Short template switch events explain mutation clusters in the human genome.

PubMed

Löytynoja, Ari; Goldman, Nick

2017-06-01

Resequencing efforts are uncovering the extent of genetic variation in humans and provide data to study the evolutionary processes shaping our genome. One recurring puzzle in both intra- and inter-species studies is the high frequency of complex mutations comprising multiple nearby base substitutions or insertion-deletions. We devised a generalized mutation model of template switching during replication that extends existing models of genome rearrangement and used this to study the role of template switch events in the origin of short mutation clusters. Applied to the human genome, our model detects thousands of template switch events during the evolution of human and chimp from their common ancestor and hundreds of events between two independently sequenced human genomes. Although many of these are consistent with a template switch mechanism previously proposed for bacteria, our model also identifies new types of mutations that create short inversions, some flanked by paired inverted repeats. The local template switch process can create numerous complex mutation patterns, including hairpin loop structures, and explains multinucleotide mutations and compensatory substitutions without invoking positive selection, speculative mechanisms, or implausible coincidence. Clustered sequence differences are challenging for current mapping and variant calling methods, and we show that many erroneous variant annotations exist in human reference data. Local template switch events may have been neglected as an explanation for complex mutations because of biases in commonly used analyses. Incorporation of our model into reference-based analysis pipelines and comparisons of de novo assembled genomes will lead to improved understanding of genome variation and evolution. © 2017 Löytynoja and Goldman; Published by Cold Spring Harbor Laboratory Press.

Clustered Mutation Signatures Reveal that Error-Prone DNA Repair Targets Mutations to Active Genes.

PubMed

Supek, Fran; Lehner, Ben

2017-07-27

Many processes can cause the same nucleotide change in a genome, making the identification of the mechanisms causing mutations a difficult challenge. Here, we show that clustered mutations provide a more precise fingerprint of mutagenic processes. Of nine clustered mutation signatures identified from >1,000 tumor genomes, three relate to variable APOBEC activity and three are associated with tobacco smoking. An additional signature matches the spectrum of translesion DNA polymerase eta (POLH). In lymphoid cells, these mutations target promoters, consistent with AID-initiated somatic hypermutation. In solid tumors, however, they are associated with UV exposure and alcohol consumption and target the H3K36me3 chromatin of active genes in a mismatch repair (MMR)-dependent manner. These regions normally have a low mutation rate because error-free MMR also targets H3K36me3 chromatin. Carcinogens and error-prone repair therefore redistribute mutations to the more important regions of the genome, contributing a substantial mutation load in many tumors, including driver mutations. Copyright © 2017 Elsevier Inc. All rights reserved.

Mutational landscape of yeast mutator strains.

PubMed

Serero, Alexandre; Jubin, Claire; Loeillet, Sophie; Legoix-Né, Patricia; Nicolas, Alain G

2014-02-04

The acquisition of mutations is relevant to every aspect of genetics, including cancer and evolution of species on Darwinian selection. Genome variations arise from rare stochastic imperfections of cellular metabolism and deficiencies in maintenance genes. Here, we established the genome-wide spectrum of mutations that accumulate in a WT and in nine Saccharomyces cerevisiae mutator strains deficient for distinct genome maintenance processes: pol32Δ and rad27Δ (replication), msh2Δ (mismatch repair), tsa1Δ (oxidative stress), mre11Δ (recombination), mec1Δ tel1Δ (DNA damage/S-phase checkpoints), pif1Δ (maintenance of mitochondrial genome and telomere length), cac1Δ cac3Δ (nucleosome deposition), and clb5Δ (cell cycle progression). This study reveals the diversity, complexity, and ultimate unique nature of each mutational spectrum, composed of punctual mutations, chromosomal structural variations, and/or aneuploidies. The mutations produced in clb5Δ/CCNB1, mec1Δ/ATR, tel1Δ/ATM, and rad27Δ/FEN1 strains extensively reshape the genome, following a trajectory dependent on previous events. It comprises the transmission of unstable genomes that lead to colony mosaicisms. This comprehensive analytical approach of mutator defects provides a model to understand how genome variations might accumulate during clonal evolution of somatic cell populations, including tumor cells.

Identification of coding and non-coding mutational hotspots in cancer genomes.

PubMed

Piraino, Scott W; Furney, Simon J

2017-01-05

The identification of mutations that play a causal role in tumour development, so called "driver" mutations, is of critical importance for understanding how cancers form and how they might be treated. Several large cancer sequencing projects have identified genes that are recurrently mutated in cancer patients, suggesting a role in tumourigenesis. While the landscape of coding drivers has been extensively studied and many of the most prominent driver genes are well characterised, comparatively less is known about the role of mutations in the non-coding regions of the genome in cancer development. The continuing fall in genome sequencing costs has resulted in a concomitant increase in the number of cancer whole genome sequences being produced, facilitating systematic interrogation of both the coding and non-coding regions of cancer genomes. To examine the mutational landscapes of tumour genomes we have developed a novel method to identify mutational hotspots in tumour genomes using both mutational data and information on evolutionary conservation. We have applied our methodology to over 1300 whole cancer genomes and show that it identifies prominent coding and non-coding regions that are known or highly suspected to play a role in cancer. Importantly, we applied our method to the entire genome, rather than relying on predefined annotations (e.g. promoter regions) and we highlight recurrently mutated regions that may have resulted from increased exposure to mutational processes rather than selection, some of which have been identified previously as targets of selection. Finally, we implicate several pan-cancer and cancer-specific candidate non-coding regions, which could be involved in tumourigenesis. We have developed a framework to identify mutational hotspots in cancer genomes, which is applicable to the entire genome. This framework identifies known and novel coding and non-coding mutional hotspots and can be used to differentiate candidate driver regions from likely passenger regions susceptible to somatic mutation.

Finding cancer driver mutations in the era of big data research.

PubMed

Poulos, Rebecca C; Wong, Jason W H

2018-04-02

In the last decade, the costs of genome sequencing have decreased considerably. The commencement of large-scale cancer sequencing projects has enabled cancer genomics to join the big data revolution. One of the challenges still facing cancer genomics research is determining which are the driver mutations in an individual cancer, as these contribute only a small subset of the overall mutation profile of a tumour. Focusing primarily on somatic single nucleotide mutations in this review, we consider both coding and non-coding driver mutations, and discuss how such mutations might be identified from cancer sequencing datasets. We describe some of the tools and database that are available for the annotation of somatic variants and the identification of cancer driver genes. We also address the use of genome-wide variation in mutation load to establish background mutation rates from which to identify driver mutations under positive selection. Finally, we describe the ways in which mutational signatures can act as clues for the identification of cancer drivers, as these mutations may cause, or arise from, certain mutational processes. By defining the molecular changes responsible for driving cancer development, new cancer treatment strategies may be developed or novel preventative measures proposed.

Experimental evolution reveals genome-wide spectrum and dynamics of mutations in the rice blast fungus, Magnaporthe oryzae.

PubMed

Jeon, Junhyun; Choi, Jaeyoung; Lee, Gir-Won; Dean, Ralph A; Lee, Yong-Hwan

2013-01-01

Knowledge on mutation processes is central to interpreting genetic analysis data as well as understanding the underlying nature of almost all evolutionary phenomena. However, studies on genome-wide mutational spectrum and dynamics in fungal pathogens are scarce, hindering our understanding of their evolution and biology. Here, we explored changes in the phenotypes and genome sequences of the rice blast fungus Magnaporthe oryzae during the forced in vitro evolution by weekly transfer of cultures on artificial media. Through combination of experimental evolution with high throughput sequencing technology, we found that mutations accumulate rapidly prior to visible phenotypic changes and that both genetic drift and selection seem to contribute to shaping mutational landscape, suggesting the buffering capacity of fungal genome against mutations. Inference of mutational effects on phenotypes through the use of T-DNA insertion mutants suggested that at least some of the DNA sequence mutations are likely associated with the observed phenotypic changes. Furthermore, our data suggest oxidative damages and UV as major sources of mutation during subcultures. Taken together, our work revealed important properties of original source of variation in the genome of the rice blast fungus. We believe that these results provide not only insights into stability of pathogenicity and genome evolution in plant pathogenic fungi but also a model in which evolution of fungal pathogens in natura can be comparatively investigated.

Accumulation of Spontaneous Mutations in the Ciliate Tetrahymena thermophila

PubMed Central

Long, Hong-An; Paixão, Tiago; Azevedo, Ricardo B. R.; Zufall, Rebecca A.

2013-01-01

Knowledge of the rate and fitness effects of mutations is essential for understanding the process of evolution. Mutations are inherently difficult to study because they are rare and are frequently eliminated by natural selection. In the ciliate Tetrahymena thermophila, mutations can accumulate in the germline genome without being exposed to selection. We have conducted a mutation accumulation (MA) experiment in this species. Assuming that all mutations are deleterious and have the same effect, we estimate that the deleterious mutation rate per haploid germline genome per generation is U = 0.0047 (95% credible interval: 0.0015, 0.0125), and that germline mutations decrease fitness by s = 11% when expressed in a homozygous state (95% CI: 4.4%, 27%). We also estimate that deleterious mutations are partially recessive on average (h = 0.26; 95% CI: –0.022, 0.62) and that the rate of lethal mutations is <10% of the deleterious mutation rate. Comparisons between the observed evolutionary responses in the germline and somatic genomes and the results from individual-based simulations of MA suggest that the two genomes have similar mutational parameters. These are the first estimates of the deleterious mutation rate and fitness effects from the eukaryotic supergroup Chromalveolata and are within the range of those of other eukaryotes. PMID:23934880

MutSpec: a Galaxy toolbox for streamlined analyses of somatic mutation spectra in human and mouse cancer genomes.

PubMed

Ardin, Maude; Cahais, Vincent; Castells, Xavier; Bouaoun, Liacine; Byrnes, Graham; Herceg, Zdenko; Zavadil, Jiri; Olivier, Magali

2016-04-18

The nature of somatic mutations observed in human tumors at single gene or genome-wide levels can reveal information on past carcinogenic exposures and mutational processes contributing to tumor development. While large amounts of sequencing data are being generated, the associated analysis and interpretation of mutation patterns that may reveal clues about the natural history of cancer present complex and challenging tasks that require advanced bioinformatics skills. To make such analyses accessible to a wider community of researchers with no programming expertise, we have developed within the web-based user-friendly platform Galaxy a first-of-its-kind package called MutSpec. MutSpec includes a set of tools that perform variant annotation and use advanced statistics for the identification of mutation signatures present in cancer genomes and for comparing the obtained signatures with those published in the COSMIC database and other sources. MutSpec offers an accessible framework for building reproducible analysis pipelines, integrating existing methods and scripts developed in-house with publicly available R packages. MutSpec may be used to analyse data from whole-exome, whole-genome or targeted sequencing experiments performed on human or mouse genomes. Results are provided in various formats including rich graphical outputs. An example is presented to illustrate the package functionalities, the straightforward workflow analysis and the richness of the statistics and publication-grade graphics produced by the tool. MutSpec offers an easy-to-use graphical interface embedded in the popular Galaxy platform that can be used by researchers with limited programming or bioinformatics expertise to analyse mutation signatures present in cancer genomes. MutSpec can thus effectively assist in the discovery of complex mutational processes resulting from exogenous and endogenous carcinogenic insults.

Linkage disequilibrium between STRPs and SNPs across the human genome.

PubMed

Payseur, Bret A; Place, Michael; Weber, James L

2008-05-01

Patterns of linkage disequilibrium (LD) reveal the action of evolutionary processes and provide crucial information for association mapping of disease genes. Although recent studies have described the landscape of LD among single nucleotide polymorphisms (SNPs) from across the human genome, associations involving other classes of molecular variation remain poorly understood. In addition to recombination and population history, mutation rate and process are expected to shape LD. To test this idea, we measured associations between short-tandem-repeat polymorphisms (STRPs), which can mutate rapidly and recurrently, and SNPs in 721 regions across the human genome. We directly compared STRP-SNP LD with SNP-SNP LD from the same genomic regions in the human HapMap populations. The intensity of STRP-SNP LD, measured by the average of D', was reduced, consistent with the action of recurrent mutation. Nevertheless, a higher fraction of STRP-SNP pairs than SNP-SNP pairs showed significant LD, on both short (up to 50 kb) and long (cM) scales. These results reveal the substantial effects of mutational processes on LD at STRPs and provide important measures of the potential of STRPs for association mapping of disease genes.

C. elegans whole-genome sequencing reveals mutational signatures related to carcinogens and DNA repair deficiency

PubMed Central

Meier, Bettina; Cooke, Susanna L.; Weiss, Joerg; Bailly, Aymeric P.; Alexandrov, Ludmil B.; Marshall, John; Raine, Keiran; Maddison, Mark; Anderson, Elizabeth; Stratton, Michael R.; Campbell, Peter J.

2014-01-01

Mutation is associated with developmental and hereditary disorders, aging, and cancer. While we understand some mutational processes operative in human disease, most remain mysterious. We used Caenorhabditis elegans whole-genome sequencing to model mutational signatures, analyzing 183 worm populations across 17 DNA repair-deficient backgrounds propagated for 20 generations or exposed to carcinogens. The baseline mutation rate in C. elegans was approximately one per genome per generation, not overtly altered across several DNA repair deficiencies over 20 generations. Telomere erosion led to complex chromosomal rearrangements initiated by breakage–fusion–bridge cycles and completed by simultaneously acquired, localized clusters of breakpoints. Aflatoxin B1 induced substitutions of guanines in a GpC context, as observed in aflatoxin-induced liver cancers. Mutational burden increased with impaired nucleotide excision repair. Cisplatin and mechlorethamine, DNA crosslinking agents, caused dose- and genotype-dependent signatures among indels, substitutions, and rearrangements. Strikingly, both agents induced clustered rearrangements resembling “chromoanasynthesis,” a replication-based mutational signature seen in constitutional genomic disorders, suggesting that interstrand crosslinks may play a pathogenic role in such events. Cisplatin mutagenicity was most pronounced in xpf-1 mutants, suggesting that this gene critically protects cells against platinum chemotherapy. Thus, experimental model systems combined with genome sequencing can recapture and mechanistically explain mutational signatures associated with human disease. PMID:25030888

Spontaneous Mutation Rate in the Smallest Photosynthetic Eukaryotes

PubMed Central

Krasovec, Marc; Eyre-Walker, Adam; Sanchez-Ferandin, Sophie

2017-01-01

Abstract Mutation is the ultimate source of genetic variation, and knowledge of mutation rates is fundamental for our understanding of all evolutionary processes. High throughput sequencing of mutation accumulation lines has provided genome wide spontaneous mutation rates in a dozen model species, but estimates from nonmodel organisms from much of the diversity of life are very limited. Here, we report mutation rates in four haploid marine bacterial-sized photosynthetic eukaryotic algae; Bathycoccus prasinos, Ostreococcus tauri, Ostreococcus mediterraneus, and Micromonas pusilla. The spontaneous mutation rate between species varies from μ = 4.4 × 10−10 to 9.8 × 10−10 mutations per nucleotide per generation. Within genomes, there is a two-fold increase of the mutation rate in intergenic regions, consistent with an optimization of mismatch and transcription-coupled DNA repair in coding sequences. Additionally, we show that deviation from the equilibrium GC content increases the mutation rate by ∼2% to ∼12% because of a GC bias in coding sequences. More generally, the difference between the observed and equilibrium GC content of genomes explains some of the inter-specific variation in mutation rates. PMID:28379581

Merkel Cell Polyomavirus Exhibits Dominant Control of the Tumor Genome and Transcriptome in Virus-Associated Merkel Cell Carcinoma.

PubMed

Starrett, Gabriel J; Marcelus, Christina; Cantalupo, Paul G; Katz, Joshua P; Cheng, Jingwei; Akagi, Keiko; Thakuria, Manisha; Rabinowits, Guilherme; Wang, Linda C; Symer, David E; Pipas, James M; Harris, Reuben S; DeCaprio, James A

2017-01-03

Merkel cell polyomavirus is the primary etiological agent of the aggressive skin cancer Merkel cell carcinoma (MCC). Recent studies have revealed that UV radiation is the primary mechanism for somatic mutagenesis in nonviral forms of MCC. Here, we analyze the whole transcriptomes and genomes of primary MCC tumors. Our study reveals that virus-associated tumors have minimally altered genomes compared to non-virus-associated tumors, which are dominated by UV-mediated mutations. Although virus-associated tumors contain relatively small mutation burdens, they exhibit a distinct mutation signature with observable transcriptionally biased kataegic events. In addition, viral integration sites overlap focal genome amplifications in virus-associated tumors, suggesting a potential mechanism for these events. Collectively, our studies indicate that Merkel cell polyomavirus is capable of hijacking cellular processes and driving tumorigenesis to the same severity as tens of thousands of somatic genome alterations. A variety of mutagenic processes that shape the evolution of tumors are critical determinants of disease outcome. Here, we sequenced the entire genome of virus-positive and virus-negative primary Merkel cell carcinomas (MCCs), revealing distinct mutation spectra and corresponding expression profiles. Our studies highlight the strong effect that Merkel cell polyomavirus has on the divergent development of viral MCC compared to the somatic alterations that typically drive nonviral tumorigenesis. A more comprehensive understanding of the distinct mutagenic processes operative in viral and nonviral MCCs has implications for the effective treatment of these tumors. Copyright © 2017 Starrett et al.

Genome-wide chemical mutagenesis screens allow unbiased saturation of the cancer genome and identification of drug resistance mutations.

PubMed

Brammeld, Jonathan S; Petljak, Mia; Martincorena, Inigo; Williams, Steven P; Alonso, Luz Garcia; Dalmases, Alba; Bellosillo, Beatriz; Robles-Espinoza, Carla Daniela; Price, Stacey; Barthorpe, Syd; Tarpey, Patrick; Alifrangis, Constantine; Bignell, Graham; Vidal, Joana; Young, Jamie; Stebbings, Lucy; Beal, Kathryn; Stratton, Michael R; Saez-Rodriguez, Julio; Garnett, Mathew; Montagut, Clara; Iorio, Francesco; McDermott, Ultan

2017-04-01

Drug resistance is an almost inevitable consequence of cancer therapy and ultimately proves fatal for the majority of patients. In many cases, this is the consequence of specific gene mutations that have the potential to be targeted to resensitize the tumor. The ability to uniformly saturate the genome with point mutations without chromosome or nucleotide sequence context bias would open the door to identify all putative drug resistance mutations in cancer models. Here, we describe such a method for elucidating drug resistance mechanisms using genome-wide chemical mutagenesis allied to next-generation sequencing. We show that chemically mutagenizing the genome of cancer cells dramatically increases the number of drug-resistant clones and allows the detection of both known and novel drug resistance mutations. We used an efficient computational process that allows for the rapid identification of involved pathways and druggable targets. Such a priori knowledge would greatly empower serial monitoring strategies for drug resistance in the clinic as well as the development of trials for drug-resistant patients. © 2017 Brammeld et al.; Published by Cold Spring Harbor Laboratory Press.

The landscape of cancer genes and mutational processes in breast cancer

PubMed Central

Stephens, Philip J.; Tarpey, Patrick S.; Davies, Helen; Loo, Peter Van; Greenman, Chris; Wedge, David C.; Nik-Zainal, Serena; Martin, Sancha; Varela, Ignacio; Bignell, Graham R.; Yates, Lucy R.; Papaemmanuil, Elli; Beare, David; Butler, Adam; Cheverton, Angela; Gamble, John; Hinton, Jonathan; Jia, Mingming; Jayakumar, Alagu; Jones, David; Latimer, Calli; Lau, King Wai; McLaren, Stuart; McBride, David J.; Menzies, Andrew; Mudie, Laura; Raine, Keiran; Rad, Roland; Chapman, Michael Spencer; Teague, Jon; Easton, Douglas; Langerød, Anita; OSBREAC; Lee, Ming Ta Michael; Shen, Chen-Yang; Tee, Benita Tan Kiat; Huimin, Bernice Wong; Broeks, Annegien; Vargas, Ana Cristina; Turashvili, Gulisa; Martens, John; Fatima, Aquila; Miron, Penelope; Chin, Suet-Feung; Thomas, Gilles; Boyault, Sandrine; Mariani, Odette; Lakhani, Sunil R.; van de Vijver, Marc; van ’t Veer, Laura; Foekens, John; Desmedt, Christine; Sotiriou, Christos; Tutt, Andrew; Caldas, Carlos; Reis-Filho, Jorge S.; Aparicio, Samuel A. J. R.; Salomon, Anne Vincent; Børresen-Dale, Anne-Lise; Richardson, Andrea L.; Campbell, Peter J.; Futreal, P. Andrew; Stratton, Michael R.

2012-01-01

All cancers carry somatic mutations in their genomes. A subset, known as driver mutations, confer clonal selective advantage on cancer cells and are causally implicated in oncogenesis1, and the remainder are passenger mutations. The driver mutations and mutational processes operative in breast cancer have not yet been comprehensively explored. Here we examine the genomes of 100 tumours for somatic copy number changes and mutations in the coding exons of protein-coding genes. The number of somatic mutations varied markedly between individual tumours. We found strong correlations between mutation number, age at which cancer was diagnosed and cancer histological grade, and observed multiple mutational signatures, including one present in about ten per cent of tumours characterized by numerous mutations of cytosine at TpC dinucleotides. Driver mutations were identified in several new cancer genes including AKT2, ARID1B, CASP8, CDKN1B, MAP3K1, MAP3K13, NCOR1, SMARCD1 and TBX3. Among the 100 tumours, we found driver mutations in at least 40 cancer genes and 73 different combinations of mutated cancer genes. The results highlight the substantial genetic diversity underlying this common disease. PMID:22722201

Opposing Forces of A/T-Biased Mutations and G/C-Biased Gene Conversions Shape the Genome of the Nematode Pristionchus pacificus

PubMed Central

Weller, Andreas M.; Rödelsperger, Christian; Eberhardt, Gabi; Molnar, Ruxandra I.; Sommer, Ralf J.

2014-01-01

Base substitution mutations are a major source of genetic novelty and mutation accumulation line (MAL) studies revealed a nearly universal AT bias in de novo mutation spectra. While a comparison of de novo mutation spectra with the actual nucleotide composition in the genome suggests the existence of general counterbalancing mechanisms, little is known about the evolutionary and historical details of these opposing forces. Here, we correlate MAL-derived mutation spectra with patterns observed from population resequencing. Variation observed in natural populations has already been subject to evolutionary forces. Distinction between rare and common alleles, the latter of which are close to fixation and of presumably older age, can provide insight into mutational processes and their influence on genome evolution. We provide a genome-wide analysis of de novo mutations in 22 MALs of the nematode Pristionchus pacificus and compare the spectra with natural variants observed in resequencing of 104 natural isolates. MALs show an AT bias of 5.3, one of the highest values observed to date. In contrast, the AT bias in natural variants is much lower. Specifically, rare derived alleles show an AT bias of 2.4, whereas common derived alleles close to fixation show no AT bias at all. These results indicate the existence of a strong opposing force and they suggest that the GC content of the P. pacificus genome is in equilibrium. We discuss GC-biased gene conversion as a potential mechanism acting against AT-biased mutations. This study provides insight into genome evolution by combining MAL studies with natural variation. PMID:24414549

C. elegans whole-genome sequencing reveals mutational signatures related to carcinogens and DNA repair deficiency.

PubMed

Meier, Bettina; Cooke, Susanna L; Weiss, Joerg; Bailly, Aymeric P; Alexandrov, Ludmil B; Marshall, John; Raine, Keiran; Maddison, Mark; Anderson, Elizabeth; Stratton, Michael R; Gartner, Anton; Campbell, Peter J

2014-10-01

Mutation is associated with developmental and hereditary disorders, aging, and cancer. While we understand some mutational processes operative in human disease, most remain mysterious. We used Caenorhabditis elegans whole-genome sequencing to model mutational signatures, analyzing 183 worm populations across 17 DNA repair-deficient backgrounds propagated for 20 generations or exposed to carcinogens. The baseline mutation rate in C. elegans was approximately one per genome per generation, not overtly altered across several DNA repair deficiencies over 20 generations. Telomere erosion led to complex chromosomal rearrangements initiated by breakage-fusion-bridge cycles and completed by simultaneously acquired, localized clusters of breakpoints. Aflatoxin B1 induced substitutions of guanines in a GpC context, as observed in aflatoxin-induced liver cancers. Mutational burden increased with impaired nucleotide excision repair. Cisplatin and mechlorethamine, DNA crosslinking agents, caused dose- and genotype-dependent signatures among indels, substitutions, and rearrangements. Strikingly, both agents induced clustered rearrangements resembling "chromoanasynthesis," a replication-based mutational signature seen in constitutional genomic disorders, suggesting that interstrand crosslinks may play a pathogenic role in such events. Cisplatin mutagenicity was most pronounced in xpf-1 mutants, suggesting that this gene critically protects cells against platinum chemotherapy. Thus, experimental model systems combined with genome sequencing can recapture and mechanistically explain mutational signatures associated with human disease. © 2014 Meier et al.; Published by Cold Spring Harbor Laboratory Press.

Mapping Second Chromosome Mutations to Defined Genomic Regions in Drosophila melanogaster

PubMed Central

Kahsai, Lily; Cook, Kevin R.

2017-01-01

Hundreds of Drosophila melanogaster stocks are currently maintained at the Bloomington Drosophila Stock Center with mutations that have not been associated with sequence-defined genes. They have been preserved because they have interesting loss-of-function phenotypes. The experimental value of these mutations would be increased by tying them to specific genomic intervals so that geneticists can more easily associate them with annotated genes. Here, we report the mapping of 85 second chromosome complementation groups in the Bloomington collection to specific, small clusters of contiguous genes or individual genes in the sequenced genome. This information should prove valuable to Drosophila geneticists interested in processes associated with particular phenotypes and those searching for mutations affecting specific sequence-defined genes. PMID:29066472

Tumor Hypoxia and Genetic Alterations in Sporadic Cancers

PubMed Central

Koi, Minoru; Boland, C.R.

2011-01-01

The cancer genome contains many gene alterations. How cancer cells acquire these alterations is a matter for discussion. One hypothesis is that cancer cells obtain mutations in genome stability genes at an early stage of tumor development, which results in genetic instability and generates a gene pool that enhances cellular proliferation and survival. Another hypothesis puts its emphasis on the natural selection of gene mutations for fitness. Recent data for systematic cancer genome sequencing shows that mutations in stability genes are rare in human sporadic cancers. Instead, many “passenger” mutations that do not drive the carcinogenesis process have been found in the cancer genome. Both the hypotheses mentioned above fall short in explaining recent data. Recently, many studies demonstrate the role of the tumor microenvironment, especially hypoxia and reoxygenation, in genetic instability. In this review, literature will be presented which supports a third hypothesis, i.e. that hypoxia/re-oxygenation induces genetic instability. PMID:21272156

Pathway and network analysis of cancer genomes.

PubMed

Creixell, Pau; Reimand, Jüri; Haider, Syed; Wu, Guanming; Shibata, Tatsuhiro; Vazquez, Miguel; Mustonen, Ville; Gonzalez-Perez, Abel; Pearson, John; Sander, Chris; Raphael, Benjamin J; Marks, Debora S; Ouellette, B F Francis; Valencia, Alfonso; Bader, Gary D; Boutros, Paul C; Stuart, Joshua M; Linding, Rune; Lopez-Bigas, Nuria; Stein, Lincoln D

2015-07-01

Genomic information on tumors from 50 cancer types cataloged by the International Cancer Genome Consortium (ICGC) shows that only a few well-studied driver genes are frequently mutated, in contrast to many infrequently mutated genes that may also contribute to tumor biology. Hence there has been large interest in developing pathway and network analysis methods that group genes and illuminate the processes involved. We provide an overview of these analysis techniques and show where they guide mechanistic and translational investigations.

Increasing Nucleosome Occupancy Is Correlated with an Increasing Mutation Rate so Long as DNA Repair Machinery Is Intact

PubMed Central

Taylor, Jared F.; Khattab, Omar S.; Chen, Yu-Han; Chen, Yumay; Jacobsen, Steven E.; Wang, Ping H.

2015-01-01

Deciphering the multitude of epigenomic and genomic factors that influence the mutation rate is an area of great interest in modern biology. Recently, chromatin has been shown to play a part in this process. To elucidate this relationship further, we integrated our own ultra-deep sequenced human nucleosomal DNA data set with a host of published human genomic and cancer genomic data sets. Our results revealed, that differences in nucleosome occupancy are associated with changes in base-specific mutation rates. Increasing nucleosome occupancy is associated with an increasing transition to transversion ratio and an increased germline mutation rate within the human genome. Additionally, cancer single nucleotide variants and microindels are enriched within nucleosomes and both the coding and non-coding cancer mutation rate increases with increasing nucleosome occupancy. There is an enrichment of cancer indels at the theoretical start (74 bp) and end (115 bp) of linker DNA between two nucleosomes. We then hypothesized that increasing nucleosome occupancy decreases access to DNA by DNA repair machinery and could account for the increasing mutation rate. Such a relationship should not exist in DNA repair knockouts, and we thus repeated our analysis in DNA repair machinery knockouts to test our hypothesis. Indeed, our results revealed no correlation between increasing nucleosome occupancy and increasing mutation rate in DNA repair knockouts. Our findings emphasize the linkage of the genome and epigenome through the nucleosome whose properties can affect genome evolution and genetic aberrations such as cancer. PMID:26308346

Whole-genome landscapes of major melanoma subtypes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hayward, Nicholas K.; Wilmott, James S.; Waddell, Nicola

Melanoma of the skin is a common cancer only in Europeans, whereas it arises in internal body surfaces (mucosal sites) and on the hands and feet (acral sites) in people throughout the world. We report analysis of whole-genome sequences from cutaneous, acral and mucosal subtypes of melanoma. The heavily mutated landscape of coding and non-coding mutations in cutaneous melanoma resolved novel signatures of mutagenesis attributable to ultraviolet radiation. But, acral and mucosal melanomas were dominated by structural changes and mutation signatures of unknown aetiology, not previously identified in melanoma. The number of genes affected by recurrent mutations disrupting non-coding sequencesmore » was similar to that affected by recurrent mutations to coding sequences. Significantly mutated genes included BRAF, CDKN2A, NRAS and TP53 in cutaneous melanoma, BRAF, NRAS and NF1 in acral melanoma and SF3B1 in mucosal melanoma. Mutations affecting the TERT promoter were the most frequent of all; however, neither they nor ATRX mutations, which correlate with alternative telomere lengthening, were associated with greater telomere length. In most cases, melanomas had potentially actionable mutations, most in components of the mitogen-activated protein kinase and phosphoinositol kinase pathways. The whole-genome mutation landscape of melanoma reveals diverse carcinogenic processes across its subtypes, some unrelated to sun exposure, and extends potential involvement of the non-coding genome in its pathogenesis.« less

Whole-genome landscapes of major melanoma subtypes

DOE PAGES

Hayward, Nicholas K.; Wilmott, James S.; Waddell, Nicola; ...

2017-05-03

Melanoma of the skin is a common cancer only in Europeans, whereas it arises in internal body surfaces (mucosal sites) and on the hands and feet (acral sites) in people throughout the world. We report analysis of whole-genome sequences from cutaneous, acral and mucosal subtypes of melanoma. The heavily mutated landscape of coding and non-coding mutations in cutaneous melanoma resolved novel signatures of mutagenesis attributable to ultraviolet radiation. But, acral and mucosal melanomas were dominated by structural changes and mutation signatures of unknown aetiology, not previously identified in melanoma. The number of genes affected by recurrent mutations disrupting non-coding sequencesmore » was similar to that affected by recurrent mutations to coding sequences. Significantly mutated genes included BRAF, CDKN2A, NRAS and TP53 in cutaneous melanoma, BRAF, NRAS and NF1 in acral melanoma and SF3B1 in mucosal melanoma. Mutations affecting the TERT promoter were the most frequent of all; however, neither they nor ATRX mutations, which correlate with alternative telomere lengthening, were associated with greater telomere length. In most cases, melanomas had potentially actionable mutations, most in components of the mitogen-activated protein kinase and phosphoinositol kinase pathways. The whole-genome mutation landscape of melanoma reveals diverse carcinogenic processes across its subtypes, some unrelated to sun exposure, and extends potential involvement of the non-coding genome in its pathogenesis.« less

Mutation of Breast Cancer Cell Genomic DNA by APOBEC3B

DTIC Science & Technology

2013-09-01

lethal prostate cancers. Proc. Natl Acad. Sci. USA 108, 17087–17092 (2011). 5. Parsons, D. W. et al. The genetic landscape of the childhood cancer...7. Stransky, N. et al. The mutational landscape of head and neck squamous cell carcinoma. Science 333, 1157–1160 (2011). 8. Nik-Zainal, S. et al...Mutational processes molding the genomes of 21 breast cancers. Cell 149, 979–993 (2012). 9. Stephens, P. J. et al. The landscape of cancer genes and

Thiol peroxidase deficiency leads to increased mutational load and decreased fitness in Saccharomyces cerevisiae.

PubMed

Kaya, Alaattin; Lobanov, Alexei V; Gerashchenko, Maxim V; Koren, Amnon; Fomenko, Dmitri E; Koc, Ahmet; Gladyshev, Vadim N

2014-11-01

Thiol peroxidases are critical enzymes in the redox control of cellular processes that function by reducing low levels of hydroperoxides and regulating redox signaling. These proteins were also shown to regulate genome stability, but how their dysfunction affects the actual mutations in the genome is not known. Saccharomyces cerevisiae has eight thiol peroxidases of glutathione peroxidase and peroxiredoxin families, and the mutant lacking all these genes (∆8) is viable. In this study, we employed two independent ∆8 isolates to analyze the genome-wide mutation spectrum that results from deficiency in these enzymes. Deletion of these genes was accompanied by a dramatic increase in point mutations, many of which clustered in close proximity and scattered throughout the genome, suggesting strong mutational bias. We further subjected multiple lines of wild-type and ∆8 cells to long-term mutation accumulation, followed by genome sequencing and phenotypic characterization. ∆8 lines showed a significant increase in nonrecurrent point mutations and indels. The original ∆8 cells exhibited reduced growth rate and decreased life span, which were further reduced in all ∆8 mutation accumulation lines. Although the mutation spectrum of the two independent isolates was different, similar patterns of gene expression were observed, suggesting the direct contribution of thiol peroxidases to the observed phenotypes. Expression of a single thiol peroxidase could partially restore the growth phenotype of ∆8 cells. This study shows how deficiency in nonessential, yet critical and conserved oxidoreductase function, leads to increased mutational load and decreased fitness. Copyright © 2014 by the Genetics Society of America.

Genomic Evolution of Breast Cancer Metastasis and Relapse

DOE PAGES

Yates, Lucy R.; Knappskog, Stian; Wedge, David; ...

2017-08-14

Patterns of genomic evolution between primary and metastatic breast cancer have not been studied in large numbers, despite patients with metastatic breast cancer having dismal survival. We sequenced whole genomes or a panel of 365 genes on 299 samples from 170 patients with locally relapsed or metastatic breast cancer. Several lines of analysis indicate that clones seeding metastasis or relapse disseminate late from primary tumors, but continue to acquire mutations, mostly accessing the same mutational processes active in the primary tumor. Most distant metastases acquired driver mutations not seen in the primary tumor, drawing from a wider repertoire of cancermore » genes than early drivers. Lastly, these include a number of clinically actionable alterations and mutations inactivating SWI-SNF and JAK2-STAT3 pathways.« less

Genomic Evolution of Breast Cancer Metastasis and Relapse

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yates, Lucy R.; Knappskog, Stian; Wedge, David

Patterns of genomic evolution between primary and metastatic breast cancer have not been studied in large numbers, despite patients with metastatic breast cancer having dismal survival. We sequenced whole genomes or a panel of 365 genes on 299 samples from 170 patients with locally relapsed or metastatic breast cancer. Several lines of analysis indicate that clones seeding metastasis or relapse disseminate late from primary tumors, but continue to acquire mutations, mostly accessing the same mutational processes active in the primary tumor. Most distant metastases acquired driver mutations not seen in the primary tumor, drawing from a wider repertoire of cancermore » genes than early drivers. Lastly, these include a number of clinically actionable alterations and mutations inactivating SWI-SNF and JAK2-STAT3 pathways.« less

Understanding mutagenesis through delineation of mutational signatures in human cancer

DOE PAGES

Petljak, Mia; Alexandrov, Ludmil B.

2016-05-04

Each individual cell within a human body acquires a certain number of somatic mutations during a course of its lifetime. These mutations originate from a wide spectra of both endogenous and exogenous mutational processes that leave distinct patterns of mutations, termed mutational signatures, embedded within the genomes of all cells. In recent years, the vast amount of data produced by sequencing of cancer genomes was coupled with novel mathematical models and computational tools to generate the first comprehensive map of mutational signatures in human cancer. Up to date, >30 distinct mutational signatures have been identified, and etiologies have been proposedmore » for many of them. This paper provides a brief historical background on examination of mutational patterns in human cancer, summarizes the knowledge accumulated since introducing the concept of mutational signatures and discusses their future potential applications and perspectives within the field.« less

Determinants of spontaneous mutation in the bacterium Escherichia coli as revealed by whole-genome sequencing

PubMed Central

Foster, Patricia L.; Lee, Heewook; Popodi, Ellen; Townes, Jesse P.; Tang, Haixu

2015-01-01

A complete understanding of evolutionary processes requires that factors determining spontaneous mutation rates and spectra be identified and characterized. Using mutation accumulation followed by whole-genome sequencing, we found that the mutation rates of three widely diverged commensal Escherichia coli strains differ only by about 50%, suggesting that a rate of 1–2 × 10−3 mutations per generation per genome is common for this bacterium. Four major forces are postulated to contribute to spontaneous mutations: intrinsic DNA polymerase errors, endogenously induced DNA damage, DNA damage caused by exogenous agents, and the activities of error-prone polymerases. To determine the relative importance of these factors, we studied 11 strains, each defective for a major DNA repair pathway. The striking result was that only loss of the ability to prevent or repair oxidative DNA damage significantly impacted mutation rates or spectra. These results suggest that, with the exception of oxidative damage, endogenously induced DNA damage does not perturb the overall accuracy of DNA replication in normally growing cells and that repair pathways may exist primarily to defend against exogenously induced DNA damage. The thousands of mutations caused by oxidative damage recovered across the entire genome revealed strong local-sequence biases of these mutations. Specifically, we found that the identity of the 3′ base can affect the mutability of a purine by oxidative damage by as much as eightfold. PMID:26460006

Genome Sequencing and Analysis of the Tasmanian Devil and Its Transmissible Cancer

PubMed Central

Murchison, Elizabeth P.; Schulz-Trieglaff, Ole B.; Ning, Zemin; Alexandrov, Ludmil B.; Bauer, Markus J.; Fu, Beiyuan; Hims, Matthew; Ding, Zhihao; Ivakhno, Sergii; Stewart, Caitlin; Ng, Bee Ling; Wong, Wendy; Aken, Bronwen; White, Simon; Alsop, Amber; Becq, Jennifer; Bignell, Graham R.; Cheetham, R. Keira; Cheng, William; Connor, Thomas R.; Cox, Anthony J.; Feng, Zhi-Ping; Gu, Yong; Grocock, Russell J.; Harris, Simon R.; Khrebtukova, Irina; Kingsbury, Zoya; Kowarsky, Mark; Kreiss, Alexandre; Luo, Shujun; Marshall, John; McBride, David J.; Murray, Lisa; Pearse, Anne-Maree; Raine, Keiran; Rasolonjatovo, Isabelle; Shaw, Richard; Tedder, Philip; Tregidgo, Carolyn; Vilella, Albert J.; Wedge, David C.; Woods, Gregory M.; Gormley, Niall; Humphray, Sean; Schroth, Gary; Smith, Geoffrey; Hall, Kevin; Searle, Stephen M.J.; Carter, Nigel P.; Papenfuss, Anthony T.; Futreal, P. Andrew; Campbell, Peter J.; Yang, Fengtang; Bentley, David R.; Evers, Dirk J.; Stratton, Michael R.

2012-01-01

Summary The Tasmanian devil (Sarcophilus harrisii), the largest marsupial carnivore, is endangered due to a transmissible facial cancer spread by direct transfer of living cancer cells through biting. Here we describe the sequencing, assembly, and annotation of the Tasmanian devil genome and whole-genome sequences for two geographically distant subclones of the cancer. Genomic analysis suggests that the cancer first arose from a female Tasmanian devil and that the clone has subsequently genetically diverged during its spread across Tasmania. The devil cancer genome contains more than 17,000 somatic base substitution mutations and bears the imprint of a distinct mutational process. Genotyping of somatic mutations in 104 geographically and temporally distributed Tasmanian devil tumors reveals the pattern of evolution and spread of this parasitic clonal lineage, with evidence of a selective sweep in one geographical area and persistence of parallel lineages in other populations. PaperClip PMID:22341448

Pathways Impacted by Genomic Alterations in Pulmonary Carcinoid Tumors.

PubMed

Asiedu, Michael K; Thomas, Charles F; Dong, Jie; Schulte, Sandra C; Khadka, Prasidda; Sun, Zhifu; Kosari, Farhad; Jen, Jin; Molina, Julian; Vasmatzis, George; Kuang, Ray; Aubry, Marie Christine; Yang, Ping; Wigle, Dennis A

2018-04-01

Purpose: Pulmonary carcinoid tumors account for up to 5% of all lung malignancies in adults, comprise 30% of all carcinoid malignancies, and are defined histologically as typical carcinoid (TC) and atypical carcinoid (AC) tumors. The role of specific genomic alterations in the pathogenesis of pulmonary carcinoid tumors remains poorly understood. We sought to identify genomic alterations and pathways that are deregulated in these tumors to find novel therapeutic targets for pulmonary carcinoid tumors. Experimental Design: We performed integrated genomic analysis of carcinoid tumors comprising whole genome and exome sequencing, mRNA expression profiling and SNP genotyping of specimens from normal lung, TC and AC, and small cell lung carcinoma (SCLC) to fully represent the lung neuroendocrine tumor spectrum. Results: Analysis of sequencing data found recurrent mutations in cancer genes including ATP1A2, CNNM1, MACF1, RAB38, NF1, RAD51C, TAF1L, EPHB2, POLR3B , and AGFG1 The mutated genes are involved in biological processes including cellular metabolism, cell division cycle, cell death, apoptosis, and immune regulation. The top most significantly mutated genes were TMEM41B, DEFB127, WDYHV1, and TBPL1 Pathway analysis of significantly mutated and cancer driver genes implicated MAPK/ERK and amyloid beta precursor protein (APP) pathways whereas analysis of CNV and gene expression data suggested deregulation of the NF-κB and MAPK/ERK pathways. The mutation signature was predominantly C>T and T>C transitions with a minor contribution of T>G transversions. Conclusions: This study identified mutated genes affecting cancer relevant pathways and biological processes that could provide opportunities for developing targeted therapies for pulmonary carcinoid tumors. Clin Cancer Res; 24(7); 1691-704. ©2018 AACR . ©2018 American Association for Cancer Research.

RNA splicing factors as oncoproteins and tumor suppressors

PubMed Central

Dvinge, Heidi; Kim, Eunhee; Abdel-Wahab, Omar; Bradley, Robert K.

2016-01-01

Preface The recent genomic characterization of cancers has revealed recurrent somatic point mutations and copy number changes affecting genes encoding RNA splicing factors. Initial studies of these ‘spliceosomal mutations’ suggest that the proteins bearing these mutations exhibit altered splice site and/or exon recognition preferences relative to their wild-type counterparts, resulting in cancer-specific mis-splicing. Such changes in the splicing machinery may create novel vulnerabilities in cancer cells that can be therapeutically exploited using compounds that can influence the splicing process. Further studies to dissect the biochemical, genomic, and biological effects of spliceosomal mutations are critical for the development of cancer therapies targeted to these mutations. PMID:27282250

An Organismal CNV Mutator Phenotype Restricted to Early Human Development.

PubMed

Liu, Pengfei; Yuan, Bo; Carvalho, Claudia M B; Wuster, Arthur; Walter, Klaudia; Zhang, Ling; Gambin, Tomasz; Chong, Zechen; Campbell, Ian M; Coban Akdemir, Zeynep; Gelowani, Violet; Writzl, Karin; Bacino, Carlos A; Lindsay, Sarah J; Withers, Marjorie; Gonzaga-Jauregui, Claudia; Wiszniewska, Joanna; Scull, Jennifer; Stankiewicz, Paweł; Jhangiani, Shalini N; Muzny, Donna M; Zhang, Feng; Chen, Ken; Gibbs, Richard A; Rautenstrauss, Bernd; Cheung, Sau Wai; Smith, Janice; Breman, Amy; Shaw, Chad A; Patel, Ankita; Hurles, Matthew E; Lupski, James R

2017-02-23

De novo copy number variants (dnCNVs) arising at multiple loci in a personal genome have usually been considered to reflect cancer somatic genomic instabilities. We describe a multiple dnCNV (MdnCNV) phenomenon in which individuals with genomic disorders carry five to ten constitutional dnCNVs. These CNVs originate from independent formation incidences, are predominantly tandem duplications or complex gains, exhibit breakpoint junction features reminiscent of replicative repair, and show increased de novo point mutations flanking the rearrangement junctions. The active CNV mutation shower appears to be restricted to a transient perizygotic period. We propose that a defect in the CNV formation process is responsible for the "CNV-mutator state," and this state is dampened after early embryogenesis. The constitutional MdnCNV phenomenon resembles chromosomal instability in various cancers. Investigations of this phenomenon may provide unique access to understanding genomic disorders, structural variant mutagenesis, human evolution, and cancer biology. Copyright © 2017 Elsevier Inc. All rights reserved.

Multiple mutant clones in blood rarely coexist

NASA Astrophysics Data System (ADS)

Dingli, David; Pacheco, Jorge M.; Traulsen, Arne

2008-02-01

Leukemias arise due to mutations in the genome of hematopoietic (blood) cells. Hematopoiesis has a multicompartment architecture, with cells exhibiting different rates of replication and differentiation. At the root of this process, one finds a small number of stem cells, and hence the description of the mutation-selection dynamics of blood cells calls for a stochastic approach. We use stochastic dynamics to investigate to which extent acquired hematopoietic disorders are associated with mutations of single or multiple genes within developing blood cells. Our analysis considers the appearance of mutations both in the stem cell compartment as well as in more committed compartments. We conclude that in the absence of genomic instability, acquired hematopoietic disorders due to mutations in multiple genes are most likely very rare events, as multiple mutations typically require much longer development times compared to those associated with a single mutation.

Reconstruction of thermotolerant yeast by one-point mutation identified through whole-genome analyses of adaptively-evolved strains.

PubMed

Satomura, Atsushi; Miura, Natsuko; Kuroda, Kouichi; Ueda, Mitsuyoshi

2016-03-17

Saccharomyces cerevisiae is used as a host strain in bioproduction, because of its rapid growth, ease of genetic manipulation, and high reducing capacity. However, the heat produced during the fermentation processes inhibits the biological activities and growth of the yeast cells. We performed whole-genome sequencing of 19 intermediate strains previously obtained during adaptation experiments under heat stress; 49 mutations were found in the adaptation steps. Phylogenetic tree revealed at least five events in which these strains had acquired mutations in the CDC25 gene. Reconstructed CDC25 point mutants based on a parental strain had acquired thermotolerance without any growth defects. These mutations led to the downregulation of the cAMP-dependent protein kinase (PKA) signaling pathway, which controls a variety of processes such as cell-cycle progression and stress tolerance. The one-point mutations in CDC25 were involved in the global transcriptional regulation through the cAMP/PKA pathway. Additionally, the mutations enabled efficient ethanol fermentation at 39 °C, suggesting that the one-point mutations in CDC25 may contribute to bioproduction.

TARGET Researchers Identify Mutations in SIX1/2 and microRNA Processing Genes in Favorable Histology Wilms Tumor | Office of Cancer Genomics

Cancer.gov

TARGET researchers molecularly characterized favorable histology Wilms tumor (FHWT), a pediatric renal cancer. Comprehensive genome and transcript analyses revealed single-nucleotide substitution/deletion mutations in microRNA processing genes (15% of FHWT patients) and Sine Oculis Homeobox Homolog 1/2 (SIX1/2) genes (7% of FHWT patients). SIX1/2 genes play a critical role in renal development and were not previously associated with FHWT, thus presenting a novel role for SIX1/2 pathway aberrations in this disease.

Mutation as a Stress Response and the Regulation of Evolvability

PubMed Central

Galhardo, Rodrigo S.; Hastings, P. J.; Rosenberg, Susan M.

2010-01-01

Our concept of a stable genome is evolving to one in which genomes are plastic and responsive to environmental changes. Growing evidence shows that a variety of environmental stresses induce genomic instability in bacteria, yeast, and human cancer cells, generating occasional fitter mutants and potentially accelerating adaptive evolution. The emerging molecular mechanisms of stress-induced mutagenesis vary but share telling common components that underscore two common themes. The first is the regulation of mutagenesis in time by cellular stress responses, which promote random mutations specifically when cells are poorly adapted to their environments, i.e., when they are stressed. A second theme is the possible restriction of random mutagenesis in genomic space, achieved via coupling of mutation-generating machinery to local events such as DNA-break repair or transcription. Such localization may minimize accumulation of deleterious mutations in the genomes of rare fitter mutants, and promote local concerted evolution. Although mutagenesis induced by stresses other than direct damage to DNA was previously controversial, evidence for the existence of various stress-induced mutagenesis programs is now overwhelming and widespread. Such mechanisms probably fuel evolution of microbial pathogenesis and antibiotic-resistance, and tumor progression and chemotherapy resistance, all of which occur under stress, driven by mutations. The emerging commonalities in stress-induced-mutation mechanisms provide hope for new therapeutic interventions for all of these processes. PMID:17917874

Darwinism for the Genomic Age: Connecting Mutation to Diversification

PubMed Central

Hua, Xia; Bromham, Lindell

2017-01-01

A growing body of evidence suggests that rates of diversification of biological lineages are correlated with differences in genome-wide mutation rate. Given that most research into differential patterns of diversification rate have focused on species traits or ecological parameters, a connection to the biochemical processes of genome change is an unexpected observation. While the empirical evidence for a significant association between mutation rate and diversification rate is mounting, there has been less effort in explaining the factors that mediate this connection between genetic change and species richness. Here we draw together empirical studies and theoretical concepts that may help to build links in the explanatory chain that connects mutation to diversification. First we consider the way that mutation rates vary between species. We then explore how differences in mutation rates have flow-through effects to the rate at which populations acquire substitutions, which in turn influences the speed at which populations become reproductively isolated from each other due to the acquisition of genomic incompatibilities. Since diversification rate is commonly measured from phylogenetic analyses, we propose a conceptual approach for relating events of reproductive isolation to bifurcations on molecular phylogenies. As we examine each of these relationships, we consider theoretical models that might shine a light on the observed association between rate of molecular evolution and diversification rate, and critically evaluate the empirical evidence for these links, focusing on phylogenetic comparative studies. Finally, we ask whether we are getting closer to a real understanding of the way that the processes of molecular evolution connect to the observable patterns of diversification. PMID:28224003

Darwinism for the Genomic Age: Connecting Mutation to Diversification.

PubMed

Hua, Xia; Bromham, Lindell

2017-01-01

A growing body of evidence suggests that rates of diversification of biological lineages are correlated with differences in genome-wide mutation rate. Given that most research into differential patterns of diversification rate have focused on species traits or ecological parameters, a connection to the biochemical processes of genome change is an unexpected observation. While the empirical evidence for a significant association between mutation rate and diversification rate is mounting, there has been less effort in explaining the factors that mediate this connection between genetic change and species richness. Here we draw together empirical studies and theoretical concepts that may help to build links in the explanatory chain that connects mutation to diversification. First we consider the way that mutation rates vary between species. We then explore how differences in mutation rates have flow-through effects to the rate at which populations acquire substitutions, which in turn influences the speed at which populations become reproductively isolated from each other due to the acquisition of genomic incompatibilities. Since diversification rate is commonly measured from phylogenetic analyses, we propose a conceptual approach for relating events of reproductive isolation to bifurcations on molecular phylogenies. As we examine each of these relationships, we consider theoretical models that might shine a light on the observed association between rate of molecular evolution and diversification rate, and critically evaluate the empirical evidence for these links, focusing on phylogenetic comparative studies. Finally, we ask whether we are getting closer to a real understanding of the way that the processes of molecular evolution connect to the observable patterns of diversification.

Mutational signatures of DNA mismatch repair deficiency in C. elegans and human cancers.

PubMed

Meier, Bettina; Volkova, Nadezda V; Hong, Ye; Schofield, Pieta; Campbell, Peter J; Gerstung, Moritz; Gartner, Anton

2018-05-01

Throughout their lifetime, cells are subject to extrinsic and intrinsic mutational processes leaving behind characteristic signatures in the genome. DNA mismatch repair (MMR) deficiency leads to hypermutation and is found in different cancer types. Although it is possible to associate mutational signatures extracted from human cancers with possible mutational processes, the exact causation is often unknown. Here, we use C. elegans genome sequencing of pms-2 and mlh-1 knockouts to reveal the mutational patterns linked to C. elegans MMR deficiency and their dependency on endogenous replication errors and errors caused by deletion of the polymerase ε subunit pole-4 Signature extraction from 215 human colorectal and 289 gastric adenocarcinomas revealed three MMR-associated signatures, one of which closely resembles the C. elegans MMR spectrum and strongly discriminates microsatellite stable and unstable tumors (AUC = 98%). A characteristic difference between human and C. elegans MMR deficiency is the lack of elevated levels of N C G > NTG mutations in C. elegans, likely caused by the absence of cytosine (CpG) methylation in worms . The other two human MMR signatures may reflect the interaction between MMR deficiency and other mutagenic processes, but their exact cause remains unknown. In summary, combining information from genetically defined models and cancer samples allows for better aligning mutational signatures to causal mutagenic processes. © 2018 Meier et al.; Published by Cold Spring Harbor Laboratory Press.

Recurrent mutation of IGF signalling genes and distinct patterns of genomic rearrangement in osteosarcoma

DOE PAGES

Behjati, Sam; Tarpey, Patrick S.; Haase, Kerstin; ...

2017-06-23

Osteosarcoma is a primary malignancy of bone that affects children and adults. Here, we present the largest sequencing study of osteosarcoma to date, comprising 112 childhood and adult tumours encompassing all major histological subtypes. A key finding of our study is the identification of mutations in insulin-like growth factor (IGF) signalling genes in 8/112 (7%) of cases. We validate this observation using fluorescence in situ hybridization (FISH) in an additional 87 osteosarcomas, with IGF1 receptor (IGF1R) amplification observed in 14% of tumours. These findings may inform patient selection in future trials of IGF1R inhibitors in osteosarcoma. Analysing patterns of mutation,more » we identify distinct rearrangement profiles including a process characterized by chromothripsis and amplification. This process operates recurrently at discrete genomic regions and generates driver mutations. Lastly, it may represent an age-independent mutational mechanism that contributes to the development of osteosarcoma in children and adults alike.« less

Recurrent mutation of IGF signalling genes and distinct patterns of genomic rearrangement in osteosarcoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Behjati, Sam; Tarpey, Patrick S.; Haase, Kerstin

Osteosarcoma is a primary malignancy of bone that affects children and adults. Here, we present the largest sequencing study of osteosarcoma to date, comprising 112 childhood and adult tumours encompassing all major histological subtypes. A key finding of our study is the identification of mutations in insulin-like growth factor (IGF) signalling genes in 8/112 (7%) of cases. We validate this observation using fluorescence in situ hybridization (FISH) in an additional 87 osteosarcomas, with IGF1 receptor (IGF1R) amplification observed in 14% of tumours. These findings may inform patient selection in future trials of IGF1R inhibitors in osteosarcoma. Analysing patterns of mutation,more » we identify distinct rearrangement profiles including a process characterized by chromothripsis and amplification. This process operates recurrently at discrete genomic regions and generates driver mutations. Lastly, it may represent an age-independent mutational mechanism that contributes to the development of osteosarcoma in children and adults alike.« less

Rapid identification of kidney cyst mutations by whole exome sequencing in zebrafish

PubMed Central

Ryan, Sean; Willer, Jason; Marjoram, Lindsay; Bagwell, Jennifer; Mankiewicz, Jamie; Leshchiner, Ignaty; Goessling, Wolfram; Bagnat, Michel; Katsanis, Nicholas

2013-01-01

Forward genetic approaches in zebrafish have provided invaluable information about developmental processes. However, the relative difficulty of mapping and isolating mutations has limited the number of new genetic screens. Recent improvements in the annotation of the zebrafish genome coupled to a reduction in sequencing costs prompted the development of whole genome and RNA sequencing approaches for gene discovery. Here we describe a whole exome sequencing (WES) approach that allows rapid and cost-effective identification of mutations. We used our WES methodology to isolate four mutations that cause kidney cysts; we identified novel alleles in two ciliary genes as well as two novel mutants. The WES approach described here does not require specialized infrastructure or training and is therefore widely accessible. This methodology should thus help facilitate genetic screens and expedite the identification of mutants that can inform basic biological processes and the causality of genetic disorders in humans. PMID:24130329

[The role of remodeling complexes CHD1 and ISWI in spontaneous and UV-induced mutagenesis control in yeast Saccharomyces cerevisiae].

PubMed

Evstiukhina, T A; Alekseeva, E A; Fedorov, D V; Peshekhonov, V T; Korolev, V G

2017-02-01

Chromatin remodulators are special multiprotein machines capable of transforming the structure, constitution, and positioning of nucleosomes on DNA. Biochemical activities of remodeling complexes CHD1 and ISWI from the SWI2/SNF2 family are well established. They ensure correct positioning of nucleosomes along the genome, which is probably critical for genome stability, in particular, after action of polymerases, repair enzymes, and transcription. In this paper, we show that single mutations in genes ISW1, ISW2, and CHD1 weakly affect repair and mutagenic processes in yeast cells. At the same time, there are differences in the effect of these mutations on spontaneous mutation levels, which indicates certain specificity of action of protein complexes ISW1, ISW2, and CHD1 on expression of different genes that control repair and mutation processes in yeast.

Mutation Rates across Budding Yeast Chromosome VI Are Correlated with Replication Timing

PubMed Central

Lang, Gregory I.; Murray, Andrew W.

2011-01-01

Previous experimental studies suggest that the mutation rate is nonuniform across the yeast genome. To characterize this variation across the genome more precisely, we measured the mutation rate of the URA3 gene integrated at 43 different locations tiled across Chromosome VI. We show that mutation rate varies 6-fold across a single chromosome, that this variation is correlated with replication timing, and we propose a model to explain this variation that relies on the temporal separation of two processes for replicating past damaged DNA: error-free DNA damage tolerance and translesion synthesis. This model is supported by the observation that eliminating translesion synthesis decreases this variation. PMID:21666225

Construction of a mutagenesis cartridge for poliovirus genome-linked viral protein: isolation and characterization of viable and nonviable mutants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuhn, R.J.; Tada, H.; Ypma-Wong, M.F.

