Publications by authors named "Scott R Kennedy"

30 Publications

  • Page 1 of 1

PolyG-DS: An ultrasensitive polyguanine tract-profiling method to detect clonal expansions and trace cell lineage.

Proc Natl Acad Sci U S A 2021 Aug;118(31)

Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA 98195;

Polyguanine tracts (PolyGs) are short guanine homopolymer repeats that are prone to accumulating mutations when cells divide. This feature makes them especially suitable for cell lineage tracing, which has been exploited to detect and characterize precancerous and cancerous somatic evolution. PolyG genotyping, however, is challenging because of the inherent biochemical difficulties in amplifying and sequencing repetitive regions. To overcome this limitation, we developed PolyG-DS, a next-generation sequencing (NGS) method that combines the error-correction capabilities of duplex sequencing (DS) with enrichment of PolyG loci using CRISPR-Cas9-targeted genomic fragmentation. PolyG-DS markedly reduces technical artifacts by comparing the sequences derived from the complementary strands of each original DNA molecule. We demonstrate that PolyG-DS genotyping is accurate, reproducible, and highly sensitive, enabling the detection of low-frequency alleles (<0.01) in spike-in samples using a panel of only 19 PolyG markers. PolyG-DS replicated prior results based on PolyG fragment length analysis by capillary electrophoresis, and exhibited higher sensitivity for identifying clonal expansions in the nondysplastic colon of patients with ulcerative colitis. We illustrate the utility of this method for resolving the phylogenetic relationship among precancerous lesions in ulcerative colitis and for tracing the metastatic dissemination of ovarian cancer. PolyG-DS enables the study of tumor evolution without prior knowledge of tumor driver mutations and provides a tool to perform cost-effective and easily scalable ultra-accurate NGS-based PolyG genotyping for multiple applications in biology, genetics, and cancer research.
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http://dx.doi.org/10.1073/pnas.2023373118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8346827PMC
August 2021

Rate volatility and asymmetric segregation diversify mutation burden in cells with mutator alleles.

Commun Biol 2021 01 4;4(1):21. Epub 2021 Jan 4.

Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, 98195-7705, USA.

Mutations that compromise mismatch repair (MMR) or DNA polymerase ε or δ exonuclease domains produce mutator phenotypes capable of fueling cancer evolution. Here, we investigate how combined defects in these pathways expands genetic heterogeneity in cells of the budding yeast, Saccharomyces cerevisiae, using a single-cell resolution approach that tallies all mutations arising from individual divisions. The distribution of replication errors present in mother cells after the initial S-phase was broader than expected for a single uniform mutation rate across all cell divisions, consistent with volatility of the mutator phenotype. The number of mismatches that then segregated to the mother and daughter cells co-varied, suggesting that each division is governed by a different underlying genome-wide mutation rate. The distribution of mutations that individual cells inherit after the second S-phase is further broadened by the sequential actions of semiconservative replication and mitotic segregation of chromosomes. Modeling suggests that this asymmetric segregation may diversify mutation burden in mutator-driven tumors.
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http://dx.doi.org/10.1038/s42003-020-01544-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7782790PMC
January 2021

Analyzing population structure for forensic STR markers in next generation sequencing data.

Forensic Sci Int Genet 2020 11 12;49:102364. Epub 2020 Aug 12.

Department of Biostatistics, University of Washington, University Tower, 15th Floor, 4333 Brook-lyn Ave., Box 35946, Seattle, WA, USA.

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.
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http://dx.doi.org/10.1016/j.fsigen.2020.102364DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7655679PMC
November 2020

Loss of vacuolar acidity results in iron-sulfur cluster defects and divergent homeostatic responses during aging in Saccharomyces cerevisiae.

Geroscience 2020 04 23;42(2):749-764. Epub 2020 Jan 23.

Department of Biology and Biotechnology, University of Houston-Clear Lake, Houston, TX, 77058, USA.

The loss of vacuolar/lysosomal acidity is an early event during aging that has been linked to mitochondrial dysfunction. However, it is unclear how loss of vacuolar acidity results in age-related dysfunction. Through unbiased genetic screens, we determined that increased iron uptake can suppress the mitochondrial respiratory deficiency phenotype of yeast vma mutants, which have lost vacuolar acidity due to genetic disruption of the vacuolar ATPase proton pump. Yeast vma mutants exhibited nuclear localization of Aft1, which turns on the iron regulon in response to iron-sulfur cluster (ISC) deficiency. This led us to find that loss of vacuolar acidity with age in wild-type yeast causes ISC defects and a DNA damage response. Using microfluidics to investigate aging at the single-cell level, we observe grossly divergent trajectories of iron homeostasis within an isogenic and environmentally homogeneous population. One subpopulation of cells fails to mount the expected compensatory iron regulon gene expression program, and suffers progressively severe ISC deficiency with little to no activation of the iron regulon. In contrast, other cells show robust iron regulon activity with limited ISC deficiency, which allows extended passage and survival through a period of genomic instability during aging. These divergent trajectories suggest that iron regulation and ISC homeostasis represent a possible target for aging interventions.
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http://dx.doi.org/10.1007/s11357-020-00159-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7205917PMC
April 2020

Next-Generation Genotoxicology: Using Modern Sequencing Technologies to Assess Somatic Mutagenesis and Cancer Risk.

