ABSTRACT
The genetics of complex disease produce alterations in the molecular interactions of cellular pathways whose collective effect may become clear through the organized structure of molecular networks. To characterize molecular systems associated with late-onset Alzheimer's disease (LOAD), we constructed gene-regulatory networks in 1,647 postmortem brain tissues from LOAD patients and nondemented subjects, and we demonstrate that LOAD reconfigures specific portions of the molecular interaction structure. Through an integrative network-based approach, we rank-ordered these network structures for relevance to LOAD pathology, highlighting an immune- and microglia-specific module that is dominated by genes involved in pathogen phagocytosis, contains TYROBP as a key regulator, and is upregulated in LOAD. Mouse microglia cells overexpressing intact or truncated TYROBP revealed expression changes that significantly overlapped the human brain TYROBP network. Thus the causal network structure is a useful predictor of response to gene perturbations and presents a framework to test models of disease mechanisms underlying LOAD.
Subject(s)
Alzheimer Disease/genetics , Brain/metabolism , Gene Regulatory Networks , Adaptor Proteins, Signal Transducing/metabolism , Alzheimer Disease/metabolism , Animals , Bayes Theorem , Brain/pathology , Humans , Membrane Proteins/metabolism , Mice , Microglia/metabolismABSTRACT
OBJECTIVE: IBD therapies and treatments are evolving to deeper levels of remission. Molecular measures of disease may augment current endpoints including the potential for less invasive assessments. DESIGN: Transcriptome analysis on 712 endoscopically defined inflamed (Inf) and 1778 non-inflamed (Non-Inf) intestinal biopsies (n=498 Crohn's disease, n=421 UC and 243 controls) in the Mount Sinai Crohn's and Colitis Registry were used to identify genes differentially expressed between Inf and Non-Inf biopsies and to generate a molecular inflammation score (bMIS) via gene set variance analysis. A circulating MIS (cirMIS) score, reflecting intestinal molecular inflammation, was generated using blood transcriptome data. bMIS/cirMIS was validated as indicators of intestinal inflammation in four independent IBD cohorts. RESULTS: bMIS/cirMIS was strongly associated with clinical, endoscopic and histological disease activity indices. Patients with the same histologic score of inflammation had variable bMIS scores, indicating that bMIS describes a deeper range of inflammation. In available clinical trial data sets, both scores were responsive to IBD treatment. Despite similar baseline endoscopic and histologic activity, UC patients with lower baseline bMIS levels were more likely treatment responders compared with those with higher levels. Finally, among patients with UC in endoscopic and histologic remission, those with lower bMIS levels were less likely to have a disease flare over time. CONCLUSION: Transcriptionally based scores provide an alternative objective and deeper quantification of intestinal inflammation, which could augment current clinical assessments used for disease monitoring and have potential for predicting therapeutic response and patients at higher risk of disease flares.
Subject(s)
Colitis, Ulcerative , Crohn Disease , Humans , Colitis, Ulcerative/pathology , Inflammation/genetics , Inflammation/pathology , Crohn Disease/pathology , Biopsy , Biomarkers , Intestinal Mucosa/pathologyABSTRACT
Inherited alleles account for most of the genetic risk for schizophrenia. However, new (de novo) mutations, in the form of large chromosomal copy number changes, occur in a small fraction of cases and disproportionally disrupt genes encoding postsynaptic proteins. Here we show that small de novo mutations, affecting one or a few nucleotides, are overrepresented among glutamatergic postsynaptic proteins comprising activity-regulated cytoskeleton-associated protein (ARC) and N-methyl-d-aspartate receptor (NMDAR) complexes. Mutations are additionally enriched in proteins that interact with these complexes to modulate synaptic strength, namely proteins regulating actin filament dynamics and those whose messenger RNAs are targets of fragile X mental retardation protein (FMRP). Genes affected by mutations in schizophrenia overlap those mutated in autism and intellectual disability, as do mutation-enriched synaptic pathways. Aligning our findings with a parallel case-control study, we demonstrate reproducible insights into aetiological mechanisms for schizophrenia and reveal pathophysiology shared with other neurodevelopmental disorders.
