize in active enhancer regions that define the context of lineage differentiation. Finally, we demonstrated a negative prognostic role of PHF6MT, especially in association with RUNX1. The negative effects on survival were additive as PHF6MT cases with RUNX1 mutations had worse outcomes when compared to cases carrying single mutation or wild-type.

  Study phs003303

• Single molecule molecular inversion probe capture developed using the CIViC database

  This study evaluated the use of the Clinical Interpretations of Variants in Cancer database (CIViC) to develop an Open-sourced CIViC Annotation Pipeline (OpenCAP). OpenCAP is a resource that allows users to develop a clinical capture panel that can be linked back to clinical summaries within the CIViC database. This method for capture panel development and variant annotation was tested using 27 cancer samples from 5 tumor subtypes with original exome or genome sequencing. Specifically, using OpenCAP, 111 variants were identified as relevant for the capture panel and 2,027 smMIPs were designed to target these variants. The smMIPs panel design was employed on all samples to validate the capture panel design. When compared to original sequencing, the CIViC smMIPs capture panel demonstrated a 95% sensitivity for variant detection (n = 61 variants). Additionally, the CIViC smMIPs capture panel identified 182 additional clinically relevant variants that had been missed on original sequencing.

  Study phs001890

• University of Miami Udall Center of Excellence Identification of Rare Variants in PD through Whole Exome Sequencing

  The focus of this study is to identify a new level of genetic variation, i.e. rare genetic variants with a population frequency less than 5%, usually less than 1%, which are believed to provide a stronger risk per variant than those studied to date in the large genome wide association studies (GWAS). To do this we are generating whole exome sequencing data on the Illumina HiSeq. Each HiSeq produces at least 600 billion base pairs of DNA sequence in one run. Whole exome sequencing sequence data, about 50 million base pairs, or about 1.5% of the total DNA of each person's genome, is generated. We are using this data to look for new DNA variations that give risk for Parkinson disease, as well as "modifiers", that may lead to having more severe or milder disease or later or earlier ages of onset.

  Study phs000908

• Genetic Determinants of EGFR-Driven Lung Cancer Growth and Therapeutic Response In Vivo

  Oncogenic alterations in EGFR frequently co-occur with additional genetic alterations in EGFR-driven lung adenocarcinoma (LUAD), but how specific combinations of mutations affect tumor phenotypes and responses to targeted therapy is yet unknown. We leveraged a genetically engineered mouse model of EGFR mutant/Trp53-deficient LUAD to study the consequences of inactivating 10 different tumor suppressor genes on the fitness and tyorosine kinase inhibitor (TKI) sensitivity of these tumors. We found that loss of Keap1 is associated with a reduced response to therapy. In patients, we found that mutations in the KEAP1/NFE2L2/CUL3 pathway are associated with a significantly shorter time to treatment failure for EGFR TKI therapy compared to matched patients with wild-type KEAP1/NFE2L2/CUL3 tumors. We also analyzed whole exome sequencing data from tumor specimens before TKI treatment and at the time of treatment resistance for mutations in the KEAP1 pathway and these data are being submitted here.  

  Study phs002334

• Genetic Study of Inflammatory Bowel Disease (IBD) in African Americans

  Association studies have previously identified 200 genome-wide significant (GWS) IBD susceptibility loci in European ancestry populations. At least thirty-five loci have been identified in Asians and a handful appear Asian specific. AAs have a higher risk for developing disease complications in IBD and worse disease outcome, suggesting that significant differences in pathological and molecular mechanisms may exist in AAs. It was therefore imperative to use more comprehensive genotyping platforms in more highly powered samples to detect AA-specific IBD loci and associations. We conducted two independent GWAS using a total of 2345 AA IBD cases and 5002 controls population from unrelated, self-identified AAs individuals. GWAS1 included 1258 IBD cases (843 CD, 368 UC, 47 IBD-U) and 1678 controls derived from the dbGaP Health and Retirement Study (phs000428). GWAS 2 included 1087 IBD cases (803 CD, 215 UC, 69 IBD-U) and 3324 controls obtained from the Kaiser RPGEH study (phs000788).

  Study phs001571

• Differential Transcription Start Site Usage in Brain-related Samples

  RNA-Seq is an effective method to study the transcriptome, but specialized methods are required to identify 5' ends of transcripts. Several published strategies exist for this specific purpose, but their relative merits have not been systematically analyzed. Here, we directly compare the performance of six such methods - testing five with cellular RNA as well as a novel spike-in RNA assay that helps address interpretation challenges that arise from uncertainties in annotation or RNA processing. Using a single human RNA sample, we constructed and sequenced 18 libraries with these methods and one standard, control RNA-Seq library. We find that the CAGE method performed best for mRNA and that most of its unannotated peaks are supported by evidence from other genomic methods. We then applied CAGE to eight brain-related samples and revealed sample-specific transcription start site (TSS) usage as well as a transcriptome-wide shift in TSS usage between fetal and adult brain.

  Study phs001463

• The Finland-United States Investigation of NIDDM Genetics (FUSION) Study

  The Finland-United States Investigation of NIDDM Genetics (FUSION) study is a long-term effort to identify genetic variants that predispose to type 2 diabetes (T2D) or that impact the variability of T2D-related quantitative traits. The initial effort involved linkage analysis of affected-sibling-pair (ASP) families based on over 5,000 individuals living in Finland, and association fine mapping based on these family members and additional T2D cases and controls. We completed a genome-wide association scan on 1161 T2D cases and 1174 normal glucose tolerant (NGT) controls. Individual-level data is available for the 919 T2D cases and 787 NGT controls who reconsented to the use of their data or are deceased (phs000100). In addition, we selected these 919 T2D cases and a matched set of 919 NGT controls (774 overlapping with GWAS) for targeted sequencing of 78 genes associated with glucose, insulin, and/or lipids. 400 of these T2D cases were also chosen for whole-exome sequencing (phs000702).

  Study phs000867

• Fine Mapping of Eight Psoriasis Susceptibility Loci

  Psoriasis is a common immune-mediate disease identified through scaly patches of the skin. In this study we collected genotype information on 5,067 samples of European ancestry as a follow-up study of our initial GWAS (Nair et al. 2009). We designed an Illumina iSelect custom genotyping array with 2,269 tagging SNPs in the eight known psoriasis susceptibility regions (MHC, IL12B, IL23R, IL23A, TNFAIP3, IL13, RNF114, and TNIP1). The custom array also included 5,463 SNPs outside the eight known susceptibility regions, which were selected based on their association p-values in a meta-analysis of HapMap-imputed genotypes of two published GWAS-CASP (Nair et al. 2009) and (Ellinghaus et al. 2010). After quality control we analyzed 4,806 samples (2,699 cases and 2,107 controls). Please note: The accession number for this dbGaP study was incorrectly listed in the relevant publication (Das et al., 2015, Eur. J. Hum. Genet 23:844-853) as phs000019.v1.p1.

  Study phs001298

• Neuropsychiatric Genetics of African Populations - Psychosis (NeuroGAP-Psychosis)

  The NeuroGAP-Psychosis study was conducted in South Africa, Kenya, Uganda and Ethiopia as a response to the lack of genetic diversity in large genomic studies of psychiatric disorders. The study aims to expand knowledge of the genetic and environmental risk factors for neuropsychiatric disorders in Africa through large-scale sample collection and analysis. The initial design is a case-control study in which cases are individuals with schizophrenia or bipolar disorder and controls are age, sex and ancestry matched with individuals from the same geographic location. Cases are patients who are recruited from clinical facilities, both inpatient and outpatient. Controls are people who present for treatment for general medical conditions at health facilities. Data on phenotype, mental disorders, history of physical health problems, substance use and history of past traumatic events was collected from all participants. Additionally, a sample of saliva is collected for DNA extraction. DNA samples were sent to the Broad Institute for genotyping using Illumina Infinium Global Screening Array.  