Environ Mol Mutagen 2020 01 11;61(1):135-151. Epub 2019 Nov 11.

Department of Pathology, University of Washington, Seattle, Washington.

Mutations have a profound effect on human health, particularly through an increased risk of carcinogenesis and genetic disease. The strong correlation between mutagenesis and carcinogenesis has been a driving force behind genotoxicity research for more than 50 years. The stochastic and infrequent nature of mutagenesis makes it challenging to observe and to study. Indeed, decades have been spent developing increasingly sophisticated assays and methods to study these low-frequency genetic errors, in hopes of better predicting which chemicals may be carcinogens, understanding their mode of action, and informing guidelines to prevent undue human exposure. While effective, widely used genetic selection-based technologies have a number of limitations that have hampered major advancements in the field of genotoxicity. Emerging new tools, in the form of enhanced next-generation sequencing platforms and methods, are changing this paradigm. In this review, we discuss rapidly evolving sequencing tools and technologies, such as error-corrected sequencing and single cell analysis, which we anticipate will fundamentally reshape the field. In addition, we consider a variety emerging applications for these new technologies, including the detection of DNA adducts, inference of mutational processes based on genomic site and local sequence contexts, and evaluation of genome engineering fidelity, as well as other cutting-edge challenges for the next 50 years of environmental and molecular mutagenesis research. Environ. Mol. Mutagen. 61:135-151, 2020. © 2019 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.
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http://dx.doi.org/10.1002/em.22342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7003768PMC
January 2020

Cancer-Associated Mutations but No Cancer: Insights into the Early Steps of Carcinogenesis and Implications for Early Cancer Detection.

Trends Cancer 2019 09 22;5(9):531-540. Epub 2019 Aug 22.

Department of Pathology, University of Washington, Seattle, WA, USA. Electronic address:

Cancer is a disease of aging fueled by the accumulation of somatic mutations. While mutations in tumors are well characterized, little is known about the early mutational processes that initiate tumorigenesis. Recent advances in next-generation sequencing (NGS) have enabled the detection of mutations in normal tissue, revealing an unanticipated high level of age-related somatic mutations affecting most individuals and tissues. Surprisingly, many of these mutations are similar to mutations commonly found in tumors, suggesting an ongoing process of positive selection and clonal expansion akin to what occurs in cancer, but within normal tissue. Here we discuss some of the most important biological and clinical implications of these novel findings, with a special focus on their impact for cancer detection and prediction.
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http://dx.doi.org/10.1016/j.trecan.2019.07.007DOI Listing
September 2019

Mitochondrially-targeted APOBEC1 is a potent mtDNA mutator affecting mitochondrial function and organismal fitness in Drosophila.

Nat Commun 2019 07 23;10(1):3280. Epub 2019 Jul 23.

MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge Biomedical Campus, Hills Road, Cambridge, CB2 0XY, UK.

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.
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http://dx.doi.org/10.1038/s41467-019-10857-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6650417PMC
July 2019

Abnormal oxidative metabolism in a quiet genomic background underlies clear cell papillary renal cell carcinoma.

Elife 2019 04 1;8. Epub 2019 Apr 1.

Department of Medicine, Molecular Oncology, Siteman Cancer Center, Washington University, St. Louis, United States.

While genomic sequencing routinely identifies oncogenic alterations for the majority of cancers, many tumors harbor no discernable driver lesion. Here, we describe the exceptional molecular phenotype of a genomically quiet kidney tumor, clear cell papillary renal cell carcinoma (CCPAP). In spite of a largely wild-type nuclear genome, CCPAP tumors exhibit severe depletion of mitochondrial DNA (mtDNA) and RNA and high levels of oxidative stress, reflecting a shift away from respiratory metabolism. Moreover, CCPAP tumors exhibit a distinct metabolic phenotype uniquely characterized by accumulation of the sugar alcohol sorbitol. Immunohistochemical staining of primary CCPAP tumor specimens recapitulates both the depletion of mtDNA-encoded proteins and a lipid-depleted metabolic phenotype, suggesting that the cytoplasmic clarity in CCPAP is primarily related to the presence of glycogen. These results argue for non-genetic profiling as a tool for the study of cancers of unknown driver.
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http://dx.doi.org/10.7554/eLife.38986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6459676PMC
April 2019

Defining the impact of mutation accumulation on replicative lifespan in yeast using cancer-associated mutator phenotypes.

Proc Natl Acad Sci U S A 2019 02 4;116(8):3062-3071. Epub 2019 Feb 4.