Subject(s)
Models, Neurological , Mutation/genetics , Nerve Net/metabolism , Neural Pathways/metabolism , Schizophrenia/genetics , Schizophrenia/physiopathology , Synapses/metabolism , Child Development Disorders, Pervasive/genetics , Cytoskeletal Proteins/metabolism , Exome/genetics , Fragile X Mental Retardation Protein/metabolism , Humans , Intellectual Disability/genetics , Mutation Rate , Nerve Net/physiopathology , Nerve Tissue Proteins/metabolism , Neural Pathways/physiopathology , Phenotype , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Schizophrenia/metabolism , Substrate SpecificityABSTRACT
BACKGROUND: Exosomes and other extracellular vesicles (EVs) have emerged as an important mechanism of cell-to-cell communication. However, previous studies either did not fully resolve what genetic materials were shuttled by exosomes or only focused on a specific set of miRNAs and mRNAs. A more systematic method is required to identify the genetic materials that are potentially transferred during cell-to-cell communication through EVs in an unbiased manner. RESULTS: In this work, we present a novel next generation of sequencing (NGS) based approach to identify EV mediated mRNA exchanges between co-cultured adipocyte and macrophage cells. We performed molecular and genomic profiling and jointly considered data from RNA sequencing (RNA-seq) and genotyping to track the "sequence varying mRNAs" transferred between cells. We identified 8 mRNAs being transferred from macrophages to adipocytes and 21 mRNAs being transferred in the opposite direction. These mRNAs represented biological functions including extracellular matrix, cell adhesion, glycoprotein, and signal peptides. CONCLUSIONS: Our study sheds new light on EV mediated RNA communications between adipocyte and macrophage cells, which may play a significant role in developing insulin resistance in diabetic patients. This work establishes a new method that is applicable to examining genetic material exchanges in many cellular systems and has the potential to be extended to in vivo studies as well.
Subject(s)
Cell Communication , Extracellular Vesicles/metabolism , RNA, Messenger/metabolism , Adipocytes/metabolism , Cell Line , Coculture Techniques , Gene Expression , Genotyping Techniques , High-Throughput Nucleotide Sequencing , Humans , Macrophages/metabolism , RNA Transport , Sequence Analysis, RNAABSTRACT
BACKGROUND & AIMS: According to the clonal model of tumor evolution, trunk alterations arise at early stages and are ubiquitous. Through the characterization of early stages of hepatocarcinogenesis, we aimed to identify trunk alterations in hepatocellular carcinoma (HCC) and study their intra- and inter-tumor distribution in advanced lesions. METHODS: A total of 151 samples representing the multistep process of hepatocarcinogenesis were analyzed by targeted-sequencing and a single nucleotide polymorphism array. Genes altered in early lesions (31 dysplastic nodules [DNs] and 38 small HCCs [sHCC]) were defined as trunk. Their distribution was explored in: a) different regions of large tumors (43 regions, 21 tumors), and b) different nodules of the same patient (39 tumors, 17 patients). Multinodular lesions were classified as intrahepatic metastases (IMs) or synchronous tumors based on chromosomal aberrations. RESULTS: TERT promoter mutations (10.5%) and broad copy-number aberrations in chromosomes 1 and 8 (3-7%) were identified as trunk gatekeepers in DNs and were maintained in sHCCs. Trunk drivers identified in sHCCs included TP53 (23%) and CTNNB1 (11%) mutations, and focal amplifications or deletions in known drivers (6%). Overall, TERT, TP53 and CTNNB1 mutations were the most frequent trunk events and at least one was present in 51% of sHCCs. Around 90% of mutations in these genes were ubiquitous among different regions of large tumors. In multinodular HCCs, 35% of patients harbored IMs; 85% of mutations in TERT, TP53 and/or CTNNB1 were retained in primary and metastatic tumors. CONCLUSIONS: Trunk events in early stages (TERT, TP53, CTNNB1 mutations) were ubiquitous across different regions of the same tumor and between primary and metastatic nodules in >85% of cases. This concept supports the knowledge that single biopsies would suffice to capture trunk mutations in HCC. LAY SUMMARY: Trunk alterations arise at early stages of cancer and are shared among all malignant cells of the tumor. In order to identify trunk alterations in HCC, we characterized early stages of hepatocarcinogenesis represented by dysplastic nodules and small lesions. Mutations in TERT, TP53 and CTNNB1 genes were the most frequent. Analyses in more advanced lesions showed that mutations in these same genes were shared between different regions of the same tumor and between primary and metastatic tumors, suggesting their trunk role in this disease.
