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1.
Nucleic Acids Res ; 50(15): 8626-8642, 2022 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-35947695

RESUMO

Mitochondrial DNA (mtDNA) is prone to mutation in aging and over evolutionary time, yet the processes that regulate the accumulation of de novo mtDNA mutations and modulate mtDNA heteroplasmy are not fully elucidated. Mitochondria lack certain DNA repair processes, which could contribute to polymerase error-induced mutations and increase susceptibility to chemical-induced mtDNA mutagenesis. We conducted error-corrected, ultra-sensitive Duplex Sequencing to investigate the effects of two known nuclear genome mutagens, cadmium and Aflatoxin B1, on germline mtDNA mutagenesis in Caenorhabditis elegans. Detection of thousands of mtDNA mutations revealed pervasive heteroplasmy in C. elegans and that mtDNA mutagenesis is dominated by C:G → A:T mutations generally attributed to oxidative damage. However, there was no effect of either exposure on mtDNA mutation frequency, spectrum, or trinucleotide context signature despite a significant increase in nuclear mutation rate after aflatoxin B1 exposure. Mitophagy-deficient mutants pink-1 and dct-1 accumulated significantly higher levels of mtDNA damage compared to wild-type C. elegans after exposures. However, there were only small differences in mtDNA mutation frequency, spectrum, or trinucleotide context signature compared to wild-type after 3050 generations, across all treatments. These findings suggest mitochondria harbor additional previously uncharacterized mechanisms that regulate mtDNA mutational processes across generations.


Assuntos
Caenorhabditis elegans , DNA Mitocondrial , Animais , DNA Mitocondrial/genética , Caenorhabditis elegans/genética , Cádmio/toxicidade , Aflatoxina B1/toxicidade , Acúmulo de Mutações , Mitocôndrias/genética , Mutação , Células Germinativas
2.
Nucleic Acids Res ; 49(19): 11103-11118, 2021 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-34614167

RESUMO

Mutations in mitochondrial DNA (mtDNA) cause maternally inherited diseases, while somatic mutations are linked to common diseases of aging. Although mtDNA mutations impact health, the processes that give rise to them are under considerable debate. To investigate the mechanism by which de novo mutations arise, we analyzed the distribution of naturally occurring somatic mutations across the mouse and human mtDNA obtained by Duplex Sequencing. We observe distinct mutational gradients in G→A and T→C transitions delimited by the light-strand origin and the mitochondrial Control Region (mCR). The gradient increases unequally across the mtDNA with age and is lost in the absence of DNA polymerase γ proofreading activity. In addition, high-resolution analysis of the mCR shows that important regulatory elements exhibit considerable variability in mutation frequency, consistent with them being mutational 'hot-spots' or 'cold-spots'. Collectively, these patterns support genome replication via a deamination prone asymmetric strand-displacement mechanism as the fundamental driver of mutagenesis in mammalian DNA. Moreover, the distribution of mtDNA single nucleotide polymorphisms in humans and the distribution of bases in the mtDNA across vertebrate species mirror this gradient, indicating that replication-linked mutations are likely the primary source of inherited polymorphisms that, over evolutionary timescales, influences genome composition during speciation.


Assuntos
Envelhecimento/genética , Replicação do DNA , DNA Mitocondrial/genética , Genoma Mitocondrial , Mutação em Linhagem Germinativa , Mitocôndrias/genética , Acúmulo de Mutações , Envelhecimento/metabolismo , Animais , Mapeamento Cromossômico , DNA Polimerase gama/deficiência , DNA Polimerase gama/genética , DNA Mitocondrial/metabolismo , Especiação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Taxa de Mutação , Polimorfismo de Nucleotídeo Único
3.
Genome Res ; 28(10): 1589-1599, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30232196