Department of Pathology, University of Washington, Seattle, WA 98195-7705;

Mutations accumulate within somatic cells and have been proposed to contribute to aging. It is unclear what level of mutation burden may be required to consistently reduce cellular lifespan. Human cancers driven by a mutator phenotype represent an intriguing model to test this hypothesis, since they carry the highest mutation burdens of any human cell. However, it remains technically challenging to measure the replicative lifespan of individual mammalian cells. Here, we modeled the consequences of cancer-related mutator phenotypes on lifespan using yeast defective for mismatch repair (MMR) and/or leading strand (Polε) or lagging strand (Polδ) DNA polymerase proofreading. Only haploid mutator cells with significant lifetime mutation accumulation (MA) exhibited shorter lifespans. Diploid strains, derived by mating haploids of various genotypes, carried variable numbers of fixed mutations and a range of mutator phenotypes. Some diploid strains with fewer than two mutations per megabase displayed a 25% decrease in lifespan, suggesting that moderate numbers of random heterozygous mutations can increase mortality rate. As mutation rates and burdens climbed, lifespan steadily eroded. Strong diploid mutator phenotypes produced a form of genetic anticipation with regard to aging, where the longer a lineage persisted, the shorter lived cells became. Using MA lines, we established a relationship between mutation burden and lifespan, as well as population doubling time. Our observations define a threshold of random mutation burden that consistently decreases cellular longevity in diploid yeast cells. Many human cancers carry comparable mutation burdens, suggesting that while cancers appear immortal, individual cancer cells may suffer diminished lifespan due to accrued mutation burden.
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http://dx.doi.org/10.1073/pnas.1815966116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6386679PMC
February 2019

Deleterious mitochondrial DNA point mutations are overrepresented in Drosophila expressing a proofreading-defective DNA polymerase γ.

PLoS Genet 2018 11 19;14(11):e1007805. Epub 2018 Nov 19.

Department of Genome Sciences, University of Washington, Seattle, WA, United States of America.

Mitochondrial DNA (mtDNA) mutations cause severe maternally inherited syndromes and the accumulation of somatic mtDNA mutations is implicated in aging and common diseases. However, the mechanisms that influence the frequency and pathogenicity of mtDNA mutations are poorly understood. To address this matter, we created a Drosophila mtDNA mutator strain expressing a proofreading-deficient form of the mitochondrial DNA polymerase. Mutator flies have a dramatically increased somatic mtDNA mutation frequency that correlates with the dosage of the proofreading-deficient polymerase. Mutator flies also exhibit mitochondrial dysfunction, shortened lifespan, a progressive locomotor deficit, and loss of dopaminergic neurons. Surprisingly, the frequency of nonsynonymous, pathogenic, and conserved-site mutations in mutator flies exceeded predictions of a neutral mutational model, indicating the existence of a positive selection mechanism that favors deleterious mtDNA variants. We propose from these findings that deleterious mtDNA mutations are overrepresented because they selectively evade quality control surveillance or because they are amplified through compensatory mitochondrial biogenesis.
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http://dx.doi.org/10.1371/journal.pgen.1007805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6289449PMC
November 2018

Mitochondrial DNA Mutations are Associated with Ulcerative Colitis Preneoplasia but Tend to be Negatively Selected in Cancer.

Mol Cancer Res 2019 02 16;17(2):488-498. Epub 2018 Nov 16.

Department of Pathology, University of Washington, Seattle, Washington.

The role of mitochondrial DNA (mtDNA) mutations in cancer remains controversial. Ulcerative colitis is an inflammatory bowel disease that increases the risk of colorectal cancer and involves mitochondrial dysfunction, making it an ideal model to study the role of mtDNA in tumorigenesis. Our goal was to comprehensively characterize mtDNA mutations in ulcerative colitis tumorigenesis using Duplex Sequencing, an ultra-accurate next-generation sequencing method. We analyzed 46 colon biopsies from non-ulcerative colitis control patients and ulcerative colitis patients with and without cancer, including biopsies at all stages of dysplastic progression. mtDNA was sequenced at a median depth of 1,364x. Mutations were classified by mutant allele frequency: clonal > 0.95, subclonal 0.01-0.95, and very low frequency (VLF) < 0.01. We identified 208 clonal and subclonal mutations and 56,764 VLF mutations. Mutations were randomly distributed across the mitochondrial genome. Clonal and subclonal mutations increased in number and pathogenicity in early dysplasia, but decreased in number and pathogenicity in cancer. Most clonal, subclonal, and VLF mutations were C>T transitions in the heavy strand of mtDNA, which likely arise from DNA replication errors. A subset of VLF mutations were C>A transversions, which are probably due to oxidative damage. VLF transitions and indels were less abundant in the non-D-loop region and decreased with progression. Our results indicate that mtDNA mutations are frequent in ulcerative colitis preneoplasia but negatively selected in cancers. IMPLICATIONS: While mtDNA mutations might contribute to early ulcerative colitis tumorigenesis, they appear to be selected against in cancer, suggesting that functional mitochondria might be required for malignant transformation in ulcerative colitis.
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http://dx.doi.org/10.1158/1541-7786.MCR-18-0520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6481604PMC
February 2019

Targeted genome fragmentation with CRISPR/Cas9 enables fast and efficient enrichment of small genomic regions and ultra-accurate sequencing with low DNA input (CRISPR-DS).