Subject(s)
Carcinoma, Hepatocellular/genetics , Liver Neoplasms/genetics , Mutation , Carcinoma, Hepatocellular/pathology , DNA Copy Number Variations , Humans , Liver Neoplasms/pathology , Promoter Regions, Genetic , Telomerase/genetics , Tumor Suppressor Protein p53/genetics , beta Catenin/geneticsABSTRACT
BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is the second most lethal cancer caused by lack of effective therapies. Although promising, HCC molecular classification, which enriches potential responders to specific therapies, has not yet been assessed in clinical trials of anti-HCC drugs. We aimed to overcome these challenges by developing clinicopathological surrogate indices of HCC molecular classification. METHODS: Hepatocellular carcinoma classification defined in our previous transcriptome meta-analysis (S1, S2 and S3 subclasses) was implemented in an FDA-approved diagnostic platform (Elements assay, NanoString). Ninety-six HCC tumours (training set) were assayed to develop molecular subclass-predictive indices based on clinicopathological features, which were independently validated in 99 HCC tumours (validation set). Molecular deregulations associated with the histopathological features were determined by pathway analysis. Sample sizes for HCC clinical trials enriched with specific molecular subclasses were determined. RESULTS: Hepatocellular carcinoma subclass-predictive indices were steatohepatitic (SH)-HCC variant and immune cell infiltrate for S1 subclass, macrotrabecular/compact pattern, lack of pseudoglandular pattern, and high serum alpha-foetoprotein (>400 ng/ml) for S2 subclass, and microtrabecular pattern, lack of SH-HCC and clear cell variants, and lower histological grade for S3 subclass. Macrotrabecular/compact pattern, a predictor of S2 subclass, was associated with the activation of therapeutically targetable oncogene YAP and stemness markers EPCAM/KRT19. BMP4 was associated with pseudoglandular pattern. Subclass-predictive indices-based patient enrichment reduced clinical trial sample sizes from 121, 184 and 53 to 30, 43 and 22 for S1, S2 and S3 subclass-targeting therapies respectively. CONCLUSIONS: Hepatocellular carcinoma molecular subclasses can be enriched by clinicopathological indices tightly associated with deregulation of therapeutically targetable molecular pathways.
Subject(s)
Antigens, Neoplasm/analysis , Bone Morphogenetic Protein 4/analysis , Carcinoma, Hepatocellular , Cell Adhesion Molecules/analysis , Keratin-19/analysis , Liver Neoplasms , Aged , Amino Acid Sequence , Carcinoma, Hepatocellular/classification , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Drug Discovery , Epithelial Cell Adhesion Molecule , Female , Gene Expression Profiling , Genome-Wide Association Study , Humans , Liver Neoplasms/classification , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Male , Middle Aged , Molecular Sequence Data , Neoplasm Grading , Prognosis , alpha-Fetoproteins/analysisABSTRACT
OBJECTIVE: The number of patients with HCV-related cirrhosis is increasing, leading to a rising risk of complications and death. Prognostic stratification in patients with early-stage cirrhosis is still challenging. We aimed to develop and validate a clinically useful prognostic index based on genomic and clinical variables to identify patients at high risk of disease progression. DESIGN: We developed a prognostic index, comprised of a 186-gene signature validated in our previous genome-wide profiling study, bilirubin (>1â mg/dL) and platelet count (<100,000/mm(3)), in an Italian HCV cirrhosis cohort (training cohort, n=216, median follow-up 10â years). The gene signature test was implemented using a digital transcript counting (nCounter) assay specifically developed for clinical use and the prognostic index was evaluated using archived specimens from an independent cohort of HCV-related cirrhosis in the USA (validation cohort, n=145, median follow-up 8â years). RESULTS: In the training cohort, the prognostic index was associated with hepatic decompensation (HR=2.71, p=0.003), overall death (HR=6.00, p<0.001), hepatocellular carcinoma (HR=3.31, p=0.001) and progression of Child-Turcotte-Pugh class (HR=6.70, p<0.001). The patients in the validation cohort were stratified into high-risk (16%), intermediate-risk (42%) or low-risk (42%) groups by the prognostic index. The high-risk group had a significantly increased risk of hepatic decompensation (HR=7.36, p<0.001), overall death (HR=3.57, p=0.002), liver-related death (HR=6.49, p<0.001) and all liver-related adverse events (HR=4.98, p<0.001). CONCLUSIONS: A genomic and clinical prognostic index readily available for clinical use was successfully validated, warranting further clinical evaluation for prognostic prediction and clinical trial stratification and enrichment for preventive interventions.
