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• Slim Initiative in Genomic Medicine for the Americas (SIGMA): Mexico City Diabetes Study (MCDS)

  The ~52,000 sample Type 2 Diabetes Exome Sequencing project is a collaboration of six consortia with various funding mechanisms that have joined together to investigate genetic variants for type 2 diabetes (T2D) using the largest T2D case/control sample set compiled to date. This includes samples from: Type 2 Diabetes Genetic Exploration by Next-generation sequencing in multi-Ethnic Samples (T2D-GENES) Genetics of Type 2 Diabetes (GoT2D) Exome Sequencing Project (ESP) Slim Initiative in Genomic Medicine for the Americas: Type 2 Diabetes (SIGMA T2D) Lundbeck Foundation Centre for Applied Medical Genomics in Personalised Disease Prediction, Prevention, and Care (LuCAMP) Progress in Diabetes Genetics in Youth (ProDIGY) This data generated from the SIGMA Mexico City Diabetes Study was part of the Slim Initiative in Genomic Medicine for the Americas: Type 2 Diabetes (SIGMA T2D), which is an international research consortium funded by the Carlos Slim Foundation that seeks to identify the genetic risk factors for type 2 diabetes (T2D) in Mexico and Latin America and translate those findings into improved treatment and prevention of diabetes. The SIGMA T2D project has sequenced and genotyped more than 13,000 samples from Mexican and Mexican Americans. The individuals were obtained from over 20 cohorts across the 6 consortia that are listed in Table 1. The strategy was to perform deep exome sequencing of individuals, 24,991 with T2D and 24,953 controls, divided among five ancestry groups: Europeans, East Asians, South Asians, American Hispanics, and African Americans. The T2D-GENES, ProDIGY and SIGMA studies, sequencing was performed at the Broad Institute using the Agilent v2 capture reagent or Illumina Rapid Capture on Illumina HiSeq machines. Please note that while we summarize the full sample list in publications and below, two of the cohorts below are not in dbGaP, due to the samples not being consented for deposition. This includes the Kooperative Gesundheitsforschung in der Region Augsburg (KORA) study and Lundbeck Foundation Centre for Applied Medical Genomics in Personalised Disease Prediction, Prevention, and Care (LuCamp) study. The Exome Sequencing Project (ESP) was deposited in dbGaP as part of their initial study and the phs numbers for that project can be found here: https://esp.gs.washington.edu/drupal/dbGaP_Releases Table 1. 52,000 sample T2D Case/Control Whole Exome Sequencing Studies Ancestry Consortia Study Countries of Origin # Cases # Controls African American T2D-GENES Project 1 Jackson Heart Study US 500 526 African American T2D-GENES Project 1 Wake Forest School of Medicine Study US 518 530 African American ESP Exome Sequencing Project (ESP) US 467 1374 African American T2D-GENES Follow up study BioMe Biobank Program (BioMe) US 1297 1256 East Asian T2D-GENES Project 1 Korea Association Research Project Korea 526 561 East Asian T2D-GENES Project 1& Follow up Study Singapore Diabetes Cohort Study; Singapore Prospective Study Program Singapore (Chinese) 1486 1568 East Asian T2D-GENES Follow up study Korea SNUH South Korea 450 475 East Asian T2D-GENES Follow up study Research Studies in Hong Kong (Hong Kong) Hong Kong 493 485 European T2D-GENES Project 1 Ashkenazi US, Israel 506 355 European T2D-GENES Project 1 Metabolic Syndrome in Men Study (METSIM) Finland 484 498 European GoT2D Finland-United States Investigation of NIDDM Genetics (FUSION) Study Finland 472 476 European GoT2D Kooperative Gesundheitsforschung in der Region Augsburg (KORA) Germany 97 90 European GoT2D UK Type 2 Diabetes Genetics Consortium (UKT2D) UK 322 320 European GoT2D Malmo-Botnia Study Finland, Sweden 478 443 European LuCamp Lundbeck Foundation Centre for Applied Medical Genomics in Personalised Disease Prediction, Prevention, and Care (LuCamp) Denmark 997 997 European ESP Exome Sequencing Project (ESP) US 390 2843 European T2D-GENES Follow up study Genetics of Diabetes and Audit Research Tayside Study (GoDARTS) Scotland, UK 960 966 European T2D-GENES Follow up study Framingham Heart Study (FHS) US 396 596 Hispanic T2D-GENES Project 1 San Antonio Family Heart Study, San Antonio Family Diabetes/ Gallbladder Study, Veterans Administration Genetic Epidemiology Study, and the Investigation of Nephropathy and Diabetes Study Family Component US 272 218 Hispanic T2D-GENES Project 1 & SIGMAv2 Starr County, Texas US 1762 1738 Hispanic SIGMAv1 Mexico City Diabetes Study Mexico 281 549 Hispanic SIGMAv1 & v2 Multiethnic Cohort (MEC) US 1476 1443 Hispanic SIGMAv1 & v2 UNAM/INCMNSZ Diabetes Study (UIDS) Mexico 1998 1977 Hispanic SIGMAv1 & v2 Diabetes in Mexico Study (DMS) Mexico 1522 1546 Multi ethnic ProDIGY Treatment Options for Type 2 Diabetes in Adolescents and Youth (TODAY) US 3097 0 Multi ethnic ProDIGY SEARCH for Diabetes in Youth (SEARCH) US 553 0 South Asian T2D-GENES Project 1 London Life Sciences Population Study (LOLIPOP) UK (Indian Asian) 531 538 South Asian T2D-GENES Project 1 & Follow up study Singapore Indian Eye Study Singapore (Indian Asian) 1640 1478 South Asian T2D-GENES Follow up study Pakistan Genomic Resource (PGR) Pakistan 914 932

  Study phs001375

• Clonal dynamics of normal haematopoiesis across the human lifespan

  Age-related loss of function in the human haematopoietic system is well documented, manifesting as reduced regenerative capacity, age-related cytopenias and immune dysfunction. However, the cellular and population level changes that underpin both this functional decline and the increased risk of clonal haematopoiesis and blood cancer in the elderly remain elusive. Here we performed whole genome sequencing on >3350 single haematopoietic stem cell / multipotent progenitors (HSC/MPP) derived colonies across 10 haematologically normal subjects aged 0 to 81. We found that HSC/MPPs accumulated 17 single nucleotide variants per year post birth and had a reduction in telomere length of 50bp per year throughout young adult life. We reconstructed phylogenies of the sampled HSC/MPPs to interrogate changes in clonal dynamics through life. Haematopoiesis in adults aged less than 65 was predominantly polyclonal, with few known driver mutations. In contrast, individuals aged over 75 displayed a profound change in clonal structure, with frequent clonal expansions, many unexplained by known driver mutations. The ratio of non-synonymous to synonymous mutations revealed widespread positive selection, estimating around 1000 driver mutations in the dataset (10-fold more than the number of known drivers). We identified novel genes ZNF318 and HIST2H3D as being under positive selection, despite not being enriched in myeloid malignancies. Our data show that HSC clonal dynamics is more complex than previously thought. One implication is that by old age, the majority of HSCs carry at least one of a number of largely undescribed driver mutations, which may underlie aspects of their functional decline.

  Dataset EGAD00001007851

• African American Multiple Myeloma GWAS

  The case set is from a case-control study designed to identify common genetic risk factors for multiple myeloma in African Americans, the population with the highest risk for this cancer. We conducted two GWAS and combined each of these with convenience controls consisting of unaffected African American participants in cohorts with existing GWAS data. We then conducted a meta-analysis of the two sets. Cases were persons of African ancestry with smoldering or active multiple myeloma identified at participating oncology clinics or through SEER registries, as part of the African American Multiple Myeloma Study (AAMMS) diagnosed from Jan 1, 1988 through July 31, 2016. The majority of the samples were collected from incident and prevalent cases diagnosed since Jan 1, 2008 (80.9%), with a minority obtained from biobanks from cases diagnosed prior to 2008 (19.1%). Additional samples were obtained from the Multiethnic Cohort (USC and University of Hawaii) (n=40), the University of California at San Francisco Multiple Myeloma Study (n=27), and from the Multiple Myeloma Research Consortium for secondary analysis (samples originally provided to MMRC by 8 additional sites (n=84)). We have identified the phenotype (smoldering myeloma, plasma cell multiple myeloma, or myeloma not otherwise specified (myeloma NOS) when myeloma phenotype was not known), sex and age at diagnosis in this data set. The initial GWAS set consisted of 1308 (1,305 passed QC) cases with DNA samples, with a GWAS performed on the Illumina Human Core. Controls consisted of 7,078 unaffected African American subjects who were participants in the African American Prostate and Breast Consortium, with existing GWAS data from the Illumina1M Duo BeadChip. The second GWAS set consisted of 529 African American multiple myeloma patients with samples (406 from University of Arkansas, results not contained in this dataset because NCI funds were not used for the collection and genotyping) with GWAS data resulting from the Illumina Mega-BeadChip v1.1. Controls were 2,390 unaffected African American participants in the Multiethnic Cohort with existing GWAS data from the same array. After QC and removal of duplicates within sets, sex mismatches, and removal of plasmacytoma cases (ICD-0 code 9731), this deposited data set contains the typed GWAS data for the Illumina Human Core (set 1) (n=1298), and for the MegaBead Chip v1.1 (set 2) (n=123), with the University of Arkansas samples removed, for a total of 1,421 case genotypes. Note that 13 samples that overlap set 1 and set 2 were included.

  Study phs001632

• Phase II trial of targeted immune-depleting chemotherapy and reduced-intensity allogeneic hematopoietic stem cell transplantation using 8/8 and 7/8 HLA-matched unrelated donors and utilizing two graft-versus-host disease prophylaxis regimens for the treatment of leukemias, lymphomas, and pre-malignant blood disorders

  Background: Major problems with stem cell transplantation (SCT) for cancer treatment are a lack of suitable donors for patients without an HLA tissue-matched sibling and graft-versus-host disease (GVHD), a serious side effects of immune-suppressing chemotherapy that is given to bring the cancer under control before SCT. In GVHD, the patient's immune system attacks the transplanted donor cells. This study will try to improve the results of SCT from unrelated HLA-matched donors using targeted immune-depleting chemotherapy to bring the cancer under control before transplantation and to lower the chance of graft rejection, followed by reduced-intensity transplant chemotherapy to make the procedure less toxic. Objectives: To evaluate the safety and effectiveness of targeted immune-depleting chemotherapy followed by reduced-intensity transplant chemotherapy in patients with advanced cancers of the blood and immune system. To evaluate the safety and effectiveness of two different drug combinations to prevent GVHD. Both regimens have been successful in preventing GVHD, but they work by different mechanisms and affect the rebuilding of the immune system after the transplant. Eligibility: People 18 to 74 years of age with advanced or high-risk cancers of the blood and immune system who do not have a suitable HLA-matched sibling. Design: All patients receive chemotherapy before transplant to treat the cancer and suppress immune function. All patients receive a conditioning regimen of cyclophosphamide for 4 days and fludarabine for 4 days before SCT to prepare for the transplant. Patients are randomly assigned to one of two combination drug treatments to prevent GHVD as follows: Group 1: Tacrolimus starting 3 days before SCT and continuing for 6 months, plus methotrexate on days 1, 3, 6, and 11 post-SCT, plus sirolimus starting 3 days before the SCT and continuing through day 14 following SCT. Group 2: Alemtuzumab for 4 days starting 8 days before SCT, plus cyclosporine starting 1 day before SCT and continuing for 6 months. Patients receive the donor's stem cells and immune cells 2 days after the conditioning regimen. Patients are followed at the clinic regularly for the first 6 months after SCT, and then less often for at least 5 years. Some visits may include bone marrow aspirates and biopsies, blood draws, and other tests to monitor disease status. A skin biopsy, oral mucosa biopsy, and saliva collection are done to study chronic GVHD.

  Study phs002021

• Starr County Health Studies' Genetics of Diabetes Study

  An initial observation in the 1970s that Starr County, Texas, had the highest diabetes-specific mortality of any of the 254 Texas counties led to the establishment of a field office in Rio Grande City, Texas (the county seat of Starr County) and the initiation of a series of studies to understand the epidemiology and genetics of type 2 diabetes, its complications and related conditions in this predominantly (97%) Mexican American population. The field office opened in February 1981 and has operated continuously since then. During this time, three systematic surveys of the population have been conducted as well as family and longitudinal studies. Culturally sensitive diabetes education programs have been developed and implemented and also a weight loss intervention. In total, more than 23,000 Mexican American individuals have been examined with more than 200,000 aliquots of biological specimens stored. Collectively, these studies are known as the Starr County Health Studies. The present Genetics of Diabetes Study has the goal of identifying loci/alleles that underlie susceptibility to type 2 diabetes in this high risk population using genome wide markers and association testing. The study makes use of two primary resources that have been developed in order to identify 1,000 type 2 diabetes cases and 1,000 disease free controls. In the first instance we have identified a subset of unrelated cases from the total cases that we have identified. Where multiply affected sibships were available, the youngest onset case with detailed phenotyping (including assessment of diabetic retinopathy) was selected. Controls came from a recent survey to establish a representative sample of the Starr County population. Blocks were randomly selected, households on selected blocks enumerated and one individual randomly selected from each household for a detailed examination. The examination included an oral glucose tolerance test. Those included as controls are those with no prior diagnosis of diabetes and a negative oral glucose tolerance test. This collection of cases and controls provides the opportunity to identify susceptibility loci for type 2 diabetes and its complications, specifically diabetic retinopathy and albuminuria. The data set also allows the examination of impaired fasting glucose and impaired glucose tolerance. The samples are split between those born in the United States and those born in Mexico and this gives a natural contrast for examining genotype and environmental interactions. It is anticipated that the understanding that will come will lead to strategies for delaying and preventing the onset of diabetes and its complications.

  Study phs000143

• Discovery, Biology, and Risk of Inherited Variants in Breast Cancer (DRIVE) - OncoArray Genotypes

  Discovery, Biology, and Risk of Inherited Variants in Breast Cancer (DRIVE) was one of five projects funded in 2010 as part of the NCI's Genetic Associations and Mechanisms in Oncology (GAME-ON) initiative (http://epi.grants.cancer.gov/gameon/). GAME-ON's overall goal was to foster an intra-disciplinary and collaborative approach to the translation of promising research leads deriving from the initial wave of cancer GWAS. Specific goals included replication of previous GWAS findings and identification of new susceptibility loci through meta analyses of existing GWAS data and fine mapping of identified loci to better pinpoint causal variants; and identify germline variants that are associated with risk of multiple cancers. The other four funded GAME-ON projects were: the ColoRectal TransdisciplinaryStudy (CORECT), Elucidating Loci Involved in Prostate Cancer Susceptibility (ELLIPSE), Follow-up of Ovarian Cancer Genetic Association and Interaction Study (FOCI), and Transdisciplinary Research in Cancer of the Lung (TRICL). To identify additional cancer risk loci, improve the precision of fine-mapping, and facilitate cross-cancer analyses, the GAME-ON projects and other consortia formed the OncoArray network (http://epi.grants.cancer.gov/oncoarray/), which developed and genotyped a new custom genotyping array (the "OncoArray") in large numbers of cancer cases and controls (over 400,000 samples). The OncoArray is a custom array manufactured by Illumina. The array includes a backbone of approximately 260,000 SNPs that provide genome-wide coverage of most common variants, together with markers of interest for each of the five GAME-ON cancers identified through genome-wide association studies (GWAS), fine-mapping of known susceptibility regions, sequencing studies, and other approaches. The array also includes loci of interest identified through studies of other cancer types, and other loci of interest to multiple cancer types (including loci associated with cancer related phenotypes, drug metabolism and radiation response). Additionally, SNPs relating to quantitative phenotypes such as body mass index (BMI), height, and breast density that correlate with common cancer risks are also included. The DRIVE data included under this dbGAP submission include OncoArray data from 60,015 breast cancer cases and controls genotyped at the Center for Inherited Disease Research (CIDR), University of Cambridge, National Cancer Institute, University of Copenhagen, University of Southern California and Mayo Clinic. Details on an additional approx. 80,000 breast cancer cases and controls genotyped at other centers can be found at http://bcac.ccge.medschl.cam.ac.uk/bcacdata/oncoarray/.

  Study phs001265

• Genetics of Human Inherited Retinal Diseases (GHIRD)

  The goal of this project is to identify novel mutations and genes involved in human retinal disorders, a stated priority of the National Eye Institute. To accomplish this, additional genes whose mutations cause inherited retinal diseases (IRD) will be identified by combining targeted gene panel sequencing, whole exome sequencing, and whole genome sequencing. We have collected about 1050 patient families around the world. In this proposal, we will identify the underlying mutations in these patients using a combination of targeted gene panel sequencing, whole exome sequencing, whole genome sequencing, bioinformatics, statistics, and functional studies. Genes targeted for gene panel sequencing are: ABCA4; ASIC2; JAG1; ABCC6; ARL3; ATP1B2; BBS1; BBS2; BBS4; OPN1SW; CAPN5; C21orf2; CA4; CACNA1F; CDH3; CHM; ERCC8; CLCN2; CLCN3; CLCN7; TPP1; CLN3; CLN5; CNGB1; CNGA1; CNGA3; COL2A1; COL9A1; COL11A1; CRX; VCAN; CTSD; TIMM8A; DMD; CLN8; ERCC6; EFEMP1; OPN1MW; GNAT1; GNAT2; GNGT1; GRM6; GUCA1A; GUCA1B; GUCY2F; GUCY2D; HARS; CFH; HK1; IDH3B; IMPDH1; IMPG1; KCNJ13; KIF11; LRP5; MAK; MITF; TRPM1; MTTP; MVK; MYO7A; NDP; NEK2; NEUROD1; NPHP1; NRL; OAT; OPA1; OTX2; PAX2; PAX6; PDCL; PCYT1A; PDE6A; PDE6C; PDE6G; PDE6H; PDE6B; PEX1; PEX6; PEX7; PEX10; PEX12; PEX13; PEX14; PFDN5; PGK1; PHYH; PIN1; PLA2G5; PPT1; PRKCZ; PEX19; PEX2; PEX5; RB1; RBP3; RBP4; OPN1LW; RDH5; PRPH2; RGR; RHO; GRK1; RLBP1; ROM1; RP9; RP1; RP2; RPGR; RPE65; RS1; SAG; ATXN7; CCL2; SLC6A6; ELOVL4; TEAD1; NR2F1; TIMP3; NR2E1; TTPA; TUB; TULP1; UCHL3; USH2A; CLRN1; VLDLR; BEST1; WFS1; ALMS1; MKKS; FZD4; OFD1; PEX3; CTSF; GBF1; ADAM9; RGS9; PEX11B; PROM1; UNC119; PRPF4; PRPF3; AIFM1; SLC24A1; LRAT; ASIC3; RAB28; PEX16; ITM2B; SLC4A7; IQCB1; CROCC; IFT140; DHX38; MFN2; NR2E3; USH1C; TOPORS; MERTK; FBLN5; CIB2; PRPF8; SDCCAG8; IFT27; TREX1; RRAS2; CEP164; ATF6; TRIM32; RIMS1; SNRNP200; CLUAP1; EMC1; ZNF423; TTLL5; AGTPBP1; RPGRIP1L; CRB1; TSPAN12; ARL2BP; AIPL1; PRPF6; FSCN2; ABHD12; PRPF31; TCTN3; IFT172; CNNM4; NPHP3; INVS; BBS9; FLVCR1; RDH8; IMPG2; WDPCP; RDH11; TMEM216; TMEM138; LZTFL1; CNGB3; AHI1; MKS1; CLN6; BBS7; POMGNT1; PEX26; SPATA7; KIZ; KLHL7; INPP5E; MDM1; CABP4; RPGRIP1; MCOLN1; CC2D2A; KIAA1549; SLC7A14; WDR19; NYX; CDH23; SEMA4A; MPP5; NMNAT1; TMEM237; PCDH15; C5orf42; GNPTAB; TMEM231; TCTN1; SRD5A3; BBS10; ZNF408; TTC21B; CSPP1; LPCAT1; DHDDS; PDZD7; PANK2; CEP290; OPA3; DNAJC5; PITPNM3; MFRP; HMCN1; ADGRV1; ARL6; FAM161A; TMEM126A; GNPTG; GJA10; RAX2; TUBGCP6; TMEM67; ACBD5; C12orf65; CDHR1; BBIP1; REEP6; CACNA2D4; RP1L1; CEP41; C1QTNF5; NXNL1; TTC8; USH1G; BBS5; ZNF513; RDH12; SLC38A8; C8orf37; VPS13B; NXNL2; BBS12; ADGRA3; LCA5; KCNV2; ADAMTS18; ARL13B; ATOH7; MFSD8; NPHP4; CCDC66; CYP4V2; CRB2; RD3; LRIT3; EYS; KIF7; CERKL; RGS9BP; C2orf71; GDF6; DTHD1; GPR179; CISD2; CCR2; PRCD; WHRN; ADGRA3. The current dbGaP study release makes available all sequencing-derived vcf data of study participants.

  Study phs001517

• Heart Failure Network: Functional Impact of GLP-1 for Heart Failure Treatment (HFN FIGHT-BioLINCC)

  Objectives To test whether therapy with a GLP-1 agonist improves clinical stability following hospitalization for acute heart failure. Background Heart failure was the leading cause of hospitalization in the United States with more than 4 million admissions per year from 2003–2009. Abnormal cardiac metabolism contributes to the pathophysiology of advanced heart failure with reduced left ventricular ejection fraction (LVEF). As heart failure progresses, these abnormalities become more pronounced and are observed in both patients with and without type 2 diabetes. At this time of this study, there was no heart failure therapy that targeted these metabolic abnormalities; however, in earlier clinical studies, glucagon-like peptide 1 (GLP-1) agonists showed cardioprotective effects in patients with advanced heart failure, irrespective of type 2 diabetes status. Glucagon-like peptide 1 is an endogenous incretin hormone that improves insulin sensitivity with minimal risk of hypoglycemia. This study was created to determine if use of GLP-1 agonists improved clinical stability in recently hospitalized patients with acute heart failure and reduced LVEF. Participants There were 300 participants. Design Participants were identified by hospital admission records and were enrolled during either the last 24 hours of his or her hospitalization for heart failure or the 2-week interval after the hospitalization. Baseline evaluations were conducted, which included echocardiographic measures, the 6-minute walk test, the Kansas City Cardiomyopathy Questionnaire (KCCQ), and blood tests. After the baseline evaluation, patients were randomized in a 1:1 ratio to receive either the GLP-1 agonist liraglutide or placebo as a daily subcutaneous injection. The protocol involved titration of study drug dosage as tolerated every 14 days from 0.6 mg/d to 1.2 mg/d to 1.8 mg/d during the first 30 days of the trial (as tolerated). Follow-up testing was performed at 30-, 90-, and 180-day study visits. The primary end point was a global rank score in which all participants, regardless of treatment assignment, were ranked across 3 hierarchical tiers: time to death, time to rehospitalization for heart failure, and time-averaged proportional change in N-terminal pro-B-type natriuretic peptide (NT-proBNP) level from baseline to 180 days. Conclusions The GLP-1 agonist liraglutide did not improve post-hospitalization clinical stability in patients with advanced heart failure and reduced LVEF. There was no significant between-group difference in the global rank scores and furthermore, there were no significant differences detected in any of the secondary endpoints.

  Study phs003542

• Rapid Acceleration of Diagnostics - Underserved Populations (RADx-UP): CO-CREATE-Ex: Community-engaged Optimization of COVID-19 Rapid Evaluation And TEsting Experiences

  Cases of COVID-19 infection in San Diego have persistently remained high in the county's central and southern communities near the US/Mexico border, home to primarily Latino residents who have been one of the ethnic minority communities most impacted by COVID-19. To address this, our Phase I RADx-UP project partnered with San Ysidro Health (SYH), a federally qualified health center, and the Global Action Research Center (Global ARC), a social change organization, to co-create and demonstrate the impact of a PCR-based COVID-19 testing program in San Ysidro. To date, we have administered approximately 15,000 PCR tests for >10,000 community members (92% Latino) and received requests from SYH to scale-out the testing program to additional primary care clinic sites. Responsive to RFA-OD-22-006, we will continue working with our community (Global ARC) and clinical (SYH) partners to refine, specify, implement, and evaluate an implementation strategy bundle that optimizes COVID-19 testing, expanding beyond current PCR testing to focus on FDA-authorized COVID-19 rapid antigen testing. Use of self-administered rapid antigen tests will allow participants to build capacity to self-test and use health information technology to access additional clinic services. Based on initial implementation mapping with the project's established Community and Scientific Advisory Board, three implementation strategies were prioritized to accelerate sustained uptake of rapid COVID-19 testing. These three strategies include: (1) current, walk-up onsite free testing; (2) promotores-led COVID-19 test counseling and preventive care reminders; (3) vending machines that dispense FDA-authorized self-testing kits. Our primary objectives are to: 1) refine and operationalize our multi-component implementation strategy bundle and a related set of measures of success for rapid FDA- authorized COVID-19 testing and 2) implement and evaluate impact of our innovative, multilevel, and multicomponent implementation strategy bundle to optimize COVID-19 rapid testing among underserved, Latino communities using a roll-out implementation optimization study design across four clinics over 18 months. The COVID-19 pandemic has underscored the urgent and essential need to engage invested community parties continuously and meaningfully in public health research to reduce health disparities. This proposal is well-positioned to provide a set of strategies and measures of success that can be used in settings serving underserved communities while maintaining flexibility to adjust to the changing COVID-19 landscape.

