RESUMO
SF3B1, SRSF2, and U2AF1 are the most frequently mutated splicing factor genes in the myelodysplastic syndromes (MDS). We have performed a comprehensive and systematic analysis to determine the effect of these commonly mutated splicing factors on pre-mRNA splicing in the bone marrow stem/progenitor cells and in the erythroid and myeloid precursors in splicing factor mutant MDS. Using RNA-seq, we determined the aberrantly spliced genes and dysregulated pathways in CD34+ cells of 84 patients with MDS. Splicing factor mutations result in different alterations in splicing and largely affect different genes, but these converge in common dysregulated pathways and cellular processes, focused on RNA splicing, protein synthesis, and mitochondrial dysfunction, suggesting common mechanisms of action in MDS. Many of these dysregulated pathways and cellular processes can be linked to the known disease pathophysiology associated with splicing factor mutations in MDS, whereas several others have not been previously associated with MDS, such as sirtuin signaling. We identified aberrantly spliced events associated with clinical variables, and isoforms that independently predict survival in MDS and implicate dysregulation of focal adhesion and extracellular exosomes as drivers of poor survival. Aberrantly spliced genes and dysregulated pathways were identified in the MDS-affected lineages in splicing factor mutant MDS. Functional studies demonstrated that knockdown of the mitosis regulators SEPT2 and AKAP8, aberrantly spliced target genes of SF3B1 and SRSF2 mutations, respectively, led to impaired erythroid cell growth and differentiation. This study illuminates the effect of the common spliceosome mutations on the MDS phenotype and provides novel insights into disease pathophysiology.
Assuntos
Mutação , Síndromes Mielodisplásicas/genética , Fatores de Processamento de RNA/genética , Splicing de RNA , Spliceossomos/genética , Estudos de Coortes , Reparo do DNA , Regulação da Expressão Gênica , Humanos , Síndromes Mielodisplásicas/epidemiologia , Fosfoproteínas/genética , Fatores de Processamento de Serina-Arginina/genética , Fator de Processamento U2AF/genética , Análise de SobrevidaRESUMO
For the analysis of gene function in vivo, gene overexpression in the mouse provides an alternative to loss-of-function knock-out approaches and can help reveal phenotypes where compensatory mechanisms are at play. Furthermore, when multiple lines overexpressing a gene-of-interest at varying levels are studied, the consequences of differences in gene dosage can be explored. Despite these advantages, inherent shortcomings in the methodologies used for the generation of gain-of-function transgenic mouse models have limited their application to functional gene analysis, and the necessity for multiple lines comes at a significant animal and financial cost. The targeting of transgenic overexpression constructs at single copy into neutral genomic loci is the preferred method for the generation of such models, which avoids the unpredictable outcomes associated with conventional random integration. However, despite the increased reliability that targeted transgenic methodologies provide, only one expression level results, as defined by the promoter used. Here, we report a new versatile overexpression allele, the promoter-switch allele, which couples PhiC31 integrase-targeted transgenesis with Flp recombinase promoter switching and Cre recombinase activation. These recombination switches allow the conversion of different overexpression alleles, combining the advantages of transgenic targeting with tunable transgene expression. With this approach, phenotype severity can be correlated with transgene expression in a single mouse model, providing a cost-effective solution amenable to systematic gain-of-function studies.
Assuntos
Expressão Gênica , Transgenes , Alelos , Animais , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Especificidade de Órgãos , Regiões Promotoras GenéticasRESUMO
CRISPR/Cas is a microbial adaptive immune system that uses RNA-guided nucleases to cleave foreign genetic elements. The CRISPR/Cas9 method has been engineered from the type II prokaryotic CRISPR system and uses a single-guide RNA to target the Cas9 nuclease to a specific genomic sequence. Cas9 induces double-stranded DNA breaks which are repaired either by imperfect non-homologous end joining to generate insertions or deletions (indels) or, if a repair template is provided, by homology-directed repair. Due to its specificity, simplicity and versatility, the CRISPR/Cas9 system has recently emerged as a powerful tool for genome engineering in various species. This technology can be used to investigate the function of a gene of interest or to correct gene mutations in cells via genome editing, paving the way for future gene therapy approaches. Improvements to the efficiency of CRISPR repair, in particular to increase the rate of gene correction and to reduce undesired off-target effects, and the development of more effective delivery methods will be required for its broad therapeutic application.
Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Engenharia Genética/métodos , Terapia Genética/métodos , Edição de RNA , Animais , Proteínas Associadas a CRISPR/metabolismo , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , HumanosRESUMO
Objectives: Intracerebral hemorrhage (ICH) occurs mostly in the striatum. In ICH, blood prolactin level increases 3-fold. The effects of intracerebroventricular injection (ICV) of prolactin on motor disorders will be investigated. Materials and Methods: This study was performed on 32 male Wistar rats in 4 groups: sham, ICH, and prolactin with 1 µg/2 µl (P1) and 2 µg/2 µl (P2) doses. Results: The weight of animals on days 1 (PË0.01), 3, and 7 (PË0.05) in the sham and P2 groups increased compared with the ICH group. Neurological Deficit Score (NDS) in ICH and P1 groups decreased, and increased compared with sham and ICH groups (PË0.001), respectively. NDS in the P1 group increased compared with the P2 group on days 1 (PË0.0 5), 3, and 7 (PË0.001). The duration time of rotarod in ICH and P1 groups decreased and increased compared with sham and ICH groups (PË0.001), respectively. The duration time of rotarod in the P1 group on days 3 and 7 increased compared with the P2 group (PË0.001). Travel distance in days 1(PË0.01), 3(PË0.001), and 7(PË0.01) decreased in the ICH group. Prolactin receptor (PRL receptor) expression in ICH, P1, and P2 groups increased compared with sham and ICH groups (PË0.001). Glial fibrillary acidic protein (GFAP) expression (PË0.001) and apolipoprotein E (APOE) (PË0.01) expression in the ICH group increased compared with the sham group. GFAP and APOE expression in the P1 group increased compared with the ICH group (PË0.001). APOE expression in the P1 group increased compared with the P2 group (PË0.001). Conclusion: According to the results, prolactin reduces movement disorders.
RESUMO
The BCL2-inhibitor, Venetoclax (VEN), has shown significant anti-leukemic efficacy in combination with the DNMT-inhibitor, Azacytidine (AZA). To explore the mechanisms underlying the selective sensitivity of mutant leukemia cells to VEN and AZA, we used cell-based isogenic models containing a common leukemia-associated mutation in the epigenetic regulator ASXL1. KBM5 cells with CRISPR/Cas9-mediated correction of the ASXL1G710X mutation showed reduced leukemic growth, increased myeloid differentiation, and decreased HOXA and BCL2 gene expression in vitro compared to uncorrected KBM5 cells. Increased expression of the anti-apoptotic gene, BCL2, was also observed in bone marrow CD34+ cells from ASXL1 mutant MDS patients compared to CD34+ cells from wild-type MDS cases. ATAC-sequencing demonstrated open chromatin at the BCL2 promoter in the ASXL1 mutant KBM5 cells. BH3 profiling demonstrated increased dependence of mutant cells on BCL2. Upon treatment with VEN, mutant cells demonstrated increased growth inhibition. In addition, genome-wide methylome analysis of primary MDS samples and isogenic cell lines demonstrated increased gene-body methylation in ASXL1 mutant cells, with consequently increased sensitivity to AZA. These data mechanistically link the common leukemia-associated mutation ASXL1 to enhanced sensitivity to VEN and AZA via epigenetic upregulation of BCL2 expression and widespread alterations in DNA methylation.
Assuntos
Antineoplásicos/farmacologia , Azacitidina/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Proteínas Repressoras/genética , Sulfonamidas/farmacologia , Linhagem Celular Tumoral , Epigênese Genética/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Mutação/efeitos dos fármacos , Mutação Puntual/efeitos dos fármacosRESUMO
Evidence suggests that circadian rhythms are regulated through diffusible signals generated by the suprachiasmatic nucleus (SCN). Vasoactive intestinal peptide (VIP) is located in SCN neurons positioned to receive photic input from the retinohypothalamic tract and transmit information to other SCN cells and adjacent hypothalamic areas. Studies using knockout mice indicate that VIP is essential for synchrony among SCN cells and for the expression of normal circadian rhythms. To test the hypothesis that VIP is also an SCN output signal, we recorded wheel-running activity rhythms in hamsters and continuously infused the VIP receptor agonist BAY 55-9837 in the third ventricle for 28 days. Unlike other candidate output signals, infusion of BAY 55-9837 did not affect activity levels. Instead, BAY 55-9837 lengthened the circadian period by 0.69 +/- 0.04 h (P < 0.0002 compared with controls). Period returned to baseline after infusions. We analyzed the effect of BAY 55-9837 on cultured SCN from PER2::LUC mice to determine if lengthening of the period by BAY 55-9837 is a direct effect on the SCN. Application of 10 muM BAY 55-9837 to SCN in culture lengthened the period of PER2 luciferase expression (24.73 +/- 0.24 h) compared with control SCN (23.57 +/- 0.26, P = 0.01). In addition, rhythm amplitude was significantly increased, consistent with increased synchronization of SCN neurons. The effect of BAY 55-9837 in vivo on period is similar to the effect of constant light. The present results suggest that VIP-VPAC2 signaling in the SCN may play two roles, synchronizing SCN neurons and setting the period of the SCN as a whole.