1988-01-01

By following a strategy of genetic analysis of poliovirus, the authors have constructed a synthetic mutagenesis cartridge spanning the genome-linked viral protein coding region and flanking cleavage sites in an infectious cDNA clone of the type I (Mahoney) genome. The insertion of new restriction sites within the infectious clone has allowed them to replace the wild-type sequences with short complementary pairs of synthetic oligonucleotides containing various mutations. A set of mutations have been made that create methionine codons within the genome-linked viral protein region. The resulting viruses have growth characteristics similar to wild type. Experiments that led to an alterationmore » of the tyrosine residue responsible for the linkage to RNA have resulted in nonviable virus. In one mutant, proteolytic processing assayed in vitro appeared unimpaired by the mutation. They suggest that the position of the tyrosine residue is important for genome-linked viral protein function(s).« less

APOBEC3B upregulation and genomic mutation patterns in serous ovarian carcinoma

PubMed Central

Leonard, Brandon; Hart, Steven N.; Burns, Michael B.; Carpenter, Michael A.; Temiz, Nuri A.; Rathore, Anurag; Vogel, Rachel Isaksson; Nikas, Jason B.; Law, Emily K.; Brown, William L.; Li, Ying; Zhang, Yuji; Maurer, Matthew J.; Oberg, Ann L.; Cunningham, Julie M.; Shridhar, Viji; Bell, Debra A.; April, Craig; Bentley, David; Bibikova, Marina; Cheetham, R. Keira; Fan, Jian-Bing; Grocock, Russell; Humphray, Sean; Kingsbury, Zoya; Peden, John; Chien, Jeremy; Swisher, Elizabeth M.; Hartmann, Lynn C.; Kalli, Kimberly R.; Goode, Ellen L.; Sicotte, Hugues; Kaufmann, Scott H.; Harris, Reuben S.

2013-01-01

Ovarian cancer is a clinically and molecularly heterogeneous disease. The driving forces behind this variability are unknown. Here we report wide variation in expression of the DNA cytosine deaminase APOBEC3B, with elevated expression in a majority of ovarian cancer cell lines (3 standard deviations above the mean of normal ovarian surface epithelial cells) and high grade primary ovarian cancers. APOBEC3B is active in the nucleus of several ovarian cancer cell lines and elicits a biochemical preference for deamination of cytosines in 5′TC dinucleotides. Importantly, examination of whole-genome sequence from 16 ovarian cancers reveals that APOBEC3B expression correlates with total mutation load as well as elevated levels of transversion mutations. In particular, high APOBEC3B expression correlates with C-to-A and C-to-G transversion mutations within 5′TC dinucleotide motifs in early-stage high grade serous ovarian cancer genomes, suggesting that APOBEC3B-catalyzed genomic uracil lesions are further processed by downstream DNA ‘repair’ enzymes including error-prone translesion polymerases. These data identify a potential role for APOBEC3B in serous ovarian cancer genomic instability. PMID:24154874

Comparative genomics and transcriptional profiles of Saccharopolyspora erythraea NRRL 2338 and a classically improved erythromycin over-producing strain

PubMed Central

2012-01-01

Background The molecular mechanisms altered by the traditional mutation and screening approach during the improvement of antibiotic-producing microorganisms are still poorly understood although this information is essential to design rational strategies for industrial strain improvement. In this study, we applied comparative genomics to identify all genetic changes occurring during the development of an erythromycin overproducer obtained using the traditional mutate-and- screen method. Results Compared with the parental Saccharopolyspora erythraea NRRL 2338, the genome of the overproducing strain presents 117 deletion, 78 insertion and 12 transposition sites, with 71 insertion/deletion sites mapping within coding sequences (CDSs) and generating frame-shift mutations. Single nucleotide variations are present in 144 CDSs. Overall, the genomic variations affect 227 proteins of the overproducing strain and a considerable number of mutations alter genes of key enzymes in the central carbon and nitrogen metabolism and in the biosynthesis of secondary metabolites, resulting in the redirection of common precursors toward erythromycin biosynthesis. Interestingly, several mutations inactivate genes coding for proteins that play fundamental roles in basic transcription and translation machineries including the transcription anti-termination factor NusB and the transcription elongation factor Efp. These mutations, along with those affecting genes coding for pleiotropic or pathway-specific regulators, affect global expression profile as demonstrated by a comparative analysis of the parental and overproducer expression profiles. Genomic data, finally, suggest that the mutate-and-screen process might have been accelerated by mutations in DNA repair genes. Conclusions This study helps to clarify the mechanisms underlying antibiotic overproduction providing valuable information about new possible molecular targets for rationale strain improvement. PMID:22401291

Identifying structural variation in haploid microbial genomes from short-read resequencing data using breseq.

PubMed

Barrick, Jeffrey E; Colburn, Geoffrey; Deatherage, Daniel E; Traverse, Charles C; Strand, Matthew D; Borges, Jordan J; Knoester, David B; Reba, Aaron; Meyer, Austin G

2014-11-29

Mutations that alter chromosomal structure play critical roles in evolution and disease, including in the origin of new lifestyles and pathogenic traits in microbes. Large-scale rearrangements in genomes are often mediated by recombination events involving new or existing copies of mobile genetic elements, recently duplicated genes, or other repetitive sequences. Most current software programs for predicting structural variation from short-read DNA resequencing data are intended primarily for use on human genomes. They typically disregard information in reads mapping to repeat sequences, and significant post-processing and manual examination of their output is often required to rule out false-positive predictions and precisely describe mutational events. We have implemented an algorithm for identifying structural variation from DNA resequencing data as part of the breseq computational pipeline for predicting mutations in haploid microbial genomes. Our method evaluates the support for new sequence junctions present in a clonal sample from split-read alignments to a reference genome, including matches to repeat sequences. Then, it uses a statistical model of read coverage evenness to accept or reject these predictions. Finally, breseq combines predictions of new junctions and deleted chromosomal regions to output biologically relevant descriptions of mutations and their effects on genes. We demonstrate the performance of breseq on simulated Escherichia coli genomes with deletions generating unique breakpoint sequences, new insertions of mobile genetic elements, and deletions mediated by mobile elements. Then, we reanalyze data from an E. coli K-12 mutation accumulation evolution experiment in which structural variation was not previously identified. Transposon insertions and large-scale chromosomal changes detected by breseq account for ~25% of spontaneous mutations in this strain. In all cases, we find that breseq is able to reliably predict structural variation with modest read-depth coverage of the reference genome (>40-fold). Using breseq to predict structural variation should be useful for studies of microbial epidemiology, experimental evolution, synthetic biology, and genetics when a reference genome for a closely related strain is available. In these cases, breseq can discover mutations that may be responsible for important or unintended changes in genomes that might otherwise go undetected.

Genome-Wide Motif Statistics are Shaped by DNA Binding Proteins over Evolutionary Time Scales

NASA Astrophysics Data System (ADS)

Qian, Long; Kussell, Edo

The composition of genomes with respect to short DNA motifs impacts the ability of DNA binding proteins to locate and bind their target sites. Since nonfunctional DNA binding can be detrimental to cellular functions and ultimately to organismal fitness, organisms could benefit from reducing the number of nonfunctional binding sites genome wide. Using in vitro measurements of binding affinities for a large collection of DNA binding proteins, in multiple species, we detect a significant global avoidance of weak binding sites in genomes. The underlying evolutionary process leaves a distinct genomic hallmark in that similar words have correlated frequencies, which we detect in all species across domains of life. We hypothesize that natural selection against weak binding sites contributes to this process, and using an evolutionary model we show that the strength of selection needed to maintain global word compositions is on the order of point mutation rates. Alternative contributions may come from interference of protein-DNA binding with replication and mutational repair processes, which operates with similar rates. We conclude that genome-wide word compositions have been molded by DNA binding proteins through tiny evolutionary steps over timescales spanning millions of generations.

PTEN in the maintenance of genome integrity: From DNA replication to chromosome segregation.

PubMed

Hou, Sheng-Qi; Ouyang, Meng; Brandmaier, Andrew; Hao, Hongbo; Shen, Wen H

2017-10-01

Faithful DNA replication and accurate chromosome segregation are the key machineries of genetic transmission. Disruption of these processes represents a hallmark of cancer and often results from loss of tumor suppressors. PTEN is an important tumor suppressor that is frequently mutated or deleted in human cancer. Loss of PTEN has been associated with aneuploidy and poor prognosis in cancer patients. In mice, Pten deletion or mutation drives genomic instability and tumor development. PTEN deficiency induces DNA replication stress, confers stress tolerance, and disrupts mitotic spindle architecture, leading to accumulation of structural and numerical chromosome instability. Therefore, PTEN guards the genome by controlling multiple processes of chromosome inheritance. Here, we summarize current understanding of the PTEN function in promoting high-fidelity transmission of genetic information. We also discuss the PTEN pathways of genome maintenance and highlight potential targets for cancer treatment. © 2017 WILEY Periodicals, Inc.

Genomic instability and tumorigenic induction in immortalized human bronchial epithelial cells by heavy ions

NASA Astrophysics Data System (ADS)

Hei, T. K.; Piao, C. Q.; Wu, L. J.; Willey, J. C.; Hall, E. J.

1998-11-01

Carcinogenesis is postulated to be a progressive multistage process characterized by an increase in genomic instability and clonal selection with each mutational event endowing a selective growth advantage. Genomic instability as manifested by the amplification of specific gene fragments is common among tumor and transformed cells. In the present study, immortalized human bronchial (BEP2D) cells were irradiated with graded doses of either 1GeV/nucleon 56Fe ions or 150 keV/μm alpha particles. Transformed cells developed through a series of successive steps before becoming tumorigenic in nude mice. Tumorigenic cells showed neither ras mutations nor deletion in the p16 tumor suppressor gene. In contrast, they harbored mutations in the p53 gene and over-expressed cyclin D1. Genomic instability among transformed cells at various stage of the carcinogenic process was examined based on frequencies of PALA resistance. Incidence of genomic instability was highest among established tumor cell lines relative to transformed, non-tumorigenic and control cell lines. Treatment of BEP2D cells with a 4 mM dose of the aminothiol WR-1065 significantly reduced their neoplastic transforming response to 56Fe particles. This model provides an opportunity to study the cellular and molecular mechanisms involved in malignant transformation of human epithelial cells by heavy ions.

Coordinated Changes in Mutation and Growth Rates Induced by Genome Reduction.

PubMed

Nishimura, Issei; Kurokawa, Masaomi; Liu, Liu; Ying, Bei-Wen

2017-07-05

Genome size is determined during evolution, but it can also be altered by genetic engineering in laboratories. The systematic characterization of reduced genomes provides valuable insights into the cellular properties that are quantitatively described by the global parameters related to the dynamics of growth and mutation. In the present study, we analyzed a small collection of W3110 Escherichia coli derivatives containing either the wild-type genome or reduced genomes of various lengths to examine whether the mutation rate, a global parameter representing genomic plasticity, was affected by genome reduction. We found that the mutation rates of these cells increased with genome reduction. The correlation between genome length and mutation rate, which has been reported for the evolution of bacteria, was also identified, intriguingly, for genome reduction. Gene function enrichment analysis indicated that the deletion of many of the genes encoding membrane and transport proteins play a role in the mutation rate changes mediated by genome reduction. Furthermore, the increase in the mutation rate with genome reduction was highly associated with a decrease in the growth rate in a nutrition-dependent manner; thus, poorer media showed a larger change that was of higher significance. This negative correlation was strongly supported by experimental evidence that the serial transfer of the reduced genome improved the growth rate and reduced the mutation rate to a large extent. Taken together, the global parameters corresponding to the genome, growth, and mutation showed a coordinated relationship, which might be an essential working principle for balancing the cellular dynamics appropriate to the environment. IMPORTANCE Genome reduction is a powerful approach for investigating the fundamental rules for living systems. Whether genetically disturbed genomes have any specific properties that are different from or similar to those of natively evolved genomes has been under investigation. In the present study, we found that Escherichia coli cells with reduced genomes showed accelerated nucleotide substitution errors (mutation rates), although these cells retained the normal DNA mismatch repair systems. Intriguingly, this finding of correlation between reduced genome size and a higher mutation rate was consistent with the reported evolution of mutation rates. Furthermore, the increased mutation rate was quantitatively associated with a decreased growth rate, indicating that the global parameters related to the genome, growth, and mutation, which represent the amount of genetic information, the efficiency of propagation, and the fidelity of replication, respectively, are dynamically coordinated. Copyright © 2017 Nishimura et al.

Mutational analyses of molecularly cloned satellite tobacco mosaic virus during serial passage in plants: Evidence for hotspots of genetic change

USGS Publications Warehouse

Kurath, G.; Dodds, J.A.

1995-01-01

The high level of genetic diversity and rapid evolution of viral RNA genomes are well documented, but few studies have characterized the rate and nature of ongoing genetic change over time under controlled experimental conditions, especially in plant hosts. The RNA genome of satellite tobacco mosaic virus (STMV) was used as an effective model for such studies because of advantageous features of its genome structure and because the extant genetic heterogeneity of STMV has been characterized previously. In the present study, the process of genetic change over time was studied by monitoring multiple serial passage lines of STMV populations for changes in their consensus sequences. A total of 42 passage lines were initiated by inoculation of tobacco plants with a helper tobamovirus and one of four STMV RNA inocula that were transcribed from full-length infectious STMV clones or extracted from purified STMV type strain virions. Ten serial passages were carried out for each line and the consensus genotypes of progeny STMV populations were assessed for genetic change by RNase protection analyses of the entire 1,059-nt STMV genome. Three different types of genetic change were observed, including the fixation of novel mutations in 9 of 42 lines, mutation at the major heterogeneity site near nt 751 in 5 of the 19 lines inoculated with a single genotype, and selection of a single major genotype in 6 of the 23 lines inoculated with mixed genotypes. Sequence analyses showed that the majority of mutations were single base substitutions. The distribution of mutation sites included three clusters in which mutations occurred at or very near the same site, suggesting hot spots of genetic change in the STMV genome. The diversity of genetic changes in sibling lines is clear evidence for the important role of chance and random sampling events in the process of genetic diversification of STMV virus populations.

In Darwinian evolution, feedback from natural selection leads to biased mutations.

PubMed

Caporale, Lynn Helena; Doyle, John

2013-12-01

Natural selection provides feedback through which information about the environment and its recurring challenges is captured, inherited, and accumulated within genomes in the form of variations that contribute to survival. The variation upon which natural selection acts is generally described as "random." Yet evidence has been mounting for decades, from such phenomena as mutation hotspots, horizontal gene transfer, and highly mutable repetitive sequences, that variation is far from the simplifying idealization of random processes as white (uniform in space and time and independent of the environment or context).  This paper focuses on what is known about the generation and control of mutational variation, emphasizing that it is not uniform across the genome or in time, not unstructured with respect to survival, and is neither memoryless nor independent of the (also far from white) environment. We suggest that, as opposed to frequentist methods, Bayesian analysis could capture the evolution of nonuniform probabilities of distinct classes of mutation, and argue not only that the locations, styles, and timing of real mutations are not correctly modeled as generated by a white noise random process, but that such a process would be inconsistent with evolutionary theory. © 2013 New York Academy of Sciences.

Rates of spontaneous mutation.

PubMed Central

Drake, J W; Charlesworth, B; Charlesworth, D; Crow, J F

1998-01-01

Rates of spontaneous mutation per genome as measured in the laboratory are remarkably similar within broad groups of organisms but differ strikingly among groups. Mutation rates in RNA viruses, whose genomes contain ca. 10(4) bases, are roughly 1 per genome per replication for lytic viruses and roughly 0.1 per genome per replication for retroviruses and a retrotransposon. Mutation rates in microbes with DNA-based chromosomes are close to 1/300 per genome per replication; in this group, therefore, rates per base pair vary inversely and hugely as genome sizes vary from 6 x 10(3) to 4 x 10(7) bases or base pairs. Mutation rates in higher eukaryotes are roughly 0.1-100 per genome per sexual generation but are currently indistinguishable from 1/300 per cell division per effective genome (which excludes the fraction of the genome in which most mutations are neutral). It is now possible to specify some of the evolutionary forces that shape these diverse mutation rates. PMID:9560386

The rate and character of spontaneous mutation in an RNA virus.

PubMed Central

Malpica, José M; Fraile, Aurora; Moreno, Ignacio; Obies, Clara I; Drake, John W; García-Arenal, Fernando

2002-01-01

Estimates of spontaneous mutation rates for RNA viruses are few and uncertain, most notably due to their dependence on tiny mutation reporter sequences that may not well represent the whole genome. We report here an estimate of the spontaneous mutation rate of tobacco mosaic virus using an 804-base cognate mutational target, the viral MP gene that encodes the movement protein (MP). Selection against newly arising mutants was countered by providing MP function from a transgene. The estimated genomic mutation rate was on the lower side of the range previously estimated for lytic animal riboviruses. We also present the first unbiased riboviral mutational spectrum. The proportion of base substitutions is the same as that in a retrovirus but is lower than that in most DNA-based organisms. Although the MP mutant frequency was 0.02-0.05, 35% of the sequenced mutants contained two or more mutations. Therefore, the mutation process in populations of TMV and perhaps of riboviruses generally differs profoundly from that in populations of DNA-based microbes and may be strongly influenced by a subpopulation of mutator polymerases. PMID:12524327

Serial processing of biological reactions using flow-through microfluidic devices: coupled PCR/LDR for the detection of low-abundant DNA point mutations.

PubMed

Hashimoto, Masahiko; Barany, Francis; Xu, Feng; Soper, Steven A

2007-09-01

We have fabricated a flow-through biochip consisting of passive elements for the analysis of single base mutations in genomic DNA using polycarbonate (PC) as the substrate. The biochip was configured to carry out two processing steps on the input sample, a primary polymerase chain reaction (PCR) followed by an allele-specific ligation detection reaction (LDR) for scoring the presence of low abundant point mutations in genomic DNA. The operation of the device was demonstrated by detecting single nucleotide polymorphisms in gene fragments (K-ras) that carry high diagnostic value for colorectal cancers. The effect of carryover from the primary PCR on the subsequent LDR was investigated in terms of LDR yield and fidelity. We found that a post-PCR treatment step prior to the LDR phase of the assay was not essential. As a consequence, a thermal cycling microchip was used for a sequential PCR/LDR in a simple continuous-flow format, in which the following three steps were carried out: (1) exponential amplification of the gene fragments from genomic DNA; (2) mixing of the resultant PCR product(s) with an LDR cocktail via a Y-shaped passive micromixer; and (3) ligation of two primers (discriminating primer that carried the complement base to the mutation locus being interrogated and a common primer) only when the particular mutation was present in the genomic DNA. We successfully demonstrated the ability to detect one mutant DNA in 1000 normal sequences with the integrated microfluidic system. The PCR/LDR assay using the microchip performed the entire assay at a relatively fast processing speed: 18.7 min for 30 rounds of PCR, 4.1 min for 13 rounds of LDR (total processing time = ca. 22.8 min) and could screen multiple mutations simultaneously in a multiplexed format. In addition, the low cost of the biochip due to the fact that it was fabricated from polymers using replication technologies and consisted of passive elements makes the platform amenable to clinical diagnostics, where one-time use devices are required to eliminate false positives resulting from carryover contamination.

Coordinated Changes in Mutation and Growth Rates Induced by Genome Reduction

PubMed Central

Nishimura, Issei; Kurokawa, Masaomi; Liu, Liu

2017-01-01

ABSTRACT Genome size is determined during evolution, but it can also be altered by genetic engineering in laboratories. The systematic characterization of reduced genomes provides valuable insights into the cellular properties that are quantitatively described by the global parameters related to the dynamics of growth and mutation. In the present study, we analyzed a small collection of W3110 Escherichia coli derivatives containing either the wild-type genome or reduced genomes of various lengths to examine whether the mutation rate, a global parameter representing genomic plasticity, was affected by genome reduction. We found that the mutation rates of these cells increased with genome reduction. The correlation between genome length and mutation rate, which has been reported for the evolution of bacteria, was also identified, intriguingly, for genome reduction. Gene function enrichment analysis indicated that the deletion of many of the genes encoding membrane and transport proteins play a role in the mutation rate changes mediated by genome reduction. Furthermore, the increase in the mutation rate with genome reduction was highly associated with a decrease in the growth rate in a nutrition-dependent manner; thus, poorer media showed a larger change that was of higher significance. This negative correlation was strongly supported by experimental evidence that the serial transfer of the reduced genome improved the growth rate and reduced the mutation rate to a large extent. Taken together, the global parameters corresponding to the genome, growth, and mutation showed a coordinated relationship, which might be an essential working principle for balancing the cellular dynamics appropriate to the environment. PMID:28679744

Deriving a Mutation Index of Carcinogenicity Using Protein Structure and Protein Interfaces

PubMed Central

Hakas, Jarle; Pearl, Frances; Zvelebil, Marketa

2014-01-01

With the advent of Next Generation Sequencing the identification of mutations in the genomes of healthy and diseased tissues has become commonplace. While much progress has been made to elucidate the aetiology of disease processes in cancer, the contributions to disease that many individual mutations make remain to be characterised and their downstream consequences on cancer phenotypes remain to be understood. Missense mutations commonly occur in cancers and their consequences remain challenging to predict. However, this knowledge is becoming more vital, for both assessing disease progression and for stratifying drug treatment regimes. Coupled with structural data, comprehensive genomic databases of mutations such as the 1000 Genomes project and COSMIC give an opportunity to investigate general principles of how cancer mutations disrupt proteins and their interactions at the molecular and network level. We describe a comprehensive comparison of cancer and neutral missense mutations; by combining features derived from structural and interface properties we have developed a carcinogenicity predictor, InCa (Index of Carcinogenicity). Upon comparison with other methods, we observe that InCa can predict mutations that might not be detected by other methods. We also discuss general limitations shared by all predictors that attempt to predict driver mutations and discuss how this could impact high-throughput predictions. A web interface to a server implementation is publicly available at http://inca.icr.ac.uk/. PMID:24454733

Amino acid changes in disease-associated variants differ radically from variants observed in the 1000 genomes project dataset.

PubMed

de Beer, Tjaart A P; Laskowski, Roman A; Parks, Sarah L; Sipos, Botond; Goldman, Nick; Thornton, Janet M

2013-01-01

The 1000 Genomes Project data provides a natural background dataset for amino acid germline mutations in humans. Since the direction of mutation is known, the amino acid exchange matrix generated from the observed nucleotide variants is asymmetric and the mutabilities of the different amino acids are very different. These differences predominantly reflect preferences for nucleotide mutations in the DNA (especially the high mutation rate of the CpG dinucleotide, which makes arginine mutability very much higher than other amino acids) rather than selection imposed by protein structure constraints, although there is evidence for the latter as well. The variants occur predominantly on the surface of proteins (82%), with a slight preference for sites which are more exposed and less well conserved than random. Mutations to functional residues occur about half as often as expected by chance. The disease-associated amino acid variant distributions in OMIM are radically different from those expected on the basis of the 1000 Genomes dataset. The disease-associated variants preferentially occur in more conserved sites, compared to 1000 Genomes mutations. Many of the amino acid exchange profiles appear to exhibit an anti-correlation, with common exchanges in one dataset being rare in the other. Disease-associated variants exhibit more extreme differences in amino acid size and hydrophobicity. More modelling of the mutational processes at the nucleotide level is needed, but these observations should contribute to an improved prediction of the effects of specific variants in humans.

Base changes in tumour DNA have the power to reveal the causes and evolution of cancer

DOE PAGES

Hollstein, M.; Alexandrov, L. B.; Wild, C. P.; ...

2016-06-06

Next-generation sequencing (NGS) technology has demonstrated that the cancer genomes are peppered with mutations. Although most somatic tumour mutations are unlikely to have any role in the cancer process per se, the spectra of DNA sequence changes in tumour mutation catalogues have the potential to identify the mutagens, and to reveal the mutagenic processes responsible for human cancer. Very recently, a novel approach for data mining of the vast compilations of tumour NGS data succeeded in separating and precisely defining at least 30 distinct patterns of sequence change hidden in mutation databases. At least half of these mutational signatures canmore » be readily assigned to known human carcinogenic exposures or endogenous mechanisms of mutagenesis. A quantum leap in our knowledge of mutagenesis in human cancers has resulted, stimulating a flurry of research activity. We trace here the major findings leading first to the hypothesis that carcinogenic insults leave characteristic imprints on the DNA sequence of tumours, and culminating in empirical evidence from NGS data that well-defined carcinogen mutational signatures are indeed present in tumour genomic DNA from a variety of cancer types. The notion that tumour DNAs can divulge environmental sources of mutation is now a well-accepted fact. This approach to cancer aetiology has also incriminated various endogenous, enzyme-driven processes that increase the somatic mutation load in sporadic cancers. The tasks now confronting the field of molecular epidemiology are to assign mutagenic processes to orphan and newly discovered tumour mutation patterns, and to determine whether avoidable cancer risk factors influence signatures produced by endogenous enzymatic mechanisms. As a result, innovative research with experimental models and exploitation of the geographical heterogeneity in cancer incidence can address these challenges.« less

Base changes in tumour DNA have the power to reveal the causes and evolution of cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hollstein, M.; Alexandrov, L. B.; Wild, C. P.

Next-generation sequencing (NGS) technology has demonstrated that the cancer genomes are peppered with mutations. Although most somatic tumour mutations are unlikely to have any role in the cancer process per se, the spectra of DNA sequence changes in tumour mutation catalogues have the potential to identify the mutagens, and to reveal the mutagenic processes responsible for human cancer. Very recently, a novel approach for data mining of the vast compilations of tumour NGS data succeeded in separating and precisely defining at least 30 distinct patterns of sequence change hidden in mutation databases. At least half of these mutational signatures canmore » be readily assigned to known human carcinogenic exposures or endogenous mechanisms of mutagenesis. A quantum leap in our knowledge of mutagenesis in human cancers has resulted, stimulating a flurry of research activity. We trace here the major findings leading first to the hypothesis that carcinogenic insults leave characteristic imprints on the DNA sequence of tumours, and culminating in empirical evidence from NGS data that well-defined carcinogen mutational signatures are indeed present in tumour genomic DNA from a variety of cancer types. The notion that tumour DNAs can divulge environmental sources of mutation is now a well-accepted fact. This approach to cancer aetiology has also incriminated various endogenous, enzyme-driven processes that increase the somatic mutation load in sporadic cancers. The tasks now confronting the field of molecular epidemiology are to assign mutagenic processes to orphan and newly discovered tumour mutation patterns, and to determine whether avoidable cancer risk factors influence signatures produced by endogenous enzymatic mechanisms. As a result, innovative research with experimental models and exploitation of the geographical heterogeneity in cancer incidence can address these challenges.« less

De novo mutations in the genome organizer CTCF cause intellectual disability.

PubMed

Gregor, Anne; Oti, Martin; Kouwenhoven, Evelyn N; Hoyer, Juliane; Sticht, Heinrich; Ekici, Arif B; Kjaergaard, Susanne; Rauch, Anita; Stunnenberg, Hendrik G; Uebe, Steffen; Vasileiou, Georgia; Reis, André; Zhou, Huiqing; Zweier, Christiane

2013-07-11

An increasing number of genes involved in chromatin structure and epigenetic regulation has been implicated in a variety of developmental disorders, often including intellectual disability. By trio exome sequencing and subsequent mutational screening we now identified two de novo frameshift mutations and one de novo missense mutation in CTCF in individuals with intellectual disability, microcephaly, and growth retardation. Furthermore, an individual with a larger deletion including CTCF was identified. CTCF (CCCTC-binding factor) is one of the most important chromatin organizers in vertebrates and is involved in various chromatin regulation processes such as higher order of chromatin organization, enhancer function, and maintenance of three-dimensional chromatin structure. Transcriptome analyses in all three individuals with point mutations revealed deregulation of genes involved in signal transduction and emphasized the role of CTCF in enhancer-driven expression of genes. Our findings indicate that haploinsufficiency of CTCF affects genomic interaction of enhancers and their regulated gene promoters that drive developmental processes and cognition. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Somatic Mutation Patterns in Hemizygous Genomic Regions Unveil Purifying Selection during Tumor Evolution

PubMed Central

Basu, Swaraj; Larsson, Erik

2016-01-01

Identification of cancer driver genes using somatic mutation patterns indicative of positive selection has become a major goal in cancer genomics. However, cancer cells additionally depend on a large number of genes involved in basic cellular processes. While such genes should in theory be subject to strong purifying (negative) selection against damaging somatic mutations, these patterns have been elusive and purifying selection remains inadequately explored in cancer. Here, we hypothesized that purifying selection should be evident in hemizygous genomic regions, where damaging mutations cannot be compensated for by healthy alleles. Using a 7,781-sample pan-cancer dataset, we first confirmed this in POLR2A, an essential gene where hemizygous deletions are known to confer elevated sensitivity to pharmacological suppression. We next used this principle to identify several genes and pathways that show patterns indicative of purifying selection to avoid deleterious mutations. These include the POLR2A interacting protein INTS10 as well as genes involved in mRNA splicing, nonsense-mediated mRNA decay and other RNA processing pathways. Many of these genes belong to large protein complexes, and strong overlaps were observed with recent functional screens for gene essentiality in human cells. Our analysis supports that purifying selection acts to preserve the remaining function of many hemizygously deleted essential genes in tumors, indicating vulnerabilities that might be exploited by future therapeutic strategies. PMID:28027311

Single quantum dot analysis enables multiplexed point mutation detection by gap ligase chain reaction.

PubMed

Song, Yunke; Zhang, Yi; Wang, Tza-Huei

2013-04-08

Gene point mutations present important biomarkers for genetic diseases. However, existing point mutation detection methods suffer from low sensitivity, specificity, and a tedious assay processes. In this report, an assay technology is proposed which combines the outstanding specificity of gap ligase chain reaction (Gap-LCR), the high sensitivity of single-molecule coincidence detection, and the superior optical properties of quantum dots (QDs) for multiplexed detection of point mutations in genomic DNA. Mutant-specific ligation products are generated by Gap-LCR and subsequently captured by QDs to form DNA-QD nanocomplexes that are detected by single-molecule spectroscopy (SMS) through multi-color fluorescence burst coincidence analysis, allowing for multiplexed mutation detection in a separation-free format. The proposed assay is capable of detecting zeptomoles of KRAS codon 12 mutation variants with near 100% specificity. Its high sensitivity allows direct detection of KRAS mutation in crude genomic DNA without PCR pre-amplification. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Transient overexpression of DNA adenine methylase enables efficient and mobile genome engineering with reduced off-target effects

PubMed Central

Lennen, Rebecca M.; Nilsson Wallin, Annika I.; Pedersen, Margit; Bonde, Mads; Luo, Hao; Herrgård, Markus J.; Sommer, Morten O. A.

2016-01-01

Homologous recombination of single-stranded oligonucleotides is a highly efficient process for introducing precise mutations into the genome of E. coli and other organisms when mismatch repair (MMR) is disabled. This can result in the rapid accumulation of off-target mutations that can mask desired phenotypes, especially when selections need to be employed following the generation of combinatorial libraries. While the use of inducible mutator phenotypes or other MMR evasion tactics have proven useful, reported methods either require non-mobile genetic modifications or costly oligonucleotides that also result in reduced efficiencies of replacement. Therefore a new system was developed, Transient Mutator Multiplex Automated Genome Engineering (TM-MAGE), that solves problems encountered in other methods for oligonucleotide-mediated recombination. TM-MAGE enables nearly equivalent efficiencies of allelic replacement to the use of strains with fully disabled MMR and with an approximately 12- to 33-fold lower off-target mutation rate. Furthermore, growth temperatures are not restricted and a version of the plasmid can be readily removed by sucrose counterselection. TM-MAGE was used to combinatorially reconstruct mutations found in evolved salt-tolerant strains, enabling the identification of causative mutations and isolation of strains with up to 75% increases in growth rate and greatly reduced lag times in 0.6 M NaCl. PMID:26496947

Seamless editing of the chloroplast genome in plants.

PubMed

Martin Avila, Elena; Gisby, Martin F; Day, Anil

2016-07-29

Gene editing technologies enable the precise insertion of favourable mutations and performance enhancing trait genes into chromosomes whilst excluding all excess DNA from modified genomes. The technology gives rise to a new class of biotech crops which is likely to have widespread applications in agriculture. Despite progress in the nucleus, the seamless insertions of point mutations and non-selectable foreign genes into the organelle genomes of crops have not been described. The chloroplast genome is an attractive target to improve photosynthesis and crop performance. Current chloroplast genome engineering technologies for introducing point mutations into native chloroplast genes leave DNA scars, such as the target sites for recombination enzymes. Seamless editing methods to modify chloroplast genes need to address reversal of site-directed point mutations by template mediated repair with the vast excess of wild type chloroplast genomes that are present early in the transformation process. Using tobacco, we developed an efficient two-step method to edit a chloroplast gene by replacing the wild type sequence with a transient intermediate. This was resolved to the final edited gene by recombination between imperfect direct repeats. Six out of 11 transplastomic plants isolated contained the desired intermediate and at the second step this was resolved to the edited chloroplast gene in five of six plants tested. Maintenance of a single base deletion mutation in an imperfect direct repeat of the native chloroplast rbcL gene showed the limited influence of biased repair back to the wild type sequence. The deletion caused a frameshift, which replaced the five C-terminal amino acids of the Rubisco large subunit with 16 alternative residues resulting in a ~30-fold reduction in its accumulation. We monitored the process in vivo by engineering an overlapping gusA gene downstream of the edited rbcL gene. Translational coupling between the overlapping rbcL and gusA genes resulted in relatively high GUS accumulation (~0.5 % of leaf protein). Editing chloroplast genomes using transient imperfect direct repeats provides an efficient method for introducing point mutations into chloroplast genes. Moreover, we describe the first synthetic operon allowing expression of a downstream overlapping gene by translational coupling in chloroplasts. Overlapping genes provide a new mechanism for co-ordinating the translation of foreign proteins in chloroplasts.

Scanning the Effects of Ethyl Methanesulfonate on the Whole Genome of Lotus japonicus Using Second-Generation Sequencing Analysis

PubMed Central

Mohd-Yusoff, Nur Fatihah; Ruperao, Pradeep; Tomoyoshi, Nurain Emylia; Edwards, David; Gresshoff, Peter M.; Biswas, Bandana; Batley, Jacqueline

2015-01-01

Genetic structure can be altered by chemical mutagenesis, which is a common method applied in molecular biology and genetics. Second-generation sequencing provides a platform to reveal base alterations occurring in the whole genome due to mutagenesis. A model legume, Lotus japonicus ecotype Miyakojima, was chemically mutated with alkylating ethyl methanesulfonate (EMS) for the scanning of DNA lesions throughout the genome. Using second-generation sequencing, two individually mutated third-generation progeny (M3, named AM and AS) were sequenced and analyzed to identify single nucleotide polymorphisms and reveal the effects of EMS on nucleotide sequences in these mutant genomes. Single-nucleotide polymorphisms were found in every 208 kb (AS) and 202 kb (AM) with a bias mutation of G/C-to-A/T changes at low percentage. Most mutations were intergenic. The mutation spectrum of the genomes was comparable in their individual chromosomes; however, each mutated genome has unique alterations, which are useful to identify causal mutations for their phenotypic changes. The data obtained demonstrate that whole genomic sequencing is applicable as a high-throughput tool to investigate genomic changes due to mutagenesis. The identification of these single-point mutations will facilitate the identification of phenotypically causative mutations in EMS-mutated germplasm. PMID:25660167

Genomic mutation consequence calculator.

PubMed

Major, John E

2007-11-15

The genomic mutation consequence calculator (GMCC) is a tool that will reliably and quickly calculate the consequence of arbitrary genomic mutations. GMCC also reports supporting annotations for the specified genomic region. The particular strength of the GMCC is it works in genomic space, not simply in spliced transcript space as some similar tools do. Within gene features, GMCC can report on the effects on splice site, UTR and coding regions in all isoforms affected by the mutation. A considerable number of genomic annotations are also reported, including: genomic conservation score, known SNPs, COSMIC mutations, disease associations and others. The manual interface also offers link outs to various external databases and resources. In batch mode, GMCC returns a csv file which can easily be parsed by the end user. GMCC is intended to support the many tumor resequencing efforts, but can be useful to any study investigating genomic mutations.

Comparative Analysis of Syntenic Genes in Grass Genomes Reveals Accelerated Rates of Gene Structure and Coding Sequence Evolution in Polyploid Wheat1[W][OA

PubMed Central

Akhunov, Eduard D.; Sehgal, Sunish; Liang, Hanquan; Wang, Shichen; Akhunova, Alina R.; Kaur, Gaganpreet; Li, Wanlong; Forrest, Kerrie L.; See, Deven; Šimková, Hana; Ma, Yaqin; Hayden, Matthew J.; Luo, Mingcheng; Faris, Justin D.; Doležel, Jaroslav; Gill, Bikram S.

2013-01-01

Cycles of whole-genome duplication (WGD) and diploidization are hallmarks of eukaryotic genome evolution and speciation. Polyploid wheat (Triticum aestivum) has had a massive increase in genome size largely due to recent WGDs. How these processes may impact the dynamics of gene evolution was studied by comparing the patterns of gene structure changes, alternative splicing (AS), and codon substitution rates among wheat and model grass genomes. In orthologous gene sets, significantly more acquired and lost exonic sequences were detected in wheat than in model grasses. In wheat, 35% of these gene structure rearrangements resulted in frame-shift mutations and premature termination codons. An increased codon mutation rate in the wheat lineage compared with Brachypodium distachyon was found for 17% of orthologs. The discovery of premature termination codons in 38% of expressed genes was consistent with ongoing pseudogenization of the wheat genome. The rates of AS within the individual wheat subgenomes (21%–25%) were similar to diploid plants. However, we uncovered a high level of AS pattern divergence between the duplicated homeologous copies of genes. Our results are consistent with the accelerated accumulation of AS isoforms, nonsynonymous mutations, and gene structure rearrangements in the wheat lineage, likely due to genetic redundancy created by WGDs. Whereas these processes mostly contribute to the degeneration of a duplicated genome and its diploidization, they have the potential to facilitate the origin of new functional variations, which, upon selection in the evolutionary lineage, may play an important role in the origin of novel traits. PMID:23124323

Lineage dynamics and mutation-selection balance in non-adapting asexual populations

NASA Astrophysics Data System (ADS)

Pénisson, Sophie; Sniegowski, Paul D.; Colato, Alexandre; Gerrish, Philip J.

2013-02-01

In classical population genetics, mutation-selection balance refers to the equilibrium frequency of a deleterious allele established and maintained under two opposing forces: recurrent mutation, which tends to increase the frequency of the allele; and selection, which tends to decrease its frequency. In a haploid population, if μ denotes the per capita rate of production of the deleterious allele by mutation and s denotes the selective disadvantage of carrying the allele, then the classical mutation-selection balance frequency of the allele is approximated by μ/s. This calculation assumes that lineages carrying the mutant allele in question—the ‘focal allele’—do not accumulate deleterious mutations linked to the focal allele. In principle, indirect selection against the focal allele caused by such additional mutations can decrease the frequency of the focal allele below the classical mutation-selection balance. This effect of indirect selection will be strongest in an asexual population, in which the entire genome is in linkage. Here, we use an approach based on a multitype branching process to investigate this effect, analyzing lineage dynamics under mutation, direct selection, and indirect selection in a non-adapting asexual population. We find that the equilibrium balance between recurrent mutation to the focal allele and the forces of direct and indirect selection against the focal allele is closely approximated by γμ/(s + U) (s = 0 if the focal allele is neutral), where γ ≈ eθθ-(ω+θ)(ω + θ)(Γ(ω + θ) - Γ(ω + θ,θ)), \\theta =U/\\tilde {s}, and \\omega =s/\\tilde {s}; U denotes the genomic deleterious mutation rate and \\tilde {s} denotes the geometric mean selective disadvantage of deleterious mutations elsewhere on the genome. This mutation-selection balance for asexual populations can remain surprisingly invariant over wide ranges of the mutation rate.

Identification of novel RNA secondary structures within the hepatitis C virus genome reveals a cooperative involvement in genome packaging

PubMed Central

Stewart, H.; Bingham, R.J.; White, S. J.; Dykeman, E. C.; Zothner, C.; Tuplin, A. K.; Stockley, P. G.; Twarock, R.; Harris, M.

2016-01-01

The specific packaging of the hepatitis C virus (HCV) genome is hypothesised to be driven by Core-RNA interactions. To identify the regions of the viral genome involved in this process, we used SELEX (systematic evolution of ligands by exponential enrichment) to identify RNA aptamers which bind specifically to Core in vitro. Comparison of these aptamers to multiple HCV genomes revealed the presence of a conserved terminal loop motif within short RNA stem-loop structures. We postulated that interactions of these motifs, as well as sub-motifs which were present in HCV genomes at statistically significant levels, with the Core protein may drive virion assembly. We mutated 8 of these predicted motifs within the HCV infectious molecular clone JFH-1, thereby producing a range of mutant viruses predicted to possess altered RNA secondary structures. RNA replication and viral titre were unaltered in viruses possessing only one mutated structure. However, infectivity titres were decreased in viruses possessing a higher number of mutated regions. This work thus identified multiple novel RNA motifs which appear to contribute to genome packaging. We suggest that these structures act as cooperative packaging signals to drive specific RNA encapsidation during HCV assembly. PMID:26972799

Developmental genes significantly afflicted by aberrant promoter methylation and somatic mutation predict overall survival of late-stage colorectal cancer

PubMed Central

An, Ning; Yang, Xue; Cheng, Shujun; Wang, Guiqi; Zhang, Kaitai

2015-01-01

Carcinogenesis is an exceedingly complicated process, which involves multi-level dysregulations, including genomics (majorly caused by somatic mutation and copy number variation), DNA methylomics, and transcriptomics. Therefore, only looking into one molecular level of cancer is not sufficient to uncover the intricate underlying mechanisms. With the abundant resources of public available data in the Cancer Genome Atlas (TCGA) database, an integrative strategy was conducted to systematically analyze the aberrant patterns of colorectal cancer on the basis of DNA copy number, promoter methylation, somatic mutation and gene expression. In this study, paired samples in each genomic level were retrieved to identify differentially expressed genes with corresponding genetic or epigenetic dysregulations. Notably, the result of gene ontology enrichment analysis indicated that the differentially expressed genes with corresponding aberrant promoter methylation or somatic mutation were both functionally concentrated upon developmental process, suggesting the intimate association between development and carcinogenesis. Thus, by means of random walk with restart, 37 significant development-related genes were retrieved from a priori-knowledge based biological network. In five independent microarray datasets, Kaplan–Meier survival and Cox regression analyses both confirmed that the expression of these genes was significantly associated with overall survival of Stage III/IV colorectal cancer patients. PMID:26691761

Developmental genes significantly afflicted by aberrant promoter methylation and somatic mutation predict overall survival of late-stage colorectal cancer.

PubMed

An, Ning; Yang, Xue; Cheng, Shujun; Wang, Guiqi; Zhang, Kaitai

2015-12-22

Carcinogenesis is an exceedingly complicated process, which involves multi-level dysregulations, including genomics (majorly caused by somatic mutation and copy number variation), DNA methylomics, and transcriptomics. Therefore, only looking into one molecular level of cancer is not sufficient to uncover the intricate underlying mechanisms. With the abundant resources of public available data in the Cancer Genome Atlas (TCGA) database, an integrative strategy was conducted to systematically analyze the aberrant patterns of colorectal cancer on the basis of DNA copy number, promoter methylation, somatic mutation and gene expression. In this study, paired samples in each genomic level were retrieved to identify differentially expressed genes with corresponding genetic or epigenetic dysregulations. Notably, the result of gene ontology enrichment analysis indicated that the differentially expressed genes with corresponding aberrant promoter methylation or somatic mutation were both functionally concentrated upon developmental process, suggesting the intimate association between development and carcinogenesis. Thus, by means of random walk with restart, 37 significant development-related genes were retrieved from a priori-knowledge based biological network. In five independent microarray datasets, Kaplan-Meier survival and Cox regression analyses both confirmed that the expression of these genes was significantly associated with overall survival of Stage III/IV colorectal cancer patients.

Genetic instability in budding and fission yeast—sources and mechanisms

PubMed Central

Skoneczna, Adrianna; Kaniak, Aneta; Skoneczny, Marek

2015-01-01

Cells are constantly confronted with endogenous and exogenous factors that affect their genomes. Eons of evolution have allowed the cellular mechanisms responsible for preserving the genome to adjust for achieving contradictory objectives: to maintain the genome unchanged and to acquire mutations that allow adaptation to environmental changes. One evolutionary mechanism that has been refined for survival is genetic variation. In this review, we describe the mechanisms responsible for two biological processes: genome maintenance and mutation tolerance involved in generations of genetic variations in mitotic cells of both Saccharomyces cerevisiae and Schizosaccharomyces pombe. These processes encompass mechanisms that ensure the fidelity of replication, DNA lesion sensing and DNA damage response pathways, as well as mechanisms that ensure precision in chromosome segregation during cell division. We discuss various factors that may influence genome stability, such as cellular ploidy, the phase of the cell cycle, transcriptional activity of a particular region of DNA, the proficiency of DNA quality control systems, the metabolic stage of the cell and its respiratory potential, and finally potential exposure to endogenous or environmental stress. PMID:26109598

Genetic instability in budding and fission yeast-sources and mechanisms.

PubMed

Skoneczna, Adrianna; Kaniak, Aneta; Skoneczny, Marek

2015-11-01

Cells are constantly confronted with endogenous and exogenous factors that affect their genomes. Eons of evolution have allowed the cellular mechanisms responsible for preserving the genome to adjust for achieving contradictory objectives: to maintain the genome unchanged and to acquire mutations that allow adaptation to environmental changes. One evolutionary mechanism that has been refined for survival is genetic variation. In this review, we describe the mechanisms responsible for two biological processes: genome maintenance and mutation tolerance involved in generations of genetic variations in mitotic cells of both Saccharomyces cerevisiae and Schizosaccharomyces pombe. These processes encompass mechanisms that ensure the fidelity of replication, DNA lesion sensing and DNA damage response pathways, as well as mechanisms that ensure precision in chromosome segregation during cell division. We discuss various factors that may influence genome stability, such as cellular ploidy, the phase of the cell cycle, transcriptional activity of a particular region of DNA, the proficiency of DNA quality control systems, the metabolic stage of the cell and its respiratory potential, and finally potential exposure to endogenous or environmental stress. © FEMS 2015.

An Old Story Retold: Loss of G1 Control Defines A Distinct Genomic Subtype of Esophageal Squamous Cell Carcinoma.

PubMed

Wang, Qiyan; Bai, Jian; Abliz, Amir; Liu, Ying; Gong, Kenan; Li, Jingjing; Shi, Wenjie; Pan, Yaqi; Liu, Fangfang; Lai, Shujuan; Yang, Haijun; Lu, Changdong; Zhang, Lixin; Chen, Wei; Xu, Ruiping; Cai, Hong; Ke, Yang; Zeng, Changqing

2015-08-01

Esophageal squamous cell carcinoma (ESCC) has a high mortality rate. To determine the molecular basis of ESCC development, this study sought to identify characteristic genome-wide alterations in ESCC, including exonic mutations and structural alterations. The clinical implications of these genetic alterations were also analyzed. Exome sequencing and verification were performed for nine pairs of ESCC and the matched blood samples, followed by validation with additional samples using Sanger sequencing. Whole-genome SNP arrays were employed to detect copy number alteration (CNA) and loss of heterozygosity (LOH) in 55 cases, including the nine ESCC samples subjected to exome sequencing. A total of 108 non-synonymous somatic mutations (NSSMs) in 102 genes were verified in nine patients. The chromatin modification process was found to be enriched in our gene ontology (GO) analysis. Tumor genomes with TP53 mutations were significantly more unstable than those without TP53 mutations. In terms of the landscape of genomic alterations, deletion of 9p21.3 covering CDKN2A/2B (30.9%), amplification of 11q13.3 covering CCND1 (30.9%), and TP53 point mutation (50.9%) occurred in two-thirds of the cases. These results suggest that the deregulation of the G1 phase during the cell cycle is a key event in ESCC. Furthermore, six minimal common regions were found to be significantly altered in ESCC samples and three of them, 9p21.3, 7p11.2, and 3p12.1, were associated with lymph node metastasis. With the high correlation of TP53 mutation and genomic instability in ESCC, the amplification of CCND1, the deletion of CDKN2A/2B, and the somatic mutation of TP53 appear to play pivotal roles via G1 deregulation and therefore helps to classify this cancer into different genomic subtypes. These findings provide clinical significance that could be useful in future molecular diagnoses and therapeutic targeting. Copyright © 2015 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

Ion Torrent sequencing as a tool for mutation discovery in the flax (Linum usitatissimum L.) genome.

PubMed

Galindo-González, Leonardo; Pinzón-Latorre, David; Bergen, Erik A; Jensen, Dustin C; Deyholos, Michael K

2015-01-01

Detection of induced mutations is valuable for inferring gene function and for developing novel germplasm for crop improvement. Many reverse genetics approaches have been developed to identify mutations in genes of interest within a mutagenized population, including some approaches that rely on next-generation sequencing (e.g. exome capture, whole genome resequencing). As an alternative to these genome or exome-scale methods, we sought to develop a scalable and efficient method for detection of induced mutations that could be applied to a small number of target genes, using Ion Torrent technology. We developed this method in flax (Linum usitatissimum), to demonstrate its utility in a crop species. We used an amplicon-based approach in which DNA samples from an ethyl methanesulfonate (EMS)-mutagenized population were pooled and used as template in PCR reactions to amplify a region of each gene of interest. Barcodes were incorporated during PCR, and the pooled amplicons were sequenced using an Ion Torrent PGM. A pilot experiment with known SNPs showed that they could be detected at a frequency > 0.3% within the pools. We then selected eight genes for which we wanted to discover novel mutations, and applied our approach to screen 768 individuals from the EMS population, using either the Ion 314 or Ion 316 chips. Out of 29 potential mutations identified after processing the NGS reads, 16 mutations were confirmed using Sanger sequencing. The methodology presented here demonstrates the utility of Ion Torrent technology in detecting mutation variants in specific genome regions for large populations of a species such as flax. The methodology could be scaled-up to test >100 genes using the higher capacity chips now available from Ion Torrent.

Genomic instability--an evolving hallmark of cancer.

PubMed

Negrini, Simona; Gorgoulis, Vassilis G; Halazonetis, Thanos D

2010-03-01

Genomic instability is a characteristic of most cancers. In hereditary cancers, genomic instability results from mutations in DNA repair genes and drives cancer development, as predicted by the mutator hypothesis. In sporadic (non-hereditary) cancers the molecular basis of genomic instability remains unclear, but recent high-throughput sequencing studies suggest that mutations in DNA repair genes are infrequent before therapy, arguing against the mutator hypothesis for these cancers. Instead, the mutation patterns of the tumour suppressor TP53 (which encodes p53), ataxia telangiectasia mutated (ATM) and cyclin-dependent kinase inhibitor 2A (CDKN2A; which encodes p16INK4A and p14ARF) support the oncogene-induced DNA replication stress model, which attributes genomic instability and TP53 and ATM mutations to oncogene-induced DNA damage.

Clinical implications of somatic mutations in aplastic anemia and myelodysplastic syndrome in genomic age.

PubMed

Maciejewski, Jaroslaw P; Balasubramanian, Suresh K

2017-12-08

Recent technological advances in genomics have led to the discovery of new somatic mutations and have brought deeper insights into clonal diversity. This discovery has changed not only the understanding of disease mechanisms but also the diagnostics and clinical management of bone marrow failure. The clinical applications of genomics include enhancement of current prognostic schemas, prediction of sensitivity or refractoriness to treatments, and conceptualization and selective application of targeted therapies. However, beyond these traditional clinical aspects, complex hierarchical clonal architecture has been uncovered and linked to the current concepts of leukemogenesis and stem cell biology. Detection of clonal mutations, otherwise typical of myelodysplastic syndrome, in the course of aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria has led to new pathogenic concepts in these conditions and created a new link between AA and its clonal complications, such as post-AA and paroxysmal nocturnal hemoglobinuria. Distinctions among founder vs subclonal mutations, types of clonal evolution (linear or branching), and biological features of individual mutations (sweeping, persistent, or vanishing) will allow for better predictions of the biologic impact they impart in individual cases. As clonal markers, mutations can be used for monitoring clonal dynamics of the stem cell compartment during physiologic aging, disease processes, and leukemic evolution. © 2016 by The American Society of Hematology. All rights reserved.

Mutational signatures reveal the role of RAD52 in p53-independent p21-driven genomic instability.

PubMed

Galanos, Panagiotis; Pappas, George; Polyzos, Alexander; Kotsinas, Athanassios; Svolaki, Ioanna; Giakoumakis, Nickolaos N; Glytsou, Christina; Pateras, Ioannis S; Swain, Umakanta; Souliotis, Vassilis L; Georgakilas, Alexandros G; Geacintov, Nicholas; Scorrano, Luca; Lukas, Claudia; Lukas, Jiri; Livneh, Zvi; Lygerou, Zoi; Chowdhury, Dipanjan; Sørensen, Claus Storgaard; Bartek, Jiri; Gorgoulis, Vassilis G

2018-03-16

Genomic instability promotes evolution and heterogeneity of tumors. Unraveling its mechanistic basis is essential for the design of appropriate therapeutic strategies. In a previous study, we reported an unexpected oncogenic property of p21 WAF1/Cip1 , showing that its chronic expression in a p53-deficient environment causes genomic instability by deregulation of the replication licensing machinery. We now demonstrate that p21 WAF1/Cip1 can further fuel genomic instability by suppressing the repair capacity of low- and high-fidelity pathways that deal with nucleotide abnormalities. Consequently, fewer single nucleotide substitutions (SNSs) occur, while formation of highly deleterious DNA double-strand breaks (DSBs) is enhanced, crafting a characteristic mutational signature landscape. Guided by the mutational signatures formed, we find that the DSBs are repaired by Rad52-dependent break-induced replication (BIR) and single-strand annealing (SSA) repair pathways. Conversely, the error-free synthesis-dependent strand annealing (SDSA) repair route is deficient. Surprisingly, Rad52 is activated transcriptionally in an E2F1-dependent manner, rather than post-translationally as is common for DNA repair factor activation. Our results signify the importance of mutational signatures as guides to disclose the repair history leading to genomic instability. We unveil how chronic p21 WAF1/Cip1 expression rewires the repair process and identifies Rad52 as a source of genomic instability and a candidate therapeutic target.

Development and validation of a comprehensive genomic diagnostic tool for myeloid malignancies

PubMed Central

McKerrell, Thomas; Moreno, Thaidy; Ponstingl, Hannes; Bolli, Niccolo; Dias, João M. L.; Tischler, German; Colonna, Vincenza; Manasse, Bridget; Bench, Anthony; Bloxham, David; Herman, Bram; Fletcher, Danielle; Park, Naomi; Quail, Michael A.; Manes, Nicla; Hodkinson, Clare; Baxter, Joanna; Sierra, Jorge; Foukaneli, Theodora; Warren, Alan J.; Chi, Jianxiang; Costeas, Paul; Rad, Roland; Huntly, Brian; Grove, Carolyn; Ning, Zemin; Tyler-Smith, Chris; Varela, Ignacio; Scott, Mike; Nomdedeu, Josep; Mustonen, Ville

2016-01-01

The diagnosis of hematologic malignancies relies on multidisciplinary workflows involving morphology, flow cytometry, cytogenetic, and molecular genetic analyses. Advances in cancer genomics have identified numerous recurrent mutations with clear prognostic and/or therapeutic significance to different cancers. In myeloid malignancies, there is a clinical imperative to test for such mutations in mainstream diagnosis; however, progress toward this has been slow and piecemeal. Here we describe Karyogene, an integrated targeted resequencing/analytical platform that detects nucleotide substitutions, insertions/deletions, chromosomal translocations, copy number abnormalities, and zygosity changes in a single assay. We validate the approach against 62 acute myeloid leukemia, 50 myelodysplastic syndrome, and 40 blood DNA samples from individuals without evidence of clonal blood disorders. We demonstrate robust detection of sequence changes in 49 genes, including difficult-to-detect mutations such as FLT3 internal-tandem and mixed-lineage leukemia (MLL) partial-tandem duplications, and clinically significant chromosomal rearrangements including MLL translocations to known and unknown partners, identifying the novel fusion gene MLL-DIAPH2 in the process. Additionally, we identify most significant chromosomal gains and losses, and several copy neutral loss-of-heterozygosity mutations at a genome-wide level, including previously unreported changes such as homozygosity for DNMT3A R882 mutations. Karyogene represents a dependable genomic diagnosis platform for translational research and for the clinical management of myeloid malignancies, which can be readily adapted for use in other cancers. PMID:27121471

Condensin II mutation causes T-cell lymphoma through tissue-specific genome instability

PubMed Central

Woodward, Jessica; Taylor, Gillian C.; Soares, Dinesh C.; Boyle, Shelagh; Sie, Daoud; Read, David; Chathoth, Keerthi; Vukovic, Milica; Tarrats, Nuria; Jamieson, David; Campbell, Kirsteen J.; Blyth, Karen; Acosta, Juan Carlos; Ylstra, Bauke; Arends, Mark J.; Kranc, Kamil R.; Jackson, Andrew P.; Bickmore, Wendy A.

2016-01-01

Chromosomal instability is a hallmark of cancer, but mitotic regulators are rarely mutated in tumors. Mutations in the condensin complexes, which restructure chromosomes to facilitate segregation during mitosis, are significantly enriched in cancer genomes, but experimental evidence implicating condensin dysfunction in tumorigenesis is lacking. We report that mice inheriting missense mutations in a condensin II subunit (Caph2nes) develop T-cell lymphoma. Before tumors develop, we found that the same Caph2 mutation impairs ploidy maintenance to a different extent in different hematopoietic cell types, with ploidy most severely perturbed at the CD4+CD8+ T-cell stage from which tumors initiate. Premalignant CD4+CD8+ T cells show persistent catenations during chromosome segregation, triggering DNA damage in diploid daughter cells and elevated ploidy. Genome sequencing revealed that Caph2 single-mutant tumors are near diploid but carry deletions spanning tumor suppressor genes, whereas P53 inactivation allowed Caph2 mutant cells with whole-chromosome gains and structural rearrangements to form highly aggressive disease. Together, our data challenge the view that mitotic chromosome formation is an invariant process during development and provide evidence that defective mitotic chromosome structure can promote tumorigenesis. PMID:27737961

A family-based probabilistic method for capturing de novo mutations from high-throughput short-read sequencing data.

PubMed

Cartwright, Reed A; Hussin, Julie; Keebler, Jonathan E M; Stone, Eric A; Awadalla, Philip

2012-01-06

Recent advances in high-throughput DNA sequencing technologies and associated statistical analyses have enabled in-depth analysis of whole-genome sequences. As this technology is applied to a growing number of individual human genomes, entire families are now being sequenced. Information contained within the pedigree of a sequenced family can be leveraged when inferring the donors' genotypes. The presence of a de novo mutation within the pedigree is indicated by a violation of Mendelian inheritance laws. Here, we present a method for probabilistically inferring genotypes across a pedigree using high-throughput sequencing data and producing the posterior probability of de novo mutation at each genomic site examined. This framework can be used to disentangle the effects of germline and somatic mutational processes and to simultaneously estimate the effect of sequencing error and the initial genetic variation in the population from which the founders of the pedigree arise. This approach is examined in detail through simulations and areas for method improvement are noted. By applying this method to data from members of a well-defined nuclear family with accurate pedigree information, the stage is set to make the most direct estimates of the human mutation rate to date.