RESUMO

Next-generation sequencing methods suffer from low recovery, uneven coverage, and false mutations. DNA fragmentation by sonication is a major contributor to these problems because it produces randomly sized fragments, PCR amplification bias, and end artifacts. In addition, oligonucleotide-based hybridization capture, a common target enrichment method, has limited efficiency for small genomic regions, contributing to low recovery. This becomes a critical problem in clinical applications, which value cost-effective approaches focused on the sequencing of small gene panels. To address these issues, we developed a targeted genome fragmentation approach based on CRISPR/Cas9 digestion that produces DNA fragments of similar length. These fragments can be enriched by a simple size selection, resulting in targeted enrichment of up to approximately 49,000-fold. Additionally, homogenous length fragments significantly reduce PCR amplification bias and maximize read usability. We combined this novel target enrichment approach with Duplex Sequencing, which uses double-strand molecular tagging to correct for sequencing errors. The approach, termed CRISPR-DS, enables efficient target enrichment of small genomic regions, even coverage, ultra-accurate sequencing, and reduced DNA input. As proof of principle, we applied CRISPR-DS to the sequencing of the exonic regions of TP53 and performed side-by-side comparisons with standard Duplex Sequencing. CRISPR-DS detected previously reported pathogenic TP53 mutations present as low as 0.1% in peritoneal fluid of women with ovarian cancer, while using 10- to 100-fold less DNA than standard Duplex Sequencing. Whether used as standalone enrichment or coupled with high-accuracy sequencing methods, CRISPR-based fragmentation offers a simple solution for fast and efficient small target enrichment.


Assuntos
Sistemas CRISPR-Cas , Neoplasias Ovarianas/genética , Análise de Sequência de DNA/métodos , Proteína Supressora de Tumor p53/genética , DNA/genética , Fragmentação do DNA , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos
4.
Ann Neurol ; 80(2): 301-6, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27315116

RESUMO

Mitochondrial dysfunction and oxidative damage are commonly associated with early stage Alzheimer disease (AD). The accumulation of somatic mutations in mitochondrial DNA (mtDNA) has been hypothesized to be a driver of these phenotypes, but the detection of increased mutation loads has been difficult due to a lack of sensitive methods. We used an ultrasensitive next generation sequencing technique to measure the mutation load of the entire mitochondrial genome. Here, we report a significant increase in the mtDNA mutation frequency in the hippocampus of early stage AD, with the cause of these mutations being consistent with replication errors and not oxidative damage. Ann Neurol 2016;80:301-306.


Assuntos
Doença de Alzheimer/genética , DNA Mitocondrial/genética , Mutação , Estresse Oxidativo , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Genoma Mitocondrial/genética , Sequenciamento de Nucleotídeos em Larga Escala , Hipocampo/metabolismo , Humanos , Masculino , Lobo Parietal/metabolismo
5.
Acta Neuropathol ; 128(5): 639-650, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24997849

RESUMO

Extracellular α-synuclein is important in the pathogenesis of Parkinson's disease (PD) and also as a potential biomarker when tested in the cerebrospinal fluid (CSF). The performance of blood plasma or serum α-synuclein as a biomarker has been found to be inconsistent and generally ineffective, largely due to the contribution of peripherally derived α-synuclein. In this study, we discovered, via an intracerebroventricular injection of radiolabeled α-synuclein into mouse brain, that CSF α-synuclein was readily transported to blood, with a small portion being contained in exosomes that are relatively specific to the central nervous system (CNS). Consequently, we developed a technique to evaluate the levels of α-synuclein in these exosomes in individual plasma samples. When applied to a large cohort of clinical samples (267 PD, 215 controls), we found that in contrast to CSF α-synuclein concentrations, which are consistently reported to be lower in PD patients compared to controls, the levels of plasma exosomal α-synuclein were substantially higher in PD patients, suggesting an increased efflux of the protein to the peripheral blood of these patients. Furthermore, although no association was observed between plasma exosomal and CSF α-synuclein, a significant correlation between plasma exosomal α-synuclein and disease severity (r = 0.176, p = 0.004) was observed, and the diagnostic sensitivity and specificity achieved by plasma exosomal α-synuclein were comparable to those determined by CSF α-synuclein. Further studies are clearly needed to elucidate the mechanism involved in the transport of CNS α-synuclein to the periphery, which may lead to a more convenient and robust assessment of PD clinically.


