Search Results - EGA European Genome-Phenome Archive

Genomic Sequencing of Cervical Cancers

Cervical cancer is responsible for 10-15% of cancer related deaths in women worldwide. The etiological role of infection with high-risk human papilloma viruses (HPV) in carcinomas of the cervix is well established. In general, the development of cervical carcinomas follows a progression from persistent HPV infection through precancerous lesions to invasive cancer. Previous studies have implicated somatic mutations in PIK3CA, PTEN, TP53, STK11 and KRAS as well as chromosome-arm level copy number alterations in the pathogenesis of cervical carcinomas. Here, we report whole exome sequencing analysis of 118 cervical carcinoma-normal paired samples from patients in Norway and Mexico, as well as transcriptome sequencing of 80 cases and whole genome sequencing of 13 tumor-normal pairs. Novel somatic mutations include recurrent E322K substitutions in the MAPK1 gene encoding the ERK2 kinase and inactivating mutations in the HLA-B gene. In addition, recurrent somatic mutations in FBXW7, EP300, and NFE2L2 are novel in the context of primary cervical carcinomas. Analysis of HPV integration sites revealed recurrent integration into the RAD51B locus as well as co-occurrence of HPV genome integration and copy number gains within several genomic loci. These findings shed new light on the pathogenesis of cervical carcinomas and suggest potential novel therapeutic targets.

Study phs000600

Single Cell and Tissue Level Functional Genomics Analysis of Astrocyte-Related Mechanisms in Taupathy

This data is the first part of a larger single cell and tissue level functional genomics analysis of dementias with tau pathology.  Cellular resolution will clarify the role of glial genes and pathways in neurodegenerative disease. This includes understanding the role of microglia and astrocytes in neurodegenerative disorders characterized by Tau protein pathology. To achieve a refined view of microglial neuroinflammatory signaling in tau-associated neurodegeneration, we performed a functional genomic analysis that integrated time-course gene expression data from mouse models expressing human mutant MAPT (the tau gene) with single nuclear sequencing data obtained from patients with frontotemporal dementia with Tau protein pathology (Pick’s disease). This included microglia-specific gene expression data obtained from single nuclear sequencing of postmortem frozen precentral gyrus samples collected from 16 total individuals, including 8 subjects with clinical diagnosis of behavior variant frontotemporal dementia and neuropathological diagnosis of Pick’s disease (Tau protein pathology) and controls (n=8). Our findings present detailed molecular profiles of disease-associated microglial transitions over the course of tau-associated neurodegeneration. Furthermore, we demonstrate overlap between microglia profiles obtained from precentral gyrus of patients with Pick’s disease with those up-regulated during the neurodegenerative stage in mice expressing human MAPT mutations.

Study phs002197

Whole genome sequencing of core-binding factor leukemia

Pediatric de novo acute myeloid leukemia (AML) is a heterogeneous disease that can be divided into clinically distinct subtypes based on the presence of specific chromosomal abnormalities or gene alterations. One of the best characterized subtypes of AML involves leukemias with alterations of the core-binding factor (CBF)-complex, which comprises the FAB subtypes M2 and M4Eo and associates with a favorable outcome. Patients with the AML M2 subtype harbor a translocation between chromosomes 8 and 21 [t(8;21)] that yields the chimeric fusion gene RUNX1(AML1)-RUNX1T1(ETO), while patients with AML M4Eo express the chimeric fusion gene CBFβ-SMMHC(MYH11) as a result of an inversion/translocation event of chromosome 16 [inv(16)/t(16;16)]. In an effort to define the total complement of genetic changes in CBF-leukemia, we performed paired-end whole genome sequencing (WGS) on diagnostic leukemia blasts and matched germ line samples from 17 pediatric CBF-leukemia patients using the Illumina platform. Somatic alterations, including single nucleotide variations (SNVs) and structural variations (SVs), including insertions, deletions, inversions, and inter- and intra-chromosomal rearrangements, were detected using complementary analysis pipelines (Bambino, CREST and CONSERTING). Recurrent screening of identified mutations will be performed in a cohort of approximately 94 cases of CBF-leukemias.