Subject(s)
Hepacivirus/genetics , Hepatitis C, Chronic/complications , Liver Cirrhosis/etiology , RNA, Viral/genetics , Disease Progression , Female , Follow-Up Studies , Hepatitis C, Chronic/diagnosis , Hepatitis C, Chronic/virology , Humans , Incidence , Liver Cirrhosis/diagnosis , Liver Cirrhosis/epidemiology , Male , Middle Aged , Prognosis , Prospective Studies , Risk Factors , United States/epidemiologyABSTRACT
PURPOSE: Health-care professionals need to be trained to work with whole-genome sequencing (WGS) in their practice. Our aim was to explore how students responded to a novel genome analysis course that included the option to analyze their own genomes. METHODS: This was an observational cohort study. Questionnaires were administered before (T3) and after the genome analysis course (T4), as well as 6 months later (T5). In-depth interviews were conducted at T5. RESULTS: All students (n = 19) opted to analyze their own genomes. At T5, 12 of 15 students stated that analyzing their own genomes had been useful. Ten reported they had applied their knowledge in the workplace. Technical WGS knowledge increased (mean of 63.8% at T3, mean of 72.5% at T4; P = 0.005). In-depth interviews suggested that analyzing their own genomes may increase students' motivation to learn and their understanding of the patient experience. Most (but not all) of the students reported low levels of WGS results-related distress and low levels of regret about their decision to analyze their own genomes. CONCLUSION: Giving students the option of analyzing their own genomes may increase motivation to learn, but some students may experience personal WGS results-related distress and regret. Additional evidence is required before considering incorporating optional personal genome analysis into medical education on a large scale.
Subject(s)
Genome, Human , Genomics , High-Throughput Nucleotide Sequencing , Students/psychology , Attitude of Health Personnel , Cohort Studies , Decision Making , Female , Genomics/methods , Humans , Longitudinal Studies , Male , Students, Medical/psychology , Surveys and QuestionnairesABSTRACT
DNA replication, repair, transcription and chromatin structure are intricately associated nuclear processes, but the molecular links between these events are often obscure. In this study, we have surveyed the protein complexes that bind at ß-globin locus control region, and purified and characterized the function of one such multiprotein complex from human erythroleukemic K562 cells. We further validated the existence of this complex in human CD34+ cell-derived normal erythroid cells. This complex contains ILF2/ILF3 transcription factors, p300 acetyltransferase and proteins associated with DNA replication, transcription and repair. RNAi knockdown of ILF2, a DNA-binding component of this complex, abrogates the recruitment of the complex to its cognate DNA sequence and inhibits transcription, histone acetylation and usage of the origin of DNA replication at the ß-globin locus. These results imply a direct link between mammalian DNA replication, transcription and histone acetylation mediated by a single multiprotein complex.
Subject(s)
DNA Repair/physiology , DNA Replication/physiology , DNA-Binding Proteins/genetics , Multiprotein Complexes/genetics , Transcription, Genetic/physiology , beta-Globins/metabolism , Cell Line, Tumor , DNA Repair/genetics , DNA Replication/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/physiology , Humans , Multiprotein Complexes/metabolism , Multiprotein Complexes/physiology , Nuclear Factor 45 Protein/genetics , Nuclear Factor 45 Protein/metabolism , Nuclear Factor 90 Proteins/genetics , Nuclear Factor 90 Proteins/metabolism , Oligonucleotides/genetics , RNA Interference , Transcription, Genetic/genetics , beta-Globins/genetics , p300-CBP Transcription Factors/genetics , p300-CBP Transcription Factors/metabolismABSTRACT
PU.1 is a hematopoietic transcription factor that is required for the development of myeloid and B cells. PU.1 is also expressed in erythroid progenitors, where it blocks erythroid differentiation by binding to and inhibiting the main erythroid promoting factor, GATA-1. However, other mechanisms by which PU.1 affects the fate of erythroid progenitors have not been thoroughly explored. Here, we used ChIP-Seq analysis for PU.1 and gene expression profiling in erythroid cells to show that PU.1 regulates an extensive network of genes that constitute major pathways for controlling growth and survival of immature erythroid cells. By analyzing fetal liver erythroid progenitors from mice with low PU.1 expression, we also show that the earliest erythroid committed cells are dramatically reduced in vivo. Furthermore, we find that PU.1 also regulates many of the same genes and pathways in other blood cells, leading us to propose that PU.1 is a multifaceted factor with overlapping, as well as distinct, functions in several hematopoietic lineages.