  Study phs003686

• Framingham Heart Study-Cohort (FHS-Cohort) - Imaging

  The Framingham Heart Study (FHS) is a population-based, observational cohort study initiated in 1948 to prospectively investigate the determinants of cardiovascular disease to guide public health prevention. The FHS began by recruiting an Original Cohort of 5,209 men and women between the ages of 30 and 62 from the town of Framingham, Massachusetts, who had not yet developed overt symptoms of cardiovascular disease or suffered a heart attack or stroke. The Original cohort Exam 1 took place between 1948 and 1953. Since that time the cohort has had a total of 32 biennial exams (ending in 2014) and event follow-up through 2022. In 1971, the FHS added the Offspring cohort, comprising 5124 children whose parents were enrolled in the Original cohort and the spouses of the children. This cohort on average has been examined every three to four years. However, there was an eight year gap between Exam 1 and Exam 2 and a seven year gap between Exam 7 and Exam 8. The latest exam (10) was completed in 2022. In 2002, the transgenerational FHS design was facilitated with the recruitment of 4095 children of the Offspring cohort (Third Generation), and 103 spouses of the Offspring who were not previously enrolled in the study (New Offspring Spouses, NOS). These cohorts have completed 3 exams through 2019. To reflect the changing demographic characteristics of the greater Framingham community, the FHS additionally recruited and enrolled 2 cohorts comprising racial and ethnic minority groups, termed Omni-1 and Omni-2, (n = 506 and 410, respectively) in 1995 and 2002, respectively. These cohorts included individuals of African American, Hispanic, Asian, Indian, Native American, and Pacific Islander descent. The OMNI-1 cohort have completed 5 exams through 2022. The OMNI-2 cohort have completed 3 exams through 2019. Data available for request include Echocardiogram images, available from the following exams in each cohort. Original Cohort: exams 18-32; Offspring cohort: exams 3-10; Third Generation, NOS and OMNI-2 cohorts: exams 1-3; OMNI-1 cohort: exams 1-5.CT image data at one or two timepoints were added for 4427 participants in the offspring, third generation, and OMNI1 and OMNI2 cohorts.Summary level phenotypes for the Framingham Cohort study participants can be viewed at the top-level study page phs000007 Framingham Cohort. Individual level phenotype data and molecular data for all Framingham top-level study and substudies are available by requesting Authorized Access to the Framingham Cohort study phs000007.

  Study phs003593

• TNBC ctDNA Targeted Panel

  Circulating tumour DNA (ctDNA) detection in liquid biopsy is an emerging alternative to tissue biopsy, but its utility in treatment response monitoring and prognosis in triple negative breast cancer (TNBC) remains to be well understood. In this study, we determined the presence of early stage TNBC ctDNA detectable actionable mutations with a clinically validated hotspot treatment indication panel. Sequencing of plasma DNA from 130 TNBC patients collected within 7 months of primary treatment completion revealed that 7.7% had detectable residual disease with a hotspot panel. Among neoadjuvant treated patients, we observed a trend where patients with incomplete pathologic response and positive ctDNA within 7 months of treatment completion were at risk of reduced progression free survival. We propose that a high risk subset of early TNBC patients treated in NAT protocols may be identifiable by combining tissue response and sensitive ctDNA detection.

  Study EGAS00001006937

• Response to tumor-infiltrating lymphocyte adoptive therapy is associated with preexisting CD8+ T-myeloid cell networks in melanoma

  Adoptive cell therapy (ACT) using ex vivo–expanded tumor-infiltrating lymphocytes (TILs) can eliminate or shrink metastatic melanoma, but its long-term efficacy remains limited to a fraction of patients. Using longitudinal samples from 13 patients with metastatic melanoma treated with TIL-ACT in a phase 1 clinical study, we interrogated cellular states within the tumor microenvironment (TME) and their interactions. We performed bulk and single-cell RNA sequencing, whole-exome sequencing, and spatial proteomic analyses in pre- and post-ACT tumor tissues, finding that ACT responders exhibited higher basal tumor cell–intrinsic immunogenicity and mutational burden. Compared with nonresponders, CD8+ TILs exhibited increased cytotoxicity, exhaustion, and costimulation, whereas myeloid cells had increased type I interferon signaling in responders. Cell-cell interaction prediction analyses corroborated by spatial neighborhood analyses revealed that responders had rich baseline intratumoral and stromal tumor–reactive T cell networks with activated myeloid populations. Successful TIL-ACT therapy further reprogrammed the myeloid compartment and increased TIL-myeloid networks. Our systematic target discovery study identifies potential T-myeloid cell network–based biomarkers that could improve patient selection and guide the design of ACT clinical trials.

  Study EGAS50000001217

• Genome Diversity in Africa Project: Benin (2021-02-16)

  Globally, human populations show structured genetic diversity as a result of geographical dispersion, selection and drift. Understanding this genetic variation can provide insights into the evolutionary processes that shape both human adaptation and variation in disease. Populations from SSA have the highest levels of genetic diversity. This characteristic, in addition to historical genetic admixture, can lead to complexities in the design of studies assessing the genetic determinants of disease and human variation. However, such studies of African populations are also likely to provide new opportunities to discover novel disease susceptibility loci and variants and refine gene-disease association signals. A systematic assessment of genetic diversity within SSA would facilitate genomic epidemiological studies in the region. The Genome Diversity in Africa Project (GDAP) aims to produce a comprehensive catalogue of human genetic variation in SSA, including single nucleotide polymorphisms (SNPs), structural variants, and haplotypes. This resource will make a substantial contribution to understanding patterns of genetic diversity within and among populations in SSA, as well as providing a global resource to help design, implement and interpret genomic studies in SSA populations and studies comprising globally diverse populations, complementing existing genomic resources. Specifically, we plan to carry out high depth whole genome sequencing of up to 2000 individuals across Africa (25 individuals from each ethnolinguistic group). Our scientific objectives are to: 1) develop a resource that provides a comprehensive catalogue of genetic variation in populations from SSA accessible to the global scientific community; 2) characterise population genetic diversity, structure, gene flow and admixture across SSA; 3) develop a cost-efficient, next-generation genotype array for diverse populations across SSA; and 4) facilitate whole genome-sequencing association studies of complex traits and diseases by developing a reference panel for imputation and resource for enhancing fine-mapping disease susceptibility loci. These scientific objectives will be supported by cross-cutting operational activities, including network and management of the consortium, research ethics, and research capacity building in statistical genetics and bioinformatics 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/ . This dataset contains all the data available for this study on 2021-02-16.

  Dataset EGAD00001006970

• Whole genome sequencing of retinoblastoma reveals the diversity of rearrangements disrupting RB1 and uncovers a treatment related mutational signature

  Background: The development of retinoblastoma is thought to require pathological genetic changes in both alleles of the RB1 gene. However, cases exist where RB1 mutations are undetectable suggesting alternative pathways to malignancy. Methods: We applied comprehensive whole genome sequencing (WGS) and transcriptomics to sporadic retinoblastomas derived from twenty patients attending our clinic, contrasting these results to that obtained through customary clinical testing. We sought RB1 and other driver mutations, investigated mutation burden, mutational signatures and phylogenetic relatedness in one case of bilateral retinoblastoma. Results: At least one RB1 mutation was identified in all retinoblastomas. We confirmed RB1 mutations previously identified by clinical screening, identified three new RB1 mutations and provided clarity to the mechanism behind a further six mutations. Eight tumours carried structural rearrangements involving RB1 ranging from relatively simple to extremely complex rearrangement patterns, including a chromothripsis-like pattern in one tumour. Potential driver mutations included mutations in BCOR (5/20) and amplification of MYCN (2/20) and MDM4 (1/20). We show that RB1 mutations are not mutually exclusive of MYCN amplifications, and further reveal that all tumours demonstrate increased MYCN expression suggesting a universal role in retinoblastoma tumorigenesis.  Bilateral tumours obtained from one patient harboured conserved germline but divergent somatic RB1 mutations, indicating independent evolution. In-keeping with previous WGS of paediatric cancers, the mutation burden in retinoblastomas was extremely low. Mutational signature analysis showed a predominance of signatures associated with cell division and an absence of ultraviolet-related DNA damage. In a tumour exposed to chemotherapy prior to enucleation, a profound platinum-related mutational signature was observed. Conclusions: WGS provides a complete picture of the genomic landscape of retinoblastomas, allowing the discovery of mutations otherwise undetected by conventional clinical screening approaches. The presence of at least one RB1 mutation in all retinoblastomas and the relative paucity of driver mutations in other genes suggests mutations beyond RB1, MYCN and BCOR are rare. Whilst most RB1 mutations are identifiable by clinical screening, the increased resolution and ability to detect otherwise elusive rearrangements of RB1 by WGS, confirming whether they are somatic or germline, has important repercussions on clinical management and advice on recurrence risks.

  Dataset EGAD00001006431

• Targeted and Whole Exome Sequencing for Validation of PGDx elio tissue complete

  This dataset consists of 116 tumor and normal samples analyzed with whole exome sequencing on the HiSeq2500 instruments with 100bp paired-end reads as well as 760 tumor and normal samples analyzed with the PGDx elio tissue complete assay. The PGDx elio tissue complete assay is a hybrid capture approach targeting 500+ genes with sequencing on the NextSeq instruments with 150bp paired-end reads. The bam files provided have been adapter masked and contain duplicate reads.

  Dataset EGAD00001008099

• Brain Arteriovenous Malformation Genetics Study

  The Predictors of Spontaneous Cerebral AVM Hemorrhage study was designed to identify genetic markers that predict adverse outcomes in brain arteriovenous malformation (BAVM) patients, examine race-ethnicity influences on BAVM hemorrhage risk, and use genome-wide association analysis to identify novel genetic predictors of AVM and hemorrhage using the Affymetrix GeneChip. We performed the first genome-wide association study (GWAS) (PMID: 26818729) of sporadic BAVM to investigate association of common single nucleotide polymorphisms (SNPs) with risk of sporadic BAVM in the international, multicenter Genetics of Arteriovenous Malformation (GEN-AVM) consortium. The study included a Caucasian discovery cohort of 515 BAVM cases and 1,191 controls genotyped using Affymetrix genome-wide SNP arrays. The Phase 1 discovery cohort comprised of 338 BAVM cases (154 male) and 504 controls genotyped using the Affymetrix Genome-Wide Human SNP Array 6.0. The molecular data (individual genetic data) for the Phase 1 discovery cohort 338 BAVM cases (self-reported Caucasian) recruited at University of California, San Francisco, or Kaiser Permanente of Northern California (KPNC) are included in the dbGaP Study Report: Brain Arteriovenous Malformation Genetics Study, with the additional BAVM case phenotypic and sample attribute characteristics: sex, race, age at BAVM diagnosis, hemorrhagic presentation, BAVM size. Written informed consent was obtained from all participants and the study was approved by the respective Institutional Review Boards. The molecular dataset for this study in dbGaP is based on the genotypes obtained for the initial set of 906,600 SNPs for cases and controls called together using Birdseed v2 algorithm in Affymetrix Genotyping Console Software. The final QC'd dataset includes 717,335 markers as SNPs with MAF<0.01 or deviating from Hardy-Weinberg equilibrium (P<0.00001) in controls were excluded. After QC, the average genotyping call rate was 99%. The discovery phase GWAS identified 57 top BAVM-associated SNPs which were tested in a replication cohort including 608 BAVM cases and 744 controls. The molecular dataset for these 338 BAVM cases was also included in a report investigating common and rare genome-wide copy number variation for association with BAVM (PMID: 24098321), which included a total sample size of 371 sporadic Caucasian BAVM cases and 563 Caucasian controls. This study identified a CNV region on 1p36.13 (NBPF1 locus) that was significantly enriched with duplications in BAVM cases compared to controls, although this association did not replicate in an independent cohort of 184 sporadic BAVM cases and 182 controls. This resource will help researchers to better understand the genetic risk factors for BAVM and/or hemorrhage. These data may be applied to examine the genetic contributions to arteriovenous malformation, stroke, and related vascular disorders and may help generate hypotheses regarding the genetics of cerebrovascular disease.

  Study phs002069

• The Genomic Landscape of Mongolian Hepatocellular Carcinoma

  Background and Hypotheses: Mongolia has the highest reported incidence of - and mortality from - hepatocellular carcinoma (HCC) in the world, which is between three and seven times higher than that observed in other high-incidence populations, such as South Korea, Thailand, and China. Although its causative factors and underlying tumor biology remain unknown, risk factors such as chronic hepatitis B virus and/or hepatitis C virus infection are highly prevalent. Moreover, Mongolia has an extraordinarily high prevalence of hepatitis delta virus (HDV), more than ten times above the global estimate, which we hypothesize as a major driver of Mongolian hepatocarcinogenesis. By performing a comprehensive and integrative genomic characterization, we aimed to identify robust molecular subclasses and driver features informative of the etiology and progression of the disease. This study, the first one of this nature ever to be conducted on a Mongolian cohort, identifies similarities to previous studies performed on other populations (which reflect general characteristics of liver carcinogenesis) as well as unique features of Mongolian HCC. Study Design and Methods: Clinical information and paired tumor/non-tumor liver tissue samples were obtained from 76 HCC patients undergoing surgery at the National Cancer Center of Mongolia. Transcriptome sequencing and whole exome sequencing were performed on all tumor and non-tumor samples, followed by bioinformatics processing and quality control. Consensus clustering and regularized Cox regression analyses were performed on transcriptome data. Driver mutations and mutational signatures were assessed. These results were compared to those from 373 HCC patients of different races and ethnicities and diverse etiologies. Furthermore, molecular subclass gene signatures from multiple previous studies were assessed for validation. Results and Conclusions: Using a transcriptomics-based unsupervised approach, we identified four molecular subclasses among Mongolian HCC cases, which were associated with different patient survival outcomes. These molecular subclasses were mapped to signatures reported in other HCC cohorts, showing significant associations consistent with survival outcomes. A supervised analysis determined risk scores strongly associated with survival, consistent with the four molecular subclasses. Mutational signature analysis using recently published COSMIC and Environmental Agents Compendia identified signatures with distinct prevalence among HDV+ and HVD- groups. Signatures differentially associated with HDV+ include mutational patterns linked to alkylating agents (such as temozolomide), tobacco chewing and exposures to 1,8-Dinitropyrene and furan, whereas HDV- appeared differentially associated with aristolochic acid II. In addition to common driver mutations (TP53, CTNNB1) frequently found in pan-cancer analysis, Mongolian HCC exhibits several unique drivers (most notably GTF2IRD2B, PNRC2, and SPTA1), the latter of which may be linked to HDV infection. These results suggest the existence of new molecular mechanisms at play in Mongolian hepatocarcinogenesis.

  Study phs002000

• Neoantigen-Targeted CD8+ T-Cell Responses 1 With PD-1 Blockade Therapy

  Background: The primary target of T-cell responses induced against cancer cells are peptides derived from non-synonymous mutations presented by HLA (neoantigens). However, the large diversity of HLA alleles and restricted availability of clinical samples have limited the study of the specificity and the evolution over time of the neoantigen-specific T cell responses induced after treatment with immune checkpoint blockade (ICB) immunotherapy. Methods: We applied a newly developed neoepitope-specific T cell isolation technology to perform a longitudinal landscape analysis of the neoepitope-specific T cells in peripheral blood and tumor from 11 patients with metastatic melanoma; 7 with response and 4 with no response to ICB. Briefly, first using computational prediction, the putative neoantigens in each patient were ranked based on affinity for the patient's HLA and expression levels. Then hundreds of barcoded capture reagents consisting of the patient HLA class I subtypes loaded with the corresponding predicted neoantigen were made and used to isolate neoepitope-specific T cells. Once isolated, the TCRs and the barcodes were sequenced. Finally, the tumor reactivity of the isolated neoepitope-specific TCRs (neoTCRs) was assessed upon co-culture of autologous melanoma cell lines from each patient with primary human T cells expressing the neoTCRs generated using a CRISPR-based non-viral gene editing to replace the endogenous TCRs. Results: The tumor mutation burden ranged between 3507 and 177 for patients with response to therapy and 977 to 31 for patients without response to therapy. We screened a median of 172 predicted neoantigen-HLA per patient across their 6 HLA molecules and isolated neoTCRs in all 11 patients. The number of mutations targeted ranged between 13 and 1. We assessed the neoTCR-tumor reactivity in samples from 3 patients with response and 3 patients without response. 39 of the 62 neoTCRs demonstrated specific recognition and cytotoxicity to patient-matched melanoma cell lines. Multiple T cell clonotypes recognized a limited number of mutations in 7 patients with response (median of 41 different neoTCR clonotypes per patient), and the T cell specificities were recurrently detected in the blood and the tumor over time. Samples from 4 patients without response to therapy also demonstrated neoepitope-specific T cell responses in blood and tumor to a similarly restricted number of mutations, but lacked TCR polyclonality (median 6.5 neoTCR clonotypes per patient) and were not recurrently detected in sequential samples. Conclusion: Effective ICB therapy is associated with polyclonal neoepitope-specific T-cell responses in the tumor and blood that recognize a limited number of immunodominant mutations and are recurrently detected over time.

  Study phs003153

• CSER: Genomic Sequencing to Aid Diagnosis in Pediatric and Prenatal Practice: Examining Clinical Utility, Ethical Implications, Payer Coverage, and Data Integration in a Diverse Population

  Congenital abnormalities and developmental disorders affect 3-5% of live born infants and children. Despite advances in both pre- and post-natal treatment, the utility of genetic testing in diagnosing the etiology underlying such conditions in order to guide management has been frustratingly limited. Traditional genetic testing with specific gene tests, or even gene panels, is diagnostic in only a small percentage of cases. Recent technological advances in next generation sequencing (NGS) have led to the ability to sequence and interpret the entire exome relatively quickly, allowing a diagnosis in 25-30% or more of cases of developmental disorders when other genetic tests have not yielded a result. Although whole exome sequencing (WES) holds great promise for improved diagnosis leading to better clinical outcomes, challenges remain in determining how best to apply and utilize sequence data. Fulfilling the promise of WES also requires investigation of ELSI (ethical, legal, social) concerns, given skepticism in some communities that research will benefit them; economic considerations that ultimately determine access to and equitable use of WES; and a need to share clinical genetic results with families and across health care systems to enable better prognostication and management of rare conditions in community settings. We propose a Program in Prenatal and Pediatric Genomic Sequencing (P3EGS) at UCSF to examine the diagnostic and clinical utility of WES. P3EGS will recruit and study affected individuals and their parents, including pregnancies in which the fetus has a confirmed structural anomaly and children with previously undiagnosed developmental disorders that are likely of genetic etiology. Following consent and collection of standardized phenotypic data, the families will undergo WES as part of clinical care. To achieve diversity, patient ascertainment and recruitment will occur at four UCSF sites that serve a broad range of underrepresented minorities (target of 75%) and span the full socio-economic spectrum, including the underserved. Our specific aims will: 1) examine the clinical utility of WES, including assessment of a variety of health-related and reproductive outcomes, in undiagnosed individuals; 2) address ethical, social and economic issues in the delivery of genomic sequencing results to ancestrally and economically diverse populations through (2.1) a mixed methods, longitudinal empirical study of clinical interactions and experiences, (2.2) an economic analysis of insurance coverage, price and reimbursement of multigene tests, and (2.3) creation of an Ethics Advisory Board to respond to emerging issues and establishment of authentic stakeholder engagement; and 3) pilot a user-friendly web-based patient/provider application integrating genomic and clinical data as a shared evidence base to support result communication, interpretation and clinical decision making; the application will be based on the “Bioscreen” model created and successfully implemented at UCSF.

  Study phs002324

• Inflammatory Bowel Disease Exome Sequencing Study

  The Broad Institute and Massachusetts General Hospital (MGH) are launching a new initiative to perform large-scale exome sequencing in inflammatory bowel disease (Crohn's disease and ulcerative colitis). Given the considerable unmet therapeutic need in inflammatory bowel disease (IBD), the recent emergence of rapid and efficient genome sequencing technologies and the compelling evidence for the role of genetics in these disorders have motivated the founding of a collaborative sequencing effort geared toward the discovery of high-impact genetic variants influencing IBD risk that can serve as guides to future therapeutic development and diagnostic tools. The initiative will be directed by experienced IBD researchers from MGH and the Broad Institute but aims to develop a collaborative exome sequencing network that will partner with researchers worldwide. Two large pilot exome sequencing projects are being launched immediately as part of this initiative based on established genetic study designs that enrich for the discovery of rare, high-impact risk and protective variants. 1) Early-onset pediatric IBD: a major focus of the work will surround full-exome sequencing of the earliest-onset childhood IBD cases. It has long been recognized in many diseases that penetrant genetic risk factors are much more likely to be found in cases with unusually early onset. The Broad Institute has completed more than 50,000 exomes in the past two years; and their technical expertise in generating and processing such sequencing data, along with population genetic variation patterns from these experiments, will provide a foundation for obtaining the best outcome in the interpretation of these cases. Samples with IBD onset before 6 years of age - and in some cases going up to age 10 - collected by IBD researchers around the world are welcomed for inclusion in this study. 2) Adult-onset IBD case-control study: Genetic mapping has provided dramatic insights into IBD pathogenesis in recent years, but a substantial amount of heritability - in particular the role of strong-acting rare mutations - is yet to be elucidated. To deliver those insights, IBD cases with an unusually low burden of known genetic risk factors (particularly emphasizing those with positive family history and/or from isolated or enriched populations) will be contrasted with population controls that have an extremely high burden of known IBD risk factors, enhancing discovery of critical protective variants that may provide the best clues for therapeutic development. This project will also be initiated as an international collaboration, particularly among researchers with sample sets with Immunochip genotyping that will enable the immediate genetic identification of these enriched target populations. This project will be supported by the NHGRI Large-Scale Sequencing Program, with all generated data made publicly available to the research community through standard NIH databases.

  Study phs001076

• National Epidemiologic Survey on Alcohol and Related Conditions-III (NESARC-III)

  The National Epidemiologic Survey on Alcohol and Related Conditions-III (NESARC-III) is a nationally representative cross-sectional survey of the U.S. general population, which was sponsored by the National Institute on Alcohol Abuse and Alcoholism (NIAAA) and conducted in 2012-2013. The NESARC-III's target population was the U.S. noninstitutionalized civilian population, 18 years and older, including residents of selected group quarters (e.g., group homes, workers' dormitories). Multistage probability sampling was used to randomly select respondents. Primary sampling units (PSUs) were individual counties or groups of contiguous counties, secondary sampling units (SSU) comprised groups of Census-defined blocks, and tertiary sampling units were households within SSUs. Finally, eligible adults within sampled households were randomly selected. Hispanics, Blacks, and Asians were oversampled and resulted in a total of 36,309 respondents. All respondents provided informed consent for survey participation. Data were adjusted for oversampling and screener - and person-level nonresponse, then weighted through post-stratification to represent the US civilian population based on the 2012 American Community Survey. These weighting adjustments were found to compensate adequately for nonresponse. Interviewer field methods involved initial structured home study, in-person training, ongoing supervision, and random respondent callbacks to verify data. The NESARC-III collected information on alcohol and drug consumption, alcohol use disorder, other substance use disorders (e.g., nicotine, cannabis and opioid), and associated physical and psychiatric adverse consequences. In addition to a full array of sociodemographic characteristics such as race/ethnicity, gender, age, risk and environmental factors including adverse childhood experiences (e.g., sexual, physical abuse and neglect) and family history for alcohol, drug use disorders and other psychiatric disorders (e.g., mood, anxiety, personality disorders and PTSD) were included (phenotypic variables=4,320). As part of the NESARC-III, 22,848 informative samples were genotyped on Affymetrix Axiom Exome Array consisting of 319,283 SNPs and 103,404 custom-selected SNPs Array (refer to the SNP annotation file). The latter array was selected based on the addiction associated genes, the results of 5 GWAS of alcohol and other psychiatric disorders, and animal models with addiction phenotypes. After filtering out poor quality SNPs using the standard Affymetrix pipeline there are 295,218 SNPs in the NESARC-III genetic data. The 22,848 subjects in this study were all deeply and consistently phenotyped via structured diagnostic interview. Controls group can be selected for specific disorders from a pool of all samples. Here are numbers of cases for major DSM-5 mental disorders in NESARC-III genetic data: Alcohol use disorder (7,075); Nicotine use disorder (6,641); Drug use disorder (2,570); Major depressive disorder (5,214); Persistent depression(1,429); Bipolar I (543); Anxiety disorders (4,226); PTSD (1,674); Personality disorders (4,014); Eating Disorders (457).

  Study phs001590

• Heart Failure Network Aldosterone Targeted Neurohormonal Combined with Natriuresis Therapy - (HFN ATHENA-BioLINCC)

  ObjectiveTo assess whether high-dose spironolactone treatment for patients with acute heart failure lowers natriuretic peptide levels and improves outcomes better than usual care.BackgroundAcute heart failure (AHF) accounts for more than a million hospitalizations in the United States annually. The role of low-dose mineralocorticoid receptors antagonists (MRAs) therapy as a neurohormonal antagonist is well established for the treatment of chronic heart failure and reduced ejection fraction. However, the role of high-dose MRA therapy in AHF remained uncertain. A previous clinical trial suggested that the benefits of high-dose MRA therapy in AHF included lower natriuretic peptide levels, less congestion, better renal function, and less need for an intravenous diuretic. The use of intravenous loop diuretics can intensify secondary hyperaldosteronism among AHF patients, and hyperaldosteronism directly contributes to diuretic resistance in AHF. Elevated aldosterone levels in AHF are associated with an increased risk of cardiovascular mortality and HF readmission. Hospitalizations for heart failure are associated with increased risk of mortality and/or readmission.The HFN-ATHENA trial was conducted to determine if mineralocorticoid receptor antagonists administered at high doses relieved congestion, decreased diuretic resistance, and mitigated the effects of adverse neurohormonal activation in AHF.SubjectsThe ATHENA trial enrolled 360 participants. Of these, 178 participants were randomized to usual care treatment (placebo or low-dose spironolactone) and 182 participants were randomized to high-dose spironolactone.DesignThe study intervention was initiated within 24 hours of patients receiving the first dose of intravenous diuretics. Participants not taking spironolactone at enrollment were randomized to 100 mg spironolactone or a placebo. Participants taking low-dose spironolactone before their hospital admission were randomized to 100 mg or 25 mg per day in the usual care treatment arm. Prescription of all other medications, including diuretics, was left at the discretion of the treating physician. The study drug was discontinued after 96 hours and further MRA use was left to the treating physician's discretion. The primary end point was the proportional change in the log NT-proBNP levels from randomization to 96 hours (or at the hospital discharge if the discharge occurred earlier than 96 hours). Secondary endpoints included: (1) a clinical congestion score; (2) dyspnea relief; (3) daily cumulative net urine output for up to 96 hours; (4) net weight change from baseline to 96 hours or discharge (whichever came first); (5) furosemide equivalents of the loop diuretic dosage at discharge; and (6) the development of in-hospital worsening HF, with signs and symptoms requiring additional therapy.ConclusionsThere was no significant difference in the primary or secondary endpoints between the high-dose treatment arm and the usual care treatment arm.

  Study phs003506

• Whole transcriptome RNA sequencing as comprehensive diagnostic tool for acute myeloid leukemia.