Study phs000414

International Multi-Center ADHD Genetics Project

The goal of the project is to complete a 600,000 tag SNP genome-wide association scan of 958 parent-child trios from the International Multisite ADHD Genetics (IMAGE) project, in order to assess the association of SNP markers with ADHD, analyze quantitative ADHD phenotypes, complete copy number analyses, assess parent of origin effects and season of birth effects, and test for epistasis among apparently uncorrelated genes. Acquiring DNA Samples All consent forms stipulate that the samples can only be used by researchers who have been approved by the National Institute of Mental Health (NIMH), National Institutes of Health. All consent forms, except those used at the Zürich site (N=141 subjects), explicitly indicate that the samples may be used by researchers from commercial enterprises seeking to benefit financially from the analysis of the samples. The Zürich consent does not prohibit such use. The Zürich consent form also included an "opt out" that allowed the subjects to indicate that they did not want their samples stored at the NIMH repository or used by researchers external to the project. No subjects enrolled in the project opted out. Consent groups and participant set ADHD (ADHD): 2758 (924 trios)

Study phs000016

Cross-Species Single-Cell Analysis of Pancreatic Ductal Adenocarcinoma Reveals Antigen-Presenting Cancer-Associated Fibroblasts

Cancer-associated fibroblasts (CAFs) are major players in the progression and drug resistance of pancreatic ductal adenocarcinoma (PDAC). CAFs constitute a diverse cell population consisting of several recently described subtypes, although the extent of CAF heterogeneity has remained undefined. Here we employ single-cell RNA-sequencing to thoroughly characterize the neoplastic and tumor microenvironment content of human PDAC tumors. Six human PDAC tumor specimens from six patients were collected, and processed for single-cell RNA-sequencing analysis. Adjacent-normal pancreas tissue was also collected from two of the patients. Tumor samples were digested, and fluorescence-activated cell sorting was used to isolate viable cells. For one tumor sample, viable, CD45-negative, CD31-negative, and EpCAM-negative cells were also isolated to enrich for CAFs. The 10X Chromium platform was then used to isolate single cells for RNA-sequencing analysis. This work has demonstrated the differences in immune cell populations between adjacent-normal and tumor tissues, and identified subpopulations of epithelial cells and CAFs present in PDAC tumors. This high-throughput analysis is a resource to better understand the cell populations present in PDAC, and may ultimately aid in the development of more effective therapies for this deadly malignancy.

Study phs001840

Integrated Genomic Analysis of Periampullary Tumors at The Human Genome Sequencing Center, Baylor College of Medicine (HGSC-BCM)

Adenocarcinomas arising in the complex environment of the ampulla of Vater constitute a histopathological heterogenous group, presumably originating from the different epithelial cellular constituents present at the site: pancreas, bile duct, and intestinal duodenum. These tumors have been described in many different ways: intra-ampullary, periampullary, intra-ampullary papillary-tubular neoplasm, ampullary-ductal, periampullary-duodenal, and ampullary-not otherwise specified. These varied classifications reflect the difficulty in classifying these tumors into specific groups. Only the tumors clearly localized in the bile duct or duodenum are identified as distal cholangiocarcinomas (CAC) or duodenal adenocarcinomas (DUOAC). The current classification is based on macroscopic features that may distinguish the epithelium of origin, microscopic features, clinicopathological criteria, histopathology and expression of differential markers. This classification is subjective and prone to inter-observer variability and significantly impacts on treatment selection and therapeutic development. In order to define subtypes of periampullary cancer with clinical relevance, we performed whole exome sequencing and copy number analysis of 160 cancers arising in the periampullary region, 62 of these clearly arising from either the bile duct (n = 44), or the duodenum (n = 18) and 98 periampullary cancers (AMPAC) where the epithelium of origin could not be clearly defined.