Genome-wide annotation of mutations in a phenotyped mutant library provides an efficient platform for discovery of casual gene mutations

USDA-ARS?s Scientific Manuscript database

Ethyl methanesulfonate (EMS) efficiently generates high-density mutations in genomes. Conventionally, these mutations are identified by techniques that can detect single-nucleotide mismatches in heteroduplexes of individual PCR amplicons. We applied whole-genome sequencing to 256-phenotyped mutant l...

Comparative genomic analysis identified a mutation related to enhanced heterologous protein production in the filamentous fungus Aspergillus oryzae.

PubMed

Jin, Feng-Jie; Katayama, Takuya; Maruyama, Jun-Ichi; Kitamoto, Katsuhiko

2016-11-01

Genomic mapping of mutations using next-generation sequencing technologies has facilitated the identification of genes contributing to fundamental biological processes, including human diseases. However, few studies have used this approach to identify mutations contributing to heterologous protein production in industrial strains of filamentous fungi, such as Aspergillus oryzae. In a screening of A. oryzae strains that hyper-produce human lysozyme (HLY), we previously isolated an AUT1 mutant that showed higher production of various heterologous proteins; however, the underlying factors contributing to the increased heterologous protein production remained unclear. Here, using a comparative genomic approach performed with whole-genome sequences, we attempted to identify the genes responsible for the high-level production of heterologous proteins in the AUT1 mutant. The comparative sequence analysis led to the detection of a gene (AO090120000003), designated autA, which was predicted to encode an unknown cytoplasmic protein containing an alpha/beta-hydrolase fold domain. Mutation or deletion of autA was associated with higher production levels of HLY. Specifically, the HLY yields of the autA mutant and deletion strains were twofold higher than that of the control strain during the early stages of cultivation. Taken together, these results indicate that combining classical mutagenesis approaches with comparative genomic analysis facilitates the identification of novel genes involved in heterologous protein production in filamentous fungi.

An Evolutionary Approach for Identifying Driver Mutations in Colorectal Cancer

PubMed Central

Leder, Kevin; Riester, Markus; Iwasa, Yoh; Lengauer, Christoph; Michor, Franziska

2015-01-01

The traditional view of cancer as a genetic disease that can successfully be treated with drugs targeting mutant onco-proteins has motivated whole-genome sequencing efforts in many human cancer types. However, only a subset of mutations found within the genomic landscape of cancer is likely to provide a fitness advantage to the cell. Distinguishing such “driver” mutations from innocuous “passenger” events is critical for prioritizing the validation of candidate mutations in disease-relevant models. We design a novel statistical index, called the Hitchhiking Index, which reflects the probability that any observed candidate gene is a passenger alteration, given the frequency of alterations in a cross-sectional cancer sample set, and apply it to a mutational data set in colorectal cancer. Our methodology is based upon a population dynamics model of mutation accumulation and selection in colorectal tissue prior to cancer initiation as well as during tumorigenesis. This methodology can be used to aid in the prioritization of candidate mutations for functional validation and contributes to the process of drug discovery. PMID:26379039

Extraordinary genome stability in the ciliate Paramecium tetraurelia

PubMed Central

Sung, Way; Tucker, Abraham E.; Doak, Thomas G.; Choi, Eunjin; Thomas, W. Kelley; Lynch, Michael

2012-01-01

Mutation plays a central role in all evolutionary processes and is also the basis of genetic disorders. Established base-substitution mutation rates in eukaryotes range between ∼5 × 10−10 and 5 × 10−8 per site per generation, but here we report a genome-wide estimate for Paramecium tetraurelia that is more than an order of magnitude lower than any previous eukaryotic estimate. Nevertheless, when the mutation rate per cell division is extrapolated to the length of the sexual cycle for this protist, the measure obtained is comparable to that for multicellular species with similar genome sizes. Because Paramecium has a transcriptionally silent germ-line nucleus, these results are consistent with the hypothesis that natural selection operates on the cumulative germ-line replication fidelity per episode of somatic gene expression, with the germ-line mutation rate per cell division evolving downward to the lower barrier imposed by random genetic drift. We observe ciliate-specific modifications of widely conserved amino acid sites in DNA polymerases as one potential explanation for unusually high levels of replication fidelity. PMID:23129619

Genome-Wide Motif Statistics are Shaped by DNA Binding Proteins over Evolutionary Time Scales

NASA Astrophysics Data System (ADS)

Qian, Long; Kussell, Edo

2016-10-01

The composition of a genome with respect to all possible short DNA motifs impacts the ability of DNA binding proteins to locate and bind their target sites. Since nonfunctional DNA binding can be detrimental to cellular functions and ultimately to organismal fitness, organisms could benefit from reducing the number of nonfunctional DNA binding sites genome wide. Using in vitro measurements of binding affinities for a large collection of DNA binding proteins, in multiple species, we detect a significant global avoidance of weak binding sites in genomes. We demonstrate that the underlying evolutionary process leaves a distinct genomic hallmark in that similar words have correlated frequencies, a signal that we detect in all species across domains of life. We consider the possibility that natural selection against weak binding sites contributes to this process, and using an evolutionary model we show that the strength of selection needed to maintain global word compositions is on the order of point mutation rates. Likewise, we show that evolutionary mechanisms based on interference of protein-DNA binding with replication and mutational repair processes could yield similar results and operate with similar rates. On the basis of these modeling and bioinformatic results, we conclude that genome-wide word compositions have been molded by DNA binding proteins acting through tiny evolutionary steps over time scales spanning millions of generations.

Compactness of viral genomes: effect of disperse and localized random mutations

NASA Astrophysics Data System (ADS)

Lošdorfer Božič, Anže; Micheletti, Cristian; Podgornik, Rudolf; Tubiana, Luca

2018-02-01

Genomes of single-stranded RNA viruses have evolved to optimize several concurrent properties. One of them is the architecture of their genomic folds, which must not only feature precise structural elements at specific positions, but also allow for overall spatial compactness. The latter was shown to be disrupted by random synonymous mutations, a disruption which can consequently negatively affect genome encapsidation. In this study, we use three mutation schemes with different degrees of locality to mutate the genomes of phage MS2 and Brome Mosaic virus in order to understand the observed sensitivity of the global compactness of their folds. We find that mutating local stretches of their genomes’ sequence or structure is less disruptive to their compactness compared to inducing randomly-distributed mutations. Our findings are indicative of a mechanism for the conservation of compactness acting on a global scale of the genomes, and have several implications for understanding the interplay between local and global architecture of viral RNA genomes.

SPOP mutation leads to genomic instability in prostate cancer

PubMed Central

Boysen, Gunther; Barbieri, Christopher E; Prandi, Davide; Blattner, Mirjam; Chae, Sung-Suk; Dahija, Arun; Nataraj, Srilakshmi; Huang, Dennis; Marotz, Clarisse; Xu, Limei; Huang, Julie; Lecca, Paola; Chhangawala, Sagar; Liu, Deli; Zhou, Pengbo; Sboner, Andrea; de Bono, Johann S

2015-01-01

Genomic instability is a fundamental feature of human cancer often resulting from impaired genome maintenance. In prostate cancer, structural genomic rearrangements are a common mechanism driving tumorigenesis. However, somatic alterations predisposing to chromosomal rearrangements in prostate cancer remain largely undefined. Here, we show that SPOP, the most commonly mutated gene in primary prostate cancer modulates DNA double strand break (DSB) repair, and that SPOP mutation is associated with genomic instability. In vivo, SPOP mutation results in a transcriptional response consistent with BRCA1 inactivation resulting in impaired homology-directed repair (HDR) of DSB. Furthermore, we found that SPOP mutation sensitizes to DNA damaging therapeutic agents such as PARP inhibitors. These results implicate SPOP as a novel participant in DSB repair, suggest that SPOP mutation drives prostate tumorigenesis in part through genomic instability, and indicate that mutant SPOP may increase response to DNA-damaging therapeutics. DOI: http://dx.doi.org/10.7554/eLife.09207.001 PMID:26374986

Genome-Wide Mutation Avalanches Induced in Diploid Yeast Cells by a Base Analog or an APOBEC Deaminase

PubMed Central

Lada, Artem G.; Stepchenkova, Elena I.; Waisertreiger, Irina S. R.; Noskov, Vladimir N.; Dhar, Alok; Eudy, James D.; Boissy, Robert J.; Hirano, Masayuki; Rogozin, Igor B.; Pavlov, Youri I.

2013-01-01

Genetic information should be accurately transmitted from cell to cell; conversely, the adaptation in evolution and disease is fueled by mutations. In the case of cancer development, multiple genetic changes happen in somatic diploid cells. Most classic studies of the molecular mechanisms of mutagenesis have been performed in haploids. We demonstrate that the parameters of the mutation process are different in diploid cell populations. The genomes of drug-resistant mutants induced in yeast diploids by base analog 6-hydroxylaminopurine (HAP) or AID/APOBEC cytosine deaminase PmCDA1 from lamprey carried a stunning load of thousands of unselected mutations. Haploid mutants contained almost an order of magnitude fewer mutations. To explain this, we propose that the distribution of induced mutation rates in the cell population is uneven. The mutants in diploids with coincidental mutations in the two copies of the reporter gene arise from a fraction of cells that are transiently hypersensitive to the mutagenic action of a given mutagen. The progeny of such cells were never recovered in haploids due to the lethality caused by the inactivation of single-copy essential genes in cells with too many induced mutations. In diploid cells, the progeny of hypersensitive cells survived, but their genomes were saturated by heterozygous mutations. The reason for the hypermutability of cells could be transient faults of the mutation prevention pathways, like sanitization of nucleotide pools for HAP or an elevated expression of the PmCDA1 gene or the temporary inability of the destruction of the deaminase. The hypothesis on spikes of mutability may explain the sudden acquisition of multiple mutational changes during evolution and carcinogenesis. PMID:24039593

Cis-regulatory somatic mutations and gene-expression alteration in B-cell lymphomas.

PubMed

Mathelier, Anthony; Lefebvre, Calvin; Zhang, Allen W; Arenillas, David J; Ding, Jiarui; Wasserman, Wyeth W; Shah, Sohrab P

2015-04-23

With the rapid increase of whole-genome sequencing of human cancers, an important opportunity to analyze and characterize somatic mutations lying within cis-regulatory regions has emerged. A focus on protein-coding regions to identify nonsense or missense mutations disruptive to protein structure and/or function has led to important insights; however, the impact on gene expression of mutations lying within cis-regulatory regions remains under-explored. We analyzed somatic mutations from 84 matched tumor-normal whole genomes from B-cell lymphomas with accompanying gene expression measurements to elucidate the extent to which these cancers are disrupted by cis-regulatory mutations. We characterize mutations overlapping a high quality set of well-annotated transcription factor binding sites (TFBSs), covering a similar portion of the genome as protein-coding exons. Our results indicate that cis-regulatory mutations overlapping predicted TFBSs are enriched in promoter regions of genes involved in apoptosis or growth/proliferation. By integrating gene expression data with mutation data, our computational approach culminates with identification of cis-regulatory mutations most likely to participate in dysregulation of the gene expression program. The impact can be measured along with protein-coding mutations to highlight key mutations disrupting gene expression and pathways in cancer. Our study yields specific genes with disrupted expression triggered by genomic mutations in either the coding or the regulatory space. It implies that mutated regulatory components of the genome contribute substantially to cancer pathways. Our analyses demonstrate that identifying genomically altered cis-regulatory elements coupled with analysis of gene expression data will augment biological interpretation of mutational landscapes of cancers.

Polycistronic tRNA and CRISPR guide-RNA enables highly efficient multiplexed genome engineering in human cells

PubMed Central

Dong, Fengping; Xie, Kabin; Chen, Yueying; Yang, Yinong; Mao, Yingwei

2016-01-01

CRISPR/Cas9 has been widely used for genomic editing in many organisms. Many human diseases are caused by multiple mutations. The CRISPR/Cas9 system provides a potential tool to introduce multiple mutations in a genome. To mimic complicated genomic variants in human diseases, such as multiple gene deletions or mutations, two or more small guide RNAs (sgRNAs) need to be introduced all together. This can be achieved by separate Pol III promoters in a construct. However, limited enzyme sites and increased insertion size lower the efficiency to make a construct. Here, we report a strategy to quickly assembly multiple sgRNAs in one construct using a polycistronic-tRNA-gRNA (PTG) strategy. Taking advantage of the endogenous tRNA processing system in mammalian cells, we efficiently express multiple sgRNAs driven using only one Pol III promoter. Using an all-in-one construct carrying PTG, we disrupt the deacetylase domain in multiple histone deacetylases (HDACs) in human cells simultaneously. We demonstrate that multiple HDAC deletions significantly affect the activation of the Wnt-signaling pathway. Thus, this method enables to efficiently target multiple genes and provide a useful tool to establish mutated cells mimicking human diseases. PMID:27890617

Polycistronic tRNA and CRISPR guide-RNA enables highly efficient multiplexed genome engineering in human cells.

PubMed

Dong, Fengping; Xie, Kabin; Chen, Yueying; Yang, Yinong; Mao, Yingwei

2017-01-22

CRISPR/Cas9 has been widely used for genomic editing in many organisms. Many human diseases are caused by multiple mutations. The CRISPR/Cas9 system provides a potential tool to introduce multiple mutations in a genome. To mimic complicated genomic variants in human diseases, such as multiple gene deletions or mutations, two or more small guide RNAs (sgRNAs) need to be introduced all together. This can be achieved by separate Pol III promoters in a construct. However, limited enzyme sites and increased insertion size lower the efficiency to make a construct. Here, we report a strategy to quickly assembly multiple sgRNAs in one construct using a polycistronic-tRNA-gRNA (PTG) strategy. Taking advantage of the endogenous tRNA processing system in mammalian cells, we efficiently express multiple sgRNAs driven using only one Pol III promoter. Using an all-in-one construct carrying PTG, we disrupt the deacetylase domain in multiple histone deacetylases (HDACs) in human cells simultaneously. We demonstrate that multiple HDAC deletions significantly affect the activation of the Wnt-signaling pathway. Thus, this method enables to efficiently target multiple genes and provide a useful tool to establish mutated cells mimicking human diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

Circularization of the HIV-1 genome facilitates strand transfer during reverse transcription

PubMed Central

Beerens, Nancy; Kjems, Jørgen

2010-01-01

Two obligatory DNA strand transfers take place during reverse transcription of a retroviral RNA genome. The first strand transfer involves a jump from the 5′ to the 3′ terminal repeat (R) region positioned at each end of the viral genome. The process depends on base pairing between the cDNA synthesized from the 5′ R region and the 3′ R RNA. The tertiary conformation of the viral RNA genome may facilitate strand transfer by juxtaposing the 5′ R and 3′ R sequences that are 9 kb apart in the linear sequence. In this study, RNA sequences involved in an interaction between the 5′ and 3′ ends of the HIV-1 genome were mapped by mutational analysis. This interaction appears to be mediated mainly by a sequence in the extreme 3′ end of the viral genome and in the gag open reading frame. Mutation of 3′ R sequences was found to inhibit the 5′–3′ interaction, which could be restored by a complementary mutation in the 5′ gag region. Furthermore, we find that circularization of the HIV-1 genome does not affect the initiation of reverse transcription, but stimulates the first strand transfer during reverse transcription in vitro, underscoring the functional importance of the interaction. PMID:20430859

Exact Solution of Mutator Model with Linear Fitness and Finite Genome Length

NASA Astrophysics Data System (ADS)

Saakian, David B.

2017-08-01

We considered the infinite population version of the mutator phenomenon in evolutionary dynamics, looking at the uni-directional mutations in the mutator-specific genes and linear selection. We solved exactly the model for the finite genome length case, looking at the quasispecies version of the phenomenon. We calculated the mutator probability both in the statics and dynamics. The exact solution is important for us because the mutator probability depends on the genome length in a highly non-trivial way.

Whole-Genome and Epigenomic Landscapes of Etiologically Distinct Subtypes of Cholangiocarcinoma.

PubMed

Jusakul, Apinya; Cutcutache, Ioana; Yong, Chern Han; Lim, Jing Quan; Huang, Mi Ni; Padmanabhan, Nisha; Nellore, Vishwa; Kongpetch, Sarinya; Ng, Alvin Wei Tian; Ng, Ley Moy; Choo, Su Pin; Myint, Swe Swe; Thanan, Raynoo; Nagarajan, Sanjanaa; Lim, Weng Khong; Ng, Cedric Chuan Young; Boot, Arnoud; Liu, Mo; Ong, Choon Kiat; Rajasegaran, Vikneswari; Lie, Stefanus; Lim, Alvin Soon Tiong; Lim, Tse Hui; Tan, Jing; Loh, Jia Liang; McPherson, John R; Khuntikeo, Narong; Bhudhisawasdi, Vajaraphongsa; Yongvanit, Puangrat; Wongkham, Sopit; Totoki, Yasushi; Nakamura, Hiromi; Arai, Yasuhito; Yamasaki, Satoshi; Chow, Pierce Kah-Hoe; Chung, Alexander Yaw Fui; Ooi, London Lucien Peng Jin; Lim, Kiat Hon; Dima, Simona; Duda, Dan G; Popescu, Irinel; Broet, Philippe; Hsieh, Sen-Yung; Yu, Ming-Chin; Scarpa, Aldo; Lai, Jiaming; Luo, Di-Xian; Carvalho, André Lopes; Vettore, André Luiz; Rhee, Hyungjin; Park, Young Nyun; Alexandrov, Ludmil B; Gordân, Raluca; Rozen, Steven G; Shibata, Tatsuhiro; Pairojkul, Chawalit; Teh, Bin Tean; Tan, Patrick

2017-10-01

Cholangiocarcinoma (CCA) is a hepatobiliary malignancy exhibiting high incidence in countries with endemic liver-fluke infection. We analyzed 489 CCAs from 10 countries, combining whole-genome (71 cases), targeted/exome, copy-number, gene expression, and DNA methylation information. Integrative clustering defined 4 CCA clusters-fluke-positive CCAs (clusters 1/2) are enriched in ERBB2 amplifications and TP53 mutations; conversely, fluke-negative CCAs (clusters 3/4) exhibit high copy-number alterations and PD-1 / PD-L2 expression, or epigenetic mutations ( IDH1/2, BAP1 ) and FGFR / PRKA -related gene rearrangements. Whole-genome analysis highlighted FGFR2 3' untranslated region deletion as a mechanism of FGFR2 upregulation. Integration of noncoding promoter mutations with protein-DNA binding profiles demonstrates pervasive modulation of H3K27me3-associated sites in CCA. Clusters 1 and 4 exhibit distinct DNA hypermethylation patterns targeting either CpG islands or shores-mutation signature and subclonality analysis suggests that these reflect different mutational pathways. Our results exemplify how genetics, epigenetics, and environmental carcinogens can interplay across different geographies to generate distinct molecular subtypes of cancer. Significance: Integrated whole-genome and epigenomic analysis of CCA on an international scale identifies new CCA driver genes, noncoding promoter mutations, and structural variants. CCA molecular landscapes differ radically by etiology, underscoring how distinct cancer subtypes in the same organ may arise through different extrinsic and intrinsic carcinogenic processes. Cancer Discov; 7(10); 1116-35. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 1047 . ©2017 American Association for Cancer Research.

Radiation-induced genomic instability: radiation quality and dose response

NASA Technical Reports Server (NTRS)

Smith, Leslie E.; Nagar, Shruti; Kim, Grace J.; Morgan, William F.

2003-01-01

Genomic instability is a term used to describe a phenomenon that results in the accumulation of multiple changes required to convert a stable genome of a normal cell to an unstable genome characteristic of a tumor. There has been considerable recent debate concerning the importance of genomic instability in human cancer and its temporal occurrence in the carcinogenic process. Radiation is capable of inducing genomic instability in mammalian cells and instability is thought to be the driving force responsible for radiation carcinogenesis. Genomic instability is characterized by a large collection of diverse endpoints that include large-scale chromosomal rearrangements and aberrations, amplification of genetic material, aneuploidy, micronucleus formation, microsatellite instability, and gene mutation. The capacity of radiation to induce genomic instability depends to a large extent on radiation quality or linear energy transfer (LET) and dose. There appears to be a low dose threshold effect with low LET, beyond which no additional genomic instability is induced. Low doses of both high and low LET radiation are capable of inducing this phenomenon. This report reviews data concerning dose rate effects of high and low LET radiation and their capacity to induce genomic instability assayed by chromosomal aberrations, delayed lethal mutations, micronuclei and apoptosis.

Mobile Genome Express (MGE): A comprehensive automatic genetic analyses pipeline with a mobile device.

PubMed

Yoon, Jun-Hee; Kim, Thomas W; Mendez, Pedro; Jablons, David M; Kim, Il-Jin

2017-01-01

The development of next-generation sequencing (NGS) technology allows to sequence whole exomes or genome. However, data analysis is still the biggest bottleneck for its wide implementation. Most laboratories still depend on manual procedures for data handling and analyses, which translates into a delay and decreased efficiency in the delivery of NGS results to doctors and patients. Thus, there is high demand for developing an automatic and an easy-to-use NGS data analyses system. We developed comprehensive, automatic genetic analyses controller named Mobile Genome Express (MGE) that works in smartphones or other mobile devices. MGE can handle all the steps for genetic analyses, such as: sample information submission, sequencing run quality check from the sequencer, secured data transfer and results review. We sequenced an Actrometrix control DNA containing multiple proven human mutations using a targeted sequencing panel, and the whole analysis was managed by MGE, and its data reviewing program called ELECTRO. All steps were processed automatically except for the final sequencing review procedure with ELECTRO to confirm mutations. The data analysis process was completed within several hours. We confirmed the mutations that we have identified were consistent with our previous results obtained by using multi-step, manual pipelines.

Genomic Profiling on an Unselected Solid Tumor Population Reveals a Highly Mutated Wnt/β-Catenin Pathway Associated with Oncogenic EGFR Mutations.

PubMed

Jiang, Jingrui; Protopopov, Alexei; Sun, Ruobai; Lyle, Stephen; Russell, Meaghan

2018-04-09

Oncogenic epidermal growth factor receptors (EGFRs) can recruit key effectors in diverse cellular processes to propagate oncogenic signals. Targeted and combinational therapeutic strategies have been successfully applied for treating EGFR-driven cancers. However, a main challenge in EGFR therapies is drug resistance due to mutations, oncogenic shift, alternative signaling, and other potential mechanisms. To further understand the genetic alterations associated with oncogenic EGFRs and to provide further insight into optimal and personalized therapeutic strategies, we applied a proprietary comprehensive next-generation sequencing (NGS)-based assay of 435 genes to systematically study the genomic profiles of 1565 unselected solid cancer patient samples. We found that activating EGFR mutations were predominantly detected in lung cancer, particularly in non-small cell lung cancer (NSCLC). The mutational landscape of EGFR-driven tumors covered most key signaling pathways and biological processes. Strikingly, the Wnt/β-catenin pathway was highly mutated (48 variants detected in 46% of the EGFR-driven tumors), and its variant number topped that in the TP53/apoptosis and PI3K-AKT-mTOR pathways. Furthermore, an analysis of mutation distribution revealed a differential association pattern of gene mutations between EGFR exon 19del and EGFR L858R. Our results confirm the aggressive nature of the oncogenic EGFR-driven tumors and reassure that a combinational strategy should have advantages over an EGFR-targeted monotherapy and holds great promise for overcoming drug resistance.

A simple and efficient method to visualize and quantify the efficiency of chromosomal mutations from genome editing

PubMed Central

Fu, Liezhen; Wen, Luan; Luu, Nga; Shi, Yun-Bo

2016-01-01

Genome editing with designer nucleases such as TALEN and CRISPR/Cas enzymes has broad applications. Delivery of these designer nucleases into organisms induces various genetic mutations including deletions, insertions and nucleotide substitutions. Characterizing those mutations is critical for evaluating the efficacy and specificity of targeted genome editing. While a number of methods have been developed to identify the mutations, none other than sequencing allows the identification of the most desired mutations, i.e., out-of-frame insertions/deletions that disrupt genes. Here we report a simple and efficient method to visualize and quantify the efficiency of genomic mutations induced by genome-editing. Our approach is based on the expression of a two-color fusion protein in a vector that allows the insertion of the edited region in the genome in between the two color moieties. We show that our approach not only easily identifies developing animals with desired mutations but also efficiently quantifies the mutation rate in vivo. Furthermore, by using LacZα and GFP as the color moieties, our approach can even eliminate the need for a fluorescent microscope, allowing the analysis with simple bright field visualization. Such an approach will greatly simplify the screen for effective genome-editing enzymes and identify the desired mutant cells/animals. PMID:27748423

Copy-number analysis and inference of subclonal populations in cancer genomes using Sclust.

PubMed

Cun, Yupeng; Yang, Tsun-Po; Achter, Viktor; Lang, Ulrich; Peifer, Martin

2018-06-01

The genomes of cancer cells constantly change during pathogenesis. This evolutionary process can lead to the emergence of drug-resistant mutations in subclonal populations, which can hinder therapeutic intervention in patients. Data derived from massively parallel sequencing can be used to infer these subclonal populations using tumor-specific point mutations. The accurate determination of copy-number changes and tumor impurity is necessary to reliably infer subclonal populations by mutational clustering. This protocol describes how to use Sclust, a copy-number analysis method with a recently developed mutational clustering approach. In a series of simulations and comparisons with alternative methods, we have previously shown that Sclust accurately determines copy-number states and subclonal populations. Performance tests show that the method is computationally efficient, with copy-number analysis and mutational clustering taking <10 min. Sclust is designed such that even non-experts in computational biology or bioinformatics with basic knowledge of the Linux/Unix command-line syntax should be able to carry out analyses of subclonal populations.

Complementation between polymerase- and exonuclease-deficient mitochondrial DNA polymerase mutants in genomically engineered flies

PubMed Central

Bratic, Ana; Kauppila, Timo E. S.; Macao, Bertil; Grönke, Sebastian; Siibak, Triinu; Stewart, James B.; Baggio, Francesca; Dols, Jacqueline; Partridge, Linda; Falkenberg, Maria; Wredenberg, Anna; Larsson, Nils-Göran

2015-01-01

Replication errors are the main cause of mitochondrial DNA (mtDNA) mutations and a compelling approach to decrease mutation levels would therefore be to increase the fidelity of the catalytic subunit (POLγA) of the mtDNA polymerase. Here we genomically engineer the tamas locus, encoding fly POLγA, and introduce alleles expressing exonuclease- (exo−) and polymerase-deficient (pol−) POLγA versions. The exo− mutant leads to accumulation of point mutations and linear deletions of mtDNA, whereas pol− mutants cause mtDNA depletion. The mutant tamas alleles are developmentally lethal but can complement each other in trans resulting in viable flies with clonally expanded mtDNA mutations. Reconstitution of human mtDNA replication in vitro confirms that replication is a highly dynamic process where POLγA goes on and off the template to allow complementation during proofreading and elongation. The created fly models are valuable tools to study germ line transmission of mtDNA and the pathophysiology of POLγA mutation disease. PMID:26554610

Transformation of Chloroplast Ribosomal RNA Genes in Chlamydomonas: Molecular and Genetic Characterization of Integration Events

PubMed Central

Newman, S. M.; Boynton, J. E.; Gillham, N. W.; Randolph-Anderson, B. L.; Johnson, A. M.; Harris, E. H.

1990-01-01

Transformation of chloroplast ribosomal RNA (rRNA) genes in Chlamydomonas has been achieved by the biolistic process using cloned chloroplast DNA fragments carrying mutations that confer antibiotic resistance. The sites of exchange employed during the integration of the donor DNA into the recipient genome have been localized using a combination of antibiotic resistance mutations in the 16S and 23S rRNA genes and restriction fragment length polymorphisms that flank these genes. Complete or nearly complete replacement of a region of the chloroplast genome in the recipient cell by the corresponding sequence from the donor plasmid was the most common integration event. Exchange events between the homologous donor and recipient sequences occurred preferentially near the vector:insert junctions. Insertion of the donor rRNA genes and flanking sequences into one inverted repeat of the recipient genome was followed by intramolecular copy correction so that both copies of the inverted repeat acquired identical sequences. Increased frequencies of rRNA gene transformants were achieved by reducing the copy number of the chloroplast genome in the recipient cells and by decreasing the heterology between donor and recipient DNA sequences flanking the selectable markers. In addition to producing bona fide chloroplast rRNA transformants, the biolistic process induced mutants resistant to low levels of streptomycin, typical of nuclear mutations in Chlamydomonas. PMID:1981764

Mutational Dynamics of Aroid Chloroplast Genomes

PubMed Central

Ahmed, Ibrar; Biggs, Patrick J.; Matthews, Peter J.; Collins, Lesley J.; Hendy, Michael D.; Lockhart, Peter J.

2012-01-01

A characteristic feature of eukaryote and prokaryote genomes is the co-occurrence of nucleotide substitution and insertion/deletion (indel) mutations. Although similar observations have also been made for chloroplast DNA, genome-wide associations have not been reported. We determined the chloroplast genome sequences for two morphotypes of taro (Colocasia esculenta; family Araceae) and compared these with four publicly available aroid chloroplast genomes. Here, we report the extent of genome-wide association between direct and inverted repeats, indels, and substitutions in these aroid chloroplast genomes. We suggest that alternative but not mutually exclusive hypotheses explain the mutational dynamics of chloroplast genome evolution. PMID:23204304

Functional Annotations of Paralogs: A Blessing and a Curse

PubMed Central

Zallot, Rémi; Harrison, Katherine J.; Kolaczkowski, Bryan; de Crécy-Lagard, Valérie

2016-01-01

Gene duplication followed by mutation is a classic mechanism of neofunctionalization, producing gene families with functional diversity. In some cases, a single point mutation is sufficient to change the substrate specificity and/or the chemistry performed by an enzyme, making it difficult to accurately separate enzymes with identical functions from homologs with different functions. Because sequence similarity is often used as a basis for assigning functional annotations to genes, non-isofunctional gene families pose a great challenge for genome annotation pipelines. Here we describe how integrating evolutionary and functional information such as genome context, phylogeny, metabolic reconstruction and signature motifs may be required to correctly annotate multifunctional families. These integrative analyses can also lead to the discovery of novel gene functions, as hints from specific subgroups can guide the functional characterization of other members of the family. We demonstrate how careful manual curation processes using comparative genomics can disambiguate subgroups within large multifunctional families and discover their functions. We present the COG0720 protein family as a case study. We also discuss strategies to automate this process to improve the accuracy of genome functional annotation pipelines. PMID:27618105

The Glyphosate-Based Herbicide Roundup Does not Elevate Genome-Wide Mutagenesis of Escherichia coli.

PubMed

Tincher, Clayton; Long, Hongan; Behringer, Megan; Walker, Noah; Lynch, Michael

2017-10-05

Mutations induced by pollutants may promote pathogen evolution, for example by accelerating mutations conferring antibiotic resistance. Generally, evaluating the genome-wide mutagenic effects of long-term sublethal pollutant exposure at single-nucleotide resolution is extremely difficult. To overcome this technical barrier, we use the mutation accumulation/whole-genome sequencing (MA/WGS) method as a mutagenicity test, to quantitatively evaluate genome-wide mutagenesis of Escherichia coli after long-term exposure to a wide gradient of the glyphosate-based herbicide (GBH) Roundup Concentrate Plus. The genome-wide mutation rate decreases as GBH concentration increases, suggesting that even long-term GBH exposure does not compromise the genome stability of bacteria. Copyright © 2017 Tincher et al.

Detecting and Characterizing Genomic Signatures of Positive Selection in Global Populations

PubMed Central

Liu, Xuanyao; Ong, Rick Twee-Hee; Pillai, Esakimuthu Nisha; Elzein, Abier M.; Small, Kerrin S.; Clark, Taane G.; Kwiatkowski, Dominic P.; Teo, Yik-Ying

2013-01-01

Natural selection is a significant force that shapes the architecture of the human genome and introduces diversity across global populations. The question of whether advantageous mutations have arisen in the human genome as a result of single or multiple mutation events remains unanswered except for the fact that there exist a handful of genes such as those that confer lactase persistence, affect skin pigmentation, or cause sickle cell anemia. We have developed a long-range-haplotype method for identifying genomic signatures of positive selection to complement existing methods, such as the integrated haplotype score (iHS) or cross-population extended haplotype homozygosity (XP-EHH), for locating signals across the entire allele frequency spectrum. Our method also locates the founder haplotypes that carry the advantageous variants and infers their corresponding population frequencies. This presents an opportunity to systematically interrogate the whole human genome whether a selection signal shared across different populations is the consequence of a single mutation process followed subsequently by gene flow between populations or of convergent evolution due to the occurrence of multiple independent mutation events either at the same variant or within the same gene. The application of our method to data from 14 populations across the world revealed that positive-selection events tend to cluster in populations of the same ancestry. Comparing the founder haplotypes for events that are present across different populations revealed that convergent evolution is a rare occurrence and that the majority of shared signals stem from the same evolutionary event. PMID:23731540

Slow but not low: genomic comparisons reveal slower evolutionary rate and higher dN/dS in conifers compared to angiosperms.

PubMed

Buschiazzo, Emmanuel; Ritland, Carol; Bohlmann, Jörg; Ritland, Kermit

2012-01-20

Comparative genomics can inform us about the processes of mutation and selection across diverse taxa. Among seed plants, gymnosperms have been lacking in genomic comparisons. Recent EST and full-length cDNA collections for two conifers, Sitka spruce (Picea sitchensis) and loblolly pine (Pinus taeda), together with full genome sequences for two angiosperms, Arabidopsis thaliana and poplar (Populus trichocarpa), offer an opportunity to infer the evolutionary processes underlying thousands of orthologous protein-coding genes in gymnosperms compared with an angiosperm orthologue set. Based upon pairwise comparisons of 3,723 spruce and pine orthologues, we found an average synonymous genetic distance (dS) of 0.191, and an average dN/dS ratio of 0.314. Using a fossil-established divergence time of 140 million years between spruce and pine, we extrapolated a nucleotide substitution rate of 0.68 × 10(-9) synonymous substitutions per site per year. When compared to angiosperms, this indicates a dramatically slower rate of nucleotide substitution rates in conifers: on average 15-fold. Coincidentally, we found a three-fold higher dN/dS for the spruce-pine lineage compared to the poplar-Arabidopsis lineage. This joint occurrence of a slower evolutionary rate in conifers with higher dN/dS, and possibly positive selection, showcases the uniqueness of conifer genome evolution. Our results are in line with documented reduced nucleotide diversity, conservative genome evolution and low rates of diversification in conifers on the one hand and numerous examples of local adaptation in conifers on the other hand. We propose that reduced levels of nucleotide mutation in large and long-lived conifer trees, coupled with large effective population size, were the main factors leading to slow substitution rates but retention of beneficial mutations.

A natural allele of Nxf1/TAP supresses retrovirus insertional mutations

PubMed Central

Floyd, Jennifer A.; Gold, David A.; Concepcion, Dorothy; Poon, Tiffany H.; Wang, Xiaobo; Keithley, Elizabeth; Chen, Dan; Ward, Erica J.; Chinn, Steven B.; Friedman, Rick A.; Yu, Hon-Tsen; Moriwaki, Kazuo; Shiroishi, Toshihiko; Hamilton, Bruce A.

2009-01-01

Endogenous retroviruses have shaped the evolution of mammalian genomes. Host genes that control the effects of retrovirus insertions are therefore of great interest. The Modifier-of-vibrator-1 locus controls level of correctly processed mRNA from genes mutated by endogenous retrovirus insertions into introns, including the pitpnvb tremor mutation and the Eya1BOR model of human branchiootorenal syndrome. Positional complementation cloning identifies Mvb1 as the nuclear export factor Nxf1, providing an unexpected link between mRNA export receptor and pre-mRNA processing. Population structure of the suppressing allele in wild M. m. castaneus suggests selective advantage. A congenic Mvb1CAST allele is a useful tool for modifying gene expression from existing mutations and could be used to manipulate engineered mutations containing retroviral elements. PMID:14517553

Detection of inherited mutations for breast and ovarian cancer using genomic capture and massively parallel sequencing

PubMed Central

Walsh, Tom; Lee, Ming K.; Casadei, Silvia; Thornton, Anne M.; Stray, Sunday M.; Pennil, Christopher; Nord, Alex S.; Mandell, Jessica B.; Swisher, Elizabeth M.; King, Mary-Claire

2010-01-01

Inherited loss-of-function mutations in the tumor suppressor genes BRCA1, BRCA2, and multiple other genes predispose to high risks of breast and/or ovarian cancer. Cancer-associated inherited mutations in these genes are collectively quite common, but individually rare or even private. Genetic testing for BRCA1 and BRCA2 mutations has become an integral part of clinical practice, but testing is generally limited to these two genes and to women with severe family histories of breast or ovarian cancer. To determine whether massively parallel, “next-generation” sequencing would enable accurate, thorough, and cost-effective identification of inherited mutations for breast and ovarian cancer, we developed a genomic assay to capture, sequence, and detect all mutations in 21 genes, including BRCA1 and BRCA2, with inherited mutations that predispose to breast or ovarian cancer. Constitutional genomic DNA from subjects with known inherited mutations, ranging in size from 1 to >100,000 bp, was hybridized to custom oligonucleotides and then sequenced using a genome analyzer. Analysis was carried out blind to the mutation in each sample. Average coverage was >1200 reads per base pair. After filtering sequences for quality and number of reads, all single-nucleotide substitutions, small insertion and deletion mutations, and large genomic duplications and deletions were detected. There were zero false-positive calls of nonsense mutations, frameshift mutations, or genomic rearrangements for any gene in any of the test samples. This approach enables widespread genetic testing and personalized risk assessment for breast and ovarian cancer. PMID:20616022

The Nature and Evolution of Genomic Diversity in the Mycobacterium tuberculosis Complex.

PubMed

Brites, Daniela; Gagneux, Sebastien

2017-01-01

The Mycobacterium tuberculosis Complex (MTBC) consists of a clonal group of several mycobacterial lineages pathogenic to a range of different mammalian hosts. In this chapter, we discuss the origins and the evolutionary forces shaping the genomic diversity of the human-adapted MTBC. Advances in whole-genome sequencing have brought invaluable insights into the macro-evolution of the MTBC, and the biogeographical distribution of the different MTBC lineages, the phylogenetic relationships between these lineages. Moreover, micro-evolutionary processes start to be better understood, including those influencing bacterial mutation rates and those governing the fate of new mutations emerging within patients during treatment. Current genomic and epidemiological evidence reflect the fact that, through ecological specialization, the MTBC affecting humans became an obligate and extremely well-adapted human pathogen. Identifying the adaptive traits of human-adapted MTBC and unraveling the bacterial loci that interact with human genomic variation might help identify new targets for developing better vaccines and designing more effective treatments.

[Induced germ line genomic instability at mini- and micro-satellites in animals].

PubMed

Bezlepkin, V G; Gaziev, A I

2001-01-01

The recent data on the phenomenon of the induced germline genomic instability at mini- and microsatellites in animals were considered. Natural hypervariability of the minisatellites and microsatellites and their abundance in eukaryotic genome provide it's utility as the useful genetic markers for evaluation of the germline mutation frequency induced by treatment with different type of genotoxic factors at the low doses. High sensitivity of assays and possibility for direct determinations of the mutations, without the necessity to use extrapolation, are ensured. Some discussion is presented on the role of non-targeted mechanisms for the radiation-prone DNA lesions in the induction of germline genomic instability and also on the involving in this process the recombination events upon meiosis or during the early development stages of embryos. It is proposed that quantitative determination of germline genomic instability rate may be used as an acceptable variant for the genetic risk assessment and as indicator of increased probability for cancer and other pathologies at the offspring born to irradiated parents.

Noncoding Genomics in Gastric Cancer and the Gastric Precancerous Cascade: Pathogenesis and Biomarkers

PubMed Central

Garcia-Bloj, Benjamin; Fry, Jacqueline; Wichmann, Ignacio

2015-01-01

Gastric cancer is the fifth most common cancer and the third leading cause of cancer-related death, whose patterns vary among geographical regions and ethnicities. It is a multifactorial disease, and its development depends on infection by Helicobacter pylori (H. pylori) and Epstein-Barr virus (EBV), host genetic factors, and environmental factors. The heterogeneity of the disease has begun to be unraveled by a comprehensive mutational evaluation of primary tumors. The low-abundance of mutations suggests that other mechanisms participate in the evolution of the disease, such as those found through analyses of noncoding genomics. Noncoding genomics includes single nucleotide polymorphisms (SNPs), regulation of gene expression through DNA methylation of promoter sites, miRNAs, other noncoding RNAs in regulatory regions, and other topics. These processes and molecules ultimately control gene expression. Potential biomarkers are appearing from analyses of noncoding genomics. This review focuses on noncoding genomics and potential biomarkers in the context of gastric cancer and the gastric precancerous cascade. PMID:26379360

Replication-associated mutational asymmetry in the human genome.

PubMed

Chen, Chun-Long; Duquenne, Lauranne; Audit, Benjamin; Guilbaud, Guillaume; Rappailles, Aurélien; Baker, Antoine; Huvet, Maxime; d'Aubenton-Carafa, Yves; Hyrien, Olivier; Arneodo, Alain; Thermes, Claude

2011-08-01

During evolution, mutations occur at rates that can differ between the two DNA strands. In the human genome, nucleotide substitutions occur at different rates on the transcribed and non-transcribed strands that may result from transcription-coupled repair. These mutational asymmetries generate transcription-associated compositional skews. To date, the existence of such asymmetries associated with replication has not yet been established. Here, we compute the nucleotide substitution matrices around replication initiation zones identified as sharp peaks in replication timing profiles and associated with abrupt jumps in the compositional skew profile. We show that the substitution matrices computed in these regions fully explain the jumps in the compositional skew profile when crossing initiation zones. In intergenic regions, we observe mutational asymmetries measured as differences between complementary substitution rates; their sign changes when crossing initiation zones. These mutational asymmetries are unlikely to result from cryptic transcription but can be explained by a model based on replication errors and strand-biased repair. In transcribed regions, mutational asymmetries associated with replication superimpose on the previously described mutational asymmetries associated with transcription. We separate the substitution asymmetries associated with both mechanisms, which allows us to determine for the first time in eukaryotes, the mutational asymmetries associated with replication and to reevaluate those associated with transcription. Replication-associated mutational asymmetry may result from unequal rates of complementary base misincorporation by the DNA polymerases coupled with DNA mismatch repair (MMR) acting with different efficiencies on the leading and lagging strands. Replication, acting in germ line cells during long evolutionary times, contributed equally with transcription to produce the present abrupt jumps in the compositional skew. These results demonstrate that DNA replication is one of the major processes that shape human genome composition.

Evolution of Local Mutation Rate and Its Determinants.

PubMed

Terekhanova, Nadezhda V; Seplyarskiy, Vladimir B; Soldatov, Ruslan A; Bazykin, Georgii A

2017-05-01

Mutation rate varies along the human genome, and part of this variation is explainable by measurable local properties of the DNA molecule. Moreover, mutation rates differ between orthologous genomic regions of different species, but the drivers of this change are unclear. Here, we use data on human divergence from chimpanzee, human rare polymorphism, and human de novo mutations to predict the substitution rate at orthologous regions of non-human mammals. We show that the local mutation rates are very similar between human and apes, implying that their variation has a strong underlying cryptic component not explainable by the known genomic features. Mutation rates become progressively less similar in more distant species, and these changes are partially explainable by changes in the local genomic features of orthologous regions, most importantly, in the recombination rate. However, they are much more rapid, implying that the cryptic component underlying the mutation rate is more ephemeral than the known genomic features. These findings shed light on the determinants of mutation rate evolution. local mutation rate, molecular evolution, recombination rate. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

GenomeGems: evaluation of genetic variability from deep sequencing data

PubMed Central

2012-01-01

Background Detection of disease-causing mutations using Deep Sequencing technologies possesses great challenges. In particular, organizing the great amount of sequences generated so that mutations, which might possibly be biologically relevant, are easily identified is a difficult task. Yet, for this assignment only limited automatic accessible tools exist. Findings We developed GenomeGems to gap this need by enabling the user to view and compare Single Nucleotide Polymorphisms (SNPs) from multiple datasets and to load the data onto the UCSC Genome Browser for an expanded and familiar visualization. As such, via automatic, clear and accessible presentation of processed Deep Sequencing data, our tool aims to facilitate ranking of genomic SNP calling. GenomeGems runs on a local Personal Computer (PC) and is freely available at http://www.tau.ac.il/~nshomron/GenomeGems. Conclusions GenomeGems enables researchers to identify potential disease-causing SNPs in an efficient manner. This enables rapid turnover of information and leads to further experimental SNP validation. The tool allows the user to compare and visualize SNPs from multiple experiments and to easily load SNP data onto the UCSC Genome browser for further detailed information. PMID:22748151

On the molecular mechanism of GC content variation among eubacterial genomes.

PubMed

Wu, Hao; Zhang, Zhang; Hu, Songnian; Yu, Jun

2012-01-10

As a key parameter of genome sequence variation, the GC content of bacterial genomes has been investigated for over half a century, and many hypotheses have been put forward to explain this GC content variation and its relationship to other fundamental processes. Previously, we classified eubacteria into dnaE-based groups (the dimeric combination of DNA polymerase III alpha subunits), according to a hypothesis where GC content variation is essentially governed by genome replication and DNA repair mechanisms. Further investigation led to the discovery that two major mutator genes, polC and dnaE2, may be responsible for genomic GC content variation. Consequently, an in-depth analysis was conducted to evaluate various potential intrinsic and extrinsic factors in association with GC content variation among eubacterial genomes. Mutator genes, especially those with dominant effects on the mutation spectra, are biased towards either GC or AT richness, and they alter genomic GC content in the two opposite directions. Increased bacterial genome size (or gene number) appears to rely on increased genomic GC content; however, it is unclear whether the changes are directly related to certain environmental pressures. Certain environmental and bacteriological features are related to GC content variation, but their trends are more obvious when analyzed under the dnaE-based grouping scheme. Most terrestrial, plant-associated, and nitrogen-fixing bacteria are members of the dnaE1|dnaE2 group, whereas most pathogenic or symbiotic bacteria in insects, and those dwelling in aquatic environments, are largely members of the dnaE1|polV group. Our studies provide several lines of evidence indicating that DNA polymerase III α subunit and its isoforms participating in either replication (such as polC) or SOS mutagenesis/translesion synthesis (such as dnaE2), play dominant roles in determining GC variability. Other environmental or bacteriological factors, such as genome size, temperature, oxygen requirement, and habitat, either play subsidiary roles or rely indirectly on different mutator genes to fine-tune the GC content. These results provide a comprehensive insight into mechanisms of GC content variation and the robustness of eubacterial genomes in adapting their ever-changing environments over billions of years.

Mutational burdens and evolutionary ages of thyroid follicular adenoma are comparable to those of follicular carcinoma

PubMed Central

Jung, Seung-Hyun; Kim, Min Sung; Jung, Chan Kwon; Park, Hyun-Chun; Kim, So Youn; Liu, Jieying; Bae, Ja-Seong; Lee, Sung Hak; Kim, Tae-Min; Lee, Sug Hyung; Chung, Yeun-Jun

2016-01-01

Follicular thyroid adenoma (FTA) precedes follicular thyroid carcinoma (FTC) by definition with a favorable prognosis compared to FTC. However, the genetic mechanism of FTA to FTC progression remains unknown. For this, it is required to disclose FTA and FTC genomes in mutational and evolutionary perspectives. We performed whole-exome sequencing and copy number profiling of 14 FTAs and 13 FTCs, which exhibited previously-known gene mutations (NRAS, HRAS, BRAF, TSHR and EIF1AX) and copy number alterations (CNAs) (22q loss and 1q gain) in follicular tumors. In addition, we found eleven potential cancer-related genes with mutations (EZH1, SPOP, NF1, TCF12, IGF2BP3, KMT2C, CNOT1, BRIP1, KDM5C, STAG2 and MAP4K3) that have not been reported in thyroid follicular tumors. Of note, FTA genomes showed comparable levels of mutations to FTC in terms of the number, sequence composition and functional consequences (potential driver mutations) of mutations. Analyses of evolutionary ages using somatic mutations as molecular clocks further identified that FTA genomes were as old as FTC genomes. Whole-transcriptome sequencing did not find any gene fusions with potential significance. Our data indicate that FTA genomes may be as old as FTC genomes, thus suggesting that follicular thyroid tumor genomes during the transition from FTA to FTC may stand stable at genomic levels in contrast to the discernable changes at pathologic and clinical levels. Also, the data suggest a possibility that the mutational profiles obtained from early biopsies may be useful for the molecular diagnosis and therapeutics of follicular tumor patients. PMID:27626165

Mutational burdens and evolutionary ages of thyroid follicular adenoma are comparable to those of follicular carcinoma.

PubMed

Jung, Seung-Hyun; Kim, Min Sung; Jung, Chan Kwon; Park, Hyun-Chun; Kim, So Youn; Liu, Jieying; Bae, Ja-Seong; Lee, Sung Hak; Kim, Tae-Min; Lee, Sug Hyung; Chung, Yeun-Jun

2016-10-25

Follicular thyroid adenoma (FTA) precedes follicular thyroid carcinoma (FTC) by definition with a favorable prognosis compared to FTC. However, the genetic mechanism of FTA to FTC progression remains unknown. For this, it is required to disclose FTA and FTC genomes in mutational and evolutionary perspectives. We performed whole-exome sequencing and copy number profiling of 14 FTAs and 13 FTCs, which exhibited previously-known gene mutations (NRAS, HRAS, BRAF, TSHR and EIF1AX) and copy number alterations (CNAs) (22q loss and 1q gain) in follicular tumors. In addition, we found eleven potential cancer-related genes with mutations (EZH1, SPOP, NF1, TCF12, IGF2BP3, KMT2C, CNOT1, BRIP1, KDM5C, STAG2 and MAP4K3) that have not been reported in thyroid follicular tumors. Of note, FTA genomes showed comparable levels of mutations to FTC in terms of the number, sequence composition and functional consequences (potential driver mutations) of mutations. Analyses of evolutionary ages using somatic mutations as molecular clocks further identified that FTA genomes were as old as FTC genomes. Whole-transcriptome sequencing did not find any gene fusions with potential significance. Our data indicate that FTA genomes may be as old as FTC genomes, thus suggesting that follicular thyroid tumor genomes during the transition from FTA to FTC may stand stable at genomic levels in contrast to the discernable changes at pathologic and clinical levels. Also, the data suggest a possibility that the mutational profiles obtained from early biopsies may be useful for the molecular diagnosis and therapeutics of follicular tumor patients.

Mapping mitochondrial heteroplasmy in a Leydig tumor by laser capture micro-dissection and cycling temperature capillary electrophoresis.

PubMed

Refinetti, Paulo; Arstad, Christian; Thilly, William G; Morgenthaler, Stephan; Ekstrøm, Per Olaf

2017-01-01

The growth of tumor cells is accompanied by mutations in nuclear and mitochondrial genomes creating marked genetic heterogeneity. Tumors also contain non-tumor cells of various origins. An observed somatic mitochondrial mutation would have occurred in a founding cell and spread through cell division. Micro-anatomical dissection of a tumor coupled with assays for mitochondrial point mutations permits new insights into this growth process. More generally, the ability to detect and trace, at a histological level, somatic mitochondrial mutations in human tissues and tumors, makes these mutations into markers for lineage tracing. A tumor was first sampled by a large punch biopsy and scanned for any significant degree of heteroplasmy in a set of sequences containing known mutational hotspots of the mitochondrial genome. A heteroplasmic tumor was sliced at a 12 μm thickness and placed on membranes. Laser capture micro-dissection was used to take 25000 μm 2 subsamples or spots. After DNA amplification, cycling temperature capillary electrophoresis (CTCE) was used on the laser captured samples to quantify mitochondrial mutant fractions. Of six testicular tumors studied, one, a Leydig tumor, was discovered to carry a detectable degree of heteroplasmy for two separate point mutations: a C → T mutation at bp 64 and a T → C mutation found at bp 152. From this tumor, 381 spots were sampled with laser capture micro-dissection. The ordered distribution of spots exhibited a wide range of fractions of the mutant sequences from 0 to 100% mutant copies. The two mutations co-distributed in the growing tumor indicating they were present on the same genome copies in the founding cell. Laser capture microdissection of sliced tumor samples coupled with CTCE-based point mutation assays provides an effective and practical means to obtain maps of mitochondrial mutational heteroplasmy within human tumors.

The Fusarium Graminearum Genome Reveals a Link Between Localized Polymorphism and Pathogen Specialization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cuomo, Christina A.; Guldener, Ulrich; Xu, Jin Rong

2007-09-07

We sequenced and annotated the genome of the filamentous fungus Fusarium graminearum, a major pathogen of cultivated cereals. Very few repetitive sequences were detected, and the process of repeat-induced point mutation, in which duplicated sequences are subject to extensive mutation, may partially account for the reduced repeat content and apparent low number of paralogous (ancestrally duplicated) genes. A second strain of F. graminearum contained more than 10,000 single-nucleotide polymorphisms, which were frequently located near telomeres and within other discrete chromosomal segments. Many highly polymorphic regions contained sets of genes implicated in plant-fungus interactions and were unusually divergent, with higher ratesmore » of recombination. These regions of genome innovation may result from selection due to interactions of F. graminearum with its plant hosts.« less

Genomic and epigenomic heterogeneity of hepatocellular carcinoma

PubMed Central

Lin, De-Chen; Mayakonda, Anand; Dinh, Huy Q.; Huang, Pinbo; Lin, Lehang; Liu, Xiaoping; Ding, Ling-wen; Wang, Jie; Berman, Benjamin P.; Song, Er-Wei; Yin, Dong; Koeffler, H. Phillip

2017-01-01

Understanding the intratumoral heterogeneity of hepatocellular carcinoma (HCC) is instructive for developing personalized therapy and identifying molecular biomarkers. Here we applied whole-exome sequencing to 69 samples from 11 patients to resolve the genetic architecture of subclonal diversification. Spatial genomic diversity was found in all 11 HCC cases, with 29% of driver mutations being heterogeneous, including TERT, ARID1A, NOTCH2, and STAG2. Similar with other cancer types, TP53 mutations were always shared between all tumor regions i.e. located on the “trunk” of the evolutionary tree. In addition, we found that variants within several drug targets such as KIT, SYK and PIK3CA were mutated in a fully clonal manner, indicating their therapeutic potentials for HCC. Temporal dissection of mutational signatures suggested that mutagenic processes associated with exposure to aristolochic acid and aflatoxin might play a more important role in early, as opposed to late, stages of HCC development. Moreover, we observed extensive intratumoral epigenetic heterogeneity in HCC based on multiple independent analytical methods and showed that intratumoral methylation heterogeneity might play important roles in the biology of HCC cells. Our results also demonstrated prominent heterogeneity of intratumoral methylation even in a stable HCC genome. Together, these findings highlight widespread intratumoral heterogeneity at both the genomic and epigenomic levels in HCC and provide an important molecular foundation for better understanding the pathogenesis of this malignancy. PMID:28302680

The Impact of Environmental and Endogenous Damage on Somatic Mutation Load in Human Skin Fibroblasts

PubMed Central

Saini, Natalie; Chan, Kin; Grimm, Sara A.; Dai, Shuangshuang; Fargo, David C.; Kaufmann, William K.; Taylor, Jack A.; Lee, Eunjung; Cortes-Ciriano, Isidro; Park, Peter J.; Schurman, Shepherd H.; Malc, Ewa P.; Mieczkowski, Piotr A.

2016-01-01

Accumulation of somatic changes, due to environmental and endogenous lesions, in the human genome is associated with aging and cancer. Understanding the impacts of these processes on mutagenesis is fundamental to understanding the etiology, and improving the prognosis and prevention of cancers and other genetic diseases. Previous methods relying on either the generation of induced pluripotent stem cells, or sequencing of single-cell genomes were inherently error-prone and did not allow independent validation of the mutations. In the current study we eliminated these potential sources of error by high coverage genome sequencing of single-cell derived clonal fibroblast lineages, obtained after minimal propagation in culture, prepared from skin biopsies of two healthy adult humans. We report here accurate measurement of genome-wide magnitude and spectra of mutations accrued in skin fibroblasts of healthy adult humans. We found that every cell contains at least one chromosomal rearrangement and 600–13,000 base substitutions. The spectra and correlation of base substitutions with epigenomic features resemble many cancers. Moreover, because biopsies were taken from body parts differing by sun exposure, we can delineate the precise contributions of environmental and endogenous factors to the accrual of genetic changes within the same individual. We show here that UV-induced and endogenous DNA damage can have a comparable impact on the somatic mutation loads in skin fibroblasts. Trial Registration ClinicalTrials.gov NCT01087307 PMID:27788131

Multi-Species Genome Comparison Sheds New Light on Evolutionary Processes, Cancer Mutations

ERIC Educational Resources Information Center

Journal of College Science Teaching, 2005

2005-01-01

An international team that includes researchers from the National Human Genome Research Institute (NHGRI), part of the National Institutes of Health (NIH), has discovered that mammalian chromosomes have evolved by breaking at specific sites rather than randomly as long thought--and that many of the breakage hot spots are also involved in human…

Cancer Genes in Lung Cancer

PubMed Central

El-Telbany, Ahmed

2012-01-01

Cancer is now known as a disease of genomic alterations. Mutational analysis and genomics profiling in recent years have advanced the field of lung cancer genetics/genomics significantly. It is becoming more accepted now that the identification of genomic alterations in lung cancer can impact therapeutics, especially when the alterations represent “oncogenic drivers” in the processes of tumorigenesis and progression. In this review, we will highlight the key driver oncogenic gene mutations and fusions identified in lung cancer. The review will summarize and report the available demographic and clinicopathological data as well as molecular details behind various lung cancer gene alterations in the context of race. We hope to shed some light into the disparities in the incidence of various genetic mutations among lung cancer patients of different racial backgrounds. As molecularly targeted therapy continues to advance in lung cancer, racial differences in specific genetic/genomic alterations can have an important impact in the choices of therapeutics and in our understanding of the drug sensitivity/resistance profile. The most relevant genes in lung cancer described in this review include the following: EGFR, KRAS, MET, LKB1, BRAF, PIK3CA, ALK, RET, and ROS1. Commonly identified genetic/genomic alterations such as missense or nonsense mutations, small insertions or deletions, alternative splicing, and chromosomal fusion rearrangements were discussed. Relevance in current targeted therapeutic drugs was mentioned when appropriate. We also highlighted various targeted therapeutics that are currently under clinical development, such as the MET inhibitors and antibodies. With the advent of next-generation sequencing, the landscape of genomic alterations in lung cancer is expected to be much transformed and detailed in upcoming years. These genomic landscape differences in the context of racial disparities should be emphasized both in tumorigenesis and in drug sensitivity/resistance. It is hoped that such effort will help to diminish racial disparities in lung cancer outcome in the future. PMID:23264847

Comparative genomic and proteomic analyses of Clostridium acetobutylicum Rh8 and its parent strain DSM 1731 revealed new understandings on butanol tolerance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bao, Guanhui; University of Chinese Academy of Sciences, Beijing; Dong, Hongjun

Highlights: • Genomes of a butanol tolerant strain and its parent strain were deciphered. • Comparative genomic and proteomic was applied to understand butanol tolerance. • None differentially expressed proteins have mutations in its corresponding genes. • Mutations in ribosome might be responsible for the global difference of proteomics. - Abstract: Clostridium acetobutylicum strain Rh8 is a butanol-tolerant mutant which can tolerate up to 19 g/L butanol, 46% higher than that of its parent strain DSM 1731. We previously performed comparative cytoplasm- and membrane-proteomic analyses to understand the mechanism underlying the improved butanol tolerance of strain Rh8. In this work,more » we further extended this comparison to the genomic level. Compared with the genome of the parent strain DSM 1731, two insertion sites, four deletion sites, and 67 single nucleotide variations (SNVs) are distributed throughout the genome of strain Rh8. Among the 67 SNVs, 16 SNVs are located in the predicted promoters and intergenic regions; while 29 SNVs are located in the coding sequence, affecting a total of 21 proteins involved in transport, cell structure, DNA replication, and protein translation. The remaining 22 SNVs are located in the ribosomal genes, affecting a total of 12 rRNA genes in different operons. Analysis of previous comparative proteomic data indicated that none of the differentially expressed proteins have mutations in its corresponding genes. Rchange Algorithms analysis indicated that the mutations occurred in the ribosomal genes might change the ribosome RNA thermodynamic characteristics, thus affect the translation strength of these proteins. Take together, the improved butanol tolerance of C. acetobutylicum strain Rh8 might be acquired through regulating the translational process to achieve different expression strength of genes involved in butanol tolerance.« less

Genomes of the Mouse Collaborative Cross.

