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1.
Cell ; 175(4): 1014-1030.e19, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30343900

RESUMO

Although current immune-checkpoint therapy (ICT) mainly targets lymphoid cells, it is associated with a broader remodeling of the tumor micro-environment. Here, using complementary forms of high-dimensional profiling, we define differences across all hematopoietic cells from syngeneic mouse tumors during unrestrained tumor growth or effective ICT. Unbiased assessment of gene expression of tumor-infiltrating cells by single-cell RNA sequencing (scRNAseq) and longitudinal assessment of cellular protein expression by mass cytometry (CyTOF) revealed significant remodeling of both the lymphoid and myeloid intratumoral compartments. Surprisingly, we observed multiple subpopulations of monocytes/macrophages, distinguishable by the markers CD206, CX3CR1, CD1d, and iNOS, that change over time during ICT in a manner partially dependent on IFNγ. Our data support the hypothesis that this macrophage polarization/activation results from effects on circulatory monocytes and early macrophages entering tumors, rather than on pre-polarized mature intratumoral macrophages.


Assuntos
Linfócitos/imunologia , Células Mieloides/imunologia , Neoplasias/imunologia , Análise de Célula Única , Transcriptoma , Animais , Linhagem Celular Tumoral , Citometria de Fluxo , Imunoterapia/métodos , Interferon gama/imunologia , Ativação de Macrófagos , Masculino , Espectrometria de Massas , Camundongos , Células Precursoras de Monócitos e Macrófagos/imunologia , Neoplasias/terapia
2.
Cell ; 168(5): 801-816.e13, 2017 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-28215704

RESUMO

DNMT3A mutations occur in ∼25% of acute myeloid leukemia (AML) patients. The most common mutation, DNMT3AR882H, has dominant negative activity that reduces DNA methylation activity by ∼80% in vitro. To understand the contribution of DNMT3A-dependent methylation to leukemogenesis, we performed whole-genome bisulfite sequencing of primary leukemic and non-leukemic cells in patients with or without DNMT3AR882 mutations. Non-leukemic hematopoietic cells with DNMT3AR882H displayed focal methylation loss, suggesting that hypomethylation antedates AML. Although virtually all AMLs with wild-type DNMT3A displayed CpG island hypermethylation, this change was not associated with gene silencing and was essentially absent in AMLs with DNMT3AR882 mutations. Primary hematopoietic stem cells expanded with cytokines were hypermethylated in a DNMT3A-dependent manner, suggesting that hypermethylation may be a response to, rather than a cause of, cellular proliferation. Our findings suggest that hypomethylation is an initiating phenotype in AMLs with DNMT3AR882, while DNMT3A-dependent CpG island hypermethylation is a consequence of AML progression.


Assuntos
Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Leucemia Mieloide Aguda/genética , Células da Medula Óssea/patologia , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Epigênese Genética , Humanos , Leucemia Mieloide Aguda/patologia , Mutação , Análise de Sequência de DNA
3.
Nat Immunol ; 20(8): 980-991, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31209406

RESUMO

Innate lymphoid cells (ILCs) are tissue-resident lymphocytes categorized on the basis of their core regulatory programs and the expression of signature cytokines. Human ILC3s that produce the cytokine interleukin-22 convert into ILC1-like cells that produce interferon-γ in vitro, but whether this conversion occurs in vivo remains unclear. In the present study we found that ILC3s and ILC1s in human tonsils represented the ends of a spectrum that included additional discrete subsets. RNA velocity analysis identified an intermediate ILC3-ILC1 cluster, which had strong directionality toward ILC1s. In humanized mice, the acquisition of ILC1 features by ILC3s showed tissue dependency. Chromatin studies indicated that the transcription factors Aiolos and T-bet cooperated to repress regulatory elements active in ILC3s. A transitional ILC3-ILC1 population was also detected in the human intestine. We conclude that ILC3s undergo conversion into ILC1-like cells in human tissues in vivo, and that tissue factors and Aiolos were required for this process.


