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• Cell and Circuit-Specific Exploration of HIV Neurogenomics in Context of Opiate and Cocaine Misuse

  Utilizing frozen postmortem brain tissue from the Manhattan HIV Brain Bank, we performed cell-type-specific, genome-wide sequencing of three different types: 1) single-nucleus RNA-sequencing (snRNA-seq), 2) in situ Hi-C, and 3) HIV integration site sequencing (IS-seq). We assayed tissue from three different donor types: 1) HIV-uninfected control (HIV-), HIV-infected (HIV+), and HIV-infected with HIV encephalitis (HIVE), who served as a positive control for active CNS viral infection. The diagnosis of HIVE was made by a board-certified neuropathologist on routine histopathology, based on the presence of a microglial nodule encephalitis with characteristic multinucleated syncytial cells and staining for HIV p24 antigen. Other HIV-infected individuals (the HIV+ group) did not have this histologic evidence of active HIV brain replication, and were variable with regard to cART status and virologic control prior to death. Donor characteristics, including ante-mortem plasma HIV loads, CD4 counts, and cART status, were documented. Quality controls were used for each assay: For snRNA-seq we applied filters to remove low quality nuclei from analysis and performed experiments in which mouse tissue was mixed with human tissue to determine a reliable HIV read count threshold. For Hi-C, we used paired HIV- and HIVE samples and processed all samples in parallel, at the same time. Correlation analysis was used to ensure biological replicates were aligned. For IS-seq, each time the assay was run, a positive and negative control were included. This allowed us to ensure a lack of contamination in IS-seq libraries and that, when a particular sample failed to yield library, this was due to low levels of HIV rather than a failure of the assay. Altogether, we built an integrative dataset from 79 sequencing files, including snRNA-seq, and cell type specific Hi-C, IS-seq, and ChIP-seq datasets. Human autopsy brain samples were collected by the Manhattan HIV Brain Bank (MHBB, U24MH100931), using protocols under the supervision of the Icahn School of Medicine at Mount Sinai (ISMMS) Institutional Review Board. Written informed consent was obtained either from decedents or their primary next of kin. Major Findings: Reorganization of open/repressive (A/B) compartment structures in HIVE microglia encompassing 6.4% of the genome was linked to transcriptional activation of interferon (IFN) signaling and cell migratory pathways and was partially recapitulated by IFN-gamma stimulation of cultured microglia. In contrast, decreased expression and repressive compartmentalization of genes regulating neuronal health and signaling was seen in both HIVE and HIV+ microglia. IS-seq recovered 1,221 integration sites in the brain that displayed distinct genomic patterns as compared to peripheral lymphocyte integration and were enriched for chromosomal domains newly mobilized into a permissive chromatin environment in HIVE microglia. Viral transcription occurred in a subset of highly activated microglia comprising 0.003% of all nuclei in HIVE brain. Our findings point to a disruption of microglia-neuronal interactions in the HIV infected brain and an interrelation of retroviral integration and expression with interferon-associated remodeling of the microglial 3D genome during progression to HIVE. Search terms: HIV, postmortem brain, microglia, substance abuse, viral integration, chromosomal conformation capture, Hi-C, chromatin, single nuclei RNA-seq, Irf8, NeuN, HIV encephalitis, neuron-microglia interaction, cytokine

  Study phs003080

• Effect of ETS2 modulation on chromatin accessibility and enhancer activity in inflammatory (TPP) macrophages

