RESUMO
Change history In this Letter, there are several errors regarding the assignments of mtDNA haplotypes for a subset of egg donors from our study. These errors have not been corrected online.
RESUMO
Common fragile sites (CFSs) are genomic regions that are unstable under conditions of replicative stress. Although the characteristics of CFSs that render them vulnerable to stress are associated mainly with replication, the cellular pathways that protect CFSs during replication remain unclear. Here, we identify and describe a role for FANCD2 as a trans-acting facilitator of CFS replication, in the absence of exogenous replicative stress. In the absence of FANCD2, replication forks stall within the AT-rich fragility core of CFS, leading to dormant origin activation. Furthermore, FANCD2 deficiency is associated with DNA:RNA hybrid formation at CFS-FRA16D, and inhibition of DNA:RNA hybrid formation suppresses replication perturbation. In addition, we also found that FANCD2 reduces the number of potential sites of replication initiation. Our data demonstrate that FANCD2 protein is required to ensure efficient CFS replication and provide mechanistic insight into how FANCD2 regulates CFS stability.
Assuntos
Sítios Frágeis do Cromossomo , Replicação do DNA , DNA/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , RNA/genética , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Linhagem Celular Transformada , DNA/metabolismo , Anemia de Fanconi , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Proteína do Grupo de Complementação A da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Instabilidade Genômica , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Humanos , Linfócitos/citologia , Linfócitos/metabolismo , RNA/metabolismoRESUMO
Maternally inherited mitochondrial (mt)DNA mutations can cause fatal or severely debilitating syndromes in children, with disease severity dependent on the specific gene mutation and the ratio of mutant to wild-type mtDNA (heteroplasmy) in each cell and tissue. Pathogenic mtDNA mutations are relatively common, with an estimated 778 affected children born each year in the United States. Mitochondrial replacement therapies or techniques (MRT) circumventing mother-to-child mtDNA disease transmission involve replacement of oocyte maternal mtDNA. Here we report MRT outcomes in several families with common mtDNA syndromes. The mother's oocytes were of normal quality and mutation levels correlated with those in existing children. Efficient replacement of oocyte mutant mtDNA was performed by spindle transfer, resulting in embryos containing >99% donor mtDNA. Donor mtDNA was stably maintained in embryonic stem cells (ES cells) derived from most embryos. However, some ES cell lines demonstrated gradual loss of donor mtDNA and reversal to the maternal haplotype. In evaluating donor-to-maternal mtDNA interactions, it seems that compatibility relates to mtDNA replication efficiency rather than to mismatch or oxidative phosphorylation dysfunction. We identify a polymorphism within the conserved sequence box II region of the D-loop as a plausible cause of preferential replication of specific mtDNA haplotypes. In addition, some haplotypes confer proliferative and growth advantages to cells. Hence, we propose a matching paradigm for selecting compatible donor mtDNA for MRT.
Assuntos
DNA Mitocondrial/genética , DNA Mitocondrial/uso terapêutico , Herança Materna/genética , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Terapia de Substituição Mitocondrial/métodos , Mutação , Oócitos/metabolismo , Blastocisto/citologia , Blastocisto/metabolismo , Linhagem Celular , Sequência Conservada/genética , DNA Mitocondrial/biossíntese , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Haplótipos/genética , Humanos , Masculino , Meiose , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/prevenção & controle , Doação de Oócitos , Oócitos/citologia , Oócitos/patologia , Fosforilação Oxidativa , Linhagem , Polimorfismo GenéticoRESUMO
Hematopoiesis, the formation of blood cells, involves the hierarchical differentiation of immature blast cells into mature, functional cell types and lineages of the immune system. Hematopoietic stem cells precisely regulate self-renewal versus differentiation to balance the production of blood cells and maintenance of the stem cell pool. The canonical view of acute myeloid leukemia (AML) is that it results from a combination of molecular events in a hematopoietic stem cell that block differentiation and drive proliferation. These events result in the accumulation of primitive hematopoietic blast cells in the blood and bone marrow. We used mathematical modeling to determine the impact of varying differentiation rates on myeloblastic accumulation. Our model shows that, instead of the commonly held belief that AML results from a complete block of differentiation of the hematopoietic stem cell, even a slight skewing of the fraction of cells that differentiate would produce an accumulation of blasts. We confirmed this model by interphase fluorescent in situ hybridization (FISH) and sequencing of purified cell populations from patients with AML, which showed that different leukemia-causing molecular abnormalities typically thought to block differentiation were consistently present in mature myeloid cells such as neutrophils and monocytes at similar levels to those in immature myeloid cells. These findings suggest reduced or skewed, rather than blocked, differentiation is responsible for the development of AML. Approaches that restore normal regulation of hematopoiesis could be effective treatment strategies.