PubMed

Srivastava, Anuj; Morgan, Andrew P; Najarian, Maya L; Sarsani, Vishal Kumar; Sigmon, J Sebastian; Shorter, John R; Kashfeen, Anwica; McMullan, Rachel C; Williams, Lucy H; Giusti-Rodríguez, Paola; Ferris, Martin T; Sullivan, Patrick; Hock, Pablo; Miller, Darla R; Bell, Timothy A; McMillan, Leonard; Churchill, Gary A; de Villena, Fernando Pardo-Manuel

2017-06-01

The Collaborative Cross (CC) is a multiparent panel of recombinant inbred (RI) mouse strains derived from eight founder laboratory strains. RI panels are popular because of their long-term genetic stability, which enhances reproducibility and integration of data collected across time and conditions. Characterization of their genomes can be a community effort, reducing the burden on individual users. Here we present the genomes of the CC strains using two complementary approaches as a resource to improve power and interpretation of genetic experiments. Our study also provides a cautionary tale regarding the limitations imposed by such basic biological processes as mutation and selection. A distinct advantage of inbred panels is that genotyping only needs to be performed on the panel, not on each individual mouse. The initial CC genome data were haplotype reconstructions based on dense genotyping of the most recent common ancestors (MRCAs) of each strain followed by imputation from the genome sequence of the corresponding founder inbred strain. The MRCA resource captured segregating regions in strains that were not fully inbred, but it had limited resolution in the transition regions between founder haplotypes, and there was uncertainty about founder assignment in regions of limited diversity. Here we report the whole genome sequence of 69 CC strains generated by paired-end short reads at 30× coverage of a single male per strain. Sequencing leads to a substantial improvement in the fine structure and completeness of the genomes of the CC. Both MRCAs and sequenced samples show a significant reduction in the genome-wide haplotype frequencies from two wild-derived strains, CAST/EiJ and PWK/PhJ. In addition, analysis of the evolution of the patterns of heterozygosity indicates that selection against three wild-derived founder strains played a significant role in shaping the genomes of the CC. The sequencing resource provides the first description of tens of thousands of new genetic variants introduced by mutation and drift in the CC genomes. We estimate that new SNP mutations are accumulating in each CC strain at a rate of 2.4 ± 0.4 per gigabase per generation. The fixation of new mutations by genetic drift has introduced thousands of new variants into the CC strains. The majority of these mutations are novel compared to currently sequenced laboratory stocks and wild mice, and some are predicted to alter gene function. Approximately one-third of the CC inbred strains have acquired large deletions (>10 kb) many of which overlap known coding genes and functional elements. The sequence of these mice is a critical resource to CC users, increases threefold the number of mouse inbred strain genomes available publicly, and provides insight into the effect of mutation and drift on common resources. Copyright © 2017 Srivastava et al.

Genomes of the Mouse Collaborative Cross

PubMed Central

Srivastava, Anuj; Morgan, Andrew P.; Najarian, Maya L.; Sarsani, Vishal Kumar; Sigmon, J. Sebastian; Shorter, John R.; Kashfeen, Anwica; McMullan, Rachel C.; Williams, Lucy H.; Giusti-Rodríguez, Paola; Ferris, Martin T.; Sullivan, Patrick; Hock, Pablo; Miller, Darla R.; Bell, Timothy A.; McMillan, Leonard; Churchill, Gary A.; de Villena, Fernando Pardo-Manuel

2017-01-01

The Collaborative Cross (CC) is a multiparent panel of recombinant inbred (RI) mouse strains derived from eight founder laboratory strains. RI panels are popular because of their long-term genetic stability, which enhances reproducibility and integration of data collected across time and conditions. Characterization of their genomes can be a community effort, reducing the burden on individual users. Here we present the genomes of the CC strains using two complementary approaches as a resource to improve power and interpretation of genetic experiments. Our study also provides a cautionary tale regarding the limitations imposed by such basic biological processes as mutation and selection. A distinct advantage of inbred panels is that genotyping only needs to be performed on the panel, not on each individual mouse. The initial CC genome data were haplotype reconstructions based on dense genotyping of the most recent common ancestors (MRCAs) of each strain followed by imputation from the genome sequence of the corresponding founder inbred strain. The MRCA resource captured segregating regions in strains that were not fully inbred, but it had limited resolution in the transition regions between founder haplotypes, and there was uncertainty about founder assignment in regions of limited diversity. Here we report the whole genome sequence of 69 CC strains generated by paired-end short reads at 30× coverage of a single male per strain. Sequencing leads to a substantial improvement in the fine structure and completeness of the genomes of the CC. Both MRCAs and sequenced samples show a significant reduction in the genome-wide haplotype frequencies from two wild-derived strains, CAST/EiJ and PWK/PhJ. In addition, analysis of the evolution of the patterns of heterozygosity indicates that selection against three wild-derived founder strains played a significant role in shaping the genomes of the CC. The sequencing resource provides the first description of tens of thousands of new genetic variants introduced by mutation and drift in the CC genomes. We estimate that new SNP mutations are accumulating in each CC strain at a rate of 2.4 ± 0.4 per gigabase per generation. The fixation of new mutations by genetic drift has introduced thousands of new variants into the CC strains. The majority of these mutations are novel compared to currently sequenced laboratory stocks and wild mice, and some are predicted to alter gene function. Approximately one-third of the CC inbred strains have acquired large deletions (>10 kb) many of which overlap known coding genes and functional elements. The sequence of these mice is a critical resource to CC users, increases threefold the number of mouse inbred strain genomes available publicly, and provides insight into the effect of mutation and drift on common resources. PMID:28592495

A Gene Gravity Model for the Evolution of Cancer Genomes: A Study of 3,000 Cancer Genomes across 9 Cancer Types.

PubMed

Cheng, Feixiong; Liu, Chuang; Lin, Chen-Ching; Zhao, Junfei; Jia, Peilin; Li, Wen-Hsiung; Zhao, Zhongming

2015-09-01

Cancer development and progression result from somatic evolution by an accumulation of genomic alterations. The effects of those alterations on the fitness of somatic cells lead to evolutionary adaptations such as increased cell proliferation, angiogenesis, and altered anticancer drug responses. However, there are few general mathematical models to quantitatively examine how perturbations of a single gene shape subsequent evolution of the cancer genome. In this study, we proposed the gene gravity model to study the evolution of cancer genomes by incorporating the genome-wide transcription and somatic mutation profiles of ~3,000 tumors across 9 cancer types from The Cancer Genome Atlas into a broad gene network. We found that somatic mutations of a cancer driver gene may drive cancer genome evolution by inducing mutations in other genes. This functional consequence is often generated by the combined effect of genetic and epigenetic (e.g., chromatin regulation) alterations. By quantifying cancer genome evolution using the gene gravity model, we identified six putative cancer genes (AHNAK, COL11A1, DDX3X, FAT4, STAG2, and SYNE1). The tumor genomes harboring the nonsynonymous somatic mutations in these genes had a higher mutation density at the genome level compared to the wild-type groups. Furthermore, we provided statistical evidence that hypermutation of cancer driver genes on inactive X chromosomes is a general feature in female cancer genomes. In summary, this study sheds light on the functional consequences and evolutionary characteristics of somatic mutations during tumorigenesis by propelling adaptive cancer genome evolution, which would provide new perspectives for cancer research and therapeutics.

A Gene Gravity Model for the Evolution of Cancer Genomes: A Study of 3,000 Cancer Genomes across 9 Cancer Types

PubMed Central

Lin, Chen-Ching; Zhao, Junfei; Jia, Peilin; Li, Wen-Hsiung; Zhao, Zhongming

2015-01-01

Cancer development and progression result from somatic evolution by an accumulation of genomic alterations. The effects of those alterations on the fitness of somatic cells lead to evolutionary adaptations such as increased cell proliferation, angiogenesis, and altered anticancer drug responses. However, there are few general mathematical models to quantitatively examine how perturbations of a single gene shape subsequent evolution of the cancer genome. In this study, we proposed the gene gravity model to study the evolution of cancer genomes by incorporating the genome-wide transcription and somatic mutation profiles of ~3,000 tumors across 9 cancer types from The Cancer Genome Atlas into a broad gene network. We found that somatic mutations of a cancer driver gene may drive cancer genome evolution by inducing mutations in other genes. This functional consequence is often generated by the combined effect of genetic and epigenetic (e.g., chromatin regulation) alterations. By quantifying cancer genome evolution using the gene gravity model, we identified six putative cancer genes (AHNAK, COL11A1, DDX3X, FAT4, STAG2, and SYNE1). The tumor genomes harboring the nonsynonymous somatic mutations in these genes had a higher mutation density at the genome level compared to the wild-type groups. Furthermore, we provided statistical evidence that hypermutation of cancer driver genes on inactive X chromosomes is a general feature in female cancer genomes. In summary, this study sheds light on the functional consequences and evolutionary characteristics of somatic mutations during tumorigenesis by propelling adaptive cancer genome evolution, which would provide new perspectives for cancer research and therapeutics. PMID:26352260

2004 Environmental Mutagen Society Annual Meeting - Genes, Mutations and Disease: The Environmental Connection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Samson, Leona D.

2004-08-23

The Meeting consisted of 9 Symposia, 4 Keynote Lectures, 3 Platform Sessions and 4 Poster Sessions. In addition there were Breakfast Meetings for Special Interest Groups designed to inform attendees about the latest advances in environmental mutagenesis research. Several of the topics to be covered at this broad meeting will be of interest to the Department of Energy, Office of Science. The relevance of this meeting to the DOE derives from the fact that low dose radiation may represent one of the most significant sources of human mutations that are attributable to the environment. The EMS membership, and those whomore » attended the EMS Annual Meeting were interested in both chemical and radiation induced biological effects, such as cell death, mutation, teratogenesis, carcinogenesis and aging. These topics thate were presented at the 2004 EMS Annual meeting that were of clear interest to DOE include: human variation in cancer susceptibility, unusual mechanisms of mutation, germ and stem cell mutagenesis, recombination and the maintenance of genomic stability, multiple roles for DNA mismatch repair, DNA helicases, mutation, cancer and aging, Genome-wide transcriptional responses to environmental change, Telomeres and genomic stability: when ends don?t meet, systems biology approach to cell phenotypic decision processes, and the surprising biology of short RNAs. Poster and platform sessions addressed topics related to environmental mutagen exposure, DNA repair, mechanisms of mutagenesis, epidemiology, genomic and proteomics and bioinformatics. These sessions were designed to give student, postdocs and more junior scientists a chance to present their work.« less

The Repeat Expansion Diseases: the dark side of DNA repair?

PubMed Central

Zhao, Xiao-Nan; Usdin, Karen

2015-01-01

DNA repair normally protects the genome against mutations that threaten genome integrity and thus cell viability. However, growing evidence suggests that in the case of the Repeat Expansion Diseases, disorders that result from an increase in the size of a disease-specific microsatellite, the disease-causing mutation is actually the result of aberrant DNA repair. A variety of proteins from different DNA repair pathways have thus far been implicated in this process. This review will summarize recent findings from patients and from mouse models of these diseases that shed light on how these pathways may interact to cause repeat expansion. PMID:26002199

Somatic mutations reveal asymmetric cellular dynamics in the early human embryo

DOE PAGES

Ju, Young Seok; Martincorena, Inigo; Gerstung, Moritz; ...

2017-03-22

Somatic cells acquire mutations throughout the course of an individual’s life. Mutations occurring early in embryogenesis are often present in a substantial proportion of, but not all, cells in postnatal humans and thus have particular characteristics and effects. Depending on their location in the genome and the proportion of cells they are present in, these mosaic mutations can cause a wide range of genetic disease syndromes and predispose carriers to cancer. They have a high chance of being transmitted to offspring as de novo germline mutations and, in principle, can provide insights into early human embryonic cell lineages and theirmore » contributions to adult tissues. Although it is known that gross chromosomal abnormalities are remarkably common in early human embryos, our understanding of early embryonic somatic mutations is very limited. Here we use whole-genome sequences of normal blood from 241 adults to identify 163 early embryonic mutations. We estimate that approximately three base substitution mutations occur per cell per cell-doubling event in early human embryogenesis and these are mainly attributable to two known mutational signatures. We used the mutations to reconstruct developmental lineages of adult cells and demonstrate that the two daughter cells of many early embryonic cell-doubling events contribute asymmetrically to adult blood at an approximately 2:1 ratio. As a result, this study therefore provides insights into the mutation rates, mutational processes and developmental outcomes of cell dynamics that operate during early human embryogenesis.« less

Somatic mutations reveal asymmetric cellular dynamics in the early human embryo

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ju, Young Seok; Martincorena, Inigo; Gerstung, Moritz

Somatic cells acquire mutations throughout the course of an individual’s life. Mutations occurring early in embryogenesis are often present in a substantial proportion of, but not all, cells in postnatal humans and thus have particular characteristics and effects. Depending on their location in the genome and the proportion of cells they are present in, these mosaic mutations can cause a wide range of genetic disease syndromes and predispose carriers to cancer. They have a high chance of being transmitted to offspring as de novo germline mutations and, in principle, can provide insights into early human embryonic cell lineages and theirmore » contributions to adult tissues. Although it is known that gross chromosomal abnormalities are remarkably common in early human embryos, our understanding of early embryonic somatic mutations is very limited. Here we use whole-genome sequences of normal blood from 241 adults to identify 163 early embryonic mutations. We estimate that approximately three base substitution mutations occur per cell per cell-doubling event in early human embryogenesis and these are mainly attributable to two known mutational signatures. We used the mutations to reconstruct developmental lineages of adult cells and demonstrate that the two daughter cells of many early embryonic cell-doubling events contribute asymmetrically to adult blood at an approximately 2:1 ratio. As a result, this study therefore provides insights into the mutation rates, mutational processes and developmental outcomes of cell dynamics that operate during early human embryogenesis.« less

A Constant Rate of Spontaneous Mutation in DNA-Based Microbes

NASA Astrophysics Data System (ADS)

Drake, John W.

1991-08-01

In terms of evolution and fitness, the most significant spontaneous mutation rate is likely to be that for the entire genome (or its nonfrivolous fraction). Information is now available to calculate this rate for several DNA-based haploid microbes, including bacteriophages with single- or double-stranded DNA, a bacterium, a yeast, and a filamentous fungus. Their genome sizes vary by ≈6500-fold. Their average mutation rates per base pair vary by ≈16,000-fold, whereas their mutation rates per genome vary by only ≈2.5-fold, apparently randomly, around a mean value of 0.0033 per DNA replication. The average mutation rate per base pair is inversely proportional to genome size. Therefore, a nearly invariant microbial mutation rate appears to have evolved. Because this rate is uniform in such diverse organisms, it is likely to be determined by deep general forces, perhaps by a balance between the usually deleterious effects of mutation and the physiological costs of further reducing mutation rates.

Detecting and characterizing genomic signatures of positive selection in global populations.

PubMed

Liu, Xuanyao; Ong, Rick Twee-Hee; Pillai, Esakimuthu Nisha; Elzein, Abier M; Small, Kerrin S; Clark, Taane G; Kwiatkowski, Dominic P; Teo, Yik-Ying

2013-06-06

Natural selection is a significant force that shapes the architecture of the human genome and introduces diversity across global populations. The question of whether advantageous mutations have arisen in the human genome as a result of single or multiple mutation events remains unanswered except for the fact that there exist a handful of genes such as those that confer lactase persistence, affect skin pigmentation, or cause sickle cell anemia. We have developed a long-range-haplotype method for identifying genomic signatures of positive selection to complement existing methods, such as the integrated haplotype score (iHS) or cross-population extended haplotype homozygosity (XP-EHH), for locating signals across the entire allele frequency spectrum. Our method also locates the founder haplotypes that carry the advantageous variants and infers their corresponding population frequencies. This presents an opportunity to systematically interrogate the whole human genome whether a selection signal shared across different populations is the consequence of a single mutation process followed subsequently by gene flow between populations or of convergent evolution due to the occurrence of multiple independent mutation events either at the same variant or within the same gene. The application of our method to data from 14 populations across the world revealed that positive-selection events tend to cluster in populations of the same ancestry. Comparing the founder haplotypes for events that are present across different populations revealed that convergent evolution is a rare occurrence and that the majority of shared signals stem from the same evolutionary event. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Promoting Cas9 degradation reduces mosaic mutations in non-human primate embryos

PubMed Central

Tu, Zhuchi; Yang, Weili; Yan, Sen; Yin, An; Gao, Jinquan; Liu, Xudong; Zheng, Yinghui; Zheng, Jiezhao; Li, Zhujun; Yang, Su; Li, Shihua; Guo, Xiangyu; Li, Xiao-Jiang

2017-01-01

CRISPR-Cas9 is a powerful new tool for genome editing, but this technique creates mosaic mutations that affect the efficiency and precision of its ability to edit the genome. Reducing mosaic mutations is particularly important for gene therapy and precision genome editing. Although the mechanisms underlying the CRSIPR/Cas9-mediated mosaic mutations remain elusive, the prolonged expression and activity of Cas9 in embryos could contribute to mosaicism in DNA mutations. Here we report that tagging Cas9 with ubiquitin-proteasomal degradation signals can facilitate the degradation of Cas9 in non-human primate embryos. Using embryo-splitting approach, we found that shortening the half-life of Cas9 in fertilized zygotes reduces mosaic mutations and increases its ability to modify genomes in non-human primate embryos. Also, injection of modified Cas9 in one-cell embryos leads to live monkeys with the targeted gene modifications. Our findings suggest that modifying Cas9 activity can be an effective strategy to enhance precision genome editing. PMID:28155910

Rates and Genomic Consequences of Spontaneous Mutational Events in Drosophila melanogaster

PubMed Central

Schrider, Daniel R.; Houle, David; Lynch, Michael; Hahn, Matthew W.

2013-01-01

Because spontaneous mutation is the source of all genetic diversity, measuring mutation rates can reveal how natural selection drives patterns of variation within and between species. We sequenced eight genomes produced by a mutation-accumulation experiment in Drosophila melanogaster. Our analysis reveals that point mutation and small indel rates vary significantly between the two different genetic backgrounds examined. We also find evidence that ∼2% of mutational events affect multiple closely spaced nucleotides. Unlike previous similar experiments, we were able to estimate genome-wide rates of large deletions and tandem duplications. These results suggest that, at least in inbred lines like those examined here, mutational pressures may result in net growth rather than contraction of the Drosophila genome. By comparing our mutation rate estimates to polymorphism data, we are able to estimate the fraction of new mutations that are eliminated by purifying selection. These results suggest that ∼99% of duplications and deletions are deleterious—making them 10 times more likely to be removed by selection than nonsynonymous mutations. Our results illuminate not only the rates of new small- and large-scale mutations, but also the selective forces that they encounter once they arise. PMID:23733788

CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula.

PubMed

Curtin, Shaun J; Xiong, Yer; Michno, Jean-Michel; Campbell, Benjamin W; Stec, Adrian O; Čermák, Tomas; Starker, Colby; Voytas, Daniel F; Eamens, Andrew L; Stupar, Robert M

2018-06-01

Processing of double-stranded RNA precursors into small RNAs is an essential regulator of gene expression in plant development and stress response. Small RNA processing requires the combined activity of a functionally diverse group of molecular components. However, in most of the plant species, there are insufficient mutant resources to functionally characterize each encoding gene. Here, mutations in loci encoding protein machinery involved in small RNA processing in soya bean and Medicago truncatula were generated using the CRISPR/Cas9 and TAL-effector nuclease (TALEN) mutagenesis platforms. An efficient CRISPR/Cas9 reagent was used to create a bi-allelic double mutant for the two soya bean paralogous Double-stranded RNA-binding2 (GmDrb2a and GmDrb2b) genes. These mutations, along with a CRISPR/Cas9-generated mutation of the M. truncatula Hua enhancer1 (MtHen1) gene, were determined to be germ-line transmissible. Furthermore, TALENs were used to generate a mutation within the soya bean Dicer-like2 gene. CRISPR/Cas9 mutagenesis of the soya bean Dicer-like3 gene and the GmHen1a gene was observed in the T 0 generation, but these mutations failed to transmit to the T 1 generation. The irregular transmission of induced mutations and the corresponding transgenes was investigated by whole-genome sequencing to reveal a spectrum of non-germ-line-targeted mutations and multiple transgene insertion events. Finally, a suite of combinatorial mutant plants were generated by combining the previously reported Gmdcl1a, Gmdcl1b and Gmdcl4b mutants with the Gmdrb2ab double mutant. Altogether, this study demonstrates the synergistic use of different genome engineering platforms to generate a collection of useful mutant plant lines for future study of small RNA processing in legume crops. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Genome editing of crops: A renewed opportunity for food security.

PubMed

Georges, Fawzy; Ray, Heather

2017-01-02

Genome editing of crop plants is a rapidly advancing technology whereby targeted mutations can be introduced into a plant genome in a highly specific manner and with great precision. For the most part, the technology does not incorporate transgenic modifications and is far superior to conventional chemical mutagenesis. In this study we bring into focus some of the underlying differences between the 3 existing technologies: classical plant breeding, genetic modification and genome editing. We discuss some of the main achievements from each area and highlight their common characteristics and individual limitations, while emphasizing the unique capabilities of genome editing. We subsequently examine the possible regulatory mechanisms which governments may be inclined to use in assessing the status of genome edited products. If assessed on the basis of their phenotype rather than the process by which they are obtained, these products will be categorized as equivalent to those produced by classical mutagenesis. This would mean that genome edited products will not be subject to the restrictions imposed on genetically modified products, except in some cases where the mutation involves a large sequence insertion into the genome. We conclude by examining the potential of societal acceptance of genome editing technology, reinforced by a scientific perspective on promoting such acceptance.

Genome editing of crops: A renewed opportunity for food security

PubMed Central

Georges, Fawzy

2017-01-01

ABSTRACT Genome editing of crop plants is a rapidly advancing technology whereby targeted mutations can be introduced into a plant genome in a highly specific manner and with great precision. For the most part, the technology does not incorporate transgenic modifications and is far superior to conventional chemical mutagenesis. In this study we bring into focus some of the underlying differences between the 3 existing technologies: classical plant breeding, genetic modification and genome editing. We discuss some of the main achievements from each area and highlight their common characteristics and individual limitations, while emphasizing the unique capabilities of genome editing. We subsequently examine the possible regulatory mechanisms which governments may be inclined to use in assessing the status of genome edited products. If assessed on the basis of their phenotype rather than the process by which they are obtained, these products will be categorized as equivalent to those produced by classical mutagenesis. This would mean that genome edited products will not be subject to the restrictions imposed on genetically modified products, except in some cases where the mutation involves a large sequence insertion into the genome. We conclude by examining the potential of societal acceptance of genome editing technology, reinforced by a scientific perspective on promoting such acceptance. PMID:28075688

Genome sequence of a diabetes-prone rodent reveals a mutation hotspot around the ParaHox gene cluster.

PubMed

Hargreaves, Adam D; Zhou, Long; Christensen, Josef; Marlétaz, Ferdinand; Liu, Shiping; Li, Fang; Jansen, Peter Gildsig; Spiga, Enrico; Hansen, Matilde Thye; Pedersen, Signe Vendelbo Horn; Biswas, Shameek; Serikawa, Kyle; Fox, Brian A; Taylor, William R; Mulley, John Frederick; Zhang, Guojie; Heller, R Scott; Holland, Peter W H

2017-07-18

The sand rat Psammomys obesus is a gerbil species native to deserts of North Africa and the Middle East, and is constrained in its ecology because high carbohydrate diets induce obesity and type II diabetes that, in extreme cases, can lead to pancreatic failure and death. We report the sequencing of the sand rat genome and discovery of an unusual, extensive, and mutationally biased GC-rich genomic domain. This highly divergent genomic region encompasses several functionally essential genes, and spans the ParaHox cluster which includes the insulin-regulating homeobox gene Pdx1. The sequence of sand rat Pdx1 has been grossly affected by GC-biased mutation, leading to the highest divergence observed for this gene across the Bilateria. In addition to genomic insights into restricted caloric intake in a desert species, the discovery of a localized chromosomal region subject to elevated mutation suggests that mutational heterogeneity within genomes could influence the course of evolution.

Translational Genomics: Practical Applications of the Genomic Revolution in Breast Cancer.

PubMed

Yates, Lucy R; Desmedt, Christine

2017-06-01

The genomic revolution has fundamentally changed our perception of breast cancer. It is now apparent from DNA-based massively parallel sequencing data that at the genomic level, every breast cancer is unique and shaped by the mutational processes to which it was exposed during its lifetime. More than 90 breast cancer driver genes have been identified as recurrently mutated, and many occur at low frequency across the breast cancer population. Certain cancer genes are associated with traditionally defined histologic subtypes, but genomic intertumoral heterogeneity exists even between cancers that appear the same under the microscope. Most breast cancers contain subclonal populations, many of which harbor driver alterations, and subclonal structure is typically remodeled over time, across metastasis and as a consequence of treatment interventions. Genomics is deepening our understanding of breast cancer biology, contributing to an accelerated phase of targeted drug development and providing insights into resistance mechanisms. Genomics is also providing tools necessary to deliver personalized cancer medicine, but a number of challenges must still be addressed. Clin Cancer Res; 23(11); 2630-9. ©2017 AACR See all articles in this CCR Focus section, "Breast Cancer Research: From Base Pairs to Populations." ©2017 American Association for Cancer Research.

Genome Sequencing and Comparative Genomics of the Broad Host-Range Pathogen Rhizoctonia solani AG8

PubMed Central

Hane, James K.; Anderson, Jonathan P.; Williams, Angela H.; Sperschneider, Jana; Singh, Karam B.

2014-01-01

Rhizoctonia solani is a soil-borne basidiomycete fungus with a necrotrophic lifestyle which is classified into fourteen reproductively incompatible anastomosis groups (AGs). One of these, AG8, is a devastating pathogen causing bare patch of cereals, brassicas and legumes. R. solani is a multinucleate heterokaryon containing significant heterozygosity within a single cell. This complexity posed significant challenges for the assembly of its genome. We present a high quality genome assembly of R. solani AG8 and a manually curated set of 13,964 genes supported by RNA-seq. The AG8 genome assembly used novel methods to produce a haploid representation of its heterokaryotic state. The whole-genomes of AG8, the rice pathogen AG1-IA and the potato pathogen AG3 were observed to be syntenic and co-linear. Genes and functions putatively relevant to pathogenicity were highlighted by comparing AG8 to known pathogenicity genes, orthology databases spanning 197 phytopathogenic taxa and AG1-IA. We also observed SNP-level “hypermutation” of CpG dinucleotides to TpG between AG8 nuclei, with similarities to repeat-induced point mutation (RIP). Interestingly, gene-coding regions were widely affected along with repetitive DNA, which has not been previously observed for RIP in mononuclear fungi of the Pezizomycotina. The rate of heterozygous SNP mutations within this single isolate of AG8 was observed to be higher than SNP mutation rates observed across populations of most fungal species compared. Comparative analyses were combined to predict biological processes relevant to AG8 and 308 proteins with effector-like characteristics, forming a valuable resource for further study of this pathosystem. Predicted effector-like proteins had elevated levels of non-synonymous point mutations relative to synonymous mutations (dN/dS), suggesting that they may be under diversifying selection pressures. In addition, the distant relationship to sequenced necrotrophs of the Ascomycota suggests the R. solani genome sequence may prove to be a useful resource in future comparative analysis of plant pathogens. PMID:24810276

SpliceDisease database: linking RNA splicing and disease.

PubMed

Wang, Juan; Zhang, Jie; Li, Kaibo; Zhao, Wei; Cui, Qinghua

2012-01-01

RNA splicing is an important aspect of gene regulation in many organisms. Splicing of RNA is regulated by complicated mechanisms involving numerous RNA-binding proteins and the intricate network of interactions among them. Mutations in cis-acting splicing elements or its regulatory proteins have been shown to be involved in human diseases. Defects in pre-mRNA splicing process have emerged as a common disease-causing mechanism. Therefore, a database integrating RNA splicing and disease associations would be helpful for understanding not only the RNA splicing but also its contribution to disease. In SpliceDisease database, we manually curated 2337 splicing mutation disease entries involving 303 genes and 370 diseases, which have been supported experimentally in 898 publications. The SpliceDisease database provides information including the change of the nucleotide in the sequence, the location of the mutation on the gene, the reference Pubmed ID and detailed description for the relationship among gene mutations, splicing defects and diseases. We standardized the names of the diseases and genes and provided links for these genes to NCBI and UCSC genome browser for further annotation and genomic sequences. For the location of the mutation, we give direct links of the entry to the respective position/region in the genome browser. The users can freely browse, search and download the data in SpliceDisease at http://cmbi.bjmu.edu.cn/sdisease.

Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast.

PubMed

Huang, Mingtao; Bai, Yunpeng; Sjostrom, Staffan L; Hallström, Björn M; Liu, Zihe; Petranovic, Dina; Uhlén, Mathias; Joensson, Haakan N; Andersson-Svahn, Helene; Nielsen, Jens

2015-08-25

There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant α-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories.

Analysis of ATP6 sequence diversity in the Triticum-Aegilops group of species reveals the crucial role of rearrangement in mitochondrial genome evolution

USDA-ARS?s Scientific Manuscript database

Mutation and chromosomal rearrangements are the two main forces of increasing genetic diversity for natural selection to act upon, and ultimately drive the evolutionary process. Although genome evolution is a function of both forces, simultaneously, the ratio of each can be varied among different ge...

Importance of DNA repair in tumor suppression

NASA Astrophysics Data System (ADS)

Brumer, Yisroel; Shakhnovich, Eugene I.

2004-12-01

The transition from a normal to cancerous cell requires a number of highly specific mutations that affect cell cycle regulation, apoptosis, differentiation, and many other cell functions. One hallmark of cancerous genomes is genomic instability, with mutation rates far greater than those of normal cells. In microsatellite instability (MIN tumors), these are often caused by damage to mismatch repair genes, allowing further mutation of the genome and tumor progression. These mutation rates may lie near the error catastrophe found in the quasispecies model of adaptive RNA genomes, suggesting that further increasing mutation rates will destroy cancerous genomes. However, recent results have demonstrated that DNA genomes exhibit an error threshold at mutation rates far lower than their conservative counterparts. Furthermore, while the maximum viable mutation rate in conservative systems increases indefinitely with increasing master sequence fitness, the semiconservative threshold plateaus at a relatively low value. This implies a paradox, wherein inaccessible mutation rates are found in viable tumor cells. In this paper, we address this paradox, demonstrating an isomorphism between the conservatively replicating (RNA) quasispecies model and the semiconservative (DNA) model with post-methylation DNA repair mechanisms impaired. Thus, as DNA repair becomes inactivated, the maximum viable mutation rate increases smoothly to that of a conservatively replicating system on a transformed landscape, with an upper bound that is dependent on replication rates. On a specific single fitness peak landscape, the repair-free semiconservative system is shown to mimic a conservative system exactly. We postulate that inactivation of post-methylation repair mechanisms is fundamental to the progression of a tumor cell and hence these mechanisms act as a method for the prevention and destruction of cancerous genomes.

Differential analysis between somatic mutation and germline variation profiles reveals cancer-related genes.

PubMed

Przytycki, Pawel F; Singh, Mona

2017-08-25

A major aim of cancer genomics is to pinpoint which somatically mutated genes are involved in tumor initiation and progression. We introduce a new framework for uncovering cancer genes, differential mutation analysis, which compares the mutational profiles of genes across cancer genomes with their natural germline variation across healthy individuals. We present DiffMut, a fast and simple approach for differential mutational analysis, and demonstrate that it is more effective in discovering cancer genes than considerably more sophisticated approaches. We conclude that germline variation across healthy human genomes provides a powerful means for characterizing somatic mutation frequency and identifying cancer driver genes. DiffMut is available at https://github.com/Singh-Lab/Differential-Mutation-Analysis .

Mutational and structural analysis of diffuse large B-cell lymphoma using whole genome sequencing | Office of Cancer Genomics

Cancer.gov

Abstract: Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous cancer comprising at least two molecular subtypes that differ in gene expression and distribution of mutations. Recently, application of genome/exome sequencing and RNA-seq to DLBCL has revealed numerous genes that are recurrent targets of somatic point mutation in this disease.

The ability of human nuclear DNA to cause false positive low-abundance heteroplasmy calls varies across the mitochondrial genome.

PubMed

Albayrak, Levent; Khanipov, Kamil; Pimenova, Maria; Golovko, George; Rojas, Mark; Pavlidis, Ioannis; Chumakov, Sergei; Aguilar, Gerardo; Chávez, Arturo; Widger, William R; Fofanov, Yuriy

2016-12-12

Low-abundance mutations in mitochondrial populations (mutations with minor allele frequency ≤ 1%), are associated with cancer, aging, and neurodegenerative disorders. While recent progress in high-throughput sequencing technology has significantly improved the heteroplasmy identification process, the ability of this technology to detect low-abundance mutations can be affected by the presence of similar sequences originating from nuclear DNA (nDNA). To determine to what extent nDNA can cause false positive low-abundance heteroplasmy calls, we have identified mitochondrial locations of all subsequences that are common or similar (one mismatch allowed) between nDNA and mitochondrial DNA (mtDNA). Performed analysis revealed up to a 25-fold variation in the lengths of longest common and longest similar (one mismatch allowed) subsequences across the mitochondrial genome. The size of the longest subsequences shared between nDNA and mtDNA in several regions of the mitochondrial genome were found to be as low as 11 bases, which not only allows using these regions to design new, very specific PCR primers, but also supports the hypothesis of the non-random introduction of mtDNA into the human nuclear DNA. Analysis of the mitochondrial locations of the subsequences shared between nDNA and mtDNA suggested that even very short (36 bases) single-end sequencing reads can be used to identify low-abundance variation in 20.4% of the mitochondrial genome. For longer (76 and 150 bases) reads, the proportion of the mitochondrial genome where nDNA presence will not interfere found to be 44.5 and 67.9%, when low-abundance mutations at 100% of locations can be identified using 417 bases long single reads. This observation suggests that the analysis of low-abundance variations in mitochondria population can be extended to a variety of large data collections such as NCBI Sequence Read Archive, European Nucleotide Archive, The Cancer Genome Atlas, and International Cancer Genome Consortium.

Genome-Wide Mutation Rate Response to pH Change in the Coral Reef Pathogen Vibrio shilonii AK1.

PubMed

Strauss, Chloe; Long, Hongan; Patterson, Caitlyn E; Te, Ronald; Lynch, Michael

2017-08-22

Recent application of mutation accumulation techniques combined with whole-genome sequencing (MA/WGS) has greatly promoted studies of spontaneous mutation. However, such explorations have rarely been conducted on marine organisms, and it is unclear how marine habitats have influenced genome stability. This report resolves the mutation rate and spectrum of the coral reef pathogen Vibrio shilonii , which causes coral bleaching and endangers the biodiversity maintained by coral reefs. We found that its mutation rate and spectrum are highly similar to those of other studied bacteria from various habitats, despite the saline environment. The mutational properties of this marine bacterium are thus controlled by other general evolutionary forces such as natural selection and genetic drift. We also found that as pH drops, the mutation rate decreases and the mutation spectrum is biased in the direction of generating G/C nucleotides. This implies that evolutionary features of this organism and perhaps other marine microbes might be altered by the increasingly acidic ocean water caused by excess CO 2 emission. Nonetheless, further exploration is needed as the pH range tested in this study was rather narrow and many other possible mutation determinants, such as carbonate increase, are associated with ocean acidification. IMPORTANCE This study explored the pH dependence of a bacterial genome-wide mutation rate. We discovered that the genome-wide rates of appearance of most mutation types decrease linearly and that the mutation spectrum is biased in generating more G/C nucleotides with pH drop in the coral reef pathogen V. shilonii . Copyright © 2017 Strauss et al.

Population-Scale Sequencing Data Enable Precise Estimates of Y-STR Mutation Rates

PubMed Central

Willems, Thomas; Gymrek, Melissa; Poznik, G. David; Tyler-Smith, Chris; Erlich, Yaniv

2016-01-01

Short tandem repeats (STRs) are mutation-prone loci that span nearly 1% of the human genome. Previous studies have estimated the mutation rates of highly polymorphic STRs by using capillary electrophoresis and pedigree-based designs. Although this work has provided insights into the mutational dynamics of highly mutable STRs, the mutation rates of most others remain unknown. Here, we harnessed whole-genome sequencing data to estimate the mutation rates of Y chromosome STRs (Y-STRs) with 2–6 bp repeat units that are accessible to Illumina sequencing. We genotyped 4,500 Y-STRs by using data from the 1000 Genomes Project and the Simons Genome Diversity Project. Next, we developed MUTEA, an algorithm that infers STR mutation rates from population-scale data by using a high-resolution SNP-based phylogeny. After extensive intrinsic and extrinsic validations, we harnessed MUTEA to derive mutation-rate estimates for 702 polymorphic STRs by tracing each locus over 222,000 meioses, resulting in the largest collection of Y-STR mutation rates to date. Using our estimates, we identified determinants of STR mutation rates and built a model to predict rates for STRs across the genome. These predictions indicate that the load of de novo STR mutations is at least 75 mutations per generation, rivaling the load of all other known variant types. Finally, we identified Y-STRs with potential applications in forensics and genetic genealogy, assessed the ability to differentiate between the Y chromosomes of father-son pairs, and imputed Y-STR genotypes. PMID:27126583

An Upper Limit on the Functional Fraction of the Human Genome.

PubMed

Graur, Dan

2017-07-01

For the human population to maintain a constant size from generation to generation, an increase in fertility must compensate for the reduction in the mean fitness of the population caused, among others, by deleterious mutations. The required increase in fertility due to this mutational load depends on the number of sites in the genome that are functional, the mutation rate, and the fraction of deleterious mutations among all mutations in functional regions. These dependencies and the fact that there exists a maximum tolerable replacement level fertility can be used to put an upper limit on the fraction of the human genome that can be functional. Mutational load considerations lead to the conclusion that the functional fraction within the human genome cannot exceed 25%, and is probably considerably lower. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Experimental Induction of Genome Chaos.

PubMed

Ye, Christine J; Liu, Guo; Heng, Henry H

2018-01-01

Genome chaos, or karyotype chaos, represents a powerful survival strategy for somatic cells under high levels of stress/selection. Since the genome context, not the gene content, encodes the genomic blueprint of the cell, stress-induced rapid and massive reorganization of genome topology functions as a very important mechanism for genome (karyotype) evolution. In recent years, the phenomenon of genome chaos has been confirmed by various sequencing efforts, and many different terms have been coined to describe different subtypes of the chaotic genome including "chromothripsis," "chromoplexy," and "structural mutations." To advance this exciting field, we need an effective experimental system to induce and characterize the karyotype reorganization process. In this chapter, an experimental protocol to induce chaotic genomes is described, following a brief discussion of the mechanism and implication of genome chaos in cancer evolution.

A Mismatch EndoNuclease Array-Based Methodology (MENA) for Identifying Known SNPs or Novel Point Mutations.

PubMed

Comeron, Josep M; Reed, Jordan; Christie, Matthew; Jacobs, Julia S; Dierdorff, Jason; Eberl, Daniel F; Manak, J Robert

2016-04-05

Accurate and rapid identification or confirmation of single nucleotide polymorphisms (SNPs), point mutations and other human genomic variation facilitates understanding the genetic basis of disease. We have developed a new methodology (called MENA (Mismatch EndoNuclease Array)) pairing DNA mismatch endonuclease enzymology with tiling microarray hybridization in order to genotype both known point mutations (such as SNPs) as well as identify previously undiscovered point mutations and small indels. We show that our assay can rapidly genotype known SNPs in a human genomic DNA sample with 99% accuracy, in addition to identifying novel point mutations and small indels with a false discovery rate as low as 10%. Our technology provides a platform for a variety of applications, including: (1) genotyping known SNPs as well as confirming newly discovered SNPs from whole genome sequencing analyses; (2) identifying novel point mutations and indels in any genomic region from any organism for which genome sequence information is available; and (3) screening panels of genes associated with particular diseases and disorders in patient samples to identify causative mutations. As a proof of principle for using MENA to discover novel mutations, we report identification of a novel allele of the beethoven (btv) gene in Drosophila, which encodes a ciliary cytoplasmic dynein motor protein important for auditory mechanosensation.

Reduced mutation rate in exons due to differential mismatch repair

PubMed Central

Mularoni, Loris; Muiños, Ferran; Gonzalez-Perez, Abel; López-Bigas, Núria

2017-01-01

While recent studies have revealed higher than anticipated heterogeneity of mutation rate across genomic regions, mutations in exons and introns are assumed to be generated at the same rate. Here we find fewer somatic mutations in exons than expected based on their sequence content, and demonstrate that this is not due to purifying selection. Moreover, we show that it is caused by higher mismatch repair activity in exonic than in intronic regions. Our findings have important implications for our understanding of mutational and DNA repair processes, our knowledge of the evolution of eukaryotic genes, and practical ramifications for the study of the evolution of both tumors and species. PMID:29106418

Genome instabilities arising from ribonucleotides in DNA.

PubMed

Klein, Hannah L

2017-08-01

Genomic DNA is transiently contaminated with ribonucleotide residues during the process of DNA replication through misincorporation by the replicative DNA polymerases α, δ and ε, and by the normal replication process on the lagging strand, which uses RNA primers. These ribonucleotides are efficiently removed during replication by RNase H enzymes and the lagging strand synthesis machinery. However, when ribonucleotides remain in DNA they can distort the DNA helix, affect machineries for DNA replication, transcription and repair, and can stimulate genomic instabilities which are manifest as increased mutation, recombination and chromosome alterations. The genomic instabilities associated with embedded ribonucleotides are considered here, along with a discussion of the origin of the lesions that stimulate particular classes of instabilities. Copyright © 2017 Elsevier B.V. All rights reserved.

A strongly selected mutation in the HIV-1 genome is independent of T cell responses and neutralizing antibodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Donglai; Wang, Chu; Hora, Bhavna

Mutations rapidly accumulate in the HIV-1 genome after infection. Some of those mutations are selected by host immune responses and often cause viral fitness losses. This study is to investigate whether strongly selected mutations that are not associated with immune responses result in fitness losses. Strongly selected mutations were identified by analyzing 5'-half HIV-1 genome (gag/pol) sequences from longitudinal samples of subject CH0131. The K43R mutation in the gag gene was first detected at day 91 post screening and was fixed in the viral population at day 273 while the synonymous N323tc mutation was first detected at day 177 andmore » fixed at day 670. No conventional or cryptic T cell responses were detected against either mutation sites by ELISpot analysis. However, when fitness costs of both mutations were measured by introducing each mutation into their cognate transmitted/founder (T/F) viral genome, the K43R mutation caused a significant fitness loss while the N323tc mutation had little impact on viral fitness. In conclusion, the rapid fixation, the lack of detectable immune responses and the significant fitness cost of the K43R mutation suggests that it was strongly selected by host factors other than T cell responses and neutralizing antibodies.« less

A strongly selected mutation in the HIV-1 genome is independent of T cell responses and neutralizing antibodies

DOE PAGES

Liu, Donglai; Wang, Chu; Hora, Bhavna; ...

2017-10-10

Mutations rapidly accumulate in the HIV-1 genome after infection. Some of those mutations are selected by host immune responses and often cause viral fitness losses. This study is to investigate whether strongly selected mutations that are not associated with immune responses result in fitness losses. Strongly selected mutations were identified by analyzing 5'-half HIV-1 genome (gag/pol) sequences from longitudinal samples of subject CH0131. The K43R mutation in the gag gene was first detected at day 91 post screening and was fixed in the viral population at day 273 while the synonymous N323tc mutation was first detected at day 177 andmore » fixed at day 670. No conventional or cryptic T cell responses were detected against either mutation sites by ELISpot analysis. However, when fitness costs of both mutations were measured by introducing each mutation into their cognate transmitted/founder (T/F) viral genome, the K43R mutation caused a significant fitness loss while the N323tc mutation had little impact on viral fitness. In conclusion, the rapid fixation, the lack of detectable immune responses and the significant fitness cost of the K43R mutation suggests that it was strongly selected by host factors other than T cell responses and neutralizing antibodies.« less

Mapping Challenging Mutations by Whole-Genome Sequencing

PubMed Central

Smith, Harold E.; Fabritius, Amy S.; Jaramillo-Lambert, Aimee; Golden, Andy

2016-01-01

Whole-genome sequencing provides a rapid and powerful method for identifying mutations on a global scale, and has spurred a renewed enthusiasm for classical genetic screens in model organisms. The most commonly characterized category of mutation consists of monogenic, recessive traits, due to their genetic tractability. Therefore, most of the mapping methods for mutation identification by whole-genome sequencing are directed toward alleles that fulfill those criteria (i.e., single-gene, homozygous variants). However, such approaches are not entirely suitable for the characterization of a variety of more challenging mutations, such as dominant and semidominant alleles or multigenic traits. Therefore, we have developed strategies for the identification of those classes of mutations, using polymorphism mapping in Caenorhabditis elegans as our model for validation. We also report an alternative approach for mutation identification from traditional recombinant crosses, and a solution to the technical challenge of sequencing sterile or terminally arrested strains where population size is limiting. The methods described herein extend the applicability of whole-genome sequencing to a broader spectrum of mutations, including classes that are difficult to map by traditional means. PMID:26945029

Phylomedicine: An evolutionary telescope to explore and diagnose the universe of disease mutations

PubMed Central

Kumar, Sudhir; Dudley, Joel T.; Filipski, Alan; Liu, Li

2011-01-01

Modern technologies have made the sequencing of personal genomes routine. They have revealed thousands of nonsynonymous (amino-acid altering) single nucleotide variants (nSNVs) of protein coding DNA per genome. What do these variants foretell about an individual’s predisposition to diseases? The experimental technologies required to carry out such evaluations at a genomic scale are not yet available. Fortunately, the process of natural selection has lent us an almost infinite set of tests in nature. During the long-term evolution, new mutations and existing variations have been evaluated for their biological consequences in countless species, and outcomes were readily revealed by multispecies genome comparisons. We review studies that have investigated evolutionary characteristics and in silico functional diagnoses of nSNVs found in thousands of disease-associated genes. We conclude that the patterns of long-term evolutionary conservation and permissible divergence are essential and instructive modalities for functional assessment of human genetic variations. PMID:21764165

Cancer genomics: technology, discovery, and translation.

PubMed

Tran, Ben; Dancey, Janet E; Kamel-Reid, Suzanne; McPherson, John D; Bedard, Philippe L; Brown, Andrew M K; Zhang, Tong; Shaw, Patricia; Onetto, Nicole; Stein, Lincoln; Hudson, Thomas J; Neel, Benjamin G; Siu, Lillian L

2012-02-20

In recent years, the increasing awareness that somatic mutations and other genetic aberrations drive human malignancies has led us within reach of personalized cancer medicine (PCM). The implementation of PCM is based on the following premises: genetic aberrations exist in human malignancies; a subset of these aberrations drive oncogenesis and tumor biology; these aberrations are actionable (defined as having the potential to affect management recommendations based on diagnostic, prognostic, and/or predictive implications); and there are highly specific anticancer agents available that effectively modulate these targets. This article highlights the technology underlying cancer genomics and examines the early results of genome sequencing and the challenges met in the discovery of new genetic aberrations. Finally, drawing from experiences gained in a feasibility study of somatic mutation genotyping and targeted exome sequencing led by Princess Margaret Hospital-University Health Network and the Ontario Institute for Cancer Research, the processes, challenges, and issues involved in the translation of cancer genomics to the clinic are discussed.

Frequency and type of inheritable mutations induced by γ rays in rice as revealed by whole genome sequencing.

PubMed

Li, Shan; Zheng, Yun-Chao; Cui, Hai-Rui; Fu, Hao-Wei; Shu, Qing-Yao; Huang, Jian-Zhong

Mutation breeding is based on the induction of genetic variations; hence knowledge of the frequency and type of induced mutations is of paramount importance for the design and implementation of a mutation breeding program. Although γ ray irradiation has been widely used since the 1960s in the breeding of about 200 economically important plant species, molecular elucidation of its genetic effects has so far been achieved largely by analysis of target genes or genomic regions. In the present study, the whole genomes of six γ-irradiated M 2 rice plants were sequenced; a total of 144-188 million high-quality (Q>20) reads were generated for each M 2 plant, resulting in genome coverage of >45 times for each plant. Single base substitution (SBS) and short insertion/deletion (Indel) mutations were detected at the average frequency of 7.5×10 -6 -9.8×10 -6 in the six M 2 rice plants (SBS being about 4 times more frequent than Indels). Structural and copy number variations, though less frequent than SBS and Indel, were also identified and validated. The mutations were scattered in all genomic regions across 12 rice chromosomes without apparent hotspots. The present study is the first genome-wide single-nucleotide resolution study on the feature and frequency of γ irradiation-induced mutations in a seed propagated crop; the findings are of practical importance for mutation breeding of rice and other crop species.

The Number of Point Mutations in Induced Pluripotent Stem Cells and Nuclear Transfer Embryonic Stem Cells Depends on the Method and Somatic Cell Type Used for Their Generation.

PubMed

Araki, Ryoko; Mizutani, Eiji; Hoki, Yuko; Sunayama, Misato; Wakayama, Sayaka; Nagatomo, Hiroaki; Kasama, Yasuji; Nakamura, Miki; Wakayama, Teruhiko; Abe, Masumi

2017-05-01

Induced pluripotent stem cells hold great promise for regenerative medicine but point mutations have been identified in these cells and have raised serious concerns about their safe use. We generated nuclear transfer embryonic stem cells (ntESCs) from both mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) and by whole genome sequencing found fewer mutations compared with iPSCs generated by retroviral gene transduction. Furthermore, TTF-derived ntESCs showed only a very small number of point mutations, approximately 80% less than the number observed in iPSCs generated using retrovirus. Base substitution profile analysis confirmed this greatly reduced number of point mutations. The point mutations in iPSCs are therefore not a Yamanaka factor-specific phenomenon but are intrinsic to genome reprogramming. Moreover, the dramatic reduction in point mutations in ntESCs suggests that most are not essential for genome reprogramming. Our results suggest that it is feasible to reduce the point mutation frequency in iPSCs by optimizing various genome reprogramming conditions. We conducted whole genome sequencing of ntES cells derived from MEFs or TTFs. We thereby succeeded in establishing TTF-derived ntES cell lines with far fewer point mutations. Base substitution profile analysis of these clones also indicated a reduced point mutation frequency, moving from a transversion-predominance to a transition-predominance. Stem Cells 2017;35:1189-1196. © 2017 AlphaMed Press.

West Nile virus (WNV) genome RNAs with up to three adjacent mutations that disrupt long distance 5'-3' cyclization sequence basepairs are viable

DOE Office of Scientific and Technical Information (OSTI.GOV)

Basu, Mausumi; Brinton, Margo A., E-mail: mbrinton@gsu.ed

2011-03-30

Mosquito-borne flavivirus genomes contain conserved 5' and 3' cyclization sequences (CYC) that facilitate long distance RNA-RNA interactions. In previous studies, flavivirus replicon RNA replication was completely inhibited by single or multiple mismatching CYC nt substitutions. In the present study, full-length WNV genomes with one, two or three mismatching CYC substitutions showed reduced replication efficiencies but were viable and generated revertants with increased replication efficiency. Several different three adjacent mismatching CYC substitution mutant RNAs were rescued by a second site mutation that created an additional basepair (nts 147-10913) on the internal genomic side of the 5'-3' CYC. The finding that full-lengthmore » genomes with up to three mismatching CYC mutations are viable and can be rescued by a single nt spontaneous mutation indicates that more than three adjacent CYC basepair substitutions would be required to increase the safety of vaccine genomes by creating mismatches in inter-genomic recombinants.« less

Extreme Recombination Frequencies Shape Genome Variation and Evolution in the Honeybee, Apis mellifera

PubMed Central

Wallberg, Andreas; Glémin, Sylvain; Webster, Matthew T.

2015-01-01

Meiotic recombination is a fundamental cellular process, with important consequences for evolution and genome integrity. However, we know little about how recombination rates vary across the genomes of most species and the molecular and evolutionary determinants of this variation. The honeybee, Apis mellifera, has extremely high rates of meiotic recombination, although the evolutionary causes and consequences of this are unclear. Here we use patterns of linkage disequilibrium in whole genome resequencing data from 30 diploid honeybees to construct a fine-scale map of rates of crossing over in the genome. We find that, in contrast to vertebrate genomes, the recombination landscape is not strongly punctate. Crossover rates strongly correlate with levels of genetic variation, but not divergence, which indicates a pervasive impact of selection on the genome. Germ-line methylated genes have reduced crossover rate, which could indicate a role of methylation in suppressing recombination. Controlling for the effects of methylation, we do not infer a strong association between gene expression patterns and recombination. The site frequency spectrum is strongly skewed from neutral expectations in honeybees: rare variants are dominated by AT-biased mutations, whereas GC-biased mutations are found at higher frequencies, indicative of a major influence of GC-biased gene conversion (gBGC), which we infer to generate an allele fixation bias 5 – 50 times the genomic average estimated in humans. We uncover further evidence that this repair bias specifically affects transitions and favours fixation of CpG sites. Recombination, via gBGC, therefore appears to have profound consequences on genome evolution in honeybees and interferes with the process of natural selection. These findings have important implications for our understanding of the forces driving molecular evolution. PMID:25902173

Genetic drift and mutational hazard in the evolution of salamander genomic gigantism.

PubMed

Mohlhenrich, Erik Roger; Mueller, Rachel Lockridge

2016-12-01

Salamanders have the largest nuclear genomes among tetrapods and, excepting lungfishes, among vertebrates as a whole. Lynch and Conery (2003) have proposed the mutational-hazard hypothesis to explain variation in genome size and complexity. Under this hypothesis, noncoding DNA imposes a selective cost by increasing the target for degenerative mutations (i.e., the mutational hazard). Expansion of noncoding DNA, and thus genome size, is driven by increased levels of genetic drift and/or decreased mutation rates; the former determines the efficiency with which purifying selection can remove excess DNA, whereas the latter determines the level of mutational hazard. Here, we test the hypothesis that salamanders have experienced stronger long-term, persistent genetic drift than frogs, a related clade with more typically sized vertebrate genomes. To test this hypothesis, we compared dN/dS and Kr/Kc values of protein-coding genes between these clades. Our results do not support this hypothesis; we find that salamanders have not experienced stronger genetic drift than frogs. Additionally, we find evidence consistent with a lower nucleotide substitution rate in salamanders. This result, along with previous work showing lower rates of small deletion and ectopic recombination in salamanders, suggests that a lower mutational hazard may contribute to genomic gigantism in this clade. © 2016 The Author(s). Evolution © 2016 The Society for the Study of Evolution.

Recurrent DGCR8, DROSHA, and SIX homeodomain mutations in favorable histology Wilms tumors | Office of Cancer Genomics

Cancer.gov

We report the most common single-nucleotide substitution/deletion mutations in favorable histology Wilms tumors (FHWTs) to occur within SIX1/2 (7% of 534 tumors) and microRNA processing genes (miRNAPGs) DGCR8 and DROSHA (15% of 534 tumors). Comprehensive analysis of 77 FHWTs indicates that tumors with SIX1/2 and/or miRNAPG mutations show a pre-induction metanephric mesenchyme gene expression pattern and are significantly associated with both perilobar nephrogenic rests and 11p15 imprinting aberrations.

Genome-wide survey of artificial mutations induced by ethyl methanesulfonate and gamma rays in tomato.

PubMed

Shirasawa, Kenta; Hirakawa, Hideki; Nunome, Tsukasa; Tabata, Satoshi; Isobe, Sachiko

2016-01-01

Genome-wide mutations induced by ethyl methanesulfonate (EMS) and gamma irradiation in the tomato Micro-Tom genome were identified by a whole-genome shotgun sequencing analysis to estimate the spectrum and distribution of whole-genome DNA mutations and the frequency of deleterious mutations. A total of ~370 Gb of paired-end reads for four EMS-induced mutants and three gamma-ray-irradiated lines as well as a wild-type line were obtained by next-generation sequencing technology. Using bioinformatics analyses, we identified 5920 induced single nucleotide variations and insertion/deletion (indel) mutations. The predominant mutations in the EMS mutants were C/G to T/A transitions, while in the gamma-ray mutants, C/G to T/A transitions, A/T to T/A transversions, A/T to G/C transitions and deletion mutations were equally common. Biases in the base composition flanking mutations differed between the mutagenesis types. Regarding the effects of the mutations on gene function, >90% of the mutations were located in intergenic regions, and only 0.2% were deleterious. In addition, we detected 1,140,687 spontaneous single nucleotide polymorphisms and indel polymorphisms in wild-type Micro-Tom lines. We also found copy number variation, deletions and insertions of chromosomal segments in both the mutant and wild-type lines. The results provide helpful information not only for mutation research, but also for mutant screening methodology with reverse-genetic approaches. © 2015 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Synaptic scaffold evolution generated components of vertebrate cognitive complexity

PubMed Central

Nithianantharajah, J.; Komiyama, N.H.; McKechanie, A.; Johnstone, M.; Blackwood, D. H.; St Clair, D.; Emes, R.D.; van de Lagemaat, L. N.; Saksida, L.M.; Bussey, T.J.; Grant, S.G.N.

2014-01-01

The origins and evolution of higher cognitive functions including complex forms of learning, attention and executive functions are unknown. A potential mechanism driving the evolution of vertebrate cognition early in the vertebrate lineage (550 My ago) was genome duplication and subsequent diversification of postsynaptic genes. Here we report the first genetic analysis of a vertebrate gene family in cognitive functions measured using computerized touchscreens. Comparison of mice carrying mutations in all four Dlg paralogs show simple associative learning required Dlg4, while Dlg2 and Dlg3 diversified to play opposing roles in complex cognitive processes. Exploiting the translational utility of touchscreens in humans and mice, testing Dlg2 mutations in both species showed Dlg2’s role in complex learning, cognitive flexibility and attention has been highly conserved over 100 My. Dlg family mutations underlie psychiatric disorders suggesting genome evolution expanded the complexity of vertebrate cognition at the cost of susceptibility to mental illness. PMID:23201973

Technical approaches for mouse models of human disease.