Genome Res 2018 10 19;28(10):1589-1599. Epub 2018 Sep 19.

Department of Pathology, University of Washington, Seattle, Washington 98195, USA.

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 and performed side-by-side comparisons with standard Duplex Sequencing. CRISPR-DS detected previously reported pathogenic 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.
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http://dx.doi.org/10.1101/gr.235291.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6169890PMC
October 2018

Aging and the rise of somatic cancer-associated mutations in normal tissues.

PLoS Genet 2018 01 4;14(1):e1007108. Epub 2018 Jan 4.

Department of Pathology, University of Washington, Seattle, Washington, United States of America.

DNA mutations are inevitable. Despite proficient DNA repair mechanisms, somatic cells accumulate mutations during development and aging, generating cells with different genotypes within the same individual, a phenomenon known as somatic mosaicism. While the existence of somatic mosaicism has long been recognized, in the last five years, advances in sequencing have provided unprecedented resolution to characterize the extent and nature of somatic genetic variation. Collectively, these new studies are revealing a previously uncharacterized aging phenotype: the accumulation of clones with cancer driver mutations. Here, we summarize the most recent findings, which converge in the novel notion that cancer-associated mutations are prevalent in normal tissue and accumulate with aging.
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http://dx.doi.org/10.1371/journal.pgen.1007108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5754046PMC
January 2018

Normally lethal amino acid substitutions suppress an ultramutator DNA Polymerase δ variant.

Sci Rep 2017 04 18;7:46535. Epub 2017 Apr 18.

Department of Pathology, University of Washington, Seattle, Washington 98195, USA.

In yeast, the pol3-01,L612M double mutant allele, which causes defects in DNA polymerase delta (Pol δ) proofreading (pol3-01) and nucleotide selectivity (pol3-L612M), confers an "ultramutator" phenotype that rapidly drives extinction of haploid and diploid MMR-proficient cells. Here, we investigate antimutator mutations that encode amino acid substitutions in Pol δ that suppress this lethal phenotype. We find that most of the antimutator mutations individually suppress the pol3-01 and pol3-L612M mutator phenotypes. The locations of many of the amino acid substitutions in Pol δ resemble those of previously identified antimutator substitutions; however, two novel mutations encode substitutions (R674G and Q697R) of amino acids in the fingers domain that coordinate the incoming dNTP. These mutations are lethal without pol3-L612M and markedly change the mutation spectra produced by the pol3-01,L612M mutator allele, suggesting that they alter nucleotide selection to offset the pol3-L612M mutator phenotype. Consistent with this hypothesis, mutations and drug treatments that perturb dNTP pool levels disproportionately influence the viability of pol3-L612M,R674G and pol3-L612M,Q697R cells. Taken together, our findings suggest that mutation rate can evolve through genetic changes that alter the balance of dNTP binding and dissociation from DNA polymerases.
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http://dx.doi.org/10.1038/srep46535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5394481PMC
April 2017

Werner syndrome through the lens of tissue and tumour genomics.

Sci Rep 2016 08 25;6:32038. Epub 2016 Aug 25.

Department of Pathology, University of Washington, Seattle, WA USA.

Werner syndrome (WS) is the canonical adult human progeroid ('premature aging') syndrome. Patients with this autosomal recessive Mendelian disorder display constitutional genomic instability and an elevated risk of important age-associated diseases including cancer. Remarkably few analyses of WS patient tissue and tumors have been performed to provide insight into WS disease pathogenesis or the high risk of neoplasia. We used autopsy tissue from four mutation-typed WS patients to characterize pathologic and genomic features of WS, and to determine genomic features of three neoplasms arising in two of these patients. The results of these analyses provide new information on WS pathology and genomics; provide a first genomic characterization of neoplasms arising in WS; and provide new histopathologic and genomic data to test several popular models of WS disease pathogenesis.
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http://dx.doi.org/10.1038/srep32038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4997333PMC
August 2016

A mitochondrial DNA hypomorph of cytochrome oxidase specifically impairs male fertility in Drosophila melanogaster.

Elife 2016 08 2;5. Epub 2016 Aug 2.

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States.