Subject(s)
Erythroid Precursor Cells/cytology , Gene Regulatory Networks , Proto-Oncogene Proteins/genetics , Trans-Activators/genetics , Transcription, Genetic , Animals , Cell Differentiation , Cell Line , Cell Lineage , Chromatin Immunoprecipitation , Erythroid Precursor Cells/metabolism , Mice , Proto-Oncogene Proteins/metabolism , Trans-Activators/metabolismABSTRACT
The DNA methylation status of human X chromosomes from male and female neutrophils was identified by high-throughput sequencing of HpaII and MspI digested fragments. In the intergenic and intragenic regions on the X chromosome, the sites outside CpG islands were heavily hypermethylated to the same degree in both genders. Nearly half of X chromosome promoters were either hypomethylated or hypermethylated in both females and males. Nearly one third of X chromosome promoters were a mixture of hypomethylated and heterogeneously methylated sites in females and were hypomethylated in males. Thus, a large fraction of genes that are silenced on the inactive X chromosome are hypomethylated in their promoter regions. These genes frequently belong to the evolutionarily younger strata of the X chromosome. The promoters that were hypomethylated at more than two sites contained most of the genes that escaped silencing on the inactive X chromosome. The overall levels of expression of X-linked genes were indistinguishable in females and males, regardless of the methylation state of the inactive X chromosome. Thus, in addition to DNA methylation, other factors are involved in the fine tuning of gene dosage compensation in neutrophils.
Subject(s)
Chromosomes, Human, X/genetics , DNA Methylation , Gene Expression Regulation , Gene Expression , Genes, X-Linked , Female , Humans , Male , Neutrophils/metabolism , Promoter Regions, Genetic , Sex FactorsABSTRACT
Neuroacanthocytoses are neurodegenerative disorders marked by phenotypic and genetic heterogeneity. There are several associated genetic loci, and many defects, including gene deletions and insertions, and missense, nonsense, and splicing mutations, have been found spread over hundreds of kilobases of genomic DNA. In some cases, specific diagnosis is unclear, particularly in the early stages of disease or when there is an atypical presentation. Determination of the precise genetic defect allows assignment of the diagnosis and permits carrier detection and genetic counseling. The objective of this report was to utilize exome sequencing for genetic diagnosis in the neuroacanthocytosis syndromes. Genomic DNA from 2 patients with clinical features of chorea-acanthocytosis was subjected to targeted exon capture. Captured DNA was subjected to ultrahigh throughput next-generation sequencing. Sequencing data were assembled, filtered against known human variant genetic databases, and results were analyzed. Both patients were compound heterozygotes for mutations in the VPS13A gene, the gene associated with chorea-acanthocytosis. Patient 1 had a 4-bp deletion that removes the 5' donor splice site of exon 58 and a nucleotide substitution that disrupts the 5' donor splice site of exon 70. Patient 2 had a dinucleotide deletion in exon 16 and a dinucleotide insertion in exon 33. No mutations were identified in the XK, PANK2, or JPH3 gene loci. Exome sequencing is a valuable diagnostic tool in the neuroacanthocytosis syndromes. These studies may provide a better understanding of the function of the associated proteins and provide insight into the pathogenesis of these disorders.
Subject(s)
Exome/genetics , Genetic Testing/methods , Mutation/genetics , Neuroacanthocytosis/diagnosis , Neuroacanthocytosis/genetics , Vesicular Transport Proteins/genetics , Adult , Female , Genetic Heterogeneity , Humans , Male , Middle Aged , Sequence Analysis, DNAABSTRACT
CONTEXT: Pituitary corticotroph adenomas are rare tumors that can be associated with excess adrenocorticotropin (ACTH) and adrenal cortisol production, resulting in the clinically debilitating endocrine condition Cushing disease. A subset of corticotroph tumors behave aggressively, and genomic drivers behind the development of these tumors are largely unknown. OBJECTIVE: To investigate genomic drivers of corticotroph tumors at risk for aggressive behavior. DESIGN: Whole-exome sequencing of patient-matched corticotroph tumor and normal deoxyribonucleic acid (DNA) from a patient cohort enriched for tumors at risk for aggressive behavior. SETTING: Tertiary care center. PATIENTS: Twenty-seven corticotroph tumors from 22 patients were analyzed. Twelve tumors were macroadenomas, of which 6 were silent ACTH tumors, 2 were Crooke's cell tumors, and 1 was a corticotroph carcinoma. INTERVENTION: Whole-exome sequencing. MAIN OUTCOME MEASURE: Somatic mutation genomic biomarkers. RESULTS: We found recurrent somatic mutations in USP8 and TP53 genes, both with higher allelic fractions than other somatic mutations. These mutations were mutually exclusive, with TP53 mutations occurring only in USP8 wildtype (WT) tumors, indicating they may be independent driver genes. USP8-WT tumors were characterized by extensive somatic copy number variation compared with USP8-mutated tumors. Independent of molecular driver status, we found an association between invasiveness, macroadenomas, and aneuploidy. CONCLUSIONS: Our data suggest that corticotroph tumors may be categorized into a USP8-mutated, genome-stable subtype versus a USP8-WT, genome-disrupted subtype, the latter of which has a TP53-mutated subtype with high level of chromosome instability. These findings could help identify high risk corticotroph tumors, namely those with widespread CNV, that may need closer monitoring and more aggressive treatment.