Assuntos
alfa-Sinucleína/sangue , alfa-Sinucleína/líquido cefalorraquidiano , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Encéfalo/metabolismo , Estudos de Casos e Controles , Estudos de Coortes , Exossomos/metabolismo , Feminino , Humanos , Masculino , Espectrometria de Massas , Camundongos , Microscopia Eletrônica , Pessoa de Meia-Idade , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Doença de Parkinson/sangue , Doença de Parkinson/líquido cefalorraquidiano , Curva ROC
6.
Elife ; 122023 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-36799304

RESUMO

Accumulation of somatic mutations in the mitochondrial genome (mtDNA) has long been proposed as a possible mechanism of mitochondrial and tissue dysfunction that occurs during aging. A thorough characterization of age-associated mtDNA somatic mutations has been hampered by the limited ability to detect low-frequency mutations. Here, we used Duplex Sequencing on eight tissues of an aged mouse cohort to detect >89,000 independent somatic mtDNA mutations and show significant tissue-specific increases during aging across all tissues examined which did not correlate with mitochondrial content and tissue function. G→A/C→T substitutions, indicative of replication errors and/or cytidine deamination, were the predominant mutation type across all tissues and increased with age, whereas G→T/C→A substitutions, indicative of oxidative damage, were the second most common mutation type, but did not increase with age regardless of tissue. We also show that clonal expansions of mtDNA mutations with age is tissue- and mutation type-dependent. Unexpectedly, mutations associated with oxidative damage rarely formed clones in any tissue and were significantly reduced in the hearts and kidneys of aged mice treated at late age with elamipretide or nicotinamide mononucleotide. Thus, the lack of accumulation of oxidative damage-linked mutations with age suggests a life-long dynamic clearance of either the oxidative lesions or mtDNA genomes harboring oxidative damage.


Assuntos
Envelhecimento , DNA Mitocondrial , Camundongos , Animais , DNA Mitocondrial/genética , Envelhecimento/genética , Mitocôndrias/genética , Mitocôndrias/patologia , Estresse Oxidativo/genética , Mutação
7.
Forensic Sci Int Genet ; 49: 102364, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32805606

RESUMO

Match probabilities calculated during the evaluation of DNA evidence profiles rely on appropriate values of the population structure quantity θ. NGS-based methods will enhance forensic identification and with the transformation to such methods comes the need to facilitate NGS-based population genetics analysis. If NGS data are to be used for match probabilities there needs to be a way to accommodate population structure, which requires values for θ for those data. Such estimates have not been available. This study assesses population structure for sequence-based data using a relatively new approach applied to STR data over 27 loci in five different geographic groups. Matching proportions between individuals or groups are used to obtain locus-specific θ estimates as well as estimates per geographic group and a global measure. The results demonstrate similar effects of sequencing data on θ estimates compared to what has been seen for CE-based results.


Assuntos
Marcadores Genéticos , Genética Populacional , Sequenciamento de Nucleotídeos em Larga Escala , Repetições de Microssatélites , Análise de Sequência de DNA , Alelos , Impressões Digitais de DNA , Genótipo , Humanos , Grupos Raciais/genética
8.
Nat Commun ; 10(1): 3280, 2019 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-31337756

RESUMO

Somatic mutations in the mitochondrial genome (mtDNA) have been linked to multiple disease conditions and to ageing itself. In Drosophila, knock-in of a proofreading deficient mtDNA polymerase (POLG) generates high levels of somatic point mutations and also small indels, but surprisingly limited impact on organismal longevity or fitness. Here we describe a new mtDNA mutator model based on a mitochondrially-targeted cytidine deaminase, APOBEC1. mito-APOBEC1 acts as a potent mutagen which exclusively induces C:G>T:A transitions with no indels or mtDNA depletion. In these flies, the presence of multiple non-synonymous substitutions, even at modest heteroplasmy, disrupts mitochondrial function and dramatically impacts organismal fitness. A detailed analysis of the mutation profile in the POLG and mito-APOBEC1 models reveals that mutation type (quality) rather than quantity is a critical factor in impacting organismal fitness. The specificity for transition mutations and the severe phenotypes make mito-APOBEC1 an excellent mtDNA mutator model for ageing research.


Assuntos
Desaminase APOBEC-1/fisiologia , DNA Mitocondrial/química , Drosophila/genética , Desaminase APOBEC-1/genética , Desaminase APOBEC-1/metabolismo , Animais , Drosophila/fisiologia , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Modelos Genéticos , Mutação , Organismos Geneticamente Modificados
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