Due to their strict maternal inheritance in most animals and plants, mitochondrial genomes are predicted to accumulate mutations that are beneficial or neutral in females but harmful in males. Although a few male-harming mtDNA mutations have been identified, consistent with this 'Mother's Curse', their effect on females has been largely unexplored. Here, we identify COII(G177S), a mtDNA hypomorph of cytochrome oxidase II, which specifically impairs male fertility due to defects in sperm development and function without impairing other male or female functions. COII(G177S) represents one of the clearest examples of a 'male-harming' mtDNA mutation in animals and suggest that the hypomorphic mtDNA mutations like COII(G177S) might specifically impair male gametogenesis. Intriguingly, some D. melanogaster nuclear genetic backgrounds can fully rescue COII(G177S) -associated sterility, consistent with previously proposed models that nuclear genomes can regulate the phenotypic manifestation of mtDNA mutations.
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http://dx.doi.org/10.7554/eLife.16923DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4970871PMC
August 2016

Mitochondrial DNA mutations increase in early stage Alzheimer disease and are inconsistent with oxidative damage.

Ann Neurol 2016 08 13;80(2):301-6. Epub 2016 Jul 13.

Department of Pathology, University of Washington, Seattle, WA.

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.
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http://dx.doi.org/10.1002/ana.24709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4982791PMC
August 2016

Ultra-deep sequencing detects ovarian cancer cells in peritoneal fluid and reveals somatic TP53 mutations in noncancerous tissues.

Proc Natl Acad Sci U S A 2016 May 5;113(21):6005-10. Epub 2016 May 5.

Department of Pathology, University of Washington, Seattle, WA 98195;

Current sequencing methods are error-prone, which precludes the identification of low frequency mutations for early cancer detection. Duplex sequencing is a sequencing technology that decreases errors by scoring mutations present only in both strands of DNA. Our aim was to determine whether duplex sequencing could detect extremely rare cancer cells present in peritoneal fluid from women with high-grade serous ovarian carcinomas (HGSOCs). These aggressive cancers are typically diagnosed at a late stage and are characterized by TP53 mutations and peritoneal dissemination. We used duplex sequencing to analyze TP53 mutations in 17 peritoneal fluid samples from women with HGSOC and 20 from women without cancer. The tumor TP53 mutation was detected in 94% (16/17) of peritoneal fluid samples from women with HGSOC (frequency as low as 1 mutant per 24,736 normal genomes). Additionally, we detected extremely low frequency TP53 mutations (median mutant fraction 1/13,139) in peritoneal fluid from nearly all patients with and without cancer (35/37). These mutations were mostly deleterious, clustered in hotspots, increased with age, and were more abundant in women with cancer than in controls. The total burden of TP53 mutations in peritoneal fluid distinguished cancers from controls with 82% sensitivity (14/17) and 90% specificity (18/20). Age-associated, low frequency TP53 mutations were also found in 100% of peripheral blood samples from 15 women with and without ovarian cancer (none with hematologic disorder). Our results demonstrate the ability of duplex sequencing to detect rare cancer cells and provide evidence of widespread, low frequency, age-associated somatic TP53 mutation in noncancerous tissue.
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http://dx.doi.org/10.1073/pnas.1601311113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4889384PMC
May 2016

Endogenous Parkin Preserves Dopaminergic Substantia Nigral Neurons following Mitochondrial DNA Mutagenic Stress.

Neuron 2015 Jul;87(2):371-81

Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address:

Parkinson's disease (PD) is a neurodegenerative disease caused by the loss of dopaminergic neurons in the substantia nigra. PARK2 mutations cause early-onset forms of PD. PARK2 encodes an E3 ubiquitin ligase, Parkin, that can selectively translocate to dysfunctional mitochondria to promote their removal by autophagy. However, Parkin knockout (KO) mice do not display signs of neurodegeneration. To assess Parkin function in vivo, we utilized a mouse model that accumulates dysfunctional mitochondria caused by an accelerated generation of mtDNA mutations (Mutator mice). In the absence of Parkin, dopaminergic neurons in Mutator mice degenerated causing an L-DOPA reversible motor deficit. Other neuronal populations were unaffected. Phosphorylated ubiquitin was increased in the brains of Mutator mice, indicating PINK1-Parkin activation. Parkin loss caused mitochondrial dysfunction and affected the pathogenicity but not the levels of mtDNA somatic mutations. A systemic loss of Parkin synergizes with mitochondrial dysfunction causing dopaminergic neuron death modeling PD pathogenic processes.
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http://dx.doi.org/10.1016/j.neuron.2015.06.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4803114PMC
July 2015

Volatility of Mutator Phenotypes at Single Cell Resolution.

PLoS Genet 2015 Apr 13;11(4):e1005151. Epub 2015 Apr 13.

Department of Pathology, University of Washington, Seattle, Washington, United States of America.