Study phs000895

Trisomy21: Risk Factors for Chromosome Nondisjunction (T21NDJ)

The overall goal of the project is to identify genetic risk factors associated with chromosome 21 nondisjunction in the oocyte. The dataset derives from multi-site collection of live birth probands with Down syndrome due to standard trisomy 21 (T21) and their biological parents. The type of nondisjunction error (NDJ)(maternal or paternal) in all cases has been determined to be maternal in origin based on the chromosome 21 variants contributed from parent to proband. The Center of Inherited Disease with support from NICHD has conducted genome-wide genotyping using the Illuminia Human OmniExpress Plus Exome array on approximately 800 women who have been identified through their offspring with DS and have been characterized as having a maternal meiois I (MI) or meiosis II (MII) nondisjunction error. Genotypes from biological fathers of the offspring with DS can be used with the data on mothers to better define the type of nondisjunction error (MI or MII) and to refine the chromosome 21 recombination profile. We provide the type of nondisjunction error, knowing that this will be updated based on the new panel. We do not provide the recombination profile, as this can be best defined using the new comprehensive set of SNPs in the OmniExpress panel.

Study phs000718

NIDDK IBD Genetics Consortium Repository Immunochip

The Immunochip is a custom Illumina Infinium chip comprising 196,524 SNPs and small indels selected primarily based on GWAS analysis of 12 autoimmune and inflammatory diseases. The chip has two purposes: fine mapping of 289 established associations corresponding to 187 distinct loci, and deep replication of suggestive, but not yet proven, associations. Fine-mapping regions were defined as 0.2cM centered on GWAS hit SNPs, and all SNPs and short indels in these regions from the 1000 Genomes Project low coverage pilot CEU samples, as well as variants discovered in resequencing experiments conducted by groups collaborating in the chip design were selected for inclusion. Replication of autoimmune and inflammatory GWAS (including Crohn's disease and ulcerative colitis) contributed the bulk of the remaining SNP lists. Approximately 25,000 SNPs were included as replication of unrelated diseases as part of the WTCCC2 project, which serve as useful null SNPs for these analyses. In total, approximately 240,000 SNPs were selected for inclusion, with an assay design success rate of ~80%. Reprinted from Jostins et al. (2012), with permission from Springer Nature (see https://www.nature.com/reprints/permission-requests.html). Jostins, L., et al. (2012) Host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease. Nature, 491, 119-24. PMID: 23128233.

Study phs001721

Germline hypomorphic CARD11 mutations in severe atopic disease

We described five patients from four unrelated families who had severe atopic dermatitis and susceptibility to mild skin viral infections. Some patients also exhibited other allergic symptoms such as asthma and food allergy. We performed targeted sequencing for affected patients A-I of family A and B-I of family B, whole exome sequencing for D-I, D-II.1 and healthy mother D-II.3 of family D, and whole genome sequencing for C-I and healthy mother C-II.2 of family C. We identified three different novel damaging missense mutations of CARD11 (GeneID:84433) in patients A-I (L194P), B-I (R975W), and C-I (E57D). In patients D-I and D-II.1, a dup183_196 (p.K196_V197insMKEERDSYNDELVK) mutation was found in the coil-coil domain of the protein. These mutations act as dominant negatives impeding the scaffolding function of the wild type protein, which leads to downstream NF-kB and mTORC1 signaling defects after TCR stimulation. Sanger sequencing later identified the corresponding CARD11 mutations in the affected family members (C-II.1, D-II.2) that are not included in the dbGaP submission. Hypomorphic CARD11 dominant negative mutants are novel causes of severe atopic disease.

Study phs001369

Discovery of Non-ETS Gene Fusions in Human Prostate Cancer using Next Generation RNA Sequencing