Assuntos
Ritmo Circadiano/fisiologia , Receptores de Peptídeo Intestinal Vasoativo/metabolismo , Peptídeo Intestinal Vasoativo/metabolismo , Animais , Ritmo Circadiano/efeitos dos fármacos , Cricetinae , Hipotálamo/metabolismo , Luz , Masculino , Mesocricetus , Camundongos , Camundongos Knockout/metabolismo , Atividade Motora , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/fisiologia , Fragmentos de Peptídeos , Receptores de Peptídeo Intestinal Vasoativo/fisiologia , Transdução de Sinais/fisiologia , Núcleo Supraquiasmático/metabolismo , Núcleo Supraquiasmático/fisiologia , Peptídeo Intestinal Vasoativo/farmacologiaRESUMO
AIMS: Brain injuries based on their causes are divided into two categories, TBI and NTBI. TBI is caused by damages such as head injury, but non-physical injury causes NTBI. Prolactin is one of the blood factors that increase during brain injury. It has been assumed to play a regenerative role in post-injury recovery. MATERIALS AND METHODS: In this review, various valid papers from electronic sources (including Web of Science, Scopus, PubMed, SID, Google Scholar, and ISI databases) used, which in them the protective effect of prolactin on brain injury investigated. KEY FINDINGS: Inflammation following brain injury with the production of pro-inflammatory cytokines in the affected area can even lead to excitotoxicity and cell death in the damaged area. Medical brain damage treatments are long-term, and can have several side effects. Therefore, it is better to consider medication treatments that have fewer side effects and greater efficacy. Research suggests that prolactin has numerous regenerative effects on brain injury, and prevents cell death. Prolactin is one of the hormones produced in the body; therefore it has fewer side effects and may be more effective because it increases during brain injury. SIGNIFICANCE: Prolactin can be used peripherally and centrally, and exerts its neuro regenerative effects against further damage post-TBI and NTBI.
Assuntos
Lesões Encefálicas/tratamento farmacológico , Inflamação/prevenção & controle , Prolactina/uso terapêutico , Animais , Lesões Encefálicas/patologia , Citocinas/metabolismo , Humanos , Inflamação/metabolismoRESUMO
The circadian (near 24-h) clock is involved in the temporal organisation of physiological and biochemical activities of many organisms, including humans. The clock functions through the rhythmic transcription and translation of several genes, forming an oscillatory feedback loop. Genetic analysis has shown that the circadian clock exists in both a central circadian pacemaker (i.e. the suprachiasmatic nucleus of the hypothalamus), as well as in most peripheral tissues. In particular, the circadian clockwork genes are expressed in all female and male reproductive tissues studied so far, as well as in the conceptus itself. The current data clearly show a robust rhythm in female reproductive tissues, but whether rhythmicity also exists in male reproductive tissues remains uncertain. Although the conceptus also expresses most of the canonical circadian genes, the rhythmicity of their expression is still under investigation. Published data indicate that environmental and genetic manipulations influence reproductive function and fecundity, suggesting an important role for the circadian clock in reproduction, and possibly early development.
