RESUMO
Somatic mutations in DNMT3A, which encodes a de novo DNA methyltransferase, are found in â¼30% of normal karyotype acute myeloid leukemia (AML) cases. Most mutations are heterozygous and alter R882 within the catalytic domain (most commonly R882H), suggesting the possibility of dominant-negative consequences. The methyltransferase activity of R882H DNMT3A is reduced by â¼80% compared with the WT enzyme. In vitro mixing of WT and R882H DNMT3A does not affect the WT activity, but coexpression of the two proteins in cells profoundly inhibits the WT enzyme by disrupting its ability to homotetramerize. AML cells with the R882H mutation have severely reduced de novo methyltransferase activity and focal hypomethylation at specific CpGs throughout AML cell genomes.
Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Mutação , Alelos , Sequência de Aminoácidos , DNA (Citosina-5-)-Metiltransferases/química , Metilação de DNA , DNA Metiltransferase 3A , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Prognóstico , Conformação ProteicaRESUMO
To assess the genetic consequences of induced pluripotent stem cell (iPSC) reprogramming, we sequenced the genomes of ten murine iPSC clones derived from three independent reprogramming experiments, and compared them to their parental cell genomes. We detected hundreds of single nucleotide variants (SNVs) in every clone, with an average of 11 in coding regions. In two experiments, all SNVs were unique for each clone and did not cluster in pathways, but in the third, all four iPSC clones contained 157 shared genetic variants, which could also be detected in rare cells (<1 in 500) within the parental MEF pool. These data suggest that most of the genetic variation in iPSC clones is not caused by reprogramming per se, but is rather a consequence of cloning individual cells, which "captures" their mutational history. These findings have implications for the development and therapeutic use of cells that are reprogrammed by any method.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação , Animais , Diferenciação Celular , Proliferação de Células , Células Clonais , DNA/análise , DNA/genética , Instabilidade Genômica , Regeneração Tecidual Guiada/métodos , Regeneração Tecidual Guiada/normas , Células-Tronco Pluripotentes Induzidas/patologia , Camundongos , Polimorfismo de Nucleotídeo Único , Medicina RegenerativaRESUMO
Most patients with acute myeloid leukaemia (AML) die from progressive disease after relapse, which is associated with clonal evolution at the cytogenetic level. To determine the mutational spectrum associated with relapse, we sequenced the primary tumour and relapse genomes from eight AML patients, and validated hundreds of somatic mutations using deep sequencing; this allowed us to define clonality and clonal evolution patterns precisely at relapse. In addition to discovering novel, recurrently mutated genes (for example, WAC, SMC3, DIS3, DDX41 and DAXX) in AML, we also found two major clonal evolution patterns during AML relapse: (1) the founding clone in the primary tumour gained mutations and evolved into the relapse clone, or (2) a subclone of the founding clone survived initial therapy, gained additional mutations and expanded at relapse. In all cases, chemotherapy failed to eradicate the founding clone. The comparison of relapse-specific versus primary tumour mutations in all eight cases revealed an increase in transversions, probably due to DNA damage caused by cytotoxic chemotherapy. These data demonstrate that AML relapse is associated with the addition of new mutations and clonal evolution, which is shaped, in part, by the chemotherapy that the patients receive to establish and maintain remissions.
Assuntos
Evolução Clonal/genética , Genoma Humano/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Células Clonais/patologia , Dano ao DNA/efeitos dos fármacos , Análise Mutacional de DNA , Genes Neoplásicos/genética , Genoma Humano/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Mutagênese/efeitos dos fármacos , Mutagênese/genética , Recidiva , Reprodutibilidade dos TestesRESUMO
Previous research efforts have developed and validated various scales potentially useful in evaluating service learning outcomes. The developmental efforts reported for the four scales examined in this study did not include the test-retest reliabilities that would provide assurance to service learning researchers of the long-term stability and therefore usefulness of these measures. Summary estimates of 13-wk. test-retest reliabilities for the scales Civic Participation, Self-efficacy Toward Service, Attitude Toward Helping Others, and College Education's Role in Addressing Social Issues provide service learning researchers with evidence of stability of the scales over the typical duration of service learning courses.