PubMed

Justice, Monica J; Siracusa, Linda D; Stewart, A Francis

2011-05-01

The mouse is the leading organism for disease research. A rich resource of genetic variation occurs naturally in inbred and special strains owing to spontaneous mutations. However, one can also obtain desired gene mutations by using the following processes: targeted mutations that eliminate function in the whole organism or in a specific tissue; forward genetic screens using chemicals or transposons; or the introduction of exogenous transgenes as DNAs, bacterial artificial chromosomes (BACs) or reporter constructs. The mouse is the only mammal that provides such a rich resource of genetic diversity coupled with the potential for extensive genome manipulation, and is therefore a powerful application for modeling human disease. This poster review outlines the major genome manipulations available in the mouse that are used to understand human disease: natural variation, reverse genetics, forward genetics, transgenics and transposons. Each of these applications will be essential for understanding the diversity that is being discovered within the human population.

On the molecular mechanism of GC content variation among eubacterial genomes

PubMed Central

2012-01-01

Background As a key parameter of genome sequence variation, the GC content of bacterial genomes has been investigated for over half a century, and many hypotheses have been put forward to explain this GC content variation and its relationship to other fundamental processes. Previously, we classified eubacteria into dnaE-based groups (the dimeric combination of DNA polymerase III alpha subunits), according to a hypothesis where GC content variation is essentially governed by genome replication and DNA repair mechanisms. Further investigation led to the discovery that two major mutator genes, polC and dnaE2, may be responsible for genomic GC content variation. Consequently, an in-depth analysis was conducted to evaluate various potential intrinsic and extrinsic factors in association with GC content variation among eubacterial genomes. Results Mutator genes, especially those with dominant effects on the mutation spectra, are biased towards either GC or AT richness, and they alter genomic GC content in the two opposite directions. Increased bacterial genome size (or gene number) appears to rely on increased genomic GC content; however, it is unclear whether the changes are directly related to certain environmental pressures. Certain environmental and bacteriological features are related to GC content variation, but their trends are more obvious when analyzed under the dnaE-based grouping scheme. Most terrestrial, plant-associated, and nitrogen-fixing bacteria are members of the dnaE1|dnaE2 group, whereas most pathogenic or symbiotic bacteria in insects, and those dwelling in aquatic environments, are largely members of the dnaE1|polV group. Conclusion Our studies provide several lines of evidence indicating that DNA polymerase III α subunit and its isoforms participating in either replication (such as polC) or SOS mutagenesis/translesion synthesis (such as dnaE2), play dominant roles in determining GC variability. Other environmental or bacteriological factors, such as genome size, temperature, oxygen requirement, and habitat, either play subsidiary roles or rely indirectly on different mutator genes to fine-tune the GC content. These results provide a comprehensive insight into mechanisms of GC content variation and the robustness of eubacterial genomes in adapting their ever-changing environments over billions of years. Reviewers This paper was reviewed by Nicolas Galtier, Adam Eyre-Walker, and Eugene Koonin. PMID:22230424

Scarless Cas9 Assisted Recombineering (no-SCAR) in Escherichia coli, an Easy-to-Use System for Genome Editing.

PubMed

Reisch, Christopher R; Prather, Kristala L J

2017-01-05

The discovery and development of genome editing systems that leverage the site-specific DNA endonuclease system CRISPR/Cas9 has fundamentally changed the ease and speed of genome editing in many organisms. In eukaryotes, the CRISPR/Cas9 system utilizes a "guide" RNA to enable the Cas9 nuclease to make a double-strand break at a particular genome locus, which is repaired by non-homologous end joining (NHEJ) repair enzymes, often generating random mutations in the process. A specific alteration of the target genome can also be generated by supplying a DNA template in vivo with a desired mutation, which is incorporated by homology-directed repair. However, E. coli lacks robust systems for double-strand break repair. Thus, in contrast to eukaryotes, targeting E. coli chromosomal DNA with Cas9 causes cell death. However, Cas9-mediated killing of bacteria can be exploited to select against cells with a specified genotype within a mixed population. In combination with the well described λ-Red system for recombination in E. coli, we created a highly efficient system for marker-free and scarless genome editing. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Slow but not low: genomic comparisons reveal slower evolutionary rate and higher dN/dS in conifers compared to angiosperms

PubMed Central

2012-01-01

Background Comparative genomics can inform us about the processes of mutation and selection across diverse taxa. Among seed plants, gymnosperms have been lacking in genomic comparisons. Recent EST and full-length cDNA collections for two conifers, Sitka spruce (Picea sitchensis) and loblolly pine (Pinus taeda), together with full genome sequences for two angiosperms, Arabidopsis thaliana and poplar (Populus trichocarpa), offer an opportunity to infer the evolutionary processes underlying thousands of orthologous protein-coding genes in gymnosperms compared with an angiosperm orthologue set. Results Based upon pairwise comparisons of 3,723 spruce and pine orthologues, we found an average synonymous genetic distance (dS) of 0.191, and an average dN/dS ratio of 0.314. Using a fossil-established divergence time of 140 million years between spruce and pine, we extrapolated a nucleotide substitution rate of 0.68 × 10-9 synonymous substitutions per site per year. When compared to angiosperms, this indicates a dramatically slower rate of nucleotide substitution rates in conifers: on average 15-fold. Coincidentally, we found a three-fold higher dN/dS for the spruce-pine lineage compared to the poplar-Arabidopsis lineage. This joint occurrence of a slower evolutionary rate in conifers with higher dN/dS, and possibly positive selection, showcases the uniqueness of conifer genome evolution. Conclusions Our results are in line with documented reduced nucleotide diversity, conservative genome evolution and low rates of diversification in conifers on the one hand and numerous examples of local adaptation in conifers on the other hand. We propose that reduced levels of nucleotide mutation in large and long-lived conifer trees, coupled with large effective population size, were the main factors leading to slow substitution rates but retention of beneficial mutations. PMID:22264329

Mechanisms of viral mutation.

PubMed

Sanjuán, Rafael; Domingo-Calap, Pilar

2016-12-01

The remarkable capacity of some viruses to adapt to new hosts and environments is highly dependent on their ability to generate de novo diversity in a short period of time. Rates of spontaneous mutation vary amply among viruses. RNA viruses mutate faster than DNA viruses, single-stranded viruses mutate faster than double-strand virus, and genome size appears to correlate negatively with mutation rate. Viral mutation rates are modulated at different levels, including polymerase fidelity, sequence context, template secondary structure, cellular microenvironment, replication mechanisms, proofreading, and access to post-replicative repair. Additionally, massive numbers of mutations can be introduced by some virus-encoded diversity-generating elements, as well as by host-encoded cytidine/adenine deaminases. Our current knowledge of viral mutation rates indicates that viral genetic diversity is determined by multiple virus- and host-dependent processes, and that viral mutation rates can evolve in response to specific selective pressures.

Identifying pathways affected by cancer mutations.

PubMed

Iengar, Prathima

2017-12-16

Mutations in 15 cancers, sourced from the COSMIC Whole Genomes database, and 297 human pathways, arranged into pathway groups based on the processes they orchestrate, and sourced from the KEGG pathway database, have together been used to identify pathways affected by cancer mutations. Genes studied in ≥15, and mutated in ≥10 samples of a cancer have been considered recurrently mutated, and pathways with recurrently mutated genes have been considered affected in the cancer. Novel doughnut plots have been presented which enable visualization of the extent to which pathways and genes, in each pathway group, are targeted, in each cancer. The 'organismal systems' pathway group (including organism-level pathways; e.g., nervous system) is the most targeted, more than even the well-recognized signal transduction, cell-cycle and apoptosis, and DNA repair pathway groups. The important, yet poorly-recognized, role played by the group merits attention. Pathways affected in ≥7 cancers yielded insights into processes affected. Copyright © 2017 Elsevier Inc. All rights reserved.

Multiple primary tumors of the upper aerodigestive tract: is there a role for constitutional mutations in the p53 gene?

PubMed

Gallo, O; Sardi, I; Pepe, G; Franchi, A; Attanasio, M; Giusti, B; Bocciolini, C; Abbate, R

1999-07-19

Head-and-neck cancer (HNC) patients have a high risk of developing second primary tumors of the upper aerodigestive tract, the main cause of death. Although the roles of tobacco and diet in multiple head-and-neck carcinogenesis have been thoroughly investigated, little is known about individual genetic susceptibility factors involved in this process. Genomic instability, reflecting the propensity and the susceptibility of the genome to acquire multiple alterations, could be considered a driving force behind multiple carcinogenesis. Mutation of the p53 tumor-suppressor gene has been proposed to play an important role in this process. Therefore, we evaluated the incidence of inherited p53 germ-line alteration(s) in a population of 24 consecutive HNC patients and their first-degree relatives affected by multiple malignancies as well as the occurrence of p53 somatic acquired mutation(s) in 16 cancers, including first and second primaries from 5 HNCs of the same group. Mutations in exons 4-11 of the p53 gene were investigated using SSCP-PCR analysis and DNA sequencing. Analysis was extended to the peripheral blood and cancer biopsies available from first-degree relatives of cancer-prone families with p53 germ-line mutations. p53 germ-line mutations were identified in the peripheral blood and corresponding cancers of 3 HNC patients who had multiple malignancies. The only missense mutation detected was mapped in exon 6; it is a GTG to GAG substitution with an amino acid change from Val to Glu at codon 197. The remaining 2 p53 germ-line mutations were single-nucleotide substitutions without amino acid change in exon 6 (codon 213, CGA to CGG) and in exon 8 (codon 295, CCT to CCC), respectively. These mutations were found in HNC patients with a family history of cancer. Abnormal expression of wild-type p53 protein in normal and pathological tissues from patients with the same sense single-nucleotide substitutions was detected by immuno-histochemistry.

Clinical genomics information management software linking cancer genome sequence and clinical decisions.

PubMed

Watt, Stuart; Jiao, Wei; Brown, Andrew M K; Petrocelli, Teresa; Tran, Ben; Zhang, Tong; McPherson, John D; Kamel-Reid, Suzanne; Bedard, Philippe L; Onetto, Nicole; Hudson, Thomas J; Dancey, Janet; Siu, Lillian L; Stein, Lincoln; Ferretti, Vincent

2013-09-01

Using sequencing information to guide clinical decision-making requires coordination of a diverse set of people and activities. In clinical genomics, the process typically includes sample acquisition, template preparation, genome data generation, analysis to identify and confirm variant alleles, interpretation of clinical significance, and reporting to clinicians. We describe a software application developed within a clinical genomics study, to support this entire process. The software application tracks patients, samples, genomic results, decisions and reports across the cohort, monitors progress and sends reminders, and works alongside an electronic data capture system for the trial's clinical and genomic data. It incorporates systems to read, store, analyze and consolidate sequencing results from multiple technologies, and provides a curated knowledge base of tumor mutation frequency (from the COSMIC database) annotated with clinical significance and drug sensitivity to generate reports for clinicians. By supporting the entire process, the application provides deep support for clinical decision making, enabling the generation of relevant guidance in reports for verification by an expert panel prior to forwarding to the treating physician. Copyright © 2013 Elsevier Inc. All rights reserved.

MutationAligner: a resource of recurrent mutation hotspots in protein domains in cancer

PubMed Central

Gauthier, Nicholas Paul; Reznik, Ed; Gao, Jianjiong; Sumer, Selcuk Onur; Schultz, Nikolaus; Sander, Chris; Miller, Martin L.

2016-01-01

The MutationAligner web resource, available at http://www.mutationaligner.org, enables discovery and exploration of somatic mutation hotspots identified in protein domains in currently (mid-2015) more than 5000 cancer patient samples across 22 different tumor types. Using multiple sequence alignments of protein domains in the human genome, we extend the principle of recurrence analysis by aggregating mutations in homologous positions across sets of paralogous genes. Protein domain analysis enhances the statistical power to detect cancer-relevant mutations and links mutations to the specific biological functions encoded in domains. We illustrate how the MutationAligner database and interactive web tool can be used to explore, visualize and analyze mutation hotspots in protein domains across genes and tumor types. We believe that MutationAligner will be an important resource for the cancer research community by providing detailed clues for the functional importance of particular mutations, as well as for the design of functional genomics experiments and for decision support in precision medicine. MutationAligner is slated to be periodically updated to incorporate additional analyses and new data from cancer genomics projects. PMID:26590264

Genome-Wide Biases in the Rate and Molecular Spectrum of Spontaneous Mutations in Vibrio cholerae and Vibrio fischeri.

PubMed

Dillon, Marcus M; Sung, Way; Sebra, Robert; Lynch, Michael; Cooper, Vaughn S

2017-01-01

The vast diversity in nucleotide composition and architecture among bacterial genomes may be partly explained by inherent biases in the rates and spectra of spontaneous mutations. Bacterial genomes with multiple chromosomes are relatively unusual but some are relevant to human health, none more so than the causative agent of cholera, Vibrio cholerae Here, we present the genome-wide mutation spectra in wild-type and mismatch repair (MMR) defective backgrounds of two Vibrio species, the low-%GC squid symbiont V. fischeri and the pathogen V. cholerae, collected under conditions that greatly minimize the efficiency of natural selection. In apparent contrast to their high diversity in nature, both wild-type V. fischeri and V. cholerae have among the lowest rates for base-substitution mutations (bpsms) and insertion-deletion mutations (indels) that have been measured, below 10 - 3 /genome/generation. Vibrio fischeri and V. cholerae have distinct mutation spectra, but both are AT-biased and produce a surprising number of multi-nucleotide indels. Furthermore, the loss of a functional MMR system caused the mutation spectra of these species to converge, implying that the MMR system itself contributes to species-specific mutation patterns. Bpsm and indel rates varied among genome regions, but do not explain the more rapid evolutionary rates of genes on chromosome 2, which likely result from weaker purifying selection. More generally, the very low mutation rates of Vibrio species correlate inversely with their immense population sizes and suggest that selection may not only have maximized replication fidelity but also optimized other polygenic traits relative to the constraints of genetic drift. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Investigation of mutations in the HBB gene using the 1,000 genomes database.

PubMed

Carlice-Dos-Reis, Tânia; Viana, Jaime; Moreira, Fabiano Cordeiro; Cardoso, Greice de Lemos; Guerreiro, João; Santos, Sidney; Ribeiro-Dos-Santos, Ândrea

2017-01-01

Mutations in the HBB gene are responsible for several serious hemoglobinopathies, such as sickle cell anemia and β-thalassemia. Sickle cell anemia is one of the most common monogenic diseases worldwide. Due to its prevalence, diverse strategies have been developed for a better understanding of its molecular mechanisms. In silico analysis has been increasingly used to investigate the genotype-phenotype relationship of many diseases, and the sequences of healthy individuals deposited in the 1,000 Genomes database appear to be an excellent tool for such analysis. The objective of this study is to analyze the variations in the HBB gene in the 1,000 Genomes database, to describe the mutation frequencies in the different population groups, and to investigate the pattern of pathogenicity. The computational tool SNPEFF was used to align the data from 2,504 samples of the 1,000 Genomes database with the HG19 genome reference. The pathogenicity of each amino acid change was investigated using the databases CLINVAR, dbSNP and HbVar and five different predictors. Twenty different mutations were found in 209 healthy individuals. The African group had the highest number of individuals with mutations, and the European group had the lowest number. Thus, it is concluded that approximately 8.3% of phenotypically healthy individuals from the 1,000 Genomes database have some mutation in the HBB gene. The frequency of mutated genes was estimated at 0.042, so that the expected frequency of being homozygous or compound heterozygous for these variants in the next generation is approximately 0.002. In total, 193 subjects had a non-synonymous mutation, which 186 (7.4%) have a deleterious mutation. Considering that the 1,000 Genomes database is representative of the world's population, it can be estimated that fourteen out of every 10,000 individuals in the world will have a hemoglobinopathy in the next generation.

Evidence for Hitchhiking of Deleterious Mutations within the Human Genome

PubMed Central

Chun, Sung; Fay, Justin C.

2011-01-01

Deleterious mutations present a significant obstacle to adaptive evolution. Deleterious mutations can inhibit the spread of linked adaptive mutations through a population; conversely, adaptive substitutions can increase the frequency of linked deleterious mutations and even result in their fixation. To assess the impact of adaptive mutations on linked deleterious mutations, we examined the distribution of deleterious and neutral amino acid polymorphism in the human genome. Within genomic regions that show evidence of recent hitchhiking, we find fewer neutral but a similar number of deleterious SNPs compared to other genomic regions. The higher ratio of deleterious to neutral SNPs is consistent with simulated hitchhiking events and implies that positive selection eliminates some deleterious alleles and increases the frequency of others. The distribution of disease-associated alleles is also altered in hitchhiking regions. Disease alleles within hitchhiking regions have been associated with auto-immune disorders, metabolic diseases, cancers, and mental disorders. Our results suggest that positive selection has had a significant impact on deleterious polymorphism and may be partly responsible for the high frequency of certain human disease alleles. PMID:21901107

The binding of histone deacetylases and the integrity of zinc finger-like motifs of the E7 protein are essential for the life cycle of human papillomavirus type 31.

PubMed

Longworth, Michelle S; Laimins, Laimonis A

2004-04-01

The E7 oncoprotein of high-risk human papillomaviruses (HPVs) binds to and alters the action of cell cycle regulatory proteins such as members of the retinoblastoma (Rb) family of proteins as well as the histone deacetylases (HDACs). To examine the significance of the binding of E7 to HDACs in the viral life cycle, a mutational analysis of the E7 open reading frame was performed in the context of the complete HPV type 31 (HPV-31) genome. Human foreskin keratinocytes were transfected with wild-type HPV-31 genomes or HPV-31 genomes containing mutations in HDAC binding sequences as well as in the C-terminal zinc finger-like domain, and stable cell lines were isolated. All mutant genomes, except those with E7 mutations in the HDAC binding site, were found to be stably maintained extrachromosomally at an early passage following transfection. Upon further passage in culture, genomes containing mutations to the Rb binding domain as well as the zinc finger-like region quickly lost the ability to maintain episomal genomes. Genomes containing mutations abolishing E7 binding to HDACs or to Rb or mutations to the zinc finger-like motifs failed to extend the life span of transfected keratinocytes and caused cells to arrest at the same time as the untransfected keratinocytes. When induced to differentiate by suspension in methylcellulose, cells maintaining genomes with mutations in the Rb binding domain or the zinc finger-like motifs were impaired in their abilities to activate late viral functions. This study demonstrates that the interaction of E7 with HDACs and the integrity of the zinc finger-like motifs are essential for extending the life span of keratinocytes and for stable maintenance of viral genomes.

Disruption of Transcriptional Coactivator Sub1 Leads to Genome-Wide Re-distribution of Clustered Mutations Induced by APOBEC in Active Yeast Genes

PubMed Central

Dhar, Alok; Polev, Dmitrii E.; Masharsky, Alexey E.; Rogozin, Igor B.; Pavlov, Youri I.

2015-01-01

Mutations in genomes of species are frequently distributed non-randomly, resulting in mutation clusters, including recently discovered kataegis in tumors. DNA editing deaminases play the prominent role in the etiology of these mutations. To gain insight into the enigmatic mechanisms of localized hypermutagenesis that lead to cluster formation, we analyzed the mutational single nucleotide variations (SNV) data obtained by whole-genome sequencing of drug-resistant mutants induced in yeast diploids by AID/APOBEC deaminase and base analog 6-HAP. Deaminase from sea lamprey, PmCDA1, induced robust clusters, while 6-HAP induced a few weak ones. We found that PmCDA1, AID, and APOBEC1 deaminases preferentially mutate the beginning of the actively transcribed genes. Inactivation of transcription initiation factor Sub1 strongly reduced deaminase-induced can1 mutation frequency, but, surprisingly, did not decrease the total SNV load in genomes. However, the SNVs in the genomes of the sub1 clones were re-distributed, and the effect of mutation clustering in the regions of transcription initiation was even more pronounced. At the same time, the mutation density in the protein-coding regions was reduced, resulting in the decrease of phenotypically detected mutants. We propose that the induction of clustered mutations by deaminases involves: a) the exposure of ssDNA strands during transcription and loss of protection of ssDNA due to the depletion of ssDNA-binding proteins, such as Sub1, and b) attainment of conditions favorable for APOBEC action in subpopulation of cells, leading to enzymatic deamination within the currently expressed genes. This model is applicable to both the initial and the later stages of oncogenic transformation and explains variations in the distribution of mutations and kataegis events in different tumor cells. PMID:25941824

Comparison of space flight and heavy ion radiation induced genomic/epigenomic mutations in rice (Oryza sativa)

NASA Astrophysics Data System (ADS)

Shi, Jinming; Lu, Weihong; Sun, Yeqing

2014-04-01

Rice seeds, after space flight and low dose heavy ion radiation treatment were cultured on ground. Leaves of the mature plants were obtained for examination of genomic/epigenomic mutations by using amplified fragment length polymorphism (AFLP) and methylation sensitive amplification polymorphism (MSAP) method, respectively. The mutation sites were identified by fragment recovery and sequencing. The heritability of the mutations was detected in the next generation. Results showed that both space flight and low dose heavy ion radiation can induce significant alterations on rice genome and epigenome (P < 0.05). For both genetic and epigenetic assays, while there was no significant difference in mutation rates and their ability to be inherited to the next generation, the site of mutations differed between the space flight and radiation treated groups. More than 50% of the mutation sites were shared by two radiation treated groups, radiated with different LET value and dose, while only about 20% of the mutation sites were shared by space flight group and radiation treated group. Moreover, in space flight group, we found that DNA methylation changes were more prone to occur on CNG sequence than CG sequence. Sequencing results proved that both space flight and heavy ion radiation induced mutations were widely spread on rice genome including coding region and repeated region. Our study described and compared the characters of space flight and low dose heavy ion radiation induced genomic/epigenomic mutations. Our data revealed the mechanisms of application of space environment for mutagenesis and crop breeding. Furthermore, this work implicated that the nature of mutations induced under space flight conditions may involve factors beyond ion radiation.

Somatic POLE exonuclease domain mutations are early events in sporadic endometrial and colorectal carcinogenesis, determining driver mutational landscape, clonal neoantigen burden and immune response.

PubMed

Temko, Daniel; Van Gool, Inge C; Rayner, Emily; Glaire, Mark; Makino, Seiko; Brown, Matthew; Chegwidden, Laura; Palles, Claire; Depreeuw, Jeroen; Beggs, Andrew; Stathopoulou, Chaido; Mason, John; Baker, Ann-Marie; Williams, Marc; Cerundolo, Vincenzo; Rei, Margarida; Taylor, Jenny C; Schuh, Anna; Ahmed, Ahmed; Amant, Frédéric; Lambrechts, Diether; Smit, Vincent Thbm; Bosse, Tjalling; Graham, Trevor A; Church, David N; Tomlinson, Ian

2018-03-31

Genomic instability, which is a hallmark of cancer, is generally thought to occur in the middle to late stages of tumourigenesis, following the acquisition of permissive molecular aberrations such as TP53 mutation or whole genome doubling. Tumours with somatic POLE exonuclease domain mutations are notable for their extreme genomic instability (their mutation burden is among the highest in human cancer), distinct mutational signature, lymphocytic infiltrate, and excellent prognosis. To what extent these characteristics are determined by the timing of POLE mutations in oncogenesis is unknown. Here, we have shown that pathogenic POLE mutations are detectable in non-malignant precursors of endometrial and colorectal cancer. Using genome and exome sequencing, we found that multiple driver mutations in POLE-mutant cancers show the characteristic POLE mutational signature, including those in genes conventionally regarded as initiators of tumourigenesis. In POLE-mutant cancers, the proportion of monoclonal predicted neoantigens was similar to that in other cancers, but the absolute number was much greater. We also found that the prominent CD8 + T-cell infiltrate present in POLE-mutant cancers was evident in their precursor lesions. Collectively, these data indicate that somatic POLE mutations are early, quite possibly initiating, events in the endometrial and colorectal cancers in which they occur. The resulting early onset of genomic instability may account for the striking immune response and excellent prognosis of these tumours, as well as their early presentation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

A new look at an old virus: patterns of mutation accumulation in the human H1N1 influenza virus since 1918

PubMed Central

2012-01-01

Background The H1N1 influenza A virus has been circulating in the human population for over 95 years, first manifesting itself in the pandemic of 1917–1918. Initial mortality was extremely high, but dropped exponentially over time. Influenza viruses have high mutation rates, and H1N1 has undergone significant genetic changes since 1918. The exact nature of H1N1 mutation accumulation over time has not been fully explored. Methods We have made a comprehensive historical analysis of mutational changes within H1N1 by examining over 4100 fully-sequenced H1N1 genomes. This has allowed us to examine the genetic changes arising within H1N1 from 1918 to the present. Results We document multiple extinction events, including the previously known extinction of the human H1N1 lineage in the 1950s, and an apparent second extinction of the human H1N1 lineage in 2009. These extinctions appear to be due to a continuous accumulation of mutations. At the time of its disappearance in 2009, the human H1N1 lineage had accumulated over 1400 point mutations (more than 10% of the genome), including approximately 330 non-synonymous changes (7.4% of all codons). The accumulation of both point mutations and non-synonymous amino acid changes occurred at constant rates (μ = 14.4 and 2.4 new mutations/year, respectively), and mutations accumulated uniformly across the entire influenza genome. We observed a continuous erosion over time of codon-specificity in H1N1, including a shift away from host (human, swine, and bird [duck]) codon preference patterns. Conclusions While there have been numerous adaptations within the H1N1 genome, most of the genetic changes we document here appear to be non-adaptive, and much of the change appears to be degenerative. We suggest H1N1 has been undergoing natural genetic attenuation, and that significant attenuation may even occur during a single pandemic. This process may play a role in natural pandemic cessation and has apparently contributed to the exponential decline in mortality rates over time, as seen in all major human influenza strains. These findings may be relevant to the development of strategies for managing influenza pandemics and strain evolution. PMID:23062055

Suicidal function of DNA methylation in age-related genome disintegration.

PubMed

Mazin, Alexander L

2009-10-01

This article is dedicated to the 60th anniversary of 5-methylcytosine discovery in DNA. Cytosine methylation can affect genetic and epigenetic processes, works as a part of the genome-defense system and has mutagenic activity; however, the biological functions of this enzymatic modification are not well understood. This review will put forward the hypothesis that the host-defense role of DNA methylation in silencing and mutational destroying of retroviruses and other intragenomic parasites was extended during evolution to most host genes that have to be inactivated in differentiated somatic cells, where it acquired a new function in age-related self-destruction of the genome. The proposed model considers DNA methylation as the generator of 5mC>T transitions that induce 40-70% of all spontaneous somatic mutations of the multiple classes at CpG and CpNpG sites and flanking nucleotides in the p53, FIX, hprt, gpt human genes and some transgenes. The accumulation of 5mC-dependent mutations explains: global changes in the structure of the vertebrate genome throughout evolution; the loss of most 5mC from the DNA of various species over their lifespan and the Hayflick limit of normal cells; the polymorphism of methylation sites, including asymmetric mCpNpN sites; cyclical changes of methylation and demethylation in genes. The suicidal function of methylation may be a special genetic mechanism for increasing DNA damage and the programmed genome disintegration responsible for cell apoptosis and organism aging and death.

Characterizing genomic differences of human cancer stratified by the TP53 mutation status.

PubMed

Wang, Mengyao; Yang, Chao; Zhang, Xiuqing; Li, Xiangchun

2018-06-01

The key roles of the TP53 mutation in cancer have been well established. TP53 is the most frequently mutated gene, and its inactivation is widespread among human cancer types. However, the landscape of genomic alterations in human cancers stratified by the TP53 mutation has not yet been described. We obtained somatic mutation and copy number change data of 6551 regular-mutated samples from the Cancer Genome Atlas (TCGA) and compared significantly mutated genes (SMGs), copy number alterations, mutational signatures and mutational strand asymmetries between cancer samples with and without the TP53 mutation. We identified 126 SMGs, 30 of which were statistically significant in both the TP53 mutant and wild-type groups. Several SMGs, such as VHL, SMAD4 and PTEN, showed a mutation bias towards the TP53 wild-type group, whereas ATRX, IDH1 and RB1 were more prevalent in the TP53 mutant group. Five mutational signatures were extracted from the combined TCGA dataset on which mutational asymmetry analysis was performed, revealing that the TP53 mutant group exhibited substantially greater replication and transcription biases. Furthermore, we found that alterations of multiple genes in a merged mutually exclusive network composed of BRAF, EGFR, PAK1, PIK3CA, PTEN, APC and TERT were related to shortened survival in the TP53 wild-type group. In summary, we characterized the genomic differences and similarities underlying human cancers stratified by the TP53 mutation and identified multi-gene alterations of a merged mutually exclusive network to be a poor prognostic factor for the TP53 wild-type group.

RNA Recombination Enhances Adaptability and Is Required for Virus Spread and Virulence.

PubMed

Xiao, Yinghong; Rouzine, Igor M; Bianco, Simone; Acevedo, Ashley; Goldstein, Elizabeth Faul; Farkov, Mikhail; Brodsky, Leonid; Andino, Raul

2016-04-13

Mutation and recombination are central processes driving microbial evolution. A high mutation rate fuels adaptation but also generates deleterious mutations. Recombination between two different genomes may resolve this paradox, alleviating effects of clonal interference and purging deleterious mutations. Here we demonstrate that recombination significantly accelerates adaptation and evolution during acute virus infection. We identified a poliovirus recombination determinant within the virus polymerase, mutation of which reduces recombination rates without altering replication fidelity. By generating a panel of variants with distinct mutation rates and recombination ability, we demonstrate that recombination is essential to enrich the population in beneficial mutations and purge it from deleterious mutations. The concerted activities of mutation and recombination are key to virus spread and virulence in infected animals. These findings inform a mathematical model to demonstrate that poliovirus adapts most rapidly at an optimal mutation rate determined by the trade-off between selection and accumulation of detrimental mutations. Copyright © 2016 Elsevier Inc. All rights reserved.

Efficient CRISPR/Cas9-based gene knockout in watermelon.

PubMed

Tian, Shouwei; Jiang, Linjian; Gao, Qiang; Zhang, Jie; Zong, Mei; Zhang, Haiying; Ren, Yi; Guo, Shaogui; Gong, Guoyi; Liu, Fan; Xu, Yong

2017-03-01

CRISPR/Cas9 system can precisely edit genomic sequence and effectively create knockout mutations in T0 generation watermelon plants. Genome editing offers great advantage to reveal gene function and generate agronomically important mutations to crops. Recently, RNA-guided genome editing system using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been applied to several plant species, achieving successful targeted mutagenesis. Here, we report the genome of watermelon, an important fruit crop, can also be precisely edited by CRISPR/Cas9 system. ClPDS, phytoene desaturase in watermelon, was selected as the target gene because its mutant bears evident albino phenotype. CRISPR/Cas9 system performed genome editing, such as insertions or deletions at the expected position, in transfected watermelon protoplast cells. More importantly, all transgenic watermelon plants harbored ClPDS mutations and showed clear or mosaic albino phenotype, indicating that CRISPR/Cas9 system has technically 100% of genome editing efficiency in transgenic watermelon lines. Furthermore, there were very likely no off-target mutations, indicated by examining regions that were highly homologous to sgRNA sequences. Our results show that CRISPR/Cas9 system is a powerful tool to effectively create knockout mutations in watermelon.

Germline mutations affecting the histone H4 core cause a developmental syndrome by altering DNA damage response and cell cycle control.

PubMed

Tessadori, Federico; Giltay, Jacques C; Hurst, Jane A; Massink, Maarten P; Duran, Karen; Vos, Harmjan R; van Es, Robert M; Scott, Richard H; van Gassen, Koen L I; Bakkers, Jeroen; van Haaften, Gijs

2017-11-01

Covalent modifications of histones have an established role as chromatin effectors, as they control processes such as DNA replication and transcription, and repair or regulate nucleosomal structure. Loss of modifications on histone N tails, whether due to mutations in genes belonging to histone-modifying complexes or mutations directly affecting the histone tails, causes developmental disorders or has a role in tumorigenesis. More recently, modifications affecting the globular histone core have been uncovered as being crucial for DNA repair, pluripotency and oncogenesis. Here we report monoallelic missense mutations affecting lysine 91 in the histone H4 core (H4K91) in three individuals with a syndrome of growth delay, microcephaly and intellectual disability. Expression of the histone H4 mutants in zebrafish embryos recapitulates the developmental anomalies seen in the patients. We show that the histone H4 alterations cause genomic instability, resulting in increased apoptosis and cell cycle progression anomalies during early development. Mechanistically, our findings indicate an important role for the ubiquitination of H4K91 in genomic stability during embryonic development.

Mutation signatures of carcinogen exposure: genome-wide detection and new opportunities for cancer prevention

PubMed Central

2014-01-01

Exposure to environmental mutagens is an important cause of human cancer, and measures to reduce mutagenic and carcinogenic exposures have been highly successful at controlling cancer. Until recently, it has been possible to connect the chemical characteristics of mutagens to actual mutations observed in human tumors only indirectly. Now, next-generation sequencing technology enables us to observe in detail the DNA-sequence-level effects of well-known mutagens, such as ultraviolet radiation and tobacco smoke, as well as endogenous mutagenic processes, such as those involving activated DNA cytidine deaminases (APOBECs). We can also observe the effects of less well-known but potent mutagens, including those recently found to be present in some herbal remedies. Crucially, we can now tease apart the superimposed effects of several mutational exposures and processes and determine which ones occurred during the development of individual tumors. Here, we review advances in detecting these mutation signatures and discuss the implications for surveillance and prevention of cancer. The number of sequenced tumors from diverse cancer types and multiple geographic regions is growing explosively, and the genomes of these tumors will bear the signatures of even more diverse mutagenic exposures. Thus, we envision development of wide-ranging compendia of mutation signatures from tumors and a concerted effort to experimentally elucidate the signatures of a large number of mutagens. This information will be used to link signatures observed in tumors to the exposures responsible for them, which will offer unprecedented opportunities for prevention. PMID:25031618

Mitochondrial genetic codes evolve to match amino acid requirements of proteins.

PubMed

Swire, Jonathan; Judson, Olivia P; Burt, Austin

2005-01-01

Mitochondria often use genetic codes different from the standard genetic code. Now that many mitochondrial genomes have been sequenced, these variant codes provide the first opportunity to examine empirically the processes that produce new genetic codes. The key question is: Are codon reassignments the sole result of mutation and genetic drift? Or are they the result of natural selection? Here we present an analysis of 24 phylogenetically independent codon reassignments in mitochondria. Although the mutation-drift hypothesis can explain reassignments from stop to an amino acid, we found that it cannot explain reassignments from one amino acid to another. In particular--and contrary to the predictions of the mutation-drift hypothesis--the codon involved in such a reassignment was not rare in the ancestral genome. Instead, such reassignments appear to take place while the codon is in use at an appreciable frequency. Moreover, the comparison of inferred amino acid usage in the ancestral genome with the neutral expectation shows that the amino acid gaining the codon was selectively favored over the amino acid losing the codon. These results are consistent with a simple model of weak selection on the amino acid composition of proteins in which codon reassignments are selected because they compensate for multiple slightly deleterious mutations throughout the mitochondrial genome. We propose that the selection pressure is for reduced protein synthesis cost: most reassignments give amino acids that are less expensive to synthesize. Taken together, our results strongly suggest that mitochondrial genetic codes evolve to match the amino acid requirements of proteins.

Reverse Engineering Field Isolates of Myxoma Virus Demonstrates that Some Gene Disruptions or Losses of Function Do Not Explain Virulence Changes Observed in the Field

PubMed Central

Liu, June; Cattadori, Isabella M.; Sim, Derek G.; Eden, John-Sebastian; Read, Andrew F.

2017-01-01

ABSTRACT The coevolution of myxoma virus (MYXV) and wild European rabbits in Australia and Europe is a paradigm for the evolution of a pathogen in a new host species. Genomic analyses have identified the mutations that have characterized this evolutionary process, but defining causal mutations in the pathways from virulence to attenuation and back to virulence has not been possible. Using reverse genetics, we examined the roles of six selected mutations found in Australian field isolates of MYXV that fall in known or potential virulence genes. Several of these mutations occurred in genes previously identified as virulence genes in whole-gene knockout studies. Strikingly, no single or double mutation among the mutations tested had an appreciable impact on virulence. This suggests either that virulence evolution was defined by amino acid changes other than those analyzed here or that combinations of multiple mutations, possibly involving epistatic interactions or noncoding sequences, have been critical in the ongoing evolution of MYXV virulence. In sum, our results show that single-gene knockout studies of a progenitor virus can have little power to predict the impact of individual mutations seen in the field. The genetic determinants responsible for this canonical case of virulence evolution remain to be determined. IMPORTANCE The species jump of myxoma virus (MYXV) from the South American tapeti to the European rabbit populations of Australia and Europe is a canonical example of host-pathogen coevolution. Detailed molecular studies have identified multiple genes in MYXV that are critical for virulence, and genome sequencing has revealed the evolutionary history of MYXV in Australia and Europe. However, it has not been possible to categorically identify the key mutations responsible for the attenuation of or reversion to virulence during this evolutionary process. Here we use reverse genetics to examine the role of mutations in viruses isolated early and late in the Australian radiation of MYXV. Surprisingly, none of the candidate mutations that we identified as likely having roles in attenuation proved to be important for virulence. This indicates that considerable caution is warranted when interpreting the possible role of individual mutations during virulence evolution. PMID:28768866

Statistical Methods for Identifying Sequence Motifs Affecting Point Mutations

PubMed Central

Zhu, Yicheng; Neeman, Teresa; Yap, Von Bing; Huttley, Gavin A.

2017-01-01

Mutation processes differ between types of point mutation, genomic locations, cells, and biological species. For some point mutations, specific neighboring bases are known to be mechanistically influential. Beyond these cases, numerous questions remain unresolved, including: what are the sequence motifs that affect point mutations? How large are the motifs? Are they strand symmetric? And, do they vary between samples? We present new log-linear models that allow explicit examination of these questions, along with sequence logo style visualization to enable identifying specific motifs. We demonstrate the performance of these methods by analyzing mutation processes in human germline and malignant melanoma. We recapitulate the known CpG effect, and identify novel motifs, including a highly significant motif associated with A→G mutations. We show that major effects of neighbors on germline mutation lie within ±2 of the mutating base. Models are also presented for contrasting the entire mutation spectra (the distribution of the different point mutations). We show the spectra vary significantly between autosomes and X-chromosome, with a difference in T→C transition dominating. Analyses of malignant melanoma confirmed reported characteristic features of this cancer, including statistically significant strand asymmetry, and markedly different neighboring influences. The methods we present are made freely available as a Python library https://bitbucket.org/pycogent3/mutationmotif. PMID:27974498

Whole-exome sequencing identifies recurrent AKT1 mutations in sclerosing hemangioma of lung

PubMed Central

Jung, Seung-Hyun; Kim, Min Sung; Lee, Sung-Hak; Park, Hyun-Chun; Choi, Hyun Joo; Maeng, Leeso; Min, Ki Ouk; Kim, Jeana; Park, Tae In; Shin, Ok Ran; Kim, Tae-Jung; Xu, Haidong; Lee, Kyo Young; Kim, Tae-Min; Song, Sang Yong; Lee, Charles; Chung, Yeun-Jun; Lee, Sug Hyung

2016-01-01

Pulmonary sclerosing hemangioma (PSH) is a benign tumor with two cell populations (epithelial and stromal cells), for which genomic profiles remain unknown. We conducted exome sequencing of 44 PSHs and identified recurrent somatic mutations of AKT1 (43.2%) and β-catenin (4.5%). We used a second subset of 24 PSHs to confirm the high frequency of AKT1 mutations (overall 31/68, 45.6%; p.E17K, 33.8%) and recurrent β-catenin mutations (overall 3 of 68, 4.4%). Of the PSHs without AKT1 mutations, two exhibited AKT1 copy gain. AKT1 mutations existed in both epithelial and stromal cells. In two separate PSHs from one patient, we observed two different AKT1 mutations, indicating they were not disseminated but independent arising tumors. Because the AKT1 mutations were not found to co-occur with β-catenin mutations (or any other known driver alterations) in any of the PSHs studied, we speculate that this may be the single-most common driver alteration to develop PSHs. Our study revealed genomic differences between PSHs and lung adenocarcinomas, including a high rate of AKT1 mutation in PSHs. These genomic features of PSH identified in the present study provide clues to understanding the biology of PSH and for differential genomic diagnosis of lung tumors. PMID:27601661

The impact of photoperiod insensitive Ppd-1a mutations on the photoperiod pathway across the three genomes of hexaploid wheat (Triticum aestivum).

PubMed

Shaw, Lindsay M; Turner, Adrian S; Laurie, David A

2012-07-01

Flowering time is a trait that has been extensively altered during wheat domestication, enabling it to be highly productive in diverse environments and providing a rich source of variation for studying adaptation mechanisms. Hexaploid wheat is ancestrally a long-day plant, but many environments require varieties with photoperiod insensitivity (PI) that can flower in short days. PI results from mutations in the Ppd-1 gene on the A, B or D genomes, with individual mutations conferring different degrees of earliness. The basis of this is poorly understood. Using a common genetic background, the effects of A, B and D genome PI mutations on genes of the circadian clock and photoperiod pathway were studied using genome-specific expression assays. Ppd-1 PI mutations did not affect the clock or immediate clock outputs, but affected TaCO1 and TaFT1, with a reduction in TaCO1 expression as TaFT1 expression increased. Therefore, although Ppd-1 is related to PRR genes of the Arabidopsis circadian clock, Ppd-1 affects flowering by an alternative route, most likely by upregulating TaFT1 with a feedback effect that reduces TaCO1 expression. Individual genes in the circadian clock and photoperiod pathway were predominantly expressed from one genome, and there was no genome specificity in Ppd-1 action. Lines combining PI mutations on two or three genomes had enhanced earliness with higher levels, but not earlier induction, of TaFT1, showing that there is a direct quantitative relationship between Ppd-1 mutations, TaFT1 expression and flowering. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

How resilient is the soybean genome? Insights from fast neutron mutagenesis

USDA-ARS?s Scientific Manuscript database

Previously, we described the development of a fast neutron mutant population resource in soybean and identified mutations of interest through phenotypic screening. Here, we consider the resiliency of the soybean genome by examining genomic rearrangements and mutations that arise from fast neutron ra...

The evolutionary processes of mitochondrial and chloroplast genomes differ from those of nuclear genomes

NASA Astrophysics Data System (ADS)

Korpelainen, Helena

2004-11-01

This paper first introduces our present knowledge of the origin of mitochondria and chloroplasts, and the organization and inheritance patterns of their genomes, and then carries on to review the evolutionary processes influencing mitochondrial and chloroplast genomes. The differences in evolutionary phenomena between the nuclear and cytoplasmic genomes are highlighted. It is emphasized that varying inheritance patterns and copy numbers among different types of genomes, and the potential advantage achieved through the transfer of many cytoplasmic genes to the nucleus, have important implications for the evolution of nuclear, mitochondrial and chloroplast genomes. Cytoplasmic genes transferred to the nucleus have joined the more strictly controlled genetic system of the nuclear genome, including also sexual recombination, while genes retained within the cytoplasmic organelles can be involved in selection and drift processes both within and among individuals. Within-individual processes can be either intra- or intercellular. In the case of heteroplasmy, which is attributed to mutations or biparental inheritance, within-individual selection on cytoplasmic DNA may provide a mechanism by which the organism can adapt rapidly. The inheritance of cytoplasmic genomes is not universally maternal. The presence of a range of inheritance patterns indicates that different strategies have been adopted by different organisms. On the other hand, the variability occasionally observed in the inheritance mechanisms of cytoplasmic genomes reduces heritability and increases environmental components in phenotypic features and, consequently, decreases the potential for adaptive evolution.

Transcription-associated mutational pressure in the Parvovirus B19 genome: Reactivated genomes contribute to the variability of viral populations.

PubMed

Khrustalev, Vladislav Victorovich; Ermalovich, Marina Anatolyevna; Hübschen, Judith M; Khrustaleva, Tatyana Aleksandrovna

2017-12-21

In this study we used non-overlapping parts of the two long open reading frames coding for nonstructural (NS) and capsid (VP) proteins of all available sequences of the Parvovirus B19 subgenotype 1a genome and found out that the rates of A to G, C to T and A to T mutations are higher in the first long reading frame (NS) of the virus than in the second one (VP). This difference in mutational pressure directions for two parts of the same viral genome can be explained by the fact of transcription of just the first long reading frame during the lifelong latency in nonerythroid cells. Adenine deamination (producing A to G and A to T mutations) and cytosine deamination (producing C to T mutations) occur more frequently in transcriptional bubbles formed by DNA "plus" strand of the first open reading frame. These mutations can be inherited only in case of reactivation of the infectious virus due to the help of Adenovirus that allows latent Parvovirus B19 to start transcription of the second reading frame and then to replicate its genome by the rolling circle mechanism using the specific origin. Results of this study provide evidence that the genomes reactivated from latency make significant contributions to the variability of Parvovirus B19. Copyright © 2017 Elsevier Ltd. All rights reserved.

Error-free versus mutagenic processing of genomic uracil--relevance to cancer.

PubMed

Krokan, Hans E; Sætrom, Pål; Aas, Per Arne; Pettersen, Henrik Sahlin; Kavli, Bodil; Slupphaug, Geir

2014-07-01

Genomic uracil is normally processed essentially error-free by base excision repair (BER), with mismatch repair (MMR) as an apparent backup for U:G mismatches. Nuclear uracil-DNA glycosylase UNG2 is the major enzyme initiating BER of uracil of U:A pairs as well as U:G mismatches. Deficiency in UNG2 results in several-fold increases in genomic uracil in mammalian cells. Thus, the alternative uracil-removing glycosylases, SMUG1, TDG and MBD4 cannot efficiently complement UNG2-deficiency. A major function of SMUG1 is probably to remove 5-hydroxymethyluracil from DNA with general back-up for UNG2 as a minor function. TDG and MBD4 remove deamination products U or T mismatched to G in CpG/mCpG contexts, but may have equally or more important functions in development, epigenetics and gene regulation. Genomic uracil was previously thought to arise only from spontaneous cytosine deamination and incorporation of dUMP, generating U:G mismatches and U:A pairs, respectively. However, the identification of activation-induced cytidine deaminase (AID) and other APOBEC family members as DNA-cytosine deaminases has spurred renewed interest in the processing of genomic uracil. Importantly, AID triggers the adaptive immune response involving error-prone processing of U:G mismatches, but also contributes to B-cell lymphomagenesis. Furthermore, mutational signatures in a substantial fraction of other human cancers are consistent with APOBEC-induced mutagenesis, with U:G mismatches as prime suspects. Mutations can be caused by replicative polymerases copying uracil in U:G mismatches, or by translesion polymerases that insert incorrect bases opposite abasic sites after uracil-removal. In addition, kataegis, localized hypermutations in one strand in the vicinity of genomic rearrangements, requires APOBEC protein, UNG2 and translesion polymerase REV1. What mechanisms govern error-free versus error prone processing of uracil in DNA remains unclear. In conclusion, genomic uracil is an essential intermediate in adaptive immunity and innate antiviral responses, but may also be a fundamental cause of a wide range of malignancies. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

A little bit of sex matters for genome evolution in asexual plants.

PubMed

Hojsgaard, Diego; Hörandl, Elvira

2015-01-01

Genome evolution in asexual organisms is theoretically expected to be shaped by various factors: first, hybrid origin, and polyploidy confer a genomic constitution of highly heterozygous genotypes with multiple copies of genes; second, asexuality confers a lack of recombination and variation in populations, which reduces the efficiency of selection against deleterious mutations; hence, the accumulation of mutations and a gradual increase in mutational load (Muller's ratchet) would lead to rapid extinction of asexual lineages; third, allelic sequence divergence is expected to result in rapid divergence of lineages (Meselson effect). Recent transcriptome studies on the asexual polyploid complex Ranunculus auricomus using single-nucleotide polymorphisms confirmed neutral allelic sequence divergence within a short time frame, but rejected a hypothesis of a genome-wide accumulation of mutations in asexuals compared to sexuals, except for a few genes related to reproductive development. We discuss a general model that the observed incidence of facultative sexuality in plants may unmask deleterious mutations with partial dominance and expose them efficiently to purging selection. A little bit of sex may help to avoid genomic decay and extinction.

MutationAligner: a resource of recurrent mutation hotspots in protein domains in cancer.

PubMed

Gauthier, Nicholas Paul; Reznik, Ed; Gao, Jianjiong; Sumer, Selcuk Onur; Schultz, Nikolaus; Sander, Chris; Miller, Martin L

2016-01-04

The MutationAligner web resource, available at http://www.mutationaligner.org, enables discovery and exploration of somatic mutation hotspots identified in protein domains in currently (mid-2015) more than 5000 cancer patient samples across 22 different tumor types. Using multiple sequence alignments of protein domains in the human genome, we extend the principle of recurrence analysis by aggregating mutations in homologous positions across sets of paralogous genes. Protein domain analysis enhances the statistical power to detect cancer-relevant mutations and links mutations to the specific biological functions encoded in domains. We illustrate how the MutationAligner database and interactive web tool can be used to explore, visualize and analyze mutation hotspots in protein domains across genes and tumor types. We believe that MutationAligner will be an important resource for the cancer research community by providing detailed clues for the functional importance of particular mutations, as well as for the design of functional genomics experiments and for decision support in precision medicine. MutationAligner is slated to be periodically updated to incorporate additional analyses and new data from cancer genomics projects. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Independent Polled Mutations Leading to Complex Gene Expression Differences in Cattle

PubMed Central

Wiedemar, Natalie; Tetens, Jens; Jagannathan, Vidhya; Menoud, Annie; Neuenschwander, Samuel; Bruggmann, Rémy; Thaller, Georg; Drögemüller, Cord

2014-01-01

The molecular regulation of horn growth in ruminants is still poorly understood. To investigate this process, we collected 1019 hornless (polled) animals from different cattle breeds. High-density SNP genotyping confirmed the presence of two different polled associated haplotypes in Simmental and Holstein cattle co-localized on BTA 1. We refined the critical region of the Simmental polled mutation to 212 kb and identified an overlapping region of 932 kb containing the Holstein polled mutation. Subsequently, whole genome sequencing of polled Simmental and Holstein cows was used to determine polled associated genomic variants. By genotyping larger cohorts of animals with known horn status we found a single perfectly associated insertion/deletion variant in Simmental and other beef cattle confirming the recently published possible Celtic polled mutation. We identified a total of 182 sequence variants as candidate mutations for polledness in Holstein cattle, including an 80 kb genomic duplication and three SNPs reported before. For the first time we showed that hornless cattle with scurs are obligate heterozygous for one of the polled mutations. This is in contrast to published complex inheritance models for the bovine scurs phenotype. Studying differential expression of the annotated genes and loci within the mapped region on BTA 1 revealed a locus (LOC100848215), known in cow and buffalo only, which is higher expressed in fetal tissue of wildtype horn buds compared to tissue of polled fetuses. This implicates that the presence of this long noncoding RNA is a prerequisite for horn bud formation. In addition, both transcripts associated with polledness in goat and sheep (FOXL2 and RXFP2), show an overexpression in horn buds confirming their importance during horn development in cattle. PMID:24671182

Somatic mutation load of estrogen receptor-positive breast tumors predicts overall survival: an analysis of genome sequence data.

PubMed

Haricharan, Svasti; Bainbridge, Matthew N; Scheet, Paul; Brown, Powel H

2014-07-01

Breast cancer is one of the most commonly diagnosed cancers in women. While there are several effective therapies for breast cancer and important single gene prognostic/predictive markers, more than 40,000 women die from this disease every year. The increasing availability of large-scale genomic datasets provides opportunities for identifying factors that influence breast cancer survival in smaller, well-defined subsets. The purpose of this study was to investigate the genomic landscape of various breast cancer subtypes and its potential associations with clinical outcomes. We used statistical analysis of sequence data generated by the Cancer Genome Atlas initiative including somatic mutation load (SML) analysis, Kaplan-Meier survival curves, gene mutational frequency, and mutational enrichment evaluation to study the genomic landscape of breast cancer. We show that ER(+), but not ER(-), tumors with high SML associate with poor overall survival (HR = 2.02). Further, these high mutation load tumors are enriched for coincident mutations in both DNA damage repair and ER signature genes. While it is known that somatic mutations in specific genes affect breast cancer survival, this study is the first to identify that SML may constitute an important global signature for a subset of ER(+) tumors prone to high mortality. Moreover, although somatic mutations in individual DNA damage genes affect clinical outcome, our results indicate that coincident mutations in DNA damage response and signature ER genes may prove more informative for ER(+) breast cancer survival. Next generation sequencing may prove an essential tool for identifying pathways underlying poor outcomes and for tailoring therapeutic strategies.

Integrative proteomics, genomics, and translational immunology approaches reveal mutated forms of Proteolipid Protein 1 (PLP1) and mutant-specific immune response in multiple sclerosis.

PubMed

Qendro, Veneta; Bugos, Grace A; Lundgren, Debbie H; Glynn, John; Han, May H; Han, David K

2017-03-01

In order to gain mechanistic insights into multiple sclerosis (MS) pathogenesis, we utilized a multi-dimensional approach to test the hypothesis that mutations in myelin proteins lead to immune activation and central nervous system autoimmunity in MS. Mass spectrometry-based proteomic analysis of human MS brain lesions revealed seven unique mutations of PLP1; a key myelin protein that is known to be destroyed in MS. Surprisingly, in-depth genomic analysis of two MS patients at the genomic DNA and mRNA confirmed mutated PLP1 in RNA, but not in the genomic DNA. Quantification of wild type and mutant PLP RNA levels by qPCR further validated the presence of mutant PLP RNA in the MS patients. To seek evidence linking mutations in abundant myelin proteins and immune-mediated destruction of myelin, specific immune response against mutant PLP1 in MS patients was examined. Thus, we have designed paired, wild type and mutant peptide microarrays, and examined antibody response to multiple mutated PLP1 in sera from MS patients. Consistent with the idea of different patients exhibiting unique mutation profiles, we found that 13 out of 20 MS patients showed antibody responses against specific but not against all the mutant-PLP1 peptides. Interestingly, we found mutant PLP-directed antibody response against specific mutant peptides in the sera of pre-MS controls. The results from integrative proteomic, genomic, and immune analyses reveal a possible mechanism of mutation-driven pathogenesis in human MS. The study also highlights the need for integrative genomic and proteomic analyses for uncovering pathogenic mechanisms of human diseases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Within-host whole genome analysis of an antibiotic resistant Pseudomonas aeruginosa strain sub-type in cystic fibrosis.

PubMed

Sherrard, Laura J; Tai, Anna S; Wee, Bryan A; Ramsay, Kay A; Kidd, Timothy J; Ben Zakour, Nouri L; Whiley, David M; Beatson, Scott A; Bell, Scott C

2017-01-01

A Pseudomonas aeruginosa AUST-02 strain sub-type (M3L7) has been identified in Australia, infects the lungs of some people with cystic fibrosis and is associated with antibiotic resistance. Multiple clonal lineages may emerge during treatment with mutations in chromosomally encoded antibiotic resistance genes commonly observed. Here we describe the within-host diversity and antibiotic resistance of M3L7 during and after antibiotic treatment of an acute pulmonary exacerbation using whole genome sequencing and show both variation and shared mutations in important genes. Eleven isolates from an M3L7 population (n = 134) isolated over 3 months from an individual with cystic fibrosis underwent whole genome sequencing. A phylogeny based on core genome SNPs identified three distinct phylogenetic groups comprising two groups with higher rates of mutation (hypermutators) and one non-hypermutator group. Genomes were screened for acquired antibiotic resistance genes with the result suggesting that M3L7 resistance is principally driven by chromosomal mutations as no acquired mechanisms were detected. Small genetic variations, shared by all 11 isolates, were found in 49 genes associated with antibiotic resistance including frame-shift mutations (mexA, mexT), premature stop codons (oprD, mexB) and mutations in quinolone-resistance determining regions (gyrA, parE). However, whole genome sequencing also revealed mutations in 21 genes that were acquired following divergence of groups, which may also impact the activity of antibiotics and multi-drug efflux pumps. Comparison of mutations with minimum inhibitory concentrations of anti-pseudomonal antibiotics could not easily explain all resistance profiles observed. These data further demonstrate the complexity of chronic and antibiotic resistant P. aeruginosa infection where a multitude of co-existing genotypically diverse sub-lineages might co-exist during and after intravenous antibiotic treatment.

Therapeutic Genome Editing: Prospects and Challenges

PubMed Central

Cox, David Benjamin Turitz; Platt, Randall Jeffrey; Zhang, Feng

2015-01-01

Recent advances in the development of genome editing technologies based on programmable nucleases have significantly improved our ability to make precise changes in the genomes of eukaryotic cells. Genome editing is already broadening our ability to elucidate the contribution of genetics to disease by facilitating the creation of more accurate cellular and animal models of pathological processes. A particularly tantalizing application of programmable nucleases is the potential to directly correct genetic mutations in affected tissues and cells to treat diseases that are refractory to traditional therapies. Here we discuss current progress towards developing programmable nuclease-based therapies as well as future prospects and challenges. PMID:25654603

A high-throughput next-generation sequencing-based method for detecting the mutational fingerprint of carcinogens

PubMed Central

Besaratinia, Ahmad; Li, Haiqing; Yoon, Jae-In; Zheng, Albert; Gao, Hanlin; Tommasi, Stella

2012-01-01

Many carcinogens leave a unique mutational fingerprint in the human genome. These mutational fingerprints manifest as specific types of mutations often clustering at certain genomic loci in tumor genomes from carcinogen-exposed individuals. To develop a high-throughput method for detecting the mutational fingerprint of carcinogens, we have devised a cost-, time- and labor-effective strategy, in which the widely used transgenic Big Blue® mouse mutation detection assay is made compatible with the Roche/454 Genome Sequencer FLX Titanium next-generation sequencing technology. As proof of principle, we have used this novel method to establish the mutational fingerprints of three prominent carcinogens with varying mutagenic potencies, including sunlight ultraviolet radiation, 4-aminobiphenyl and secondhand smoke that are known to be strong, moderate and weak mutagens, respectively. For verification purposes, we have compared the mutational fingerprints of these carcinogens obtained by our newly developed method with those obtained by parallel analyses using the conventional low-throughput approach, that is, standard mutation detection assay followed by direct DNA sequencing using a capillary DNA sequencer. We demonstrate that this high-throughput next-generation sequencing-based method is highly specific and sensitive to detect the mutational fingerprints of the tested carcinogens. The method is reproducible, and its accuracy is comparable with that of the currently available low-throughput method. In conclusion, this novel method has the potential to move the field of carcinogenesis forward by allowing high-throughput analysis of mutations induced by endogenous and/or exogenous genotoxic agents. PMID:22735701

A high-throughput next-generation sequencing-based method for detecting the mutational fingerprint of carcinogens.