Assuntos
Crise Blástica/patologia , Diferenciação Celular/fisiologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Modelos Biológicos , Adolescente , Adulto , Idoso , Morte Celular , Feminino , Regulação Leucêmica da Expressão Gênica , Hematopoese , Células-Tronco Hematopoéticas/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Células Mieloides/patologia , Fatores de Transcrição/genéticaRESUMO
Rosette-forming glioneuronal tumor (RGNT) most commonly occurs adjacent to the fourth ventricle and therefore rarely presents with epilepsy. Recent reports describe RGNT occurrence in other anatomical locations with considerable morphologic and genetic overlap with the epilepsy-associated dysembryoplastic neuroepithelial tumor (DNET). Examples of RGNT or DNET with anaplastic change are rare, and typically occur in the setting of radiation treatment. We present the case of a 5-year-old girl with seizures, who underwent near total resection of a cystic temporal lobe lesion. Pathology showed morphologic and immunohistochemical features of RGNT, albeit with focally overlapping DNET-like patterns. Resections of residual or recurrent tumor were performed 1 year and 5 years after the initial resection, but no adjuvant radiation or chemotherapy was given. Ten years after the initial resection, surveillance imaging identified new and enhancing nodules, leading to another gross total resection. This specimen showed areas similar to the original tumor, but also high-grade foci with oligodendroglial morphology, increased cellularity, palisading necrosis, microvascular proliferation, and up to 13 mitotic figures per 10 high power fields. Ancillary studies the status by sequencing showed wild-type of the isocitrate dehydrogenase 1 (IDH1), IDH2, and human histone 3.3 (H3F3A) genes, and BRAF studies were negative for mutation or rearrangement. Fluorescence in situ hybridization (FISH) showed codeletion of 1p and 19q limited to the high-grade regions. By immunohistochemistry there was loss of nuclear alpha-thalassemia mental retardation syndrome, X-linked (ATRX) expression only in the high-grade region. Next-generation sequencing showed an fibroblast growth factor receptor receptor 1 (FGFR1) kinase domain internal tandem duplication in three resection specimens. ATRX mutation in the high-grade tumor was confirmed by sequencing which showed a frameshift mutation (p.R1427fs), while the apparent 1p/19q-codeletion by FISH was due to loss of chromosome arm 1p and only partial loss of 19q. Exceptional features of this case include the temporal lobe location, 1p/19q loss by FISH without true whole-arm codeletion, and anaplastic transformation associated with ATRX mutation without radiation or chemotherapy.
Assuntos
Neoplasias Encefálicas/patologia , Transformação Celular Neoplásica/genética , Neoplasias Neuroepiteliomatosas/patologia , Lobo Temporal/patologia , Proteína Nuclear Ligada ao X/genética , Neoplasias Encefálicas/complicações , Neoplasias Encefálicas/genética , Pré-Escolar , Epilepsia/etiologia , Feminino , Humanos , Mutação , Recidiva Local de Neoplasia/complicações , Recidiva Local de Neoplasia/patologia , Neoplasias Neuroepiteliomatosas/complicações , Neoplasias Neuroepiteliomatosas/genéticaRESUMO
OBJECTIVE: The purpose of this study was to describe current practices for onboarding travel nurses (TRNs) and identify TRNs' specific onboarding needs. BACKGROUND: Onboarding must be streamlined and organized for TRNs to provide safe patient care. METHODS: Cross-sectional descriptive survey was used with 306 TRNs throughout United States who were recruited electronically from a closed social media group page. RESULTS: The TRNs identified critical information, including unit patient ratios, onboarding schedule 7 to 14 days before travel assignment start, and login IDs/accesses on day 1. Travel nurse onboarding and competency assessment checklists should be specific to the unit/facility where they will work. CONCLUSION: Findings from this study have the potential to support hospitals in the development of streamlined and tailored TRN onboarding to support regulatory compliance and patient safety as well as realize significant cost savings for TRN onboarding.