  BackgroundAcute myeloid leukemia (AML) is characterized by uncontrolled proliferation of malignant hematopoietic cells in the bone marrow that are arrested in differentiation. In AML pathogenesis, hematopoietic progenitor cells acquire multiple genetic aberrations often occurring in the same set of genes that ultimately lead to malignant transformation. In the WHO classification 2016, six AML classes with different prognosis are identified by chromosomal translocations measured by standard cytogenetics. All balanced translocations produce fusion genes, except for t(3;3)/inv(3;3), which lead to overexpression of MECOM/EVI1 associated with poor prognosis. For accurate risk assessment of cytogenetically normal AML, four genes need to be screened to distinguish AML with mutated NPM1 in the absence of FLT3 mutations and AML with bi-allelic CEBPA mutations, which have a favorable prognosis, and AML with RUNX1 mutations which have a poor prognosis. Recently, a full genomic classification system has been proposed by Papaemmanuil et al. (NEJM 2016). In this system, the same six classes with chromosomal translocations are identified by standard cytogenetics as well as five additional classes characterized by genetic mutations in 14 different genes. AimThe aim of the study was to investigate whether whole transcriptome RNA-sequencing (RNAseq) can be used as single technology for classification of AML. MethodA panel of 100 AML were analyzed and a bio-informatics pipeline called HAMLET (Human AML Expedited Transcriptomics) was developed in which 4 modules are integrated to detect (1) fusion genes, (2) small variants in 13 recurrently mutated genes, (3) internal tandem duplications in FLT3 (FLT3-ITD) and partial tandem duplications in KMT2A (KMT2A-PTD) and (4) overexpression of MECOM/EVI1. All mutations that were called by HAMLET were validated by diagnostic data as available for all 100 AML or targeted PCR followed by Sanger or next generation sequencing on all positive AML and at least an equal number of negative cases. Results & discussionThe data showed that HAMLET accurately identified all genetic aberrations with high sensitivity and specificity. In addition, in 7 AML, fusion transcripts were detected that are not measured by standard cytogenetics including three cases that lack any class-defining lesion according to Papaemmanuil et al. Moreover, overexpression of MECOM/EVI1 was detected in two AML with inv(3) as well as in five cases without inv(3)/t(3;3), and a gene signature was measured to distinguish AML with CEBPA mutations with favorable prognosis. In conclusion, HAMLET provides a comprehensive and flexible pipeline for RNAseq analysis to retrieve all relevant information for current classification of AML as well as additional information that may improve classification in the future.

  Study EGAS00001003096

• sn-RNAseq profiling of the impact of a cytokine storm model in human cardiac organoids

  We developed conditions to model conditions of diastolic dysfunction caused by inflammatory factors in human cardiac organoids. Our organoid model is based on conditions promoting maturation (Mills et al., PNAS, 2017 and Voges et al., In Revision) and incorporating human pluripotent stem cell derived cardiomyocytes, epicardial cells, fibroblasts, endothelial cells and pericytes. Following 15 days of differentiation (Voges et al., Development, 2017) we formed human cardiac organoids incorporating both our multicellular differentiation (Mills et al., PNAS, 2017) and additional endothelial cells (Voges et al., In Revision). After another 12 days of maturation in 5 days maturation medium and 5 days weaning medium (Voges et al., In Revision) human cardiac organoids were either treated with weaning medium or “cardiac cytokine storm” comprised of 100 ng/ml IFNg, 10 ng/ml IL-1 β and 10 µg/ml poly(I:C). After 48 hours human cardiac organoids were harvested and snap frozen in -80C before nuclei isolation and single nuclei RNA sequencing.Pooled hCO (~40) were homogenized in 4 mL lysis buffer (300 mM sucrose, 10 mM Tris-HCl (pH = 8), 5 mM CaCl2, 5 mM magnesium acetate, 2 mM EDTA, 0.5 mM EGTA, 1 mM DTT)(all Sigma-Aldrich) with 30 strokes of a dounce tissue grinder (Wheaton). Large pieces of hCO were allowed to settle and homogenate was passed through pre-wetted 40 µm cell strainers (Becton Dickinson). Remaining hCO material in the douncer was resuspended in 4 mL and the douncing and filtering steps were repeated twice. All steps of the homogenization were performed on ice. The filtered homogenate was centrifuged at 1500 x g for 5 min at 4oC. Nuclei pellets were then re-suspended in PBS. A fraction of resuspended nuclei were then stained with hoechst nuclear stain (1:500 dilution) and counted on a haemocytometer under fluorescent microscope.The nuclei were then re-centrifuged (1500 x g for 5 min at 4°C) and resuspended at a density to load ~5,000 nuclei per sample. Cell were loaded into the Chromium Controller (10X Genomics) for gel bead emulsion (GEM) formation. Library preparation was conducted according to the manufacturer’s recommended protocol using the Chromium Next GEM Single Cell 3’ GEM, Library & Gel Bead Kit v3.1. Libraries were sequenced on the NextSeq 500/550 v2 (Illumina) with 150 bp reads.In the snRNA-seq we note the lower than expected percentage of non-myocytes and the loss of endothelial cells (Mills et al., PNAS, 2017 and Voges et al., In Revision), indicating the protocol requires further optimization for hCO samples.

  Study EGAS00001005174

• Convergent somatic mutations in effectors of insulin signalling in chronic liver disease

  Chronic liver disease is associated with metabolic dysregulation, liver failure and hepatocellular carcinoma. We analysed somatic mutations from 1202 genomes across 32 liver samples, including normal controls, alcohol-related and non-alcoholic fatty liver disease. Five of 27 patients with liver disease carried hotspot driver mutations in FOXO1, the major transcription factor downstream of insulin signalling. FOXO1 mutations were independently acquired by up to 5 distinct clones within the same patient’s sample, and impaired insulin-mediated nuclear export of FOXO1. GPAM, which produces storage triacylglycerol from dietary calories, also had significant excess of mutations, similarly exhibiting convergent evolution within biopsies. Telomeres were shorter in diseased than normal liver, with attrition more pronounced in larger clones. Multiple independent acquisitions of drivers within one small liver sample imply that such mutations could affect hundreds of grams of tissue across the whole organ, potentially contributing to systemic metabolic dysfunction.

  Dataset EGAD00001006255

• NIPT samples for systematic evaluation of NIPT aneuploidy detection software tools

  This dataset contains two sets of samples. The reference sample set consists of a total of 669 samples that had been reported previously to be euploid by the NIPTIFY screening test. The validation sample set is based on a previously published validation study by Zilina et al. (1), consisting of 423 samples, of which 259 were high-risk pregnancies that had undergone diagnostic invasive prenatal analysis (1). All samples were sequenced with Illumina NextSeq 500 platform, producing 85 bp single-end reads with an average per-sample coverage of 0.32× at the University of Tartu, Institute of Genomics Core Facility, according to the manufacturer’s standard protocols, as described previously (1). This study was performed with the approval of the Research Ethics Committee of the University of Tartu (#315/T-13). 1. Zilina O, Rekker K, Kaplinski L, Sauk M, Paluoja P, Teder H, et al. Creating basis for introducing non‐invasive prenatal testing in the Estonian public health setting. Prenat Diagn [Internet]. 2019 Dec 6;39(13):1262-8. Available from: https://onlinelibrary.wiley.com/doi/abs/10.1002/pd.5578

  Dataset EGAD00001007712

• Genetics causes of male infertility in 185 patient-parent trios from Netherlands and UK

  The dataset is composed by the raw and processed sequencing data generated from 185 Patients affected by azoospermia or severe oligozoospermia recruited from the Netherlands and the UK.

  Dataset EGAD00001007862

• Neutrophils and emergency granulopoiesis drive immune suppression and an extreme response endotype during sepsis

  This dataset contains raw sequencing files associated with the paper "Neutrophils and emergency granulopoiesis drive immune suppression and an extreme response endotype during sepsis" (https://doi.org/10.1038/s41590-023-01490-5). It is composed of two experiments, which are as follows: 1. Single-cell profiling of whole blood leukocytes: This experiment comprises sequencing files generated using the BD Rhapsody single-cell multi-omics profiling (RNA + protein) platform. This platform was used to profile the whole blood leukocyte population in a cohort of sepsis patients, cardiac surgery controls and healthy controls. There are 48 samples and four files per sample: two paired-end FASTQ files (R1 and R2) corresponding to the RNA-seq library, and two paired-end FASTQ files (R1 and R2) corresponding to the protein profiling (ADT-based AbSeq) library. 2. Single-cell profiling of circulating HSPCs: This experiment comprises sequencing files generated using the 10X single-cell multi-omics (RNA-seq + ATAC-seq) profiling platform. This platform was used to profile circulating HSPCs in a cohort of sepsis patients and healthy controls. There are 5 samples (or plexes), each of which consists of a pool of individuals for whom cells were multiplexed and sequenced as a single sample. There are four files per sample: two paired-end FASTQ files (R1 and R2) corresponding to the RNA-seq library, and two paired-end FASTQ files (R1 and R2) corresponding to the ATAC-seq library. Because these samples consists of multiplexed pools, index files are also provided. These can be used for sample deconvolution using the Cell Ranger pipeline. NOTE: Due to EGA's constrains in the number of files permitted per sample, index files (I1 and I2) for the HSPC data set are provided as a separate experiment named "FASTQ index files (I1 and I2) for deconvolution of 10X single-cell multiomics libraries".

  Dataset EGAD00001010927

• The Dutch Microbiome Project Batch 2

  The Dutch Microbiome Project (DMP) data includes shotgun metagenomic sequencing of faecal samples 8,208 Dutch individuals. Paired-end sequencing was performed using Illumina HiSeq 2000 platform. Data is archived in two batches to facilitate easier data access and upload to EGA. Batch 2 of DMP includes ~400 samples.

  Dataset EGAD00001007501

• Framingham Heart Study-Cohort (FHS-Cohort) - BioLINCC

  Data Access NOTE Please refer to the "Authorized Access" section below for information about how access to the data from this accession differs from many other dbGaP accessions. Related Studies Other Framingham data available include: Imaging studies (Framingham Heart Study-Cohort (FHS-Cohort)-Imaging, phs003593), Genetics and genomics (Framingham Cohort, phs000007), Collaborative Cohort of Cohorts for COVID-19 Research (C4R): (Framingham Heart Study, phs002911), and as a component of the Sleep Heart Health Study (SHHS-BioLINCC, phs003637). Available Data Original cohort: data now include examination data from the first 32 clinical exams, selected ancillary data, and event follow-up through 2018. Offspring/Omni1 cohort: Data available include Framingham Offspring examination data from the first 9 clinical exams and selected ancillary data and event follow-up through 2019. Also included are the first 4 exams from the OMNI 1 cohort. Third Generation/Omni2/New Offspring Cohort: Data available include Framingham Generation 3 examination data from the first 3 clinical exams, selected ancillary data and event follow-up through 2019. Also included are the OMNI 2 and New Offspring (NOS) cohorts. A genetic pedigree is not provided for the Framingham phenotype only data. Objectives The objectives of the Framingham Study are to study the incidence and prevalence of cardiovascular disease (CVD) and its risk factors, trends in CVD incidence and its risk factors over time, and familial patterns of CVD and risk factors. Other important objectives include the estimation of incidence rates of disease and description of the natural history of cardiovascular disease, including the sequence of clinical signs and systems that precede the clinically recognizable syndrome and the consequences and course of clinically manifest disease. Background Original cohort: The original cohort of the Framingham Study began in 1948 under the U.S. Public Health Service and was transferred under the direct operations of the new National Heart Institute, NIH, in 1949. Participants were sampled from Framingham, Massachusetts, including both men and women. This was the first prospective study of cardiovascular disease and identified the concept of risk factors and their joint effects. Offspring/Omni1 cohort: With the aging of the Framingham cohort and with interest in familiar aggregation and heritability, a new cohort consisting of the offspring of the original cohort was sampled. Spouses of offspring were also included. This new sample, began enrollment in 1971 and constituted a second generation of participants, permitting new assessment of risk factors and outcomes, and provided a resource for the genetic analyses which were yet to come. The Offspring participants have had repeated examinations, though at typically longer intervals than the original cohort. Third Generation/Omni2/New Offspring Cohort: Thirty-one years after enrollment began for the second generation of the Framingham Heart Study (Framingham Offspring Study), Framingham investigators began enrolling adults with at least one parent enrolled in the Offspring study into the Framingham Generation 3 cohort. The addition of the third generation was expected to facilitate investigation of secular trends in risk factors and indicators of health and disease within families, to enhance statistical power to detect genetic and environmental determinants of complex diseases, and to clarify how subclinical cardiovascular disease predicts occurrence of overt clinical events. Participants Original cohort: At entry to the study in 1948-1952, the study recruited 5,209 men and women, ages 28-62 years, living in Framingham, MA. Offspring/Omni1 cohort: 5,124 men and women, ages 5-70 years at entry consisting of offspring of the original Framingham cohort (and spouses of the offspring). In 1994, the Omni Cohort 1 enrolled 507 men and women of African-American, Hispanic, Asian, Indian, Pacific Islander and Native American origins, who at the time of enrollment were residents of Framingham and the surrounding towns. Third Generation/Omni2/New Offspring Cohort: 4095 men and women, almost all who self reported their ethnicity as white, ages 19+ years at entry, with at least one parent in the Framingham Offspring study, participated in the Gen III cohort. The New Offspring Cohort enrolled spouses of Offspring participants that were not otherwise enrolled and had at least two biological children participating in Gen III. 103 New Offspring Spouses (47 men and 56 women) participated. The OMNI 2 cohort enrolled additional ethnically diverse participants, including many individuals related to the participants of Omni Cohort 1 and also individuals unrelated to Omni Cohort 1 members for a total of 410 new participants. Design The cardiovascular disease conditions under investigation include coronary heart disease (angina pectoris, myocardial infarction, coronary insufficiency and sudden and non-sudden death), stroke, hypertension, peripheral arterial disease and congestive heart failure. Original cohort: The Framingham Study is a longitudinal investigation of constitutional and environmental factors influencing the development of CVD in men and women. Examination of participants has taken place every two years and the cohort has been followed for morbidity and mortality over that time period. Offspring/Omni1 cohort: By 1975, a sample of 5,124 men and women, consisting of offspring of the original Framingham cohort (and spouses of the offspring) had participated in the study. Additional studies of these subjects have continued under research contracts. Third Generation/Omni2/New Offspring Cohort: The children of Offspring Cohort participants were initially identified who would be 20 years of age or older by the end of the enrollment period. A higher priority for recruitment was assigned to individuals who belonged to large extended families, in order to complement phenotypic and genotypic information already obtained from prior generations. The baseline examination was begun in 2002 and completed in 2005.

  Study phs003594

• Institut Curie Neuroblastoma Whole Genome Sequencing Diagnosis Relapse

  Neuroblastoma, a clinically heterogeneous pediatric cancer, is characterized by distinct genomic profiles but few recurrent mutations. As neuroblastoma is expected to have high degree of genetic heterogeneity, study of neuroblastoma's clonal evolution with deep coverage whole-genome sequencing of diagnosis and relapse samples will lead to a better understanding of the molecular events associated with relapse. Samples were included in this study if sufficient DNA from constitutional, diagnosis and relapse tumors was available for WGS. Whole genome sequencing was performed on trios (constitutional, diagnose and relapse DNA) from eight patients using Illumina Hi-seq2500 leading to paired-ends (PE) 90x90 for 6 of them and 100x100 for two. Expected coverage for sample NB0175 100x100bp was 30X for tumor and constitutional samples. For the seven other patients expected coverage was 80X for tumor samples with PE 100x100, 100X in the other tumor samples and 50X for all constitutional samples (see table 1). Following alignment with BWA (Li et al., Oxford J, 2009 Jul) allowing up to 4% of mismatches, bam files were cleaned up according to the Genome Analysis Toolkit (GATK) recommendations (Van der Auwera et al., Current Protocols in Bioinformatics, 2013, picard-1.45, GenomeAnalysisTK-2.2-16). Variant calling was performed in parallel using 3 variant callers: GenomeAnalysisTK-2.2-16, Samtools-0.1.18 and MuTect-1.1.4 (McKenna et al., Genome Res, 2010; Li et al., Oxford J, 2009 Aug; Cibulskis et al., Nature, 2013). Annovar-v2012-10-23 with cosmic-v64 and dbsnp-v137 were used for the annotation and RefSeq for the structural annotation. For GATK and Samtools, single nucleotide variants (SNVs) with a quality under 30, a depth of coverage under 6 or with less than 2 reads supporting the variant were filter out. MuTect with parameters following GATK and Samtools thresholds have been used to filter our irrelevant variants. .SNVs within and around exons of coding genes overlapping splice sites.. Then,variants reported in more than 1% of the population in the 1000 genomes (1000gAprl_2012) or Exome Sequencing Project (ESP6500) have been discarded in order to filter polymorphisms. Finally, synonymous variants were filtered out. MuTect focuses on somatic by filtering with constitutional sample. Mpileup comparison between constitutional and somatic DNAs allowed us to focus also on tumor specific SNVs with GATK and Samtools. Finally, every SNV called by our pipeline and also supported in any constitutional samples were filtered our in order to prevent putative constitutional DNA coverage deficiency. Then we analyzed CNVs (copy number variants) with HMMcopy-v0.1.1 (Gavin et al., Genome Res, 2012) and control-FREEC-v6.7 (Boeva et al., Bioinformatics 2011) with a respective window of 2000bp and 1000 bp, and auto-correction of normal contamination of tumor samples for Control-FREEC. Finally we explored Structural variants (SVs) including deletions, inversions, tandem duplications and translocations using DELLY-v0.5.5 with standard parameters (Rausch et al., Oxford J, 2012). In tumors, at least 10 supporting reads were required to make a call and 5 supporting reads for the sample NB0175 with a coverage of only 40X (see table 2). To predict SVs in constitutional samples for subsequent somatic filtering, only 2 supporting reads were required in order not to miss one. To identify somatic events, all the SVs in each normal sample were first flanked by 500 bp in both directions and any SVs called in a tumor sample which was in the combined flanked regions of respective normal sample was removed (see graph 1). Deletions with more than 5 genes impacted or larger than 1Mb and inversions or tandem duplications covering more than 4 genes, were removed. We focused on exonic and splicing events for deletions, inversions, and tandem duplications. For translocation, we keep all SVs that occurred in intronic, exonic, 5'UTR, upstream or splicing regions. Bioinformatics detection of variations with Deep sequencing approach Once PE reads merged and adaptors trimmed by SeqPrep with default parameters, merged reads were aligned via the BWA (Li H. and Durbin R. 2009 PMID 19451168) allowing up to 1 differences in the 22-base-long seeds and reporting only unique alignments. Only reads having a mapping quality 20 or more have been further analysed. Variant calling software was not used, since we aimed to predict variations at low frequencies, observed in less than 1% of reads. Such variants require a custom approach. Using DepthOfCoverage functions of the Genome Analysis Toolkit (GATK) v2.13.2 (McKenna A, et al., 2010 Genome Research PMID: 20644199), we focused on high quality coverage of bases A, C, G and T at the targeted variant position. Depth of coverage of each base following a mapping quality higher than 20 and a base quality higher than 10 have been taken into account in order to focus only on high quality data. Aiming to determine the background level of variability at the studied regions, 10 control samples were included in the analysis. The same approach and filtering criteria have been applied as introduced above over the entire amplicons. In order to highlight variants, for each sample the frequencies of each bases at each amplicon position were then compared to those observed in the set of controls. Statistical analyses were performed with the R statistical software (http://www.R-project.org). Fisher’s exact two-sided tests with a Bonferroni correction were performed to compare percentages of bases between the data sets, i.e. for a given base between a case and the controls. Finally, significant variations were filtered-in once (i) a significant increase in the percentage of avariant base and (ii) a significant decrease in the percentage of it's reference base following our p.values criteria was observed (p.val < 0.05).

  Study EGAS00001001184

• University of Pennsylvania CAR T Cell Responding and Non-responding Patients

  The adoptive transfer of autologous T cells genetically modified to express a CD19-specific, 4-1BB/CD3zeta-signaling chimeric antigen receptor (CAR; CTL019) has shown remarkable activity in patients with B acute lymphoblastic leukemia. Similar therapy can induce long-term remissions for relapsed/refractory chronic lymphocytic leukemia (CLL) patients, but in only a small subset of subjects. The determinants of response and resistance to CTL019 therapy of CLL are not fully understood. We employed next generation sequencing of RNA (RNA-seq) to identify predictive indicators of response to CTL019 treatment. We performed RNA-seq on leukapheresis and manufactured infusion product T cells from patients with heavily pre-treated and high-risk disease. To characterize potency, we also performed RNA-seq on the cellular infusion product after CAR-specific stimulation. Our findings indicate that durable remission in CLL is associated with gene expression signatures of early memory T cell differentiation (e.g., STAT3), while T cells from poorly- or non-responding patients exhibited elevated expression of key regulators of late memory as well as effector T cell differentiation, apoptosis, aerobic glycolysis, hypoxia and exhaustion. These gene expression signatures, along with additional immunological biomarkers, may be used to identify which patients are most likely to respond to cellular therapies and suggest manufacturing modifications that might potentiate the generation of maximally efficacious infusion products. In a follow-up study, we report multiplex CRISPR-Cas9 gene editing on T cells in humans to test the hypothesis that disrupting genes encoding the endogenous T cell receptor (TCR), TCRα (TRAC) and TCRβ (TRBC), as well as PD-1 (PDCD1) would enhance the efficacy of autologous T cells engineered to express a transgenic NY-ESO TCR. We used single cell RNA-seq to comprehensively characterize the transcriptomic phenotype of this T cell product and its evolution over time in a patient with sarcoma. Notably, this patient's transferred T cells that persisted in vivo demonstrated increases in expression of genes associated with a central memory state (IL7R, TCF7). These findings may guide future trials involving administration of genetically redirected, genome -edited T cells.In this follow-up study, we investigated whether CRISPR-Cas9-mediated disruption of PDCD1 and/or CTLA-4 could restore CART dysfunction in healthy donors and/or CLL patients. We show that depletion of CTLA-4 rescues the function of T cells from leukemia patients that previously failed CART cell treatment. We performed single-cell RNA sequencing and characterized the transcriptomic landscape of CTLA-4 KO CART19 cells that showed higher proliferation and anti-tumor efficacy by permitting unopposed CD28 signaling and maintenance of CAR expression on the T cell surface under stress-test conditions of chronic antigen exposure. Our findings reveal that selective disruption of CTLA-4 invigorates dysfunctional patient T cells, providing a strategy for increasing patient responses to CART cell therapy.

  Study phs001707

• Coagulation and Fibrinolysis in a Pediatric Insulin Titration Trial

  Summary of the Design and Aims: The Coagulation and Fibrinolysis in a Pediatric Insulin Titration Trial (CAF-PINT) is an ancillary study to the HALF PINT (NCT01565941) randomized, controlled trial that was originally designed to study changes in inflammatory and thrombosis pathways in the setting of an interventional trial. The parent HALF PINT study was designed to study the impact of tight glycemic control (TGC) using an explicit insulin titration algorithm to safely achieve a normal glucose in target range of 80-110 mg/dL versus 150-180mg/dL on clinical outcomes among critically ill hyperglycemic children with Heart and Lung Failure. The CAF PINT study collected blood samples pre randomization and at 2 and 4 days after randomization. Blood samples collected in EDTA tubes were centrifuged at local sites to separate the plasma from cell pellets and frozen prior to shipment. In addition, 0.5 mL of whole blood collected in PAXgene tubes modified for use in pediatrics frozen 24 hours after collection prior to shipment.Population Information: Setting: Twenty-one academic children's hospitals (HALF-PINT) and 1 academic children's hospital (IIT-SBPP). Participants: Critically ill hyperglycemic children requiring mechanical ventilation/vasoactive support (n=293).Interventions and Exposures: Patients in HALF PINT were randomized to achieve a low target glucose (80-110 mg/dL) vs. a higher target (150-180 mg/dL). The primary trial was stopped early due to low likelihood of achieving a statistically significant difference in the primary outcomes. Molecular Technologies Employed: Plasma biomarkers were assayed on thawed plasma using a Luminex panel. RNA was extracted from PAXgene Blood RNA Kits modified for pediatric use. Extracted RNA was sequenced with the Novaseq S4 system (Illumina) to generate 2 x 150 base paired end reads to a target depth of 50 million read-pairs per sample.Principal Findings of the Study: We tested for heterogeneity of treatment effects according to baseline blood inflammatory proteins using logistic regression with individual biomarkers as interaction terms and Cox regression analysis using biomarker-derived latent classes. Children with higher inflammatory proteins including TNFR-1, IL-6, IL-8, and IL-10 had lower mortality when treated with insulin to a target glucose of 80-110 mg/dL as compared to 150-180 mg/dL (interaction p<0.05). Causal forest estimates ranged from a 40% mortality reduction to a 15% mortality increase with TGC, depending on each patient’s individual biomarker profile. Latent class categorization strongly interacted with TGC with respect to mortality (Cox regression interaction p=0.005). Specifically, patients with higher inflammation benefited from TGC (6% mortality with TGC vs. 48% mortality, p=0.004) whereas hypoinflamed patients showed a trend towards harm (14% mortality with TGC vs. 7% mortality, p=0.055).Data available through dbGaP: Transcriptomic (Gene Counts) and phenotype data

  Study phs003016

• Heart Failure Network: Inorganic Nitrite Delivery to Improve Exercise Capacity in HFpEF (HFN INDIE-BioLINCC)

  Data Access NOTEPlease refer to the “Authorized Access” section below for information about how access to the data from this accession differs from many other dbGaP accessions.ObjectiveTo determine the effect of inhaled, nebulized inorganic nitrite on exercise capacity in patients with heart failure with preserved ejection fraction. Background Approximately half of patients with heart failure have a preserved ejection fraction (HFpEF). However, there are no proven effective medical treatments for this syndrome. Evidence suggests that impairments in nitric oxide availability have a potentially important role in the pathophysiology of HFpEF. Unlike organic nitrates, inorganic nitrite is converted to nitric oxide in the presence of hypoxia and acidosis, conditions that develop during exercise. Because the cardiac, vascular, and skeletal muscle abnormalities that limit physical capacity and contribute to symptoms in patients with HFpEF characteristically develop during exercise, inorganic nitrite may provide the best way to target nitric oxide delivery precisely at the time of greatest need. The HFN-INDIE trial was initiated to test the hypothesis that compared to placebo, longer-term use of inhaled, nebulized inorganic nitrite would enhance peak exercise capacity in patients with HFpEF. Participants A total of 105 participants were randomized. 53 were randomized to receive nitrite first and 52 were randomized to receive placebo first. Design HFN-INDIE was a multicenter, randomized, double-blind, placebo-controlled, crossover study. After enrollment, patients underwent baseline studies to determine eligibility. All patients were required to display objective exercise limitation, evidenced by reduced peak oxygen consumption (V̇O2) on cardiopulmonary exercise testing of less than 75% predicted, with a respiratory exchange ratio indicative of maximal effort (≥1.0). Following qualifying exercise testing, eligible participants received an open-label, single-dose run-in of inhaled, nebulized sodium nitrite (80 mg) to assess tolerability, symptoms, and orthostatic vital signs. Patients developing hypotension (systolic blood pressure The prespecified primary end point was peak V̇o2, measured as the highest 30-second average during upright cycle ergometry, during the 4-week period in which patients were receiving inorganic nitrite as compared with placebo. Accelerometry, health-related quality-of-life scores on the self-administered Kansas City Cardiomyopathy Questionnaire (score range, 0-100, with higher scores indicating better quality of life), echocardiographic indicators of cardiac filling pressures measured at trough drug levels (E/e' ratio, estimated pulmonary artery systolic pressure, and left atrial volume index; lower scores indicate better health for all), ventilatory efficiency (VE/V̇co2, lower indicating better health), exercise time (higher indicating better health), and NT-proBNP levels (lower indicating better health) were also collected. Conclusions Among patients with HFpEF, administration of inhaled inorganic nitrite for 4 weeks, compared with placebo, did not result in significant improvement in exercise capacity. Borlaug et al., 2018, PMID: 30398602.