Half of prostate cancers harbor gene fusions between TMPRSS2 and members of the ETS transcription factor family. To date little is known about the presence of non-ETS fusion events in prostate cancer. We employed next-generation transcriptome sequencing (RNA-Seq) in order to explore the whole transcriptome of 25 human prostate cancer samples for the presence of chimeric fusion transcripts. We generated more than 1 billion sequence reads and used a novel computational approach (FusionSeq) in order to identify novel gene fusion candidates with high confidence. In total, we discovered and characterized seven new cancer-specific gene fusions, two involving the ETS genes ETV1 and ERG, and five involving non-ETS genes such as CDKN1A (p21), CD9 and IKBKB (IKK-beta), genes known to exhibit key biological roles in cellular homeostasis or assumed to be critical in tumorigenesis of other tumor entities, as well as the oncogene PIGU and the tumor suppressor gene RSRC2. The novel gene fusions are found to be of low frequency but interestingly, the non-ETS fusions were all present in prostate cancer harboring the TMPRSS2-ERG gene fusion. Future work will focus on determining if the ETS rearrangements in prostate cancer are associated or directly predispose to a rearrangement prone phenotype.

Study phs000310

Somatic Mutation Profile by Next Generation Sequencing in HER2+ Breast Cancer

The HER pathway is the driving force behind 30% of human breast cancers. It is important to understand how targeted therapies block different cellular pathways, and mechanisms of escape from this blockage. Therapies directed at HER2 establish a successful treatment paradigm, but de novo and acquired resistance exist. The HER signaling system is a complex network with four receptors and eleven ligands, a phosphorylation signaling cascade, and many transcription factors, all complicated by both positive and negative feedback circuits. Analysis of genomes, exomes and transcriptomes by next generation sequencing is aimed at uncovering the genetic factors responsible for patient responses to HER2-directed therapies. We are sequencing HER2-overexpressing cancers, in order to identify potential somatic changes that may better select patients who will benefit from therapy, to determine new targets that may overcome resistance, and to improve outcomes with known current HER2-targeted therapies. Whole exome capture sequencing will determine somatic mutation profiles in HER2-overexpressing tumors, to comprehensively characterize the somatic alterations, with the goal of identifying those patients most likely to respond, as well as discovering new targets that may overcome resistance to HER2-directed therapy. We augment the whole exome data with RNA-seq data to determine expression levels of somatic mutations we discover.

Study phs000770

Targeted MitoExome Sequencing of Mitochondrial OXPHOS Diseases (Massachusetts General Hospital)

Human disorders of mitochondrial oxidative phosphorylation (OXPHOS) represent a devastating collection of inherited diseases. These disorders impact at least 1:5000 live births, and are characterized by multi-organ system involvement. They are characterized by remarkable locus heterogeneity, with mutations in the mtDNA as well as in over 77 nuclear genes identified to date. It is estimated that additional genes may be mutated in these disorders. To discover the genetic causes of mitochondrial OXPHOS diseases, we performed targeted, deep sequencing of the entire mitochondrial genome (mtDNA) and the coding exons of over 1000 nuclear genes encoding the mitochondrial proteome. We applied this 'MitoExome' sequencing to 124 unrelated patients with a wide range of OXPHOS disease presentations from the Massachusetts General Hospital Mitochondrial Disorders Clinic. The 2.3Mb targeted region was captured by hybrid selection and Illumina sequenced with paired 76bp reads. The total set of 1605 targeted nuclear genes included 1013 genes with strong evidence of mitochondrial localization from the MitoCarta database, 377 genes with weaker evidence of mitochondrial localization from the MitoP2 database and other sources, and 215 genes known to cause other inborn errors of metabolism. Approximately 88% of targeted bases were well-covered (>20X), with mean 200X coverage per targeted base.

Study phs000339

Rapid Evolution of a Skin Lightening Allele in Southern African KhoeSan

The genetic architecture and polygenicity of skin pigmentation is explored in KhoeSan communities, including Nama individuals whose phenotype and genotype data are provided in this dataset. By pairing genotypes and quantitative spectrophotometric skin pigmentation phenotypes, we show that skin pigmentation is highly heritable in KhoeSan, yet known pigmentation loci only explains a small fraction of the phenotypic variance. Using genome-wide association analyses, we identified both canonical and non-canonical skin pigmentation loci. We show that this phenotype is more polygenic and complex than previously characterized. (Martin et al., Cell 2017. PMID: 29195075) Following up on the top associated signal with large effect size in the gene SLC24A5, we demonstrate that the canonical Eurasian nonsynonymous allele was introduced into KhoeSan via a recent migration ~2 kya and was under extremely strong selection. The derived allele was present at high frequency despite controlling for gene flow. With high-throughput sequences in the captured SLC24A5 region, we show that the most common derived haplotype is identical amongst Europeans, eastern African and KhoeSan. Using 4-population demographic simulations with selection, we show that the allele was introduced into the KhoeSan only 2,000 ya via a back-to-Africa migration and then experienced a selective sweep.