Assuntos
Capacitação em Serviço/organização & administração , Recursos Humanos de Enfermagem Hospitalar/normas , Segurança do Paciente/normas , Seleção de Pessoal/normas , Admissão e Escalonamento de Pessoal/normas , Enfermagem Itinerante/estatística & dados numéricos , Enfermagem Itinerante/normas , Adulto , Estudos Transversais , Feminino , Previsões , Humanos , Capacitação em Serviço/tendências , Masculino , Pessoa de Meia-Idade , Recursos Humanos de Enfermagem Hospitalar/tendências , Segurança do Paciente/estatística & dados numéricos , Seleção de Pessoal/tendências , Admissão e Escalonamento de Pessoal/tendências , Enfermagem Itinerante/tendências , Estados UnidosRESUMO
Fanconi anemia (FA) is an inherited bone marrow failure disorder associated with a high incidence of leukemia and solid tumors. Bone marrow transplantation is currently the only curative therapy for the hematopoietic complications of this disorder. However, long-term morbidity and mortality remain very high, and new therapeutics are badly needed. Here we show that the widely used diabetes drug metformin improves hematopoiesis and delays tumor formation in Fancd2-/- mice. Metformin is the first compound reported to improve both of these FA phenotypes. Importantly, the beneficial effects are specific to FA mice and are not seen in the wild-type controls. In this preclinical model of FA, metformin outperformed the current standard of care, oxymetholone, by improving peripheral blood counts in Fancd2-/- mice significantly faster. Metformin increased the size of the hematopoietic stem cell compartment and enhanced quiescence in hematopoietic stem and progenitor cells. In tumor-prone Fancd2-/-Trp53+/- mice, metformin delayed the onset of tumors and significantly extended the tumor-free survival time. In addition, we found that metformin and the structurally related compound aminoguanidine reduced DNA damage and ameliorated spontaneous chromosome breakage and radials in human FA patient-derived cells. Our results also indicate that aldehyde detoxification might be one of the mechanisms by which metformin reduces DNA damage in FA cells.
Assuntos
Carcinogênese/patologia , Anemia de Fanconi/tratamento farmacológico , Anemia de Fanconi/patologia , Hematopoese/efeitos dos fármacos , Metformina/farmacologia , Aldeídos/metabolismo , Animais , Contagem de Células Sanguíneas , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/patologia , Carcinogênese/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Quebra Cromossômica , Dano ao DNA , Dieta , Anemia de Fanconi/sangue , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Guanidinas/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Humanos , Inativação Metabólica/efeitos dos fármacos , Metformina/administração & dosagem , Camundongos , Poli I-C/farmacologiaRESUMO
Mononucleated and binucleated polyploid hepatocytes (4n, 8n, 16n and higher) are found in all mammalian species, but the functional significance of this conserved phenomenon remains unknown. Polyploidization occurs through failed cytokinesis, begins at weaning in rodents and increases with age. Previously, we demonstrated that the opposite event, ploidy reversal, also occurs in polyploid hepatocytes generated by artificial cell fusion. This raised the possibility that somatic 'reductive mitoses' can also happen in normal hepatocytes. Here we show that multipolar mitotic spindles form frequently in mouse polyploid hepatocytes and can result in one-step ploidy reversal to generate offspring with halved chromosome content. Proliferating hepatocytes produce a highly diverse population of daughter cells with multiple numerical chromosome imbalances as well as uniparental origins. Our findings support a dynamic model of hepatocyte polyploidization, ploidy reversal and aneuploidy, a phenomenon that we term the 'ploidy conveyor'. We propose that this mechanism evolved to generate genetic diversity and permits adaptation of hepatocytes to xenobiotic or nutritional injury.
