RESUMEN
Genome-wide (GWAS) and copy number variant (CNV) association studies have reproducibly identified numerous risk alleles associated with bipolar disorder (BD), major depressive disorder (MDD), and schizophrenia (SCZ), but biological characterization of these alleles lags gene discovery, owing to the inaccessibility of live human brain cells and inadequate animal models for human psychiatric conditions. Human-derived induced pluripotent stem cells (iPSCs) provide a renewable cellular reagent that can be differentiated into living, disease-relevant cells and 3D brain organoids carrying the full complement of genetic variants present in the donor germline. Experimental studies of iPSC-derived cells allow functional characterization of risk alleles, establishment of causal relationships between genes and neurobiology, and screening for novel therapeutics. Here we report the creation and availability of an iPSC resource comprising clinical, genomic, and cellular data obtained from genetically isolated families with BD and related conditions. Results from the first 324 study participants, 61 of whom have validated pluripotent clones, show enrichment of rare single nucleotide variants and CNVs overlapping many known risk genes and pathogenic CNVs. This growing iPSC resource is available to scientists pursuing functional genomic studies of BD and related conditions.
Asunto(s)
Trastorno Depresivo Mayor , Células Madre Pluripotentes Inducidas , Trastornos Psicóticos , Esquizofrenia , Animales , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Trastorno Depresivo Mayor/genética , Trastorno Depresivo Mayor/metabolismo , Trastornos Psicóticos/metabolismo , Esquizofrenia/genética , Esquizofrenia/metabolismo , Genómica , Estudio de Asociación del Genoma CompletoRESUMEN
Aneuploidy syndromes impact multiple organ systems but understanding of tissue-specific aneuploidy effects remains limited-especially for the comparison between peripheral tissues and relatively inaccessible tissues like brain. Here, we address this gap in knowledge by studying the transcriptomic effects of chromosome X, Y, and 21 aneuploidies in lymphoblastoid cell lines, fibroblasts and iPSC-derived neuronal cells (LCLs, FCL, and iNs, respectively). We root our analyses in sex chromosome aneuploidies, which offer a uniquely wide karyotype range for dosage effect analysis. We first harness a large LCL RNA-seq dataset from 197 individuals with one of 6 sex chromosome dosages (SCDs: XX, XXX, XY, XXY, XYY, and XXYY) to i) validate theoretical models of SCD sensitivity and ii) define an expanded set of 41 genes that show obligate dosage sensitivity to SCD and are all in cis (i.e., reside on the X or Y chromosome). We then use multiple complementary analyses to show that cis effects of SCD in LCLs are preserved in both FCLs (n = 32) and iNs (n = 24), whereas trans effects (i.e., those on autosomal gene expression) are mostly not preserved. Analysis of additional datasets confirms that the greater cross-cell type reproducibility of cis vs. trans effects is also seen in trisomy 21 cell lines. These findings i) expand our understanding of X, Y, and 21 chromosome dosage effects on human gene expression and ii) suggest that LCLs may provide a good model system for understanding cis effects of aneuploidy in harder-to-access cell types.
Asunto(s)
Aneuploidia , Síndrome de Down , Humanos , Reproducibilidad de los Resultados , Síndrome de Down/genética , Cromosomas Sexuales , Expresión GénicaRESUMEN
Sex chromosome aneuploidies (SCAs) are common genetic syndromes characterized by the presence of an aberrant number of X and Y chromosomes due to meiotic defects. These conditions impact the structure and function of diverse tissues, but the proximal effects of SCAs on genome organization are unknown. Here, to determine the consequences of SCAs on global genome organization, we have analyzed multiple architectural features of chromosome organization in a comprehensive set of primary cells from SCA patients with various ratios of X and Y chromosomes by use of imaging-based high-throughput chromosome territory mapping (HiCTMap). We find that X chromosome supernumeracy does not affect the size, volume, or nuclear position of the Y chromosome or an autosomal chromosome. In contrast, the active X chromosome undergoes architectural changes as a function of increasing X copy number as measured by a decrease in size and an increase in circularity, which is indicative of chromatin compaction. In Y chromosome supernumeracy, Y chromosome size is reduced suggesting higher chromatin condensation. The radial positioning of chromosomes is unaffected in SCA karyotypes. Taken together, these observations document changes in genome architecture in response to alterations in sex chromosome numbers and point to trans-effects of dosage compensation on chromosome organization.