Study phs001753

Generation of a Cellular Atlas of Human Adipose Tissue

White adipose tissue (WAT), once regarded as morphologically and functionally bland, is now recognized to be dynamic, plastic, heterogenous, and involved in a wide array of biological processes including energy homeostasis, glucose and lipid handling, blood pressure control, and host defense.  High fat feeding and other metabolic stressors cause dramatic changes in adipose morphology, physiology, and cellular composition, and alterations in adiposity are associated with insulin resistance, dyslipidemia, and Type 2 diabetes (T2D). Here we provide detailed cellular atlases of human and murine subcutaneous and visceral white fat at single-cell resolution across a range of body weight. We identify subpopulations of adipocytes, adipose stem and progenitor cells (ASPCs), vascular, and immune cells and demonstrate commonalities and differences across species and dietary conditions. We link specific cell types to increased risk of metabolic disease, and we provide an initial blueprint for a comprehensive set of interactions between individual cell types in the adipose niche in leanness and obesity. We also utilize bulk RNA sequencing data of enzymatically digested human adipocytes to scale these findings to a larger cohort. These data comprise an extensive resource for the exploration of genes, traits, and cell types in the function of WAT across species, depots, and nutritional conditions. 

Study phs002766

A Chromatin Accessibility Atlas of the Developing Human Telencephalon

Gene expression differs between cell types and regions within complex tissues such as the developing brain. To discover regulatory elements underlying this specificity, we generated genome-wide maps of chromatin accessibility in nine anatomically-defined regions of the developing human telencephalon. Additionally, we defined the histone modification landscape of the prefrontal cortex and generated chromatin accessibility maps of its upper and deep layers. We predicted a subset of open chromatin regions (18%) that are most likely to be active enhancers, many of which are dynamic with 26% differing between early and late mid-gestation and 28% present in only one brain region. These predicted regulatory elements (pREs) are enriched proximal to genes with expression differences across developmental stages, regions, and cortical laminae; they harbor distinct sequence motifs that suggest potential upstream regulators. We leveraged this atlas to predict and validate novel regulatory elements of genes that control cortex laminar identity and genes associated with autism spectrum disorder (ASD). These include enhancers proximal to FEZF2 and BCL11A that were validated in mouse, and an enhancer of ASD gene SLC6A1 containing two functional de novo mutations in individuals with ASD whose enhancer function we validated via CRISPRa. We also include data for transcription factor binding in mid-fetal cortex tissues, using CUT&RUN methods.

Study phs002033

Joint-Specific TF Regulation in RA

The mechanisms responsible for the distribution and severity of joint involvement in rheumatoid arthritis (RA) are not known. To explore whether site-specific fibroblast-like synoviocyte (FLS) biology might be associated with location-specific synovitis and explain the predilection for hand (wrist/metacarpal phalangeal joints) involvement in RA, we generated bulk, whole-genome transcriptomic and chromatin accessibility data from FLS. Samples were matched in age, sex, and were homogeneous in that all had significant joint damage with longstanding disease requiring surgical intervention. Analysis revealed joint-specific patterns of FLS phenotypes, with proliferative, migratory, proinflammatory, and matrix-degrading characteristics observed in resting FLS derived from the hand joints compared with hip or knee. TNF-stimulation amplified these differences, with greater enrichment of proinflammatory and proliferative genes in hand FLS compared with hip and knee FLS. Hand FLS also had the greatest expression of markers associated with an ‘activated' state relative to the ‘resting' state, with the greatest cytokine and MMP expression in TNF-stimulated hand FLS. Predicted differences in proliferation and migration were biologically validated with hand FLS exhibiting greater migration and cell growth than hip or knee FLS. Distinctive joint-specific FLS biology associated with a more aggressive inflammatory response might contribute to the distribution and severity of joint involvement in RA.