Assuntos
Variação Genética , Hepatócitos/citologia , Hepatócitos/metabolismo , Modelos Genéticos , Poliploidia , Adaptação Fisiológica , Aneuploidia , Animais , Segregação de Cromossomos , Citometria de Fluxo , Hibridização in Situ Fluorescente , Cariotipagem , Masculino , Camundongos , Mitose , Fuso Acromático/metabolismoRESUMO
Chromosome aberrations (aneuploidies mostly) are the cause of the majority of spontaneous abortions in humans. However, little is known about defects in the underlying molecular mechanisms resulting in chromosome aberrations and following failure of preimplantation embryo development, initiation of implantation and postimplantation pregnancy loss. We suggest that defects of the spindle assembly checkpoint (SAC) are responsible for aneuploidy and the following abortions. To develop our hypothesis, we modeled this process in the mouse after inactivation of protein BubR1, one of the key players of SAC. We found that soon after implantation, more than 50 % of cells of BubR1 (-/-) embryos were aneuploid and had an increased level of premature sister chromatid separation (PSCS). Aneuploid cells do not have a predominant gain or loss of some specific chromosomes, but they have mosaic variegated aneuploidy (MVA), which is characterised by random mixture of different chromosomes. MVA leads to growth retardation, stochastic massive apoptosis, disruption of bilateral symmetry, and embryo death between embryonic days 7.5 to 13.5. Analysis published human data revealed that human recurrent pregnancy loss (RPL) embryos and rare infant patients carrying BubR1 mutations that have been described so far have the PSCS and MVA as in BubR1 deficient/insufficient mice. Based on this data, we predict that deficiency/insufficiency of BubR1 and other components of the SAC in human are responsible for a significant fraction of both early and late RPLs.
Assuntos
Aneuploidia , Proteínas de Ciclo Celular/deficiência , Perda do Embrião/genética , Embrião de Mamíferos/anormalidades , Mosaicismo/embriologia , Proteínas Serina-Treonina Quinases/deficiência , Animais , Proteínas de Ciclo Celular/metabolismo , Bandeamento Cromossômico , Embrião de Mamíferos/patologia , Feminino , Marcação de Genes , Haploinsuficiência/genética , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitose , Fenótipo , Gravidez , Proteínas Serina-Treonina Quinases/metabolismo , Cariotipagem EspectralRESUMO
Fanconi anemia (FA) is a rare inherited bone marrow failure syndrome (IBMFS). Affected individuals must be distinguished from relatives, patients with mosaicism must be identified, and patients with other IBMFS classified as non-FA. The diagnostic feature of FA is increased chromosomal breakage in blood lymphocytes cultured with diepoxybutane or mitomycin C. Here, we sought a method to uniquely identify patients with FA with mosaicism, using cells from participants in the National Cancer Institute IBMFS cohort. Lymphocytes were treated with diepoxybutane or mitomycin C, and metaphases scored for breaks and radials. Analyses included the percentage of cells with any aberration, breaks per cell, and breaks per aberrant cell. There were 26 patients with FA (4 mosaics), 46 FA relatives, and 62 patients with a non-FA IBMFS. By all analytic methods, patients with FA were abnormal compared with other groups. Those with FA mosaicism had more breakage than relatives or patients with non-FA IBMFS, but there was some individual overlap. The choices of clastogen are laboratory-dependent, but there was no method or analysis of lymphocytes that clearly distinguished all individuals mosaic for FA from relatives or patients with other IBMFS. Thus, genotyping remains the best method for providing absolute clarity.
Assuntos
Quebra Cromossômica , Anemia de Fanconi/genética , Hemoglobinúria Paroxística/genética , Mosaicismo , Adolescente , Adulto , Idoso , Anemia Aplástica , Doenças da Medula Óssea , Transtornos da Insuficiência da Medula Óssea , Criança , Pré-Escolar , Estudos de Coortes , Compostos de Epóxi/farmacologia , Feminino , Triagem de Portadores Genéticos , Genótipo , Humanos , Lactente , Linfócitos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Mitomicina/farmacologia , Mutagênicos/farmacologia , Adulto JovemRESUMO
Biallelic mutations in BLM cause Bloom syndrome (BS), a genome instability disorder characterized by growth retardation, sun sensitivity and a predisposition to cancer. As evidence of decreased genome stability, BS cells demonstrate not only elevated levels of spontaneous sister chromatid exchanges (SCEs), but also exhibit chromosomal radial formation. The molecular nature and mechanism of radial formation is not known, but radials have been thought to be DNA recombination intermediates between homologs that failed to resolve. However, we find that radials in BS cells occur over 95% between non-homologous chromosomes, and occur non-randomly throughout the genome. BLM must be phosphorylated at T99 and T122 for certain cell cycle checkpoints, but it is not known whether these modifications are necessary to suppress radial formation. We find that exogenous BLM constructs preventing phosphorylation at T99 and T122 are not able to suppress radial formation in BS cells, but are able to inhibit SCE formation. These findings indicate that BLM functions in 2 distinct pathways requiring different modifications. In one pathway, for which the phosphorylation marks appear dispensable, BLM functions to suppress SCE formation. In a second pathway, T99 and T122 phosphorylations are essential for suppression of chromosomal radial formation, both those formed spontaneously and those formed following interstrand crosslink damage.