Assuntos
Imunidade Inata/imunologia , Interferon gama/metabolismo , Interleucinas/metabolismo , Mucosa Intestinal/imunologia , Linfócitos/imunologia , Tonsila Palatina/imunologia , Animais , Diferenciação Celular/imunologia , Células Cultivadas , Criança , Pré-Escolar , Humanos , Fator de Transcrição Ikaros/metabolismo , Mucosa Intestinal/citologia , Linfócitos/classificação , Linfócitos/citologia , Camundongos , Proteínas com Domínio T/metabolismo , Interleucina 22
5.
Cell ; 159(4): 800-13, 2014 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-25417157

RESUMO

We sequenced the MSY (male-specific region of the Y chromosome) of the C57BL/6J strain of the laboratory mouse Mus musculus. In contrast to theories that Y chromosomes are heterochromatic and gene poor, the mouse MSY is 99.9% euchromatic and contains about 700 protein-coding genes. Only 2% of the MSY derives from the ancestral autosomes that gave rise to the mammalian sex chromosomes. Instead, all but 45 of the MSY's genes belong to three acquired, massively amplified gene families that have no homologs on primate MSYs but do have acquired, amplified homologs on the mouse X chromosome. The complete mouse MSY sequence brings to light dramatic forces in sex chromosome evolution: lineage-specific convergent acquisition and amplification of X-Y gene families, possibly fueled by antagonism between acquired X-Y homologs. The mouse MSY sequence presents opportunities for experimental studies of a sex-specific chromosome in its entirety, in a genetically tractable model organism.


Assuntos
Evolução Biológica , Cromossomos de Mamíferos , Camundongos Endogâmicos C57BL/genética , Análise de Sequência de DNA , Cromossomo Y , Animais , Centrômero , Cromossomos Artificiais Bacterianos/genética , Feminino , Humanos , Masculino , Filogenia , Primatas/genética , Cromossomo X
6.
Nature ; 617(7960): 312-324, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37165242

RESUMO

Here the Human Pangenome Reference Consortium presents a first draft of the human pangenome reference. The pangenome contains 47 phased, diploid assemblies from a cohort of genetically diverse individuals1. These assemblies cover more than 99% of the expected sequence in each genome and are more than 99% accurate at the structural and base pair levels. Based on alignments of the assemblies, we generate a draft pangenome that captures known variants and haplotypes and reveals new alleles at structurally complex loci. We also add 119 million base pairs of euchromatic polymorphic sequences and 1,115 gene duplications relative to the existing reference GRCh38. Roughly 90 million of the additional base pairs are derived from structural variation. Using our draft pangenome to analyse short-read data reduced small variant discovery errors by 34% and increased the number of structural variants detected per haplotype by 104% compared with GRCh38-based workflows, which enabled the typing of the vast majority of structural variant alleles per sample.


Assuntos
Genoma Humano , Genômica , Humanos , Diploide , Genoma Humano/genética , Haplótipos/genética , Análise de Sequência de DNA , Genômica/normas , Padrões de Referência , Estudos de Coortes , Alelos , Variação Genética
8.
Nature ; 604(7906): 437-446, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35444317

RESUMO

The human reference genome is the most widely used resource in human genetics and is due for a major update. Its current structure is a linear composite of merged haplotypes from more than 20 people, with a single individual comprising most of the sequence. It contains biases and errors within a framework that does not represent global human genomic variation. A high-quality reference with global representation of common variants, including single-nucleotide variants, structural variants and functional elements, is needed. The Human Pangenome Reference Consortium aims to create a more sophisticated and complete human reference genome with a graph-based, telomere-to-telomere representation of global genomic diversity. Here we leverage innovations in technology, study design and global partnerships with the goal of constructing the highest-possible quality human pangenome reference. Our goal is to improve data representation and streamline analyses to enable routine assembly of complete diploid genomes. With attention to ethical frameworks, the human pangenome reference will contain a more accurate and diverse representation of global genomic variation, improve gene-disease association studies across populations, expand the scope of genomics research to the most repetitive and polymorphic regions of the genome, and serve as the ultimate genetic resource for future biomedical research and precision medicine.