  Study phs002528

• NHLBI TOPMed: Walk-PHaSST Sickle Cell Disease (SCD)

  Walk-PHaSST study is a multi-center clinical trial to evaluate the effects of sildenafil on Sickle Cell Disease (SCD) population with low exercise capacity associated with an increased Doppler-estimated pulmonary artery systolic pressure (as assessed by the tricuspid regurgitant velocity (TRV)). It is a double-blind, placebo-controlled trial to assess the safety, tolerability, and efficacy of sildenafil in patients with SCD who had both an elevated TRV and decreased exercise capacity. The screening phase of the study enrolled 720 subjects and 74 of them were randomized for the clinical trial. For the screening cohort, we have collected general demographics and race/ethnicity, hemoglobin genotype, physical examination, laboratory screening, transthoracic Doppler echocardiography, and 6 minute walk test in 9 US and 1 UK site. The walk-PHaSST biorepository have banked samples from 610 of the screening subjects. A total of 592 subjects from the screening phase are included in the TOPMed program for WGS.

  Study phs001514

• Genomic Studies for Understanding Etiology of Esophageal Adenocarcinoma (EsophagealAdenocarcinoma_Chinese)

  While the incidence of esophageal adenocarcinoma (EAC) has risen drastically in Western countries over the last 40 years, a similar trend has not been observed for EAC in China. Here we analyzed mutational spectrum, copy number alterations, and structural variants from whole-genome sequencing of ten Chinese EAC tumor samples and their matched normals, and compared them to EAC tumor specimens from Western countries previously reported. The mutational burden in Chinese EAC was significantly lower than that found in EAC from Western countries. The hallmark A>C mutational signature observed at high frequency in EAC from Western countries, which linked to acid reflux, is completely absent in Chinese samples. Furthermore, none of Chinese samples showed evidence of chromothripsis and genome doubling that often found in EAC from Western countries. In summary, Chinese EAC tumor samples revealed distinct genomic profiles and signatures, suggesting that Chinese EAC may arise from a different etiological pathway.

  Study phs001696

• Predictive impact of rare genomic copy number variations in siblings of individuals with autism spectrum disorders

  Identification of genetic biomarkers associated with autism spectrum disorders (ASDs) could improve recurrence prediction for families with a child with ASD. Here, we describe clinical microarray findings for 253 longitudinally phenotyped ASD families from the Baby Siblings Research Consortium (BSRC), encompassing 288 infant siblings. By age 3, 103 siblings (35.8%) were diagnosed with ASD and 54 (18.8%) were developing atypically. Thirteen siblings have copy number variants (CNVs) involving ASD-relevant genes: 6 with ASD, 5 atypically developing, and 2 typically developing. Within these families, an ASD-related CNV in a sibling has a positive predictive value (PPV) for ASD or atypical development of 0.83; the Simons Simplex Collection of ASD families shows similar PPVs. Polygenic risk analyses suggest that common genetic variants may also contribute to ASD. CNV findings would have been pre-symptomatically predictive of ASD or atypical development in 11 (7%) of the 157 BSRC siblings who were eventually diagnosed clinically.

  Study phs001876

• Total exRNA Profiles from Plasma, Saliva, and Urine of Healthy Subjects

  We sequenced small RNAs from 183 plasma samples, 204 urine samples and 46 saliva samples from 55 college athletes ages 18-25 years. Many of the participants provided more than one sample, weeks or months apart, allowing us to assess variability in an individual's exRNA expression levels over time. Several individuals provided all three biofluid types at one time, producing data on individual expression levels across several biofluid types. Here we provide a systematic analysis of small exRNAs present in each biofluid, as well as an analysis of exogenous RNAs. We find that a large number of RNA fragments in plasma (63%) and urine (54%) have sequences that are assigned to YRNA and tRNA fragments respectively. Surprisingly, while many miRNAs can be detected, there are few miRNAs that are consistently detected in all samples from a single biofluid. Additionally, we performed whole transcriptome sequencing on 134 plasma and 115 urine samples and identified circRNA.

  Study phs001258

• Intervention with Micronutrients and Long-Term Impact in Brazil-RECODISA

  Intervention with Micronutrients and Long-Term Impact in Brazil, a partnership between the Federal University of Ceará, Brazil and the University of Virginia, USA, enrolled infants in the age group of 2-36 months to investigate trends in gastrointestinal infections and their effects on growth and development and to identify genetic factors contributing to these diseases.A total of 1044 children from six urban communities in Brazil's semiarid region, were enrolled during active surveillance; those with diarrhea (three or more liquid stools in the last 24 hours at enrollment) were defined as a cases and matched with controls having no diarrhea during the past two weeks (at enrollment) to evaluate their growth and development. A subset of 664 children with their genetic and associated phenotypic data is included in this submission. More information on this project and its related projects can be found here: Studies of diarrheal susceptibility, growth, and development, in pediatric populations in Northeastern Brazil

  Study phs003174

• National Eye Institute (NEI) Genetic Epidemiology of Age-Related Macular Degeneration in the Old Order Amish

  Identifying genetic risk loci for Age-Related Macular Degeneration is important. Most of the loci identified to date have been found through the interrogation of common variants. This study initiates the identification of rare variants in a founder population, the Amish, and will use genotyping of known risk loci and ultimately use the whole exome chip to assess the association of Age-related Macular Degeneration with coding variants. We will further refine the Age-Related Macular Degeneration phenotype through the use of modern imaging, the OCT, to visualize the early anatomic signs of Age-Related Macular Degeneration. We hypothesize there are endophenotypes associated with specific genotypes that can be used to determine Age-related Macular Degeneration progression. These endophenotypes are hypothesized to be influenced by a combination of common and rare variants. We will relate these endophenotypes to genotypes to define the role of genetics in the progression of Age-Related Macular Degeneration and a new risk profile incorporating genotypic information.

  Study phs001361

• Idiopathic Multicentric Castleman Disease Whole Genome Sequencing Project

  Multicentric Castleman disease (MCD) is a rare lymphoproliferative disorder driven by excessive production of inflammatory cytokines. Patients can exhibit a wide range of clinical features from mild flu-like symptoms to multiple organ failure and death. Human herpesvirus-8 (HHV-8) is a well-established cause of MCD in individuals who are immunosuppressed due to human immunodeficiency virus infection or other causes. On the other hand, it is not known what causes MCD in otherwise healthy individuals with no evidence of exposure to HHV-8 (idiopathic MCD or iMCD). Although iMCD is most common in 40-60 years old adults, it affects people of all ages and early-onset (childhood or young adulthood) cases have been reported. Germline mutations may underlie iMCD in such cases. Patients with early-onset iMCD and their family members were enrolled in the study. To identify germline mutations that may underlie iMCD, whole-genome sequencing was performed in patients and their biological parents from whom DNA samples were available.

  Study phs001706

• Single-Cell Analysis of Human Adipogenesis

  The data in this study consists of RNA sequencing reads and read counts from single-cell and single-nuclei RNA sequencing experiments performed on two cell lines to investigate the transcriptional landscape of human adipogenesis in both the brown and white adipose tissue lineage. The two cell lines are human preadipocytes collected and isolated from a defined anatomical location in the neck of a single individual. This depot is known to harbor white adipose tissue (WAT) and brown adipose tissue (BAT). These white and brown preadipocytes were immortalized for the purposes of studying human adipogenesis in vitro. In this study we induced differentiation of these two preadipocyte lines and performed RNA sequencing on over 135,000 single cells spanning a 20-day time course from unperturbed preadipocytes to mature adipocytes from both brown and white lineages. Additionally, we performed both single-cell and single-nuclei RNA sequencing to comprehensively compare the advantages and disadvantages of either technique.