Assuntos
Síndrome de Bloom/genética , Instabilidade Cromossômica , RecQ Helicases/metabolismo , Troca de Cromátide Irmã , Síndrome de Bloom/metabolismo , Células Cultivadas , Cromossomos Humanos/genética , Humanos , Método de Monte Carlo , Mutação , Fosforilação , RecQ Helicases/genéticaRESUMO
Bone marrow failure is a nearly universal complication of Fanconi anemia. The proteins encoded by FANC genes are involved in DNA damage responses through the formation of a multisubunit nuclear complex that facilitates the E3 ubiquitin ligase activity of FANCL. However, it is not known whether loss of E3 ubiquitin ligase activity accounts for the hematopoietic stem cell defects characteristic of Fanconi anemia. Here we provide evidence that FANCL increases the activity and expression of ß-catenin, a key pluripotency factor in hematopoietic stem cells. We show that FANCL ubiquitinates ß-catenin with atypical ubiquitin chain extension known to have nonproteolytic functions. Specifically, ß-catenin modified with lysine-11 ubiquitin chain extension efficiently activates a lymphocyte enhancer-binding factor-T cell factor reporter. We also show that FANCL-deficient cells display diminished capacity to activate ß-catenin leading to reduced transcription of Wnt-responsive targets c-Myc and Cyclin D1. Suppression of FANCL expression in normal human CD34(+) stem and progenitor cells results in fewer ß-catenin active cells and inhibits expansion of multilineage progenitors. Together, these results suggest that diminished Wnt/ß-catenin signaling may be an underlying molecular defect in FANCL-deficient hematopoietic stem cells leading to their accelerated loss.
Assuntos
Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , beta Catenina/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Ciclina D1/metabolismo , Anemia de Fanconi/etiologia , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Anemia de Fanconi/patologia , Proteína do Grupo de Complementação C da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação C da Anemia de Fanconi/genética , Proteína do Grupo de Complementação C da Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação L da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação L da Anemia de Fanconi/genética , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Células HEK293 , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Camundongos , Camundongos Knockout , Modelos Biológicos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/patologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fatores de Transcrição TCF/metabolismo , Ubiquitinação , beta Catenina/químicaRESUMO
Tumor-associated macrophages (TAMs) residing in the tumor microenvironment (TME) are characterized by their pivotal roles in tumor progression, antitumor immunity, and TME remodeling. However, a thorough comparative characterization of tumor-TAM crosstalk across IDH-defined categories of glioma remains elusive, likely contributing to mixed outcomes in clinical trials. We delineated the phenotypic heterogeneity of TAMs across IDH-stratified gliomas. Notably, two TAM subsets with a mesenchymal phenotype were enriched in IDH-WT glioblastoma (GBM) and correlated with poorer patient survival and reduced response to anti-PD-1 immune checkpoint inhibitor (ICI). We proposed SLAMF9 receptor as a potential therapeutic target. Inference of gene regulatory networks identified PPARG, ELK1, and MXI1 as master transcription factors of mesenchymal BMD-TAMs. Our analyses of reciprocal tumor-TAM interactions revealed distinct crosstalk in IDH-WT tumors, including ANXA1-FPR1/3, FN1-ITGAVB1, VEGFA-NRP1, and TNFSF12-TNFRSF12A with known contribution to immunosuppression, tumor proliferation, invasion and TAM recruitment. Spatially resolved transcriptomics further elucidated the architectural organization of highlighted communications. Furthermore, we demonstrated significant upregulation of ANXA1, FN1, NRP1, and TNFRSF12A genes in IDH-WT tumors using bulk RNA-seq and RT-qPCR. Longitudinal expression analysis of candidate genes revealed no difference between primary and recurrent tumors indicating that the interactive network of malignant states with TAMs does not drastically change upon recurrence. Collectively, our study offers insights into the unique cellular composition and communication of TAMs in glioma TME, revealing novel vulnerabilities for therapeutic interventions in IDH-WT GBM.