Mutator phenotypes accelerate the evolutionary process of neoplastic transformation. Historically, the measurement of mutation rates has relied on scoring the occurrence of rare mutations in target genes in large populations of cells. Averaging mutation rates over large cell populations assumes that new mutations arise at a constant rate during each cell division. If the mutation rate is not constant, an expanding mutator population may contain subclones with widely divergent rates of evolution. Here, we report mutation rate measurements of individual cell divisions of mutator yeast deficient in DNA polymerase ε proofreading and base-base mismatch repair. Our data are best fit by a model in which cells can assume one of two distinct mutator states, with mutation rates that differ by an order of magnitude. In error-prone cell divisions, mutations occurred on the same chromosome more frequently than expected by chance, often in DNA with similar predicted replication timing, consistent with a spatiotemporal dimension to the hypermutator state. Mapping of mutations onto predicted replicons revealed that mutations were enriched in the first half of the replicon as well as near termination zones. Taken together, our findings show that individual genome replication events exhibit an unexpected volatility that may deepen our understanding of the evolution of mutator-driven malignancies.
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http://dx.doi.org/10.1371/journal.pgen.1005151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4395103PMC
April 2015

Detecting ultralow-frequency mutations by Duplex Sequencing.

Nat Protoc 2014 Nov 9;9(11):2586-606. Epub 2014 Oct 9.

1] Department of Pathology, University of Washington, Seattle, USA. [2] Department of Biochemistry, University of Washington, Seattle, USA.

Duplex Sequencing (DS) is a next-generation sequencing methodology capable of detecting a single mutation among >1 × 10(7) wild-type nucleotides, thereby enabling the study of heterogeneous populations and very-low-frequency genetic alterations. DS can be applied to any double-stranded DNA sample, but it is ideal for small genomic regions of <1 Mb in size. The method relies on the ligation of sequencing adapters harboring random yet complementary double-stranded nucleotide sequences to the sample DNA of interest. Individually labeled strands are then PCR-amplified, creating sequence 'families' that share a common tag sequence derived from the two original complementary strands. Mutations are scored only if the variant is present in the PCR families arising from both of the two DNA strands. Here we provide a detailed protocol for efficient DS adapter synthesis, library preparation and target enrichment, as well as an overview of the data analysis workflow. The protocol typically takes 1-3 d.
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http://dx.doi.org/10.1038/nprot.2014.170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4271547PMC
November 2014

Oxidative stress is not a major contributor to somatic mitochondrial DNA mutations.

PLoS Genet 2014 Feb 6;10(2):e1003974. Epub 2014 Feb 6.

Department of Genome Sciences, University of Washington, Seattle, Washington, United States of America.

The accumulation of somatic mitochondrial DNA (mtDNA) mutations is implicated in aging and common diseases of the elderly, including cancer and neurodegenerative disease. However, the mechanisms that influence the frequency of somatic mtDNA mutations are poorly understood. To develop a simple invertebrate model system to address this matter, we used the Random Mutation Capture (RMC) assay to characterize the age-dependent frequency and distribution of mtDNA mutations in the fruit fly Drosophila melanogaster. Because oxidative stress is a major suspect in the age-dependent accumulation of somatic mtDNA mutations, we also used the RMC assay to explore the influence of oxidative stress on the somatic mtDNA mutation frequency. We found that many of the features associated with mtDNA mutations in vertebrates are conserved in Drosophila, including a comparable somatic mtDNA mutation frequency (∼10(-5)), an increased frequency of mtDNA mutations with age, and a prevalence of transition mutations. Only a small fraction of the mtDNA mutations detected in young or old animals were G∶C to T∶A transversions, a signature of oxidative damage, and loss-of-function mutations in the mitochondrial superoxide dismutase, Sod2, had no detectable influence on the somatic mtDNA mutation frequency. Moreover, a loss-of-function mutation in Ogg1, which encodes a DNA repair enzyme that removes oxidatively damaged deoxyguanosine residues (8-hydroxy-2'-deoxyguanosine), did not significantly influence the somatic mtDNA mutation frequency of Sod2 mutants. Together, these findings indicate that oxidative stress is not a major cause of somatic mtDNA mutations. Our data instead suggests that somatic mtDNA mutations arise primarily from errors that occur during mtDNA replication. Further studies using Drosophila should aid in the identification of factors that influence the frequency of somatic mtDNA mutations.
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http://dx.doi.org/10.1371/journal.pgen.1003974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3916223PMC
February 2014

DNA replication error-induced extinction of diploid yeast.

Genetics 2014 Mar 3;196(3):677-91. Epub 2014 Jan 3.

Department of Pathology, University of Washington, Seattle, Washington 98195.