PubMed

Besaratinia, Ahmad; Li, Haiqing; Yoon, Jae-In; Zheng, Albert; Gao, Hanlin; Tommasi, Stella

2012-08-01

Many carcinogens leave a unique mutational fingerprint in the human genome. These mutational fingerprints manifest as specific types of mutations often clustering at certain genomic loci in tumor genomes from carcinogen-exposed individuals. To develop a high-throughput method for detecting the mutational fingerprint of carcinogens, we have devised a cost-, time- and labor-effective strategy, in which the widely used transgenic Big Blue mouse mutation detection assay is made compatible with the Roche/454 Genome Sequencer FLX Titanium next-generation sequencing technology. As proof of principle, we have used this novel method to establish the mutational fingerprints of three prominent carcinogens with varying mutagenic potencies, including sunlight ultraviolet radiation, 4-aminobiphenyl and secondhand smoke that are known to be strong, moderate and weak mutagens, respectively. For verification purposes, we have compared the mutational fingerprints of these carcinogens obtained by our newly developed method with those obtained by parallel analyses using the conventional low-throughput approach, that is, standard mutation detection assay followed by direct DNA sequencing using a capillary DNA sequencer. We demonstrate that this high-throughput next-generation sequencing-based method is highly specific and sensitive to detect the mutational fingerprints of the tested carcinogens. The method is reproducible, and its accuracy is comparable with that of the currently available low-throughput method. In conclusion, this novel method has the potential to move the field of carcinogenesis forward by allowing high-throughput analysis of mutations induced by endogenous and/or exogenous genotoxic agents.

Genome-Wide Estimates of Mutation Rates and Spectrum in Schizosaccharomyces pombe Indicate CpG Sites are Highly Mutagenic Despite the Absence of DNA Methylation

PubMed Central

Behringer, Megan G.; Hall, David W.

2015-01-01

We accumulated mutations for 1952 generations in 79 initially identical, haploid lines of the fission yeast Schizosaccharomyces pombe, and then performed whole-genome sequencing to determine the mutation rates and spectrum. We captured 696 spontaneous mutations across the 79 mutation accumulation (MA) lines. We compared the mutation spectrum and rate to a recently published equivalent experiment on the same species, and to another model ascomycetous yeast, the budding yeast Saccharomyces cerevisiae. While the two species are approximately 600 million years diverged from each other, they share similar life histories, genome size and genomic G/C content. We found that Sc. pombe and S. cerevisiae have similar mutation rates, but Sc. pombe exhibits a stronger insertion bias. Intriguingly, we observed an increased mutation rate at cytosine nucleotides, specifically CpG nucleotides, which is also seen in S. cerevisiae. However, the absence of methylation in Sc. pombe and the pattern of mutation at these sites, primarily C → A as opposed to C → T, strongly suggest that the increased mutation rate is not caused by deamination of methylated cytosines. This result implies that the high mutability of CpG dinucleotides in other species may be caused in part by a methylation-independent mechanism. Many of our findings mirror those seen in the recent study, despite the use of different passaging conditions, indicating that MA is a reliable method for estimating mutation rates and spectra. PMID:26564949

Comprehensive genomic profiles of small cell lung cancer

PubMed Central

George, Julie; Lim, Jing Shan; Jang, Se Jin; Cun, Yupeng; Ozretić, Luka; Kong, Gu; Leenders, Frauke; Lu, Xin; Fernández-Cuesta, Lynnette; Bosco, Graziella; Müller, Christian; Dahmen, Ilona; Jahchan, Nadine S.; Park, Kwon-Sik; Yang, Dian; Karnezis, Anthony N.; Vaka, Dedeepya; Torres, Angela; Wang, Maia Segura; Korbel, Jan O.; Menon, Roopika; Chun, Sung-Min; Kim, Deokhoon; Wilkerson, Matt; Hayes, Neil; Engelmann, David; Pützer, Brigitte; Bos, Marc; Michels, Sebastian; Vlasic, Ignacija; Seidel, Danila; Pinther, Berit; Schaub, Philipp; Becker, Christian; Altmüller, Janine; Yokota, Jun; Kohno, Takashi; Iwakawa, Reika; Tsuta, Koji; Noguchi, Masayuki; Muley, Thomas; Hoffmann, Hans; Schnabel, Philipp A.; Petersen, Iver; Chen, Yuan; Soltermann, Alex; Tischler, Verena; Choi, Chang-min; Kim, Yong-Hee; Massion, Pierre P.; Zou, Yong; Jovanovic, Dragana; Kontic, Milica; Wright, Gavin M.; Russell, Prudence A.; Solomon, Benjamin; Koch, Ina; Lindner, Michael; Muscarella, Lucia A.; la Torre, Annamaria; Field, John K.; Jakopovic, Marko; Knezevic, Jelena; Castaños-Vélez, Esmeralda; Roz, Luca; Pastorino, Ugo; Brustugun, Odd-Terje; Lund-Iversen, Marius; Thunnissen, Erik; Köhler, Jens; Schuler, Martin; Botling, Johan; Sandelin, Martin; Sanchez-Cespedes, Montserrat; Salvesen, Helga B.; Achter, Viktor; Lang, Ulrich; Bogus, Magdalena; Schneider, Peter M.; Zander, Thomas; Ansén, Sascha; Hallek, Michael; Wolf, Jürgen; Vingron, Martin; Yatabe, Yasushi; Travis, William D.; Nürnberg, Peter; Reinhardt, Christian; Perner, Sven; Heukamp, Lukas; Büttner, Reinhard; Haas, Stefan A.; Brambilla, Elisabeth; Peifer, Martin; Sage, Julien; Thomas, Roman K.

2016-01-01

We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Δex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer. PMID:26168399

Interactome INSIDER: a structural interactome browser for genomic studies.

PubMed

Meyer, Michael J; Beltrán, Juan Felipe; Liang, Siqi; Fragoza, Robert; Rumack, Aaron; Liang, Jin; Wei, Xiaomu; Yu, Haiyuan

2018-01-01

We present Interactome INSIDER, a tool to link genomic variant information with structural protein-protein interactomes. Underlying this tool is the application of machine learning to predict protein interaction interfaces for 185,957 protein interactions with previously unresolved interfaces in human and seven model organisms, including the entire experimentally determined human binary interactome. Predicted interfaces exhibit functional properties similar to those of known interfaces, including enrichment for disease mutations and recurrent cancer mutations. Through 2,164 de novo mutagenesis experiments, we show that mutations of predicted and known interface residues disrupt interactions at a similar rate and much more frequently than mutations outside of predicted interfaces. To spur functional genomic studies, Interactome INSIDER (http://interactomeinsider.yulab.org) enables users to identify whether variants or disease mutations are enriched in known and predicted interaction interfaces at various resolutions. Users may explore known population variants, disease mutations, and somatic cancer mutations, or they may upload their own set of mutations for this purpose.

ActiveDriverDB: human disease mutations and genome variation in post-translational modification sites of proteins

PubMed Central

Krassowski, Michal; Paczkowska, Marta; Cullion, Kim; Huang, Tina; Dzneladze, Irakli; Ouellette, B F Francis; Yamada, Joseph T; Fradet-Turcotte, Amelie

2018-01-01

Abstract Interpretation of genetic variation is needed for deciphering genotype-phenotype associations, mechanisms of inherited disease, and cancer driver mutations. Millions of single nucleotide variants (SNVs) in human genomes are known and thousands are associated with disease. An estimated 21% of disease-associated amino acid substitutions corresponding to missense SNVs are located in protein sites of post-translational modifications (PTMs), chemical modifications of amino acids that extend protein function. ActiveDriverDB is a comprehensive human proteo-genomics database that annotates disease mutations and population variants through the lens of PTMs. We integrated >385,000 published PTM sites with ∼3.6 million substitutions from The Cancer Genome Atlas (TCGA), the ClinVar database of disease genes, and human genome sequencing projects. The database includes site-specific interaction networks of proteins, upstream enzymes such as kinases, and drugs targeting these enzymes. We also predicted network-rewiring impact of mutations by analyzing gains and losses of kinase-bound sequence motifs. ActiveDriverDB provides detailed visualization, filtering, browsing and searching options for studying PTM-associated mutations. Users can upload mutation datasets interactively and use our application programming interface in pipelines. Integrative analysis of mutations and PTMs may help decipher molecular mechanisms of phenotypes and disease, as exemplified by case studies of TP53, BRCA2 and VHL. The open-source database is available at https://www.ActiveDriverDB.org. PMID:29126202

Whole Genome Sequence Analysis of Mutations Accumulated in rad27Δ Yeast Strains with Defects in the Processing of Okazaki Fragments Indicates Template-Switching Events

PubMed Central

Omer, Sumita; Lavi, Bar; Mieczkowski, Piotr A.; Covo, Shay; Hazkani-Covo, Einat

2017-01-01

Okazaki fragments that are formed during lagging strand DNA synthesis include an initiating primer consisting of both RNA and DNA. The RNA fragment must be removed before the fragments are joined. In Saccharomyces cerevisiae, a key player in this process is the structure-specific flap endonuclease, Rad27p (human homolog FEN1). To obtain a genomic view of the mutational consequence of loss of RAD27, a S. cerevisiae rad27Δ strain was subcultured for 25 generations and sequenced using Illumina paired-end sequencing. Out of the 455 changes observed in 10 colonies isolated the two most common types of events were insertions or deletions (INDELs) in simple sequence repeats (SSRs) and INDELs mediated by short direct repeats. Surprisingly, we also detected a previously neglected class of 21 template-switching events. These events were presumably generated by quasi-palindrome to palindrome correction, as well as palindrome elongation. The formation of these events is best explained by folding back of the stalled nascent strand and resumption of DNA synthesis using the same nascent strand as a template. Evidence of quasi-palindrome to palindrome correction that could be generated by template switching appears also in yeast genome evolution. Out of the 455 events, 55 events appeared in multiple isolates; further analysis indicates that these loci are mutational hotspots. Since Rad27 acts on the lagging strand when the leading strand should not contain any gaps, we propose a mechanism favoring intramolecular strand switching over an intermolecular mechanism. We note that our results open new ways of understanding template switching that occurs during genome instability and evolution. PMID:28974572

Comparative biology approaches for charged particle exposures and cancer development processes

NASA Astrophysics Data System (ADS)

Kronenberg, Amy; Gauny, Stacey; Kwoh, Ely; Sudo, Hiroko; Wiese, Claudia; Dan, Cristian; Turker, Mitchell

Comparative biology studies can provide useful information for the extrapolation of results be-tween cells in culture and the more complex environment of the tissue. In other circumstances, they provide a method to guide the interpretation of results obtained for cells from differ-ent species. We have considered several key cancer development processes following charged particle exposures using comparative biology approaches. Our particular emphases have been mutagenesis and genomic instability. Carcinogenesis requires the accumulation of mutations and most of htese mutations occur on autosomes. Two loci provide the greatest avenue for the consideration of charged particle-induced mutation involving autosomes: the TK1 locus in human cells and the APRT locus in mouse cells. Each locus can provide information on a wide variety of mutational changes, from small intragenic mutations through multilocus dele-tions and extensive tracts of mitotic recombination. In addition, the mouse model can provide a direct measurement of chromosome loss which cannot be accomplished in the human cell system. Another feature of the mouse APRT model is the ability to examine effects for cells exposed in vitro with those obtained for cells exposed in situ. We will provide a comparison of the results obtained for the TK1 locus following 1 GeV/amu Fe ion exposures to the human lymphoid cells with those obtained for the APRT locus for mouse kidney epithelial cells (in vitro or in situ). Substantial conservation of mechanisms is found amongst these three exposure scenarios, with some differences attributable to the specific conditions of exposure. A similar approach will be applied to the consideraiton of proton-induced autosomal mutations in the three model systems. A comparison of the results obtained for Fe ions vs. protons in each case will highlight LET-specificc differences in response. Another cancer development process that is receiving considerable interest is genomic instability. We have examined this process following exposure to sparsely and densely ionizing charged particles in human lymphoid cells and in human epithelial cells. A comparison of the results in these systems can reveal similari-ties and differences as a function of cell type and LET. Last, we will approach the question of the relevance of genomic instability in the context of charged particle mutagenesis. In many models, it has been difficult to link these two processes. We will present data regarding the mechanistic associations between these processes. Taken together, these studies will allow the definition of conserved pathways that are likely to contribute strongly to the cancer risks for astronauts exposed to charged particle radiations. Supported by NASA grant NNJ07HC721 to A. Kronenberg and NASA grant NNX10AC12G to M. Turker.

Novel nuclear-cytoplasmic interaction in wheat (Triticum aestivum) induces vigorous plants

USDA-ARS?s Scientific Manuscript database

Interspecific hybridization can be considered an accelerator of evolution, otherwise a slow process, solely dependent on mutation and recombination. Upon interspecific hybridization, several novel interactions between nuclear and cytoplasmic genomes emerge which provide additional sources of diversi...

New Implications on Genomic Adaptation Derived from the Helicobacter pylori Genome Comparison

PubMed Central

Lara-Ramírez, Edgar Eduardo; Segura-Cabrera, Aldo; Guo, Xianwu; Yu, Gongxin; García-Pérez, Carlos Armando; Rodríguez-Pérez, Mario A.

2011-01-01

Background Helicobacter pylori has a reduced genome and lives in a tough environment for long-term persistence. It evolved with its particular characteristics for biological adaptation. Because several H. pylori genome sequences are available, comparative analysis could help to better understand genomic adaptation of this particular bacterium. Principal Findings We analyzed nine H. pylori genomes with emphasis on microevolution from a different perspective. Inversion was an important factor to shape the genome structure. Illegitimate recombination not only led to genomic inversion but also inverted fragment duplication, both of which contributed to the creation of new genes and gene family, and further, homological recombination contributed to events of inversion. Based on the information of genomic rearrangement, the first genome scaffold structure of H. pylori last common ancestor was produced. The core genome consists of 1186 genes, of which 22 genes could particularly adapt to human stomach niche. H. pylori contains high proportion of pseudogenes whose genesis was principally caused by homopolynucleotide (HPN) mutations. Such mutations are reversible and facilitate the control of gene expression through the change of DNA structure. The reversible mutations and a quasi-panmictic feature could allow such genes or gene fragments frequently transferred within or between populations. Hence, pseudogenes could be a reservoir of adaptation materials and the HPN mutations could be favorable to H. pylori adaptation, leading to HPN accumulation on the genomes, which corresponds to a special feature of Helicobacter species: extremely high HPN composition of genome. Conclusion Our research demonstrated that both genome content and structure of H. pylori have been highly adapted to its particular life style. PMID:21387011

Segmenting the human genome based on states of neutral genetic divergence.

PubMed

Kuruppumullage Don, Prabhani; Ananda, Guruprasad; Chiaromonte, Francesca; Makova, Kateryna D

2013-09-03

Many studies have demonstrated that divergence levels generated by different mutation types vary and covary across the human genome. To improve our still-incomplete understanding of the mechanistic basis of this phenomenon, we analyze several mutation types simultaneously, anchoring their variation to specific regions of the genome. Using hidden Markov models on insertion, deletion, nucleotide substitution, and microsatellite divergence estimates inferred from human-orangutan alignments of neutrally evolving genomic sequences, we segment the human genome into regions corresponding to different divergence states--each uniquely characterized by specific combinations of divergence levels. We then parsed the mutagenic contributions of various biochemical processes associating divergence states with a broad range of genomic landscape features. We find that high divergence states inhabit guanine- and cytosine (GC)-rich, highly recombining subtelomeric regions; low divergence states cover inner parts of autosomes; chromosome X forms its own state with lowest divergence; and a state of elevated microsatellite mutability is interspersed across the genome. These general trends are mirrored in human diversity data from the 1000 Genomes Project, and departures from them highlight the evolutionary history of primate chromosomes. We also find that genes and noncoding functional marks [annotations from the Encyclopedia of DNA Elements (ENCODE)] are concentrated in high divergence states. Our results provide a powerful tool for biomedical data analysis: segmentations can be used to screen personal genome variants--including those associated with cancer and other diseases--and to improve computational predictions of noncoding functional elements.

New Views on Strand Asymmetry in Insect Mitochondrial Genomes

PubMed Central

Wei, Shu-Jun; Shi, Min; Chen, Xue-Xin; Sharkey, Michael J.; van Achterberg, Cornelis; Ye, Gong-Yin; He, Jun-Hua

2010-01-01

Strand asymmetry in nucleotide composition is a remarkable feature of animal mitochondrial genomes. Understanding the mutation processes that shape strand asymmetry is essential for comprehensive knowledge of genome evolution, demographical population history and accurate phylogenetic inference. Previous studies found that the relative contributions of different substitution types to strand asymmetry are associated with replication alone or both replication and transcription. However, the relative contributions of replication and transcription to strand asymmetry remain unclear. Here we conducted a broad survey of strand asymmetry across 120 insect mitochondrial genomes, with special reference to the correlation between the signs of skew values and replication orientation/gene direction. The results show that the sign of GC skew on entire mitochondrial genomes is reversed in all species of three distantly related families of insects, Philopteridae (Phthiraptera), Aleyrodidae (Hemiptera) and Braconidae (Hymenoptera); the replication-related elements in the A+T-rich regions of these species are inverted, confirming that reversal of strand asymmetry (GC skew) was caused by inversion of replication origin; and finally, the sign of GC skew value is associated with replication orientation but not with gene direction, while that of AT skew value varies with gene direction, replication and codon positions used in analyses. These findings show that deaminations during replication and other mutations contribute more than selection on amino acid sequences to strand compositions of G and C, and that the replication process has a stronger affect on A and T content than does transcription. Our results may contribute to genome-wide studies of replication and transcription mechanisms. PMID:20856815

Genome Mutational and Transcriptional Hotspots Are Traps for Duplicated Genes and Sources of Adaptations.

PubMed

Fares, Mario A; Sabater-Muñoz, Beatriz; Toft, Christina

2017-05-01

Gene duplication generates new genetic material, which has been shown to lead to major innovations in unicellular and multicellular organisms. A whole-genome duplication occurred in the ancestor of Saccharomyces yeast species but 92% of duplicates returned to single-copy genes shortly after duplication. The persisting duplicated genes in Saccharomyces led to the origin of major metabolic innovations, which have been the source of the unique biotechnological capabilities in the Baker's yeast Saccharomyces cerevisiae. What factors have determined the fate of duplicated genes remains unknown. Here, we report the first demonstration that the local genome mutation and transcription rates determine the fate of duplicates. We show, for the first time, a preferential location of duplicated genes in the mutational and transcriptional hotspots of S. cerevisiae genome. The mechanism of duplication matters, with whole-genome duplicates exhibiting different preservation trends compared to small-scale duplicates. Genome mutational and transcriptional hotspots are rich in duplicates with large repetitive promoter elements. Saccharomyces cerevisiae shows more tolerance to deleterious mutations in duplicates with repetitive promoter elements, which in turn exhibit higher transcriptional plasticity against environmental perturbations. Our data demonstrate that the genome traps duplicates through the accelerated regulatory and functional divergence of their gene copies providing a source of novel adaptations in yeast. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Genome-Wide Biases in the Rate and Molecular Spectrum of Spontaneous Mutations in Vibrio cholerae and Vibrio fischeri

DTIC Science & Technology

2016-10-15

Mutation rates may be nonuniform because of greater rates of damage, asymmetric nucleotide pools, structural differences affecting polymerase fidelity...strains of these two significant bacterial species. In the MMR-deficient strains, mutation rates were nonuniform among genome regions and varied in pat

Computational Simulation and Analysis of Mutations: Nucleotide Fixation, Allelic Age and Rare Genetic Variations in Population

ERIC Educational Resources Information Center

Qiu, Shuhao

2015-01-01

In order to investigate the complexity of mutations, a computational approach named Genome Evolution by Matrix Algorithms ("GEMA") has been implemented. GEMA models genomic changes, taking into account hundreds of mutations within each individual in a population. By modeling of entire human chromosomes, GEMA precisely mimics real…

Somatic POLE mutations cause an ultramutated giant cell high-grade glioma subtype with better prognosis

PubMed Central

Erson-Omay, E. Zeynep; Çağlayan, Ahmet Okay; Schultz, Nikolaus; Weinhold, Nils; Omay, S. Bülent; Özduman, Koray; Köksal, Yavuz; Li, Jie; Serin Harmancı, Akdes; Clark, Victoria; Carrión-Grant, Geneive; Baranoski, Jacob; Çağlar, Caner; Barak, Tanyeri; Coşkun, Süleyman; Baran, Burçin; Köse, Doğan; Sun, Jia; Bakırcıoğlu, Mehmet; Moliterno Günel, Jennifer; Pamir, M. Necmettin; Mishra-Gorur, Ketu; Bilguvar, Kaya; Yasuno, Katsuhito; Vortmeyer, Alexander; Huttner, Anita J.; Sander, Chris; Günel, Murat

2015-01-01

Background Malignant high-grade gliomas (HGGs), including the most aggressive form, glioblastoma multiforme, show significant clinical and genomic heterogeneity. Despite recent advances, the overall survival of HGGs and their response to treatment remain poor. In order to gain further insight into disease pathophysiology by correlating genomic landscape with clinical behavior, thereby identifying distinct HGG molecular subgroups associated with improved prognosis, we performed a comprehensive genomic analysis. Methods We analyzed and compared 720 exome-sequenced gliomas (136 from Yale, 584 from The Cancer Genome Atlas) based on their genomic, histological, and clinical features. Results We identified a subgroup of HGGs (6 total, 4 adults and 2 children) that harbored a statistically significantly increased number of somatic mutations (mean = 9257.3 vs 76.2, P = .002). All of these “ultramutated” tumors harbored somatic mutations in the exonuclease domain of the polymerase epsilon gene (POLE), displaying a distinctive genetic profile, characterized by genomic stability and increased C-to-A transversions. Histologically, they all harbored multinucleated giant or bizarre cells, some with predominant infiltrating immune cells. One adult and both pediatric patients carried homozygous germline mutations in the mutS homolog 6 (MSH6) gene. In adults, POLE mutations were observed in patients younger than 40 years and were associated with a longer progression-free survival. Conclusions We identified a genomically, histologically, and clinically distinct subgroup of HGGs that harbored somatic POLE mutations and carried an improved prognosis. Identification of distinctive molecular and pathological HGG phenotypes has implications not only for improved classification but also for potential targeted treatments. PMID:25740784

Genomic Context Analysis of de Novo STXBP1 Mutations Identifies Evidence of Splice Site DNA-Motif Associated Hotspots.

PubMed

Uddin, Mohammed; Woodbury-Smith, Marc; Chan, Ada J S; Albanna, Ammar; Minassian, Berge; Boelman, Cyrus; Scherer, Stephen W

2018-03-28

Mutations within STXBP1 have been associated with a range of neurodevelopmental disorders implicating the pleotropic impact of this gene. Although the frequency of de novo mutations within STXBP1 for selective cohorts with early onset epileptic encephalopathy is more than 1%, there is no evidence for a hotspot within the gene. In this study, we analyzed the genomic context of de novo STXBP1 mutations to examine whether certain motifs indicated a greater risk of mutation. Through a comprehensive context analysis of 136 de novo /rare mutation (SNV/Indels) sites in this gene, strikingly 26.92% of all SNV mutations occurred within 5bp upstream or downstream of a 'GTA' motif ( P < 0.0005). This implies a genomic context modulated mutagenesis. Moreover, 51.85% (14 out of 27) of the 'GTA' mutations are splicing compared to 14.70% (20 out of 136) of all reported mutations within STXBP1 We also noted that 11 of these 14 'GTA' associated mutations are de novo in origin. Our analysis provides strong evidence of DNA motif modulated mutagenesis for STXBP1 de novo splicing mutations. Copyright © 2018 Uddin et al.

The genomic basis of adaptation to the fitness cost of rifampicin resistance in Pseudomonas aeruginosa

PubMed Central

Toll-Riera, Macarena; Heilbron, Karl

2016-01-01

Antibiotic resistance carries a fitness cost that must be overcome in order for resistance to persist over the long term. Compensatory mutations that recover the functional defects associated with resistance mutations have been argued to play a key role in overcoming the cost of resistance, but compensatory mutations are expected to be rare relative to generally beneficial mutations that increase fitness, irrespective of antibiotic resistance. Given this asymmetry, population genetics theory predicts that populations should adapt by compensatory mutations when the cost of resistance is large, whereas generally beneficial mutations should drive adaptation when the cost of resistance is small. We tested this prediction by determining the genomic mechanisms underpinning adaptation to antibiotic-free conditions in populations of the pathogenic bacterium Pseudomonas aeruginosa that carry costly antibiotic resistance mutations. Whole-genome sequencing revealed that populations founded by high-cost rifampicin-resistant mutants adapted via compensatory mutations in three genes of the RNA polymerase core enzyme, whereas populations founded by low-cost mutants adapted by generally beneficial mutations, predominantly in the quorum-sensing transcriptional regulator gene lasR. Even though the importance of compensatory evolution in maintaining resistance has been widely recognized, our study shows that the roles of general adaptation in maintaining resistance should not be underestimated and highlights the need to understand how selection at other sites in the genome influences the dynamics of resistance alleles in clinical settings. PMID:26763710

How life changes itself: the Read-Write (RW) genome.

PubMed

Shapiro, James A

2013-09-01

The genome has traditionally been treated as a Read-Only Memory (ROM) subject to change by copying errors and accidents. In this review, I propose that we need to change that perspective and understand the genome as an intricately formatted Read-Write (RW) data storage system constantly subject to cellular modifications and inscriptions. Cells operate under changing conditions and are continually modifying themselves by genome inscriptions. These inscriptions occur over three distinct time-scales (cell reproduction, multicellular development and evolutionary change) and involve a variety of different processes at each time scale (forming nucleoprotein complexes, epigenetic formatting and changes in DNA sequence structure). Research dating back to the 1930s has shown that genetic change is the result of cell-mediated processes, not simply accidents or damage to the DNA. This cell-active view of genome change applies to all scales of DNA sequence variation, from point mutations to large-scale genome rearrangements and whole genome duplications (WGDs). This conceptual change to active cell inscriptions controlling RW genome functions has profound implications for all areas of the life sciences. © 2013 Elsevier B.V. All rights reserved.

Perspective on Oncogenic Processes at the End of the Beginning of Cancer Genomics.

PubMed

Ding, Li; Bailey, Matthew H; Porta-Pardo, Eduard; Thorsson, Vesteinn; Colaprico, Antonio; Bertrand, Denis; Gibbs, David L; Weerasinghe, Amila; Huang, Kuan-Lin; Tokheim, Collin; Cortés-Ciriano, Isidro; Jayasinghe, Reyka; Chen, Feng; Yu, Lihua; Sun, Sam; Olsen, Catharina; Kim, Jaegil; Taylor, Alison M; Cherniack, Andrew D; Akbani, Rehan; Suphavilai, Chayaporn; Nagarajan, Niranjan; Stuart, Joshua M; Mills, Gordon B; Wyczalkowski, Matthew A; Vincent, Benjamin G; Hutter, Carolyn M; Zenklusen, Jean Claude; Hoadley, Katherine A; Wendl, Michael C; Shmulevich, Llya; Lazar, Alexander J; Wheeler, David A; Getz, Gad

2018-04-05

The Cancer Genome Atlas (TCGA) has catalyzed systematic characterization of diverse genomic alterations underlying human cancers. At this historic junction marking the completion of genomic characterization of over 11,000 tumors from 33 cancer types, we present our current understanding of the molecular processes governing oncogenesis. We illustrate our insights into cancer through synthesis of the findings of the TCGA PanCancer Atlas project on three facets of oncogenesis: (1) somatic driver mutations, germline pathogenic variants, and their interactions in the tumor; (2) the influence of the tumor genome and epigenome on transcriptome and proteome; and (3) the relationship between tumor and the microenvironment, including implications for drugs targeting driver events and immunotherapies. These results will anchor future characterization of rare and common tumor types, primary and relapsed tumors, and cancers across ancestry groups and will guide the deployment of clinical genomic sequencing. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Minimal Contribution of APOBEC3-Induced G-to-A Hypermutation to HIV-1 Recombination and Genetic Variation

PubMed Central

Nikolaitchik, Olga A.; Burdick, Ryan C.; Gorelick, Robert J.; Keele, Brandon F.; Hu, Wei-Shau; Pathak, Vinay K.

2016-01-01

Although the predominant effect of host restriction APOBEC3 proteins on HIV-1 infection is to block viral replication, they might inadvertently increase retroviral genetic variation by inducing G-to-A hypermutation. Numerous studies have disagreed on the contribution of hypermutation to viral genetic diversity and evolution. Confounding factors contributing to the debate include the extent of lethal (stop codon) and sublethal hypermutation induced by different APOBEC3 proteins, the inability to distinguish between G-to-A mutations induced by APOBEC3 proteins and error-prone viral replication, the potential impact of hypermutation on the frequency of retroviral recombination, and the extent to which viral recombination occurs in vivo, which can reassort mutations in hypermutated genomes. Here, we determined the effects of hypermutation on the HIV-1 recombination rate and its contribution to genetic variation through recombination to generate progeny genomes containing portions of hypermutated genomes without lethal mutations. We found that hypermutation did not significantly affect the rate of recombination, and recombination between hypermutated and wild-type genomes only increased the viral mutation rate by 3.9 × 10−5 mutations/bp/replication cycle in heterozygous virions, which is similar to the HIV-1 mutation rate. Since copackaging of hypermutated and wild-type genomes occurs very rarely in vivo, recombination between hypermutated and wild-type genomes does not significantly contribute to the genetic variation of replicating HIV-1. We also analyzed previously reported hypermutated sequences from infected patients and determined that the frequency of sublethal mutagenesis for A3G and A3F is negligible (4 × 10−21 and1 × 10−11, respectively) and its contribution to viral mutations is far below mutations generated during error-prone reverse transcription. Taken together, we conclude that the contribution of APOBEC3-induced hypermutation to HIV-1 genetic variation is substantially lower than that from mutations during error-prone replication. PMID:27186986

Minimal Contribution of APOBEC3-Induced G-to-A Hypermutation to HIV-1 Recombination and Genetic Variation.

PubMed

Delviks-Frankenberry, Krista A; Nikolaitchik, Olga A; Burdick, Ryan C; Gorelick, Robert J; Keele, Brandon F; Hu, Wei-Shau; Pathak, Vinay K

2016-05-01

Although the predominant effect of host restriction APOBEC3 proteins on HIV-1 infection is to block viral replication, they might inadvertently increase retroviral genetic variation by inducing G-to-A hypermutation. Numerous studies have disagreed on the contribution of hypermutation to viral genetic diversity and evolution. Confounding factors contributing to the debate include the extent of lethal (stop codon) and sublethal hypermutation induced by different APOBEC3 proteins, the inability to distinguish between G-to-A mutations induced by APOBEC3 proteins and error-prone viral replication, the potential impact of hypermutation on the frequency of retroviral recombination, and the extent to which viral recombination occurs in vivo, which can reassort mutations in hypermutated genomes. Here, we determined the effects of hypermutation on the HIV-1 recombination rate and its contribution to genetic variation through recombination to generate progeny genomes containing portions of hypermutated genomes without lethal mutations. We found that hypermutation did not significantly affect the rate of recombination, and recombination between hypermutated and wild-type genomes only increased the viral mutation rate by 3.9 × 10-5 mutations/bp/replication cycle in heterozygous virions, which is similar to the HIV-1 mutation rate. Since copackaging of hypermutated and wild-type genomes occurs very rarely in vivo, recombination between hypermutated and wild-type genomes does not significantly contribute to the genetic variation of replicating HIV-1. We also analyzed previously reported hypermutated sequences from infected patients and determined that the frequency of sublethal mutagenesis for A3G and A3F is negligible (4 × 10-21 and1 × 10-11, respectively) and its contribution to viral mutations is far below mutations generated during error-prone reverse transcription. Taken together, we conclude that the contribution of APOBEC3-induced hypermutation to HIV-1 genetic variation is substantially lower than that from mutations during error-prone replication.

Single genome retrieval of context-dependent variability in mutation rates for human germline.

PubMed

Sahakyan, Aleksandr B; Balasubramanian, Shankar

2017-01-13

Accurate knowledge of the core components of substitution rates is of vital importance to understand genome evolution and dynamics. By performing a single-genome and direct analysis of 39,894 retrotransposon remnants, we reveal sequence context-dependent germline nucleotide substitution rates for the human genome. The rates are characterised through rate constants in a time-domain, and are made available through a dedicated program (Trek) and a stand-alone database. Due to the nature of the method design and the imposed stringency criteria, we expect our rate constants to be good estimates for the rates of spontaneous mutations. Benefiting from such data, we study the short-range nucleotide (up to 7-mer) organisation and the germline basal substitution propensity (BSP) profile of the human genome; characterise novel, CpG-independent, substitution prone and resistant motifs; confirm a decreased tendency of moieties with low BSP to undergo somatic mutations in a number of cancer types; and, produce a Trek-based estimate of the overall mutation rate in human. The extended set of rate constants we report may enrich our resources and help advance our understanding of genome dynamics and evolution, with possible implications for the role of spontaneous mutations in the emergence of pathological genotypes and neutral evolution of proteomes.

MUFFINN: cancer gene discovery via network analysis of somatic mutation data.

PubMed

Cho, Ara; Shim, Jung Eun; Kim, Eiru; Supek, Fran; Lehner, Ben; Lee, Insuk

2016-06-23

A major challenge for distinguishing cancer-causing driver mutations from inconsequential passenger mutations is the long-tail of infrequently mutated genes in cancer genomes. Here, we present and evaluate a method for prioritizing cancer genes accounting not only for mutations in individual genes but also in their neighbors in functional networks, MUFFINN (MUtations For Functional Impact on Network Neighbors). This pathway-centric method shows high sensitivity compared with gene-centric analyses of mutation data. Notably, only a marginal decrease in performance is observed when using 10 % of TCGA patient samples, suggesting the method may potentiate cancer genome projects with small patient populations.

AB036. Analysis of human mitochondrial genome mutations of Vietnamese patients tentatively diagnosed with encephalomyopathy

PubMed Central

Nghia, Phan Tuan; Thai, Trinh Hong; Hue, Truong Thi; Van Minh, Nguyen; Khanh, Phung Bao; Hiep, Tran Duc; Anh, Tran Kieu; Loan, Nguyen Thi Hong; Van, Nguyen Thi Hong; Anh, Pham Van; Hung, Cao Vu; Anh, Le Ngoc

2015-01-01

Human mitochondrial genome consists of 16,569 bp, and replicates independently from the nuclear genome. Mutations in mitochondrial genome are usually causative factors of various metabolic disorders, especially those of encephalomyopathy. DNA analysis is the most reliable method for detection of mitochondrial genome mutations, and accordingly an excellent diagnostic tool for mitochondrial mutation-related diseases. In this study, 19 different mitochondrial genome mutations including A3243G, A3251G, T3271C and T3291C (MELAS); A8344G, T8356C and G8363A (MERRF); G3460A, G11778A and T14484C (LHON); T8993G/C and T9176G (Leigh); A1555G (deafness) and A4225G, G4298A, T10010C, T14727C, T14728C, T14709C (encephalomyopathy in general) were analyzed using PCR-RFLP in combination with DNA sequencing. In addition, a real-time PCR method using locked nucleic acid (LNA) Taqman probe was set up for heteroplasmy determination. Screening of 283 tentatively diagnosed encephalomyopathy patients revealed 7 cases of A3243G, 1 case of G11778A, 1 case of A1555G, 1 case of A4225G, 1 case G4298A, and 1 case of 6 bp (ACTCCT/CTCCTA) deletion. Using the LNA Taqman probe real-time PCR, the heteroplasmy of some point mutations was determined and the results support a potential relationship between heteroplasmy level and severity of the disease.

Discovery of Genomic Breakpoints Affecting Breast Cancer Progression and Prognosis

DTIC Science & Technology

2010-10-01

mutations compared to those detected by the 5Kbp method alone. Fosmid diTag method also reveals much higher proportion of gene fusions and truncations...observed highly similar structural mutational spectra affecting different sets of genes , pointing to similar histories of genomic instability against... mutations have been identified in non-BRCA1/2 multiethnic breast cancer cases (45,46), no truncating mutation of the RAP80 gene in breast cancer has

Identification of Variant-Specific Functions of PIK3CA by Rapid Phenotyping of Rare Mutations | Office of Cancer Genomics

Cancer.gov

Large-scale sequencing efforts are uncovering the complexity of cancer genomes, which are composed of causal "driver" mutations that promote tumor progression along with many more pathologically neutral "passenger" events. The majority of mutations, both in known cancer drivers and uncharacterized genes, are generally of low occurrence, highlighting the need to functionally annotate the long tail of infrequent mutations present in heterogeneous cancers.

Timing, rates and spectra of human germline mutation.

PubMed

Rahbari, Raheleh; Wuster, Arthur; Lindsay, Sarah J; Hardwick, Robert J; Alexandrov, Ludmil B; Turki, Saeed Al; Dominiczak, Anna; Morris, Andrew; Porteous, David; Smith, Blair; Stratton, Michael R; Hurles, Matthew E

2016-02-01

Germline mutations are a driving force behind genome evolution and genetic disease. We investigated genome-wide mutation rates and spectra in multi-sibling families. The mutation rate increased with paternal age in all families, but the number of additional mutations per year differed by more than twofold between families. Meta-analysis of 6,570 mutations showed that germline methylation influences mutation rates. In contrast to somatic mutations, we found remarkable consistency in germline mutation spectra between the sexes and at different paternal ages. In parental germ line, 3.8% of mutations were mosaic, resulting in 1.3% of mutations being shared by siblings. The number of these shared mutations varied significantly between families. Our data suggest that the mutation rate per cell division is higher during both early embryogenesis and differentiation of primordial germ cells but is reduced substantially during post-pubertal spermatogenesis. These findings have important consequences for the recurrence risks of disorders caused by de novo mutations.

Innate immunity and the new forward genetics.

PubMed

Beutler, Bruce

2016-12-01

As it is a hard-wired system for responses to microbes, innate immunity is particularly susceptible to classical genetic analysis. Mutations led the way to the discovery of many of the molecular elements of innate immune sensing and signaling pathways. In turn, the need for a faster way to find the molecular causes of mutation-induced phenotypes triggered a huge transformation in forward genetics. During the 1980s and 1990s, many heritable phenotypes were ascribed to mutations through positional cloning. In mice, this required three steps. First, a genetic mapping step was used to show that a given phenotype emanated from a circumscribed region of the genome. Second, a physical mapping step was undertaken, in which all of the region was cloned and its gene content determined. Finally, a concerted search for the mutation was performed. Such projects usually lasted for several years, but could produce breakthroughs in our understanding of biological processes. Publication of the annotated mouse genome sequence in 2002 made physical mapping unnecessary. More recently we devised a new technology for automated genetic mapping, which eliminated both genetic mapping and the search for mutations among candidate genes. The cause of phenotype can now be determined instantaneously. We have created more than 100,000 coding/splicing mutations. And by screening for defects of innate and adaptive immunity we have discovered many "new" proteins needed for innate immune function. Copyright © 2016 Elsevier Ltd. All rights reserved.

Innate immunity and the new forward genetics

PubMed Central

Beutler, Bruce

2016-01-01

As it is a hard-wired system for responses to microbes, innate immunity is particularly susceptible to classical genetic analysis. Mutations led the way to the discovery of many of the molecular elements of innate immune sensing and signaling pathways. In turn, the need for a faster way to find the molecular causes of mutation-induced phenotypes triggered a huge transformation in forward genetics. During the 1980s and 1990s, many heritable phenotypes were ascribed to mutations through positional cloning. In mice, this required three steps. First, a genetic mapping step was used to show that a given phenotype emanated from a circumscribed region of the genome. Second, a physical mapping step was undertaken, in which all of the region was cloned and its gene content determined. Finally, a concerted search for the mutation was performed. Such projects usually lasted for several years, but could produce breakthroughs in our understanding of biological processes. Publication of the annotated mouse genome sequence in 2002 made physical mapping unnecessary. More recently we devised a new technology for automated genetic mapping, which eliminated both genetic mapping and the search for mutations among candidate genes. The cause of phenotype can now be determined instantaneously. We have created more than 100,000 coding/splicing mutations. And by screening for defects of innate and adaptive immunity we have discovered many “new” proteins needed for innate immune function. PMID:27890263

TP53 Mutational Status Is a Potential Marker for Risk Stratification in Wilms Tumour with Diffuse Anaplasia

PubMed Central

Chagtai, Tasnim; Popov, Sergey D.; Sebire, Neil J.; Vujanic, Gordan; Perlman, Elizabeth; Anderson, James R.; Grundy, Paul; Dome, Jeffrey S.; Pritchard-Jones, Kathy

2014-01-01

Purpose The presence of diffuse anaplasia in Wilms tumours (DAWT) is associated with TP53 mutations and poor outcome. As patients receive intensified treatment, we sought to identify whether TP53 mutational status confers additional prognostic information. Patients and Methods We studied 40 patients with DAWT with anaplasia in the tissue from which DNA was extracted and analysed for TP53 mutations and 17p loss. The majority of cases were profiled by copy number (n = 32) and gene expression (n = 36) arrays. TP53 mutational status was correlated with patient event-free and overall survival, genomic copy number instability and gene expression profiling. Results From the 40 cases, 22 (55%) had TP53 mutations (2 detected only after deep-sequencing), 20 of which also had 17p loss (91%); 18 (45%) cases had no detectable mutation but three had 17p loss. Tumours with TP53 mutations and/or 17p loss (n = 25) had an increased risk of recurrence as a first event (p = 0.03, hazard ratio (HR), 3.89; 95% confidence interval (CI), 1.26–16.0) and death (p = 0.04, HR, 4.95; 95% CI, 1.36–31.7) compared to tumours lacking TP53 abnormalities. DAWT carrying TP53 mutations showed increased copy number alterations compared to those with wild-type, suggesting a more unstable genome (p = 0.03). These tumours showed deregulation of genes associated with cell cycle and DNA repair biological processes. Conclusion This study provides evidence that TP53 mutational analysis improves risk stratification in DAWT. This requires validation in an independent cohort before clinical use as a biomarker. PMID:25313908

TP53 mutational status is a potential marker for risk stratification in Wilms tumour with diffuse anaplasia.

PubMed

Maschietto, Mariana; Williams, Richard D; Chagtai, Tasnim; Popov, Sergey D; Sebire, Neil J; Vujanic, Gordan; Perlman, Elizabeth; Anderson, James R; Grundy, Paul; Dome, Jeffrey S; Pritchard-Jones, Kathy

2014-01-01

The presence of diffuse anaplasia in Wilms tumours (DAWT) is associated with TP53 mutations and poor outcome. As patients receive intensified treatment, we sought to identify whether TP53 mutational status confers additional prognostic information. We studied 40 patients with DAWT with anaplasia in the tissue from which DNA was extracted and analysed for TP53 mutations and 17p loss. The majority of cases were profiled by copy number (n = 32) and gene expression (n = 36) arrays. TP53 mutational status was correlated with patient event-free and overall survival, genomic copy number instability and gene expression profiling. From the 40 cases, 22 (55%) had TP53 mutations (2 detected only after deep-sequencing), 20 of which also had 17p loss (91%); 18 (45%) cases had no detectable mutation but three had 17p loss. Tumours with TP53 mutations and/or 17p loss (n = 25) had an increased risk of recurrence as a first event (p = 0.03, hazard ratio (HR), 3.89; 95% confidence interval (CI), 1.26-16.0) and death (p = 0.04, HR, 4.95; 95% CI, 1.36-31.7) compared to tumours lacking TP53 abnormalities. DAWT carrying TP53 mutations showed increased copy number alterations compared to those with wild-type, suggesting a more unstable genome (p = 0.03). These tumours showed deregulation of genes associated with cell cycle and DNA repair biological processes. This study provides evidence that TP53 mutational analysis improves risk stratification in DAWT. This requires validation in an independent cohort before clinical use as a biomarker.

Harnessing CRISPR-Cas systems for bacterial genome editing.

PubMed

Selle, Kurt; Barrangou, Rodolphe

2015-04-01

Manipulation of genomic sequences facilitates the identification and characterization of key genetic determinants in the investigation of biological processes. Genome editing via clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) constitutes a next-generation method for programmable and high-throughput functional genomics. CRISPR-Cas systems are readily reprogrammed to induce sequence-specific DNA breaks at target loci, resulting in fixed mutations via host-dependent DNA repair mechanisms. Although bacterial genome editing is a relatively unexplored and underrepresented application of CRISPR-Cas systems, recent studies provide valuable insights for the widespread future implementation of this technology. This review summarizes recent progress in bacterial genome editing and identifies fundamental genetic and phenotypic outcomes of CRISPR targeting in bacteria, in the context of tool development, genome homeostasis, and DNA repair. Copyright © 2015 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Szovenyi, Peter; Shaw, Jon; Yang, Xiaohan

In diploid organisms, selfing reduces the efficiency of selection in removing deleterious mutations from a population. This need not be the case for all organisms. Some plants, for example, undergo an extreme form of selfing known as intragametophytic selfing, which immediately exposes all recessive deleterious mutations in a parental genome to selective purging. Here we ask how effectively deleterious mutations are removed from such plants. Specifically, we study the extent to which deleterious mutations accumulate in a predominantly selfing and a predominantly outcrossing pair of moss species, using genome-wide transcriptome data. We find that the selfing species purge significantly moremore » non-synonymous mutations, as well as a greater proportion of radical amino acid changes which alter physicochemical properties of amino acids. Moreover, their purging of deleterious mutation is especially strong in conserved regions of protein-coding genes. Our observations show that selfing need not impede but can even accelerate the removal of deleterious mutations, and do so on a genome-wide scale.« less

Whole-genome landscape of pancreatic neuroendocrine tumours.

PubMed

Scarpa, Aldo; Chang, David K; Nones, Katia; Corbo, Vincenzo; Patch, Ann-Marie; Bailey, Peter; Lawlor, Rita T; Johns, Amber L; Miller, David K; Mafficini, Andrea; Rusev, Borislav; Scardoni, Maria; Antonello, Davide; Barbi, Stefano; Sikora, Katarzyna O; Cingarlini, Sara; Vicentini, Caterina; McKay, Skye; Quinn, Michael C J; Bruxner, Timothy J C; Christ, Angelika N; Harliwong, Ivon; Idrisoglu, Senel; McLean, Suzanne; Nourse, Craig; Nourbakhsh, Ehsan; Wilson, Peter J; Anderson, Matthew J; Fink, J Lynn; Newell, Felicity; Waddell, Nick; Holmes, Oliver; Kazakoff, Stephen H; Leonard, Conrad; Wood, Scott; Xu, Qinying; Nagaraj, Shivashankar Hiriyur; Amato, Eliana; Dalai, Irene; Bersani, Samantha; Cataldo, Ivana; Dei Tos, Angelo P; Capelli, Paola; Davì, Maria Vittoria; Landoni, Luca; Malpaga, Anna; Miotto, Marco; Whitehall, Vicki L J; Leggett, Barbara A; Harris, Janelle L; Harris, Jonathan; Jones, Marc D; Humphris, Jeremy; Chantrill, Lorraine A; Chin, Venessa; Nagrial, Adnan M; Pajic, Marina; Scarlett, Christopher J; Pinho, Andreia; Rooman, Ilse; Toon, Christopher; Wu, Jianmin; Pinese, Mark; Cowley, Mark; Barbour, Andrew; Mawson, Amanda; Humphrey, Emily S; Colvin, Emily K; Chou, Angela; Lovell, Jessica A; Jamieson, Nigel B; Duthie, Fraser; Gingras, Marie-Claude; Fisher, William E; Dagg, Rebecca A; Lau, Loretta M S; Lee, Michael; Pickett, Hilda A; Reddel, Roger R; Samra, Jaswinder S; Kench, James G; Merrett, Neil D; Epari, Krishna; Nguyen, Nam Q; Zeps, Nikolajs; Falconi, Massimo; Simbolo, Michele; Butturini, Giovanni; Van Buren, George; Partelli, Stefano; Fassan, Matteo; Khanna, Kum Kum; Gill, Anthony J; Wheeler, David A; Gibbs, Richard A; Musgrove, Elizabeth A; Bassi, Claudio; Tortora, Giampaolo; Pederzoli, Paolo; Pearson, John V; Waddell, Nicola; Biankin, Andrew V; Grimmond, Sean M

2017-03-02

The diagnosis of pancreatic neuroendocrine tumours (PanNETs) is increasing owing to more sensitive detection methods, and this increase is creating challenges for clinical management. We performed whole-genome sequencing of 102 primary PanNETs and defined the genomic events that characterize their pathogenesis. Here we describe the mutational signatures they harbour, including a deficiency in G:C > T:A base excision repair due to inactivation of MUTYH, which encodes a DNA glycosylase. Clinically sporadic PanNETs contain a larger-than-expected proportion of germline mutations, including previously unreported mutations in the DNA repair genes MUTYH, CHEK2 and BRCA2. Together with mutations in MEN1 and VHL, these mutations occur in 17% of patients. Somatic mutations, including point mutations and gene fusions, were commonly found in genes involved in four main pathways: chromatin remodelling, DNA damage repair, activation of mTOR signalling (including previously undescribed EWSR1 gene fusions), and telomere maintenance. In addition, our gene expression analyses identified a subgroup of tumours associated with hypoxia and HIF signalling.

Draft genome sequence of an extensively drug-resistant Pseudomonas aeruginosa isolate belonging to ST644 isolated from a footpad infection in a Magellanic penguin (Spheniscus magellanicus).

PubMed

Sellera, Fábio P; Fernandes, Miriam R; Moura, Quézia; Souza, Tiago A; Nascimento, Cristiane L; Cerdeira, Louise; Lincopan, Nilton

2018-03-01

The incidence of multidrug-resistant bacteria in wildlife animals has been investigated to improve our knowledge of the spread of clinically relevant antimicrobial resistance genes. The aim of this study was to report the first draft genome sequence of an extensively drug-resistant (XDR) Pseudomonas aeruginosa ST644 isolate recovered from a Magellanic penguin with a footpad infection (bumblefoot) undergoing rehabilitation process. The genome was sequenced on an Illumina NextSeq ® platform using 150-bp paired-end reads. De novo genome assembly was performed using Velvet v.1.2.10, and the whole genome sequence was evaluated using bioinformatics approaches from the Center of Genomic Epidemiology, whereas an in-house method (mapping of raw whole genome sequence reads) was used to identify chromosomal point mutations. The genome size was calculated at 6436450bp, with 6357 protein-coding sequences and the presence of genes conferring resistance to aminoglycosides, β-lactams, phenicols, sulphonamides, tetracyclines, quinolones and fosfomycin; in addition, mutations in the genes gyrA (Thr83Ile), parC (Ser87Leu), phoQ (Arg61His) and pmrB (Tyr345His), conferring resistance to quinolones and polymyxins, respectively, were confirmed. This draft genome sequence can provide useful information for comparative genomic analysis regarding the dissemination of clinically significant antibiotic resistance genes and XDR bacterial species at the human-animal interface. Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

Abnormal RNA splicing and genomic instability after induction of DNMT3A mutations by CRISPR/Cas9 gene editing.

PubMed

Banaszak, Lauren G; Giudice, Valentina; Zhao, Xin; Wu, Zhijie; Gao, Shouguo; Hosokawa, Kohei; Keyvanfar, Keyvan; Townsley, Danielle M; Gutierrez-Rodrigues, Fernanda; Fernandez Ibanez, Maria Del Pilar; Kajigaya, Sachiko; Young, Neal S

2018-03-01

DNA methyltransferase 3A (DNMT3A) mediates de novo DNA methylation. Mutations in DNMT3A are associated with hematological malignancies, most frequently acute myeloid leukemia. DNMT3A mutations are hypothesized to establish a pre-leukemic state, rendering cells vulnerable to secondary oncogenic mutations and malignant transformation. However, the mechanisms by which DNMT3A mutations contribute to leukemogenesis are not well-defined. Here, we successfully created four DNMT3A-mutated K562 cell lines with frameshift mutations resulting in truncated DNMT3A proteins. DNMT3A-mutated cell lines exhibited significantly impaired growth and increased apoptotic activity compared to wild-type (WT) cells. Consistent with previous studies, DNMT3A-mutated cells displayed impaired differentiation capacity. RNA-seq was used to compare transcriptomes of DNMT3A-mutated and WT cells; DNMT3A ablation resulted in downregulation of genes involved in spliceosome function, causing dysfunction of RNA splicing. Unexpectedly, we observed DNMT3A-mutated cells to exhibit marked genomic instability and an impaired DNA damage response compared to WT. CRISPR/Cas9-mediated DNMT3A-mutated K562 cells may be used to model effects of DNMT3A mutations in human cells. Our findings implicate aberrant splicing and induction of genomic instability as potential mechanisms by which DNMT3A mutations might predispose to malignancy. Published by Elsevier Inc.

Adaptation of Organisms by Resonance of RNA Transcription with the Cellular Redox Cycle

NASA Technical Reports Server (NTRS)

Stolc, Viktor

2012-01-01

Sequence variation in organisms differs across the genome and the majority of mutations are caused by oxidation, yet its origin is not fully understood. It has also been shown that the reduction-oxidation reaction cycle is the fundamental biochemical cycle that coordinates the timing of all biochemical processes in that cell, including energy production, DNA replication, and RNA transcription. It is shown that the temporal resonance of transcriptome biosynthesis with the oscillating binary state of the reduction-oxidation reaction cycle serves as a basis for non-random sequence variation at specific genome-wide coordinates that change faster than by accumulation of chance mutations. This work demonstrates evidence for a universal, persistent and iterative feedback mechanism between the environment and heredity, whereby acquired variation between cell divisions can outweigh inherited variation.

Tissue-specific mutation accumulation in human adult stem cells during life

NASA Astrophysics Data System (ADS)

Blokzijl, Francis; de Ligt, Joep; Jager, Myrthe; Sasselli, Valentina; Roerink, Sophie; Sasaki, Nobuo; Huch, Meritxell; Boymans, Sander; Kuijk, Ewart; Prins, Pjotr; Nijman, Isaac J.; Martincorena, Inigo; Mokry, Michal; Wiegerinck, Caroline L.; Middendorp, Sabine; Sato, Toshiro; Schwank, Gerald; Nieuwenhuis, Edward E. S.; Verstegen, Monique M. A.; van der Laan, Luc J. W.; de Jonge, Jeroen; Ijzermans, Jan N. M.; Vries, Robert G.; van de Wetering, Marc; Stratton, Michael R.; Clevers, Hans; Cuppen, Edwin; van Boxtel, Ruben

2016-10-01

The gradual accumulation of genetic mutations in human adult stem cells (ASCs) during life is associated with various age-related diseases, including cancer. Extreme variation in cancer risk across tissues was recently proposed to depend on the lifetime number of ASC divisions, owing to unavoidable random mutations that arise during DNA replication. However, the rates and patterns of mutations in normal ASCs remain unknown. Here we determine genome-wide mutation patterns in ASCs of the small intestine, colon and liver of human donors with ages ranging from 3 to 87 years by sequencing clonal organoid cultures derived from primary multipotent cells. Our results show that mutations accumulate steadily over time in all of the assessed tissue types, at a rate of approximately 40 novel mutations per year, despite the large variation in cancer incidence among these tissues. Liver ASCs, however, have different mutation spectra compared to those of the colon and small intestine. Mutational signature analysis reveals that this difference can be attributed to spontaneous deamination of methylated cytosine residues in the colon and small intestine, probably reflecting their high ASC division rate. In liver, a signature with an as-yet-unknown underlying mechanism is predominant. Mutation spectra of driver genes in cancer show high similarity to the tissue-specific ASC mutation spectra, suggesting that intrinsic mutational processes in ASCs can initiate tumorigenesis. Notably, the inter-individual variation in mutation rate and spectra are low, suggesting tissue-specific activity of common mutational processes throughout life.

Comparative genome analysis of a thermotolerant Escherichia coli obtained by Genome Replication Engineering Assisted Continuous Evolution (GREACE) and its parent strain provides new understanding of microbial heat tolerance.

PubMed

Luan, Guodong; Bao, Guanhui; Lin, Zhao; Li, Yang; Chen, Zugen; Li, Yin; Cai, Zhen

2015-12-25

Heat tolerance of microbes is of great importance for efficient biorefinery and bioconversion. However, engineering and understanding of microbial heat tolerance are difficult and insufficient because it is a complex physiological trait which probably correlates with all gene functions, genetic regulations, and cellular metabolisms and activities. In this work, a novel strain engineering approach named Genome Replication Engineering Assisted Continuous Evolution (GREACE) was employed to improve the heat tolerance of Escherichia coli. When the E. coli strain carrying a mutator was cultivated under gradually increasing temperature, genome-wide mutations were continuously generated during genome replication and the mutated strains with improved thermotolerance were autonomously selected. A thermotolerant strain HR50 capable of growing at 50°C on LB agar plate was obtained within two months, demonstrating the efficiency of GREACE in improving such a complex physiological trait. To understand the improved heat tolerance, genomes of HR50 and its wildtype strain DH5α were sequenced. Evenly distributed 361 mutations covering all mutation types were found in HR50. Closed material transportations, loose genome conformation, and possibly altered cell wall structure and transcription pattern were the main differences of HR50 compared with DH5α, which were speculated to be responsible for the improved heat tolerance. This work not only expanding our understanding of microbial heat tolerance, but also emphasizing that the in vivo continuous genome mutagenesis method, GREACE, is efficient in improving microbial complex physiological trait. Copyright © 2015 Elsevier B.V. All rights reserved.

Exploring the common molecular basis for the universal DNA mutation bias: Revival of Loewdin mutation model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu, Liang-Yu; Center for Bioinformatics, Huazhong Agricultural University, Wuhan 430070; Wang, Guang-Zhong

2011-06-10

Highlights: {yields} There exists a universal G:C {yields} A:T mutation bias in three domains of life. {yields} This universal mutation bias has not been sufficiently explained. {yields} A DNA mutation model proposed by Loewdin 40 years ago offers a common explanation. -- Abstract: Recently, numerous genome analyses revealed the existence of a universal G:C {yields} A:T mutation bias in bacteria, fungi, plants and animals. To explore the molecular basis for this mutation bias, we examined the three well-known DNA mutation models, i.e., oxidative damage model, UV-radiation damage model and CpG hypermutation model. It was revealed that these models cannot providemore » a sufficient explanation to the universal mutation bias. Therefore, we resorted to a DNA mutation model proposed by Loewdin 40 years ago, which was based on inter-base double proton transfers (DPT). Since DPT is a fundamental and spontaneous chemical process and occurs much more frequently within GC pairs than AT pairs, Loewdin model offers a common explanation for the observed universal mutation bias and thus has broad biological implications.« less

Genetic Progression of High Grade Prostatic Intraepithelial Neoplasia to Prostate Cancer.