Subject(s)
ACTH-Secreting Pituitary Adenoma/genetics , Adenoma/genetics , DNA Copy Number Variations , Endopeptidases/genetics , Endosomal Sorting Complexes Required for Transport/genetics , Tumor Suppressor Protein p53/genetics , Ubiquitin Thiolesterase/genetics , ACTH-Secreting Pituitary Adenoma/epidemiology , ACTH-Secreting Pituitary Adenoma/pathology , Adenoma/epidemiology , Adenoma/pathology , Adolescent , Adult , Case-Control Studies , Cell Transformation, Neoplastic/genetics , Cohort Studies , DNA Copy Number Variations/physiology , Female , Gene Frequency , Genetic Association Studies , Genetic Predisposition to Disease , Humans , Male , Middle Aged , Mutation , Neoplasm Invasiveness , Neoplasm Metastasis , Exome Sequencing , Young AdultABSTRACT
A library of well-characterized human induced pluripotent stem cell (hiPSC) lines from clinically healthy human subjects could serve as a useful resource of normal controls for in vitro human development, disease modeling, genotype-phenotype association studies, and drug response evaluation. We report generation and extensive characterization of a gender-balanced, racially/ethnically diverse library of hiPSC lines from 40 clinically healthy human individuals who range in age from 22 to 61 years. The hiPSCs match the karyotype and short tandem repeat identities of their parental fibroblasts, and have a transcription profile characteristic of pluripotent stem cells. We provide whole-genome sequencing data for one hiPSC clone from each individual, genomic ancestry determination, and analysis of mendelian disease genes and risks. We document similar transcriptomic profiles, single-cell RNA-sequencing-derived cell clusters, and physiology of cardiomyocytes differentiated from multiple independent hiPSC lines. This extensive characterization makes this hiPSC library a valuable resource for many studies on human biology.
Subject(s)
Health , Induced Pluripotent Stem Cells/cytology , Adult , Calcium Signaling , Cell Differentiation , Cell Line , Clone Cells , Ethnicity , Female , Gene Expression Profiling , Gene Expression Regulation , Genetic Predisposition to Disease , Genetic Variation , Heart Atria/cytology , Heart Ventricles/cytology , Humans , Male , Middle Aged , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Risk Factors , Young AdultABSTRACT
Chronic liver disease and hepatocellular carcinoma (HCC) are life-threatening diseases with limited treatment options. The lack of clinically relevant/tractable experimental models hampers therapeutic discovery. Here, we develop a simple and robust human liver cell-based system modeling a clinical prognostic liver signature (PLS) predicting long-term liver disease progression toward HCC. Using the PLS as a readout, followed by validation in nonalcoholic steatohepatitis/fibrosis/HCC animal models and patient-derived liver spheroids, we identify nizatidine, a histamine receptor H2 (HRH2) blocker, for treatment of advanced liver disease and HCC chemoprevention. Moreover, perturbation studies combined with single cell RNA-Seq analyses of patient liver tissues uncover hepatocytes and HRH2+, CLEC5Ahigh, MARCOlow liver macrophages as potential nizatidine targets. The PLS model combined with single cell RNA-Seq of patient tissues enables discovery of urgently needed targets and therapeutics for treatment of advanced liver disease and cancer prevention.