  This dataset consists of H3K27ac ChIP-seq and ATAC-seq in primary human macrophages following modulation of ETS2 expression. There are three datasets: H3K27ac ChIP-seq in ETS2-edited and unedited inflammatory (TPP) macrophages, H3K27ac ChIP-seq in ETS2-overexpressing and control M0 macrophages, and ATAC-seq in ETS2-edited and unedited inflammatory macrophages. For editing experiments, monocytes were transfected with CRISPR-Cas9 ribonucleoproteins containing a gRNA that was validated to edit ETS2, or a non-targeting control gRNA. Inflammatory macrophage differentiation was performed using conditions that model chronic inflammation (TPP): 3 days GM-CSF (50ng/mL) followed by 3 days GM-CSF, TNFa (50ng/mL), PGE2 (1mg/mL), and Pam3CSK4 (1mg/mL). For overexpression experiments, controlled overexpression of ETS2 mRNA or control mRNA (an equivalent amount of mRNA encoding the reverse complement of ETS2 – thereby controlling for the quantity, length and purine/pyrimidine composition of the transfected RNA but with a transcript that would not be translated) was induced in resting, non-activated (M0) macrophages by transfecting 500ng mRNA. To minimise non-specific activation due to the transfected RNA, in vitro transcription was performed using co-transcriptional capping (to minimise uncapped products), and incorporating modified, minimally immunogenic nucleotides (replacing uridine with N1-methyl-pseudouridine and cytidine with methylcytidine). After 18 hours, transfected cells were were activated with low dose LPS and harvested 6 hours later. For H3K27ac ChIP-seq, cells were harvested, cross-linked, quenched, lysed, and sheared. Immunoprecipitation of histone-DNA complexes was performed overnight at 4C with rotation using an anti-H3K27ac antibody and the SimpleChIP Plus Sonication ChIP kit (Cell Signaling Technology). Following reverse cross-linking, 50ng of immunoprecipitated DNA or input DNA were used to prepare sequencing libraries using the iDeal Library Preparation kit (Diagenode), according to manufacturer instructions. 10 PCR cycles were used for the amplification step and size selection was not performed. The quality and molarity of all libraries was assessed using a BioAnalyzer 2100 (Agilent) and the libraries were sequenced on an Illumina NovaSeq 6000. Raw data are provided as raw and aligned sequencing reads from H3K27ac-bound DNA and the input chromatin. ATAC-seq in TPP macrophages was performed using the Omni-ATAC protocol (Corces et al 2017) with the following modifications: cell number was increased to 75,000 cells, cell lysis time was increased to 5 minutes; volume of Tn5 transposase in the transposition mixture was doubled; duration of the transposition step was extended to 40 minutes. Sequencing libraries were prepared by PCR with the NEBNext Ultra II Q5Master Mix (NEB) as described previously (Corces et al 2017). Amplified libraries were cleaned using AMPure XP beads (Beckman Coulter). Sequencing reads were trimmed using TrimGalore! (Phred score 24), filtered to remove reads < 20 bp and aligned to the human genome (hg19) using Burrows-Wheeler Aligner (BWA). Aligned reads were converted to BAM files, sorted and indexed using SAMtools (Li et al, 2009). PCR duplicates and unmapped reads were removed, and the resulting BAM files were re-sorted and indexed. Raw data are provided as 100 bp paired-end Illumina reads from n = 3 donors for each experiment. BAM files are also provided.

  Study EGAS50000000109

• Multiple_myeloma_precursor_genomics

  Whole genome sequencing of up to 45 multiple myeloma precursor samples and matched normals pairs.

  Study EGAS00001006312

• OAC RNASeq

  Please see our related publication, 'Molecular profiling to predict immunochemotherapy outcomes in esophageal adenocarcinoma'

  Study EGAS00001006468

• Sanger sequencing of catalytic-domain encoding exons of tyrosine kinase genes from human endometrial tumor DNAs

  The purpose of the original study was to search for somatic mutations in the tyrosine kinome of serous and clear cell endometrial carcinomas (human). The study was conducted in two phases. Phase 1: A mutation discovery screen, in which ~577 exons encoding the catalytic domains of 86 tyrosine kinases were PCR-amplified and bidirectionally Sanger sequenced from 24 serous, 11 clear cell, and 5 mixed histology endometrial tumors. This was followed by alignment of sequence reads to the human reference sequence and subsequent nucleotide variant calling to identify potential somatic (tumor-specific) mutations. Potential somatic mutations were confirmed by re-amplification and sequencing of the relevant tumor DNA as well as matched non-tumor ("normal") DNA from the same case. Phase 2: A mutation prevalence screen, in which the non-catalytic regions two tyrosine kinase genes, TNK2 and DDR1, were PCR-amplified and sequenced from the 40 discovery screen tumors, and all coding exons of TNK2 and DDR1 were PCR-amplified and sequenced from another 10 clear cell, 21 serous, and 41 endometrioid endometrial tumors, in an effort to identify additional somatic mutations in each gene. Exons encoding the exonuclease domain of POLE were also sequenced to document somatic mutations.

  Study phs000841

• The National Heart, Lung, and Blood Institute (NHLBI)-funded Next Generation Genetic Association Studies (NextGen) Consortium: Phenotyping Lipid traits in iPS derived hepatocytes Study (PhLiPS Study)

  The goal of the PhLiPS study is to create a library of induced pluripotent stem cell (iPSC) lines and iPSC-derived hepatocytes of diverse genotypes for use in metabolic profiling and interrogating lipid phenotypes. These cell lines were created as a part of the Next Generation Genetic Association Studies (Next Gen) Program, which was a five-year, $80 million program to investigate functional genetic variation in humans by assessing cellular profiles that are surrogates for disease phenotypes. To achieve this, researchers from multiple institutions across the U.S. were awarded grants to derive iPSC lines from more than 1,500 individuals representing various conditions as well as healthy controls for use in functional genomic ("disease in a dish") research. This extensive panel includes a diverse set of age, gender, and ethnic backgrounds, and therefore will be an invaluable tool for evaluations across demographics. Further enhancing the utility of these cell lines are data sets such as phenotyping, GWAS, genome sequencing, gene expression and -omics analyses (e.g., lipidomic, proteomic, methylomic) that can be matched to the cell lines. The PhLiPS Study focuses on individuals free of cardiovascular disease or with lipoprotein metabolism disorders in the community served by the Hospital of the University of Pennsylvania.