  Study phs002461

• Discovery of Novel Melanoma Predisposing Mutations by Exome Sequencing

  Melanoma risk shows clear heritability, but the inherited genetic factors remain largely unknown. Rare high risk mutations in CDKN2A, CDK4, BAP1 and TERT have been described but these variants, in aggregate, account for less than 50% of the kindreds reporting a family history of melanoma. This project will seek to identify new genes which may confer melanoma susceptibility. Identifying melanoma predisposing mutations can provide new biological insights and may impact important clinical decision making regarding treatment, surveillance and preventive approaches. Hereditary melanoma patients (defined as those individuals with multiple primary melanomas and those patients with a family history of melanoma) were collected from melanoma clinics at the Massachusetts General Hospital in Boston and the A. Sygrous Hospital in Athens, Greece. All exome sequencing was performed at the Broad Institute of Harvard and MIT; samples sequence capture was performed using Agilent SureSelect Human All Exon Kit v2 and sequencing was performed on an Illumina HiSeq 2000 or 2500.

  Study phs000823

• Whole Genome, Exome, Transcriptome and Validation Sequencing of an Acute Lymphoblastic Leukemia

  We used massively parallel sequencing technology to profile the genomic DNA and RNA of tumor cells (leukemic bone marrow) and normal cells (skin biopsy) obtained from a single patient with Acute Lymphoblastic Leukemia (ALL), referred to throughout this study as 'ALL1'. Included in this study are samples obtained from a primary tumor, first relapse, second relapse and several intermediate timepoints. We identified somatic mutations present in each tumor by analysis of whole genome (WGS) and exome sequence data. Single nucleotide variants (SNVs) and small insertions and deletions were identified in both the exome and WGS data. Large copy number variations (CNVs) and structural variants (SVs) were identified in the WGS data. A custom capture reagent was designed to target most variants and used to generate deep validation sequence data. The expression status of all somatic variants was assessed by RNA-seq. The RNA-seq data was also used for gene expression analysis and gene fusion detection.

  Study phs001066

• Transcriptome study of differential expression in schizophrenia

  Schizophrenia is a common and severe psychotic disorder. While some common SNPs and rare copy number variants have been identified as being significantly associated with disease risk, the biological mechanisms remain undefined. To identify gene expression abnormalities in schizophrenia, we generated whole-genome gene expression profiles using microarrays on lymphoblastoid cell lines from a total of 413 cases and 446 controls. Regression analysis identified 95 transcripts differentially expressed by affection status at a genome-wide false discovery rate of 0.05, while simultaneously controlling for confounding effects. These transcripts represented 89 genes with functions such as neurotransmission, gene regulation, cell cycle progression, differentiation, apoptosis, and immunity. The observed differential expression of extended major histocompatibility complex region genes converges with the genetic evidence from schizophrenia genome-wide association studies, which find the same region to be the most significant schizophrenia susceptibility locus. Our analysis also provides novel candidate genes for further study to assess their potential contribution to schizophrenia.

  Study phs000775

• Rapid Acceleration of Diagnostics - Digital Health Technologies (RADx-DHT): NIH Digital Health Solutions for COVID-19: Team SAE

  The goal of this project is to develop a smartphone-based platform to monitor and support individuals with COVID-19 symptoms (who may need testing) and those who have already tested positive. The app will integrate a Bluetooth-enabled thermometer and pulse oximeter into an approach uniquely designed for low-resource settings and underserved populations. This project will focus on the American Indian/Alaskan Native (AI/AN) underserved population and will determine the feasibility of the digital health solution to collect symptom survey data and vital data including temperature, pulse, and oxygen saturation level. The complete study will include 300 participants 100% medically underserved with at least 51% from the AI/AN community. A combination of hard and soft data will be collected consisting of vital data, medical history and questionnaire data, and participant COVID-19 viral and antibody testing results collected during the study.DOI: https://rapids.ll.mit.edu/10.57895/wv88-by98

  Study phs002534

• Human Liver Cohort (HLC)

  The Human Liver Cohort (HLC) study aimed to characterize the genetic architecture of gene expression in human liver using genotyping, gene expression profiling, and enzyme activity measurements of Cytochrom P450. The HLC was assembled from a total of 780 liver samples screened. DNA samples were genotyped on the Affymetrix 500K SNP and Illumina 650Y SNP genotyping arrays. Only data from those samples which were collected postmortem are accessible through this submission. These 228 samples represent a subset of the 427 samples included in the Human Liver Cohort Publication (Schadt, Molony et al. 2008, PMID: 18462017). RNA samples were profiled on a custom Agilent 44,000 feature microarray composed of 39,280 oligonucleotide probes targeting transcripts representing 34,266 known and predicted genes, including high-confidence, noncoding RNA sequences. Gene Expression data for the samples from which it could be obtained is available in GEO under accession number GSE9588. Results and networks will be made available for download from Sage Bionetworks.

  Study phs000253

• Epigenomics of Patient Outcomes after Aneurysmal SAH

  This project was focused on understanding the pathophysiology following aneurysmal subarachnoid hemorrhage (aSAH). DNA methylation is a key mechanism for regulation of gene expression and function in the adult brain and appears to play an important role in neuroprotection during ischemic events in the brain. Delayed cerebral ischemia (DCI), occurring during the acute phase following aSAH, is a major contributor to the complications that later develop; however, the pathophysiology of DCI and the development of complications is not known. We hypothesize that characterizing the methylome representing the CNS environment post aSAH will clarify the pathophysiology associated with DCI and patient outcomes. The aims of this project focused on the daily genome-wide methylomic changes that occur in DNA representing the CNS for the first 14 days after aSAH, as well as in peripheral blood in a subset of partcipants, and determining if this methylomic data impacts DCI and the development of complications after aSAH.

  Study phs001990

• BarcUVa-Seq (Biology of Colorectal Cancer Risk Enhancers)

  This study was designed to generate a cohort of normal colon mucosal biopsies from the "average risk" population. Its primary aims included the development of an RNA-seq dataset of normal colon mucosal biopsies across three colon regions (ascending, transverse and descending). Further, we aimed to provide plausible prioritization to genetic components of colorectal cancer risk by employing methods such as expression quantitative trait loci analysis. In doing so, we provide a novel reference dataset for the study of colon cancer risk in the healthy colon epithelium. RNA-seq was performed in batches on an Illumina HiSeq 2500 (batch 1-7) or NovaSeq 6000 (batch 8) on a total of 463 samples. Matched genotyping data was generated on the Illumina OncoArray BeadChip. Phasing was carried out using Minimac 3 and imputation was performed using The Haplotype Reference Consortium panel on the Michigan Imputation Server.Genotype and RNA-seq data will be made available in dbGaP.