  Study phs003667

• The Finland-United States Investigation of NIDDM Genetics (FUSION) Study - Islet Expression and Regulation by RNAseq and ATACseq

  This study evaluates gene expression and its regulation in human pancreatic islets, a tissue relevant in the study of genetic risk factors contributing to diabetes. We obtained pancreatic tissue sections and purified pancreatic islets from deceased donors and processed these biological samples to generate various data types. We generated spatial transcriptomic data from pancreatic tissue samples using the 10X Genomics VISIUM technology with sequencing on the Illumina NovaSeq platform. We also generated multiple data types from the purified pancreatic islets using genome-wide SNP chips, bulk RNA-Seq, microRNA (miRNA)-Seq, whole genome sequence, DNA methylation (methyl)-Seq, cap analysis of gene expression (CAGE)-Seq, single cell RNA-seq, and single nuclei ATAC-seq approaches. These data include ATAC-seq of two islet subjects, RNA-seq of 31 additional subjects, genome-wide chip genotypes, and imputed genotypes of the 33 subjects released with phs001188.v1. For genotyping, 500-1000 islet equivalents (IEQ) were cultured as in Gershengorn (Science, 2004, PMID: 15564314); genomic DNA isolated from islet cultures. For RNA analyses, 2500-5000 IEQ from each islet source were used for bulk or single-cell RNA isolation. Messenger RNA was isolated with trizol extraction and 12-plex libraries were generated using the Illumina TruSeq directional mRNA-seq library protocol. Bulk RNA sequencing was performed on HiSeq2000/HiSeq2500/Hiseq4000/NovaSeq6000 sequencers using paired-end reads at the NIH Intramural Sequencing Center (NISC). miRNA libraries were prepared from total RNA from 68 samples, pooled and sequenced 50bp single-end reads on Illumina HiSeq2500. CAGE libraries were prepared from total RNA samples using the nAnT-iCAGE protocol at DNAFORM, Japan. CAGE libraries were sequenced at the NIH Intramural Sequencing Center (NISC) on the HiSeq2000 sequencer. Genotyping on the Illumina Omni2.5M array was performed at the NHGRI Genomics Core facility. Genotypes were imputed using the HRC.r1.1.2016 reference panel. To assess regions of open chromatin in islets, we performed bulk ATAC-seq on HiSeq2000 sequencers using paired-end reads at NISC. Single-nuclei ATAC-seq libraries were prepared using single-cell-combinatorial-indexing (sci-) ATAC-seq protocol and sequenced on Illumina NextSeq using paired-end reads. scRNA-seq libraries were generated using the 10X Genomics platform and sequenced on Illumina HiSeq3000 at the Genomics Technology Core of the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS). Greater than 90% of the loci associated with T2D through genome-wide association studies occur in non-coding regions, suggesting a strong regulatory component to disease susceptibility. Therefore, there is a critical need to understand the full spectrum of genetic variation and regulatory element usage in T2D-relevant tissues. To that end, this study contains multiple genomic and transcriptomic data sets from pancreas tissue sections and pancreatic islets, a resource that will contribute to our efforts to investigate the genetic risk factors for type 2 diabetes.

  Study phs001188

• Immunophenotyping in a COVID-19 Cohort (IMPACC) Transcriptomics and Genotyping Assays

  IMPACC (IMmunoPhenotyping Assessment in a COVID-19 Cohort) is a prospective longitudinal cohort study (NCT04378777) launched in May 2020 in response to the worldwide pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It was funded by the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institute of Health (NIH). Patient enrollment and sample analysis involved 20 hospitals associated with 15 U.S. biomedical research centers and 11 centralized Core immunoassay laboratories.The study aimed to enable the identification of potential biomarkers and inform future therapeutic interventions using a longitudinal approach encompassing the whole disease course for a comprehensive understanding of the contributions of the pathogen and host immune response in modulating the manifestations, severity, and post-acute sequelae of the infection. IMPACC enrolled close to 1200 adult symptomatic, molecularly confirmed hospitalized COVID-19 patients in the study within 48 hours of their hospital admission. Clinical data and biological samples (blood, mid-turbinate nasal swabs, and esophageal aspirant) were collected according to minimal risk guidelines at enrollment and on days 4, 7, 14, 21, and 28. Biological samples were collected at 3-month intervals up to 12 months after hospital discharge to assess functional and immunologic recovery measures. Sample collection, processing, and storage procedures were standardized across sites, and samples were transported to centralized assay-specific core laboratories (Core Labs) in batches for testing and analysis. The description of outcome severity was not limited to survival versus death but encompassed a clinical trajectory approach using 5 groups leveraging longitudinal data based on time in the hospital, disease severity by ordinal scale based on the degree of respiratory illness, and presence or absence of limitations at discharge. Disease severity was assessed based on the degree of respiratory illness using a 7-point ordinal scale adapted from the World Health Organization COVID-19 and NIAID disease ordinal severity scales. DNA for genetic analyses was collected from whole blood at a single time point. To identify any genomic determinants of severe COVID-19 disease, single-nucleotide polymorphism (SNP) genotyping and exome sequencing were conducted to assess genetic variants associated with individual susceptibility to severe disease. In addition, IMPACC included bulk transcriptomic analysis of upper airway (nasal swaps) samples, lower airway (esophageal aspirant) samples, and peripheral blood mononuclear cells (PBMCs) to evaluate dynamic changes in cellular composition and cellular response during the course of the disease. Reads were aligned to the GRCh38 human reference genome. Additional information for the IMPACC study has been submitted to The Immunology Database and Analysis Portal (ImmPort) under study accession SDY1760. Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dr. Steven Kleinstein (steven.kleinstein@yale.edu).

  Study phs002686

• Institut Curie Neuroblastoma Whole Genome Sequencing Diagnosis Relapse

  Neuroblastoma, a clinically heterogeneous pediatric cancer, is characterized by distinct genomic profiles but few recurrent mutations. As neuroblastoma is expected to have high degree of genetic heterogeneity, study of neuroblastoma's clonal evolution with deep coverage whole-genome sequencing of diagnosis and relapse samples will lead to a better understanding of the molecular events associated with relapse. Samples were included in this study if sufficient DNA from constitutional, diagnosis and relapse tumors was available for WGS. Whole genome sequencing was performed on trios (constitutional, diagnose and relapse DNA) from eight patients using Illumina Hi-seq2500 leading to paired-ends (PE) 90x90 for 6 of them and 100x100 for two. Expected coverage for sample NB0175 100x100bp was 30X for tumor and constitutional samples. For the seven other patients expected coverage was 80X for tumor samples with PE 100x100, 100X in the other tumor samples and 50X for all constitutional samples (see table 1). Following alignment with BWA (Li et al., Oxford J, 2009 Jul) allowing up to 4% of mismatches, bam files were cleaned up according to the Genome Analysis Toolkit (GATK) recommendations (Van der Auwera et al., Current Protocols in Bioinformatics, 2013, picard-1.45, GenomeAnalysisTK-2.2-16). Variant calling was performed in parallel using 3 variant callers: GenomeAnalysisTK-2.2-16, Samtools-0.1.18 and MuTect-1.1.4 (McKenna et al., Genome Res, 2010; Li et al., Oxford J, 2009 Aug; Cibulskis et al., Nature, 2013). Annovar-v2012-10-23 with cosmic-v64 and dbsnp-v137 were used for the annotation and RefSeq for the structural annotation. For GATK and Samtools, single nucleotide variants (SNVs) with a quality under 30, a depth of coverage under 6 or with less than 2 reads supporting the variant were filter out. MuTect with parameters following GATK and Samtools thresholds have been used to filter our irrelevant variants. .SNVs within and around exons of coding genes overlapping splice sites.. Then,variants reported in more than 1% of the population in the 1000 genomes (1000gAprl_2012) or Exome Sequencing Project (ESP6500) have been discarded in order to filter polymorphisms. Finally, synonymous variants were filtered out. MuTect focuses on somatic by filtering with constitutional sample. Mpileup comparison between constitutional and somatic DNAs allowed us to focus also on tumor specific SNVs with GATK and Samtools. Finally, every SNV called by our pipeline and also supported in any constitutional samples were filtered our in order to prevent putative constitutional DNA coverage deficiency. Then we analyzed CNVs (copy number variants) with HMMcopy-v0.1.1 (Gavin et al., Genome Res, 2012) and control-FREEC-v6.7 (Boeva et al., Bioinformatics 2011) with a respective window of 2000bp and 1000 bp, and auto-correction of normal contamination of tumor samples for Control-FREEC. Finally we explored Structural variants (SVs) including deletions, inversions, tandem duplications and translocations using DELLY-v0.5.5 with standard parameters (Rausch et al., Oxford J, 2012). In tumors, at least 10 supporting reads were required to make a call and 5 supporting reads for the sample NB0175 with a coverage of only 40X (see table 2). To predict SVs in constitutional samples for subsequent somatic filtering, only 2 supporting reads were required in order not to miss one. To identify somatic events, all the SVs in each normal sample were first flanked by 500 bp in both directions and any SVs called in a tumor sample which was in the combined flanked regions of respective normal sample was removed (see graph 1). Deletions with more than 5 genes impacted or larger than 1Mb and inversions or tandem duplications covering more than 4 genes, were removed. We focused on exonic and splicing events for deletions, inversions, and tandem duplications. For translocation, we keep all SVs that occurred in intronic, exonic, 5'UTR, upstream or splicing regions. Bioinformatics detection of variations with Deep sequencing approach Once PE reads merged and adaptors trimmed by SeqPrep with default parameters, merged reads were aligned via the BWA (Li H. and Durbin R. 2009 PMID 19451168) allowing up to 1 differences in the 22-base-long seeds and reporting only unique alignments. Only reads having a mapping quality 20 or more have been further analysed. Variant calling software was not used, since we aimed to predict variations at low frequencies, observed in less than 1% of reads. Such variants require a custom approach. Using DepthOfCoverage functions of the Genome Analysis Toolkit (GATK) v2.13.2 (McKenna A, et al., 2010 Genome Research PMID: 20644199), we focused on high quality coverage of bases A, C, G and T at the targeted variant position. Depth of coverage of each base following a mapping quality higher than 20 and a base quality higher than 10 have been taken into account in order to focus only on high quality data. Aiming to determine the background level of variability at the studied regions, 10 control samples were included in the analysis. The same approach and filtering criteria have been applied as introduced above over the entire amplicons. In order to highlight variants, for each sample the frequencies of each bases at each amplicon position were then compared to those observed in the set of controls. Statistical analyses were performed with the R statistical software (http://www.R-project.org). Fisher’s exact two-sided tests with a Bonferroni correction were performed to compare percentages of bases between the data sets, i.e. for a given base between a case and the controls. Finally, significant variations were filtered-in once (i) a significant increase in the percentage of avariant base and (ii) a significant decrease in the percentage of it's reference base following our p.values criteria was observed (p.val < 0.05).

  Dataset EGAD00001001356

• A Platform Study of Combination Immunotherapy for the Neoadjuvant and Adjuvant Treatment of Patients with Surgically Resectable Adenocarcinoma of the Pancreas

  This is a single-institutional, randomized, open label platform clinical trial testing various immunotherapy combinations. Patients with newly diagnosed and surgically resectable pancreatic ductal adenocarcinoma (PDA) and scheduled to undergo the Whipple procedure at Johns Hopkins Hospital were eligible to participate in this study. Criteria for determining resectability strictly followed National Comprehensive Cancer Network (NCCN) guidelines. The study consisted of 6 parts: parts 1-5 constituted the Prime Phase (see below), and part 6 was the Extended Treatment Phase. Part 1: Participants received one cycle of immunotherapy two weeks prior to undergoing the Whipple procedure. Part 2: Subjects underwent the Whipple procedure. Part 3: Subjects received a second cycle of immunotherapy 4-10 weeks following the Whipple procedure (2-4 weeks prior to adjuvant chemoradiation). Part 4: Subjects underwent chemoradiation. Part 5: Subjects received 4 additional 28-day cycles of immunotherapy beginning 1-2 months after completing chemoradiation for a total of six cycles (two before chemoradiation and 4 following chemoradiation). Eligible subjects were randomized to Arms A and B in a 1:1 ratio, stratified up-front by age (≤65, >65). Arm A participants received Cy/GVAX. Arm B participants received the combination of Cy/GVAX and nivolumab. GVAX Vaccine consists of equal numbers (2.5 x 108 each) of Panc 6.03 pcDNA1GM-CSF and Panc 10.05 pcDNA1GM-CSF combined into a single vaccination. Each vaccination consisted of six total intradermal injections. A single intravenous (IV) dose of 200 mg/m2 Cy (CytoxanR) was given one day prior to GVAX. Anti-PD-1 Therapeutic Antibody Nivolumab 480 mg every 4 weeks was administered as an intravenous infusion. The primary objective for Arms A and B was to compare IL17 expression (Th17) in vaccine-induced lymphoid aggregates between resected PDAs from patients treated with the combination of Cy/GVAX and anti-PD-1 blockade antibody vs. the treatment of Cy/GVAX alone. Secondary objectives were to assess the safety, overall survival (OS), and disease free survival (DFS) of patients treated with each of the study drug combinations, and to assess the effects of each of the immunotherapy study drug combinations on PD-L1/PD-1 associated pathways, vaccine-induced immune regulatory signatures, and peripheral and intratumoral antigen specific T cell responses. The exploratory objective was to explore the effects of therapy on tumor and peripheral blood and tumor infiltrating immune cells, and to explore potential molecular determinants of response, progression and disease stability. To these ends, tumor infiltrating immune cells (TIICs) were obtained from surgically resected tumors following the neoadjuvant treatment of the study immunotherapy. TIICs were sorted into CD3+CD4+ T cells, CD3+CD8+ T cells, CD3-CD19+ B cells, and CD3-CD19-CD11b+ myeloid cells by flow cytometry. The sorted immune cell subtype pellets were sent to the commercial vendor (Eurofins) for RNA extraction and sequencing. SMART-seq v4 Ultra Low Input mRNA Sample Prep Kit was used for library preparation with 100bp paired end reads. FASTQ files, that were returned from the vendor, are deposited here.

  Study phs003002

• Atherosclerosis Risk in Communities Study (ARIC-BioLINCC)

  Data Access NOTE:Please refer to the “Authorized Access” section below for information about how access to the data from this accession differs from many other dbGaP accessions. At present, the ARIC sIRB has determined that ARIC data cannot be shared with for-profit entities.Related Studies Other ARIC data available include: Imaging studies (ARIC-Imaging), Genetics and genomics (ARIC, phs000280.v8.p2), Collaborative Cohort of Cohorts for COVID-19 Research (C4R): ARIC (phs002988.v1.p1), and as a component of the Sleep Heart Health Study (SHHS-BioLINCC, phs003637.v1.p1). Available Data Data available for request include ARIC v1-v8 examination cycles, collated annual follow-up communication data for contact years 2-32, and follow-up for mortality, heart disease, and stroke events through 2019. Also included are data from ancillary studies. Objectives The objectives of ARIC are to: 1) investigate associations of factors, including those not previously measured in cohort studies, with prevalence of atherosclerosis and incidence of CHD, clinical stroke and other cardiovascular diseases; and 2) measure cardiovascular disease occurrence and trends and relate these to community levels of, and changes in, risk factors, medical care and atherosclerosis. Background At the time of project initiation, the NHLBI had long recognized the need for longitudinal studies to identify the biochemical and physiological markers and specific environmental factors which place individuals at high risk for the major atherosclerosis diseases. The development of reliable ultrasound examination of peripheral arteries enhanced the expected benefit of such studies. Community surveillance planning began for ARIC in response to recommendations of the 1978 NHLBI Workshop on the Decline in CHD Mortality and has been extended in its purpose to evaluate the large geographic differences in U.S. mortality. Participants Black and white men and women, age 45-64 at entry; sample size: 15,792. Design ARIC is a large-scale, long-term prospective study that measures associations of established and suspected coronary heart disease risk factors with both atherosclerosis and new CHD events in men and women from four geographically diverse communities. The project has two components: community surveillance of morbidity and mortality; and repeated examinations of a representative cohort of men and women in each community. The community surveillance involves abstracting hospital records and death certificates and investigating out-of-hospital deaths. The representative cohorts include approximately 4,000 persons from each community. Community surveillance data includes detailed hospital record abstraction, ECG tracings, and event adjudication. Data from out-of-hospital events in the community include physician, informant, and coroner questionnaires as well as death certificate data and event adjudication. Community surveillance ended in 2014.Cohort participants were examined four times at three year intervals between 1987 and 1998, and have been continuously contacted annually to update their medical histories. Atherosclerosis was measured by carotid ultrasonography. Risk factors studied include: blood lipids, lipoprotein cholesterols, and apolipoproteins; plasma hemostatic factors; blood chemistries and hematology; sitting, supine and standing blood pressures; anthropometry; fasting blood glucose and insulin levels; ECG findings; cigarette and alcohol use; physical activity levels; dietary aspects; and family history. Clinic visits were restarted in 2011 with ARIC visit 5.

  Study phs003738

• Resuscitation Outcomes Consortium Trial of Continuous Compressions Versus Standard CPR in Patients With out-of-Hospital Cardiac Arrest (ROC CCC-BioLINCC)

  Data Access NOTE: Please refer to the “Authorized Access” section below for information about how access to the data from this accession differs from many other dbGaP accessions.Objectives: To compare the rate of survival to hospital discharge after continuous chest compressions versus standard American Heart Association recommended cardiopulmonary resuscitation with interrupted chest compressions in patients with out-of-hospital cardiac arrest.Background: Standard cardiopulmonary resuscitation (CPR) consists of manual chest compressions to maintain blood flow and positive-pressure ventilation to maintain oxygenation until spontaneous circulation is restored. Chest compressions during standard CPR are interrupted frequently by ventilations. These interruptions reduce blood flow and potentially reduce the effectiveness of CPR. In animals with cardiac arrest, the interruption of chest compressions has been associated with decreased survival. Observational studies involving humans with out-of-hospital cardiac arrest of presumed cardiac cause have suggested that continuous compressions are associated with higher rates of survival than interrupted compressions.Participants: Of 35,904 patients with out-of-hospital cardiac arrest who were screened, 26,148 were eligible for participation in the trial and were enrolled in the trial during either the run-in phase or the active-enrollment phase. The active-enrollment phase included 23,711 patients, of whom 12,653 were assigned to the intervention group and 11,058 to the control group.Design: There were 114 participating EMS agencies across the eight participating Resuscitation Outcomes Consortium (ROC) sites, which were grouped into 47 clusters. Clusters of agencies were randomly assigned, in a 1:1 ratio, to perform continuous chest compressions or interrupted chest compressions during all the out-of-hospital cardiac arrests to which they responded. Each cluster was required to begin enrolling patients in a run-in phase to demonstrate adherence to the protocol before entering the active-enrollment phase of the trial. Twice per year, each cluster was crossed over to the other resuscitation strategy.Patients assigned to the group that received continuous chest compressions (intervention group) were to receive continuous chest compressions at a rate of 100 compressions per minute, with asynchronous positive-pressure ventilations delivered at a rate of 10 ventilations per minute. Patients assigned to the group that received interrupted chest compressions (control group) were to receive compressions that were interrupted for ventilations at a ratio of 30 compressions to two ventilations; ventilations were to be given with positive pressure during a pause in compressions of less than 5 seconds in duration. The primary outcome was the rate of survival to hospital discharge. Secondary outcomes included neurologic function at discharge, which was measured with the use of the modified Rankin scale, and adverse events. Conclusions: During the active-enrollment phase, 1129 of 12,613 patients in the continuous chest compression group and 1072 of 11,035 patients in the interrupted chest compressions group survived to hospital discharge.In patients with out-of-hospital cardiac arrest, continuous chest compressions during CPR performed by EMS providers did not result in significantly higher rates of survival or favorable neurologic function than did interrupted chest compressions.

  Study phs003901

• Inorganic Nitrite Delivery to Improve Exercise Capacity in Heart Failure with Preserved Ejection Fraction (INDIE-HFpEF): Heart Failure Network (HFN INDIE-Imaging)

  Data Access NOTE: Please refer to the "Authorized Access" section below for information about how access to the data from this accession differs from many other dbGaP accessions. Objective: To determine the effect of inhaled, nebulized inorganic nitrite on exercise capacity in patients with heart failure with preserved ejection fraction.Background: Approximately half of patients with heart failure have a preserved ejection fraction (HFpEF). However, there are no proven effective medical treatments for this syndrome. Evidence suggests that impairments in nitric oxide availability have a potentially important role in the pathophysiology of HFpEF.Unlike organic nitrates, inorganic nitrite is converted to nitric oxide in the presence of hypoxia and acidosis, conditions that develop during exercise. Because the cardiac, vascular, and skeletal muscle abnormalities that limit physical capacity and contribute to symptoms in patients with HFpEF characteristically develop during exercise, inorganic nitrite may provide the best way to target nitric oxide delivery precisely at the time of greatest need. The HFN-INDIE trial was initiated to test the hypothesis that compared to placebo, longer-term use of inhaled, nebulized inorganic nitrite would enhance peak exercise capacity in patients with HFpEF. Participants: A total of 105 participants were randomized. 53 were randomized to receive nitrite first and 52 were randomized to receive placebo first.Design: HFN-INDIE was a multicenter, randomized, double-blind, placebo-controlled, crossover study. After enrollment, patients underwent baseline studies to determine eligibility. All patients were required to display objective exercise limitation, evidenced by reduced peak oxygen consumption (V̇o2) on cardiopulmonary exercise testing of less than 75% predicted, with a respiratory exchange ratio indicative of maximal effort (≥1.0). Following qualifying exercise testing, eligible participants received an open-label, single-dose run-in of inhaled, nebulized sodium nitrite (80 mg) to assess tolerability, symptoms, and orthostatic vital signs. Patients developing hypotension (systolic blood pressure The prespecified primary end point was peak V̇o2, measured as the highest 30-second average during upright cycle ergometry, during the 4-week period in which patients were receiving inorganic nitrite as compared with placebo. Accelerometry, health-related quality-of-life scores on the self-administered Kansas City Cardiomyopathy Questionnaire (score range, 0-100, with higher scores indicating better quality of life), echocardiographic indicators of cardiac filling pressures measured at trough drug levels (E/e′ ratio, estimated pulmonary artery systolic pressure, and left atrial volume index; lower scores indicate better health for all), ventilatory efficiency (VE/V̇co2, lower indicating better health), exercise time (higher indicating better health), and NT-proBNP levels (lower indicating better health) were also collected.Data Availability: Data available from this study includes transthoracic echocardiogram images from multiple timepoints. There are 199 echocardiographic exams available, totaling over 13,100 individual echocardiogram images. Conclusions: Among patients with HFpEF, administration of inhaled inorganic nitrite for 4 weeks, compared with placebo, did not result in significant improvement in exercise capacity.Reference: Borlaug et al., 2018, PMID: 30398602.

  Study phs003804

• Lipid Research Clinics - Prevalence Study (LRC-PS-BioLINCC)

  Data Access NOTE: Please refer to the “Authorized Access” section below for information about how access to the data from this accession differs from many other dbGaP accessions. Objectives: The LRC Program began in 1971 under the sponsorship of the National Heart, Lung, and Blood Institute, National Institutes of Health. Part of this program was the Lipid Research Clinics Prevalence Study, a standardized series of cross-sectional surveys of various North American populations designed to determine the prevalence of dyslipidemias and to describe the distributions of lipids and lipoproteins in major ethnic and social groups. In addition to contributing to the aggregate analysis, each independent population-based study was designed with capabilities for separate analyses of lipid and lipoprotein distributions. The participating populations were not selected to be a probability sample representative of the North American population per se, but by virtue of their size and economic and geographical diversity, they provide a useful cross-sectional group. The Family Study was the third phase of the Lipids Research Clinics North American Population Studies. The Family Study was designed to obtain knowledge of the distribution of lipids and lipoproteins among family members and of the association of familial and genetic attributes to dyslipoproteinemias. Background: An association between serum cholesterol and coronary heart disease is well established. A system was developed for classifying hyperlipoproteinemias into six types of patterns, as a basis for characterizing lipoprotein disorders. The Lipid Research Clinics was thus created to improve the detection of and clinical management of hyperlipoproteinemias. The three primary objectives were: 1) to acquire data on the prevalence of different types of hyperlipoproteinemia in various age and ethnic groups, with special emphasis on the nature and frequency of genetic forms; 2) to collect reliable data on the prevalence and incidence of atherosclerosis in different types of hyperlipoproteinemia; and 3) to conduct an intervention trial to determine if lowering plasma lipid levels would reduce the risk of CHD. Participants: There were 60,495 participants. Conclusions: Data from this study confirm findings from earlier studies in developed countries, showing age-related differences in plasma lipid levels. However, for overall distributions, the LRC data showed slightly lower cholesterol and markedly higher trigylceride values than those previously reported for North America. Some variation in plasma lipid values was evident among the clinic populations. The large number of participants within most subgroups permitted a variety of analytic and comparative studies. For example, data from the large pediatric population revealed a drop in plasma cholesterol levels in adolescent males and females. Males aged 20-50 years had higher cholesterol levels than females in the same age group, and higher trigylceride levels between ages 20-70 years. Numbers were also significient for meaningful comparisons between lipid distributions of females who were taking sex hormones and those who were not; in females taking sex hormones, cholesterol and triglyceride levels were higher for subjects younger than 45 years, but slightly lower after age 45, than lipid levels in females not taking hormones (Circulation 1979; 60(2):427-439).