Asunto(s)
Compensación de Dosificación (Genética) , Cromosomas Sexuales/genética , Adolescente , Aneuploidia , Núcleo Celular/metabolismo , Células Cultivadas , Niño , Cromosomas Humanos Par 18/genética , Cromosomas Humanos X/genética , Cromosomas Humanos Y/genética , Femenino , Fibroblastos/metabolismo , Humanos , Masculino , ARN Largo no Codificante/metabolismo , Piel/citología , Inactivación del Cromosoma X/genética , Adulto JovenRESUMEN
The spatial organization of chromosomes in the nuclear space is an extensively studied field that relies on measurements of structural features and 3D positions of chromosomes with high precision and robustness. However, no tools are currently available to image and analyze chromosome territories in a high-throughput format. Here, we have developed High-throughput Chromosome Territory Mapping (HiCTMap), a method for the robust and rapid analysis of 2D and 3D chromosome territory positioning in mammalian cells. HiCTMap is a high-throughput imaging-based chromosome detection method which enables routine analysis of chromosome structure and nuclear position. Using an optimized FISH staining protocol in a 384-well plate format in conjunction with a bespoke automated image analysis workflow, HiCTMap faithfully detects chromosome territories and their position in 2D and 3D in a large population of cells per experimental condition. We apply this novel technique to visualize chromosomes 18, X, and Y in male and female primary human skin fibroblasts, and show accurate detection of the correct number of chromosomes in the respective genotypes. Given the ability to visualize and quantitatively analyze large numbers of nuclei, we use HiCTMap to measure chromosome territory area and volume with high precision and determine the radial position of chromosome territories using either centroid or equidistant-shell analysis. The HiCTMap protocol is also compatible with RNA FISH as demonstrated by simultaneous labeling of X chromosomes and Xist RNA in female cells. We suggest HiCTMap will be a useful tool for routine precision mapping of chromosome territories in a wide range of cell types and tissues.
Asunto(s)
Mapeo Cromosómico/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Hibridación Fluorescente in Situ/métodos , Animales , Núcleo Celular/genética , Núcleo Celular/metabolismo , Mapeo Cromosómico/instrumentación , Cromosomas Humanos Par 18/genética , Cromosomas Humanos Par 18/metabolismo , Cromosomas Humanos X/genética , Cromosomas Humanos X/metabolismo , Cromosomas Humanos Y/genética , Cromosomas Humanos Y/metabolismo , Femenino , Fibroblastos , Humanos , Procesamiento de Imagen Asistido por Computador/instrumentación , Hibridación Fluorescente in Situ/instrumentación , Masculino , Cultivo Primario de Células/métodos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Piel/citología , Coloración y Etiquetado/instrumentación , Coloración y Etiquetado/métodosRESUMEN
Therapeutic gene delivery mediated by retroviral vectors has the advantage of stable integration into the host genome. A major safety concern for gene delivery achieved by murine leukemia virus (MLV)-based retroviral vectors is the activation of adjacent cellular genes including oncogenes following integration into the host genome. Self-inactivating (SIN) vectors lacking viral enhancers/promoters in their 3' long terminal repeat (LTR) have been proposed as a means of overcoming this safety concern. However the MLV-based SIN vectors currently used by laboratories to assess insertional mutagenesis, integration site selection, and the potency of transgene expression are not uniform in the composition of their 3' LTRs. We constructed a series of SIN vectors representative of the currently employed vectors, but lacking an internal promoter. Green fluorescent protein (GFP) was used as a reporter gene. Target cells exposed to these vectors were evaluated for number of integrants and GFP expression at the messenger RNA (mRNA) level and protein level. We found that viral promoter activity in the 3' LTR is not attenuated in many currently employed SIN vectors. These results suggest that the influence of strong residual promoter activity should be taken into consideration when interpreting experimental results obtained using SIN vectors in gene therapy research.
Asunto(s)
Gammaretrovirus/genética , Vectores Genéticos/genética , Regiones Promotoras Genéticas , Animales , Variaciones en el Número de Copia de ADN , Elementos de Facilitación Genéticos , Expresión Génica , Orden Génico , Genes Reporteros , Células HEK293 , Humanos , Ratones , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Eliminación de Secuencia , Activación Transcripcional , Integración ViralRESUMEN
BACKGROUND: Gammaretroviruses and gammaretroviral vectors, in contrast to lentiviruses and lentiviral vectors, are reported to be restricted in their ability to infect growth-arrested cells. The block to this restriction has never been clearly defined. The original assessment of the inability of gammaretroviruses and gammaretroviral vectors to infect growth-arrested cells was carried out using established cell lines that had been growth-arrested by chemical means, and has been generalized to neurons, which are post-mitotic. We re-examined the capability of gammaretroviruses and their derived vectors to efficiently infect terminally differentiated neuroendocrine cells and primary cortical neurons, a target of both experimental and therapeutic interest. METHODOLOGY/PRINCIPAL FINDINGS: Using GFP expression as a marker for infection, we determined that both growth-arrested (NGF-differentiated) rat pheochromocytoma cells (PC12 cells) and primary rat cortical neurons could be efficiently transduced, and maintained long-term protein expression, after exposure to murine leukemia virus (MLV) and MLV-based retroviral vectors. Terminally differentiated PC12 cells transduced with a gammaretroviral vector encoding the anti-apoptotic protein Bcl-xL were protected from cell death induced by withdrawal of nerve growth factor (NGF), demonstrating gammaretroviral vector-mediated delivery and expression of genes at levels sufficient for therapeutic effect in non-dividing cells. Post-mitotic rat cortical neurons were also shown to be susceptible to transduction by murine replication-competent gammaretroviruses and gammaretroviral vectors. CONCLUSIONS/SIGNIFICANCE: These findings suggest that the host range of gammaretroviruses includes post-mitotic and other growth-arrested cells in mammals, and have implications for re-direction of gammaretroviral gene therapy to neurological disease.