Assuntos
Genoma Humano , Genômica , Genoma Humano/genética , Haplótipos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Análise de Sequência de DNA
9.
Nature ; 611(7936): 519-531, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36261518

RESUMO

The current human reference genome, GRCh38, represents over 20 years of effort to generate a high-quality assembly, which has benefitted society1,2. However, it still has many gaps and errors, and does not represent a biological genome as it is a blend of multiple individuals3,4. Recently, a high-quality telomere-to-telomere reference, CHM13, was generated with the latest long-read technologies, but it was derived from a hydatidiform mole cell line with a nearly homozygous genome5. To address these limitations, the Human Pangenome Reference Consortium formed with the goal of creating high-quality, cost-effective, diploid genome assemblies for a pangenome reference that represents human genetic diversity6. Here, in our first scientific report, we determined which combination of current genome sequencing and assembly approaches yield the most complete and accurate diploid genome assembly with minimal manual curation. Approaches that used highly accurate long reads and parent-child data with graph-based haplotype phasing during assembly outperformed those that did not. Developing a combination of the top-performing methods, we generated our first high-quality diploid reference assembly, containing only approximately four gaps per chromosome on average, with most chromosomes within ±1% of the length of CHM13. Nearly 48% of protein-coding genes have non-synonymous amino acid changes between haplotypes, and centromeric regions showed the highest diversity. Our findings serve as a foundation for assembling near-complete diploid human genomes at scale for a pangenome reference to capture global genetic variation from single nucleotides to structural rearrangements.


Assuntos
Mapeamento Cromossômico , Diploide , Genoma Humano , Genômica , Humanos , Mapeamento Cromossômico/normas , Genoma Humano/genética , Haplótipos/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/normas , Padrões de Referência , Genômica/métodos , Genômica/normas , Cromossomos Humanos/genética , Variação Genética/genética
10.
Cell ; 150(6): 1121-34, 2012 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-22980976

RESUMO

We report the results of whole-genome and transcriptome sequencing of tumor and adjacent normal tissue samples from 17 patients with non-small cell lung carcinoma (NSCLC). We identified 3,726 point mutations and more than 90 indels in the coding sequence, with an average mutation frequency more than 10-fold higher in smokers than in never-smokers. Novel alterations in genes involved in chromatin modification and DNA repair pathways were identified, along with DACH1, CFTR, RELN, ABCB5, and HGF. Deep digital sequencing revealed diverse clonality patterns in both never-smokers and smokers. All validated EFGR and KRAS mutations were present in the founder clones, suggesting possible roles in cancer initiation. Analysis revealed 14 fusions, including ROS1 and ALK, as well as novel metabolic enzymes. Cell-cycle and JAK-STAT pathways are significantly altered in lung cancer, along with perturbations in 54 genes that are potentially targetable with currently available drugs.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Fumar/genética , Fumar/patologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Aberrações Cromossômicas , Feminino , Perfilação da Expressão Gênica , Estudo de Associação Genômica Ampla , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação INDEL , Neoplasias Pulmonares/terapia , Masculino , Terapia de Alvo Molecular , Mutação Puntual , Proteína Reelina
11.
Cell ; 150(2): 264-78, 2012 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-22817890

RESUMO

Most mutations in cancer genomes are thought to be acquired after the initiating event, which may cause genomic instability and drive clonal evolution. However, for acute myeloid leukemia (AML), normal karyotypes are common, and genomic instability is unusual. To better understand clonal evolution in AML, we sequenced the genomes of M3-AML samples with a known initiating event (PML-RARA) versus the genomes of normal karyotype M1-AML samples and the exomes of hematopoietic stem/progenitor cells (HSPCs) from healthy people. Collectively, the data suggest that most of the mutations found in AML genomes are actually random events that occurred in HSPCs before they acquired the initiating mutation; the mutational history of that cell is "captured" as the clone expands. In many cases, only one or two additional, cooperating mutations are needed to generate the malignant founding clone. Cells from the founding clone can acquire additional cooperating mutations, yielding subclones that can contribute to disease progression and/or relapse.