  Study phs003995

• Studying Glioblastoma in a Human Organoid Tumor Transplantation Model

  This study comprises two complementary investigations that leverage innovative human model systems and cutting-edge molecular profiling to dissect glioblastoma (GBM) heterogeneity and tumor-microenvironment interactions. The first study uses a novel Human Organoid Tumor Transplantation (HOTT) model, in which freshly dissociated GBM tumor cells from five patients were transplanted into human embryonic stem cell-derived cortical organoids. This system enables the interrogation of tumor and microenvironment interactions in a human-specific, developmentally relevant context. Using single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA-seq (snRNA-seq), the authors profiled over 216,000 cells, analyzing tumor heterogeneity across multiple media conditions. Ligand-receptor analysis (CellChat), immunofluorescence, and RNAScope were used to validate findings. The study revealed that the HOTT model faithfully recapitulates primary GBM cell types and microenvironmental components, and uncovered a novel bidirectional signaling axis mediated by PTPRZ1 that influences both tumor migration and cell fate in a context-dependent manner. PTPRZ1 is identified to be important for intercellular communication between tumor and microenvironmental cells, while PDGFRB, NOTCH3, HOPX, FAM107A, CLDN5, RGS5, FOXC1, SOX18 are key markers of the neurovascular progenitor that we identify to be a multipotent, restricted progenitor in GBM. Related datasets include the inclusion of immune cells in the HOTT system, termed “iHOTT”. The second study focuses on identifying and characterizing a rare but functionally potent neurovascular progenitor (NVP) population within GBM tumors. By generating a meta-atlas from seven publicly available GBM scRNA-seq datasets, the authors identified a unique tumor-derived cell population co-expressing neural progenitor and vascular markers. This population was validated in situ and in vivo through immunostaining and lineage tracing via DNA barcoding in the HOTT system. Functional knockdown of NVPs in a murine GBM model resulted in reduced cycling cells, altered tumor composition, and increased survival. Clonal analysis showed NVPs have dual potency to give rise to both neuronal and vascular-like tumor cells, contributing significantly to tumor cell diversity. Molecular technologies used across the studies include: Single-cell and single-nucleus RNA sequencing DNA barcoding and clonal lineage tracing (CellTag) Population information: Tumor samples were obtained directly from multiple GBM patients (all IDH1 wild-type, adult), including core and peripheral tumor regions. Human cortical organoids derived from human embryonic stem cells (hESCs) served as the microenvironment in the HOTT model. Principal findings include: HOTT recapitulates the heterogeneity and cell type fidelity of GBM, while enabling the study of tumor–microenvironment signaling. PTPRZ1 has opposing roles in tumor and microenvironment, influencing GBM cell migration and fate via non-catalytic mechanisms. A novel GBM neurovascular progenitor population (NVP) was identified, validated, and shown to contribute clonally to distinct tumor cell types. Elimination of NVPs in vivo reduces tumor aggressiveness and improves survival. Data to be available through dbGaP: Raw and processed single-cell and single-nucleus RNA-seq data (HOTT model and NVP studies) CellTag barcode-labeled scRNA-seq clonal lineage data Associated metadata including patient of origin, experimental condition, and sample annotations.

  Study phs003936

• Transcriptomic analysis of liver CD8+ T cells

  Sorted single CD8+T cells expressing CD14 from human liver for SMARTSeq2. Livers processed: Kucykowicz et al STAR Prot 2022:pubmed.ncbi.nlm.nih.gov/35516846/ Published: Pallett et al Nature 2022 Tissue CD14+CD8+T-cells are reprogrammed by myeloid cells and modulated by LPS The liver is bathed in bacterial products including LPS transported from the intestinal portal vasculature, but maintains a state of tolerance exploited by persistent pathogens and tumours1,2,3,4. The cellular basis mediating this tolerance, yet allowing a switch to immunity or immunopathology, needs to be better understood for successful immunotherapy of liver diseases. Here we show that a variable proportion of CD8+T-cells compartmentalised in the human liver co-stain for CD14 and other prototypic myeloid membrane proteins; CD14+CD8+T-cells preferentially accumulate within the donor pool in liver allografts, amongst hepatic virus-specific and tumour-infiltrating responses, and in cirrhotic ascites. CD14+CD8+T-cells exhibit increased turnover, activation and constitutive immunomodulatory features with high homeostatic IL-10/IL-2 production directly ex vivo, and an enhanced capacity to rapidly produce antiviral/anti-tumour cytokines upon TCR engagement. This CD14+CD8+T-cell profile can be recapitulated by acquisition of membrane proteins, including the LPS receptor complex, from mononuclear phagocytes, resulting in augmented tumour killing by TCR-redirected T-cells in vitro. CD14+CD8+T-cells express integrins and chemokine receptors favouring interactions with the local stroma, which can promote their induction through CXCL12. LPS can also increase the frequency of CD14+CD8+T-cells in vitro and in vivo, and skew their function towards the production of chemotactic and regenerative cytokines. Thus, bacterial products in the gut-liver axis and tissue stromal factors can tune liver immunity by driving myeloid instruction of CD8+T-cells with immunomodulatory capacity. For the analysis of the single cell transcriptome two intrahepatic perfusate samples were isolated and frozen at -80 'C for storage prior to transportation to Newcastle, UK. Once thawed in pre-warmed HBSS containing 0.001% DNaseI, intrahepatic leukocytes were sorted by FACS to index classical mononuclear phagocytes (MNP) defined as Singlets, CD3-CD56-CD19-CD14hiHLA-DR+ or CD14± CD8+T-cells as follows: Singlets, CD3+CD56-CD19-CD4-CD8+V7.2- and split in to CD14± based on an isotype control. Single cells were sorted into 96-well skirted LoBind plates (Eppendorf) containing 5 µL lysis buffer (TCL buffer [Qiagen], supplemented with 1% b-mercaptoethanol [Sigma Aldrich; Merck]) on a BD Bioscience FACSFusion. The sorted plates were subsequently immediately sealed, centrifuged, and placed on dry ice to flash-freeze the cell lysate and stored at -80 'C. A modified SMART-seq2 protocol57 was performed on the single flow cytometry sorted-cells as previously described58. After cDNA generation, libraries were prepared (384 cells per library) using the Illumina Nextera XT kit (Illumina). Each library was sequenced to achieve a minimum depth of 1-2 million raw reads per cell using an Illumina HiSeq 4000 using v. 4 SBS chemistry to generate 75-bp paired-end reads.

  Study EGAS00001006885

• Diagnostic Genomic Analysis is Prognostic in AYA ALL Patients Treated on a MRD-Stratified Paediatric Protocol

  Background: Over the last decade, next generation sequencing has enabled classification of multiple new recurrent genomic drivers of acute lymphoblastic leukemia (ALL). We provide a comprehensive genomic survey of adolescent and young adult (AYA) patients treated on the Australasian Leukaemia and Lymphoma Group (ALLG) ALL06 study. ALL06 clinical trial described the efficacy and tolerability of AYA ALL patients aged 15-39 years treated on a single minimal residual disease (MRD)-stratified platform. In this study we have taken the ALL06 cohort and retrospectively performed comprehensive genomic analyses assessing response of AYA patients with various genomic subtypes treated with a paediatric approach. Methods: Genomic risk classification of B- and T-ALL patients (n=57) enrolled to the study was based on mRNA-Sequencing, MLPA, immunophenotyping and cytogenetics. Results: Using this approach, 36/40 (90%) B- and 13/17 (76.5%) T-ALL patients were classified according to genomic risk. We have linked outcome data with each patient’s genomic risk and found a strong correlation between adverse genomic risk and MRD at the end of consolidation, translating to inferior overall and relapse free survival. Patients with adverse risk genomics who achieved negative MRD status had improved responses compared to those with persistent MRD. Patients with standard risk genomics had excellent responses regardless of their MRD status. Conclusion: Our data argue strongly for incorporation of a genomic risk classification into future ALL treatment paradigms at the time of diagnosis.

  Study EGAS50000000752

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

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

  Study EGAS00001005737

• NIPT samples for systematic evaluation of BinDel, a software tool for detecting clinically significant microdeletions in low-coverage WGS-based NIPT samples

  This dataset contains three sets of samples. The first sample set contains euploid fetus pregnancies reported by NIPTIFY screening test and postnatal evaluation. Dataset was processed similarly to previously published guidelines from KU Leuven, with modifications [1]. Briefly, peripheral blood samples were collected in cell-free DNA BCT tubes (Streck, USA), and plasma was separated with standard dual centrifugation. Cell-free DNA was extracted from 3 ml plasma using MagMAX Cell-Free DNA Isolation Kit (ThermoFisher Scientific). Whole-genome libraries were prepared using the FOCUS (Fragmented DNA Compact Sequencing Assay, Competence Centre on Health Technologies, Estonia) NIPT method protocol with 12 cycles for the final PCR enrichment step. In the following quantification, equal amounts of 36 samples were pooled, and the quality and quantity of the pool were assessed on Agilent 2200 TapeStation (Agilent Technologies, USA). Whole genome sequencing was performed on the NextSeq 550 instrument (Illumina Inc.) with an average coverage of 0.32× (minimum 0.08 and maximum 0.42) and producing 85 bp single-end reads. The second sample set contains a single NIPT sample postnatally diagnosed with Prader-Willi syndrome. The sample was sequenced with Illumina NextSeq 500 platform, producing 85 bp single-end reads with an average per-sample coverage of 0.32× at the University of Tartu, Institute of Genomics Core Facility, according to the manufacturer’s standard protocols, as described previously [2]. The third sample set contains samples SC005 (SeraCare Life Sciences Inc lot #10446565), SC0042 (#10571706), and SC016 (#10560229). These are SeraCare Life Sciences Inc circulating cell-free DNA (ccfDNA) like mixture of human genomic DNA that consists of matched maternal and fetus. SC005 and SC0042 consist of matched DNA of maternal and fetus with DiGeorge Syndrome. SC016 is a custom-ordered DNA Mix with fetus DNA having a pathogenic loss of the terminal region of 20p13 and a pathogenic 3q29 duplication. SC016 was processed as the first sample set was processed, and SC0042 was processed as the second sample set was processed. Sample SC005 was processed once as was sample set 1 and once as was sample set 2 processed. This study was performed with the approval of the Research Ethics Committee of the University of Tartu (#352/M-12). 1. Bayindir B, Dehaspe L, Brison N, Brady P, Ardui S, Kammoun M, et al. Noninvasive prenatal testing using a novel analysis pipeline to screen for all autosomal fetal aneuploidies improves pregnancy management. Eur J Hum Genet. 2015;23: 1286– 1293. doi:10.1038/ejhg.2014.282 2. Žilina O, Rekker K, Kaplinski L, Sauk M, Paluoja P, Teder H, et al. Creating basis for introducing noninvasive prenatal testing in the Estonian public health setting. Prenat Diagn. 2019;39: 1262–1268. doi:10.1002/pd.5578

  Dataset EGAD00001009512

• The Environmental Determinants of Diabetes in the Young Study (TEDDY)

  The Environmental Determinants of Diabetes in the Young (TEDDY) Study investigates genetic and genetic-environmental interactions, including gestational infection or other gestational events, childhood infections and other environmental factors after birth, in relation to the development of pre-diabetic islet autoimmunity and type 1 diabetes (T1D). Beginning in 2002, a consortium of six centers assembled to participate in the development and implementation of studies to identify environmental factors that trigger the development of islet autoimmunity and T1D in genetically susceptible individuals. The TEDDY study screened around 400,000 newborns and recruited 7,749 neonates from the general population with a pre-determined T1D risk of 3% and 919 neonates with first degree relatives who have T1D and who have a pre-determined T1D risk of 10%. Thus, TEDDY proposes to follow over 8,000 participants across six clinical centers worldwide (Finland, Germany, Sweden and three in the United States) until the age of 15. Participants are followed every three months for islet autoantibody measurements with blood sampling until four years of age and then at least every six months until the age of 15. After the age of four, autoantibody positive subjects continue to be followed at 3 month intervals and autoantibody negative subjects are followed at six month intervals. In addition to the analysis of autoantibodies, additional data and sample collection is performed at each visit. The parents collect monthly stool samples in early childhood. The parents also fill out questionnaires at regular intervals in connection with study visits and record information about diet and health status in the child's TEDDY Book between visits. Continued long-term follow-up of the currently active TEDDY participants will provide important scientific information on early childhood diet, reported and measured infections, vaccinations, and psychosocial stressors that may contribute to the development of type 1 diabetes and islet autoimmunity. DNA extracted from the 9-month TEDDY blood sample was used for SNP genotyping. Samples were available for 7,082 participants. SNPs were genotyped by the Center for Public Health Genomics at the University of Virginia, using the Illumina ImmunoChip SNP microarray of around 196,000 SNPs selected from 186 regions associated with 12 autoimmune diseases (including T1D) (Hadley et al., 2015). "Data quality control (QC) steps included eliminating subjects with a low call rate (> 5% SNPs missing) and discrepancies between reported gender and prior genotyping at the HLA laboratory. Secondly, SNPs were removed from analysis due to low call rate, Hardy-Weinberg equilibrium P value < 10-6, except for chromosome 6 due to HLA eligibility requirements)" (Törn et al., 2015). QC resulted in a total of 7,023 subjects with data on 176,586 SNPs. Additional information on the TEDDY study are available in the following articles: The Environmental Determinants of Diabetes in the Young (TEDDY) Study. TEDDY Study Group. Annals of the New York Academy of Science, 2008 and TEDDY - The Environmental Determinants of Diabetes in the Young - An Observational Clinical Trial. Annals of the New York Academy of Science, 2006 Details of the TEDDY protocol can be found in The Environmental Determinants of Diabetes in the Young (TEDDY): Genetic Criteria and International Diabetes Risk Screening of 421,000 infants. Pediatric Diabetes, 2011

  Study phs001037

• Genome-Wide Association Study of Anorexia Nervosa (Price Foundation, Klarman Family Foundation, Center for Applied Genomics at the Children's Hospital of Philadelphia, Scripps Translational Sciences Institute Clinical Translational Science Award)

  Twin studies are consistent with substantial heritability in anorexia nervosa (AN), although detecting and confirming individual risk alleles has been difficult, due to small effects of single alleles and limited samples in prior studies. Methods: 1105 AN DNA samples obtained in a multisite study and 3733 pediatric patients of normal body weight were genotyped using the Illumina 610Q chip at The Center for Applied Genomics at The Children's Hospital of Philadelphia. Cases and controls were of European ancestry. Single nucleotide polymorphism (SNP) genotypes were subjected to quality control procedures, including elimination of SNPs with: call rates < 95%, minor allele frequency < 5%, and if Hardy-Weinberg equilibrium testing yielded p < 0.0001. Samples were removed if not of European ancestry or if showing cryptic relatedness. Eight AN DNA samples were deleted for low call rate, and 51 AN samples were deleted for cryptic relatedness. The statistical program, PLINK, was used to calculate a P value for each SNP passing quality control using the Cochran-Armitage trend test. The PennCNV program was used to define copy number variation (CNV). Results: We performed a genome-wide association study (GWAS) using with 1033 AN cases and 3733 controls (genomic inflation factor = 1.08). There were 11 SNPs with P<1X10-5 [(rsID, chromosome location, nearest gene, distance to nearest gene (base-pair), P value) rs6959888, 7q21.13, ZNF804B, 0, 1.63E-06; rs17725255, 20p11.23, CSRP2BP, 39396, 1.72E-06; rs10494067, 1p13.3, NTNG1, 0, 5.83E-06; rs2383378, 14q12, AKAP6, 0, 6.41E-06; rs410644, 5q14.1, SSBP2, 40462, 6.74E-06; rs4479806, 5p14.1, CDH9, 156926, 7.79E-06; rs957788, 13q33.3, FAM155A, 0, 8.11E-06; rs830998, 2q31.1, LRP2, 0, 8.68E-06; rs6782029, 3p25.3, VGLL4, 0, 9.04E-06; rs512089, 9p21.3, ELAVL2, 47984, 9.85E-06; rs3808986, 11q24.3, APLP2, 29217, 9.92E-6]. CNV analyses were performed on 1015 AN cases and 3532 controls. Overall frequencies of common or rare CNVs were similar between cases and controls. Specific rare and large (>500 kb, <1%) CNVs of interest included deletion of a large (~1.4Mbp) region of chr13q12 found in 2 AN samples and at a 10 fold lower frequency in a large sample (72,918) of subjects containing the genes SGCG, SACS, TNFRSF19, MIPEP, PCOTH, C1QTNF9B, SPATA13, MIR2276, and C1QTNF9, and an overlapping deletion and a duplication observed in 3 AN samples overlapping the CNTN6 and CNTN4 genes. Partially overlapping ~ 30 kb duplications were seen in 12 AN cases and in 1 control at the CDC5L gene on chromosome 6. Partially overlapping deletion of the 3' half of the PSTPIP1 gene was seen in 5 AN cases and no controls. Finally, a ~ 60 kb deletion of the PLEC1 gene was observed in 5 AN cases and in none of 3532 controls. Discussion: This is the first GWAS of AN. These preliminary results are consistent with absence of a major gene effect in AN. These results are consistent with genetic risk conveyed by multiple common alleles of small effect, as well as some uncommon CNVs of potentially larger effect. None of the implicated genes has a clear role in eating behavior. It must be emphasized that these results are preliminary and an additional ~ 400 AN cases will be analyzed as time and resources permit. These results require confirmation through study of independent populations of AN women.

  Study phs000679

• Rapid Acceleration of Diagnostics - Underserved Populations (RADx-UP): COVID-19 Testing and Prevention in Correctional Settings

  This study will work with diverse correctional facilities across MN, RI, WA, and FL to characterize the incidence of COVID-19 disease progression and related outcomes. The goal is to identify barriers and facilitators to develop strategies to improve testing in these under-served populations by: Increasing the reach, access, uptake, and impact of COVID-19 testing among incarcerated people and staff in prison and jails. Individuals in congregate settings have been disproportionately affected by COVID-19, with correctional settings accounting for 39 of the 50 largest cluster outbreaks in the country to date. Schema of Correctional Facilities: Correctional facilities are amplifiers of infectious diseases for structural reasons: 1) Dormitory-style living and overcrowded conditions make social distancing nearly impossible 2) They have a focus primarily on security not health 3) Their medical departments are under-resourced and often disconnected from local public health agencies. Correctional facilities must be the focus of urgent intervention to understand the incidence of COVID-19, how it spreads in correctional facilities, and durable long-term strategies to reduce transmission, including testing and vaccines. However, our work from the COVID Prison Project shows that correctional systems have implemented a range of testing strategies, without coordination, such that synthesizing the information on COVID-19 incidence, outcomes and optimal testing strategies (including repeat testing and contact tracing) has been challenging. Incarcerated people decline testing due to concerns about confidentiality, implications for release, and fear of placement in solitary confinement. Through interviews we will identify ethical concerns and potential solutions for COVID-19 testing and vaccine strategies in correctional facilities using a community-engaged strategy. The project's work from the COVID Prison Project shows that correctional systems have implemented a range of testing strategies, without coordination, such that synthesizing the information on COVID-19 incidence, outcomes and optimal testing strategies (including repeat testing and contact tracing) has been challenging. Incarcerated people decline testing due to concerns about confidentiality, implications for release, and fear of placement in solitary confinement. Through interviews we will Identify ethical concerns and potential solutions for COVID-19 testing and vaccine strategies in correctional facilities using a community-engaged strategy. Goals: 1. Identify ethical concerns and potential solutions for COVID-19 testing and vaccine strategies in correctional facilities using a community-engaged strategy. 2. Characterize incidence of COVID-19, disease progression, and related-outcomes and effectiveness of testing and contact tracing in correctional facilities. Participant Selection: a. Potential subjects must be currently incarcerated at key facilities in Washington, Rhode Island, Minnesota, and Florida. These facilities are Everglades Correctional Institution and Homestead Correctional Institution in Florida, Shakopee and Stillwater correctional facilities in Minnesota, the Rhode Island Department of Corrections, and Yakima County Jail in Washington. b. Participating correctional facilities in Florida, Minnesota, Rhode Island, and Washington will provide de-identified data on approximately 16,859 incarcerated adults and correctional staff. Data sources include deidentified data from medical records, administrative data, and policy documents pertaining to COVID-19. c. 3 series of focus groups (6, 12, 18 months) x 3 distinct groups (incarcerated people, medical providers, and correctional leaders) x 4 sites=36 total focus groups over the study period. d. Will utilize 74,812 COVID-19 tests to test and re-test a total of 16,859 incarcerated people and correctional staff for active infection over a six-month period in the FL, MN, and RI state prison systems and the Yakima County Jail in WA.

  Study phs003361

• Heart Failure Network - Nitrate's Effect on Activity Tolerance in Heart Failure with Preserved Ejection Fraction (HFN NEAT-BioLINCC)

  Data Access NOTE: Please refer to the "Authorized Access" section below for information about how access to the data from this accession differs from many other dbGaP accessions. Related StudiesEcho images are available with HFN-NEAT Imaging.ObjectivesTo determine the effect of isosorbide mononitrate on daily activity in patients with heart failure and preserved ejection fraction.Background Nitrates are commonly prescribed for symptom relief in patients with heart failure. Early studies in patients with heart failure with a reduced ejection fraction concluded that long-acting nitrates improve activity tolerance; however, approximately half of heart failure patients have preserved ejection fraction. The effects of nitrates in patients with heart failure and a preserved ejection fraction have not been extensively studied and the overall effect of nitrates on activity tolerance in such patients is uncertain. ParticipantsThere were a total of 110 participants enrolled with 51 participants assigned to receive isosorbide mono-nitrate first and placebo second, and 59 participants assigned to receive placebo first and isosorbide mononitrate second. Design Enrolled participants underwent baseline assessments, including echocardiography, quality-of-life scores, 6-minute walk test distance, and NT-proBNP levels. participants were also supplied with two kinetic activity monitors containing high-sensitivity triaxis accelerometers, to be worn 24 hours per day. The accelerometer measurements were expressed as arbitrary accelerometer units and stored every 15 minutes equaling 96 data points per day. The 15-minute cumulative accelerometer units were totaled over a 24-hour period to provide daily accelerometer units. Participants were assigned to one of two treatment groups: 6 weeks of placebo first with crossover to 6 weeks of isosorbide mononitrate, or 6 weeks of isosorbide mononitrate first with crossover to 6 weeks of placebo. The study drugs were prepared as 30-mg tablets of isosorbide mononitrate and matching placebo. During each 6-week period, participants were instructed to take no study drug for the first 2 weeks followed by one tablet (30 mg daily) for 1 week, two tablets (60 mg once daily) for 1 week, and four tablets (120 mg once daily) until the next study visit, for a treatment duration of at least 2 weeks and up to 4 weeks. After each 6-week period, participants returned to the study center to repeat end-point assessments. The primary outcome for the study was the comparison of average daily accelerometer units during the period in which participants were receiving the 120-mg dose of isosorbide mononitrate compared to the period in which participants received the placebo. Other secondary end points included the 6-minute walk distance and the post-walk Borg dyspnea score, scores on the Kansas City Cardiomyopathy Questionnaire and the Minnesota Living with Heart Failure Questionnaire, and NT-proBNP levels. In addition, participants completed a questionnaire indicating in which period (first, second, no preference) they felt better. Conclusions Participants were active for fewer hours of the day during the 120-mg phase of receipt of isosorbide mononitrate as compared with placebo. Furthermore, during all study-drug regimens combined (30 mg to 120 mg), participants were less active during receipt of isosorbide mononitrate as compared with placebo. There was no significant effect of isosorbide mononitrate on secondary outcomes.

  Study phs003548

• Heart Failure Network - Imaging from Nitrate's Effect on Activity Tolerance in Heart Failure with Preserved Ejection Fraction (HFN NEAT-Imaging)

  Data Access NOTE: Please refer to the “Authorized Access” section below for information about how access to the data from this accession differs from many other dbGaP accessions.Objectives: To determine the effect of isosorbide mononitrate on daily activity in patients with heart failure and preserved ejection fraction.Background: Nitrates are commonly prescribed for symptom relief in patients with heart failure. Early studies in patients with heart failure with a reduced ejection fraction concluded that long-acting nitrates improves activity tolerance; however, approximately half of heart failure patients have preserved ejection fraction. The effects of nitrates in patients with heart failure and a preserved ejection fraction have not been extensively studied and the overall effect of nitrates on activity tolerance in such patients is uncertain.Subjects: There were a total of 110 patients enrolled with 51 patients assigned to receive isosorbide mono-nitrate first and placebo second, and 59 patients assigned to receive placebo first and isosorbide mononitrate second.Design: Enrolled subjects underwent baseline assessments, including echocardiography, quality-of-life scores, 6-minute walk test distance, and NT-proBNP levels. Subjects were also supplied with two kinetic activity monitors containing high-sensitivity triaxis accelerometers, to be worn 24 hours per day. The accelerometer measurements were expressed as arbitrary accelerometer units and stored every 15 minutes equaling 96 data points per day. The 15-minute cumulative accelerometer units were totaled over a 24-hour period to provide daily accelerometer units.Subjects were assigned to one of two treatment groups: 6 weeks of placebo first with crossover to 6 weeks of isosorbide mononitrate, or 6 weeks of isosorbide mononitrate first with crossover to 6 weeks of placebo. The study drugs were prepared as 30-mg tablets of isosorbide mononitrate and matching placebo. During each 6-week period, patients were instructed to take no study drug for the first 2 weeks followed by one tablet (30 mg daily) for 1 week, two tablets (60 mg once daily) for 1 week, and four tablets (120 mg once daily) until the next study visit, for a treatment duration of at least 2 weeks and up to 4 weeks. After each 6-week period, patients returned to the study center to repeat end-point assessments. The primary outcome for the study was the comparison of average daily accelerometer units during the period in which patients were receiving the 120-mg dose of isosorbide mononitrate compared to the period in which patients received the placebo. Other secondary end points included the 6-minute walk distance and the post-walk Borg dyspnea score, scores on the Kansas City Cardiomyopathy Questionnaire and the Minnesota Living with Heart Failure Questionnaire, and NT-proBNP levels. In addition, subjects completed a questionnaire indicating in which period (first, second, no preference) they felt better. Conclusions: Patients were active for fewer hours of the day during the 120-mg phase of receipt of isosorbide mononitrate as compared with placebo. Furthermore, during all study-drug regimens combined (30 mg to 120 mg), patients were less active during receipt of isosorbide mononitrate as compared with placebo. There was no significant effect of isosorbide mononitrate on secondary outcomes.