  Study phs003338

• Spatial Transcriptomics Reveals Discrete Tumor Microenvironments and Autocrine Loops Within Ovarian Cancer Subclones

  Ovarian masses from patients with high grade serous ovarian carcinoma undergoing debulking surgery were subjected to spatial transcriptomics profiling using the 10x Genomics Visium (fresh frozen) platform. Copy number inference using inferCNV predicted the presence of subclones with mutually exclusive copy number alterations (CNAs) in 5 of the 8 patient samples studied. In one patient, 3 subclones were predicted. For this patient sample, a serial section of the same tumor was used to microdissect regions corresponding to each of these subclones, and a 'normal' region predicted to contain only fibroblasts and no tumor cells. These microdissected regions were processed for low pass whole genome sequencing (WGS) and CNAs recorded using ichorCNA. An oral biopsy lacking tumor cells from an unrelated donor was used as a negative control for CNA inference. The CNAs predicted by inferCNV for the three subclones were validated by the low pass WGS and no CNAs were observed in the normal region containing fibroblasts.

  Study phs003561

• Structure of the Mucosal and Stool Microbiome in Lynch Syndrome

  The gut microbiota has been associated with colorectal cancer (CRC), but causal alterations preceding CRC have not been elucidated. To prospectively assess microbiome changes prior to colorectal neoplasia, we investigated samples from 100 Lynch syndrome patients using 16S rRNA gene sequencing of colon biopsies, coupled with metagenomic and metatranscriptomic sequencing of feces. Colectomy and CRC history represented the largest effects on microbiome profiles. A subset of Clostridiaceae were depleted in stool corresponding with baseline adenomas, while Desulfovibrio was enriched in both stool and mucosal biopsies. A classifier leveraging stool metatranscriptomes resulted in modest power to predict interval development of preneoplastic colonic adenoma. Predictive transcripts corresponded with a shift in flagellin contributors and oxidative metabolic microenvironment, potentially factors in local CRC pathogenesis. This suggests that the effectiveness of prospective microbiome monitoring for adenomas may be limited but supports the potential causality of these consistent, early microbial changes in colonic neoplasia.

  Study phs002343

• NHLBI TOPMed: MWCCS: Sex Differences in the Role of Multi-Omics in HIV-Associated Carotid Artery Atherosclerosis

  The MACS-WIHS Combined Cohort Study (MWCCS) is a multicenter prospective cohort study of men and women in the U.S. that aims to understand and reduce the impact of chronic health conditions, including heart, lung, blood, and sleep (HLBS) disorders that affect people living with HIV. MWCCS, which currently has over 5000 active study participants, is designed to investigate a spectrum of questions relating to the basic science, clinical science, and epidemiology of HIV infection in the U.S., with a focus on comorbidities among men and women living with HIV. The current substudy, nested within the MWCCS, integrates detailed metabolomics and proteomics data, among other "omics" measures, to improve the understanding of the etiology of atherosclerosis and cardiovascular disease in women and men with HIV, guide mechanistic studies, and provide insights to more efficient intervention strategies. Data on 374 men are included in the current substudy.

  Study phs003651

• Spatially resolved niche and tumor microenvironmental alterations in gastric cancer peritoneal metastases

  Peritoneal metastases (PM) in gastric cancer (GC) portend a poor prognosis, yet our understanding of tumor microenvironmental (TME) characteristics associated with GCPM remain limited. Here, we analyzed intrinsic genomic alterations and transcriptomic programs predictive of GCPM in a prospective cohort of 248 patients, identifying CDH1, PIGR, and ELF3 mutations as predictors. By inspecting the spatial dynamics of the TME, we find that tumor compartment infiltration of pro-tumorigenic cell types such as inflammatory cancer-associated fibroblasts (CAFs) predict peritoneal recurrence. Next, in a cross-sectional study of 205 samples from 55 patients, distinct pathways and immune compositions in GCPM relative to liver metastases highlight the TME's significance in transcoelomic metastases. Notably, several putative therapeutic targets exhibited distinct expression patterns between PTs and PMs. Our findings highlight transcriptomic variations and niche reprogramming in the GCPM peritoneal environment, revealing roles of myeloid dendritic cells, effector memory CD8+ T cells, and CAFs in metastatic progression.

  Study EGAS50000000501

• Ultra-sensitive ctDNA monitoring required for predicting response and resistance to immunotherapy in advanced melanoma

  Circulating tumor DNA (ctDNA) monitoring in peripheral blood has shown promise for disease surveillance and prognostication of ICI response. Yet even in the metastatic context, ctDNA signals can be missed, suggesting the need for increased assay sensitivity. We used a tumor-informed ctDNA assay capable of detection down to 1 part-per-million (PPM) ctDNA to profile 188 plasma samples from 23 melanoma patients receiving ICI over several years. Residual disease was detected in ctDNA over a broad range, from 130,000 PPM down to 2.3 PPM, with approximately a quarter of detections below 50 PPM. Early on-treatment increases in ctDNA level, and both ctDNA-based molecular clearance (mCR) and molecular progression (mPD) were all prognostic of both OS and PFS. Additionally, mCR consistently preceded radiographic imaging. Our results show the importance and utility of an ultra-sensitive ctDNA assay in advanced melanoma treated by ICI with potential implications for other cancers.

  Study EGAS50000000550

• KDM6A Loss Triggers an Epigenetic Switch that Disrupts Urothelial Identity and Drives Cell Proliferation in Bladder Cancer

  Disruption of KDM6A, a histone lysine demethylase, is one of the most common somatic alternations in bladder cancer. How mutations of this chromatin modifier impact the epigenetic landscape to promote carcinogenesis is poorly understood. Here, we demonstrate that KDM6A loss triggers an epigenomic switch that disrupts urothelial identity and induces a neoplastic state characterized by increased cell proliferation. In bladder cancer cells with intact KDM6A, FOXA1 binds KDM6A at enhancers to activate genes instructing urothelial differentiation. In KDM6A-deficient cells, we observed simultaneous loss of FOXA1 target binding and genome-wide redistribution of the bZIP transcription factor ATF3, which in turn represses FOXA1 target genes and activates cell-cycle progression genes. Importantly, ATF3 depletion reverses the cell proliferation phenotype. These data establish that KDM6A loss creates an epigenetic state that drives tumor growth in an ATF3-dependent manner, forging a molecular vulnerability that can be targeted by novel therapeutic strategies.

  Study phs002801

• Single-cell RNA sequencing of human skin tumors (BCC, SCC and Melanoma)

  Cancer-associated fibroblasts (CAFs) play a key role in cancer progression and treatment outcome. Here, we elucidate the yet unresolved intra-tumoral CAF variety in three skin cancer types at molecular and spatial single-cell resolution in a large cohort. We show that two out of three CAF subtypes contribute to tumor immune surveillance with distinct mechanisms. Matrix CAFs (mCAFs) ensheath tumor nests and synthesize extracellular-matrix to prevent T cell invasion. Immunomodulatory CAFs (iCAFs), which express proinflammatory and immunomodulatory factors, are only detected in high abundance in aggressive tumors. Strikingly, iCAFs but not tumor cells are (apart from immune cells) the exclusive cell type producing chemokines and, thus, play a key role in immune cell recruitment and activation. Mechanistically, we show that cancer cells transform adjacent healthy fibroblasts into cytokine-expressing iCAFs, which subsequently recruit immune cells and modulate the immune response. In conclusion, targeting CAF variants holds promise for improved efficacy of immunotherapy.

  Study EGAS50000000365

• Role of Microglia Somatic Mutations in Neurodegenerative Diseases

  Somatic genetic heterogeneity resulting from post-zygotic DNA mutations is widespread in human tissues and can cause diseases, however few studies have investigated its role in neurodegenerative processes such as Alzheimer's Disease (AD). Here we report the selective enrichment of microglia clones carrying pathogenic variants, that are not present in neuronal, glia/stromal cells, or blood, from patients with AD in comparison to age-matched controls. Notably, microglia-specific AD-associated variants preferentially target the MAPK pathway, including recurrent CBL ring-domain mutations. These variants activate ERK and drive a microglia transcriptional program characterized by a strong neuro-inflammatory response, both in vitro and in patients. Although the natural history of AD-associated microglial clones is difficult to establish in human, microglial expression of a MAPK pathway activating variant was previously shown to cause neurodegeneration in mice, suggesting that AD-associated neuroinflammatory microglial clones may contribute to the neurodegenerative process in patients.