Assuntos
Evolução Clonal , Leucemia Mieloide Aguda/genética , Mutação , Adulto , Idoso , Análise Mutacional de DNA , Progressão da Doença , Feminino , Estudo de Associação Genômica Ampla , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/fisiopatologia , Masculino , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/genética , Recidiva , Pele/metabolismo , Adulto Jovem
12.
Nature ; 585(7823): 79-84, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32663838

RESUMO

After two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no single chromosome has been finished end to end, and hundreds of unresolved gaps persist1,2. Here we present a human genome assembly that surpasses the continuity of GRCh382, along with a gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome3, we reconstructed the centromeric satellite DNA array (approximately 3.1 Mb) and closed the 29 remaining gaps in the current reference, including new sequences from the human pseudoautosomal regions and from cancer-testis ampliconic gene families (CT-X and GAGE). These sequences will be integrated into future human reference genome releases. In addition, the complete chromosome X, combined with the ultra-long nanopore data, allowed us to map methylation patterns across complex tandem repeats and satellite arrays. Our results demonstrate that finishing the entire human genome is now within reach, and the data presented here will facilitate ongoing efforts to complete the other human chromosomes.


Assuntos
Cromossomos Humanos X/genética , Genoma Humano/genética , Telômero/genética , Centrômero/genética , Ilhas de CpG/genética , Metilação de DNA , DNA Satélite/genética , Feminino , Humanos , Mola Hidatiforme/genética , Masculino , Gravidez , Reprodutibilidade dos Testes , Testículo/metabolismo
13.
Cell ; 143(5): 837-47, 2010 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-21111241

RESUMO

Understanding the prevailing mutational mechanisms responsible for human genome structural variation requires uniformity in the discovery of allelic variants and precision in terms of breakpoint delineation. We develop a resource based on capillary end sequencing of 13.8 million fosmid clones from 17 human genomes and characterize the complete sequence of 1054 large structural variants corresponding to 589 deletions, 384 insertions, and 81 inversions. We analyze the 2081 breakpoint junctions and infer potential mechanism of origin. Three mechanisms account for the bulk of germline structural variation: microhomology-mediated processes involving short (2-20 bp) stretches of sequence (28%), nonallelic homologous recombination (22%), and L1 retrotransposition (19%). The high quality and long-range continuity of the sequence reveals more complex mutational mechanisms, including repeat-mediated inversions and gene conversion, that are most often missed by other methods, such as comparative genomic hybridization, single nucleotide polymorphism microarrays, and next-generation sequencing.


Assuntos
Genoma Humano , Variação Estrutural do Genoma , Mutação , Sequência de Bases , Conversão Gênica , Humanos , Dados de Sequência Molecular , Análise de Sequência de DNA
14.
Nature ; 572(7769): 323-328, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31367044

RESUMO

Exome-sequencing studies have generally been underpowered to identify deleterious alleles with a large effect on complex traits as such alleles are mostly rare. Because the population of northern and eastern Finland has expanded considerably and in isolation following a series of bottlenecks, individuals of these populations have numerous deleterious alleles at a relatively high frequency. Here, using exome sequencing of nearly 20,000 individuals from these regions, we investigate the role of rare coding variants in clinically relevant quantitative cardiometabolic traits. Exome-wide association studies for 64 quantitative traits identified 26 newly associated deleterious alleles. Of these 26 alleles, 19 are either unique to or more than 20 times more frequent in Finnish individuals than in other Europeans and show geographical clustering comparable to Mendelian disease mutations that are characteristic of the Finnish population. We estimate that sequencing studies of populations without this unique history would require hundreds of thousands to millions of participants to achieve comparable association power.