Assuntos
Relógios Biológicos/genética , Ritmo Circadiano/genética , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Genitália/metabolismo , Animais , Desenvolvimento Embrionário/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Humanos , MasculinoRESUMO
Induced pluripotent stem cells (iPSCs) were first described over a decade ago and are currently used in various basic biology and clinical research fields. Recent advances in the field of human iPSCs have opened the way to a better understanding of the biology of human diseases. Disease-specific iPSCs provide an unparalleled opportunity to establish novel human cell-based disease models, with the potential to enhance our understanding of the molecular mechanisms underlying human malignancies, and to accelerate the identification of effective new drugs. When combined with genome editing technologies, iPSCs represent a new approach to study single or multiple disease-causing mutations and model specific diseases in vitro. In addition, genetically corrected patient-specific iPSCs could potentially be used for stem cell based therapy. Furthermore, the reprogrammed cells share patient-specific genetic background, offering a new platform to develop personalized therapy/medicine for patients. In this review we discuss the recent advances in iPSC research technology and their potential applications in hematological diseases. Somatic cell reprogramming has presented new routes for generating patient-derived iPSCs, which can be differentiated to hematopoietic stem cells and the various downstream hematopoietic lineages. iPSC technology shows promise in the modeling of both inherited and acquired hematological disorders. A direct reprogramming and differentiation strategy is able to recapitulate hematological disorder progression and capture the earliest molecular alterations that underlie the initiation of hematological malignancies.
Assuntos
Edição de Genes , Doenças Hematológicas , Células-Tronco Pluripotentes Induzidas , Mutação , Transplante de Células-Tronco , Animais , Doenças Hematológicas/genética , Doenças Hematológicas/metabolismo , Doenças Hematológicas/patologia , Doenças Hematológicas/terapia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Células-Tronco Pluripotentes Induzidas/transplanteRESUMO
Genetically modified pigs have been considered favorable resources in xenotransplantation. Microinjection of randomly integrating transgenes into zygotes, somatic cell nuclear transfer, homologous recombination, zinc finger nucleases, transcription activator-like effector nucleases, and most recently, clustered regularly interspaced short palindromic repeats-cas9 (CRISPR/Cas9) are the techniques that have been used to generate these animals. Here, we provide an overview of the CRISPR approaches that have been used to modify genes which are vital in improving xenograft survival rate, including cytidine monophosphate-N-acetylneuraminic acid hydroxylase, B1,4N-acetylgalactosaminyltransferase, isoglobotrihexosylceramide synthase, class I MHC, von Willebrand factor, C3, and porcine endogenous retroviruses. In addition, we will mention the importance of potential candidate genes which could be targeted using CRISPR/Cas9.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Xenoenxertos , Sequências Repetidas Invertidas , Alelos , Animais , Marcadores Genéticos , HumanosRESUMO
CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGFß. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.
Assuntos
Sistemas CRISPR-Cas/genética , Engenharia Genética/métodos , Terapia Genética/métodos , Imunoterapia/métodos , Células Matadoras Naturais/metabolismo , Ribonucleoproteínas/metabolismo , HumanosRESUMO
Mutations of the splicing factor-encoding gene U2AF1 are frequent in the myelodysplastic syndromes (MDS), a myeloid malignancy, and other cancers. Patients with MDS suffer from peripheral blood cytopenias, including anemia, and an increasing percentage of bone marrow myeloblasts. We studied the impact of the common U2AF1S34F mutation on cellular function and mRNA splicing in the main cell lineages affected in MDS. We demonstrated that U2AF1S34F expression in human hematopoietic progenitors impairs erythroid differentiation and skews granulomonocytic differentiation toward granulocytes. RNA sequencing of erythroid and granulomonocytic colonies revealed that U2AF1S34F induced a higher number of cassette exon splicing events in granulomonocytic cells than in erythroid cells. U2AF1S34F altered mRNA splicing of many transcripts that were expressed in both cell types in a lineage-specific manner. In hematopoietic progenitors, the introduction of isoform changes identified in the U2AF1S34F target genes H2AFY, encoding an H2A histone variant, and STRAP, encoding serine/threonine kinase receptor-associated protein, recapitulated phenotypes associated with U2AF1S34F expression in erythroid and granulomonocytic cells, suggesting a causal link. Furthermore, we showed that isoform modulation of H2AFY and STRAP rescues the erythroid differentiation defect in U2AF1S34F MDS cells, suggesting that splicing modulators could be used therapeutically. These data have critical implications for understanding MDS phenotypic heterogeneity and support the development of therapies targeting splicing abnormalities.