  Study phs002213

• Genomic determinants of response and resistance to inotuzumab in B-ALL

  Inotuzumab Ozogamicin (InO) is an antibody-drug conjugate which delivers calicheamicin to CD22-expressing cells. In a retrospective cohort of InO treated B-cell acute lymphoblastic leukemia (B-ALL) patients, we sought to understand the genomic determinants of response to InO. Acquired CD22 mutations were observed in 11% (3/27) of post-InO relapsed samples. CD22 mutation types were diverse (frameshift, nonsense, missense and stoploss) and we identified multiple mechanisms of mutation induced CD22 escape, including protein truncation, protein destabilization, and epitope alteration. Accelerated acquisition of mutations by error-prone DNA damage repair could drive CD22 mutation and escape. Acquired loss-of-function mutations in TP53, ATM and CDKN2A were observed, suggesting compromise of the G1/S DNA damage checkpoint as a mechanism of evading InO-induced apoptosis. In conclusion, genetic alterations modulating CD22 expression and DNA damage response influence InO efficacy. The diverse mechanisms of CD22 escape advocates for a refined approach for monitoring CD22-targeted therapy.

  Study EGAS50000000067

• Genome analysis of common diseases among older Japanese adults and development of clinical genome information storage

  A comprehensive gene expression analysis of the process leading up to the onset of Alzheimer???s disease (AD) would be helpful for understanding the mechanism. We performed an RNA sequencing analysis on a cohort of 1227 Japanese blood samples, representing 424 AD patients, 543 individuals with mild cognitive impairment (MCI), and 260 cognitively normal (CN) individuals. A total of 883 and 1169 statistically significant differentially expressed genes (DEGs) were identified between CN and MCI (CN-MCI) and between MCI and AD (MCI-AD), respectively. Pathway analyses using these DEGs, followed by protein???protein interaction network analysis, revealed key roles of ribosomal genes (RPL7, RPL11, and RPL14) and phagosomes (CDC42, PTPRC, PLCG1, and ACTR2) in MCI progression, whereas immune-related genes were involved in AD progression. Given the known effectiveness of delaying MCI progression in preventing AD, the genes related to ribosomal function and phagocytosis might emerge as biomarkers for early diagnosis.

  Study JGAS000755

• Deciphering craniopharyngioma subtypes: Single-cell analysis of tumor microenvironment and immune networks

  Craniopharyngiomas, including both adamantinomatous (ACP) and squamous papillary (PCP) types, present significant challenges in treatment due to their proximity to critical pituitary structures. In this study, we conducted a single-cell RNA analysis to examine the tumor microenvironments (TME) of ACP and PCP. Single-cell clustering revealed a wide range of cell types, which were further categorized based on gene expression patterns. For ACP, these categories included developing epithelia, calcification, and immune response, while PCP showed developing epithelia, cell cycle, and immune response as prominent categories. Subclustering analysis unveiled different types of immune cells in both ACP and PCP TMEs, with specific patterns of immune responses. Interestingly, smaller tumors were associated with particular immune cell ratios. Furthermore, we identified specific cell-to-cell interactions within the tumor cells. In conclusion, our study has led to the development of a comprehensive cell atlas, shedding light on the molecular characteristics and intricate interplay of immune cells within the TMEs of ACP and PCP.

  Study JGAS000722

• INCLUDE: Down Syndrome Early Post-Natal Brain Multiome

  Neurodevelopmental deficiencies and neuronal dysfunction contribute to cognitive impairment in individuals with Down syndrome, but what molecular pathways underlie the defects that lead to intellectual disability remain unclear. Human brain development is precisely choreographed through tightly regulated transcriptional programs. Proper regulation of these transcriptional programs is necessary for developing the numerous functionally distinct regions, cell types, and connections of the brain, and forming the basis for human-specific social, cognitive, and motor abilities. These data establish a reference of molecular features of the DS developing brain. Such foundational information will provide not only a reference for any existing and future models, but also a basis for understanding disrupted neural development and impaired cognition in DS.Dataset 1 contains single-nucleus RNAseq and ATACseq from frozen postmortem human basal forebrain tissue. Dataset 2 contains single-nucleus RNAseq and ATACseq from frozen postmortem human dorsolateral prefrontal tissue. Dataset 3 contains single-nucleus RNAseq and ATACseq from frozen postmortem human caudate nucleus tissue.

  Study phs004098

• Single Cell Transcriptional Analysis of Human Adenoids Identifies Molecular Features of Airway Microfold Cells

  We aimed to characterize the cellular composition and molecular features of the human adenoid, a mucosa-associated lymphoid tissue located in the upper airway, with a focus on epithelial populations involved in immune surveillance. To this end, we performed single-nucleus RNA sequencing (snRNA-seq) on adenoid tissue obtained from 6 human donors (age 2 – 14; 3 males and 3 females) undergoing routine adenoidectomy. The donor cohort includes clinically annotated samples with no known immunodeficiency or chronic airway disease.Our analysis uncovered distinct epithelial populations, including transcriptionally defined immature and mature microfold (M) cells, and a previously unrecognized interferon-stimulated epithelial population enriched for antiviral response genes, which we term WISP cells (Waldeyer's ring Interferon-Stimulated Population). We validated key transcriptomic findings through multiplex immunofluorescence microscopy in independent donor tissues, confirming the spatial localization of WISP and M cells and their predicted interactions with specific immune subsets.

  Study phs004103

• CLL Genome

  The Chronic Lymphocytic Leukemia (CLL) Genome Project aims to identify genetic alterations involved in the development and progression of the CLL, which are still unknown, with the objective of generating a comprehensive catalogue of genetic alterations in 500 independent tumours. The CLL Genome Project, as a contributing member of the International Cancer Genome Consortium (ICGC), has the purposes of creating diagnostic tools, discovering therapeutic targets and developing new strategies that will allow a customized therapy for CLL in order to make it more precise and effective. This study consists of two datasets. EGAD00001000023 described in the Nature paper (2011), Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia, and the dataset EGA00001000044 & & EGAD00001000083 described in the Nature (2011) paper, Exome sequencing identifies recurrent mutations of the splicing factor SF3B1 gene in chronic lymphocytic leukemia.Additional data generated within the ICGC-CLL genome project can be found in studies EGAS00001000374 and EGAS00001001306

  Study EGAS00000000092

• Variant allele frequency changes in TP53 predict pembrolizumab response in patients with metastatic urothelial carcinoma

  Prognoses for patients with metastatic urothelial carcinoma (mUC) have strikingly improved with pembrolizumab, an immune checkpoint inhibitor (ICI), but clinical benefits are limited to a subset of patients. Therefore, a non-invasive biomarker to predict pembrolizumab response is urgently required. We retrospectively examined genomic alterations in 25 plasma circulating tumor DNA (ctDNA) samples using a targeted sequencing of 77 genes from 16 mUC patients during pembrolizumab treatment. Eleven (68.8%) patients had two or more genomic alterations, including TP53 mutations (as defined by ctDNA-positive status). The proportion of responders to pembrolizumab in the ctDNA-positive group was higher than the ctDNA-negative group (72.7% vs. 20.0%). Furthermore, among all detected genomic alterations, variant allele frequency decreases in TP53 during pembrolizumab treatment were mainly associated with therapeutic response. Collectively, we suggest that profiling of ctDNA in plasma, especially TP53, is useful for predicting and monitoring therapeutic responses to pembrolizumab in mUC patients.