Subject(s)
Drug Discovery , Liver/pathology , Models, Biological , Animals , Carcinogenesis/pathology , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Chemoprevention , Cohort Studies , Cyclic AMP/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Disease Models, Animal , Gene Expression Regulation, Neoplastic/drug effects , HEK293 Cells , Hepacivirus/physiology , Hepatitis C/genetics , Hepatocytes/drug effects , Hepatocytes/metabolism , Hepatocytes/pathology , Humans , Immunologic Surveillance/drug effects , Inflammation/pathology , Liver/drug effects , Liver/metabolism , Liver Cirrhosis/pathology , Liver Neoplasms/pathology , Macrophages/drug effects , Macrophages/metabolism , Macrophages/pathology , Male , Mice, Knockout , Nizatidine/pharmacology , Prognosis , Signal Transduction/drug effects , Transcriptome/geneticsABSTRACT
Next-generation sequencing (NGS) is transforming clinical research and diagnostics, vastly enhancing our ability to identify novel disease-causing genetic mutations and perform comprehensive diagnostic testing in the clinic. Whole-exome sequencing (WES) is a commonly used method which captures the majority of coding regions of the genome for sequencing, as these regions contain the majority of disease-causing mutations. The clinical applications of WES are not limited to diagnosis; the technique can be employed to help determine an optimal therapeutic strategy for a patient considering their mutation profile. WES may also be used to predict a patient's risk of developing a disease, e.g., type 2 diabetes (T2D), and can therefore be used to tailor advice for the patient about lifestyle choices that could mitigate those risks. Thus, genome sequencing strategies, such as WES, underpin the emerging field of personalized medicine. Initiatives also exist for sharing WES data in public repositories, e.g., the Exome Aggregation Consortium (ExAC) database. In time, by mining these valuable data resources, we will acquire a better understanding of the roles of both single rare mutations and specific combinations of common mutations (mutation signatures) in the pathology of complex diseases such as diabetes.Herein, we describe a protocol for performing WES on genomic DNA extracted from blood or saliva. Starting with gDNA extraction, we document preparation of a library for sequencing on Illumina instruments and the enrichment of the protein-coding regions from the library using the Roche NimbleGen SeqCap EZ Exome v3 kit; a solution-based capture method. We include details of how to efficiently purify the products of each step using the AMPure XP System and describe how to use qPCR to test the efficiency of capture, and thus determine finished library quality.
Subject(s)
Exome Sequencing , High-Throughput Nucleotide Sequencing , Exome , Exons , Gene Library , Genomic Library , High-Throughput Nucleotide Sequencing/methods , Humans , Sequence Analysis, DNA , Exome Sequencing/methods , WorkflowABSTRACT
Quiescence is a fundamental property that maintains hematopoietic stem cell (HSC) potency throughout life. Quiescent HSCs are thought to rely on glycolysis for their energy, but the overall metabolic properties of HSCs remain elusive. Using combined approaches, including single-cell RNA sequencing (RNA-seq), we show that mitochondrial membrane potential (MMP) distinguishes quiescent from cycling-primed HSCs. We found that primed, but not quiescent, HSCs relied readily on glycolysis. Notably, in vivo inhibition of glycolysis enhanced the competitive repopulation ability of primed HSCs. We further show that HSC quiescence is maintained by an abundance of large lysosomes. Repression of lysosomal activation in HSCs led to further enlargement of lysosomes while suppressing glucose uptake. This also induced increased lysosomal sequestration of mitochondria and enhanced the competitive repopulation ability of primed HSCs by over 90-fold in vivo. These findings show that restraining lysosomal activity preserves HSC quiescence and potency and may be therapeutically relevant.