Assuntos
Neoplasias Encefálicas , Glioma , Isocitrato Desidrogenase , Transcriptoma , Microambiente Tumoral , Macrófagos Associados a Tumor , Humanos , Microambiente Tumoral/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Isocitrato Desidrogenase/genética , Macrófagos Associados a Tumor/metabolismo , Macrófagos Associados a Tumor/patologia , Glioma/genética , Glioma/patologia , Glioma/metabolismo , Análise de Célula ÚnicaRESUMO
Murine hepatocytes become polyploid and then undergo ploidy reversal and become aneuploid in a dynamic process called the ploidy conveyor. Although polyploidization occurs in some types of human cells, the degree of aneuploidy in human hepatocytes is not known. We isolated hepatocytes derived from healthy human liver samples and determined chromosome number and identity using traditional karyotyping and fluorescence in situ hybridization. Similar to murine hepatocytes, human hepatocytes are highly aneuploid. Moreover, imaging studies revealed multipolar spindles and chromosome segregation defects in dividing human hepatocytes. Aneuploidy therefore does not necessarily predispose liver cells to transformation but might promote genetic diversity among hepatocytes.
Assuntos
Aneuploidia , Cromossomos Humanos , Variação Genética , Hepatócitos/patologia , Adolescente , Adulto , Idoso , Animais , Criança , Pré-Escolar , Feminino , Hepatócitos/transplante , Humanos , Hidrolases/deficiência , Hidrolases/genética , Hibridização in Situ Fluorescente , Lactente , Cariotipagem , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Quimeras de Transplante , Adulto JovemRESUMO
Genetic fumarylacetoacetate hydrolase (Fah) deficiency is unique in that healthy gene-corrected hepatocytes have a strong growth advantage and can repopulate the diseased liver. Unfortunately, similar positive selection of gene-corrected cells is absent in most inborn errors of liver metabolism and it is difficult to reach the cell replacement index required for therapeutic benefit. Therefore, methods to transiently create a growth advantage for genetically modified hepatocytes in any genetic background would be advantageous. To mimic the selective pressure of Fah deficiency in normal animals, an efficient in vivo small molecule inhibitor of FAH, 4-[(2-carboxyethyl)-hydroxyphosphinyl]-3-oxobutyrate (CEHPOBA) was developed. Microarray analysis demonstrated that pharmacological inhibition of FAH produced highly similar gene expression changes to genetic deficiency. As proof of principle, hepatocytes lacking homogentisic acid dioxygenase (Hgd) and hence resistant to FAH inhibition were transplanted into sex-mismatched wild-type recipients. Time course analyses of 4-6 weeks of CEHPOBA administration after transplantation showed a linear relationship between treatment length and replacement index. Compared to controls, recipients treated with the FAH-inhibitor had 20-100-fold increases in liver repopulation. We conclude that pharmacological inhibition of FAH is a promising approach to in vivo selection of hepatocytes.
Assuntos
Alcaptonúria/terapia , Inibidores Enzimáticos/administração & dosagem , Hepatócitos/transplante , Hidrolases/antagonistas & inibidores , Alcaptonúria/metabolismo , Animais , Butiratos/administração & dosagem , Feminino , Expressão Gênica , Terapia Genética , Hepatócitos/enzimologia , Homogentisato 1,2-Dioxigenase/genética , Hidrolases/genética , Cinética , Fígado/citologia , Fígado/metabolismo , Masculino , Camundongos , Análise em Microsséries , Compostos Organofosforados/administração & dosagemRESUMO
Range of DNA repair in response to double-strand breaks induced in human preimplantation embryos remains uncertain due to the complexity of analyzing single- or few-cell samples. Sequencing of such minute DNA input requires a whole genome amplification that can introduce artifacts, including coverage nonuniformity, amplification biases, and allelic dropouts at the target site. We show here that, on average, 26.6% of preexisting heterozygous loci in control single blastomere samples appear as homozygous after whole genome amplification indicative of allelic dropouts. To overcome these limitations, we validate on-target modifications seen in gene edited human embryos in embryonic stem cells. We show that, in addition to frequent indel mutations, biallelic double-strand breaks can also produce large deletions at the target site. Moreover, some embryonic stem cells show copy-neutral loss of heterozygosity at the cleavage site which is likely caused by interallelic gene conversion. However, the frequency of loss of heterozygosity in embryonic stem cells is lower than in blastomeres, suggesting that allelic dropouts is a common whole genome amplification outcome limiting genotyping accuracy in human preimplantation embryos.