  Study JGAS000622

• Comprehensive assay for the molecular profiling of cancer by target enrichment from formalin-fixed paraffin-embedded specimens

  Tumor molecular profiling is becoming a standard of care for patients with cancer, but the optimal platform for cancer sequencing remains undetermined. We established a comprehensive assay, the Todai OncoPanel (TOP), which consists of DNA and RNA hybridization capture-based next-generation sequencing panels. A novel method for target enrichment, named the junction capture method, was developed for the RNA panel to accurately and cost-effectively detect 365 fusion genes as well as aberrantly spliced transcripts. The TOP RNA panel can also measure the expression profiles of an additional 109 genes. The TOP DNA panel was developed to detect single nucleotide variants and insertions/deletions for 464 genes, to calculate tumor mutation burden and microsatellite instability status, and to infer chromosomal copy number. Clinically relevant somatic mutations were identified in 32.2% (59/183) of patients by prospective TOP testing, signifying the clinical utility of TOP for providing personalized medicine to cancer patients.

  Study JGAS000164

• Transcriptome alterations underlying metabolic dysfunction and liver disease in myotonic dystrophy type 1

  Myotonic dystrophy type 1 (DM1) is a multisystem disorder affecting various organs, including the skeletal muscles, eyes, heart, central nervous system, and endocrine system. Liver dysfunction and lipid metabolism abnormalities are frequently observed in DM1 but are not fully understood. To explore the molecular basis of these abnormalities, transcriptome analysis was performed on postmortem liver samples from DM1 patients DM1 patients (n = 11) and disease control patients (n = 9) using bulkRNA sequencing (RNA-seq) on the MGI DNBSEQ-G400RS. Differential gene expression and alternative splicing analyses revealed significant transcriptomic changes associated with lipid metabolism, liver fibrosis, and glucose homeostasis. Interestingly, transcriptome changes in DM1 livers also revealed partial resolution of sexual dimorphism in gene expression, along with unique sex-specific pathway alterations. A correlation between serum GGT levels and splicing abnormalities in DM1 patients was identified, suggesting a link between transcriptomic changes and clinical outcomes. This dataset provides valuable insights into the molecular mechanisms underlying DM1-related liver dysfunction and metabolic abnormalities.

  Study JGAS000814

• TONIC-Trial-cfDNA-Project

  Cell-free DNA (cfDNA) is an emerging technology to predict and monitor response to cancer treatment, including immune checkpoint blockade (ICB). However, data on cfDNA dynamics during ICB in metastatic triple negative breast cancer (mTNBC) are limited. While most applications of cfDNA involve assays that focus on mutation detection, mTNBC and multiple other cancer types are driven by copy number alterations (CNAs). We evaluate cfDNA-based copy number profile abnormality (CPA) score as a potential biomarker for monitoring ICB response in mTNBC, analyzing data from 87 patients enrolled in stage 1 and stage 2 of the TONIC-trial. We find significant concordance between cfDNA-based and tissue-based CNA profiles. Additionally, responders show a decrease in CPA scores upon ICB at week 6 (3 cycles of nivolumab). These findings underscore the potential of cfDNA-based CNA dynamics as a non-invasive biomarker for ICB early response assessment in patients with mTNBC.

  Study EGAS50000001308

• Recurrent COPA mutation drives R-spondin-independent Wnt activation in intestinal tumors

  The majority of intestinal tumors harbor mutations in canonical Wnt pathway genes such as APC, whereas the lack of such alterations in a subset of tumors implies alternative tumorigenic routes. Here, we identify recurrent in-frame deletion in COPA, frequently co-occurring with USP9X truncating mutation, in small intestinal adenoma and adenocarcinoma. Patient-derived and CRISPR-engineered small intestinal organoids carrying COPA in-frame deletions exhibit R-spondin-independent yet Wnt ligand-dependent growth, maintaining LGR5 expression without canonical Wnt drivers. Mechanistically, COPA mutation stabilizes frizzled co-receptor LRP6 irrespective of R-spondin, sustaining Wnt pathway activation under growth factor-restricted conditions. USP9X loss further potentiates this phenotype. Unlike canonical Wnt pathway members, COPA encodes the alpha-subunit of coatomer complex I, which engages in vesicle trafficking with little prior linkage to intestinal tumorigenesis. Our findings establish COPA mutation as a novel and atypical intestinal tumor driver and implicate USP9X loss as a cooperating lesion.

  Study JGAS000868

• Delineation of the heterogeneous molecular landscape of HRS cells and their biological contributions to forming comprehensive TME ecosystems.

  The tissue architecture of classic Hodgkin Lymphoma (CHL) is unique among cancers and characterized by rare malignant Hodgkin and Reed-Sternberg cells that are co-evolving in a complex ecosystem of immune cells in the tumor microenvironment. The lack of a comprehensive systems-level interrogation has hindered the description of disease heterogeneity and clinically relevant molecular subtypes. Here, we employed an integrative, multimodal approach to characterize CHL tumors using malignant cell sequencing, spatial transcriptomics and imaging mass cytometry. We identified four molecular subtypes (CST, CN913, STB, and CN2P) each characterized by distinct clinical features, mutational patterns, malignant cell gene expression profiles, and spatial architecture of immune cell populations. Functional modeling of CSF2RB mutations, a characteristic feature of CST, revealed dysregulated oncogenic signaling and changes in tumor microenvironment crosstalk. These findings highlight the significance of multi-dimensional profiling in elucidating patterns of molecular alterations that drive immune ecosystems and underlie therapeutically exploitable vulnerabilities.

  Study EGAS00001008064

• Measles oncolytic virus as an immunotherapy for recurrent/refractory pediatric medulloblastoma and atypical teratoid rhabdoid tumor

  Pediatric recurrent medulloblastoma (MB) and atypical teratoid rhabdoid tumor (ATRT) are largely incurable and warrant novel therapies. We investigated a) the safety of a measles virus variant, MV-NIS, in a pediatric phase 1 study and b) the mechanisms of MV-NIS and potential benefit of combination with immune checkpoint inhibition (ICI). Pediatric patients with recurrent MB or ATRT were treated with intratumoral injections for local recurrence or via lumbar puncture for disseminated recurrence. We evaluated local immune responses to MV-NIS with and without ICI via single-cell and bulk RNA sequencing in an intracranial, immunocompetent, syngeneic murine model. MV-NIS given intratumorally or via repeat intrathecal dosing was safe. MV-NIS prolonged survival in murine models but did not demonstrate additive benefit with ICI. No changes in tumor-infiltrating immune-cell composition or activation were observed in response to MV-NIS treatment; however, MV-NIS induced local expression of neutralizing antibodies, complement cascade, and phagocytosis-related genes.

  Study EGAS50000000811

• EATL-II STUDY

  Enteropathy-associated T-cell lymphoma (EATL), a rare and aggressive intestinal malignancy of intraepithelial T lymphocytes, comprises two disease variants (EATL-I and EATL-II) differing in clinical characteristics and pathological features. Here we report findings derived from whole exome sequencing of 15 EATL-II tumor-normal tissue pairs. The tumor suppressor gene SETD2 encoding a non-redundant H3K36-specific trimethyltransferase is altered in 14/15 cases (93%), mainly by loss-of-function mutations and/or loss of the corresponding locus (3p21.31). These alterations consistently correlate with defective H3K36 trimethylation. The JAK/STAT pathway comprises recurrent STAT5B (60%), JAK3 (46%) and SH2B3 (20%) mutations, including a STAT5B V712E activating variant. Additionally, frequent mutations in TP53, BRAF and KRAS are observed. Conversely, in EATL-I, no SETD2, STAT5B or JAK3 mutations are found, and H3K36 trimethylation is preserved. This study describes SETD2 inactivation as EATL-II molecular hallmark, supports EATL-I and -II being two distinct entities, and defines potential new targets for therapeutic intervention.