Genetic defects in DNA polymerase accuracy, proofreading, or mismatch repair (MMR) induce mutator phenotypes that accelerate adaptation of microbes and tumor cells. Certain combinations of mutator alleles synergistically increase mutation rates to levels that drive extinction of haploid cells. The maximum tolerated mutation rate of diploid cells is unknown. Here, we define the threshold for replication error-induced extinction (EEX) of diploid Saccharomyces cerevisiae. Double-mutant pol3 alleles that carry mutations for defective DNA polymerase-δ proofreading (pol3-01) and accuracy (pol3-L612M or pol3-L612G) induce strong mutator phenotypes in heterozygous diploids (POL3/pol3-01,L612M or POL3/pol3-01,L612G). Both pol3-01,L612M and pol3-01,L612G alleles are lethal in the homozygous state; cells with pol3-01,L612M divide up to 10 times before arresting at random stages in the cell cycle. Antimutator eex mutations in the pol3 alleles suppress this lethality (pol3-01,L612M,eex or pol3-01,L612G,eex). MMR defects synergize with pol3-01,L612M,eex and pol3-01,L612G,eex alleles, increasing mutation rates and impairing growth. Conversely, inactivation of the Dun1 S-phase checkpoint kinase suppresses strong pol3-01,L612M,eex and pol3-01,L612G,eex mutator phenotypes as well as the lethal pol3-01,L612M phenotype. Our results reveal that the lethal error threshold in diploids is 10 times higher than in haploids and likely determined by homozygous inactivation of essential genes. Pronounced loss of fitness occurs at mutation rates well below the lethal threshold, suggesting that mutator-driven cancers may be susceptible to drugs that exacerbate replication errors.
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http://dx.doi.org/10.1534/genetics.113.160960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3948800PMC
March 2014

Ultra-sensitive sequencing reveals an age-related increase in somatic mitochondrial mutations that are inconsistent with oxidative damage.

PLoS Genet 2013 26;9(9):e1003794. Epub 2013 Sep 26.

Department of Pathology, University of Washington, Seattle, Washington, United States of America.

Mitochondrial DNA (mtDNA) is believed to be highly vulnerable to age-associated damage and mutagenesis by reactive oxygen species (ROS). However, somatic mtDNA mutations have historically been difficult to study because of technical limitations in accurately quantifying rare mtDNA mutations. We have applied the highly sensitive Duplex Sequencing methodology, which can detect a single mutation among >10(7) wild type molecules, to sequence mtDNA purified from human brain tissue from both young and old individuals with unprecedented accuracy. We find that the frequency of point mutations increases ~5-fold over the course of 80 years of life. Overall, the mutation spectra of both groups are comprised predominantly of transition mutations, consistent with misincorporation by DNA polymerase γ or deamination of cytidine and adenosine as the primary mutagenic events in mtDNA. Surprisingly, G → T mutations, considered the hallmark of oxidative damage to DNA, do not significantly increase with age. We observe a non-uniform, age-independent distribution of mutations in mtDNA, with the D-loop exhibiting a significantly higher mutation frequency than the rest of the genome. The coding regions, but not the D-loop, exhibit a pronounced asymmetric accumulation of mutations between the two strands, with G → A and T → C mutations occurring more often on the light strand than the heavy strand. The patterns and biases we observe in our data closely mirror the mutational spectrum which has been reported in studies of human populations and closely related species. Overall our results argue against oxidative damage being a major driver of aging and suggest that replication errors by DNA polymerase γ and/or spontaneous base hydrolysis are responsible for the bulk of accumulating point mutations in mtDNA.
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http://dx.doi.org/10.1371/journal.pgen.1003794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3784509PMC
March 2014

Detection of ultra-rare mutations by next-generation sequencing.

Proc Natl Acad Sci U S A 2012 Sep 1;109(36):14508-13. Epub 2012 Aug 1.

Departments of Pathology, Genome Sciences, and Biochemistry, University of Washington School of Medicine, Seattle, WA 98195, USA.

Next-generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of ~1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when "deep sequencing" genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, we have developed a method termed Duplex Sequencing. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors result in mutations in only one strand and can thus be discounted as technical error. We determine that Duplex Sequencing has a theoretical background error rate of less than one artifactual mutation per billion nucleotides sequenced. In addition, we establish that detection of mutations present in only one of the two strands of duplex DNA can be used to identify sites of DNA damage. We apply the method to directly assess the frequency and pattern of random mutations in mitochondrial DNA from human cells.
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http://dx.doi.org/10.1073/pnas.1208715109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3437896PMC
September 2012

Somatic mutations in aging, cancer and neurodegeneration.

Mech Ageing Dev 2012 Apr 3;133(4):118-26. Epub 2011 Nov 3.

Department of Pathology, University of Washington School of Medicine, Seattle, WA 98195-7705, USA.

The somatic mutation theory of aging posits that the accumulation of mutations in the genetic material of somatic cells as a function of time results in a decrease in cellular function. In particular, the accumulation of random mutations may inactivate genes that are important for the functioning of the somatic cells of various organ systems of the adult, result in a decrease in organ function. When the organ function decreases below a critical level, death occurs. A significant amount of research has shown that somatic mutations play an important role in aging and a number of age related pathologies. In this review, we explore evidence for increases in somatic nuclear mutation burden with age and the consequences for aging, cancer, and neurodegeneration. We then review evidence for increases in mitochondrial mutation burden and the consequences for dysfunction in the disease processes.
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http://dx.doi.org/10.1016/j.mad.2011.10.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3325357PMC
April 2012

The biochemistry and fidelity of synthesis by the apicoplast genome replication DNA polymerase Pfprex from the malaria parasite Plasmodium falciparum.