Assuntos
Síndromes Mielodisplásicas/genética , Fator de Processamento U2AF/genética , Estudos de Casos e Controles , Linhagem da Célula , Proliferação de Células , Células Cultivadas , Eritropoese , Ontologia Genética , Granulócitos/fisiologia , Humanos , Mutação de Sentido Incorreto , Síndromes Mielodisplásicas/patologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Splicing de RNA , Fator de Processamento U2AF/metabolismoRESUMO
Genome editing technologies have advanced significantly over the past few years, providing a fast and effective tool to precisely manipulate the genome at specific locations. The three commonly used genome editing technologies are Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Cas9 (CRISPR/Cas9) system. ZFNs and TALENs consist of endonucleases fused to a DNA-binding domain, while the CRISPR/Cas9 system uses guide RNAs to target the bacterial Cas9 endonuclease to the desired genomic location. The double-strand breaks made by these endonucleases are repaired in the cells either by non-homologous end joining, resulting in the introduction of insertions/deletions, or, if a repair template is provided, by homology directed repair. The ZFNs, TALENs and CRISPR/Cas9 systems take advantage of these repair mechanisms for targeted genome modification and have been successfully used to manipulate the genome in human cells. These genome editing tools can be used to investigate gene function, to discover new therapeutic targets, and to develop disease models. Moreover, these genome editing technologies have great potential in gene therapy. Here, we review the latest advances in the application of genome editing technology to the study and treatment of hematological disorders.
Assuntos
Edição de Genes , Genoma , Doenças Hematológicas/genética , Animais , Técnicas Genéticas/tendências , Doenças Hematológicas/metabolismo , Doenças Hematológicas/terapia , HumanosRESUMO
Splicing is an essential cellular process which is carried out by the spliceosome in order to remove the introns and join the exons present in pre-mRNA transcripts. A variety of spliceosomal mutations have been recently identified in the myelodysplastic syndromes (MDS), a heterogeneous group of hematopoietic stem cell malignancies, revealing a new leukemogenic pathway involving spliceosomal dysfunction. Splicing factor genes are the most frequently mutated genes found in MDS, with mutations occurring in more than half of all patients. The high mutation frequency in different components of the spliceosome in MDS indicates that aberrant splicing may be a common consequence of these mutations in this disorder. RNA sequencing studies using MDS patient bone marrow cells and different mouse models have identified several downstream targets of the splicing factor mutations. Aberrant splicing of these target genes may contribute to MDS pathogenesis, however functional studies are required in order to fully determine the effects of the aberrant isoforms on disease phenotype. Splicing inhibitors are currently being developed and may be used as therapeutic agents to target aberrant pre-mRNA splicing in MDS and other cancers with splicing factor mutations. The mouse models expressing splicing factor mutations may prove particularly valuable for pre-clinical testing of these drugs.
Assuntos
Síndromes Mielodisplásicas/genética , Splicing de RNA/genética , Spliceossomos/genética , Animais , Humanos , Mutação , Síndromes Mielodisplásicas/patologiaRESUMO
Recurrent somatic mutations of the epigenetic modifier and tumor suppressor ASXL1 are common in myeloid malignancies, including chronic myeloid leukemia (CML), and are associated with poor clinical outcome. CRISPR/Cas9 has recently emerged as a powerful and versatile genome editing tool for genome engineering in various species. We have used the CRISPR/Cas9 system to correct the ASXL1 homozygous nonsense mutation present in the CML cell line KBM5, which lacks ASXL1 protein expression. CRISPR/Cas9-mediated ASXL1 homozygous correction resulted in protein re-expression with restored normal function, including down-regulation of Polycomb repressive complex 2 target genes. Significantly reduced cell growth and increased myeloid differentiation were observed in ASXL1 mutation-corrected cells, providing new insights into the role of ASXL1 in human myeloid cell differentiation. Mice xenografted with mutation-corrected KBM5 cells showed significantly longer survival than uncorrected xenografts. These results show that the sole correction of a driver mutation in leukemia cells increases survival in vivo in mice. This study provides proof-of-concept for driver gene mutation correction via CRISPR/Cas9 technology in human leukemia cells and presents a strategy to illuminate the impact of oncogenic mutations on cellular function and survival.
Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Códon sem Sentido , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Proteínas Repressoras/genética , Animais , Sequência de Bases , Proteínas Associadas a CRISPR/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Regulação Leucêmica da Expressão Gênica , Predisposição Genética para Doença , Xenoenxertos , Homozigoto , Humanos , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Dados de Sequência Molecular , Transplante de Neoplasias , Fenótipo , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Tempo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismoRESUMO
Cancer is a genetic disease, but two patients rarely have identical genotypes. Similarly, patients differ in their clinicopathological parameters, but how genotypic and phenotypic heterogeneity are interconnected is not well understood. Here we build statistical models to disentangle the effect of 12 recurrently mutated genes and 4 cytogenetic alterations on gene expression, diagnostic clinical variables and outcome in 124 patients with myelodysplastic syndromes. Overall, one or more genetic lesions correlate with expression levels of ~20% of all genes, explaining 20-65% of observed expression variability. Differential expression patterns vary between mutations and reflect the underlying biology, such as aberrant polycomb repression for ASXL1 and EZH2 mutations or perturbed gene dosage for copy-number changes. In predicting survival, genomic, transcriptomic and diagnostic clinical variables all have utility, with the largest contribution from the transcriptome. Similar observations are made on the TCGA acute myeloid leukaemia cohort, confirming the general trends reported here.
Assuntos
Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Mutação , Síndromes Mielodisplásicas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Análise Mutacional de DNA , Proteína Potenciadora do Homólogo 2 de Zeste , Feminino , Genótipo , Humanos , Leucemia Mieloide Aguda/genética , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/diagnóstico , Complexo Repressor Polycomb 2/genética , Prognóstico , Proteínas Repressoras/genética , Resultado do Tratamento , Adulto JovemRESUMO
Circadian rhythms in mammals are regulated by the hypothalamic suprachiasmatic nucleus (SCN). The generation of circadian oscillations is a cell-autonomous property, and coupling among cells is essential for the SCN to function as a pacemaker. The development of SCN anatomy and cytology has been extensively studied, but the point in development when the SCN first has the capacity to generate circadian oscillations has not been established. We therefore examined the development of circadian oscillations using per2::luc mice in which bioluminescence tracks the expression of the circadian clock protein, PER2. In vitro, hypothalamic explants first expressed consistent oscillations when isolated between 15 and 16 days postfertilization (e15). Oscillations were more robust at later ages. Explants from other brain areas did not express oscillations, indicating that the development of oscillations is not a general property of embryonic tissue. SCN explants obtained on e14 did not initially express oscillations but developed them in vitro over 4 to 6 d. Although coupling among cells is required for the long-term expression of tissue-level oscillations, explants from mice lacking the coupling peptide vasoactive intestinal peptide still developed oscillations. In the mouse, the capacity to generate molecular oscillations on e15 coincides with the completion of neurogenesis and SCN-specific transcription factor expression. Thus, within a day of its genesis at an age approximately equivalent to the end of the first trimester in humans, the SCN develops the capacity to express circadian oscillations and autonomously develops mechanisms sufficient to couple and synchronize its cells.
Assuntos
Relógios Circadianos/fisiologia , Ritmo Circadiano/fisiologia , Hipotálamo/fisiologia , Animais , Feminino , Hipotálamo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Circadianas Period/metabolismo , Peptídeo Intestinal Vasoativo/metabolismoRESUMO
Accurate pre-mRNA splicing by the spliceosome is a fundamental cellular mechanism required to remove introns that are present in most protein-coding transcripts. The recent discovery of a variety of somatic spliceosomal mutations in the myelodysplastic syndromes (MDS), a heterogeneous group of myeloid malignancies, has revealed a new leukemogenic pathway involving spliceosomal dysfunction. Spliceosome mutations are found in over half of all MDS patients and are likely founder mutations. The spliceosome mutations are highly specific to MDS and closely related conditions and, to some extent, appear to define distinct clinical phenotypes in MDS. The high frequency of mutations in different components of the RNA splicing machinery in MDS suggests that abnormal RNA splicing is the common consequence of these mutations. The identification of the downstream targets of the spliceosome mutations is an active area of research. Emerging data from the study of the MDS transcriptome suggests that spliceosomal mutations have effects on specific genes, including some previously shown to play a role in MDS pathogenesis. The effects of the spliceosomal mutations on RNA splicing and cell growth have been evaluated only in a limited context to date, however, and the determination of the impact of these mutations in primary human hematopoietic cells is essential in order to elucidate fully the molecular mechanism by which they contribute to MDS pathogenesis.