  Study EGAS00001001879

• Transcriptional and functional profiling defines human small intestinal macrophage subsets

  Macrophages (Mf) are instrumental in maintaining immune homeostasis in the intestine, yet studies on origin and heterogeneity of human intestinal Mf are scarce. Here, we identified four distinct Mf subpopulations in human small intestine (SI). Assessment of their turnover in duodenal transplants revealed that all Mf subsets were completely replaced over time; Mf1 and Mf2, phenotypically similar to peripheral blood monocytes (PBMo), were largely replaced within 3 weeks, whereas two subsets with features of mature Mf, Mf3 and Mf4, exhibited significantly slower replacement. Mf3 and Mf4 localized differently in SI; Mf3 formed a dense network in mucosal lamina propria, whereas Mf4 was enriched in submucosa. Transcriptomic analysis showed that all Mf subpopulations subsets were markedly distinct from PBMo and dendritic cells (DC). Compared to PBMo, Mf subpopulations showed reduced responsiveness to proinflammatory stimuli, but were proficient at endocytosis of particulate and soluble material. These data provide a comprehensive analysis of human SI Mf population and suggest a precursor-progeny relationship with PBMo.

  Study EGAS00001002093

• miR-200-regulated CXCL12β promotes fibroblast heterogeneity and immunosuppression in ovarian cancers

  High-grade serous epithelial ovarian cancer (HGSOC) has been recently subdivided into molecular subgroups, including the mesenchymal HGSOC associated with partial tumor-debulking surgery and poor patient survival. Consistent with stromal-related genes signatures defining mesenchymal HGSOC, we show here that stroma plays a key function in this HGSOC molecular subtype. We first highlight stromal heterogeneity in HGSOC by identifying 4 subsets of Carcinoma-Associated Fibroblast (CAF-S1-4). Mesenchymal HGSOC significantly accumulate the activated CAF-S1 subset, which exhibits immunosuppressive functions by promoting attraction, survival and activation of regulatory T-lymphocytes. The CXCL12β chemokine reliably characterizes mesenchymal HGSOC and specifically accumulates in the CAF-S1 subset through a regulation mediated by the miR-141/200a. CXCL12β promotes attraction of T-lymphocytes and is thus a key actor in CAF-S1-mediated immunosuppressive functions. In conclusion, we highlight here for the first time stromal heterogeneity and immunosuppression in mesenchymal HGSOC that could participate in the poor patient prognosis.

  Study EGAS00001002184

• Molecular Subtype-specific Biomarkers Improves Colorectal Cancer Prognostication

  Colorectal cancer (CRC) is characterized by major inter-tumor diversity that complicates the prediction of disease and treatment outcomes. Recent efforts help resolve this by sub-classification of CRC according to natural molecular subtypes, however, this strategy is not yet able to provide clinicians with improved tools for decision-making. We here present an extended framework for CRC stratification that specifically aims to improve patient prognostication. Using transcriptional profiles from 1,100 CRCs, including a novel set of >300 samples, we identify novel cancer cell and tumor archetypes and suggest the tumor microenvironment as a major prognostic determinant that can be influenced by the microbiome. Notably, our subtyping strategy allowed identification of novel archetype-specific prognostic biomarkers that provided information beyond and independent of UICC-TNM staging, MSI-status and consensus molecular subtyping. The results illustrate that our extended subtyping framework, combining subtyping and subtype-specific biomarkers, could contribute to improved patient prognostication and may form a strong basis for future studies.

  Study EGAS00001002376

• Uterine_Atlas_Endometriosis

  Uterine Atlas Endometriosis Endometriosis is a chronic inflammatory disease driven by oestrogen, affecting around 10% of women of child-bearing again world-wide. It is described by the presence of endometrium-like tissue outside of the uterus (ectopic endometriosis tissue) and debilitating chronic pain. There is a lack of understanding of endometriosis pathogenesis, as well as cellular composition of both the eutopic endometrium and ectopic endometriosis tissue of endometriosis patients.There are therefore two main aims for this study. Using single cell genomics and spatial methods we aim to: I) characterise endometrial cellular census in women with and without endometriosis II) characterise cellular census of ectopic endometriosis tissue Furthermore, we aim to establish endometrial organoids and endometriosis organoids from the samples collected and characterise these using single cell genomics and spatial methods. 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 EGAS00001004725

• Control iPSC lines with clinically annotated genetic variants for versatile multi-lineage differentiation

  Induced Pluripotent Stem Cells (iPSC) derived from healthy individuals are important controls for disease modeling studies. To create a resource of genetically annotated iPSC, we reprogrammed footprint-free lines from four volunteers in the Personal Genome Project Canada (PGPC). Multilineage directed differentiation efficiently produced functional cortical neurons, cardiomyocytes and hepatocytes. Pilot users further demonstrated line versatility by generating kidney organoids, T-cells and sensory neurons. A frameshift knockout was introduced into MYBPC3 and these cardiomyocytes exhibited a hypertrophic phenotype as expected. WGS annotation of PGPC lines revealed on average 20 coding variants. Importantly, nearly all annotated PGPC and HipSci lines harboured at least one pre-existing or acquired variant with cardiac, neurological or other disease associations. Overall, PGPC lines were efficiently differentiated by multiple users into cell types found in six tissues for disease modelling, and clinical annotation highlighted variant-preferred lines for use as unaffected controls in specific disease settings.

  Study EGAS00001003684

• Early evolutionary branching across spatial domains predisposes to clonal replacement under chemotherapy in neuroblastoma

  Neuroblastoma (NB) is one of the most lethal childhood cancers due its propensity to treatment resistance. By spatial mapping of subclone geographies before and after chemotherapy across 89 tumor regions from 12 NBs, we find that densely packed territories of closely related subclones present at diagnosis are replaced under effective treatment by islands of distantly related survivor subclones, originating from a different most recent ancestor compared to lineages dominating before treatment. Conversely, in tumors that progressed under treatment, ancestors of subclones dominating later in disease are present already at diagnosis. Chemotherapy treated xenografts and cell culture models replicates these two contrasting scenarios and shows branching evolution to be a constant feature of proliferating NB cells. Phylogenies based on whole genome sequencing of 505 individual NB cells indicate that a rich repertoire of parallel subclones, emerges already with the first oncogenic mutations and lays the foundation for clonal replacement under treatment.

  Study EGAS00001007650

• Whole genome sequencing of patients with or at risk for HCC

  Liver cancer is a major cause of cancer mortality worldwide. Screening individuals at high risk, including those with cirrhosis and viral hepatitis, provides an avenue for improved survival, but current screening methods are inadequate. In this study, we used whole-genome cell-free DNA (cfDNA) fragmentome analyses to evaluate 724 individuals from the United States, the European Union, or Hong Kong with hepatocellular carcinoma (HCC) or who were at average or high-risk for HCC. Using a machine learning model that incorporated multifeature fragmentome data, the sensitivity for detecting cancer was 88% in an average-risk population at 98% specificity and 85% among high-risk individuals at 80% specificity. We validated these results in an independent population. cfDNA fragmentation changes reflected genomic and chromatin changes in liver cancer, including from transcription factor binding sites. These findings provide a biological basis for changes in cfDNA fragmentation in patients with liver cancer and provide an accessible approach for noninvasive cancer detection.