Study phs003633

Tumor detection by analysis of both symmetric- and hemi-methylation of plasma cell-free DNA

Aberrant DNA methylation plays a critical role in tumorigenesis. While DNA methylation has been used for cancer detection and classification, DNA hemi-methylation, a novel epigenetic mark has not been analyzed extensively in cancer epigenomes. Here we report a strand-specific (ss) sequencing method (MeDIP-Seq) for plasma cell free (cf) DNA (sscf-MeDIP-Seq), which can analyze both symmetrically methylated DNA regions (DMRs) as well as hemi-methylated regions (DHMRs). Using the sscf-MeDIP-Seq method, we analyzed plasma cfDNA methylomes of 271 samples from subjects with liver cancer and brain cancer and from individuals without cancer (controls). Among them, 215 samples were chosen as the discovery cohort for the identification of DMRs and DHMRs specific in each subject group and for the training of machine learning models of multi-cancer detection (MCD) using DMRs, DHMRs and DMRs+DHMRs as inputs. These models were then used to predict the 56 samples in the validation cohort. We found that models trained with DMRs+DHMRs as inputs in general outperformed models trained with DMRs or DHMRs alone, with AUROC being 0.971, 0.981, and 0.99 in predicting control, liver and brain cancer samples in the validation cohort, respectively, by the DMR+DHMR-trained models.

Study phs003462

Melanoma Genome Sequencing Project

Melanoma is the most prevalent cause of skin cancer morbidity and mortality. In order to characterize the full range of somatic mutations that may drive the growth of melanoma, we are sequencing tumor and normal DNA from a set of roughly 150 melanomas. For the majority of samples (approximately 90% of the cases), mutations in protein coding genes will be assessed in the exonic DNA of tumor-normal pairs using hybrid capture and paired-end DNA sequencing. For a few of DNA samples (approximately 10% of the cases), the entire genomes will be analyzed to assess the possible contribution of complex structural rearrangements contributing to oncogenesis. The sequencing data are supplemented by copy number profiling on the same tumors using high-density SNP arrays. Integration of these approaches will enable the unbiased and comprehensive characterization of both known and novel recurrent DNA alterations that arise in melanoma.Versions 1-4 contain whole exome and bulk RNA sequencing data for patients with melanoma.Version 5 - We compared somatic genomic profiles from matched pre-treatment and post-resistance tumor biopsies in patients with metastatic melanoma who exhibited heterogeneous responses to immune checkpoint inhibitors (ICIs). This version includes whole exome sequencing (WES) data from 14 patients.

Study phs000452

Childhood Cancer Data Initiative (CCDI): Pediatric In Vivo Testing Program –Sarcoma, Kidney, and Liver Cancers

The goal was to complete whole exome sequencing (WES) and RNA sequencing (RNA-Seq) on patient derived xenograft samples. Cancer in children is rare with approximately 15,700 new cases diagnosed annually in children 21 years or younger in the U.S. Through use of multimodality therapy (surgery, radiation therapy, and aggressive chemotherapy), 70% of patients will be 'cured' of their disease, and 5-year Event-Free Survival (EFS) exceeds 80%. Consequently, the number of patients that can be enrolled in phase I/II clinical trials is small, and most patients will have been extensively treated, hence drug/radiation resistant. Thus, preclinical studies that accurately translate into effective clinical therapy are an essential component of pediatric drug development. Our group has contributed to studies in the Pediatric Preclinical Testing Program (PPTP) and Pediatric Preclinical Testing Consortium (PPTC) that have led to clinical studies through Children's Oncology Group (COG). Of importance, we have developed and characterized over 330 Patient Derived Xenografts (PDX), developed from tumors both at diagnosis and relapse, that can be used to facilitate pediatric drug development as directed by FDA under the Research to Accelerate Cures and Equity for Children Act (RACE for Children Act).