  Study phs001341

• Identification and Targeting of Inflammatory Macrophage-Fibroblast Crosstalk in Rheumatoid Arthritis

  Macrophages tailor their function according to the signals found in tissue microenvironments, assuming a wide spectrum of phenotypes. A detailed understanding of macrophage phenotypes in human tissues is limited. Using single-cell RNA sequencing, we defined distinct macrophage subsets in the joints of patients with the autoimmune disease rheumatoid arthritis (RA), which affects ~1% of the population. The subset we refer to as HBEGF+ inflammatory macrophages is enriched in RA tissues and is shaped by resident fibroblasts and the cytokine tumor necrosis factor (TNF). These macrophages promoted fibroblast invasiveness in an epidermal growth factor receptor-dependent manner, indicating that intercellular cross-talk in this inflamed setting reshapes both cell types and contributes to fibroblast-mediated joint destruction. In an ex vivo synovial tissue assay, most medications used to treat RA patients targeted HBEGF+ inflammatory macrophages; however, in some cases, redirecting them into a state is not expected to resolve inflammation. These data highlight how advances in our understanding of chronically inflamed human tissues and the effects of medications therein can be achieved by studies on local macrophage phenotypes and intercellular interactions. Reprinted from Kuo, Ding et al., Science Translational Medicine 2019, PMID: 31068444, with permission from American Association for the Advancement of Science.

  Study phs001340

• NHLBI TOPMed: Recipient Epidemiology and Donor Evaluation Study-III Brazil Sickle Cell Disease Cohort (REDS-BSCDC)

  Establishing a Brazilian Sickle Cell Disease Cohort and Identifying Molecular Determinants of Response to Transfusions, Genetic Determinants of Alloimmunization, and Risk Factors Associated with HIV Infection. The REDS-III Brazil SCD Cohort study focused on transfusion practices and predictors of health outcomes in patients with Sickle Cell Disease (SCD) and began in the Fall of 2013. The four primary aims of this study are: 1) Aim A - Establish a cohort of SCD patients with a comprehensive centralized electronic database of detailed clinical, laboratory and transfusion information, as well as establish a repository of blood samples to support biological studies relevant to SCD pathogenesis and transfusion complications; 2) Aim B - Characterize changes in markers of inflammation in response to transfusion by analyzing chemokine/cytokine panels in serial post transfusion specimens; 3) Aim C - Identify single nucleotide polymorphisms (SNPs) that contribute to the risk of red blood cell alloimmunization in SCD by performing a genome-wide association (GWA) study in transfused SCD patients; and, 4) Aim D - Characterize risk of HIV and HIV outcomes in the Brazilian SCD population and compare SCD outcomes among HIV sero-positive and sero-negative SCD patients. Patients are enrolled from six hospitals affiliated with the participating four REDS-III Brazil hemocenters.

  Study phs001468

• National Eye Institute (NEI) Exfoliation Genotyping Study

  The overall goal of our research is to elucidate the pathogenesis of exfoliation syndrome and the associated glaucoma, making it possible to implement effective screening and prevention strategies and to develop novel therapies. Cases genotyped for this project are from the Nurses Health Study (NHS), the Health Professionals Follow-up Study (HPFS), the Women&#39;s Genome Health Study (WGHS), the Massachusetts Eye and Ear Infirmary (MEEI), Mayo Clinic, the University of Iowa, the University of Miami and Duke University Medical Center. Exfoliation syndrome (XFS) is a common condition that is a major risk factor for high-tension open-angle glaucoma, pre-mature cataract formation, and increased risk of complications during cataract surgery. Previous studies suggest that exfoliation syndrome and the related glaucoma (XFG) are genetically complex, and one gene, LOXL1, has been identified as a major genetic risk factor. LOXL1 variants are found in up to 98% of affected patients; however, these same variants are also present in up to 80% of unaffected individuals, indicating that additional genetic and/or environmental factors are necessary for disease development. Samples genotyped for this study are a case set (1,096 samples) and 24 pedigrees (98 samples). Controls for association analyses are selected from the NEIGHBORHOOD study.

  Study phs001053

• Genetic analysis of Hirschsprung disease

  Hirschsprung disease (HSCR) is a birth defect resulting from the absence of nerve (ganglion) cells in the gastrointestinal tract. Hirschsprung disease has a population incidence of 1/5,000 live births and most often occurs as an isolated condition. However, approximately 30% of HSCR cases are associated with other birth defects such as Down syndrome, deafness, hypopigmentation, and Congenital Central Hypoventilation syndrome (CCHS). Hirschsprung disease is a genetic condition with autosomal dominant, autosomal recessive, and multigenic patterns of inheritance described. The goal of the research study is to identify genes harboring causative HSCR mutations and to better understand the complex inheritance of HSCR in families using whole genome mapping and sequencing studies with functional follow-up of candidate genes and variants. We intend to ascertain the frequency with which mutations in any human gene lead to familial and isolated forms of HSCR. Clinical information is collected to allow investigation of possible genotype - phenotype correlations. The subject population consists of individuals diagnosed with HSCR and their unaffected relatives. Individuals/families are ascertained through support groups, web-based listings of research studies and genetic testing services, an educational study website, and referrals from genetic counselors and physicians. Blood, or tissue, samples are requested from affected individuals and their unaffected relatives.

  Study phs000497