  Study phs004254

• Small molecule inhibitors of LOXL synergize with 5-AZA to restore erythropoiesis in myeloid neoplasms

  Objectives: Bone marrow (BM) stroma and microenvironmental factors in the BM milieu of myeloid neoplasms are increasingly being recognized as novel perspective therapeutic targets. Lysyl oxidases (LOX/LOXL) are crucial enzymes that cross-link collagen and contribute to the deposition of aberrant stiff extracellular matrix (ECM) in the BM of patients with myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN). Here, we studied whether inhibition of LOX/LOXL enzymes produced by BM-derived MSCs favors the differentiation of MDS/MPN hematopoietic stem cell and progenitor cells (HPSCs) and enhances the effects of 5-azacytidine (5-AZA). Methods: Primary co-cultures of MSCs and HPSCs were established from n=31 MDS/MPN patients and treated with the pan-LOX/LOXL inhibitor PXS-5505, 5-AZA or the combination of both for 4 days. At the end of co-culture, HSPC differentiation was assessed by CFU assays and flow cytometry. In some of the experiments, MSCs monolayers were pre-treated or not with PXS-5505 for 6 days and decellularized using 20 mM ammonia. Subsequently, autologous HSPCs were cultured on ECM matrices in the presence of 5-AZA or PXS-5505 + 5-AZA combination with or without inhibitors of integrin signaling such as blebbistatin, Y-27632 and αvβ3-integrin blocking antibodies. Patient-derived xenografts (PDX) were established in NSG mice based on intrafemoral co-injection of HSPCs and autologous MSCs. PDX were treated with 3 cycles of 5-AZA (75 mg/m2), PXS-5505 (30 mg/kg on alternate days for the duration of 5-AZA treatment) or combination of both. Results: MSCs of MDS and MPN patients displayed significant overexpression of LOX/LOXL transcripts as compared to healthy age-matched controls. Remarkably, LOX/LOXL inhibition in MSCs/HSPCs co-cultures increased erythroid differentiation of patient HSPCs that had suboptimal or no response to 5-AZA alone. From a total of n=31 advanced stage MDS/MPN patients, n=11 (35.5%) responded to the combination treatment, which was evidenced by a significant increase of CD235a+CD45- erythroid cell production compared to 5-AZA/PXS/untreated arms (p<0.05). Of note, combination treatment with 5-AZA + PXS-5505 facilitated differentiation of CD235+ erythroid progenitors from HSPCs sub-clones with low mutational VAFs. The presence of MSCs was necessary for the effects of the combination treatment and required direct cell contacts between MSCs and HSPCs. Moreover, PXS-5505-mediated alterations of the ECM composition alone were sufficient to induce erythroid differentiation. Inhibition of integrin-mediated signaling using blebbistatin, Y-27632 and αvβ3-integrin antibodies completely abrogated the effects of PXS-5505 induced erythroid differentiation. In vivo, the combination of PXS-5505 + 5-AZA favored erythroid differentiation of HSPCs and confirmed the in-vitro results. The combination treatment of 5-AZA + PXS-5505 induced the greatest reduction of disease burden as assessed by decrease of spleen indexes, total engraftment rates, BM fibrosis and mutational VAF profiles. Conclusions: The combination of 5-AZA + LOX/LOXL inhibitor PXS-5505 in MDS and MPN synergistically restores erythropoietic differentiation of primary HSPCs. The effect is dependent of contact interaction between HSPCs and niche components, such as MSCs and MSCs-derived ECM. Our data propose a strong rationale for a BM niche targeted combination treatment with 5-AZA + PXS-5505 in transfusion dependent MDS and MPN, which will be assessed in forthcoming clinical trials.

  Study EGAS00001006174

• Recurrent DNMT3B gene rearrangements are associated with unfavorable outcome in dicentric (9;20)-positive pediatric BCP-ALL

  The dicentric chromosome dic(9;20)(p11∼13;q11), occurs in 2% of pediatric acute lymphoblastic leukemia (ALL) cases. However, the prognostic significance of the dic(9;20) alterations and the mechanism in which these alterations drive leukemogenesis remains elusive. Our study describes the demographic, clinical, prognostic, and molecular characteristics of the dic(9;20)-positive ALL in a cohort of 57 pediatric BCP-ALL patients. Targeted RNA-seq and Array CGH analysis unraveled heterogeneous breakpoints on chromosomes 9 and 20 and frequent deletions of the IKZF1 gene. Together with the intrinsic susceptibility for deletions in CDKN2A, CDKN2B, and PAX5 genes in the dic(9;20)-positive ALL, these patients are eligible for treatment escalation in the AIEOP-BFM ALL 2017 clinical study. Survival analysis revealed poor 5-year event-free survival (pEFS) of 69% in dic(9;20)-positive ALL cases, compared to BCP-ALL patients enrolled in modern treatment studies. Strikingly, we identified six dic(9;20)-positive cases with rearrangements involving the DNMT3B gene, and poor 5-year pEFS of 25% (SE = 20%), compared to 79% (SE = 9%) for the remaining cohort (P = 0.011). Despite the loss of the methyltransferase domain of the DNMT3B gene in most cases with DNMT3B rearrangements, we did not observe major changes in the global methylation patterns. Differential gene expression analysis of DNMT3B-rearranged ALL unraveled a dysregulation of genes involved in hematopoiesis, lymphocyte maturation, and the PI3K/AKT signaling pathway. After confirmation in an independent cohort, DNMT3B-rearranged ALL cases should be considered high-risk for relapse and treated accordingly. We provide clinical data sets of Array CGH, targeted RNA-seq, total RNA-seq, whole genome bisulfite (WGBS) and whole genome DNA sequencing (WGS) obtained from bone marrow (BM) or peripheral blood (PB) mononuclear cells of 57 pediatric patients with dicentric chromosome dic(9;20) positive Acute lymphocytic leukemia (ALL), from which in 6 cases DNMT3B gene rearrangement was identified. This data is complemented by total RNA-seq and WGBS of samples from 4 additional ALL patients with a t(12;21) translocation and ETV6-RUNX1 gene fusion. DNA was isolated from BM or PB lymphocytes using Qiagen QIAamp DNA Blood Midi Kit to perform i) Array CGH of 58 dic(9;20) positive samples by hybridizing 500ng DNA using a Agilent 400K SurePrint G3 Custom CGH Human Genome Microarray (e-Array design 84704) ii) WGBS of 6 DNMT3B rearrangement positive samples and 4 ETV6-RUNX1 positive samples using Tecan TrueMethyl oxBS-Seq module for library preparation and Illumina NovaSeq 6000 platform to run 2x151 cycles iii) WGS of DNMT3B rearrangement positive samples using Illumina Lotus DNA Library Prep Kit followed by sequencing running 2x160 cycles on an Illumina NovaSeq 6000 platform. RNA was isolated from PB lymphocytes using the PerkinElmer Chemagic 360 instrument, followed by i) targeted RNA-seq of 56 dic(9;20) positive samples prepared using Illumina TruSight RNA Pan-Cancer Panel and sequenced on an Illumina MiSeq platform running 2x75 cycles ii) total RNA-seq of 6 DNMT3B rearrangement positive samples and 4 ETV6-RUNX1 positive samples utilizing TruSeq Stranded Total RNA Library Prep Gold kit and running 2x100 cycles on an Illumina NovaSeq 6000 platform.

  Study EGAS00001007383

• Persistence of circulating tumor DNA in breast cancer patients during neoadjuvant treatment is a significant predictor of poor tumor response

  Purpose:Accurate assessment of response during neoadjuvant systemic treatment (NST) poses a major clinical challenge. Therefore, a minimally-invasive assessment of tumor response based on cell-free circulating tumor DNA (ctDNA) may be beneficial to guide treatment decisions.Experimental Design: We profiled 93 genes in tissue from 193 early breast cancer patients. Patient-specific assays were designed to track ctDNA during NST from 145 patients with available plasma. ctDNA presence and levels were correlated with complete pathological response (pCR) and residual cancer burden (RCB) as well as with clinicopathologic characteristics of the tumor to identify potential proxies for ctDNA release. Moreover, we tested the predictive value of driver events identified in the tumor.Results:At baseline, ctDNA could be detected in 63/145 (43.4%) patients and persisted in 25/63 (39.7%) patients at mid-therapy (MT) and 15/63 (23.8%) patients at the end of treatment. ctDNA detection at MT was significantly associated with higher RCB (OR 0.062, 95% CI 0.01-0.48, P=0.0077). Out of 31 patients with detectable ctDNA at MT, 30 patients (96.8%) were non-responders (RCB II, n=8; RCB III, n=22) and only one patient responded to the treatment (RCB I). Considering all 145 patients with baseline (BL) plasma, none of the patients with RCB 0 and only 6.7% of patients with RCB I had ctDNA detectable at MT, while 30.6% and 29.6% of patients with RCB II/III, respectively, had a positive ctDNA result. Conclusion:Overall, our results demonstrate that the detection and persistence of ctDNA at mid-therapy may have the potential to negatively predict response to neoadjuvant treatment and identify patients who will not achieve pCR or be classified with RCB II/III.

  Study EGAS00001005798

• Clonal dynamics after allogeneic haematopoietic cell transplantation using genome-wide somatic mutations - WGS

  Allogeneic haematopoietic cell transplantation (HCT) replaces the stem cells responsible for blood production with those harvested from a donor, and is received by 40,000 patients worldwide each year. To quantify dynamics of long-term stem cell engraftment, we sequenced whole genomes of 2,824 single-cell-derived haematopoietic colonies from blood samples of 10 donor-recipient pairs taken 9-31 years after HLA-matched sibling HCT. With younger donors, 10,000-50,000 stem cells had engrafted and were still contributing to haematopoiesis at time of sampling, but estimates were 10-fold lower with older donors. Engrafted stem cells made multilineage contributions to myeloid, B-lymphoid and T-lymphoid populations, although individual clones often showed biases towards one or other mature cell type. Recipients had lower clonal diversity than matched donors, equivalent to ~10-15 years of additional ageing, arising from up to 25-fold greater expansion of stem cell clones. An HCT-related population bottleneck alone could not explain these differences: instead, phylogenetic trees evinced two distinct modes of HCT-specific selection. In 'pruning selection', cell divisions underpinning recipient-enriched clonal expansions had occurred in the donor, preceding transplant - their selective advantage derived from preferential mobilisation, harvest, survival ex vivo or initial homing. In 'growth selection', cell divisions underpinning clonal expansion occurred through proliferative advantage in the recipient's marrow after homing - clones with multiple driver mutations especially demonstrated this pattern. Uprooting stem cells from their native environment and transplanting them to foreign soil exaggerates selective pressures, distorting and accelerating the loss of clonal diversity compared to the unperturbed haematopoiesis of donors.

  Dataset EGAD00001010872

• WGS of cell-free DNA derived from plasma of patients with pediatric sarcoma and healthy controls, and lcWGS/RRBS of matched tumor tissue

  This dataset contains: i) 241 deep (median 12x) whole-genome sequencing profiles of 95 patients with Ewing sarcoma, 31 patients with other pediatric sarcomas, and 22 additional profiles from healthy controls. Sequencing was performed on a NovaSeq 6000 instrument using the NovaSeq S4 2x100 bp configuration. In addition, pilot experiments for 18 cfDNA samples were performed using Illumina HiSeq 2000/2500 machines (2x75 bp configuration). Data is provided as .fastq.gz files (2 files, .R1 and .R2, per sample). i) Low coverage whole-genome-sequencing on 43 tumor biopsy samples from patients with Ewing sarcoma with matched cfDNA samples. The samples were sequenced using a NovaSeq 6000 instrument with the NovaSeq S4 1x100 bp configuration. Data are provided as unmapped (raw) .bam files. iii) Reduced-representation bisulfite sequencing data for 38 tumor biopsy samples from patients with Ewing sarcoma with matched cfDNA samples, and 2 control samples. RRBS libraries were sequenced on Illumina HiSeq 2000/2500 machines. Data are provided as unmapped (raw) .bam files.

  Dataset EGAD00001007080

• Molecular Genetic Studies of Developmental Brain Disorders

  Study 1 2R01-NS050375 (PI: DOBYNS, William B.) The genetic basis of mid-hindbrain malformations Our general goal for this project is to advance our understanding of human developmental disorders that involve the brainstem and cerebellum - brain structures derived from the embryonic midbrain and hindbrain - that affect a minimum of 2.4 per 1000 resident births based on data from the CDC. Importantly, this large class of disorders co-occurs with more common developmental disorders such as autism, mental retardation and some forms of infantile epilepsy, and shares some of the same causes. With this renewal, we propose to expand the scope of our work beyond single phenotypes and genes to focus on delineating the critical phenotype spectra to which the most common MHM belong, and defining the underlying biological networks that are disrupted. To pursue these goals, we will use our large and growing cohort of human subjects to map additional MHM loci using SNP microarrays that provide both high-resolution autozygosity and linkage data in informative families as well as detect critical copy number variants in sporadic subjects. The causative genes will be identified using traditional Sanger or new high-throughput sequencing methods as appropriate abased on size of the critical region. We will use these and other known MHM causative genes to construct and revise model biological networks of genes and proteins, and test these genes and networks in additional patients as a candidate gene or more accurately a candidate network approach. These approaches need to be supported by ongoing active subject recruitment, as studies of comparable disorders such as mental retardation and autism have benefited from even larger numbers of subjects that we have so far collected. We need to use new high-throughput sequencing methods to more efficiently test larger critical regions, and to test entire gene networks rather than individual genes in matched cohorts of subjects. At every step; phenotype analysis, CNV analysis, model network construction and high-throughput sequencing, we will need expanded bioinformatics capabilities. Finally, we need to test the biological function of new genes and networks to support our gene identification studies. We expect that these studies will contribute immediately to more accurate diagnosis and counseling, and over time will lead to development of specific treatments for a subset of these disorders. We further expect that studies of mid-hindbrain development will have broad significance for human developmental disorders generally, providing compelling evidence for a connection between cerebellar development and other classes of developmental disorders such as autism, mental retardation and epilepsy. Study 2 R01-NS058721 (PI: DOBYNS, William B.) De novo copy number variation and gene discovery in human brain malformations Project Summary/Abstract The number of recognized brain malformations and syndromes has grown rapidly during the past several decades, yet relatively few causative genes have been identified, especially for three common malformations that have been associated with numerous cytogenetically visible chromosome deletions and duplications, and that often occur together: agenesis of the corpus callosum (ACC), cerebellar vermis hypoplasia (CVH) including Dandy-Walker malformation (DWM), and polymicrogyria (PMG). We propose to perform high-resolution array comparative genome hybridization (aCGH), emerging technology able to detect small copy number variants (CNV), in 700 probands with one or more of these three malformations. Our central hypothesis states that more than 10% of patients with ACC, CVH or PMG will have de novo CNV below the resolution of routine cytogenetic analysis, but detectable by current array platforms. We therefore expect to identify 70-100 patients with small CNV. We will distinguish CNV found in normal individuals from potentially disease-associated changes, and will confirm CNV using fluorescence in situ hybridization (FISH) and microsatellite (STRP) analysis. We will give highest priority to CNV that are de novo and involve 2 or more BACs, and secondary priority to familial and smaller CNV excluding known polymorphisms. After that, we will evaluate and rank candidate genes in the critical regions using information from public databases and our own expression studies, and perform mutation analysis of the best candidate genes from well-defined critical regions by sequencing in a large panel of subjects with phenotypes that match the phenotypes of the patients whose CNV define the critical regions. Here, we will use more refined criteria to supplement our clinical classification, such as the developmental level and presence of epilepsy or other birth defects. Any abnormalities found will be analyzed using existing data regarding polymorphisms (i.e. dbSNP), cross-species comparisons, and functional assays appropriate for the specific sequence change. Study 2A In 1995, we described a novel multiple congenital anomaly syndrome associated with facial dysmorphism (congenital ptosis, high arched eyebrows, shallow orbits, trigonocephaly), colobomas of the eyes, neuronal migration malformation (frontal predominant lissencephaly) and variable hearing loss. We hypothesized from de novo mutations and used trio-based exome sequencing to identify de novo mutations in the ACTB and ACTG1 genes. Study 2B In 1997 and 2004, we and others defined two novel developmental syndromes associated with markedly enlarged brain size, or megalencephaly, and other highly recognizable features. The megalencephaly-capillary malformation syndrome (MCAP) consists of megalencephaly and associated growth dysregulation with variable asymmetry, developmental vascular anomalies, distal limb malformations, variable cortical malformation, and a mild connective tissue dysplasia. The megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome (MPPH) resembles MCAP but lacks vascular malformations and syndactyly. We hypothesized that MCAP and MPPH result from mutations - including postzygotic events - in the same pathway, and studied them together. Using a combination of exome sequencing, Sanger sequencing, restriction-enzyme assays, and targeted ultra-deep sequencing in 50 families with MCAP or MPPH, we identified de novo germline or postzygotic mutations in three core components of the phosphatidylinositol-3-kinase/AKT pathway. These include two mutations in AKT3, a recurrent mutation in PIK3R2, and multiple mostly postzygotic mutations in PIK3CA (Rivière JB, Mirzaa GM, O'Roak BJ, Beddaoui M, Alcantara D, Conway RL, St-Onge J, Schwartzentruber JA, Gripp KW, Nikkel SM, Worthylake T, Sullivan CT, Ward TR, Butler HE, Kramer NA, Albrecht B, Armour CM, Armstrong L, Caluseriu O, Cytrynbaum C, Drolet BA, Innes AM, Lauzon JL, Lin AE, Mancini GMS, Meschino WS, Reggin JD, Saggar AK, Lerman-Sagie T, Uyanik G, Weksberg R, Zirn B, Beaulieu CL, FORGE Canada Consortium, Majewski J, Bulman DE, O'Driscoll M, Shendure J, Graham Jr. JM, Boycott KM, Dobyns WB. De novo germline and postzygotic mutations in AKT3, PIK3R2 and PIK3CA cause a spectrum of related megalencephaly syndromes. Nat. Genet. In press). Study 3 2R01-NS046616 (PI: GOLDEN, Jeffrey A) The role of ARX in normal and abnormal brain development This subcontract from the Children's Hospital of Philadelphia to the University of Chicago (UC) is intended to support research studies of the ARX and functionally related genes in human subjects with any one of several specific developmental disorders. The Co-investigator at UC (W.B. Dobyns) will identify a series of patients with mental retardation and severe infantile epilepsy, some of whom will have specific brain malformations and others who will have normal brain structure by brain imaging studies, and collect research samples from these subjects with informed consent. The studies to be performed will include mutation analysis of ARX, mutation analysis of specific downstream target genes, X inactivation studies in humans and X inactivation studies in mutant mice. The results will be analyzed to determine the significance of any changes found in the gene.

  Study phs000455

• Neural Systems, Inhibitory Control, and Methamphetamine Dependence

  The Neural Systems, Inhibitory Control, and Methamphetamine Dependence study started in 2011 and is currently finishing data collection for its last participants. The study included methamphetamine abusing individuals and healthy control participants aged 18-55 years. Participants who qualified over the telephone were scheduled to visit the London Laboratory at the UCLA Semel Institute for Neuroscience and Human Behavior for the In-Person Screening Phase. The screening procedures, usually scheduled over two visits, determined if participants were eligible to complete the subsequent study visits. Both groups completed identical screening procedures, with the exception of more in-depth drug use questions for the methamphetamine users. After providing written consent, participants completed questionnaires about their mood, medical, psychiatric and drug use history, personality and life experiences. They also provided a urine sample to determine what drugs they recently used and a breath sample to determine the carbon monoxide levels in their system. The drug screen tested for recent methamphetamine, cocaine, opiates/opioids, benzodiazepines, THC and other amphetamines. Methamphetamine participants needed to test positive for methamphetamine at admission. If methamphetamine participants tested positive for any drugs other than methamphetamine or marijuana, and if healthy control participants tested positive for any drugs other than marijuana, they were excluded from the study. Additionally, the urine samples of female participants were tested for pregnancy; pregnant females were also excluded. If no exclusionary criteria were met during the first screening visit, participants completed a second screening visit to ensure they were mentally and physically healthy enough to participate. The second screening visit included a psychiatric diagnostic interview (DSM-IV (SCID-I) for first 500 participants and the MINI International Neuropsychiatric Interview M.I.N.I for the last 50 participants). Additionally, laboratory tests and procedures were completed by the UCLA General Clinical Research Center and interpreted by a study physician. Laboratory tests included vital signs (heart rate--BPM, blood pressure, and respirations), an electrocardiogram (ECG) to record the electrical activity of the heart, and an 18-cc blood sample to perform laboratory tests which included a complete chemistry and metabolic panel, hepatic panel, Hepatitis-C tests, and HIV test. The laboratory tests from this blood sample ensured that participants' liver and kidneys were functioning normally, and that their standard blood counts (red cell, white cell, and salts) were also normal. If a Hepatitis-C (HCV) or HIV test was positive, participants were not allowed to continue in the study and their results were reported to the California State Health Department. Participants deemed eligible to continue in the methamphetamine group participated on an outpatient basis or were admitted to the UCLA Medical Center to participate on an inpatient basis for up to 10 days. Typically, detectable levels of methamphetamine in urinalysis remain for 2-3 days, therefore inpatient days 0-3 served as a "washout period" and the methamphetamine abusing inpatient participants provided daily urine samples to test for recent drug use and pregnancy (female participants only). Outpatient participants were also required to remain abstinent from all drugs except nicotine and marijuana for at least 4 days prior to study sessions. Self-reported abstinence for outpatients was verified by urine toxicology and saliva screening at every visit to UCLA before completion of study procedures. Neurocognitive assessments, about three hours each, typically took place on two separate days. The purpose of the neurocognitive sessions were to assess participants' intellectual and neurocognitive functioning. On either the same day as the neurocognitive testing or on separate days, both methamphetamine abusers and healthy controls were administered a structural MRI to collect information on brain structure.

  Study phs001197

• DMET Genes, Nicotine Metabolism and Prospective Abstinence

  This study (DA033813; PI: Andrew W Bergen; PMID:26132489) includes samples from two laboratory studies of nicotine metabolism. The Pharmacokinetics of Nicotine Metabolism in Twins study (PKTWIN; PI: Gary E Swan; PMID: 15527659) was based on recruitment from a twin registry (PMID: 23084148). The Integrated Research Project on Tobacco Use and Dependence (IRP; PI: Gary E Swan; PMID: 14578134) was based on recruitment from a pedigree-based longitudinal study of risk factors for substance use, the Smoking in Families study (SMOFAM; DA03706; PI: Hy Hops). These two laboratory studies (PKTWIN and IRP/SMOFAM) served as the Stage I dataset to interrogate Drug Metabolizing Enzyme and Transporter genes with a targeted SNP array for association with the Nicotine Metabolite Ratio (NMR, ratio of trans-3'-hydroxycotinine and cotinine), an established biomarker of nicotine metabolism. In addition to the laboratory studies, samples from eight RCTs (PMID: 23249876) with the NMR and smoking-related measures used to test SNPs identified in Stage I (PMID: 26132489). In a third stage, a lung cancer meta-analysis database (PMID: 24880342) was used to assess association of SNPs identified in Stage II with lung cancer. The objectives of the study were to identify novel genes and SNPs contributing to nicotine metabolism (Stage I), and to validate PK SNPs associated with the NMR from individuals participating in a clinical laboratory protocol with the NMR obtained from treatment-seeking smokers, and then to investigate association with prospective smoking cessation (Stage II). This study built upon existing studies of nicotine metabolism and randomized trials of smoking cessation therapies. Enhanced knowledge of the genes influencing nicotine metabolism and prospective abstinence may help personalize smoking cessation treatment and risk assessment for smoking-related diseases. For Stage I, both subject [fixed-dose NMR, covariates (age, BMI, ethnicity, sex, smoking status, and hormone use), and pedigree relationships] and sample (common DMET SNP genotype, genotyping quality control) data are available in this accession. The analysis protocol, quality control summaries, summary genotype, summary phenotype, and analysis results are available for Stage I, II and III samples (PMID: 26132489). Extensive discussion of the prior CYP2A6 association literature with the NMR, abstinence, smoking heaviness and lung cancer risk is available (PMID: 26132489). The NMR has previously been associated with CYP2A6 activity, response to smoking cessation treatments, and cigarette consumption. We searched for drug metabolizing enzyme and transporter (DMET) gene variation associated with the NMR and prospective abstinence in 2,946 participants of laboratory studies of nicotine metabolism and of clinical trials of smoking cessation therapies. Stage I was a meta-analysis of the association of 507 common single nucleotide polymorphisms (SNPs) at 173 DMET genes with the NMR in 449 participants of two laboratory studies. Nominally significant associations were identified in ten genes after adjustment for intragenic SNPs; CYP2A6 and two CYP2A6 SNPs attained experiment-wide significance adjusted for correlated SNPs (CYP2A6 PACT=4.1E-7, rs4803381 PACT=4.5E-5, rs1137115, PACT=1.2E-3). Stage II was mega-regression analyses of 10 DMET SNPs with pretreatment NMR and prospective abstinence in up to 2,497 participants from eight trials. rs4803381 and rs1137115 SNPs were associated with pretreatment NMR at genome-wide significance. In post-hoc analyses of CYP2A6 SNPs, we observed nominally significant association with: abstinence in one pharmacotherapy arm; cigarette consumption among all trial participants; and lung cancer in four case:control studies. CYP2A6 minor alleles were associated with reduced NMR, CPD, and lung cancer risk. We confirmed the major role that CYP2A6 plays in nicotine metabolism, and made novel findings with respect to genome-wide significance and associations with CPD, abstinence and lung cancer risk. Additional multivariate analyses with patient variables and genetic modeling will improve prediction of nicotine metabolism, disease risk and smoking cessation treatment prognosis.