Subject(s)
Hematopoietic Stem Cells , Mitochondria , Cell Division , Glycolysis , Hematopoietic Stem Cells/metabolism , Lysosomes , Mitochondria/metabolismABSTRACT
INTRODUCTION: The tight regulation of the cytokine network during macrophage activation is of prime importance to enable a fast and potent innate immune response against exogenous pathogens. The inflammation mediating ubiquitin-like protein HLA-F adjacent transcript number 10 (FAT10) was shown to be transcriptionally regulated by and also regulate the nuclear factor-κB (NFκB) signaling pathway. However, very little is known about the regulation of FAT10 gene expression during macrophage activation. RESULTS: RNA sequencing of interferon (IFN)γ-stimulated mouse peritoneal macrophages analyzed by ingenuity pathway analysis revealed significant involvement of tumor necrosis factor receptor 1 (TNFR1) signaling in addition to IFNγ signaling. Subsequently, IFNγ robustly upregulated FAT10 expression compared to a milder induction seen with TNFα or lipopolysaccharide (LPS) stimulation. While low dose IFNγ with TNFα synergistically elevated FAT10 expression, preincubation of macrophages with IFNγ strongly augmented TNFα-induced FAT10 expression. Moreover, a short preincubation with IFNγ, which did not elevate FAT10, was sufficient to potentiate the induction of FAT10 by TNFα. A double augmentation mechanism of TNFα signaling was demonstrated, where IFNγ rapidly induced the expression of TNFα and TNFR1, which further augmented the induction of TNFα and TNFR1 expression by TNFα. Importantly, the induction of FAT10 by IFNγ in macrophages from TNFα-deficient or TNFR1-deficient mice was completely inhibited compared to macrophages from wild type (WT) mice. Finally, we show that TNFα-induced FAT10 expression is dependent on NFκB signaling. CONCLUSION: IFNγ potentiates the TNFα/TNFR1 signaling pathway to induce FAT10 expression in mouse macrophages, mediated through NFκB network.
Subject(s)
Gene Expression Regulation/immunology , Interferon-gamma/immunology , Macrophage Activation/immunology , Macrophages/immunology , Signal Transduction/immunology , Ubiquitins/biosynthesis , Animals , Immunity, Innate/immunology , Interferon-gamma/metabolism , Macrophages/metabolism , Mice , Mice, Knockout , NF-kappa B/immunology , NF-kappa B/metabolism , Receptors, Tumor Necrosis Factor, Type I/immunology , Receptors, Tumor Necrosis Factor, Type I/metabolism , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Kinase inhibitors (KIs) represent an important class of anti-cancer drugs. Although cardiotoxicity is a serious adverse event associated with several KIs, the reasons remain poorly understood, and its prediction remains challenging. We obtain transcriptional profiles of human heart-derived primary cardiomyocyte like cell lines treated with a panel of 26 FDA-approved KIs and classify their effects on subcellular pathways and processes. Individual cardiotoxicity patient reports for these KIs, obtained from the FDA Adverse Event Reporting System, are used to compute relative risk scores. These are then combined with the cell line-derived transcriptomic datasets through elastic net regression analysis to identify a gene signature that can predict risk of cardiotoxicity. We also identify relationships between cardiotoxicity risk and structural/binding profiles of individual KIs. We conclude that acute transcriptomic changes in cell-based assays combined with drug substructures are predictive of KI-induced cardiotoxicity risk, and that they can be informative for future drug discovery.
Subject(s)
Cardiotoxicity/genetics , Cardiotoxicity/metabolism , Gene Expression Profiling/methods , Protein Kinase Inhibitors/adverse effects , Protein Kinase Inhibitors/pharmacology , Transcriptome , Antineoplastic Agents/pharmacology , Cardiotoxicity/drug therapy , Cell Line , Dose-Response Relationship, Drug , Drug Approval , Female , Gene Expression/drug effects , Humans , Male , Myocytes, Cardiac/drug effects , Regression Analysis , Risk Assessment , Risk Factors , Sequence Alignment , United States , United States Food and Drug AdministrationABSTRACT
Hepatocellular carcinoma (HCC) was the fifth leading cause of cancer death in men and eighth leading cause of death in women in the United States in 2017. In our study, we sought to identify sncRNAs in various stages of development of HCC. We obtained publicly available small RNA-seq data derived from patients with cirrhosis (n = 14), low-grade dysplastic nodules (LGDN, n = 9), high grade dysplastic nodules (HGDN, n = 6), early hepatocellular carcinoma (eHCC, n = 6), and advanced hepatocellular carcinoma (HCC, n = 20), along with healthy liver tissue samples (n = 9). All samples were analyzed for various types of non-coding RNAs using PartekFlow software. We remapped small RNA-seq to miRBase to obtain differential expressions of miRNAs and found 87 in cirrhosis, 106 in LGDN, 59 in HGDN, 80 in eHCC, and 133 in HCC. Pathway analysis of miRNAs obtained from diseased samples compared to normal samples showed signaling pathways in the microRNA dependent EMT, CD44, and others. Additionally, we analyzed the data sets for piRNAs, lncRNAs, circRNAs, and sno/mt-RNAs. We validated the in silico data using human HCC samples with NanoString miRNA global expression. Our results suggest that publically available data is a valuable resource for sncRNA identification in HCC progression (FDR set to <0.05 for all samples) and that a data mining approach is useful for biomarker development.