Study phs003160

National Cancer Institute (NCI) Early Onset Malignancies Initiative (EOMI): Molecular profiling of Breast, Colon, Kidney, Liver, Multiple Myeloma, and Prostate among Racially and Ethnically Diverse Populations

The Early Onset Malignancies Initiative (EOMI) is focused on collecting specimens from racially and ethnically diverse population groups with six different types of cancer (breast, prostate, colon, liver, kidney, and multiple myeloma). This initiative is a partnership between the Center for Research Strategy, the Center for Cancer Genomics and the Division of Cancer (DCP). DCP's NCI Community Oncology Research Program (NCORP) is a critical partner. The acquisition of the cancer and matching normal biospecimens in this initiative are from newly diagnosed early onset cancer patients who identify as Non-Hispanic Black, Hispanic, American Indian or Alaska Native, or Non-Hispanic White. The Minority/Underserved NCORPs sites will collect the samples for this project. By carrying out this initiative in the NCORP's national network of investigators, cancer care providers, and research institutions, the EOMI will harness the community-based partnerships to improve participation among these racial/ethnic groups. The NCORP investigators, as participants in NCI's clinical trials network, will annotate the biologic samples with clinical information that will enhance the ability to answer questions about molecular determinants of early disease, treatment response, and/or prognosis. The EOMI will be a step forward in addressing racial/ethnic disparities and in understanding cancer in underserved populations.

Study phs001952

Single-Cell Genomic and Transcriptomic Analysis of the Aging Human Brain

We studied human brain aging using single-cell whole genome sequencing and single-nucleus RNA sequencing of the pre-frontal cortex from 19 donors ranging in age from 0.4 years to 104 years. All donors were free of neuropathology and none received a clinical neurological diagnosis prior to death. snRNA-seq libraries were prepared in batches that always included old and young donors to mitigate batch effects in the preparations. Neurons for single-cell WGS were collected from the same tissue region as the snRNA-seq for each donor but represent independent cells. Multiplexed error-robust fluorescent in-situ hybridization (MERFISH) data was also collected from pre-frontal cortex samples of a subset of the 19 donors. Transcriptomic results identified infant-specific cell clusters, and common-down regulation of cell-essential genes involved in ribosomes, transport, homeostasis and metabolism during aging, across all cell types. scWGS identified two-age associated mutational signatures, as well as gene-length and expression-level dependent somatic mutation rates in neurons. Raw sequencing data in fastq format for single-nucleus RNA-seq and single-cell DNA-seq, as well as cell and gene count matrix, transcript metadata, and cell metadata for MERFISH will be available in dbGaP.

Study phs003445

Examination of Engineered LINE-1 Integration Events in HeLa Cells

Long Interspsesed Element-1 (LINE1 or L1) encodes two proteins (ORF1p and ORF2p) required for its mobilization (i.e. retrotransposition) in the human genome. Human ORF2p, a 150kDa protein, has endonuclease (EN) and reverse transcriptase (RT) activities. L1 EN is classified as apurinic/apyrimidin-like endonuclease (APE) and is responsible for initiating target-site primer reverse transcription (TPRT) during the L1 retrotransposition cycle. During canonical TPRT, the L1 EN activity makes a single-strand endonucleolytic nick at a double-strand DNA target sequence in genomic DNA. The endonucleolytic nick is typically made at an L1 EN degnerate consensus cleavage sequence (5'-TTTT/A-3') exposing a free 3' hydroxy group that can be used as a primer by the ORF2p L1 RT activity to reverse transcribe the associated L1 RNA. Here, we characterized over 21,000 de novo engineered L1 retrotransposition events in HeLa cells to determine if L1 exhibits overt integration preferences in the human genome. Bru-seq of HeLa cells to determine actively transcribed regions of the genome was also performed. This Bru-seq data was used to determine if engineered L1 integration events preferentially integrate into transcribed regions of the genome, and to determine which strand during transcription may present itself as preferential during L1 retrotransposition.