Assuntos
Sequenciamento do Exoma , Estudos de Associação Genética/métodos , Predisposição Genética para Doença/genética , Variação Genética/genética , Locos de Características Quantitativas/genética , Alelos , HDL-Colesterol/genética , Análise por Conglomerados , Determinação de Ponto Final , Finlândia , Mapeamento Geográfico , Humanos , Herança Multifatorial/genética , Reprodutibilidade dos Testes
16.
N Engl J Med ; 384(10): 924-935, 2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33704937

RESUMO

BACKGROUND: Genomic analysis is essential for risk stratification in patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS). Whole-genome sequencing is a potential replacement for conventional cytogenetic and sequencing approaches, but its accuracy, feasibility, and clinical utility have not been demonstrated. METHODS: We used a streamlined whole-genome sequencing approach to obtain genomic profiles for 263 patients with myeloid cancers, including 235 patients who had undergone successful cytogenetic analysis. We adapted sample preparation, sequencing, and analysis to detect mutations for risk stratification using existing European Leukemia Network (ELN) guidelines and to minimize turnaround time. We analyzed the performance of whole-genome sequencing by comparing our results with findings from cytogenetic analysis and targeted sequencing. RESULTS: Whole-genome sequencing detected all 40 recurrent translocations and 91 copy-number alterations that had been identified by cytogenetic analysis. In addition, we identified new clinically reportable genomic events in 40 of 235 patients (17.0%). Prospective sequencing of samples obtained from 117 consecutive patients was performed in a median of 5 days and provided new genetic information in 29 patients (24.8%), which changed the risk category for 19 patients (16.2%). Standard AML risk groups, as defined by sequencing results instead of cytogenetic analysis, correlated with clinical outcomes. Whole-genome sequencing was also used to stratify patients who had inconclusive results by cytogenetic analysis into risk groups in which clinical outcomes were measurably different. CONCLUSIONS: In our study, we found that whole-genome sequencing provided rapid and accurate genomic profiling in patients with AML or MDS. Such sequencing also provided a greater diagnostic yield than conventional cytogenetic analysis and more efficient risk stratification on the basis of standard risk categories. (Funded by the Siteman Cancer Research Fund and others.).


Assuntos
Análise Citogenética , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicas/genética , Sequenciamento Completo do Genoma , Estudos de Viabilidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Análise de Sobrevida , Sequenciamento Completo do Genoma/métodos
17.
Blood ; 140(14): 1607-1620, 2022 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-35675516

RESUMO

Hematopoietic stem/progenitor cells (HSPCs) reside in localized microenvironments, or niches, in the bone marrow that provide key signals regulating their activity. A fundamental property of hematopoiesis is the ability to respond to environmental cues such as inflammation. How these cues are transmitted to HSPCs within hematopoietic niches is not well established. Here, we show that perivascular bone marrow dendritic cells (DCs) express a high basal level of Toll-like receptor-1 (TLR1) and TLR2. Systemic treatment with a TLR1/2 agonist induces HSPC expansion and mobilization. It also induces marked alterations in the bone marrow microenvironment, including a decrease in osteoblast activity and sinusoidal endothelial cell numbers. TLR1/2 agonist treatment of mice in which Myd88 is deleted specifically in DCs using Zbtb46-Cre show that the TLR1/2-induced expansion of multipotent HPSCs, but not HSPC mobilization or alterations in the bone marrow microenvironment, is dependent on TLR1/2 signaling in DCs. Interleukin-1ß (IL-1ß) is constitutively expressed in both murine and human DCs and is further induced after TLR1/2 stimulation. Systemic TLR1/2 agonist treatment of Il1r1-/- mice show that TLR1/2-induced HSPC expansion is dependent on IL-1ß signaling. Single-cell RNA-sequencing of low-risk myelodysplastic syndrome bone marrow revealed that IL1B and TLR1 expression is increased in DCs. Collectively, these data suggest a model in which TLR1/2 stimulation of DCs induces secretion of IL-1ß and other inflammatory cytokines into the perivascular niche, which in turn, regulates multipotent HSPCs. Increased DC TLR1/2 signaling may contribute to altered HSPC function in myelodysplastic syndrome by increasing local IL-1ß expression.