J Mol Biol 2011 Jul 5;410(1):27-38. Epub 2011 May 5.

Department of Pathology, University of Washington, Seattle, WA 98195, USA.

Plasmodium falciparum, the major causative agent of human malaria, contains three separate genomes. The apicoplast (an intracellular organelle) contains an ∼35-kb circular DNA genome of unusually high A/T content (>86%) that is replicated by the nuclear-encoded replication complex Pfprex. Herein, we have expressed and purified the DNA polymerase domain of Pfprex [KPom1 (Klenow-like polymerase of malaria 1)] and measured its fidelity using a LacZ-based forward mutation assay. In addition, we analyzed the kinetic parameters for the incorporation of both complementary and noncomplementary nucleotides using Kpom1 lacking 3'→5' exonucleolytic activity. KPom1 exhibits a strongly biased mutational spectrum in which T→C is the most frequent single-base substitution and differs significantly from the closely related Escherichia coli DNA polymerase I. Using E. coli harboring a temperature-sensitive polymerase I allele, we established that KPom1 can complement the growth-defective phenotype at an elevated temperature. We propose that the error bias of KPom1 may be exploited in the complementation assay to identify nucleoside analogs that mimic this base-mispairing and preferentially inhibit apicoplast DNA replication.
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http://dx.doi.org/10.1016/j.jmb.2011.04.071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3117635PMC
July 2011

Templated nucleoside triphosphate binding to a noncatalytic site on RNA polymerase regulates transcription.

Proc Natl Acad Sci U S A 2011 Apr 29;108(15):6079-84. Epub 2011 Mar 29.

Department of Chemistry and, Curriculum in Applied Sciences and Engineering, University of North Carolina, Chapel Hill, NC 27599, USA.

The regulation of RNA synthesis by RNA polymerase (RNAP) is essential for proper gene expression. Crystal structures of RNAP reveal two channels: the main channel that contains the downstream DNA and a secondary channel that leads directly to the catalytic site. Although nucleoside triphosphates (NTPs) have been seen only in the catalytic site and the secondary channel in these structures, several models of transcription elongation, based on biochemical studies, propose that template-dependent binding of NTPs in the main channel regulates RNA synthesis. These models, however, remain controversial. We used transient state kinetics and a mutant of RNAP to investigate the role of the main channel in regulating nucleotide incorporation. Our data indicate that a NTP specific for the i + 2 template position can bind to a noncatalytic site and increase the rate of RNA synthesis and that the NTP bound to this site can be shuttled directly into the catalytic site. We also identify fork loop 2, which lies across from the downstream DNA, as a functional component of this site. Taken together, our data support the existence of a noncatalytic template-specific NTP binding site in the main channel that is involved in the regulation of nucleotide incorporation. NTP binding to this site could promote high-fidelity processive synthesis under a variety of environmental conditions and allow DNA sequence-mediated regulatory signals to be communicated to the active site.
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http://dx.doi.org/10.1073/pnas.1011274108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3076813PMC
April 2011

Forks, pincers, and triggers: the tools for nucleotide incorporation and translocation in multi-subunit RNA polymerases.

Curr Opin Struct Biol 2009 Dec 11;19(6):708-14. Epub 2009 Nov 11.

Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599, United States.

The central role of RNA polymerase (RNAP) is to catalyze the processive synthesis of a growing RNA transcript. Recent structural and biophysical data have led to a deeper understanding of the nucleotide addition cycle and insight into the structure-function relationships that govern transcription elongation. In this review, we discuss kinetic data on nucleotide incorporation in the context of crystal structures, which show RNAP in multiple conformations. We present a facilitated Brownian ratchet model of nucleotide incorporation, in which templated NTP binding to a non-catalytic site in the main channel promotes the conformational changes that lead to opening of the catalytic site and translocation.
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http://dx.doi.org/10.1016/j.sbi.2009.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3979713PMC
December 2009

Porous broadband antireflection coating by glancing angle deposition.

Appl Opt 2003 Aug;42(22):4573-9

Department of Electrical and Computer Engineering, Second Level Electrical and Computer Engineering Research Facility, University of Alberta, Edmonton, Alberta T6G 2V4, Canada.

We deposited graded-index SiO2 films using glancing angle deposition to produce high-transmission antireflection coatings on glass. Because of the accurate control over the thin-film microstructure provided by this technique, we were able to create graded densities with a Gaussian profile resulting in transmission values greater than 99.9% for a single-layer interface with bandwidths up to 460 nm. The graded-index layer also provides low reflectance at nonnormal angles of incidence with transmission values degrading little for incidence angles up to 30 degrees.
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http://dx.doi.org/10.1364/ao.42.004573DOI Listing
August 2003
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