PubMed

Jung, Seung-Hyun; Shin, Sun; Kim, Min Sung; Baek, In-Pyo; Lee, Ji Youl; Lee, Sung Hak; Kim, Tae-Min; Lee, Sug Hyung; Chung, Yeun-Jun

2016-05-01

Although high grade prostatic intraepithelial neoplasia (HGPIN) is considered a neoplastic lesion that precedes prostate cancer (PCA), the genomic structures of HGPIN remain unknown. Identification of the genomic landscape of HGPIN and the genomic differences between HGPIN and PCA that may drive the progression to PCA. We analyzed 20 regions of paired HGPIN and PCA from six patients using whole-exome sequencing and array-comparative genomic hybridization. Somatic mutation and copy number alteration (CNA) profiles of paired HGPIN and PCA were measured and compared. The number of total mutations and CNAs of HGPINs were significantly fewer than those of PCAs. Mutations in FOXA1 and CNAs (1q and 8q gains) were detected in both HGPIN and PCA ('common'), suggesting their roles in early PCA development. Mutations in SPOP, KDM6A, and KMT2D were 'PCA-specific', suggesting their roles in HGPIN progression to PCA. The 8p loss was either 'common' or 'PCA-specific'. In-silico estimation of evolutionary ages predicted that HGPIN genomes were much younger than PCA genomes. Our data show that PCAs are direct descendants of HGPINs in most cases that require more genomic alterations to progress to PCA. The nature of heterogeneous HGPIN population that might attenuate genomic signals should further be studied. HGPIN genomes harbor relatively fewer mutations and CNAs than PCA but require additional hits for the progression. In this study, we suggest a systemic diagram from high grade prostatic intraepithelial neoplasia (HGPIN) to prostate cancer (PCA). Our results provide a clue to explain the long latency from HGPIN to PCA and provide useful information for the genetic diagnosis of HGPIN and PCA. Copyright © 2015 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Clonal evolution of acute myeloid leukemia highlighted by latest genome sequencing studies.

PubMed

Zhang, Xuehong; Lv, Dekang; Zhang, Yu; Liu, Quentin; Li, Zhiguang

2016-09-06

Decades of years might be required for an initiated cell to become a fully-pledged, metastasized tumor. DNA mutations are accumulated during this process including background mutations that emerge scholastically, as well as driver mutations that selectively occur in a handful of cancer genes and confer the cell a growth advantage over its neighbors. A clone of tumor cells could be superseded by another clone that acquires new mutations and grows more aggressively. Tumor evolutional patterns have been studied for years using conventional approaches that focus on the investigation of a single or a couple of genes. Latest deep sequencing technology enables a global view of tumor evolution by deciphering almost all genome aberrations in a tumor. Tumor clones and the fate of each clone during tumor evolution can be depicted with the help of the concept of variant allele frequency. Here, we summarize the new insights of cancer evolutional progression in acute myeloid leukemia. Cancer evolution is currently thought to start from a clone that has accumulated the requisite somatically-acquired genetic aberrations through a series of increasingly disordered clinical and pathological phases, eventually leading to malignant transformation [1-3]. The observations in invasive colorectal cancer that usually emerges from an antecedent benign adenomatous polyp and in cervical cancer that proceeds through intraepithelial neoplasia support the idea of stepwise or linear cancerous progression [3-5]. Genetically, such progression is achieved by successive waves of clonal expansion during which cells acquire novel genomic alterations including single nucleotide variants (SNVs), small insertions and deletions (indels), and/or copy number variations (CNVs) [6]. The latest improvement in sequencing technology has allowed the deciphering of the whole exome or genome in different types of tumor and normal tissue pairs, providing detailed catalogue about genome aberrations during tumor initiation and progression, which have been reviewed in several papers [7-10]. Here, we focus on demonstrating the cancer clonal evolution pattern revealed by recent deep sequencing studies of samples from acute myeloid leukemia (AML) patients.

Reduced Mutation Rate and Increased Transformability of Transposon-Free Acinetobacter baylyi ADP1-ISx.

PubMed

Suárez, Gabriel A; Renda, Brian A; Dasgupta, Aurko; Barrick, Jeffrey E

2017-09-01

The genomes of most bacteria contain mobile DNA elements that can contribute to undesirable genetic instability in engineered cells. In particular, transposable insertion sequence (IS) elements can rapidly inactivate genes that are important for a designed function. We deleted all six copies of IS 1236 from the genome of the naturally transformable bacterium Acinetobacter baylyi ADP1. The natural competence of ADP1 made it possible to rapidly repair deleterious point mutations that arose during strain construction. In the resulting ADP1-ISx strain, the rates of mutations inactivating a reporter gene were reduced by 7- to 21-fold. This reduction was higher than expected from the incidence of new IS 1236 insertions found during a 300-day mutation accumulation experiment with wild-type ADP1 that was used to estimate spontaneous mutation rates in the strain. The extra improvement appears to be due in part to eliminating large deletions caused by IS 1236 activity, as the point mutation rate was unchanged in ADP1-ISx. Deletion of an error-prone polymerase ( dinP ) and a DNA damage response regulator ( umuD Ab [the umuD gene of A. baylyi ]) from the ADP1-ISx genome did not further reduce mutation rates. Surprisingly, ADP1-ISx exhibited increased transformability. This improvement may be due to less autolysis and aggregation of the engineered cells than of the wild type. Thus, deleting IS elements from the ADP1 genome led to a greater than expected increase in evolutionary reliability and unexpectedly enhanced other key strain properties, as has been observed for other clean-genome bacterial strains. ADP1-ISx is an improved chassis for metabolic engineering and other applications. IMPORTANCE Acinetobacter baylyi ADP1 has been proposed as a next-generation bacterial host for synthetic biology and genome engineering due to its ability to efficiently take up DNA from its environment during normal growth. We deleted transposable elements that are capable of copying themselves, inserting into other genes, and thereby inactivating them from the ADP1 genome. The resulting "clean-genome" ADP1-ISx strain exhibited larger reductions in the rates of inactivating mutations than expected from spontaneous mutation rates measured via whole-genome sequencing of lineages evolved under relaxed selection. Surprisingly, we also found that IS element activity reduces transformability and is a major cause of cell aggregation and death in wild-type ADP1 grown under normal laboratory conditions. More generally, our results demonstrate that domesticating a bacterial genome by removing mobile DNA elements that have accumulated during evolution in the wild can have unanticipated benefits. Copyright © 2017 American Society for Microbiology.

Efficient genome editing of wild strawberry genes, vector development and validation.

PubMed

Zhou, Junhui; Wang, Guoming; Liu, Zhongchi

2018-03-25

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is an effective genome editing tool for plant and animal genomes. However, there are still few reports on the successful application of CRISPR-Cas9 to horticultural plants, especially with regard to germ-line transmission of targeted mutations. Here, we report high-efficiency genome editing in the wild strawberry Fragaria vesca and its successful application to mutate the auxin biosynthesis gene TAA1 and auxin response factor 8 (ARF8). In our CRISPR system, the Arabidopsis U6 promoter AtU6-26 and the wild strawberry U6 promoter FveU6-2 were each used to drive the expression of sgRNA, and both promoters were shown to lead to high-efficiency genome editing in strawberry. To test germ-line transmission of the edited mutations and new mutations induced in the next generation, the progeny of the primary (T0) transgenic plants carrying the CRISPR construct was analysed. New mutations were detected in the progeny plants at a high efficiency, including large deletions between the two PAM sites. Further, T1 plants harbouring arf8 homozygous knockout mutations grew considerably faster than wild-type plants. The results indicate that our CRISPR vectors can be used to edit the wild strawberry genome at a high efficiency and that both sgRNA design and appropriate U6 promoters contribute to the success of genomic editing. Our results open up exciting opportunities for engineering strawberry and related horticultural crops to improve traits of economic importance. © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

The Mutational Landscape of Adenoid Cystic Carcinoma

PubMed Central

Ho, Allen S.; Kannan, Kasthuri; Roy, David M.; Morris, Luc G.T.; Ganly, Ian; Katabi, Nora; Ramaswami, Deepa; Walsh, Logan A.; Eng, Stephanie; Huse, Jason T.; Zhang, Jianan; Dolgalev, Igor; Huberman, Kety; Heguy, Adriana; Viale, Agnes; Drobnjak, Marija; Leversha, Margaret A.; Rice, Christine E.; Singh, Bhuvanesh; Iyer, N. Gopalakrishna; Leemans, C. Rene; Bloemena, Elisabeth; Ferris, Robert L.; Seethala, Raja R.; Gross, Benjamin E.; Liang, Yupu; Sinha, Rileen; Peng, Luke; Raphael, Benjamin J.; Turcan, Sevin; Gong, Yongxing; Schultz, Nikolaus; Kim, Seungwon; Chiosea, Simion; Shah, Jatin P.; Sander, Chris; Lee, William; Chan, Timothy A.

2013-01-01

Adenoid cystic carcinomas (ACCs) are among the most enigmatic of human malignancies. These aggressive salivary cancers frequently recur and metastasize despite definitive treatment, with no known effective chemotherapy regimen. Here, we determined the ACC mutational landscape and report the exome or whole genome sequences of 60 ACC tumor/normal pairs. These analyses revealed a low exonic somatic mutation rate (0.31 non-silent events/megabase) and wide mutational diversity. Interestingly, mutations selectively involved chromatin state regulators, such as SMARCA2, CREBBP, and KDM6A, suggesting aberrant epigenetic regulation in ACC oncogenesis. Mutations in genes central to DNA damage and protein kinase A signaling also implicate these processes. We observed MYB-NFIB translocations and somatic mutations in MYB-associated genes, solidifying these aberrations as critical events. Lastly, we identified recurrent mutations in the FGF/IGF/PI3K pathway that may potentially offer new avenues for therapy (30%). Collectively, our observations establish a molecular foundation for understanding and exploring new treatments for ACC. PMID:23685749

On the Sequence-Directed Nature of Human Gene Mutation: The Role of Genomic Architecture and the Local DNA Sequence Environment in Mediating Gene Mutations Underlying Human Inherited Disease

PubMed Central

Cooper, David N.; Bacolla, Albino; Férec, Claude; Vasquez, Karen M.; Kehrer-Sawatzki, Hildegard; Chen, Jian-Min

2011-01-01

Different types of human gene mutation may vary in size, from structural variants (SVs) to single base-pair substitutions, but what they all have in common is that their nature, size and location are often determined either by specific characteristics of the local DNA sequence environment or by higher-order features of the genomic architecture. The human genome is now recognized to contain ‘pervasive architectural flaws’ in that certain DNA sequences are inherently mutation-prone by virtue of their base composition, sequence repetitivity and/or epigenetic modification. Here we explore how the nature, location and frequency of different types of mutation causing inherited disease are shaped in large part, and often in remarkably predictable ways, by the local DNA sequence environment. The mutability of a given gene or genomic region may also be influenced indirectly by a variety of non-canonical (non-B) secondary structures whose formation is facilitated by the underlying DNA sequence. Since these non-B DNA structures can interfere with subsequent DNA replication and repair, and may serve to increase mutation frequencies in generalized fashion (i.e. both in the context of subtle mutations and SVs), they have the potential to serve as a unifying concept in studies of mutational mechanisms underlying human inherited disease. PMID:21853507

Mutation Detection with Next-Generation Resequencing through a Mediator Genome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wurtzel, Omri; Dori-Bachash, Mally; Pietrokovski, Shmuel

2010-12-31

The affordability of next generation sequencing (NGS) is transforming the field of mutation analysis in bacteria. The genetic basis for phenotype alteration can be identified directly by sequencing the entire genome of the mutant and comparing it to the wild-type (WT) genome, thus identifying acquired mutations. A major limitation for this approach is the need for an a-priori sequenced reference genome for the WT organism, as the short reads of most current NGS approaches usually prohibit de-novo genome assembly. To overcome this limitation we propose a general framework that utilizes the genome of relative organisms as mediators for comparing WTmore » and mutant bacteria. Under this framework, both mutant and WT genomes are sequenced with NGS, and the short sequencing reads are mapped to the mediator genome. Variations between the mutant and the mediator that recur in the WT are ignored, thus pinpointing the differences between the mutant and the WT. To validate this approach we sequenced the genome of Bdellovibrio bacteriovorus 109J, an obligatory bacterial predator, and its prey-independent mutant, and compared both to the mediator species Bdellovibrio bacteriovorus HD100. Although the mutant and the mediator sequences differed in more than 28,000 nucleotide positions, our approach enabled pinpointing the single causative mutation. Experimental validation in 53 additional mutants further established the implicated gene. Our approach extends the applicability of NGS-based mutant analyses beyond the domain of available reference genomes.« less

Tempo and mode of genomic mutations unveil human evolutionary history.

PubMed

Hara, Yuichiro

2015-01-01

Mutations that have occurred in human genomes provide insight into various aspects of evolutionary history such as speciation events and degrees of natural selection. Comparing genome sequences between human and great apes or among humans is a feasible approach for inferring human evolutionary history. Recent advances in high-throughput or so-called 'next-generation' DNA sequencing technologies have enabled the sequencing of thousands of individual human genomes, as well as a variety of reference genomes of hominids, many of which are publicly available. These sequence data can help to unveil the detailed demographic history of the lineage leading to humans as well as the explosion of modern human population size in the last several thousand years. In addition, high-throughput sequencing illustrates the tempo and mode of de novo mutations, which are producing human genetic variation at this moment. Pedigree-based human genome sequencing has shown that mutation rates vary significantly across the human genome. These studies have also provided an improved timescale of human evolution, because the mutation rate estimated from pedigree analysis is half that estimated from traditional analyses based on molecular phylogeny. Because of the dramatic reduction in sequencing cost, sequencing on-demand samples designed for specific studies is now also becoming popular. To produce data of sufficient quality to meet the requirements of the study, it is necessary to set an explicit sequencing plan that includes the choice of sample collection methods, sequencing platforms, and number of sequence reads.

Accuracy of Answers to Cell Lineage Questions Depends on Single-Cell Genomics Data Quality and Quantity.

PubMed

Spiro, Adam; Shapiro, Ehud

2016-06-01

Advances in single-cell (SC) genomics enable commensurate improvements in methods for uncovering lineage relations among individual cells, as determined by phylogenetic analysis of the somatic mutations harbored by each cell. Theoretically, complete and accurate knowledge of the genome of each cell of an individual can produce an extremely accurate cell lineage tree of that individual. However, the reality of SC genomics is that such complete and accurate knowledge would be wanting, in quality and in quantity, for the foreseeable future. In this paper we offer a framework for systematically exploring the feasibility of answering cell lineage questions based on SC somatic mutational analysis, as a function of SC genomics data quality and quantity. We take into consideration the current limitations of SC genomics in terms of mutation data quality, most notably amplification bias and allele dropouts (ADO), as well as cost, which puts practical limits on mutation data quantity obtained from each cell as well as on cell sample density. We do so by generating in silico cell lineage trees using a dedicated formal language, eSTG, and show how the ability to answer correctly a cell lineage question depends on the quality and quantity of the SC mutation data. The presented framework can serve as a baseline for the potential of current SC genomics to unravel cell lineage dynamics, as well as the potential contributions of future advancement, both biochemical and computational, for the task.

Whole-genome sequencing for comparative genomics and de novo genome assembly.

PubMed

Benjak, Andrej; Sala, Claudia; Hartkoorn, Ruben C

2015-01-01

Next-generation sequencing technologies for whole-genome sequencing of mycobacteria are rapidly becoming an attractive alternative to more traditional sequencing methods. In particular this technology is proving useful for genome-wide identification of mutations in mycobacteria (comparative genomics) as well as for de novo assembly of whole genomes. Next-generation sequencing however generates a vast quantity of data that can only be transformed into a usable and comprehensible form using bioinformatics. Here we describe the methodology one would use to prepare libraries for whole-genome sequencing, and the basic bioinformatics to identify mutations in a genome following Illumina HiSeq or MiSeq sequencing, as well as de novo genome assembly following sequencing using Pacific Biosciences (PacBio).

Large-scale analyses of synonymous substitution rates can be sensitive to assumptions about the process of mutation.

PubMed

Aris-Brosou, Stéphane; Bielawski, Joseph P

2006-08-15

A popular approach to examine the roles of mutation and selection in the evolution of genomes has been to consider the relationship between codon bias and synonymous rates of molecular evolution. A significant relationship between these two quantities is taken to indicate the action of weak selection on substitutions among synonymous codons. The neutral theory predicts that the rate of evolution is inversely related to the level of functional constraint. Therefore, selection against the use of non-preferred codons among those coding for the same amino acid should result in lower rates of synonymous substitution as compared with sites not subject to such selection pressures. However, reliably measuring the extent of such a relationship is problematic, as estimates of synonymous rates are sensitive to our assumptions about the process of molecular evolution. Previous studies showed the importance of accounting for unequal codon frequencies, in particular when synonymous codon usage is highly biased. Yet, unequal codon frequencies can be modeled in different ways, making different assumptions about the mutation process. Here we conduct a simulation study to evaluate two different ways of modeling uneven codon frequencies and show that both model parameterizations can have a dramatic impact on rate estimates and affect biological conclusions about genome evolution. We reanalyze three large data sets to demonstrate the relevance of our results to empirical data analysis.

Genome dynamics and its impact on evolution of Escherichia coli.

PubMed

Dobrindt, Ulrich; Chowdary, M Geddam; Krumbholz, G; Hacker, J

2010-08-01

The Escherichia coli genome consists of a conserved part, the so-called core genome, which encodes essential cellular functions and of a flexible, strain-specific part. Genes that belong to the flexible genome code for factors involved in bacterial fitness and adaptation to different environments. Adaptation includes increase in fitness and colonization capacity. Pathogenic as well as non-pathogenic bacteria carry mobile and accessory genetic elements such as plasmids, bacteriophages, genomic islands and others, which code for functions required for proper adaptation. Escherichia coli is a very good example to study the interdependency of genome architecture and lifestyle of bacteria. Thus, these species include pathogenic variants as well as commensal bacteria adapted to different host organisms. In Escherichia coli, various genetic elements encode for pathogenicity factors as well as factors, which increase the fitness of non-pathogenic bacteria. The processes of genome dynamics, such as gene transfer, genome reduction, rearrangements as well as point mutations contribute to the adaptation of the bacteria into particular environments. Using Escherichia coli model organisms, such as uropathogenic strain 536 or commensal strain Nissle 1917, we studied mechanisms of genome dynamics and discuss these processes in the light of the evolution of microbes.

A comprehensive assessment of somatic mutation detection in cancer using whole-genome sequencing

PubMed Central

Alioto, Tyler S.; Buchhalter, Ivo; Derdak, Sophia; Hutter, Barbara; Eldridge, Matthew D.; Hovig, Eivind; Heisler, Lawrence E.; Beck, Timothy A.; Simpson, Jared T.; Tonon, Laurie; Sertier, Anne-Sophie; Patch, Ann-Marie; Jäger, Natalie; Ginsbach, Philip; Drews, Ruben; Paramasivam, Nagarajan; Kabbe, Rolf; Chotewutmontri, Sasithorn; Diessl, Nicolle; Previti, Christopher; Schmidt, Sabine; Brors, Benedikt; Feuerbach, Lars; Heinold, Michael; Gröbner, Susanne; Korshunov, Andrey; Tarpey, Patrick S.; Butler, Adam P.; Hinton, Jonathan; Jones, David; Menzies, Andrew; Raine, Keiran; Shepherd, Rebecca; Stebbings, Lucy; Teague, Jon W.; Ribeca, Paolo; Giner, Francesc Castro; Beltran, Sergi; Raineri, Emanuele; Dabad, Marc; Heath, Simon C.; Gut, Marta; Denroche, Robert E.; Harding, Nicholas J.; Yamaguchi, Takafumi N.; Fujimoto, Akihiro; Nakagawa, Hidewaki; Quesada, Víctor; Valdés-Mas, Rafael; Nakken, Sigve; Vodák, Daniel; Bower, Lawrence; Lynch, Andrew G.; Anderson, Charlotte L.; Waddell, Nicola; Pearson, John V.; Grimmond, Sean M.; Peto, Myron; Spellman, Paul; He, Minghui; Kandoth, Cyriac; Lee, Semin; Zhang, John; Létourneau, Louis; Ma, Singer; Seth, Sahil; Torrents, David; Xi, Liu; Wheeler, David A.; López-Otín, Carlos; Campo, Elías; Campbell, Peter J.; Boutros, Paul C.; Puente, Xose S.; Gerhard, Daniela S.; Pfister, Stefan M.; McPherson, John D.; Hudson, Thomas J.; Schlesner, Matthias; Lichter, Peter; Eils, Roland; Jones, David T. W.; Gut, Ivo G.

2015-01-01

As whole-genome sequencing for cancer genome analysis becomes a clinical tool, a full understanding of the variables affecting sequencing analysis output is required. Here using tumour-normal sample pairs from two different types of cancer, chronic lymphocytic leukaemia and medulloblastoma, we conduct a benchmarking exercise within the context of the International Cancer Genome Consortium. We compare sequencing methods, analysis pipelines and validation methods. We show that using PCR-free methods and increasing sequencing depth to ∼100 × shows benefits, as long as the tumour:control coverage ratio remains balanced. We observe widely varying mutation call rates and low concordance among analysis pipelines, reflecting the artefact-prone nature of the raw data and lack of standards for dealing with the artefacts. However, we show that, using the benchmark mutation set we have created, many issues are in fact easy to remedy and have an immediate positive impact on mutation detection accuracy. PMID:26647970

Identification of mutated driver pathways in cancer using a multi-objective optimization model.

PubMed

Zheng, Chun-Hou; Yang, Wu; Chong, Yan-Wen; Xia, Jun-Feng

2016-05-01

New-generation high-throughput technologies, including next-generation sequencing technology, have been extensively applied to solve biological problems. As a result, large cancer genomics projects such as the Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium are producing large amount of rich and diverse data in multiple cancer types. The identification of mutated driver genes and driver pathways from these data is a significant challenge. Genome aberrations in cancer cells can be divided into two types: random 'passenger mutation' and functional 'driver mutation'. In this paper, we introduced a Multi-objective Optimization model based on a Genetic Algorithm (MOGA) to solve the maximum weight submatrix problem, which can be employed to identify driver genes and driver pathways promoting cancer proliferation. The maximum weight submatrix problem defined to find mutated driver pathways is based on two specific properties, i.e., high coverage and high exclusivity. The multi-objective optimization model can adjust the trade-off between high coverage and high exclusivity. We proposed an integrative model by combining gene expression data and mutation data to improve the performance of the MOGA algorithm in a biological context. Copyright © 2016 Elsevier Ltd. All rights reserved.

Polyploidy can drive rapid adaptation in yeast

NASA Astrophysics Data System (ADS)

Selmecki, Anna M.; Maruvka, Yosef E.; Richmond, Phillip A.; Guillet, Marie; Shoresh, Noam; Sorenson, Amber L.; de, Subhajyoti; Kishony, Roy; Michor, Franziska; Dowell, Robin; Pellman, David

2015-03-01

Polyploidy is observed across the tree of life, yet its influence on evolution remains incompletely understood. Polyploidy, usually whole-genome duplication, is proposed to alter the rate of evolutionary adaptation. This could occur through complex effects on the frequency or fitness of beneficial mutations. For example, in diverse cell types and organisms, immediately after a whole-genome duplication, newly formed polyploids missegregate chromosomes and undergo genetic instability. The instability following whole-genome duplications is thought to provide adaptive mutations in microorganisms and can promote tumorigenesis in mammalian cells. Polyploidy may also affect adaptation independently of beneficial mutations through ploidy-specific changes in cell physiology. Here we perform in vitro evolution experiments to test directly whether polyploidy can accelerate evolutionary adaptation. Compared with haploids and diploids, tetraploids undergo significantly faster adaptation. Mathematical modelling suggests that rapid adaptation of tetraploids is driven by higher rates of beneficial mutations with stronger fitness effects, which is supported by whole-genome sequencing and phenotypic analyses of evolved clones. Chromosome aneuploidy, concerted chromosome loss, and point mutations all provide large fitness gains. We identify several mutations whose beneficial effects are manifest specifically in the tetraploid strains. Together, these results provide direct quantitative evidence that in some environments polyploidy can accelerate evolutionary adaptation.

Genomic variation associated with local adaptation of weedy rice during de-domestication

PubMed Central

Qiu, Jie; Zhou, Yongjun; Mao, Lingfeng; Ye, Chuyu; Wang, Weidi; Zhang, Jianping; Yu, Yongyi; Fu, Fei; Wang, Yunfei; Qian, Feijian; Qi, Ting; Wu, Sanling; Sultana, Most Humaira; Cao, Ya-Nan; Wang, Yu; Timko, Michael P.; Ge, Song; Fan, Longjiang; Lu, Yongliang

2017-01-01

De-domestication is a unique evolutionary process by which domesticated crops are converted into ‘wild predecessor like' forms. Weedy rice (Oryza sativa f. spontanea) is an excellent model to dissect the molecular processes underlying de-domestication. Here, we analyse the genomes of 155 weedy and 76 locally cultivated rice accessions from four representative regions in China that were sequenced to an average 18.2 × coverage. Phylogenetic and demographic analyses indicate that Chinese weedy rice was de-domesticated independently from cultivated rice and experienced a strong genetic bottleneck. Although evolving from multiple origins, critical genes underlying convergent evolution of different weedy types can be found. Allele frequency analyses suggest that standing variations and new mutations contribute differently to japonica and indica weedy rice. We identify a Mb-scale genomic region present in weedy rice but not cultivated rice genomes that shows evidence of balancing selection, thereby suggesting that there might be more complexity inherent to the process of de-domestication. PMID:28537247

The Red Queen in mitochondria: cyto-nuclear co-evolution, hybrid breakdown and human disease

PubMed Central

Chou, Jui-Yu; Leu, Jun-Yi

2015-01-01

Cyto-nuclear incompatibility, a specific form of Dobzhansky-Muller incompatibility caused by incompatible alleles between mitochondrial and nuclear genomes, has been suggested to play a critical role during speciation. Several features of the mitochondrial genome (mtDNA), including high mutation rate, dynamic genomic structure, and uniparental inheritance, make mtDNA more likely to accumulate mutations in the population. Once mtDNA has changed, the nuclear genome needs to play catch-up due to the intimate interactions between these two genomes. In two populations, if cyto-nuclear co-evolution is driven in different directions, it may eventually lead to hybrid incompatibility. Although cyto-nuclear incompatibility has been observed in a wide range of organisms, it remains unclear what type of mutations drives the co-evolution. Currently, evidence supporting adaptive mutations in mtDNA remains limited. On the other hand, it has been known that some mutations allow mtDNA to propagate more efficiently but compromise the host fitness (described as selfish mtDNA). Arms races between such selfish mtDNA and host nuclear genomes can accelerate cyto-nuclear co-evolution and lead to a phenomenon called the Red Queen Effect. Here, we discuss how the Red Queen Effect may contribute to the frequent observation of cyto-nuclear incompatibility and be the underlying driving force of some human mitochondrial diseases. PMID:26042149

The challenges of tumor genetic diversity.

PubMed

Mroz, Edmund A; Rocco, James W

2017-05-15

The authors review and discuss the implications of genomic analyses documenting the diversity of tumors, both among patients and within individual tumors. Genetic diversity among solid tumors limits targeted therapies, because few mutations that drive tumors are both targetable and at high prevalence. Many more driver mutations and how they affect cellular signaling pathways must be identified if targeted therapy is to become widely useful. Genetic diversity within a tumor-intratumor genetic heterogeneity-makes the tumor a collection of subclones: related yet distinct cancers. Selection for pre-existing, resistant subclones by conventional or targeted therapies may explain many treatment failures. Immune therapy faces the same fundamental challenges. Nevertheless, the processes that generate and maintain heterogeneity might provide novel therapeutic targets. Addressing both types of diversity requires genomic tumor analyses linked to detailed clinical data. The trend toward sequencing restricted cancer gene panels, however, limits the ability to discover new driver mutations and assess intratumor heterogeneity. Clinical data currently collected with genomic analyses often lack critical information, substantially limiting their use in understanding tumor diversity. Now that diversity among and within tumors can no longer be ignored, research and clinical practice must adapt to take diversity into account. Cancer 2017;123:917-27. © 2016 American Cancer Society. © 2016 American Cancer Society.

Understanding genetics in neuroimaging.

PubMed

Vasquez, Marina Lipkin; Renault, Ilana Zalcberg

2015-02-01

Gene expression is a process of DNA sequence reading into protein synthesis. In cases of problems in DNA repair/apoptosis mechanisms, cells accumulate genomic abnormalities and pass them through generations of cells. The accumulation of mutations causes diseases and even tumors. In addition to cancer, many other neurologic conditions have been associated with genetic mutations. Some trials are testing patients with epigenetic treatments. Epigenetic therapy must be used with caution because epigenetic processes and changes happen constantly in normal cells, giving rise to drug off-target effects. Scientists are making progress in specifically targeting abnormal cells with minimal damage to normal ones. Copyright © 2015. Published by Elsevier Inc.

Clinical differences in patients with mitochondriocytopathies due to nuclear versus mitochondrial DNA mutations.

PubMed

Rubio-Gozalbo, M E; Dijkman, K P; van den Heuvel, L P; Sengers, R C; Wendel, U; Smeitink, J A

2000-01-01

Defects in oxidative phosphorylation (OXPHOS) are genetically unique because the different components involved in this process, respiratory chain enzyme complexes (I, III, and IV) and complex V, are encoded by nuclear and mitochondrial genome. The objective of the study was to assess whether there are clinical differences in patients suffering from OXPHOS defects caused by nuclear or mitochondrial DNA (mtDNA) mutations. We studied 16 families with > or = two siblings with a genetically established OXPHOS deficiency, four due to a nuclear gene mutation and 12 due to a mtDNA mutation. Siblings with a nuclear gene mutation showed very similar clinical pictures that became manifest in the first years (ranging from first months to early childhood). There was a severe progressive course. Seven of the eight children died in their first decade. Conversely, siblings with a mtDNA mutation had clinical pictures that varied from almost alike to very distinct. They became symptomatic at an older age (ranging from childhood to adulthood), with the exception of defects associated with Leigh or Leigh-like phenotype. The clinical course was more gradual and relatively less severe; four of the 26 patients died, one in his second year, another in her second decade and two in their sixth decade. There are differences in age at onset, severity of clinical course, outcome, and intrafamilial variability in patients affected of an OXPHOS defect due to nuclear or mtDNA mutations. Patients with nuclear mutations become symptomatic at a young age, and have a severe clinical course. Patients with mtDNA mutations show a wider clinical spectrum of age at onset and severity. These differences may be of importance regarding the choice of which genome to study in affected patients as well as with respect to genetic counseling. Copyright 2000 Wiley-Liss, Inc.

The degree of attenuation of tick-borne encephalitis virus depends on the cumulative effects of point mutations.

PubMed

Gritsun, T S; Desai, A; Gould, E A

2001-07-01

An infectious clone (pGGVs) of the tick-borne encephalitis complex virus Vasilchenko (Vs) was constructed previously. Virus recovered from pGGVs produced slightly smaller plaques than the Vs parental virus. Sequence analysis demonstrated five nucleotide differences between the original Vs virus and pGGVs; four of these mutations resulted in amino acid substitutions, while the fifth mutation was located in the 3' untranslated region (3'UTR). Two mutations were located in conserved regions and three mutations were located in variable regions of the virus genome. Reverse substitutions from the conserved regions of the genome, R(496)-->H in the envelope (E) gene and C(10884)-->T in the 3'UTR, were introduced both separately and together into the infectious clone and their biological effect on virus phenotype was evaluated. The engineered viruses with R(496) in the E protein produced plaques of smaller size than viruses with H(496) at this position. This mutation also affected the growth and neuroinvasiveness of the virus. In contrast, the consequence of a T(10884)-->C substitution within the 3'UTR was noticeable only in cytotoxicity and neuroinvasiveness tests. However, all virus mutants engineered by modification of the infectious clone, including one with two wild-type mutations, H(496) and T(10884), showed reduced neuroinvasiveness in comparison with the Vs parental virus. Therefore, although the H(496)-->R and T(10884)-->C substitutions clearly reduce virus virulence, the other mutations within the variable regions of the capsid (I(45)-->F) and the NS5 (T(2688)-->A and M(3385)-->I) genes also contribute to the process of attenuation. In terms of developing flavivirus vaccines, the impact of accumulating apparently minor mutations should be assessed in detail.

Cancer heterogeneity: converting a limitation into a source of biologic information.

PubMed

Rübben, Albert; Araujo, Arturo

2017-09-08

Analysis of spatial and temporal genetic heterogeneity in human cancers has revealed that somatic cancer evolution in most cancers is not a simple linear process composed of a few sequential steps of mutation acquisitions and clonal expansions. Parallel evolution has been observed in many early human cancers resulting in genetic heterogeneity as well as multilineage progression. Moreover, aneuploidy as well as structural chromosomal aberrations seems to be acquired in a non-linear, punctuated mode where most aberrations occur at early stages of somatic cancer evolution. At later stages, the cancer genomes seem to get stabilized and acquire only few additional rearrangements. While parallel evolution suggests positive selection of driver mutations at early stages of somatic cancer evolution, stabilization of structural aberrations at later stages suggests that negative selection takes effect when cancer cells progressively lose their tolerance towards additional mutation acquisition. Mixing of genetically heterogeneous subclones in cancer samples reduces sensitivity of mutation detection. Moreover, driver mutations present only in a fraction of cancer cells are more likely to be mistaken for passenger mutations. Therefore, genetic heterogeneity may be considered a limitation negatively affecting detection sensitivity of driver mutations. On the other hand, identification of subclones and subclone lineages in human cancers may lead to a more profound understanding of the selective forces which shape somatic cancer evolution in human cancers. Identification of parallel evolution by analyzing spatial heterogeneity may hint to driver mutations which might represent additional therapeutic targets besides driver mutations present in a monoclonal state. Likewise, stabilization of cancer genomes which can be identified by analyzing temporal genetic heterogeneity might hint to genes and pathways which have become essential for survival of cancer cell lineages at later stages of cancer evolution. These genes and pathways might also constitute patient specific therapeutic targets.

Detection of genome-wide copy number variants in myeloid malignancies using next-generation sequencing.

PubMed

Shen, Wei; Paxton, Christian N; Szankasi, Philippe; Longhurst, Maria; Schumacher, Jonathan A; Frizzell, Kimberly A; Sorrells, Shelly M; Clayton, Adam L; Jattani, Rakhi P; Patel, Jay L; Toydemir, Reha; Kelley, Todd W; Xu, Xinjie

2018-04-01

Genetic abnormalities, including copy number variants (CNV), copy number neutral loss of heterozygosity (CN-LOH) and gene mutations, underlie the pathogenesis of myeloid malignancies and serve as important diagnostic, prognostic and/or therapeutic markers. Currently, multiple testing strategies are required for comprehensive genetic testing in myeloid malignancies. The aim of this proof-of-principle study was to investigate the feasibility of combining detection of genome-wide large CNVs, CN-LOH and targeted gene mutations into a single assay using next-generation sequencing (NGS). For genome-wide CNV detection, we designed a single nucleotide polymorphism (SNP) sequencing backbone with 22 762 SNP regions evenly distributed across the entire genome. For targeted mutation detection, 62 frequently mutated genes in myeloid malignancies were targeted. We combined this SNP sequencing backbone with a targeted mutation panel, and sequenced 9 healthy individuals and 16 patients with myeloid malignancies using NGS. We detected 52 somatic CNVs, 11 instances of CN-LOH and 39 oncogenic mutations in the 16 patients with myeloid malignancies, and none in the 9 healthy individuals. All CNVs and CN-LOH were confirmed by SNP microarray analysis. We describe a genome-wide SNP sequencing backbone which allows for sensitive detection of genome-wide CNVs and CN-LOH using NGS. This proof-of-principle study has demonstrated that this strategy can provide more comprehensive genetic profiling for patients with myeloid malignancies using a single assay. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Core signaling pathways in human pancreatic cancers revealed by global genomic analyses.

PubMed

Jones, Siân; Zhang, Xiaosong; Parsons, D Williams; Lin, Jimmy Cheng-Ho; Leary, Rebecca J; Angenendt, Philipp; Mankoo, Parminder; Carter, Hannah; Kamiyama, Hirohiko; Jimeno, Antonio; Hong, Seung-Mo; Fu, Baojin; Lin, Ming-Tseh; Calhoun, Eric S; Kamiyama, Mihoko; Walter, Kimberly; Nikolskaya, Tatiana; Nikolsky, Yuri; Hartigan, James; Smith, Douglas R; Hidalgo, Manuel; Leach, Steven D; Klein, Alison P; Jaffee, Elizabeth M; Goggins, Michael; Maitra, Anirban; Iacobuzio-Donahue, Christine; Eshleman, James R; Kern, Scott E; Hruban, Ralph H; Karchin, Rachel; Papadopoulos, Nickolas; Parmigiani, Giovanni; Vogelstein, Bert; Velculescu, Victor E; Kinzler, Kenneth W

2008-09-26

There are currently few therapeutic options for patients with pancreatic cancer, and new insights into the pathogenesis of this lethal disease are urgently needed. Toward this end, we performed a comprehensive genetic analysis of 24 pancreatic cancers. We first determined the sequences of 23,219 transcripts, representing 20,661 protein-coding genes, in these samples. Then, we searched for homozygous deletions and amplifications in the tumor DNA by using microarrays containing probes for approximately 10(6) single-nucleotide polymorphisms. We found that pancreatic cancers contain an average of 63 genetic alterations, the majority of which are point mutations. These alterations defined a core set of 12 cellular signaling pathways and processes that were each genetically altered in 67 to 100% of the tumors. Analysis of these tumors' transcriptomes with next-generation sequencing-by-synthesis technologies provided independent evidence for the importance of these pathways and processes. Our data indicate that genetically altered core pathways and regulatory processes only become evident once the coding regions of the genome are analyzed in depth. Dysregulation of these core pathways and processes through mutation can explain the major features of pancreatic tumorigenesis.

Comprehensive Genomic Characterization of Upper Tract Urothelial Carcinoma.

PubMed

Moss, Tyler J; Qi, Yuan; Xi, Liu; Peng, Bo; Kim, Tae-Beom; Ezzedine, Nader E; Mosqueda, Maribel E; Guo, Charles C; Czerniak, Bogdan A; Ittmann, Michael; Wheeler, David A; Lerner, Seth P; Matin, Surena F

2017-10-01

Upper urinary tract urothelial cancer (UTUC) may have unique etiologic and genomic factors compared to bladder cancer. To characterize the genomic landscape of UTUC and provide insights into its biology using comprehensive integrated genomic analyses. We collected 31 untreated snap-frozen UTUC samples from two institutions and carried out whole-exome sequencing (WES) of DNA, RNA sequencing (RNAseq), and protein analysis. Adjusting for batch effects, consensus mutation calls from independent pipelines identified DNA mutations, gene expression clusters using unsupervised consensus hierarchical clustering (UCHC), and protein expression levels that were correlated with relevant clinical variables, The Cancer Genome Atlas, and other published data. WES identified mutations in FGFR3 (74.1%; 92% low-grade, 60% high-grade), KMT2D (44.4%), PIK3CA (25.9%), and TP53 (22.2%). APOBEC and CpG were the most common mutational signatures. UCHC of RNAseq data segregated samples into four molecular subtypes with the following characteristics. Cluster 1: no PIK3CA mutations, nonsmokers, high-grade

Uracil Accumulation and Mutagenesis Dominated by Cytosine Deamination in CpG Dinucleotides in Mice Lacking UNG and SMUG1

DOE PAGES

Alsøe, Lene; Sarno, Antonio; Carracedo, Sergio; ...

2017-08-03

Both a DNA lesion and an intermediate for antibody maturation, uracil is primarily processed by base excision repair (BER), either initiated by uracil-DNA glycosylase (UNG) or by single-strand selective monofunctional uracil DNA glycosylase (SMUG1). The relative in vivo contributions of each glycosylase remain elusive. To assess the impact of SMUG1 deficiency, we measured uracil and 5-hydroxymethyluracil, another SMUG1 substrate, in Smug1 -/ - mice. Here, we found that 5-hydroxymethyluracil accumulated in Smug1 -/ - tissues and correlated with 5-hydroxymethylcytosine levels. The highest increase was found in brain, which contained about 26-fold higher genomic 5-hydroxymethyluracil levels than the wild type. Smug1more » -/ - mice did not accumulate uracil in their genome and Ung -/ - mice showed slightly elevated uracil levels. Contrastingly, Ung -/ -Smug1 -/ - mice showed a synergistic increase in uracil levels with up to 25-fold higher uracil levels than wild type. Whole genome sequencing of UNG/SMUG1-deficient tumours revealed that combined UNG and SMUG1 deficiency leads to the accumulation of mutations, primarily C to T transitions within CpG sequences. This unexpected sequence bias suggests that CpG dinucleotides are intrinsically more mutation prone. In conclusion, we showed that SMUG1 efficiently prevent genomic uracil accumulation, even in the presence of UNG, and identified mutational signatures associated with combined UNG and SMUG1 deficiency.« less

Uracil Accumulation and Mutagenesis Dominated by Cytosine Deamination in CpG Dinucleotides in Mice Lacking UNG and SMUG1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Alsøe, Lene; Sarno, Antonio; Carracedo, Sergio

Both a DNA lesion and an intermediate for antibody maturation, uracil is primarily processed by base excision repair (BER), either initiated by uracil-DNA glycosylase (UNG) or by single-strand selective monofunctional uracil DNA glycosylase (SMUG1). The relative in vivo contributions of each glycosylase remain elusive. To assess the impact of SMUG1 deficiency, we measured uracil and 5-hydroxymethyluracil, another SMUG1 substrate, in Smug1 -/ - mice. Here, we found that 5-hydroxymethyluracil accumulated in Smug1 -/ - tissues and correlated with 5-hydroxymethylcytosine levels. The highest increase was found in brain, which contained about 26-fold higher genomic 5-hydroxymethyluracil levels than the wild type. Smug1more » -/ - mice did not accumulate uracil in their genome and Ung -/ - mice showed slightly elevated uracil levels. Contrastingly, Ung -/ -Smug1 -/ - mice showed a synergistic increase in uracil levels with up to 25-fold higher uracil levels than wild type. Whole genome sequencing of UNG/SMUG1-deficient tumours revealed that combined UNG and SMUG1 deficiency leads to the accumulation of mutations, primarily C to T transitions within CpG sequences. This unexpected sequence bias suggests that CpG dinucleotides are intrinsically more mutation prone. In conclusion, we showed that SMUG1 efficiently prevent genomic uracil accumulation, even in the presence of UNG, and identified mutational signatures associated with combined UNG and SMUG1 deficiency.« less

Gene discovery by chemical mutagenesis and whole-genome sequencing in Dictyostelium.

PubMed

Li, Cheng-Lin Frank; Santhanam, Balaji; Webb, Amanda Nicole; Zupan, Blaž; Shaulsky, Gad

2016-09-01

Whole-genome sequencing is a useful approach for identification of chemical-induced lesions, but previous applications involved tedious genetic mapping to pinpoint the causative mutations. We propose that saturation mutagenesis under low mutagenic loads, followed by whole-genome sequencing, should allow direct implication of genes by identifying multiple independent alleles of each relevant gene. We tested the hypothesis by performing three genetic screens with chemical mutagenesis in the social soil amoeba Dictyostelium discoideum Through genome sequencing, we successfully identified mutant genes with multiple alleles in near-saturation screens, including resistance to intense illumination and strong suppressors of defects in an allorecognition pathway. We tested the causality of the mutations by comparison to published data and by direct complementation tests, finding both dominant and recessive causative mutations. Therefore, our strategy provides a cost- and time-efficient approach to gene discovery by integrating chemical mutagenesis and whole-genome sequencing. The method should be applicable to many microbial systems, and it is expected to revolutionize the field of functional genomics in Dictyostelium by greatly expanding the mutation spectrum relative to other common mutagenesis methods. © 2016 Li et al.; Published by Cold Spring Harbor Laboratory Press.

Comparative Genome Analysis of Ciprofloxacin-Resistant Pseudomonas aeruginosa Reveals Genes Within Newly Identified High Variability Regions Associated With Drug Resistance Development

PubMed Central

Su, Hsun-Cheng; Khatun, Jainab; Kanavy, Dona M.

2013-01-01

The alarming rise of ciprofloxacin-resistant Pseudomonas aeruginosa has been reported in several clinical studies. Though the mutation of resistance genes and their role in drug resistance has been researched, the process by which the bacterium acquires high-level resistance is still not well understood. How does the genomic evolution of P. aeruginosa affect resistance development? Could the exposure of antibiotics to the bacteria enrich genomic variants that lead to the development of resistance, and if so, how are these variants distributed through the genome? To answer these questions, we performed 454 pyrosequencing and a whole genome analysis both before and after exposure to ciprofloxacin. The comparative sequence data revealed 93 unique resistance strain variation sites, which included a mutation in the DNA gyrase subunit A gene. We generated variation-distribution maps comparing the wild and resistant types, and isolated 19 candidates from three discrete resistance-associated high variability regions that had available transposon mutants, to perform a ciprofloxacin exposure assay. Of these region candidates with transposon disruptions, 79% (15/19) showed a reduction in the ability to gain high-level resistance, suggesting that genes within these high variability regions might enrich for certain functions associated with resistance development. PMID:23808957

Biological processes modulating longevity across primates: a phylogenetic genome-phenome analysis.

PubMed

Muntané, Gerard; Farré, Xavier; Antonio Rodríguez, Juan; Pegueroles, Cinta; Hughes, David A; de Magalhães, João Pedro; Gabaldón, Toni; Navarro, Arcadi

2018-05-21

Aging is a complex process affecting different species and individuals in different ways. Comparing genetic variation across species with their aging phenotypes will help understanding the molecular basis of aging and longevity. Although most studies on aging have so far focused on short-lived model organisms, recent comparisons of genomic, transcriptomic, and metabolomic data across lineages with different lifespans are unveiling molecular signatures associated with longevity. Here, we examine the relationship between genomic variation and maximum lifespan (MLS) across primate species. We used two different approaches. First, we searched for parallel amino acid mutations that co-occur with increases in longevity across the primate linage. Twenty-five such amino acid variants were identified, several of which have been previously reported by studies with different experimental setups and in different model organisms. The genes harboring these mutations are mainly enriched in functional categories such as Wound Healing, Blood Coagulation and Cardiovascular Disorders. We demonstrate that these pathways are highly enriched for pleiotropic effects, as predicted by the Antagonistic Pleiotropy (AP) theory of aging. A second approach was focused on changes in rates of protein evolution across the primate phylogeny. Using the Phylogenetic Generalized Least Squares (PGLS) we show that some genes exhibit strong correlations between their evolutionary rates and longevity-associated traits. These include genes in the Sphingosine 1-phosphate pathway, PI3K signaling and the Thrombin/protease-activated receptor pathway, among other cardiovascular processes. Together, these results shed light into human senescence patterns and underscore the power of comparative genomics to identify pathways related to aging and longevity.

Trade-off between Transcriptome Plasticity and Genome Evolution in Cephalopods.

PubMed

Liscovitch-Brauer, Noa; Alon, Shahar; Porath, Hagit T; Elstein, Boaz; Unger, Ron; Ziv, Tamar; Admon, Arie; Levanon, Erez Y; Rosenthal, Joshua J C; Eisenberg, Eli

2017-04-06

RNA editing, a post-transcriptional process, allows the diversification of proteomes beyond the genomic blueprint; however it is infrequently used among animals for this purpose. Recent reports suggesting increased levels of RNA editing in squids thus raise the question of the nature and effects of these events. We here show that RNA editing is particularly common in behaviorally sophisticated coleoid cephalopods, with tens of thousands of evolutionarily conserved sites. Editing is enriched in the nervous system, affecting molecules pertinent for excitability and neuronal morphology. The genomic sequence flanking editing sites is highly conserved, suggesting that the process confers a selective advantage. Due to the large number of sites, the surrounding conservation greatly reduces the number of mutations and genomic polymorphisms in protein-coding regions. This trade-off between genome evolution and transcriptome plasticity highlights the importance of RNA recoding as a strategy for diversifying proteins, particularly those associated with neural function. PAPERCLIP. Copyright © 2017 Elsevier Inc. All rights reserved.

Genome-wide comparison of ultraviolet and ethyl methanesulphonate mutagenesis methods for the brown alga Ectocarpus.

PubMed

Godfroy, Olivier; Peters, Akira F; Coelho, Susana M; Cock, J Mark

2015-12-01

Ectocarpus has emerged as a model organism for the brown algae and a broad range of genetic and genomic resources are being generated for this species. The aim of the work presented here was to evaluate two mutagenesis protocols based on ultraviolet irradiation and ethyl methanesulphonate treatment using genome resequencing to measure the number, type and distribution of mutations generated by the two methods. Ultraviolet irradiation generated a greater number of genetic lesions than ethyl methanesulphonate treatment, with more than 400 mutations being detected in the genome of the mutagenised individual. This study therefore confirms that the ultraviolet mutagenesis protocol is suitable for approaches that require a high density of mutations, such as saturation mutagenesis or Targeting Induced Local Lesions in Genomes (TILLING). Copyright © 2015 Elsevier B.V. All rights reserved.

THE GENOMIC LANDSCAPE OF PEDIATRIC AND YOUNG ADULT T-LINEAGE ACUTE LYMPHOBLASTIC LEUKEMIA

PubMed Central

Liu, Yu; Easton, John; Shao, Ying; Maciaszek, Jamie; Wang, Zhaoming; Wilkinson, Mark R.; McCastlain, Kelly; Edmonson, Michael; Pounds, Stanley B.; Shi, Lei; Zhou, Xin; Ma, Xiaotu; Sioson, Edgar; Li, Yongjin; Rusch, Michael; Gupta, Pankaj; Pei, Deqing; Cheng, Cheng; Smith, Malcolm A.; Auvil, Jaime Guidry; Gerhard, Daniela S.; Relling, Mary V.; Winick, Naomi J.; Carroll, Andrew J.; Heerema, Nyla A.; Raetz, Elizabeth; Devidas, Meenakshi; Willman, Cheryl L.; Harvey, Richard C.; Carroll, William L.; Dunsmore, Kimberly P.; Winter, Stuart S.; Wood, Brent L; Sorrentino, Brian P.; Downing, James R.; Loh, Mignon L.; Hunger, Stephen P; Zhang, Jinghui; Mullighan, Charles G.

2017-01-01

Genetic alterations activating NOTCH1 signaling and T cell transcription factors, coupled with inactivation of the INK4/ARF tumor suppressors are hallmarks of T-ALL, but detailed genome-wide sequencing of large T-ALL cohorts has not been performed. Using integrated genomic analysis of 264 T-ALL cases, we identify 106 putative driver genes, half of which were not previously described in childhood T-ALL (e.g. CCND3, CTCF, MYB, SMARCA4, ZFP36L2 and MYCN). We described new mechanisms of coding and non-coding alteration, and identify 10 recurrently altered pathways, with associations between mutated genes and pathways, and stage or subtype of T-ALL. For example, NRAS/FLT3 mutations were associated with immature T-ALL, JAK3/STAT5B mutations in HOX1 deregulated ALL, PTPN2 mutations in TLX1 T-ALL, and PIK3R1/PTEN mutations in TAL1 ALL, suggesting that different signaling pathways have distinct roles according to maturational stage. This genomic landscape provides a logical framework for the development of faithful genetic models and new therapeutic approaches. PMID:28671688

The CDC Hemophilia A Mutation Project (CHAMP) Mutation List: a New Online Resource

PubMed Central

Payne, Amanda B.; Miller, Connie H.; Kelly, Fiona M.; Soucie, J. Michael; Hooper, W. Craig

2015-01-01

Genotyping efforts in hemophilia A (HA) populations in many countries have identified large numbers of unique mutations in the Factor VIII gene (F8). To assist HA researchers conducting genotyping analyses, we have developed a listing of F8 mutations including those listed in existing locus-specific databases as well as those identified in patient populations and reported in the literature. Each mutation was reviewed and uniquely identified using Human Genome Variation Society (HGVS) nomenclature standards for coding DNA and predicted protein changes as well as traditional nomenclature based on the mature, processed protein. Listings also include the associated hemophilia severity classified by International Society of Thrombosis and Haemostasis (ISTH) criteria, associations of the mutations with inhibitors, and reference information. The mutation list currently contains 2,537 unique mutations known to cause HA. HA severity caused by the mutation is available for 2,022 mutations (80%) and information on inhibitors is available for 1,816 mutations (72%). The CDC Hemophilia A Mutation Project (CHAMP) Mutation List is available at http://www.cdc.gov/hemophiliamutations for download and search and will be updated quarterly based on periodic literature reviews and submitted reports. PMID:23280990

Similar Mutation Rates but Highly Diverse Mutation Spectra in Ascomycete and Basidiomycete Yeasts.

PubMed

Long, Hongan; Behringer, Megan G; Williams, Emily; Te, Ronald; Lynch, Michael

2016-12-01

Yeast species are extremely diverse and not monophyletic. Because the majority of yeast research focuses on ascomycetes, the mutational determinants of genetic diversity across yeast species are not well understood. By combining mutation-accumulation techniques with whole-genome sequencing, we resolved the genomic mutation rate and spectrum of the oleaginous (oil-producing) ‘red yeast’ Rhodotorula toruloides, the first such study in the fungal phylum Basidiomycota. We find that the mutation spectrum is quite different from what has been observed in all other studied unicellular eukaryotes, but similar to that in most bacteria—a predominance of transitions relative to transversions. Rhodotorula toruloides has a significantly higher A:T→G:C transition rate—possibly elevated by the abundant flanking G/C nucleotides in the GC-rich genome, as well as a much lower G:C→T:A transversion rate. In spite of these striking differences, there are substantial consistencies between R. toruloides and the ascomycete model yeasts: a spontaneous base-substitution mutation rate of 1.90 × 10 −10 per site per cell division as well as an elevated mutation rate at non-methylated 5'CpG3' sites. These results imply the evolution of variable mutation spectra in the face of similar mutation rates in yeasts.

Contribution of silent mutations to thermal adaptation of RNA bacteriophage Qβ.

PubMed

Kashiwagi, Akiko; Sugawara, Ryu; Sano Tsushima, Fumie; Kumagai, Tomofumi; Yomo, Tetsuya

2014-10-01

Changes in protein function and other biological properties, such as RNA structure, are crucial for adaptation of organisms to novel or inhibitory environments. To investigate how mutations that do not alter amino acid sequence may be positively selected, we performed a thermal adaptation experiment using the single-stranded RNA bacteriophage Qβ in which the culture temperature was increased from 37.2°C to 41.2°C and finally to an inhibitory temperature of 43.6°C in a stepwise manner in three independent lines. Whole-genome analysis revealed 31 mutations, including 14 mutations that did not result in amino acid sequence alterations, in this thermal adaptation. Eight of the 31 mutations were observed in all three lines. Reconstruction and fitness analyses of Qβ strains containing only mutations observed in all three lines indicated that five mutations that did not result in amino acid sequence changes but increased the amplification ratio appeared in the course of adaptation to growth at 41.2°C. Moreover, these mutations provided a suitable genetic background for subsequent mutations, altering the fitness contribution from deleterious to beneficial. These results clearly showed that mutations that do not alter the amino acid sequence play important roles in adaptation of this single-stranded RNA virus to elevated temperature. Recent studies using whole-genome analysis technology suggested the importance of mutations that do not alter the amino acid sequence for adaptation of organisms to novel environmental conditions. It is necessary to investigate how these mutations may be positively selected and to determine to what degree such mutations that do not alter amino acid sequences contribute to adaptive evolution. Here, we report the roles of these silent mutations in thermal adaptation of RNA bacteriophage Qβ based on experimental evolution during which Qβ showed adaptation to growth at an inhibitory temperature. Intriguingly, four synonymous mutations and one mutation in the untranslated region that spread widely in the Qβ population during the adaptation process at moderately high temperature provided a suitable genetic background to alter the fitness contribution of subsequent mutations from deleterious to beneficial at a higher temperature. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Untangling the Web: The Diverse Functions of the PIWI/piRNA Pathway

PubMed Central

Mani, Sneha Ramesh; Juliano, Celina E.

2014-01-01

SUMMARY Small RNAs impact several cellular processes through gene regulation. Argonaute proteins bind small RNAs to form effector complexes that control transcriptional and post-transcriptional gene expression. PIWI proteins belong to the Argonaute protein family, and bind PIWI-interacting RNAs (piRNAs). They are highly abundant in the germline, but are also expressed in some somatic tissues. The PIWI/piRNA pathway has a role in transposon repression in Drosophila, which occurs both by epigenetic regulation and post-transcriptional degradation of transposon mRNAs. These functions are conserved, but clear differences in the extent and mechanism of transposon repression exist between species. Mutations in piwi genes lead to the upregulation of transposon mRNAs. It is hypothesized that this increased transposon mobilization leads to genomic instability and thus sterility, although no causal link has been established between transposon upregulation and genome instability. An alternative scenario could be that piwi mutations directly affect genomic instability, and thus lead to increased transposon expression. We propose that the PIWI/piRNA pathway controls genome stability in several ways: suppression of transposons, direct regulation of chromatin architecture and regulation of genes that control important biological processes related to genome stability. The PIWI/piRNA pathway also regulates at least some, if not many, protein-coding genes, which further lends support to the idea that piwi genes may have broader functions beyond transposon repression. An intriguing possibility is that the PIWI/piRNA pathway is using transposon sequences to coordinate the expression of large groups of genes to regulate cellular function. PMID:23712694

Evaluation of Quality Assessment Protocols for High Throughput Genome Resequencing Data

PubMed Central

Chiara, Matteo; Pavesi, Giulio

2017-01-01

Large-scale initiatives aiming to recover the complete sequence of thousands of human genomes are currently being undertaken worldwide, concurring to the generation of a comprehensive catalog of human genetic variation. The ultimate and most ambitious goal of human population scale genomics is the characterization of the so-called human “variome,” through the identification of causal mutations or haplotypes. Several research institutions worldwide currently use genotyping assays based on Next-Generation Sequencing (NGS) for diagnostics and clinical screenings, and the widespread application of such technologies promises major revolutions in medical science. Bioinformatic analysis of human resequencing data is one of the main factors limiting the effectiveness and general applicability of NGS for clinical studies. The requirement for multiple tools, to be combined in dedicated protocols in order to accommodate different types of data (gene panels, exomes, or whole genomes) and the high variability of the data makes difficult the establishment of a ultimate strategy of general use. While there already exist several studies comparing sensitivity and accuracy of bioinformatic pipelines for the identification of single nucleotide variants from resequencing data, little is known about the impact of quality assessment and reads pre-processing strategies. In this work we discuss major strengths and limitations of the various genome resequencing protocols are currently used in molecular diagnostics and for the discovery of novel disease-causing mutations. By taking advantage of publicly available data we devise and suggest a series of best practices for the pre-processing of the data that consistently improve the outcome of genotyping with minimal impacts on computational costs. PMID:28736571

MCMC genome rearrangement.

PubMed

Miklós, István

2003-10-01

As more and more genomes have been sequenced, genomic data is rapidly accumulating. Genome-wide mutations are believed more neutral than local mutations such as substitutions, insertions and deletions, therefore phylogenetic investigations based on inversions, transpositions and inverted transpositions are less biased by the hypothesis on neutral evolution. Although efficient algorithms exist for obtaining the inversion distance of two signed permutations, there is no reliable algorithm when both inversions and transpositions are considered. Moreover, different type of mutations happen with different rates, and it is not clear how to weight them in a distance based approach. We introduce a Markov Chain Monte Carlo method to genome rearrangement based on a stochastic model of evolution, which can estimate the number of different evolutionary events needed to sort a signed permutation. The performance of the method was tested on simulated data, and the estimated numbers of different types of mutations were reliable. Human and Drosophila mitochondrial data were also analysed with the new method. The mixing time of the Markov Chain is short both in terms of CPU times and number of proposals. The source code in C is available on request from the author.