Asunto(s)
Gammaretrovirus/genética , Vectores Genéticos/genética , Especificidad del Huésped/fisiología , Mitosis , Neuronas/citología , Neuronas/virología , Animales , Muerte Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Corteza Cerebral/citología , ADN/metabolismo , Fase G2/efectos de los fármacos , Gammaretrovirus/efectos de los fármacos , Proteínas Fluorescentes Verdes/metabolismo , Especificidad del Huésped/efectos de los fármacos , Lentivirus/efectos de los fármacos , Lentivirus/genética , Mitosis/efectos de los fármacos , Factor de Crecimiento Nervioso/farmacología , Neuronas/efectos de los fármacos , Células PC12 , Ratas , Fase S/efectos de los fármacos , Suero , Transducción Genética , Proteína bcl-X/metabolismoRESUMEN
Human PiT2 (PiT2) is a multiple-membrane-spanning protein that functions as a type III sodium phosphate cotransporter and as the receptor for amphotropic murine leukemia virus (A-MuLV). Human PiT1 (PiT1), another type III sodium phosphate cotransporter, is a highly related protein that functions as a receptor for gibbon ape leukemia virus but not for A-MuLV. The ability of PiT1 and PiT2 to function as discrete viral receptors with unique properties presumably is reflected in critical residue differences between these two proteins. Early efforts to map the region(s) within PiT2 that is important for virus binding and/or entry relied on infection results obtained with PiT1-PiT2 chimeric cDNAs expressed in Chinese hamster ovary (CHOK1) cells. These attempts to localize the PiT2 virus-binding site were hampered because they were based on infectivity, not binding, assays, and therefore, receptors that bound but failed to facilitate virus entry could not be distinguished from receptors that did not bind virus. Using a more accurate topological model for PiT2 as well as an A-MuLV receptor-binding assay, we have identified extracellular domain one (ECD1) of the human PiT2 receptor as being important for A-MuLV binding and infection.
Asunto(s)
Virus de la Leucemia Murina/metabolismo , Virus de la Leucemia Murina/patogenicidad , Receptores Virales/química , Receptores Virales/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Línea Celular , Cricetinae , Humanos , Mesocricetus , Modelos Moleculares , Datos de Secuencia Molecular , Receptores Virales/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-ActividadRESUMEN
The mammalian gammaretroviruses gibbon ape leukemia virus (GALV) and feline leukemia virus subgroup B (FeLV-B) can use the same receptor, Pit1, to infect human cells. A highly polymorphic nine-residue sequence within Pit1, designated region A, has been proposed as the virus binding site, because mutations in this region abolish Pit1-mediated cellular infection by GALV and FeLV-B. However, a direct correlation between region A mutations deleterious for infection and loss of virus binding has not been established. We report that cells expressing a Pit1 protein harboring mutations in region A that abolish receptor function retain the ability to bind virus, indicating that Pit1 region A is not the virus binding site. Furthermore, we have now identified a second region in Pit1, comprising residues 232 to 260 (region B), that is required for both viral entry and virus binding. Epitope-tagged Pit1 proteins were used to demonstrate that mutations in region B result in improper orientation of Pit1 in the cell membrane. Compensatory mutations in region A can restore proper orientation and full receptor function to these region B mutants. Based on these results, we propose that region A of Pit1 confers competence for viral entry by influencing the topology of the authentic binding site in the membrane and hence its accessibility to a viral envelope protein. Based on glycosylation studies and results obtained by using N- and C-terminal epitope-tagged Pit1, region A and region B mutants, and the transmembrane helices predicted with the PHD PredictProtein algorithm, we propose a new Pit1 topology model.