  Study EGAS00001007249

• Coverage bias and sensitivity of variant calling for four whole-genome sequencing technologies

  Massively parallel sequencing has revolutionized research in cancer genetics and genomics and enhanced our understanding of natural human genetic variation. Recently, Lam et al. have performed a detailed comparison of two next-generation sequencing technologies with respect to their sensitivity to call single nucleotide variants (SNV) and indels. Here, we sequenced two tumor/normal pairs obtained from two paedriatic medulloblastoma patients with Life Technologies’ SOLiD 4 and 5500xl SOLiD, Illumina’s HiSeq2000, and Complete Genomics’ technology. We then compared their ability to call SNVs with high confidence. As gold standard for SNV calling, we used genotypes determined by an Affymetrix SNP array. Additionally, we performed a detailed analysis of how evenly each technology covers the genome and how the reads are distributed across functional genomic regions. Finally, we studied how a combination of data from different technologies might help to overcome the limitations in SNV calling by any of the four technologies alone.

  Study EGAS00001000274

• MGA-NUTM1 fusion in high grade spindle cell sarcoma

  NUT carcinoma (NC) with mesenchymal differentiation have rarely been described in the literature. In this report, we describe the characterization of two cases of high grade spindle cell sarcoma harboring a novel MGA-NUTM1 fusion. Whole genome sequencing identified the presence of complex rearrangements resulting in a MGA-NUTM1 fusion gene in the absence of other significant somatic mutations. The fusion protein was predicted to retain nearly the entire protein sequence of both MGA (exons 1-22) and NUTM1 (exons 3-8). In contrast to typical cases of NC, these cases were successfully treated with aggressive local control measures (surgery and radiation) and both patients remain alive without disease. These cases describe a new subtype of NUTM1-rearranged tumors warranting expansion of diagnostic testing to evaluate for the presence of MGA-NUTM1 or alternative NUTM1 gene fusions in the diagnostic workup of high grade spindle cell sarcomas or small round blue cell tumors of ambiguous lineage.

  Study EGAS00001003341

• Transcriptomic classes of BCR-ABL1 lymphoblastic leukemia

  In BCR-ABL1 lymphoblastic leukemia, treatment heterogeneity to tyrosine kinase inhibitors (TKIs), especially in the absence of kinase domain mutations in BCR-ABL1, is poorly understood. Through deep molecular profiling, we uncovered three transcriptomic subtypes of BCR-ABL1 lymphoblastic leukemia, each representing a maturation arrest at a stage of B-cell progenitor differentiation. An earlier arrest was associated with lineage promiscuity, treatment refractoriness, and poor patient outcomes. A later arrest was associated with lineage fidelity, deep and durable leukemia remissions, and improved patient outcomes. Each maturation arrest was marked by specific genomic events that control different transition points in B-cell development. Interestingly, these events were absent in BCR-ABL1+ pre-leukemic stem cells isolated from patients regardless of subtype, which supports that transcriptomic phenotypes are determined downstream of the leukemia-initialing event. Overall, our data indicate that treatment response and TKI-efficacy is an unexpected outcome of the differentiation stage at which this leukemia transforms.

  Study EGAS00001007167

• CD74-NRG1 fusions in lung adenocarcinoma

  We discovered a novel somatic gene fusion, CD74-NRG1, by transcriptome sequencing of 25 lung adenocarcinomas of never smokers. By screening 102 lung adenocarcinomas negative for known oncogenic alterations we found four additional fusion-positive tumors, all of which were of the invasive mucinous subtype. Mechanistically, CD74-NRG1 leads to extracellular expression of the EGF-like domain of NRG1 III-beta3, thereby providing the ligand for ERBB2-ERBB3 receptor complexes. Accordingly, ERBB2 and ERBB3 expression was high in the index case and expression of phospho-ERBB3 was specifically found in tumors bearing the fusion (p<0.0001). Ectopic expression of CD74-NRG1 in lung cancer cell lines expressing ERBB2 and ERBB3 activated ERBB3 and the PI3K-AKT pathway, and led to increased colony formation in soft agar. Thus, CD74-NRG1 gene fusions are activating genomic alterations in invasive mucinous adenocarcinomas and may offer a therapeutic opportunity for a lung tumor subtype with, so far, no effective treatment.

  Study EGAS00001000653

• White blood cell and cell-free DNA analyses for detection of residual disease in gastric cancer

  Liquid biopsies are providing new opportunities for detection of residual disease in cell-free DNA (cfDNA) after surgery but may be confounded through identification of alterations arising from clonal hematopoiesis. Here, we identify circulating tumor-derived DNA alterations (ctDNA) through ultrasensitive targeted sequencing analyses of matched cfDNA and white blood cells from the same patient. We apply this approach to analyze samples from patients in the CRITICS trial, a phase III randomized controlled study of perioperative chemotherapy in patients with operable gastric cancer. After filtering alterations derived from matched white-blood cells, the presence of ctDNA predicts recurrence when analyzed within nine weeks after preoperative treatment and after surgery in patients eligible for multimodal treatment. These analyses provide a facile method for distinguishing ctDNA from other cfDNA alterations and highlight the utility of ctDNA as a predictive biomarker of patient outcome to perioperative cancer therapy and surgical resection in patients with gastric cancer.

  Study EGAS00001004114

• Frequent alterations in cytoskeleton remodeling genes in primary and metastatic Chinese lung adenocarcinomas

  The landscape of genetic alterations in lung adenocarcinoma derived from Asian patients is largely uncharacterized. Here we present an integrated genomic and transcriptomic analysis of 335 primary lung adenocarcinomas and 35 corresponding lymph node metastases from Chinese patients. Altogether 13 significantly mutated genes are identified, including themost commonly mutated gene TP53 and novel mutation targets such as RHPN2, GLI3 and MRC2. TP53 mutations are furthermore significantly enriched in tumours from patients harbouring metastases. Genes regulating cytoskeleton remodelling processes are also frequently altered, especially in metastatic samples, of which the high expression level of IQGAP3 is identified as a marker for poor prognosis. Our study represents the first large-scalesequencing effort on lung adenocarcinoma in Asian patients and provides a comprehensive mutational landscape for both primary and metastatic tumours. This may thus form a basis for personalized medical care and shed light on the molecular pathogenesis of metastatic lung adenocarcinoma.

  Study EGAS00001000982

• Rare disruptive mutations in ciliary function genes contribute to testicular cancer susceptibility

  Testicular germ cell tumor (TGCT) is the most common cancer in young men1,2. Here we aimed to identify novel risk factors for TGCT using whole-exome sequencing, which was performed on 328 affected individuals from 153 families, 634 sporadic cases and 1,644 controls. We searched for genes that were recurrently affected by rare variants (minor allele frequency <0.01) with potentially damaging effects and evidence of segregation in families. 8.7% of families carried rare disruptive mutations in the cilia-microtubule genes (CMG) as compared to 0.5% of controls3 (P=2.1x10-8). The most significantly mutated CMG was DNAAF1 with biallelic inactivation and loss of DNAAF1 expression shown in tumors from carriers. DNAAF1 as a cause of TGCT was supported by a DNAAF1Hu255h(+/-) zebrafish model with 94% penetrance for TGCT compared to 14% in wildtype fish. These data implicate cilia-microtubule inactivation as a cause of TGCT development and are the first evidence for CMGs as cancer susceptibility genes.

  Study EGAS00001001789