Study phs001669

Transcriptome sequencing of human colon organoid after co-cultivation with Bifidobacterium longum

The large intestine has a dense milieu of indigenous bacteria, generating a complex ecosystem with crosstalk between individual bacteria and host cells. In vitro host cell modeling and bacterial interactions at the anaerobic interphase have elucidated the crosstalk molecular basis. Although classical cell lines derived from patients with colorectal cancer including Caco-2 cells are used, whether they adequately mimic normal colonic epithelial physiology is unclear. To address this, we performed transcriptome profiling of Caco-2 and Monolayer cells derived from healthy Human Colonic Organoid (MHCO) cultured hemi-anaerobically. Coculture with the anaerobic gut bacteria, Bifidobacterium longum subsp. longum differentiated the probiotic effects of test cells from those of physiologically normal intestinal and colorectal cancer cells. We cataloged non- or overlapping gene signatures where gene profiles of Caco-2 cells represented absorptive cells in the small intestinal epithelium, and MHCO cells showed complete colonic epithelium signature, including stem/progenitor, goblet, and enteroendocrine cells colonocytes. Characteristic gene expression changes related to lipid metabolism, inflammation, and cell-cell adhesion were observed in cocultured live Bifidobacterium longum and Caco-2 or MHCO cells. B. longum-stimulated MHCO cells exhibited barrier-enhancing characteristics, as demonstrated in clinical trials. Our data represent a valuable resource for understanding gut microbe and host cell communication.

Study JGAS000740

Bruno et al.: Interferon gamma rebalances immunopathological signatures in Chronic Granulomatous Disease through metabolic rewiring

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by recurrent life-threatening infections and hyperinflammatory complications. It is caused by mutations in the NADPH oxidase complex and the consequent loss of reactive oxygen species (ROS) production. Recombinant human interferon gamma (rIFN-γ) prophylaxis reduces the risk of severe infections, but the mechanisms behind its efficacy in CGD are still an open question, as it does not restore NADPH oxidase-dependent ROS production. Here, we show that myeloid innate immune cells of CGD patients are transcriptionally and functionally reprogrammed to a hyperactive inflammatory status, displaying an impaired in vitro induction of trained immunity. CGD monocytes have reduced intracellular amino acids concentrations and profound functional metabolic defects, both at the level of glycolysis and mitochondrial respiration. Ex vivo and in vivo treatment with IFN-γ restored these metabolic defects and reduced excessive IL-1β and IL-6 production in response to fungal stimuli in CGD monocytes. These data suggest that prophylactic rIFNγ modulates the metabolic status of innate immune cells in CGD. These data shed light on the effects of NADPH-derived ROS deficiency to the metabolic programs of immune cells and pose the basis for targeting this immunometabolic axis, potentially beyond CGD, with IFN-γ immunotherapy.

Study EGAS00001005463

Comprehensive gene analysis of colorectal cancer cases

Colorectal cancer is the third most common morbidity and the fourth leading cause of cancer death in the world.To characterize the mutation profiles of colorectal cancer with liver metastasis,we performed both whole-exosome sequencing and RNA sequencing for 12 cases of colorectal cancer with liver metastasis (group A) and 16 cases of colorectal cancer without liver metastasis (group B). As a result of driver mutation search by Mutsig pipeline, PTEN was identified as a significant mutant gene in group A alone.ADAMTS10,NELL1 and RXFP3 were identified as candidate genes for metastasis by MuTect,SID and VarScan pipeline.Furthermore, in all samples, the copy number of chromosomes 1p,4,7,8,13,15, 17p,18,20 and 22q were significantly abnormal. Chromosomes 1p,4,15,18,17p,22q were deleted and chromosomes 7, 8,13 and 20 were amplified by CNV.Pathway analysed revealed Wnt/Ca2+ and PCP pathway was involved in metastasis of cl cancer.9 kinds of fusion genes were identified in deFuse pipeline. Among them, ADAP1 fusion genes were identified in 2 samples, and ADAP1 fusion genes were further identified in 3 samples by our in-house algorithm. It is suggested that new fusion genes present in such multiple samples may be involved in the development of colorectal cancer.

Study JGAS000128

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