  Study phs000931

• Study of Melanoma Risk in Australia and the United Kingdom

  The results from a small number of melanoma GWAS have been published. Initial studies identified several pigmentation- and nevus-associated loci that mediate an effect on melanoma risk, however, these studies were somewhat limited in that sample number (hence power) was low, they used first-generation low density SNP arrays, or used pools of DNA samples. Subsequently, additional melanoma GWAS have been completed, including a "Phase 2" study by the International Melanoma Genetics Consortium (GenoMEL), a full GWAS of the Australian sample resource previously used in a pooled-DNA GWAS, and study conducted through the MD Anderson Cancer Center and the Brigham and Women's Hospital. The groups who performed these studies have recently shared data and replicated a number of new melanoma susceptibility loci. Aside from the identification of novel loci, there are two additional notable outcomes of these studies. Firstly, an examination of the observed versus expected test statistics (Q-Q plot) from the Australian study, even after removing data from SNPs within known replicated susceptibility loci, reveals that there remains an excess of positive results. Similarly, the latest GenoMEL study finds almost three times as many SNPs reaching p-values between 10-4 and 10-5 as would be expected by chance. In the light of other studies of complex traits [1], these data suggest that studies based on additional melanoma samples from the same population are likely to identify an even larger "polygenic tail" and should prove invaluable in identifying and characterizing the underlying loci. A second observation, is that the effect size for replicated associations differs very little between GWAS. Given the strong effect of ultraviolet radiation (UVR) on risk of melanoma this might be expected to affect penetrance, particularly in the Australian and English samples, which are ethnically similar, but have very different sun exposures. We hypothesize that those SNPs whose effects are strongly mediated by UVR exposure may have smaller marginal effects on risk and are therefore harder to detect initially, suggesting a whole category of genetic effects that are unexplored.Indeed, most melanoma GWAS performed to date used samples from both European and Australian populations in discovery and or replication, and thus, existing studies have been poorly powered to detect associations with a wide difference in effect between these low- and high-UVR populations. Here, we wish to pursue this hypothesis further and plan to genotype additional cases drawn from the English and Australian populations. This will serve two complementary objectives: firstly, it will greatly increase our collective power to detect additional new melanoma risk alleles; secondly, it will highlight associations that are stronger specifically within the context of either low-UVR or high-UVR exposure environments. We posit that these data will be invaluable, not just for identifying novel melanoma-associated loci, but also for addressing gene-environment interactions. We have available a collection of valuable resources to help fulfill our aims. These include a considerable number of well-characterized and ungenotyped Australian and UK melanoma cases, as well as existing genotype data from sets of Australian and UK cases and population-based controls. We propose the following Specific Aims: To extend our current collective melanoma GWAS by genotyping a large number of additional UK and Australian cases. To jointly analyze these data and identify new melanoma risk loci. To determine whether pre-existing and new melanoma risk alleles show different degrees of association between the English and Australian populations, and assess whether these differences might be due to differences in sunlight exposure.

  Study phs000519

• Detroit Research on Cancer Survivors (ROCS) and Disparities and Cancer Epidemiology - Colorectal Cancer (DANCE)

  The goals for this study were as follows:1. Enroll a total of 5,060 African Americans newly diagnosed with cancer into the Detroit Research on Cancer Survivors (Detroit ROCS) Cohort. These population-based cases will be ascertained through the Detroit Metropolitan Detroit Cancer Surveillance System (MDCSS), a founding member of the NCI SEER Program. Participation at baseline will include a written, web-based, or telephone interview, saliva and/or blood collection, and tumor block retrieval, with detailed cancer-related information coming from the MDCSS registry and medical records.2. Enroll a subgroup of colorectal cancer patients identified through the Louisiana Tumor Registry (LTR). An additional 500 colorectal cancer patients, approximately half non-Hispanic white and half African American, will be identified through the LTR and enrolled into the ROCS protocol. The survey is the same survey administered to the ROCS cohort. The Louisiana (“DANCE-CRC”) cohort will not be followed beyond baseline, and blood will not be collected (tumor tissue, however, will be collected, deidentified, and sent for processing through Dr. Rozek's laboratory at Georgetown University).The proposed cohorts will provide for a broad research agenda evaluating cancer outcomes in African Americans across a variety of factors from individual, provider, health care system and community perspectives. These factors include tumor characteristics driving individualized treatment planning, quality of life after a diagnosis and recurrence. Utilizing the MDCSS registry, we will enroll a large population-based sample of African-American cases with lung, breast, colorectal, and prostate cancers and their caregivers resulting in a cohort that will provide substantial benefit to the cancer research community. KCI has a strong history of research in the African-American community and the faculty represents a breadth of experience that includes clinical oncology, genetic epidemiology, environmental exposures, quality of life, treatment decision making, racism/discrimination, and health behaviors. Drs. Schwartz and Beebe-Dimmer have been and will continue to be generous collaborators with other institutions to ensure that this resource will be extensively utilized. The proposed overall cohort will be the only one of its kind, to our knowledge, filling a substantial gap in our understanding of the determinants driving cancer outcomes in African Americans. In addition to registry data detailing cancer specifics, yearly surveys have collected information about individualized treatment experiences, heath histories, family histories, quality of life after a diagnosis, recurrence and caregiving. Population: Adult cancer survivors (see inclusion criteria in next section) Molecular Technologies: Genotyping: Infinium Expanded Multi-Ethnic Genotyping Array (MEGAEX) DNA Sequencing: Illumina NovaSeq X Plus using XLEAP-SBS (sequencing by synthesis) Whole genome sequencingPrincipal Findings: The preliminary data collected reinforces differences by race in factors affecting cancer outcomes. Variants in DNA damage repair genes and HOXB13 may be important cancer risk factors for Black men diagnosed with prostate cancer, and are more frequently observed in men with a family history of cancer. ROCS participants reported high rates of a family history of cancer. The high rate of eligibility for Cancer genetic counseling among ROCS participants supports the need for interventions to increase referrals and uptake of genetic counseling among African Americans 60% of ROCS participants reported participating in regular physical activity (PA), with 24% reporting ≥150 min/wk. There were no differences by sex, but prostate cancer survivors were the most likely to report participating in regular PA, whereas lung cancer survivors were the least likely. Survivors who reported participating in regular PA reported higher health-related quality of life and lower depression. More African American than White survivors reported financial hardship and limiting care. More White than African American survivors reported utilizing assets, while more African American survivors reported cancer-related debt. Data available in dbGaP: Individual phenotype data Individual genotype data Individual sequencing dataSample types: Germline DNA

  Study phs003116

• Blueprint Data Access Committee.

  Access to the sequence and alignment and genotype level data produced by the Blueprint Consortium is controlled by the Blueprint Data Access Committee (DAC). Access to data will be granted to qualified investigators for appropriate use. Please email blueprint-dac@ebi.ac.uk to request the Data Access Application and Data Access Agreement.

  Dac EGAC00001000135

• Substantial somatic genomic variation and selection for BCOR mutations in human induced pluripotent stem cells

  Human Induced Pluripotent Stem Cells (hiPSC) are an established patient-specific model system where opportunities are emerging for cell-based therapies. We compared and contrasted hiPSCs derived from different tissues, skin and blood, in the same individual. We show extensive single-nucleotide mutagenesis in all hiPSC lines, although fibroblast-derived hiPSCs (F-hiPSCs) are particularly heavily mutagenized by ultraviolet (UV)-related damage. We utilized genome sequencing data on 454 F-hiPSCs and 44 blood-derived hiPSCs (B-hiPSCs) to gain further insights. Across 324 whole genome sequenced (WGS) F-hiPSCs derived by the Human Induced Pluripotent Stem Cell Initiative (HipSci), UV-related damage is present in ~72% of cell lines, sometimes causing substantial mutagenesis (range 0.25-15 per Mb). Furthermore, we find remarkable genomic heterogeneity between independent F-hiPSC clones derived from the same reprogramming process in the same donor, due to oligoclonal populations within fibroblasts. Combining WGS and exome-sequencing data of 452 HipSci F-hiPSCs, we identify 272 predicted pathogenic mutations in cancer-related genes, of which 21 genes were hit recurrently three or more times, involving 77 (17%) lines. Notably, 151 of 272 mutations were present in starting fibroblast populations suggesting that more than half of putative driver events in F-hiPSCs were acquired in vivo. In contrast, B-hiPSCs reprogrammed from erythroblasts show lower levels of genome-wide mutations (range 0.28-1.4 per Mb), no UV damage, but a strikingly high prevalence of acquired BCOR mutations in ~57% of lines, indicative of strong selection pressure. All hiPSCs had otherwise stable, diploid genomes on karyotypic pre-screening, highlighting how copy-number-based approaches do not have the required resolution to detect widespread nucleotide mutagenesis. This work strongly suggests that models for cell-based therapies require detailed nucleotide-resolution characterization prior to clinical application.

  Dataset EGAD00001007029

• The Thrifty Microbiome: The Role of the Gut Microbiota in Obesity in the Amish

  Emerging evidence that the gut microbiota may contribute in important ways to human health and disease has led us and others to hypothesize that both symbiotic and pathological relationships between gut microbes and their host may be key contributors to obesity and the metabolic complications of obesity. Our "Thrifty Microbiome Hypothesis" poses that gut microbiota play a key role in human energy homeostasis. Specifically, constituents of the gut microbial community may introduce a survival advantage to its host in times of nutrient scarcity, promoting positive energy balance by increasing efficiency of nutrient absorption and improving metabolic efficiency and energy storage. However, in the presence of excess nutrients, fat accretion and obesity may result, and in genetically predisposed individuals, increased fat mass may result in preferential abdominal obesity, ectopic fat deposition (liver, muscle), and metabolic complications of obesity (insulin resistance, hypertension, hyperlipidemia). Furthermore, in the presence of excess nutrients, a pathological transition of the gut microbial community may occur, causing leakage of bacterial products into the intestinal lymphatics and portal circulation, thereby inducing an inflammatory state, further aggravating metabolic syndrome traits and accelerating atherosclerosis. This pathological transition and the extent to which antimicrobial leakage occurs and causes inflammatory and other maladaptive sequelae of obesity may also be influenced by host factors, including genetics. In the proposed study, we will directly test the Thrifty Microbiome Hypothesis by performing detailed genomic and functional assessment of gut microbial communities in intensively phenotyped and genotyped human subjects before and after intentional manipulation of the gut microbiome. To address these hypotheses, five specific aims are proposed: (1) enroll three age- and sex-matched groups from the Old Order Amish: (i) 50 obese subjects (BMI > 30 kg/m2) with metabolic syndrome, (ii) 50 obese subjects (BMI > 30 kg/m2) without metabolic syndrome, and (iii) 50 non-obese subjects (BMI < 25 kg/m2) without metabolic syndrome and characterize the architecture of the gut microbiota from the subjects enrolled in this study by high-throughput sequencing of 16S rRNA genes; (2) characterize the gene content (metagenome) to assess the metabolic potential of the gut microbiota in 75 subjects to determine whether particular genes or pathways are correlated with disease phenotype; (3) characterize the transcriptome in 75 subjects to determine whether differences in gene expression in the gut microbiota are correlated with disease phenotype, (4) determine the effect of manipulation of the gut microbiota with antibiotics on energy homeostasis, inflammation markers, and metabolic syndrome traits in 50 obese subjects with metabolic syndrome and (5) study the relationship between gut microbiota and metabolic and cardiovascular disease traits, weight change, and host genomics in 1,000 Amish already characterized for these traits and in whom 500K Affymetrix SNP chips have already been completed. These studies will provide our deepest understanding to date of the role of gut microbes in terms of 'who's there?', 'what are they doing?', and 'how are they influencing host energy homeostasis, obesity and its metabolic complications? PUBLIC HEALTH RELEVANCE: This study aims to unravel the contribution of the bacteria that normally inhabit the human gastrointestinal tract to the development of obesity, and its more severe metabolic consequences including cardiovascular disease, insulin resistance and Type II diabetes. We will take a multidisciplinary approach to study changes in the structure and function of gut microbial communities in three sets of Old Order Amish patients from Lancaster, Pennsylvania: obese patients, obese patients with metabolic syndrome and non-obese individuals. The Old Order Amish are a genetically closed homogeneous Caucasian population of Central European ancestry ideal for genetic studies. These works have the potential to provide new mechanistic insights into the role of gut microflora in obesity and metabolic syndrome, a disease that is responsible for significant morbidity in the adult population, and may ultimately lead to novel approaches for prevention and treatment of this disorder.

  Study phs000258

• Prematurity and Respiratory Outcomes Program (PROP) Core Database Protocol (PROP-BioLINCC)

  Data Access NOTE: Please refer to the "Authorized Access" section below for information about how access to the data from this accession differs from many other dbGaP accessions. Objectives: The Prematurity and Respiratory Outcomes Program (PROP) was performed to identify suitable predictors of respiratory outcomes that may serve as surrogate endpoints in future trials of prevention and therapy of respiratory diseases in preterm infants.Background: Acute and chronic respiratory morbidities are common in premature births, and can pose significant risk to the infant's health, particularly during the first two years of life. One such condition is bronchopulmonary dysplasia (BPD) where infants require oxygen therapy due to abnormal repair and impaired lung development after acute lung injury. BPD poses a high mortality risk, even in infants that survive the initial hospitalization. Regardless of BPD diagnosis, preterm infants frequently return for medical care due to symptoms of post-prematurity respiratory disease (PRD), which includes intermittent or chronic wheezing, cough without cold, poor growth, apnea and cyanosis, and lower respiratory tract infections. Impaired lung function can persist into adulthood, contributing to chronic respiratory diseases including asthma and emphysema.However, at the time of the PROP study, there were no objective measures to predict which preterm infants would have persistent respiratory problems after discharge from the hospital. Furthermore, improved survival rates of premature infants and the high prevalence of lasting respiratory morbidities highlight the need for more comprehensive strategies to address both short-term and long-term respiratory complications. Thus, the Prematurity and Respiratory Outcomes Program (PROP) was formed to investigate multiple research hypotheses on the molecular mechanisms that contribute to respiratory disease risk of premature neonates over the first year of life. Specifically, PROP investigators hypothesized that in survivors of extreme prematurity to 36 weeks postmenstrual age (PMA), specific biologic, physiologic and clinical data obtained during the initial hospitalization will predict respiratory morbidity between discharge and 1 year corrected age. Participants: A total of 835 infants were enrolled.Design: PROP was a collaboratively developed multicenter prospective cohort study of very preterm infants from birth through the time of discharge from the NICU and up to one year corrected age.Clinical data included maternal and infant demographics, co-morbidities, and daily infant respiratory, nutritional, and medication data until discharge. These were prospectively collected from birth using medical record review and family interviews. After discharge, phone interviews were conducted with the infant's main caregiver at 3, 6, 9 and 12 months corrected age to assess domains of respiratory morbidity. At 6 and 12 months corrected age, a survey of environmental respiratory irritant exposures and an assessment for gastroesophageal reflux disease were also completed. A standardized physical exam was performed at 36-40 weeks PMA and again at one year corrected age. The exam focused on anthropometrics, vital signs, and respiratory system signs. At 34-41 weeks PMA and within one week of anticipated discharge based on physiologic stability, a set of non-invasive respiratory assessments were performed to assess physiologic biomarkers in infants that were not mechanically ventilated or receiving non-invasive positive pressure ventilation. Respiratory inductance plethysmography (RIP) was performed before and after inhaled albuterol to assess potential airway reactivity. During the RIP study, continuous pulse oximetry was performed during quiet sleep in order to analyze oxygen desaturations and apneas. Standardized oxygen requirement challenge tests were performed at about 36 weeks PMA, and at about 40 weeks PMA if the infant was still hospitalized and was not eligible for, or failed, the previous challenge. Failure was defined as SpO2 20 seconds at any point in the testing. Infants breathing ambient air or that passed the 36 week challenge test underwent a hypoxia challenge consisting of a 15-minute trial of FiO2 of 0.15. Failure was defined as SpO2 The primary outcome was respiratory morbidity, as measured by the presence or absence of substantial post-prematurity respiratory disease (PRD) up to one year corrected age. PRD was classified as a positive response in at least one of four morbidity domains during at least two separate parental interviews. The domains were respiratory medications, hospitalizations for cardiopulmonary cause, respiratory symptoms, and home technology dependence (oxygen, ventilator, or CPAP/BiPAP). Mortality from a respiratory cause was also incorporated.

  Study phs004117

• Knoll et al.: The life-saving benefit of dexamethasone in severe COVID-19 is linked to a reversal of monocyte dysregulation

  Following the demonstration of its life-saving effect in clinical trials, dexamethasone quickly became standard-of-care in the treatment of severe COVID-19. Beneficial effects were reported for patients requiring supplemental oxygen or invasive mechanical ventilation. Yet, a substantial proportion of patients still progressed to a critical condition or succumbed to the disease, despite timely initiation of glucocorticoid treatment. No molecular or cellular correlate of this beneficial treatment response has been defined. Here, we identify distinct cellular and molecular changes in circulating immune cells in patients with COVID-19 elicited in response to dexamethasone treatment. The most profound transcriptional changes in response to dexamethasone treatment were noted in monocytes and in B cells. In monocytes, we observed a reversal of hallmark signatures previously associated with COVID-19 severity, and the induction of a specific monocyte substate enriched in glucocorticoid response gene expression which we here refer to as dexamethasone response cluster. Molecular responses to dexamethasone treatment were directly linked to clinical outcome, since the reversal of pathogenic signatures and induction of glucocorticoid response genes were enriched in patients who survived the disease in response to dexamethasone treatment. Cellular responsiveness of circulating monocytes was thus identified as a correlate of clinical response to dexamethasone treatment. Changes in monocyte transcriptomes could also be linked to epigenetic alterations highlighting early differences in monocytes of dexamethasone responders and non-responders. Further, monocyte single-cell transcriptome-derived signatures were enriched in whole blood transcriptomes from patients with fatal outcome in two independent cohorts, highlighting the potential clinical value for early identification of non-responders who are refractory to dexamethasone treatment. Overall, our findings indicate that the life-saving effects of dexamethasone in COVID-19 patients are linked to a specific immunomodulatory effect based on the reversion of monocyte dysregulation. Our study elucidates the potential mechanism of action of one of the most efficient and cost-effective drugs for the treatment of COVID-19. It further highlights the potential of single-cell omics for monitoring target engagement of immunomodulatory drugs and for the stratification of patients for precision medicine approaches.

  Study EGAS00001007461

• Long-Term Oxygen Treatment Trial (LOTT-BioLINCC)

  Data Access NOTE: Please refer to the “Authorized Access” section below for information about how access to the data from this accession differs from many other dbGaP accessions. Objectives: To evaluate the efficacy of long-term treatment with supplemental oxygen in people with stable chronic obstructive pulmonary disease (COPD) and resting or exercise-induced moderate desaturation. Background: COPD is the fourth leading cause of death in the United States, with more than twelve million people currently diagnosed with the disease. In 2011, Medicare reimbursements for oxygen-related costs for people with COPD exceeded two billion dollars. The quality of life of a person with COPD decreases as the disease progresses, making treating and managing COPD in the moderate stages important. The benefits of oxygen supplementation were studied in the 1970s, but these benefits were specific to people with COPD who have severe resting hypoxemia. The LOTT trial was designed to address the effectiveness of supplemental oxygen therapy in treating people with COPD who have moderately low blood oxygen levels at rest or who have normal blood oxygen levels at rest, but have low or very low blood oxygen levels during exercise. Participants: There were a total of 1759 people screened for eligibility for the LOTT trial. Of the 1759 screened, 738 people with COPD were selected for randomization with 368 randomly assigned to the supplemental-oxygen group and 370 to the no-supplemental-oxygen group. Of the 738 people with COPD who underwent randomization, 133 (18%) had resting desaturation only, 319 (43%) had exercise-induced desaturation only, and 286 (39%) had both types of desaturation. Furthermore, in the supplemental-oxygen group, 220 people were prescribed 24-hour oxygen and 148 were prescribed oxygen during exercise and sleep only. Design: The LOTT trial was originally designed to test whether the use of supplemental oxygen would result in a longer time to death for people with COPD and moderate resting desaturation. After seven months, the trial design was judged to be infeasible; therefore, the trial was redesigned to include people with exercise-induced desaturation with a primary composite outcome of death or first hospitalization for any cause, whichever occurs first. Other outcomes measured included: death, health care utilization, COPD exacerbation, quality of life measurements, anxiety, depression, and measures of functional status. Potential participants were screened using questionnaires, a breathing test, a brief physical exam, a blood draw, and measurements of resting and walking blood oxygen levels. Based on those results, eligible participants returned for a second screening visit for further evaluations. At the end of the second visit, eligible participants were assigned randomly to supplemental oxygen therapy or no oxygen therapy. Participants assigned to supplemental oxygen therapy received stationary and/or portable oxygen systems. Participants were required to return for a one hour visit to determine how much oxygen to use while walking and to learn how to use the equipment. Participants who had low blood oxygen levels during rest were instructed to use supplemental oxygen 24 hours per day. Participants with normal resting blood oxygen levels, but low or very low blood oxygen levels during exercise were instructed to use it during physical activity and sleep. Throughout the treatment period, participants were asked to keep records of the number of oxygen tanks emptied or pounds of oxygen delivered, meter readings, and changes in equipment. Study officials contacted participants weekly for the first month, monthly for the next five months, and then every two months until the Year 1 study visit. Participants assigned to receive no oxygen treatment were contacted one week after assignment for a check-up. All participants returned for study visits once a year for up to seven years. At each of these visits, participants completed some of the same tests and questionnaires from the screening visit. Participants underwent a blood draw during the one year study visit. Participants in both treatment groups received two phone calls each year to check on status and use of oxygen. In addition, participants in both groups were asked to complete a quality of life questionnaire by mail at four months and sixteen months. Medicare claims were collected for the duration of each participant's enrollment in the study. Conclusions: In participants with stable COPD and resting or exercise-induced moderate desaturation, the prescription of long-term supplemental oxygen did not result in a longer time to death or first hospitalization than no long-term supplemental oxygen, nor did it provide sustained benefit with regard to any of the other measured outcomes. Long-Term Oxygen Treatment Trial Research Group, Albert RK, Au DH, Blackford AL, Casaburi R, Cooper JA Jr, Criner GJ, Diaz P, Fuhlbrigge AL, Gay SE, Kanner RE, MacIntyre N, Martinez FJ, Panos RJ, Piantadosi S, Sciurba F, Shade D, Stibolt T, Stoller JK, Wise R, Yusen RD, Tonascia J, Sternberg AL, Bailey W. A Randomized Trial of Long-Term Oxygen for COPD with Moderate Desaturation. N Engl J Med. 2016 Oct 27;375(17):1617-1627.