Assuntos
Células da Medula Óssea , Células Dendríticas , Células-Tronco Hematopoéticas , Interleucina-1beta , Síndromes Mielodisplásicas , Animais , Medula Óssea/metabolismo , Células da Medula Óssea/citologia , Citocinas/metabolismo , Células Dendríticas/citologia , Células-Tronco Hematopoéticas/citologia , Humanos , Interleucina-1beta/metabolismo , Camundongos , Síndromes Mielodisplásicas/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , RNA/metabolismo , Receptor 1 Toll-Like/metabolismo , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/metabolismo
18.
Proc Natl Acad Sci U S A ; 118(49)2021 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-34845035

RESUMO

Acute myeloid leukemia (AML) patients rarely have long first remissions (LFRs; >5 y) after standard-of-care chemotherapy, unless classified as favorable risk at presentation. Identification of the mechanisms responsible for long vs. more typical, standard remissions may help to define prognostic determinants for chemotherapy responses. Using exome sequencing, RNA-sequencing, and functional immunologic studies, we characterized 28 normal karyotype (NK)-AML patients with >5 y first remissions after chemotherapy (LFRs) and compared them to a well-matched group of 31 NK-AML patients who relapsed within 2 y (standard first remissions [SFRs]). Our combined analyses indicated that genetic-risk profiling at presentation (as defined by European LeukemiaNet [ELN] 2017 criteria) was not sufficient to explain the outcomes of many SFR cases. Single-cell RNA-sequencing studies of 15 AML samples showed that SFR AML cells differentially expressed many genes associated with immune suppression. The bone marrow of SFR cases had significantly fewer CD4+ Th1 cells; these T cells expressed an exhaustion signature and were resistant to activation by T cell receptor stimulation in the presence of autologous AML cells. T cell activation could be restored by removing the AML cells or blocking the inhibitory major histocompatibility complex class II receptor, LAG3. Most LFR cases did not display these features, suggesting that their AML cells were not as immunosuppressive. These findings were confirmed and extended in an independent set of 50 AML cases representing all ELN 2017 risk groups. AML cell-mediated suppression of CD4+ T cell activation at presentation is strongly associated with unfavorable outcomes in AML patients treated with standard chemotherapy.


Assuntos
Tolerância Imunológica/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Adulto , Linfócitos T CD4-Positivos/imunologia , Feminino , Humanos , Tolerância Imunológica/imunologia , Cariótipo , Leucemia Mieloide Aguda/terapia , Masculino , Pessoa de Meia-Idade , Prognóstico , Recidiva , Indução de Remissão , Fatores de Risco , Análise de Sequência de RNA/métodos , Células Th1/imunologia , Transcriptoma/genética , Resultado do Tratamento
20.
Proc Natl Acad Sci U S A ; 117(6): 3123-3134, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-31996479

RESUMO

Mutations in the DNA methyltransferase 3A (DNMT3A) gene are the most common cause of age-related clonal hematopoiesis (ARCH) in older individuals, and are among the most common initiating events for acute myeloid leukemia (AML). The most frequent DNMT3A mutation in AML patients (R882H) encodes a dominant-negative protein that reduces methyltransferase activity by ∼80% in cells with heterozygous mutations, causing a focal, canonical DNA hypomethylation phenotype; this phenotype is partially recapitulated in murine Dnmt3a-/- bone marrow cells. To determine whether the hypomethylation phenotype of Dnmt3a-/- hematopoietic cells is reversible, we developed an inducible transgene to restore expression of DNMT3A in transplanted bone marrow cells from Dnmt3a-/- mice. Partial remethylation was detected within 1 wk, but near-complete remethylation required 6 mo. Remethylation was accurate, dynamic, and highly ordered, suggesting that differentially methylated regions have unique properties that may be relevant for their functions. Importantly, 22 wk of DNMT3A addback partially corrected dysregulated gene expression, and mitigated the expansion of myeloid cells. These data show that restoring DNMT3A expression can alter the epigenetic "state" created by loss of Dnmt3a activity; this genetic proof-of-concept experiment suggests that this approach could be relevant for patients with ARCH or AML caused by loss-of-function DNMT3A mutations.


Assuntos
Células da Medula Óssea/metabolismo , DNA (Citosina-5-)-Metiltransferases , Metilação de DNA/genética , Expressão Gênica/genética , Animais , Transplante de Medula Óssea , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Hematopoese/genética , Humanos , Camundongos , Camundongos Transgênicos , Mutação/genética
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