RESUMO
Somatic mutation in neurons is linked to neurologic disease and implicated in cell-type diversification. However, the origin, extent, and patterns of genomic mutation in neurons remain unknown. We established a nuclear transfer method to clonally amplify the genomes of neurons from adult mice for whole-genome sequencing. Comprehensive mutation detection and independent validation revealed that individual neurons harbor â¼100 unique mutations from all classes but lack recurrent rearrangements. Most neurons contain at least one gene-disrupting mutation and rare (0-2) mobile element insertions. The frequency and gene bias of neuronal mutations differ from other lineages, potentially due to novel mechanisms governing postmitotic mutation. Fertile mice were cloned from several neurons, establishing the compatibility of mutated adult neuronal genomes with reprogramming to pluripotency and development.
Assuntos
Clonagem Molecular , Mutação/genética , Neurônios/fisiologia , Análise de Sequência de DNA , Fatores Etários , Animais , Animais Recém-Nascidos , Proteínas Relacionadas a Caderinas , Caderinas/genética , Caderinas/metabolismo , Divisão Celular/genética , Elementos de DNA Transponíveis/genética , Embrião de Mamíferos , Feminino , Humanos , Antígeno Ki-67/metabolismo , Camundongos , Camundongos Transgênicos , Repetições de Microssatélites/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Técnicas de Transferência Nuclear , Bulbo Olfatório/citologia , Bulbo Olfatório/embriologia , Bulbo Olfatório/crescimento & desenvolvimento , Oócitos/fisiologiaRESUMO
We used single-cell genomic approaches to map DNA copy number variation (CNV) in neurons obtained from human induced pluripotent stem cell (hiPSC) lines and postmortem human brains. We identified aneuploid neurons, as well as numerous subchromosomal CNVs in euploid neurons. Neurotypic hiPSC-derived neurons had larger CNVs than fibroblasts, and several large deletions were found in hiPSC-derived neurons but not in matched neural progenitor cells. Single-cell sequencing of endogenous human frontal cortex neurons revealed that 13 to 41% of neurons have at least one megabase-scale de novo CNV, that deletions are twice as common as duplications, and that a subset of neurons have highly aberrant genomes marked by multiple alterations. Our results show that mosaic CNV is abundant in human neurons.
Assuntos
Variações do Número de Cópias de DNA , Lobo Frontal/citologia , Mosaicismo , Células-Tronco Neurais/citologia , Neurônios/citologia , Aneuploidia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Masculino , Neurogênese , Análise de Sequência de DNA , Deleção de Sequência , Análise de Célula ÚnicaRESUMO
The biomedical utility of induced pluripotent stem cells (iPSCs) will be diminished if most iPSC lines harbor deleterious genetic mutations. Recent microarray studies have shown that human iPSCs carry elevated levels of DNA copy number variation compared with those in embryonic stem cells, suggesting that these and other classes of genomic structural variation (SV), including inversions, smaller duplications and deletions, complex rearrangements, and retroelement transpositions, may frequently arise as a consequence of reprogramming. Here we employ whole-genome paired-end DNA sequencing and sensitive mapping algorithms to identify all classes of SV in three fully pluripotent mouse iPSC lines. Despite the improved scope and resolution of this study, we find few spontaneous mutations per line (one or two) and no evidence for endogenous retroelement transposition. These results show that genome stability can persist throughout reprogramming, and argue that it is possible to generate iPSCs lacking gene-disrupting mutations using current reprogramming methods.
Assuntos
Reprogramação Celular/genética , Rearranjo Gênico/genética , Genoma/genética , Instabilidade Genômica/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Retroelementos/genética , Análise de Sequência de DNA/métodos , Animais , Sequência de Bases , Linhagem da Célula/genética , Quimera/genética , Variações do Número de Cópias de DNA/genética , Reações Falso-Negativas , Inativação Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Dados de Sequência Molecular , Mutagênese Insercional/genética , Especificidade de Órgãos/genéticaRESUMO
Many bacteria are able to efficiently bind and take up double-stranded DNA fragments, and the resulting natural transformation shapes bacterial genomes, transmits antibiotic resistance, and allows escape from immune surveillance. The genomes of many competent pathogens show evidence of extensive historical recombination between lineages, but the actual recombination events have not been well characterized. We used DNA from a clinical isolate of Haemophilus influenzae to transform competent cells of a laboratory strain. To identify which of the ~40,000 polymorphic differences had recombined into the genomes of four transformed clones, their genomes and their donor and recipient parents were deep sequenced to high coverage. Each clone was found to contain ~1000 donor polymorphisms in 3-6 contiguous runs (8.1±4.5 kb in length) that collectively comprised ~1-3% of each transformed chromosome. Seven donor-specific insertions and deletions were also acquired as parts of larger donor segments, but the presence of other structural variation flanking 12 of 32 recombination breakpoints suggested that these often disrupt the progress of recombination events. This is the first genome-wide analysis of chromosomes directly transformed with DNA from a divergent genotype, connecting experimental studies of transformation with the high levels of natural genetic variation found in isolates of the same species.