  Study phs003933

• A Randomized Trial of Combined PD-L1 and CTLA-4 Inhibition with Targeted Low-Dose or Hypofractionated Radiation for Patients with Metastatic Colorectal Cancer

  Brief SummaryThis randomized phase II trial studies the side effects of durvalumab and tremelimumab to see how well they work with or without high or low-dose radiation therapy in treating patients with colorectal (CRC) or non-small cell lung cancer (NSCLC) that has spread to other parts of the body (metastatic). Immunotherapy with durvalumab and tremelimumab may induce changes in body's immune system and may interfere with the ability of tumor cells to grow and spread. Radiation therapy uses high energy x-rays to kill tumor cells and shrink tumors. Giving durvalumab and tremelimumab with radiation therapy may work better in treating patients with colorectal or non-small cell lung cancer.Detailed DescriptionPRIMARY OBJECTIVES:I. To assess safety and tolerability of combined checkpoint blockade with MEDI4736 (durvalumab) and tremelimumab alone or with high or low-dose radiation in non-small cell lung cancer (NSCLC). (NSCLC Cohort) II. To compare the overall response (excluding the irradiated lesion[s]) between combined checkpoint blockade with MEDI4736 and tremelimumab alone or combined checkpoint blockade with low or high dose radiation. (NSCLC Cohort) III. To assess safety and tolerability of combined checkpoint blockade with MEDI4736 and tremelimumab with high or low-dose radiation. (Colorectal Cohort) IV. To determine the overall response rate (excluding the irradiated lesion[s]) with combined checkpoint blockade with MEDI4736 and tremelimumab with either low or high dose radiation. (Colorectal Cohort)SECONDARY OBJECTIVES:I. To estimate median progression-free survival and overall survival. (NSCLC Cohort) II. To determine local control within the irradiated field(s) and abscopal response rates. (NSCLC Cohort) III. To evaluate associations between PD-L1 expression as well as levels of infiltrating CD3+, CD8+ T-cells and overall response. (NSCLC Cohort) IV. To explore changes in PD-L1 expression, circulating T-cell populations, T-cell infiltration, ribonucleic acid (RNA) expression, spatial relationship of immune markers, and mutational burden as a result of low or high dose radiation. (NSCLC Cohort) V. To estimate median progression-free survival and overall survival. (Colorectal Cohort) VI. To determine local control within the irradiated field and abscopal response rates. (Colorectal Cohort) VII. To evaluate associations between PD-L1 expression as well as levels of infiltrating CD3+ CD8+ T-cells and overall response. (Colorectal Cohort) VIII. To evaluate changes between PD-L1 expression as well as levels of infiltrating CD3+, CD8+ T-cells induced by targeted low or high dose radiation. (Colorectal Cohort) IX. To explore changes in circulating T-cell populations, T-cell infiltration, RNA expression, spatial relationship of immune markers, and mutational burden as a result of low or high dose radiation. (Colorectal Cohort)OUTLINE: Patients are assigned to 1 of 2 cohorts. NOTE: This Study is only for Cohort 2. Please see phs003295 for Cohort 1COHORT 1: Patients with NSCLC are randomized to 1 of 3 arms.ARM A: Patients receive tremelimumab and durvalumab and as in Arm C. Beginning at week 2, patients receive high dose radiation therapy once per day (QD) over 10 days for up to 3 fractions.ARM B: Patients receive tremelimumab and durvalumab and as in Arm C. Beginning at week 2, patients receive low dose radiation therapy every 6 hours twice per day (BID) on weeks 2, 6, 10 and 14.ARM C: Patients receive tremelimumab intravenously (IV) and durvalumab IV over 60 minutes every 4 weeks for up to 16 weeks in the absence of disease progression or unacceptable toxicity. Patients then receive durvalumab IV over 60 minutes 4 weeks after last combination dose for up to 9 additional doses.COHORT 2: Patients with CRC are randomized to 1 of 2 arms.ARM A: Patients receive tremelimumab intravenously (IV) and durvalumab IV over 60 minutes every 4 weeks for up to 16 weeks in the absence of disease progression or unacceptable toxicity. Patients then receive durvalumab IV over 60 minutes 4 weeks after last combination dose for up to 9 additional doses. Beginning at week 2, patients receive high dose radiation therapy once per day (QD) over 10 days for up to 3 fractions.ARM B: Patients receive tremelimumab intravenously (IV) and durvalumab IV over 60 minutes every 4 weeks for up to 16 weeks in the absence of disease progression or unacceptable toxicity. Patients then receive durvalumab IV over 60 minutes 4 weeks after last combination dose for up to 9 additional doses. Beginning at week 2, patients receive low dose radiation therapy every 6 hours twice per day (BID) on weeks 2, 6, 10 and 14.After completion of study treatment, patients are followed for up to 12 weeks.Official TitleA Phase 2 Study of MEDI4736 (Durvalumab) and Tremelimumab Alone or in Combination With High or Low-Dose Radiation in Metastatic Colorectal and NSCLC Conditions Metastatic Colorectal CarcinomaMetastatic Lung Non-Small Cell CarcinomaStage IV Colorectal Cancer AJCC v7Stage IV Lung Non-Small Cell Cancer AJCC v7Stage IVA Colorectal Cancer AJCC v7Stage IVB Colorectal Cancer AJCC v7 Intervention/Treatment Biological: DurvalumabRadiation: Radiation TherapyBiological: Tremelimumab Other Study ID Numbers NCI-2016-01325NCI-2016-01325 ( Registry Identifier ) (REGISTRY: CTRP (Clinical Trial Reporting Program))17-71910021 ( Other Identifier ) (OTHER: Dana-Farber - Harvard Cancer Center LAO)10021 ( Other Identifier ) (OTHER: CTEP) Molecular Technologies/AssaysIHCmIFOlinkCyTOFRNAseqWES

  Study phs003294

• Durvalumab Plus Tremelimumab Alone or in Combination with Low-Dose or Hypofractionated Radiotherapy in Metastatic Non-Small-Cell Lung Cancer Refractory to Previous PD(L)-1 Therapy: an Open-Label, Multicentre, Randomised, Phase 2 Trial

  Brief SummaryThis randomized phase II trial studies the side effects of durvalumab and tremelimumab and to see how well they work with or without high or low-dose radiation therapy in treating patients with colorectal or non-small cell lung cancer that has spread to other parts of the body (metastatic). Immunotherapy with durvalumab and tremelimumab, may induce changes in body's immune system and may interfere with the ability of tumor cells to grow and spread. Radiation therapy uses high energy x-rays to kill tumor cells and shrink tumors. Giving durvalumab and tremelimumab with radiation therapy may work better in treating patients with colorectal or non-small cell lung cancer.Detailed DescriptionPRIMARY OBJECTIVES:I. To assess safety and tolerability of combined checkpoint blockade with MEDI4736 (durvalumab) and tremelimumab alone or with high or low-dose radiation in non-small cell lung cancer (NSCLC). (NSCLC Cohort) II. To compare the overall response (excluding the irradiated lesion[s]) between combined checkpoint blockade with MEDI4736 and tremelimumab alone or combined checkpoint blockade with low or high dose radiation. (NSCLC Cohort) III. To assess safety and tolerability of combined checkpoint blockade with MEDI4736 and tremelimumab with high or low-dose radiation. (Colorectal Cohort) IV. To determine the overall response rate (excluding the irradiated lesion[s]) with combined checkpoint blockade with MEDI4736 and tremelimumab with either low or high dose radiation. (Colorectal Cohort)SECONDARY OBJECTIVES:I. To estimate median progression-free survival and overall survival. (NSCLC Cohort) II. To determine local control within the irradiated field(s) and abscopal response rates. (NSCLC Cohort) III. To evaluate associations between PD-L1 expression as well as levels of infiltrating CD3+, CD8+ T-cells and overall response. (NSCLC Cohort) IV. To explore changes in PD-L1 expression, circulating T-cell populations, T-cell infiltration, ribonucleic acid (RNA) expression, spatial relationship of immune markers, and mutational burden as a result of low or high dose radiation. (NSCLC Cohort) V. To estimate median progression-free survival and overall survival. (Colorectal Cohort) VI. To determine local control within the irradiated field and abscopal response rates. (Colorectal Cohort) VII. To evaluate associations between PD-L1 expression as well as levels of infiltrating CD3+ CD8+ T-cells and overall response. (Colorectal Cohort) VIII. To evaluate changes between PD-L1 expression as well as levels of infiltrating CD3+, CD8+ T-cells induced by targeted low or high dose radiation. (Colorectal Cohort) IX. To explore changes in circulating T-cell populations, T-cell infiltration, RNA expression, spatial relationship of immune markers, and mutational burden as a result of low or high dose radiation. (Colorectal Cohort)OUTLINE: Patients are assigned to 1 of 2 cohorts. NOTE: This Study is only for Cohort 1. Please see phs003294 for Cohort 2COHORT 1: Patients with NSCLC are randomized to 1 of 3 arms.ARM A: Patients receive tremelimumab and durvalumab and as in Arm C. Beginning at week 2, patients receive high dose radiation therapy once per day (QD) over 10 days for up to 3 fractions.ARM B: Patients receive tremelimumab and durvalumab and as in Arm C. Beginning at week 2, patients receive low dose radiation therapy every 6 hours twice per day (BID) on weeks 2, 6, 10 and 14.ARM C: Patients receive tremelimumab intravenously (IV) and durvalumab IV over 60 minutes every 4 weeks for up to 16 weeks in the absence of disease progression or unacceptable toxicity. Patients then receive durvalumab IV over 60 minutes 4 weeks after last combination dose for up to 9 additional doses.COHORT 2: Patients with CRC are randomized to 1 of 2 arms.ARM A: Patients receive tremelimumab intravenously (IV) and durvalumab IV over 60 minutes every 4 weeks for up to 16 weeks in the absence of disease progression or unacceptable toxicity. Patients then receive durvalumab IV over 60 minutes 4 weeks after last combination dose for up to 9 additional doses. Beginning at week 2, patients receive high dose radiation therapy once per day (QD) over 10 days for up to 3 fractions.ARM B: Patients receive tremelimumab intravenously (IV) and durvalumab IV over 60 minutes every 4 weeks for up to 16 weeks in the absence of disease progression or unacceptable toxicity. Patients then receive durvalumab IV over 60 minutes 4 weeks after last combination dose for up to 9 additional doses. Beginning at week 2, patients receive low dose radiation therapy every 6 hours twice per day (BID) on weeks 2, 6, 10 and 14.After completion of study treatment, patients are followed for up to 12 weeks.Official TitleA Phase 2 Study of MEDI4736 (Durvalumab) and Tremelimumab Alone or in Combination With High or Low-Dose Radiation in Metastatic Colorectal and NSCLC Conditions Metastatic Colorectal CarcinomaMetastatic Lung Non-Small Cell CarcinomaStage IV Colorectal Cancer AJCC v7Stage IV Lung Non-Small Cell Cancer AJCC v7Stage IVA Colorectal Cancer AJCC v7Stage IVB Colorectal Cancer AJCC v7 Intervention/Treatment Biological: DurvalumabRadiation: Radiation TherapyBiological: Tremelimumab Other Study ID Numbers NCI-2016-01325NCI-2016-01325 ( Registry Identifier ) (REGISTRY: CTRP (Clinical Trial Reporting Program))17-71910021 ( Other Identifier ) (OTHER: Dana-Farber - Harvard Cancer Center LAO)10021 ( Other Identifier ) (OTHER: CTEP) Molecular Technologies / AssaysCyTOFIHCmIFOlinkRNAseqWES

  Study phs003295

• The Dutch Microbiome Project Batch 1

  The Dutch Microbiome Project (DMP) data includes shotgun metagenomic sequencing of faecal samples 8,208 Dutch individuals. Paired-end sequencing was performed using Illumina HiSeq 2000 platform. Data is archived in two batches to facilitate easier data access and upload to EGA. Batch 1 of DMP includes 4396 samples.

  Dataset EGAD00001007027

• GCAT| WGS BAM V1

  This dataset include BAM files of 808 samples from GCAT cohort. Technology used HiSeq 4000, read length 150 bp, inner mate distance 300 bp. For each sample the paired -ends are generated in separated files. Each FASTQ is splitted in multiple LANEs and grouped by the Multiplex index.

  Dataset EGAD00001008202

• HBCC Postmortem Psychiatric Molecular Studies

  This postmortem study examines molecular, genetic and epigenetic signatures in the brains of hundreds of subjects with or without mental disorders conducted by the DIRP NIMH Human Brain Collection Core (HBCC). The brain tissues are obtained under protocols approved by the CNS IRB (NCT00001260), with the permission of the next-of-kin (NOK) through the Offices of the Chief Medical Examiners (MEOs) in the District of Columbia, Northern Virginia and Central Virginia. Additional samples were obtained from the University of Maryland Brain and Tissue Bank (contracts NO1-HD-4-3368 and NO1-HD-4-3383) (http://www.medschool.umaryland.edu/btbank/ and the Stanley Medical Research Institute: http://www.stanleyresearch.org/brain-research/). Clinical characterization, neuropathological screening, toxicological analyses, and dissections of various brain regions were performed as previously described (Lipska et al. 2006; PMID: 16997002). All patients met DSM-IV criteria for a lifetime Axis I diagnosis of psychiatric disorders including schizophrenia or schizoaffective disorder, bipolar disorder and major depression. Controls had no history of psychiatric diagnoses or addictions. SNP array: Array-based genotyping was performed on most samples published in this collection. The number of SNPs assayed via Illumina chips varied between 650,000 and 5 Million. Cerebellar tissue was generally used for genotyping studies. # Diagnosis SNP Array 1 Anxiety Disorder 1 2 Autism Spectrum Disorder 13 3 Bipolar Disorder 114 4 Control 387 5 Eating Disorder (ED) 2 6 Major Depressive Disorder (MDD) 186 7 Obsessive Compulsive Disorder (OCD) 5 8 Post-Traumatic Stress Disorder (PTSD) 0 9 Schizophrenia 220 10 Other 7 11 Tic Disorder 3 12 Undetermined 1 13 Williams Syndrome 2 Table: Numbers of samples in each diagnostic category. DNA extraction: 45-80 mg of cerebellar tissue was pulverized for DNA extractions. The QIAamp DNA mini Kit (Qiagen) method was employed for tissue DNA extraction. The tissue was initially lysed using Tissue Lyser (Qiagen) and extractions were accomplished according to manufacturer's protocol. The DNA was captured in 500uL elution buffer. The concentrations were measured using Thermo Scientific's NanoDrop 1000/NanoDrop ONE. The mean yield was 128.85 uG (+/- 79.48), the mean ratio of 260/280 was 1.87 (+/- 0.105), and the mean ratio of 260/230 was 2.48 (+/-1.75). Genotyping methods: Three types of Illumina Beadarray chips were used: HumanHap650Y, Human1M-Duo, and HumanOmni5M-Quad (San Diego, California). The genotyping was done according to the manufacturer's protocol (Illumina Proprietary, Catalog # WG-901-5003, Part # 15025910 Rev.A, June 2011). Approximately, 400ng DNA was used and each DNA sample was QC tested for 260/280 ratio by nanodrop and DNA band intactness on 2% agarose gel. Briefly, the samples were whole-genome amplified, fragmented, precipitated and resuspended in appropriate hybridization buffer. Denatured samples were hybridized on prepared Bead Array Chips. After hybridization, the Bead Chip oligonucleotides were extended by a single fluorescent labeled base, which was detected by fluorescence imaging with an Illumina Bead Array Reader, iScan. Normalized bead intensity data obtained for each sample were loaded into the Illumina Genome Studio (Illumina, v.2.0.3) with cluster position files provided by Illumina, and fluorescence intensities were converted into SNP genotypes. Microarray: We generated RNA expression data using array technology for psychiatric subjects compared to non-psychiatric subjects as controls. We used tissues from three different brain regions i.e. hippocampus, dorsolateral prefrontal cortex (DLPFC), and dura mater for a large cohort of individuals (total number 552 subjects for hippocampus, 800 for DLPFC and 146 for dura). Total RNA was extracted from ~100 mg of tissue using the RNeasy kit (Qiagen) according to the manufacturer's protocol. RNA quality and quantity were examined using the Bioanalyzer (Agilent, Inc) and NanoDrop (Thermo Scientific, Inc), respectively. Samples with RNA integrity number (RIN) # Diagnosis DLPFC Hippo Dura 1 Anxiety Disorder 1 0 0 2 Autism Spectrum Disorder 14 6 0 3 Bipolar Disorder 90 49 0 4 Control 336 270 75 5 Eating Disorder (ED) 2 1 0 6 Major Depressive Disorder (MDD) 144 87 0 7 Obsessive Compulsive Disorder (OCD) 5 3 0 8 Post-Traumatic Stress Disorder (PTSD) 6 0 0 9 Schizophrenia 192 125 71 10 Other 5 6 0 11 Tic Disorder 3 3 0 12 Undetermined 1 1 0 13 Williams Syndrome 2 1 0 Table: Numbers of samples in each diagnostic category. RNA-Seq of Dorso-lateral prefrontal cortex: All brains were collected and the dorsolateral prefrontal cortical (DLPFC) samples dissected at the HBCC, DIRP, NIMH. Dorsolateral prefrontal cortex (DLPFC) specimens were dissected from right or left hemisphere of frozen coronal slabs. The study was funded by the DIRP, NIMH under contract (#HHSN 271201400099C) with Icahn School of Medicine at Mount Sinai,1106402 One Gustave L. Levy Place, Box 3500, New York NY 10029-6574. RNA extraction, library preparation and sequencing were performed under contract at Icahn School of Medicine. The Common Mind Consortium (CMC) provided project management support. RNA isolation: Total RNA from 468 HBCC samples was isolated from approximately 100 mg homogenized tissue from each sample by TRIzol/chloroform extraction and purification with the Qiagen RNeasy kit (Cat#74106) according to manufacturer's protocol. Samples were processed in randomized batches of 12. The order of extraction for schizophrenia, bipolar, and MDD disorders and control samples was assigned randomly with respect to diagnosis and all other sample characteristics. The mean total RNA yield was 24.2 ug (+/- 9.0). The RNA Integrity Number (RIN) was determined by 4200 Agilent TapeStation System. Samples with RIN DLPFC RNA-Seq quantified expression data are provided for 364 samples. Data were generated, QC'd, processed and quantified as follows: RNA library preparation and sequencing: All samples submitted to the New York Genome Center for RNAseq were prepared for sequencing in randomized batches of 94. The sequencing libraries were prepared using the KAPA Stranded RNAseq Kit with RiboErase (KAPA Biosystems). rRNA was depleted from 1ug of RNA using the KAPA RiboErase protocol that is integrated into the KAPA Stranded RNAseq Kit. The insert size and DNA concentration of the sequencing library was determined on Fragment Analyzer Automated CE System (Advanced Analytical) and Quant-iT PicoGreen (ThermoFisher) respectively. Schizophrenia Bipolar Control 89 65 210 Table: Numbers of samples in each diagnostic category. RNA-Seq of subgenual anterior cingulate cortex (sgACC): All the 200 post-mortem brain samples (61 controls; 39 bipolar disorder; 46 schizophrenia; 54 major depressive disorder) were collected by the HBCC, DIRP, NIMH. RNA Extraction and Quality Assessment: Tissue from sgACC was pulverized and stored at -80°C. Total RNA was extracted from 50-80 mg of the tissue using QIAGEN RNeasy Lipid Tissue Mini Kit (QIAGEN, Cat. # 74804) with DNase treatment (QIAGEN, Cat. # 79254). The RNA Integrity Number (RIN) for each sample was assessed with high-resolution capillary electrophoresis on the Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, California). The concentration of RNA and their 260/280 ratio (2.1+/- 0.032 SD) were determined with NanoDrop (Thermo Scientific). RNA sequencing: Stranded RNA-Seq libraries were constructed after rRNA depletion using Ribo-Zero GOLD (Illumina). RNA sequencing was performed at National Institute of Health Intramural Sequencing Center (NISC). Schizophrenia Bipolar Control MDD 46 39 61 54 Table: Numbers of samples in each diagnostic category. Whole Genome Sequencing: All brains were collected and dissected at the HBCC, DIRP, NIMH. This study generates whole genome sequencing data using sequencing of DNA in the dorsolateral prefrontal cortex (DLPFC), anterior cingulate cortex (ACC) or cerebellum of 443 individuals with schizophrenia, bipolar disorder and major depressive disorder and non-psychiatric controls. The study was funded by the DIRP, NIMH under contract (#HHSN 271201400099C) with Icahn School of Medicine at Mount Sinai,1106402 One Gustave L. Levy Place, Box 3500, New York NY 10029-6574. DNA extraction, library preparation and sequencing were performed under contract at Icahn School of Medicine. The Common Mind Consortium (CMC) provided project management support. All specimens were dissected from right or left hemisphere of frozen coronal slabs. DNA Library Preparation and Sequencing: All samples submitted to the New York Genome Center for WGS were prepared for sequencing in randomized batches of 95. The sequencing libraries were prepared using the Illumina PCR-free DNA sample preparation Kit. The insert size and DNA concentration of the sequencing library was determined on Fragment Analyzer Automated CE System (Advanced Analytical) and Quant-iT PicoGreen (ThermoFisher) respectively. A quantitative PCR assay (KAPA), with primers specific to the adapter sequence, was used to determine the yield and efficiency of the adaptor ligation process. Performed on the Illumina HiSeqX with 30X coverage. Schizophrenia Bipolar Control 115 78 230 Table: Numbers of samples in each diagnostic category. ChIP-Seq: All brains were collected and the dorsolateral prefrontal cortical (DLPFC) samples dissected at the HBCC, DIRP, NIMH. This study generates epigenetic data using sequencing of DNA after chromatin immunoprecipitation (ChIP-Seq) for marks H3K4me3 and H3K27ac in the dorsolateral prefrontal cortex (DLPFC). Dorsolateral prefrontal cortex (DLPFC) specimens were dissected from right or left hemisphere of frozen coronal slabs. The study was funded by the DIRP, NIMH under contract (#HHSN 271201400099C) with Icahn School of Medicine at Mount Sinai,1106402 One Gustave L. Levy Place, Box 3500, New York NY 10029,6574. Chromatin precipitation, library preparation and sequencing were performed under contract at Icahn School of Medicine. The Common Mind Consortium (CMC) provided project management support. Chromatin immunoprecipitation (ChIP) assays for histone marks H3K4me3 and H3K27ac were carried out using Native ChIP. Micrococcal Nuclease (MNase) (Sigma, N3755) treatment was used to digest chromatin into mononucleosomes. The following antibodies were used for chromatin pull-down: anti-H3K4me3 (Cell Signaling, Cat# 9751BC, lot 7) and anti-H3K27ac (Active Motif, Cat# 39133, Lot # 31814008). Histone modification-enriched genomic DNA fragments were recovered using Protein A/G magnetic beads (Thermo Scientific, 88803-88938 or Millipore 16-663), and then washed, eluted, and treated with RNAse A and proteinase K. Final ChIP DNA products were isolated using phenol-chloroform extraction followed by ethanol precipitation. The efficiency of each ChIP assay was validated using Qubit concentration measurement and qPCR for positive (GRIN2B, DARPP32) and negative (HBB) control genomic regions. Only ChIP assays that passed quality control were further processed for library preparation and sequencing; this included ChIP DNA that was not detectable on Qubit but showed a good signal and expected enrichment patterns in qPCR. HISTONE_MARK H3K27ac H3K4me3 Input Bipolar 56 4 7 Control 158 11 24 Schizophrenia 79 11 12 Table: Numbers of individuals in each assay grouped by histone mark or input.Long-Read Whole-Genome Sequencing (WGS) Cohort Description: Brain specimens were obtained from the Human Brain Collection Core (HBCC), part of the NIH NeuroBioBank. Samples were collected under protocols approved by the NIH CNS Institutional Review Board (IRB) (NCT03092687), with informed consent from next-of-kin (NOK). Collection was coordinated through the Offices of the Chief Medical Examiners (MEOs) in Washington, D.C., Northern Virginia, and Central Virginia. Clinical metadata and documentation are publicly available via the NIMH Data Archive (NDA) (Collection #3151) https://nda.nih.gov/edit_collection.html?id=3151 Eligibility Criteria No clinical diagnosis of major neuropsychiatric or neurodegenerative diseaseNo diagnosis of cognitive impairment during life All individuals were confirmed to be neurologically normal at time of deathDemographics Initial cohort size: 155 individuals Ancestry: All individuals self-identified as African or African-admixed Mean age at death: 44.2 years (range: 18–85 years) Sex distribution: 36.4% femaleSample Processing: Frozen frontal cortex tissue was dissected and processed according to the public protocol: https://www.protocols.io/view/processing-human-frontal-cortex-brain-tissue-for-p-kxygxzmmov8j/v2. High-molecular-weight DNA was extracted and libraries were prepared using the Oxford Nanopore Technologies (ONT) LSK-114 kit. Sequencing was performed using ONT PromethION flow cells (R10.4.1 chemistry) Data Processing and Quality Control: Basecalling: Conducted using Guppy v6.38 Read Alignment: Reads were aligned to the GRCh38 reference genome using minimap2 Sample Identity Verification: Sample identity was validated by comparing ONT-derived SNP calls with matched short-read WGS genotypes to ensure concordance and prevent sample swaps Variant Calling and Phasing: Reads were base-called with Guppy v6.38. Reads were aligned to GRCh38 using minimap2. We verified sample identity by cross-checking ONT SNV calls with the existing short-read WGS genotypes, confirming no sample switches. The napu pipeline (https://github.com/nanoporegenomics/napu_wf) produced; haplotype-resolved assemblies, joint small-variant (SNV/indel) calls, and multi-caller structural-variant sets, all reported on GRCh38 and phased where possible. Raw signal data were basecalled to obtain 5-methyl-cytosine (5mC) status; methylation tags were added to the phased BAM files. Genome-wide methylation summaries are provided in BED format.Dataset Filtering and Exclusions: All 155 samples underwent sequencing and SNP-based ancestry inference 8 samples were excluded due to ancestry inconsistent with African or African-admixed background 1 sample was excluded due to insufficient sequencing quality Final Sample Set: 146 high-quality samples from individuals of African or African-admixed ancestry were retained for downstream analyses See PMID: 39764002 for further analysis detailsDiagnosis#SamplesControl155Table: Diagnostic Summary.Note: The data derived from HBCC resources were removed from dbGAP and are now available in the NIMH Data Archive (NDA). They include genotypes, short read whole genome sequencing (WGS), epigenetics (DNA methylation, ChIP-seq for histones), RNA expression (qPCR, microarray, RNA-seq, single nucleus RNA-seq) of various brain regions in cases with schizophrenia, bipolar disorder, major depression, substance use disorders and normative controls. Please access our NDA collection (https://nda.nih.gov/edit_collection.html?id=3151) for further detail.

  Study phs000979

• BC Cancer, part of the Provincial Health Services Authority, Technology Development Office, Data Access Committee (PHSA TDO DAC)

  Access to data generated by BCCA is made available by completing the data access agreement for review by the data access committee and will be granted to qualified investigators for appropriate use.

  Dac EGAC00000000011

• GCAT| WGS FASTQ V1

  This dataset include FASTQ files of 808 samples from GCAT cohort. Technology used HiSeq 4000, read length 150 bp, inner mate distance 300 bp. For each sample the paired -ends are generated in separated files. Each FASTQ is splitted in multiple LANEs and grouped by the Multiplex index.

  Dataset EGAD00001008201

• Longitudinal Study of Fluoride and Other Factors Related to Dental Fluorosis, Dental Caries, and Bone Health

  This project is a combination of the Iowa Fluoride Study and the Iowa Bone Development Study from 1992 - 2019. The Iowa Fluoride Study (IFS) began in 1992 with recruitment of 1,882 mothers of newborns from eight Iowa hospital post-partum wards from March 1992 to February 1995. The focus of the IFS was to collect detailed fluoride, general dietary intake, and other data several times per year from age 6 weeks on and relate them to dental fluorosis and dental caries (cavities). Data collection occurred 3-5 times per year initially, and then twice per year after ~ age 4. Dental exams of the primary (baby) teeth were initiated at ~ age 5.The Iowa Bone Development Study (IBDS) grew out of the IFS in 1998 when we added a "normal bone development" focus, and soon after the age 5 dental exams, we began to collect genetic samples and accelerometry (motion sensor) data. All then-active parents of IFS-participating children were invited to have their child join the IBDS, and the majority agreed to do so. The majority of participating children received both ~ age 5 dental exams and bone densitometry assessments. The bone densitometry at age 5 was determined by dual-energy X-ray absorptiometry (DXA) of the hip, lumbar spine, and whole body.Dental exams for caries and fluorosis were performed again at age 8-9, along with DXA bone scans and accelerometry. DXA scans and accelerometry were performed again at ages 11 and 15 years, along with peripheral quantitative computed tomography (pQCT) scans of the distal tibia and radius, but no dental exams were performed.At ages 13 and 17, dental exams, DXA and pQCT scans, as well as accelerometry, were obtained.At age 19, no dental exams were conducted, but repeat DXA and pQCT scans and accelerometry were obtained, along with multi-slice detector CT (MDCT) of the distal tibia.At age 23, we conducted the final wave of exams, which included dental, DXA, pQCT, MDCT, and accelerometry assessments.In addition, parents were invited to participate once with DXA bone scans and to also provide limited questionnaire data about their own fluoride and dietary exposures and physical activity.Note: Please refer to the document Iowa Fluoride Study/Iowa Bone Development Study (IFS/IBDS) - dbGaP Data Tables: A User's Guide - Version 2.0 for a detailed reference manual on all of the clinical data submitted for this project.Note: No genetic data or specimens were submitted with this study. However, many of the child/parent participants in the study were genotyped. These genotyping data are linked to dbGaP Study Accession: phs000095 under Dr. Mary Marazita: "Dental Caries: Whole Genome Association and Gene X Environment Studies".<img style="width: 1155px;" 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

  Study phs002203