Assuntos
Blastocisto , Edição de Genes , Humanos , Blastômeros , Embrião de Mamíferos , AlelosRESUMO
Fancc suppresses cross-linker-induced genotoxicity, modulates growth-inhibitory cytokine responses, and modulates endotoxin responses. Although loss of the latter function is known to account for endotoxin-induced marrow failure in murine Fancc (mFancc)-deficient mice, some argue that cytokine and endotoxin hypersensitivities devolve simply from genomic instability. Seeking to resolve this question, we planned to ectopically express instructive human FANCC (hFANCC) mutants in murine Fancc-deficient hematopoietic stem cells. To first assure that hFANCC cDNA was competent in murine cells, we compared hFANCC and mFancc in complementation assays for cross-linking agent hypersensitivity and endotoxin hypersensitivity. We found that mFancc complemented murine Fancc-deficient cells in both assays, but that hFANCC fully suppressed only endotoxin hypersensitivity, not cross-linking agent hypersensitivity. These results support the notions that Fancc is multifunctional and that structural prerequisites for its genoprotective functions differ from those required to constrain endotoxin responses known to lead to marrow failure in Fancc-deficient mice.
Assuntos
Proteína do Grupo de Complementação C da Anemia de Fanconi/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Animais , Endotoxinas/farmacologia , Proteína do Grupo de Complementação C da Anemia de Fanconi/deficiência , Proteína do Grupo de Complementação C da Anemia de Fanconi/genética , Humanos , Hipersensibilidade Imediata/induzido quimicamente , Camundongos , Camundongos Knockout , TransgenesRESUMO
Specific cytogenetic clones might distinguish patients with unrecognized Fanconi anemia (FA) who present with acute myeloid leukemia (AML) from those with sporadic AML. Cytogenetic reports in literature cases of FA and AML were compared with de novo cases enrolled on CCG-2961. Gain of 1q, gain of 3q, monosomy 7, deleted 7q, gain of 13q, and deleted 20q were more frequent in FA AML; t(8;21), trisomy 8, t(9;11), t(6;9), and inversion 16 were exclusive to de novo AML cases. Observation of the FA AML cytogenetic clonal patterns should raise suspicion of an underlying leukemia predisposition syndrome and influence management.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos/genética , Anemia de Fanconi/genética , Anemia de Fanconi/terapia , Predisposição Genética para Doença , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Adolescente , Adulto , Criança , Pré-Escolar , Anemia de Fanconi/complicações , Feminino , Humanos , Lactente , Leucemia Mieloide Aguda/complicações , MasculinoRESUMO
An increasing interest in the molecular mechanisms governing cell division has resulted in the discovery of several groups of genes that participate in the regulation of mitosis and meiosis in eukaryotes. Inactivation of these genes in mice often leads to early embryonic lethality. To show direct causality between mutations of these genes, chromosomal instability and embryonic death, a technique enabling detailed cytogenetic analysis of embryonic cells is required. Here, we develop and test a comprehensive approach that allows complex analysis of individual early postimplantation embryos and combines polymerase chain reaction genotyping with the preparation and detailed karyotypic inspection of cells at the metaphase and anaphase stages. The method enables good chromosomal spreading and scattering of nuclei to perform routine cytogenetics (i.e., standard stain and G-banding). It also permits the application of specialized techniques such as fluorescence in situ hybridization to detect particular chromosomes and to verify the integrity of individual chromosomes. Utility of the new method is demonstrated by an analysis of embryonic day E7.5-E9.5 tissue from mice deficient in the spindle checkpoint gene Bub1b.