Clonal selection in xenografted TAM recapitulates the evolutionary process of myeloid leukemia in Down syndrome.

PubMed

Saida, Satoshi; Watanabe, Ken-ichiro; Sato-Otsubo, Aiko; Terui, Kiminori; Yoshida, Kenichi; Okuno, Yusuke; Toki, Tsutomu; Wang, RuNan; Shiraishi, Yuichi; Miyano, Satoru; Kato, Itaru; Morishima, Tatsuya; Fujino, Hisanori; Umeda, Katsutsugu; Hiramatsu, Hidefumi; Adachi, Souichi; Ito, Etsuro; Ogawa, Seishi; Ito, Mamoru; Nakahata, Tatsutoshi; Heike, Toshio

2013-05-23

Transient abnormal myelopoiesis (TAM) is a clonal preleukemic disorder that progresses to myeloid leukemia of Down syndrome (ML-DS) through the accumulation of genetic alterations. To investigate the mechanism of leukemogenesis in this disorder, a xenograft model of TAM was established using NOD/Shi-scid, interleukin (IL)-2Rγ(null) mice. Serial engraftment after transplantation of cells from a TAM patient who developed ML-DS a year later demonstrated their self-renewal capacity. A GATA1 mutation and no copy number alterations (CNAs) were detected in the primary patient sample by conventional genomic sequencing and CNA profiling. However, in serial transplantations, engrafted TAM-derived cells showed the emergence of divergent subclones with another GATA1 mutation and various CNAs, including a 16q deletion and 1q gain, which are clinically associated with ML-DS. Detailed genomic analysis identified minor subclones with a 16q deletion or this distinct GATA1 mutation in the primary patient sample. These results suggest that genetically heterogeneous subclones with varying leukemia-initiating potential already exist in the neonatal TAM phase, and ML-DS may develop from a pool of such minor clones through clonal selection. Our xenograft model of TAM may provide unique insight into the evolutionary process of leukemia.

Mutational load of the mitochondrial genome predicts pathological features and biochemical recurrence in prostate cancer.

PubMed

Kalsbeek, Anton M F; Chan, Eva F K; Grogan, Judith; Petersen, Desiree C; Jaratlerdsiri, Weerachai; Gupta, Ruta; Lyons, Ruth J; Haynes, Anne-Maree; Horvath, Lisa G; Kench, James G; Stricker, Phillip D; Hayes, Vanessa M

2016-10-05

Prostate cancer management is complicated by extreme disease heterogeneity, which is further limited by availability of prognostic biomarkers. Recognition of prostate cancer as a genetic disease has prompted a focus on the nuclear genome for biomarker discovery, with little attention given to the mitochondrial genome. While it is evident that mitochondrial DNA (mtDNA) mutations are acquired during prostate tumorigenesis, no study has evaluated the prognostic value of mtDNA variation. Here we used next-generation sequencing to interrogate the mitochondrial genomes from prostate tissue biopsies and matched blood of 115 men having undergone a radical prostatectomy for which there was a mean of 107 months clinical follow-up. We identified 74 unique prostate cancer specific somatic mtDNA variants in 50 patients, providing significant expansion to the growing catalog of prostate cancer mtDNA mutations. While no single variant or variant cluster showed recurrence across multiple patients, we observe a significant positive correlation between the total burden of acquired mtDNA variation and elevated Gleason Score at diagnosis and biochemical relapse. We add to accumulating evidence that total acquired genomic burden, rather than specific mtDNA mutations, has diagnostic value. This is the first study to demonstrate the prognostic potential of mtDNA mutational burden in prostate cancer.

Reduced Mutation Rate and Increased Transformability of Transposon-Free Acinetobacter baylyi ADP1-ISx

PubMed Central

Suárez, Gabriel A.; Renda, Brian A.; Dasgupta, Aurko

2017-01-01

ABSTRACT The genomes of most bacteria contain mobile DNA elements that can contribute to undesirable genetic instability in engineered cells. In particular, transposable insertion sequence (IS) elements can rapidly inactivate genes that are important for a designed function. We deleted all six copies of IS1236 from the genome of the naturally transformable bacterium Acinetobacter baylyi ADP1. The natural competence of ADP1 made it possible to rapidly repair deleterious point mutations that arose during strain construction. In the resulting ADP1-ISx strain, the rates of mutations inactivating a reporter gene were reduced by 7- to 21-fold. This reduction was higher than expected from the incidence of new IS1236 insertions found during a 300-day mutation accumulation experiment with wild-type ADP1 that was used to estimate spontaneous mutation rates in the strain. The extra improvement appears to be due in part to eliminating large deletions caused by IS1236 activity, as the point mutation rate was unchanged in ADP1-ISx. Deletion of an error-prone polymerase (dinP) and a DNA damage response regulator (umuDAb [the umuD gene of A. baylyi]) from the ADP1-ISx genome did not further reduce mutation rates. Surprisingly, ADP1-ISx exhibited increased transformability. This improvement may be due to less autolysis and aggregation of the engineered cells than of the wild type. Thus, deleting IS elements from the ADP1 genome led to a greater than expected increase in evolutionary reliability and unexpectedly enhanced other key strain properties, as has been observed for other clean-genome bacterial strains. ADP1-ISx is an improved chassis for metabolic engineering and other applications. IMPORTANCE Acinetobacter baylyi ADP1 has been proposed as a next-generation bacterial host for synthetic biology and genome engineering due to its ability to efficiently take up DNA from its environment during normal growth. We deleted transposable elements that are capable of copying themselves, inserting into other genes, and thereby inactivating them from the ADP1 genome. The resulting “clean-genome” ADP1-ISx strain exhibited larger reductions in the rates of inactivating mutations than expected from spontaneous mutation rates measured via whole-genome sequencing of lineages evolved under relaxed selection. Surprisingly, we also found that IS element activity reduces transformability and is a major cause of cell aggregation and death in wild-type ADP1 grown under normal laboratory conditions. More generally, our results demonstrate that domesticating a bacterial genome by removing mobile DNA elements that have accumulated during evolution in the wild can have unanticipated benefits. PMID:28667117

Bursts of retrotransposition reproduced in Arabidopsis.

PubMed

Tsukahara, Sayuri; Kobayashi, Akie; Kawabe, Akira; Mathieu, Olivier; Miura, Asuka; Kakutani, Tetsuji

2009-09-17

Retrotransposons, which proliferate by reverse transcription of RNA intermediates, comprise a major portion of plant genomes. Plants often change the genome size and organization during evolution by rapid proliferation and deletion of long terminal repeat (LTR) retrotransposons. Precise transposon sequences throughout the Arabidopsis thaliana genome and the trans-acting mutations affecting epigenetic states make it an ideal model organism with which to study transposon dynamics. Here we report the mobilization of various families of endogenous A. thaliana LTR retrotransposons identified through genetic and genomic approaches with high-resolution genomic tiling arrays and mutants in the chromatin-remodelling gene DDM1 (DECREASE IN DNA METHYLATION 1). Using multiple lines of self-pollinated ddm1 mutant, we detected an increase in copy number, and verified this for various retrotransposons in a gypsy family (ATGP3) and copia families (ATCOPIA13, ATCOPIA21, ATCOPIA93), and also for a DNA transposon of a Mutator family, VANDAL21. A burst of retrotransposition occurred stochastically and independently for each element, suggesting an additional autocatalytic process. Furthermore, comparison of the identified LTR retrotransposons in related Arabidopsis species revealed that a lineage-specific burst of retrotransposition of these elements did indeed occur in natural Arabidopsis populations. The recent burst of retrotransposition in natural population is targeted to centromeric repeats, which is presumably less harmful than insertion into genes. The ddm1-induced retrotransposon proliferations and genome rearrangements mimic the transposon-mediated genome dynamics during evolution and provide experimental systems with which to investigate the controlling molecular factors directly.

Radiation-induced genomic instability

NASA Technical Reports Server (NTRS)

Kronenberg, A.

1994-01-01

Quantitative assessment of the heritable somatic effects of ionizing radiation exposures has relied upon the assumption that radiation-induced lesions were 'fixed' in the DNA prior to the first postirradiation mitosis. Lesion conversion was thought to occur during the initial round of DNA replication or as a consequence of error-prone enzymatic processing of lesions. The standard experimental protocols for the assessment of a variety of radiation-induced endpoints (cell death, specific locus mutations, neoplastic transformation and chromosome aberrations) evaluate these various endpoints at a single snapshot in time. In contrast with the aforementioned approaches, some studies have specifically assessed radiation effects as a function of time following exposure. Evidence has accumulated in support of the hypothesis that radiation exposure induces a persistent destabilization of the genome. This instability has been observed as a delayed expression of lethal mutations, as an enhanced rate of accumulation of non-lethal heritable alterations, and as a progressive intraclonal chromosomal heterogeneity. The genetic controls and biochemical mechanisms underlying radiation-induced genomic instability have not yet been delineated. The aim is to integrate the accumulated evidence that suggests that radiation exposure has a persistent effect on the stability of the mammalian genome.

Mutagenesis of diploid mammalian genes by gene entrapment

PubMed Central

Lin, Qing; Donahue, Sarah L.; Moore-Jarrett, Tracy; Cao, Shang; Osipovich, Anna B.; Ruley, H. Earl

2006-01-01

The present study describes a genome-wide method for biallelic mutagenesis in mammalian cells. Novel poly(A) gene trap vectors, which contain features for direct cloning vector–cell fusion transcripts and for post-entrapment genome engineering, were used to generate a library of 979 mutant ES cells. The entrapment mutations generally disrupted gene expression and were readily transmitted through the germline, establishing the library as a resource for constructing mutant mice. Cells homozygous for most entrapment loci could be isolated by selecting for enhanced expression of an inserted neomycin-resistance gene that resulted from losses of heterozygosity (LOH). The frequencies of LOH measured at 37 sites in the genome ranged from 1.3 × 10−5 to 1.2 × 10−4 per cell and increased with increasing distance from the centromere, implicating mitotic recombination in the process. The ease and efficiency of obtaining homozygous mutations will (i) facilitate genetic studies of gene function in cultured cells, (ii) permit genome-wide studies of recombination events that result in LOH and mediate a type of chromosomal instability important in carcinogenesis, and (iii) provide new strategies for phenotype-driven mutagenesis screens in mammalian cells. PMID:17062627

Accessories make the outfit: Accessory Chromosomes and other dispensable DNA regions in plant-pathogenic Fungi.

PubMed

Bertazzoni, Stefania; Williams, Angela; Jones, Darcy A B; Syme, Robert A; Tan, Kar-Chun; Hane, James Kyawzwar

2018-04-17

Fungal pathogen genomes can often be divided into core and accessory regions. Accessory regions may be comprised of either accessory regions (ARs) within core chromosomes (CCs), or wholly-dispensable (accessory) chromosomes (ACs). Fungal ACs and ARs typically accumulate mutations and structural rearrangements more rapidly over time than CCs, and many harbour genes relevant to host-pathogen interactions. These regions are of particular interest in plant pathology and include host-specific virulence factors and secondary metabolite synthesis gene clusters. This review outlines known ACs and ARs in fungal genomes, methods used for their detection, their common properties that differentiate them from the core genome, and what is currently known of their various roles in pathogenicity. Reports on the evolutionary processes generating and shaping AC/AR compartments are discussed, including repeat induced point mutation (RIP) and breakage-fusion-bridge (BFB) cycles. Previously ACs have been studied extensively within key genera including Fusarium, Zymoseptoria and Alternaria, but are growing in their frequency of observation and perceived importance across a wider range of fungal species. Recent advances in sequencing technologies permit affordable genome assembly and re-sequencing of populations that will facilitate further discovery and routine screening of ACs.

Homo sapiens, Homo neanderthalensis and the Denisova specimen: New insights on their evolutionary histories using whole-genome comparisons.

PubMed

Paixão-Côrtes, Vanessa Rodrigues; Viscardi, Lucas Henrique; Salzano, Francisco Mauro; Hünemeier, Tábita; Bortolini, Maria Cátira

2012-12-01

After a brief review of the most recent findings in the study of human evolution, an extensive comparison of the complete genomes of our nearest relative, the chimpanzee (Pan troglodytes), of extant Homo sapiens, archaic Homo neanderthalensis and the Denisova specimen were made. The focus was on non-synonymous mutations, which consequently had an impact on protein levels and these changes were classified according to degree of effect. A total of 10,447 non-synonymous substitutions were found in which the derived allele is fixed or nearly fixed in humans as compared to chimpanzee. Their most frequent location was on chromosome 21. Their presence was then searched in the two archaic genomes. Mutations in 381 genes would imply radical amino acid changes, with a fraction of these related to olfaction and other important physiological processes. Eight new alleles were identified in the Neanderthal and/or Denisova genetic pools. Four others, possibly affecting cognition, occured both in the sapiens and two other archaic genomes. The selective sweep that gave rise to Homo sapiens could, therefore, have initiated before the modern/archaic human divergence.

Differential regulation of hepatitis B virus core protein expression and genome replication by a small upstream open reading frame and naturally occurring mutations in the precore region.

PubMed

Zong, Li; Qin, Yanli; Jia, Haodi; Ye, Lei; Wang, Yongxiang; Zhang, Jiming; Wands, Jack R; Tong, Shuping; Li, Jisu

2017-05-01

Hepatitis B virus (HBV) transcribes two subsets of 3.5-kb RNAs: precore RNA for hepatitis B e antigen (HBeAg) expression, and pregenomic RNA for core and P protein translation as well as genome replication. HBeAg expression could be prevented by mutations in the precore region, while an upstream open reading frame (uORF) has been proposed as a negative regulator of core protein translation. We employed replication competent HBV DNA constructs and transient transfection experiments in Huh7 cells to verify the uORF effect and to explore the alternative function of precore RNA. Optimized Kozak sequence for the uORF or extra ATG codons as present in some HBV genotypes reduced core protein expression. G1896A nonsense mutation promoted more efficient core protein expression than mutated precore ATG, while a +1 frameshift mutation was ineffective. In conclusion, various HBeAg-negative precore mutations and mutations affecting uORF differentially regulate core protein expression and genome replication. Copyright © 2017 Elsevier Inc. All rights reserved.

Microsatellites as targets of natural selection.

PubMed

Haasl, Ryan J; Payseur, Bret A

2013-02-01

The ability to survey polymorphism on a genomic scale has enabled genome-wide scans for the targets of natural selection. Theory that connects patterns of genetic variation to evidence of natural selection most often assumes a diallelic locus and no recurrent mutation. Although these assumptions are suitable to selection that targets single nucleotide variants, fundamentally different types of mutation generate abundant polymorphism in genomes. Moreover, recent empirical results suggest that mutationally complex, multiallelic loci including microsatellites and copy number variants are sometimes targeted by natural selection. Given their abundance, the lack of inference methods tailored to the mutational peculiarities of these types of loci represents a notable gap in our ability to interrogate genomes for signatures of natural selection. Previous theoretical investigations of mutation-selection balance at multiallelic loci include assumptions that limit their application to inference from empirical data. Focusing on microsatellites, we assess the dynamics and population-level consequences of selection targeting mutationally complex variants. We develop general models of a multiallelic fitness surface, a realistic model of microsatellite mutation, and an efficient simulation algorithm. Using these tools, we explore mutation-selection-drift equilibrium at microsatellites and investigate the mutational history and selective regime of the microsatellite that causes Friedreich's ataxia. We characterize microsatellite selective events by their duration and cost, note similarities to sweeps from standing point variation, and conclude that it is premature to label microsatellites as ubiquitous agents of efficient adaptive change. Together, our models and simulation algorithm provide a powerful framework for statistical inference, which can be used to test the neutrality of microsatellites and other multiallelic variants.

Microsatellites as Targets of Natural Selection

PubMed Central

Haasl, Ryan J.; Payseur, Bret A.

2013-01-01

The ability to survey polymorphism on a genomic scale has enabled genome-wide scans for the targets of natural selection. Theory that connects patterns of genetic variation to evidence of natural selection most often assumes a diallelic locus and no recurrent mutation. Although these assumptions are suitable to selection that targets single nucleotide variants, fundamentally different types of mutation generate abundant polymorphism in genomes. Moreover, recent empirical results suggest that mutationally complex, multiallelic loci including microsatellites and copy number variants are sometimes targeted by natural selection. Given their abundance, the lack of inference methods tailored to the mutational peculiarities of these types of loci represents a notable gap in our ability to interrogate genomes for signatures of natural selection. Previous theoretical investigations of mutation-selection balance at multiallelic loci include assumptions that limit their application to inference from empirical data. Focusing on microsatellites, we assess the dynamics and population-level consequences of selection targeting mutationally complex variants. We develop general models of a multiallelic fitness surface, a realistic model of microsatellite mutation, and an efficient simulation algorithm. Using these tools, we explore mutation-selection-drift equilibrium at microsatellites and investigate the mutational history and selective regime of the microsatellite that causes Friedreich’s ataxia. We characterize microsatellite selective events by their duration and cost, note similarities to sweeps from standing point variation, and conclude that it is premature to label microsatellites as ubiquitous agents of efficient adaptive change. Together, our models and simulation algorithm provide a powerful framework for statistical inference, which can be used to test the neutrality of microsatellites and other multiallelic variants. PMID:23104080

Genomic and Epigenomic Landscapes of Adult De Novo Acute Myeloid Leukemia

PubMed Central

2013-01-01

BACKGROUND Many mutations that contribute to the pathogenesis of acute myeloid leukemia (AML) are undefined. The relationships between patterns of mutations and epigenetic phenotypes are not yet clear. METHODS We analyzed the genomes of 200 clinically annotated adult cases of de novo AML, using either whole-genome sequencing (50 cases) or whole-exome sequencing (150 cases), along with RNA and microRNA sequencing and DNA-methylation analysis. RESULTS AML genomes have fewer mutations than most other adult cancers, with an average of only 13 mutations found in genes. Of these, an average of 5 are in genes that are recurrently mutated in AML. A total of 23 genes were significantly mutated, and another 237 were mutated in two or more samples. Nearly all samples had at least 1 nonsynonymous mutation in one of nine categories of genes that are almost certainly relevant for pathogenesis, including transcription-factor fusions (18% of cases), the gene encoding nucleophosmin (NPM1) (27%), tumor-suppressor genes (16%), DNA-methylation–related genes (44%), signaling genes (59%), chromatin-modifying genes (30%), myeloid transcription-factor genes (22%), cohesin-complex genes (13%), and spliceosome-complex genes (14%). Patterns of cooperation and mutual exclusivity suggested strong biologic relationships among several of the genes and categories. CONCLUSIONS We identified at least one potential driver mutation in nearly all AML samples and found that a complex interplay of genetic events contributes to AML pathogenesis in individual patients. The databases from this study are widely available to serve as a foundation for further investigations of AML pathogenesis, classification, and risk stratification. (Funded by the National Institutes of Health.) PMID:23634996

Snowball: resampling combined with distance-based regression to discover transcriptional consequences of a driver mutation

PubMed Central

Xu, Yaomin; Guo, Xingyi; Sun, Jiayang; Zhao, Zhongming

2015-01-01

Motivation: Large-scale cancer genomic studies, such as The Cancer Genome Atlas (TCGA), have profiled multidimensional genomic data, including mutation and expression profiles on a variety of cancer cell types, to uncover the molecular mechanism of cancerogenesis. More than a hundred driver mutations have been characterized that confer the advantage of cell growth. However, how driver mutations regulate the transcriptome to affect cellular functions remains largely unexplored. Differential analysis of gene expression relative to a driver mutation on patient samples could provide us with new insights in understanding driver mutation dysregulation in tumor genome and developing personalized treatment strategies. Results: Here, we introduce the Snowball approach as a highly sensitive statistical analysis method to identify transcriptional signatures that are affected by a recurrent driver mutation. Snowball utilizes a resampling-based approach and combines a distance-based regression framework to assign a robust ranking index of genes based on their aggregated association with the presence of the mutation, and further selects the top significant genes for downstream data analyses or experiments. In our application of the Snowball approach to both synthesized and TCGA data, we demonstrated that it outperforms the standard methods and provides more accurate inferences to the functional effects and transcriptional dysregulation of driver mutations. Availability and implementation: R package and source code are available from CRAN at http://cran.r-project.org/web/packages/DESnowball, and also available at http://bioinfo.mc.vanderbilt.edu/DESnowball/. Contact: zhongming.zhao@vanderbilt.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25192743

Computational approaches to identify functional genetic variants in cancer genomes

PubMed Central

Gonzalez-Perez, Abel; Mustonen, Ville; Reva, Boris; Ritchie, Graham R.S.; Creixell, Pau; Karchin, Rachel; Vazquez, Miguel; Fink, J. Lynn; Kassahn, Karin S.; Pearson, John V.; Bader, Gary; Boutros, Paul C.; Muthuswamy, Lakshmi; Ouellette, B.F. Francis; Reimand, Jüri; Linding, Rune; Shibata, Tatsuhiro; Valencia, Alfonso; Butler, Adam; Dronov, Serge; Flicek, Paul; Shannon, Nick B.; Carter, Hannah; Ding, Li; Sander, Chris; Stuart, Josh M.; Stein, Lincoln D.; Lopez-Bigas, Nuria

2014-01-01

The International Cancer Genome Consortium (ICGC) aims to catalog genomic abnormalities in tumors from 50 different cancer types. Genome sequencing reveals hundreds to thousands of somatic mutations in each tumor, but only a minority drive tumor progression. We present the result of discussions within the ICGC on how to address the challenge of identifying mutations that contribute to oncogenesis, tumor maintenance or response to therapy, and recommend computational techniques to annotate somatic variants and predict their impact on cancer phenotype. PMID:23900255

Genome-wide analysis of mutations in mutant lineages selected following fast-neutron irradiation mutagenesis of Arabidopsis thaliana

PubMed Central

Belfield, Eric J.; Gan, Xiangchao; Mithani, Aziz; Brown, Carly; Jiang, Caifu; Franklin, Keara; Alvey, Elizabeth; Wibowo, Anjar; Jung, Marko; Bailey, Kit; Kalwani, Sharan; Ragoussis, Jiannis; Mott, Richard; Harberd, Nicholas P.

2012-01-01

Ionizing radiation has long been known to induce heritable mutagenic change in DNA sequence. However, the genome-wide effect of radiation is not well understood. Here we report the molecular properties and frequency of mutations in phenotypically selected mutant lines isolated following exposure of the genetic model flowering plant Arabidopsis thaliana to fast neutrons (FNs). Previous studies suggested that FNs predominantly induce deletions longer than a kilobase in A. thaliana. However, we found a higher frequency of single base substitution than deletion mutations. While the overall frequency and molecular spectrum of fast-neutron (FN)–induced single base substitutions differed substantially from those of “background” mutations arising spontaneously in laboratory-grown plants, G:C>A:T transitions were favored in both. We found that FN-induced G:C>A:T transitions were concentrated at pyrimidine dinucleotide sites, suggesting that FNs promote the formation of mutational covalent linkages between adjacent pyrimidine residues. In addition, we found that FNs induced more single base than large deletions, and that these single base deletions were possibly caused by replication slippage. Our observations provide an initial picture of the genome-wide molecular profile of mutations induced in A. thaliana by FN irradiation and are particularly informative of the nature and extent of genome-wide mutation in lines selected on the basis of mutant phenotypes from FN-mutagenized A. thaliana populations. PMID:22499668

Linking minimum inhibitory concentrations to whole genome sequence-predicted drug resistance in Mycobacterium tuberculosis strains from Romania.

PubMed

Ruesen, Carolien; Riza, Anca Lelia; Florescu, Adriana; Chaidir, Lidya; Editoiu, Cornelia; Aalders, Nicole; Nicolosu, Dragos; Grecu, Victor; Ioana, Mihai; van Crevel, Reinout; van Ingen, Jakko

2018-06-26

Mycobacterium tuberculosis drug resistance poses a major threat to tuberculosis control. Current phenotypic tests for drug susceptibility are time-consuming, technically complex, and expensive. Whole genome sequencing is a promising alternative, though the impact of different drug resistance mutations on the minimum inhibitory concentration (MIC) remains to be investigated. We examined the genomes of 72 phenotypically drug-resistant Mycobacterium tuberculosis isolates from 72 Romanian patients for drug resistance mutations. MICs for first- and second-line drugs were determined using the MycoTB microdilution method. These MICs were compared to macrodilution critical concentration testing by the Mycobacterium Growth Indicator Tube (MGIT) platform and correlated to drug resistance mutations. Sixty-three (87.5%) isolates harboured drug resistance mutations; 48 (66.7%) were genotypically multidrug-resistant. Different drug resistance mutations were associated with different MIC ranges; katG S315T for isoniazid, and rpoB S450L for rifampicin were associated with high MICs. However, several mutations such as in rpoB, rrs and rpsL, or embB were associated with MIC ranges including the critical concentration for rifampicin, aminoglycosides or ethambutol, respectively. Different resistance mutations lead to distinct MICs, some of which may still be overcome by increased dosing. Whole genome sequencing can aid in the timely diagnosis of Mycobacterium tuberculosis drug resistance and guide clinical decision-making.

Real-time, portable genome sequencing for Ebola surveillance.

PubMed

Quick, Joshua; Loman, Nicholas J; Duraffour, Sophie; Simpson, Jared T; Severi, Ettore; Cowley, Lauren; Bore, Joseph Akoi; Koundouno, Raymond; Dudas, Gytis; Mikhail, Amy; Ouédraogo, Nobila; Afrough, Babak; Bah, Amadou; Baum, Jonathan Hj; Becker-Ziaja, Beate; Boettcher, Jan-Peter; Cabeza-Cabrerizo, Mar; Camino-Sanchez, Alvaro; Carter, Lisa L; Doerrbecker, Juiliane; Enkirch, Theresa; Dorival, Isabel Graciela García; Hetzelt, Nicole; Hinzmann, Julia; Holm, Tobias; Kafetzopoulou, Liana Eleni; Koropogui, Michel; Kosgey, Abigail; Kuisma, Eeva; Logue, Christopher H; Mazzarelli, Antonio; Meisel, Sarah; Mertens, Marc; Michel, Janine; Ngabo, Didier; Nitzsche, Katja; Pallash, Elisa; Patrono, Livia Victoria; Portmann, Jasmine; Repits, Johanna Gabriella; Rickett, Natasha Yasmin; Sachse, Andrea; Singethan, Katrin; Vitoriano, Inês; Yemanaberhan, Rahel L; Zekeng, Elsa G; Trina, Racine; Bello, Alexander; Sall, Amadou Alpha; Faye, Ousmane; Faye, Oumar; Magassouba, N'Faly; Williams, Cecelia V; Amburgey, Victoria; Winona, Linda; Davis, Emily; Gerlach, Jon; Washington, Franck; Monteil, Vanessa; Jourdain, Marine; Bererd, Marion; Camara, Alimou; Somlare, Hermann; Camara, Abdoulaye; Gerard, Marianne; Bado, Guillaume; Baillet, Bernard; Delaune, Déborah; Nebie, Koumpingnin Yacouba; Diarra, Abdoulaye; Savane, Yacouba; Pallawo, Raymond Bernard; Gutierrez, Giovanna Jaramillo; Milhano, Natacha; Roger, Isabelle; Williams, Christopher J; Yattara, Facinet; Lewandowski, Kuiama; Taylor, Jamie; Rachwal, Philip; Turner, Daniel; Pollakis, Georgios; Hiscox, Julian A; Matthews, David A; O'Shea, Matthew K; Johnston, Andrew McD; Wilson, Duncan; Hutley, Emma; Smit, Erasmus; Di Caro, Antonino; Woelfel, Roman; Stoecker, Kilian; Fleischmann, Erna; Gabriel, Martin; Weller, Simon A; Koivogui, Lamine; Diallo, Boubacar; Keita, Sakoba; Rambaut, Andrew; Formenty, Pierre; Gunther, Stephan; Carroll, Miles W

2016-02-11

The Ebola virus disease epidemic in West Africa is the largest on record, responsible for over 28,599 cases and more than 11,299 deaths. Genome sequencing in viral outbreaks is desirable to characterize the infectious agent and determine its evolutionary rate. Genome sequencing also allows the identification of signatures of host adaptation, identification and monitoring of diagnostic targets, and characterization of responses to vaccines and treatments. The Ebola virus (EBOV) genome substitution rate in the Makona strain has been estimated at between 0.87 × 10(-3) and 1.42 × 10(-3) mutations per site per year. This is equivalent to 16-27 mutations in each genome, meaning that sequences diverge rapidly enough to identify distinct sub-lineages during a prolonged epidemic. Genome sequencing provides a high-resolution view of pathogen evolution and is increasingly sought after for outbreak surveillance. Sequence data may be used to guide control measures, but only if the results are generated quickly enough to inform interventions. Genomic surveillance during the epidemic has been sporadic owing to a lack of local sequencing capacity coupled with practical difficulties transporting samples to remote sequencing facilities. To address this problem, here we devise a genomic surveillance system that utilizes a novel nanopore DNA sequencing instrument. In April 2015 this system was transported in standard airline luggage to Guinea and used for real-time genomic surveillance of the ongoing epidemic. We present sequence data and analysis of 142 EBOV samples collected during the period March to October 2015. We were able to generate results less than 24 h after receiving an Ebola-positive sample, with the sequencing process taking as little as 15-60 min. We show that real-time genomic surveillance is possible in resource-limited settings and can be established rapidly to monitor outbreaks.

Big Bang Tumor Growth and Clonal Evolution.

PubMed

Sun, Ruping; Hu, Zheng; Curtis, Christina

2018-05-01

The advent and application of next-generation sequencing (NGS) technologies to tumor genomes has reinvigorated efforts to understand clonal evolution. Although tumor progression has traditionally been viewed as a gradual stepwise process, recent studies suggest that evolutionary rates in tumors can be variable with periods of punctuated mutational bursts and relative stasis. For example, Big Bang dynamics have been reported, wherein after transformation, growth occurs in the absence of stringent selection, consistent with effectively neutral evolution. Although first noted in colorectal tumors, effective neutrality may be relatively common. Additionally, punctuated evolution resulting from mutational bursts and cataclysmic genomic alterations have been described. In this review, we contrast these findings with the conventional gradualist view of clonal evolution and describe potential clinical and therapeutic implications of different evolutionary modes and tempos. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Unclassified renal cell carcinoma: a clinicopathological, comparative genomic hybridization, and whole-genome exon sequencing study.

PubMed

Hu, Zhen-Yan; Pang, Li-Juan; Qi, Yan; Kang, Xue-Ling; Hu, Jian-Ming; Wang, Lianghai; Liu, Kun-Peng; Ren, Yuan; Cui, Mei; Song, Li-Li; Li, Hong-An; Zou, Hong; Li, Feng

2014-01-01

Unclassified renal cell carcinoma (URCC) is a rare variant of RCC, accounting for only 3-5% of all cases. Studies on the molecular genetics of URCC are limited, and hence, we report on 2 cases of URCC analyzed using comparative genome hybridization (CGH) and the genome-wide human exon GeneChip technique to identify the genomic alterations of URCC. Both URCC patients (mean age, 72 years) presented at an advanced stage and died within 30 months post-surgery. Histologically, the URCCs were composed of undifferentiated, multinucleated, giant cells with eosinophilic cytoplasm. Immunostaining revealed that both URCC cases had strong p53 protein expression and partial expression of cluster of differentiation-10 and cytokeratin. The CGH profiles showed chromosomal imbalances in both URCC cases: gains were observed in chromosomes 1p11-12, 1q12-13, 2q20-23, 3q22-23, 8p12, and 16q11-15, whereas losses were detected on chromosomes 1q22-23, 3p12-22, 5p30-ter, 6p, 11q, 16q18-22, 17p12-14, and 20p. Compared with 18 normal renal tissues, 40 mutated genes were detected in the URCC tissues, including 32 missense and 8 silent mutations. Functional enrichment analysis revealed that the missense mutation genes were involved in 11 different biological processes and pathways, including cell cycle regulation, lipid localization and transport, neuropeptide signaling, organic ether metabolism, and ATP-binding cassette transporter signaling. Our findings indicate that URCC may be a highly aggressive cancer, and the genetic alterations identified herein may provide clues regarding the tumorigenesis of URCC and serve as a basis for the development of targeted therapies against URCC in the future.

Unclassified renal cell carcinoma: a clinicopathological, comparative genomic hybridization, and whole-genome exon sequencing study

PubMed Central

Hu, Zhen-Yan; Pang, Li-Juan; Qi, Yan; Kang, Xue-Ling; Hu, Jian-Ming; Wang, Lianghai; Liu, Kun-Peng; Ren, Yuan; Cui, Mei; Song, Li-Li; Li, Hong-An; Zou, Hong; Li, Feng

2014-01-01

Unclassified renal cell carcinoma (URCC) is a rare variant of RCC, accounting for only 3-5% of all cases. Studies on the molecular genetics of URCC are limited, and hence, we report on 2 cases of URCC analyzed using comparative genome hybridization (CGH) and the genome-wide human exon GeneChip technique to identify the genomic alterations of URCC. Both URCC patients (mean age, 72 years) presented at an advanced stage and died within 30 months post-surgery. Histologically, the URCCs were composed of undifferentiated, multinucleated, giant cells with eosinophilic cytoplasm. Immunostaining revealed that both URCC cases had strong p53 protein expression and partial expression of cluster of differentiation-10 and cytokeratin. The CGH profiles showed chromosomal imbalances in both URCC cases: gains were observed in chromosomes 1p11-12, 1q12-13, 2q20-23, 3q22-23, 8p12, and 16q11-15, whereas losses were detected on chromosomes 1q22-23, 3p12-22, 5p30-ter, 6p, 11q, 16q18-22, 17p12-14, and 20p. Compared with 18 normal renal tissues, 40 mutated genes were detected in the URCC tissues, including 32 missense and 8 silent mutations. Functional enrichment analysis revealed that the missense mutation genes were involved in 11 different biological processes and pathways, including cell cycle regulation, lipid localization and transport, neuropeptide signaling, organic ether metabolism, and ATP-binding cassette transporter signaling. Our findings indicate that URCC may be a highly aggressive cancer, and the genetic alterations identified herein may provide clues regarding the tumorigenesis of URCC and serve as a basis for the development of targeted therapies against URCC in the future. PMID:25120763

The transcriptional control machinery as well as the cell wall integrity and its regulation are involved in the detoxification of the organic solvent dimethyl sulfoxide in Saccharomyces cerevisiae.

PubMed

Zhang, Lilin; Liu, Ningning; Ma, Xiao; Jiang, Linghuo

2013-03-01

In the present study, we have identified 339 dimethyl sulfoxide (DMSO)-sensitive and nine DMSO-tolerant gene mutations in Saccharomyces cerevisiae through a functional genomics approach. Twelve of these identified DMSO-sensitive mutations are of genes involved in the general control of gene expression mediated by the SWR1 complex and the RNA polymerase II mediator complex, whereas 71 of them are of genes involved in the protein trafficking and vacuolar sorting processes. In addition, twelve of these DMSO-sensitive mutations are of genes involved in the cell wall integrity (CWI) and its regulation. DMSO-tolerant mutations are of genes mainly involved in the metabolism and the gene expression control. Therefore, the transcriptional control machinery, the CWI and its regulation as well as the protein trafficking and sorting process play critical roles in the DMSO detoxification in yeast cells. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Analysis of the genetic phylogeny of multifocal prostate cancer identifies multiple independent clonal expansions in neoplastic and morphologically normal prostate tissue.

PubMed

Cooper, Colin S; Eeles, Rosalind; Wedge, David C; Van Loo, Peter; Gundem, Gunes; Alexandrov, Ludmil B; Kremeyer, Barbara; Butler, Adam; Lynch, Andrew G; Camacho, Niedzica; Massie, Charlie E; Kay, Jonathan; Luxton, Hayley J; Edwards, Sandra; Kote-Jarai, ZSofia; Dennis, Nening; Merson, Sue; Leongamornlert, Daniel; Zamora, Jorge; Corbishley, Cathy; Thomas, Sarah; Nik-Zainal, Serena; O'Meara, Sarah; Matthews, Lucy; Clark, Jeremy; Hurst, Rachel; Mithen, Richard; Bristow, Robert G; Boutros, Paul C; Fraser, Michael; Cooke, Susanna; Raine, Keiran; Jones, David; Menzies, Andrew; Stebbings, Lucy; Hinton, Jon; Teague, Jon; McLaren, Stuart; Mudie, Laura; Hardy, Claire; Anderson, Elizabeth; Joseph, Olivia; Goody, Victoria; Robinson, Ben; Maddison, Mark; Gamble, Stephen; Greenman, Christopher; Berney, Dan; Hazell, Steven; Livni, Naomi; Fisher, Cyril; Ogden, Christopher; Kumar, Pardeep; Thompson, Alan; Woodhouse, Christopher; Nicol, David; Mayer, Erik; Dudderidge, Tim; Shah, Nimish C; Gnanapragasam, Vincent; Voet, Thierry; Campbell, Peter; Futreal, Andrew; Easton, Douglas; Warren, Anne Y; Foster, Christopher S; Stratton, Michael R; Whitaker, Hayley C; McDermott, Ultan; Brewer, Daniel S; Neal, David E

2015-04-01

Genome-wide DNA sequencing was used to decrypt the phylogeny of multiple samples from distinct areas of cancer and morphologically normal tissue taken from the prostates of three men. Mutations were present at high levels in morphologically normal tissue distant from the cancer, reflecting clonal expansions, and the underlying mutational processes at work in morphologically normal tissue were also at work in cancer. Our observations demonstrate the existence of ongoing abnormal mutational processes, consistent with field effects, underlying carcinogenesis. This mechanism gives rise to extensive branching evolution and cancer clone mixing, as exemplified by the coexistence of multiple cancer lineages harboring distinct ERG fusions within a single cancer nodule. Subsets of mutations were shared either by morphologically normal and malignant tissues or between different ERG lineages, indicating earlier or separate clonal cell expansions. Our observations inform on the origin of multifocal disease and have implications for prostate cancer therapy in individual cases.

Generation of novel resistance genes using mutation and targeted gene editing

USDA-ARS?s Scientific Manuscript database

Classical breeding for virus resistance is a lengthy process and is restricted by the availability of resistance genes. Precise genome editing is a "dream technology" to improve plants for virus resistance and these tools have opened new and very promising ways to generate virus resistant plants by ...

[Detection of pathogenic mutations in Marfan syndrome by targeted next-generation semiconductor sequencing].

PubMed

Lu, Chaoxia; Wu, Wei; Xiao, Jifang; Meng, Yan; Zhang, Shuyang; Zhang, Xue

2013-06-01

To detect pathogenic mutations in Marfan syndrome (MFS) using an Ion Torrent Personal Genome Machine (PGM) and to validate the result of targeted next-generation semiconductor sequencing for the diagnosis of genetic disorders. Peripheral blood samples were collected from three MFS patients and a normal control with informed consent. Genomic DNA was isolated by standard method and then subjected to targeted sequencing using an Ion Ampliseq(TM) Inherited Disease Panel. Three multiplex PCR reactions were carried out to amplify the coding exons of 328 genes including FBN1, TGFBR1 and TGFBR2. DNA fragments from different samples were ligated with barcoded sequencing adaptors. Template preparation and emulsion PCR, and Ion Sphere Particles enrichment were carried out using an Ion One Touch system. The ion sphere particles were sequenced on a 318 chip using the PGM platform. Data from the PGM runs were processed using an Ion Torrent Suite 3.2 software to generate sequence reads. After sequence alignment and extraction of SNPs and indels, all the variants were filtered against dbSNP137. DNA sequences were visualized with an Integrated Genomics Viewer. The most likely disease-causing variants were analyzed by Sanger sequencing. The PGM sequencing has yielded an output of 855.80 Mb, with a > 100 × median sequencing depth and a coverage of > 98% for the targeted regions in all the four samples. After data analysis and database filtering, one known missense mutation (p.E1811K) and two novel premature termination mutations (p.E2264X and p.L871FfsX23) in the FBN1 gene were identified in the three MFS patients. All mutations were verified by conventional Sanger sequencing. Pathogenic FBN1 mutations have been identified in all patients with MFS, indicating that the targeted next-generation sequencing on the PGM sequencers can be applied for accurate and high-throughput testing of genetic disorders.

A comparative analysis of whole genome sequencing of esophageal adenocarcinoma pre- and post-chemotherapy

PubMed Central

Noorani, Ayesha; Lynch, Andy G.; Achilleos, Achilleas; Eldridge, Matthew; Bower, Lawrence; Weaver, Jamie M.J.; Crawte, Jason; Ong, Chin-Ann; Shannon, Nicholas; MacRae, Shona; Grehan, Nicola; Nutzinger, Barbara; O'Donovan, Maria; Hardwick, Richard; Tavaré, Simon; Fitzgerald, Rebecca C.

2017-01-01

The scientific community has avoided using tissue samples from patients that have been exposed to systemic chemotherapy to infer the genomic landscape of a given cancer. Esophageal adenocarcinoma is a heterogeneous, chemoresistant tumor for which the availability and size of pretreatment endoscopic samples are limiting. This study compares whole-genome sequencing data obtained from chemo-naive and chemo-treated samples. The quality of whole-genomic sequencing data is comparable across all samples regardless of chemotherapy status. Inclusion of samples collected post-chemotherapy increased the proportion of late-stage tumors. When comparing matched pre- and post-chemotherapy samples from 10 cases, the mutational signatures, copy number, and SNV mutational profiles reflect the expected heterogeneity in this disease. Analysis of SNVs in relation to allele-specific copy-number changes pinpoints the common ancestor to a point prior to chemotherapy. For cases in which pre- and post-chemotherapy samples do show substantial differences, the timing of the divergence is near-synchronous with endoreduplication. Comparison across a large prospective cohort (62 treatment-naive, 58 chemotherapy-treated samples) reveals no significant differences in the overall mutation rate, mutation signatures, specific recurrent point mutations, or copy-number events in respect to chemotherapy status. In conclusion, whole-genome sequencing of samples obtained following neoadjuvant chemotherapy is representative of the genomic landscape of esophageal adenocarcinoma. Excluding these samples reduces the material available for cataloging and introduces a bias toward the earlier stages of cancer. PMID:28465312

CNS germinomas are characterized by global demethylation, chromosomal instability and mutational activation of the Kit-, Ras/Raf/Erk- and Akt-pathways

PubMed Central

Schulte, Simone Laura; Waha, Andreas; Steiger, Barbara; Denkhaus, Dorota; Dörner, Evelyn; Calaminus, Gabriele; Leuschner, Ivo; Pietsch, Torsten

2016-01-01

CNS germinomas represent a unique germ cell tumor entity characterized by undifferentiated tumor cells and a high response rate to current treatment protocols. Limited information is available on their underlying genomic, epigenetic and biological alterations. We performed a genome-wide analysis of genomic copy number alterations in 49 CNS germinomas by molecular inversion profiling. In addition, CpG dinucleotide methylation was studied by immunohistochemistry for methylated cytosine residues. Mutational analysis was performed by resequencing of candidate genes including KIT and RAS family members. Ras/Erk and Akt pathway activation was analyzed by immunostaining with antibodies against phospho-Erk, phosho-Akt, phospho-mTOR and phospho-S6. All germinomas coexpressed Oct4 and Kit but showed an extensive global DNA demethylation compared to other tumors and normal tissues. Molecular inversion profiling showed predominant genomic instability in all tumors with a high frequency of regional gains and losses including high level gene amplifications. Activating mutations of KIT exons 11, 13, and 17 as well as a case with genomic KIT amplification and activating mutations or amplifications of RAS gene family members including KRAS, NRAS and RRAS2 indicated mutational activation of crucial signaling pathways. Co-activation of Ras/Erk and Akt pathways was present in 83% of germinomas. These data suggest that CNS germinoma cells display a demethylated nuclear DNA similar to primordial germ cells in early development. This finding has a striking coincidence with extensive genomic instability. In addition, mutational activation of Kit-, Ras/Raf/Erk- and Akt- pathways indicate the biological importance of these pathways and their components as potential targets for therapy. PMID:27391150

Re-sequencing transgenic plants revealed rearrangements at T-DNA inserts, and integration of a short T-DNA fragment, but no increase of small mutations elsewhere.

PubMed

Schouten, Henk J; Vande Geest, Henri; Papadimitriou, Sofia; Bemer, Marian; Schaart, Jan G; Smulders, Marinus J M; Perez, Gabino Sanchez; Schijlen, Elio

2017-03-01

Transformation resulted in deletions and translocations at T-DNA inserts, but not in genome-wide small mutations. A tiny T-DNA splinter was detected that probably would remain undetected by conventional techniques. We investigated to which extent Agrobacterium tumefaciens-mediated transformation is mutagenic, on top of inserting T-DNA. To prevent mutations due to in vitro propagation, we applied floral dip transformation of Arabidopsis thaliana. We re-sequenced the genomes of five primary transformants, and compared these to genomic sequences derived from a pool of four wild-type plants. By genome-wide comparisons, we identified ten small mutations in the genomes of the five transgenic plants, not correlated to the positions or number of T-DNA inserts. This mutation frequency is within the range of spontaneous mutations occurring during seed propagation in A. thaliana, as determined earlier. In addition, we detected small as well as large deletions specifically at the T-DNA insert sites. Furthermore, we detected partial T-DNA inserts, one of these a tiny 50-bp fragment originating from a central part of the T-DNA construct used, inserted into the plant genome without flanking other T-DNA. Because of its small size, we named this fragment a T-DNA splinter. As far as we know this is the first report of such a small T-DNA fragment insert in absence of any T-DNA border sequence. Finally, we found evidence for translocations from other chromosomes, flanking T-DNA inserts. In this study, we showed that next-generation sequencing (NGS) is a highly sensitive approach to detect T-DNA inserts in transgenic plants.

Novel Cell Culture-Adapted Genotype 2a Hepatitis C Virus Infectious Clone

PubMed Central

Date, Tomoko; Kato, Takanobu; Kato, Junko; Takahashi, Hitoshi; Morikawa, Kenichi; Akazawa, Daisuke; Murayama, Asako; Tanaka-Kaneko, Keiko; Sata, Tetsutaro; Tanaka, Yasuhito; Mizokami, Masashi

2012-01-01

Although the recently developed infectious hepatitis C virus system that uses the JFH-1 clone enables the study of whole HCV viral life cycles, limited particular HCV strains have been available with the system. In this study, we isolated another genotype 2a HCV cDNA, the JFH-2 strain, from a patient with fulminant hepatitis. JFH-2 subgenomic replicons were constructed. HuH-7 cells transfected with in vitro transcribed replicon RNAs were cultured with G418, and selected colonies were isolated and expanded. From sequencing analysis of the replicon genome, several mutations were found. Some of the mutations enhanced JFH-2 replication; the 2217AS mutation in the NS5A interferon sensitivity-determining region exhibited the strongest adaptive effect. Interestingly, a full-length chimeric or wild-type JFH-2 genome with the adaptive mutation could replicate in Huh-7.5.1 cells and produce infectious virus after extensive passages of the virus genome-replicating cells. Virus infection efficiency was sufficient for autonomous virus propagation in cultured cells. Additional mutations were identified in the infectious virus genome. Interestingly, full-length viral RNA synthesized from the cDNA clone with these adaptive mutations was infectious for cultured cells. This approach may be applicable for the establishment of new infectious HCV clones. PMID:22787209

Space environment induced mutations prefer to occur at polymorphic sites of rice genomes

NASA Astrophysics Data System (ADS)

Li, Y.; Liu, M.; Cheng, Z.; Sun, Y.

To explore the genomic characteristics of rice mutants induced by space environment, space-induced mutants 971-5, 972-4, and R955, which acquired new traits after space flight such as increased yield, reduced resistance to rice blast, and semi-dwarfism compared with their on-ground controls, 971ck, 972ck, and Bing95-503, respectively, together with other 8 japonica and 3 indica rice varieties, 17 in total, were analyzed by amplified fragment length polymorphism (AFLP) method. We chose 16 AFLP primer-pairs which generated a total of 1251 sites, of which 745 (59.6%) were polymorphic over all the genotypes. With the 16 pairs of primer combinations, 54 space-induced mutation sites were observed in 971-5, 86 in 972-4, and 5 in R955 compared to their controls, and the mutation rates were 4.3%, 6.9% and 0.4%, respectively. Interestingly, 75.9%, 84.9% and 100% of the mutation sites identified in 971-5, 972-4, and R955 occurred in polymorphic sites. This result suggests that the space environment preferentially induced mutations at polymorphic sites in rice genomes and might share a common mechanism with other types of mutagens. It also implies that polymorphic sites in genomes are potential "hotspots" for mutations induced by the space environment.

Experimental Evolution of Mycobacterium tuberculosis in Human Macrophages Results in Low-Frequency Mutations Not Associated with Selective Advantage

PubMed Central

Guerrini, Valentina; Subbian, Selvakumar; Santucci, Pierre; Canaan, Stéphane; Pozzi, Gianni

2016-01-01

Isolates of the human pathogen Mycobacterium tuberculosis recovered from clinical samples exhibit genetic heterogeneity. Such variation may result from the stressful environment encountered by the pathogen inside the macrophage, which is the host cell tubercle bacilli parasitize. To study the evolution of the M. tuberculosis genome during growth inside macrophages, we developed a model of intracellular culture in which bacteria were serially passaged in macrophage-like THP-1 cells for about 80 bacterial generations. Genome sequencing of single bacterial colonies isolated before and after the infection cycles revealed that M. tuberculosis developed mutations at a rate of about 5.7 × 10−9 / bp/ generation, consistent with mutation rates calculated during in vivo infection. Analysis of mutant growth in macrophages and in mice showed that the mutations identified after the cyclic infection conferred no advantage to the mutants relative to wild-type. Furthermore, activity testing of the recombinant protein harboring one of these mutations showed that the presence of the mutation did not affect the enzymatic activity. The serial infection protocol developed in this work to study M. tuberculosis genome microevolution can be applied to exposure to stressors to determine their effect on genome remodeling during intra-macrophage growth. PMID:27959952

Experimental Evolution of Mycobacterium tuberculosis in Human Macrophages Results in Low-Frequency Mutations Not Associated with Selective Advantage.

PubMed

Guerrini, Valentina; Subbian, Selvakumar; Santucci, Pierre; Canaan, Stéphane; Gennaro, Maria Laura; Pozzi, Gianni

2016-01-01

Isolates of the human pathogen Mycobacterium tuberculosis recovered from clinical samples exhibit genetic heterogeneity. Such variation may result from the stressful environment encountered by the pathogen inside the macrophage, which is the host cell tubercle bacilli parasitize. To study the evolution of the M. tuberculosis genome during growth inside macrophages, we developed a model of intracellular culture in which bacteria were serially passaged in macrophage-like THP-1 cells for about 80 bacterial generations. Genome sequencing of single bacterial colonies isolated before and after the infection cycles revealed that M. tuberculosis developed mutations at a rate of about 5.7 × 10-9 / bp/ generation, consistent with mutation rates calculated during in vivo infection. Analysis of mutant growth in macrophages and in mice showed that the mutations identified after the cyclic infection conferred no advantage to the mutants relative to wild-type. Furthermore, activity testing of the recombinant protein harboring one of these mutations showed that the presence of the mutation did not affect the enzymatic activity. The serial infection protocol developed in this work to study M. tuberculosis genome microevolution can be applied to exposure to stressors to determine their effect on genome remodeling during intra-macrophage growth.

Yeast mother cell-specific ageing, genetic (in)stability, and the somatic mutation theory of ageing.

PubMed

Laun, Peter; Bruschi, Carlo V; Dickinson, J Richard; Rinnerthaler, Mark; Heeren, Gino; Schwimbersky, Richard; Rid, Raphaela; Breitenbach, Michael

2007-01-01

Yeast mother cell-specific ageing is characterized by a limited capacity to produce daughter cells. The replicative lifespan is determined by the number of cell cycles a mother cell has undergone, not by calendar time, and in a population of cells its distribution follows the Gompertz law. Daughter cells reset their clock to zero and enjoy the full lifespan characteristic for the strain. This kind of replicative ageing of a cell population based on asymmetric cell divisions is investigated as a model for the ageing of a stem cell population in higher organisms. The simple fact that the daughter cells can reset their clock to zero precludes the accumulation of chromosomal mutations as the cause of ageing, because semiconservative replication would lead to the same mutations in the daughters. However, nature is more complicated than that because, (i) the very last daughters of old mothers do not reset the clock; and (ii) mutations in mitochondrial DNA could play a role in ageing due to the large copy number in the cell and a possible asymmetric distribution of damaged mitochondrial DNA between mother and daughter cell. Investigation of the loss of heterozygosity in diploid cells at the end of their mother cell-specific lifespan has shown that genomic rearrangements do occur in old mother cells. However, it is not clear if this kind of genomic instability is causative for the ageing process. Damaged material other than DNA, for instance misfolded, oxidized or otherwise damaged proteins, seem to play a major role in ageing, depending on the balance between production and removal through various repair processes, for instance several kinds of proteolysis and autophagy. We are reviewing here the evidence for genetic change and its causality in the mother cell-specific ageing process of yeast.

Yeast mother cell-specific ageing, genetic (in)stability, and the somatic mutation theory of ageing

PubMed Central

Laun, Peter; Bruschi, Carlo V.; Dickinson, J. Richard; Rinnerthaler, Mark; Heeren, Gino; Schwimbersky, Richard; Rid, Raphaela; Breitenbach, Michael

2007-01-01

Yeast mother cell-specific ageing is characterized by a limited capacity to produce daughter cells. The replicative lifespan is determined by the number of cell cycles a mother cell has undergone, not by calendar time, and in a population of cells its distribution follows the Gompertz law. Daughter cells reset their clock to zero and enjoy the full lifespan characteristic for the strain. This kind of replicative ageing of a cell population based on asymmetric cell divisions is investigated as a model for the ageing of a stem cell population in higher organisms. The simple fact that the daughter cells can reset their clock to zero precludes the accumulation of chromosomal mutations as the cause of ageing, because semiconservative replication would lead to the same mutations in the daughters. However, nature is more complicated than that because, (i) the very last daughters of old mothers do not reset the clock; and (ii) mutations in mitochondrial DNA could play a role in ageing due to the large copy number in the cell and a possible asymmetric distribution of damaged mitochondrial DNA between mother and daughter cell. Investigation of the loss of heterozygosity in diploid cells at the end of their mother cell-specific lifespan has shown that genomic rearrangements do occur in old mother cells. However, it is not clear if this kind of genomic instability is causative for the ageing process. Damaged material other than DNA, for instance misfolded, oxidized or otherwise damaged proteins, seem to play a major role in ageing, depending on the balance between production and removal through various repair processes, for instance several kinds of proteolysis and autophagy. We are reviewing here the evidence for genetic change and its causality in the mother cell-specific ageing process of yeast. PMID:17986449

Comparison against 186 canid whole-genome sequences reveals survival strategies of an ancient clonally transmissible canine tumor

PubMed Central

Decker, Brennan; Davis, Brian W.; Rimbault, Maud; Long, Adrienne H.; Karlins, Eric; Jagannathan, Vidhya; Reiman, Rebecca; Parker, Heidi G.; Drögemüller, Cord; Corneveaux, Jason J.; Chapman, Erica S.; Trent, Jeffery M.; Leeb, Tosso; Huentelman, Matthew J.; Wayne, Robert K.; Karyadi, Danielle M.; Ostrander, Elaine A.

2015-01-01

Canine transmissible venereal tumor (CTVT) is a parasitic cancer clone that has propagated for thousands of years via sexual transfer of malignant cells. Little is understood about the mechanisms that converted an ancient tumor into the world's oldest known continuously propagating somatic cell lineage. We created the largest existing catalog of canine genome-wide variation and compared it against two CTVT genome sequences, thereby separating alleles derived from the founder's genome from somatic mutations that must drive clonal transmissibility. We show that CTVT has undergone continuous adaptation to its transmissible allograft niche, with overlapping mutations at every step of immunosurveillance, particularly self-antigen presentation and apoptosis. We also identified chronologically early somatic mutations in oncogenesis- and immune-related genes that may represent key initiators of clonal transmissibility. Thus, we provide the first insights into the specific genomic aberrations that underlie CTVT's dogged perseverance in canids around the world. PMID:26232412

CRISPR/Cas9 Editing of the Bacillus subtilis Genome

PubMed Central

Burby, Peter E.; Simmons, Lyle A.

2017-01-01

A fundamental procedure for most modern biologists is the genetic manipulation of the organism under study. Although many different methods for editing bacterial genomes have been used in laboratories for decades, the adaptation of CRISPR/Cas9 technology to bacterial genetics has allowed researchers to manipulate bacterial genomes with unparalleled facility. CRISPR/Cas9 has allowed for genome edits to be more precise, while also increasing the efficiency of transferring mutations into a variety of genetic backgrounds. As a result, the advantages are realized in tractable organisms and organisms that have been refractory to genetic manipulation. Here, we describe our method for editing the genome of the bacterium Bacillus subtilis. Our method is highly efficient, resulting in precise, markerless mutations. Further, after generating the editing plasmid, the mutation can be quickly introduced into several genetic backgrounds, greatly increasing the speed with which genetic analyses may be performed. PMID:28706963

Pan-Cancer Analysis of Mutation Hotspots in Protein Domains.

PubMed

Miller, Martin L; Reznik, Ed; Gauthier, Nicholas P; Aksoy, Bülent Arman; Korkut, Anil; Gao, Jianjiong; Ciriello, Giovanni; Schultz, Nikolaus; Sander, Chris

2015-09-23

In cancer genomics, recurrence of mutations in independent tumor samples is a strong indicator of functional impact. However, rare functional mutations can escape detection by recurrence analysis owing to lack of statistical power. We enhance statistical power by extending the notion of recurrence of mutations from single genes to gene families that share homologous protein domains. Domain mutation analysis also sharpens the functional interpretation of the impact of mutations, as domains more succinctly embody function than entire genes. By mapping mutations in 22 different tumor types to equivalent positions in multiple sequence alignments of domains, we confirm well-known functional mutation hotspots, identify uncharacterized rare variants in one gene that are equivalent to well-characterized mutations in another gene, detect previously unknown mutation hotspots, and provide hypotheses about molecular mechanisms and downstream effects of domain mutations. With the rapid expansion of cancer genomics projects, protein domain hotspot analysis will likely provide many more leads linking mutations in proteins to the cancer phenotype. Copyright © 2015 Elsevier Inc. All rights reserved.

The Spectrum of Replication Errors in the Absence of Error Correction Assayed Across the Whole Genome of Escherichia coli.

PubMed

Niccum, Brittany A; Lee, Heewook; MohammedIsmail, Wazim; Tang, Haixu; Foster, Patricia L

2018-06-15

When the DNA polymerase that replicates the Escherichia coli chromosome, DNA Pol III, makes an error, there are two primary defenses against mutation: proofreading by the epsilon subunit of the holoenzyme and mismatch repair. In proofreading deficient strains, mismatch repair is partially saturated and the cell's response to DNA damage, the SOS response, may be partially induced. To investigate the nature of replication errors, we used mutation accumulation experiments and whole genome sequencing to determine mutation rates and mutational spectra across the entire chromosome of strains deficient in proofreading, mismatch repair, and the SOS response. We report that a proofreading-deficient strain has a mutation rate 4,000-fold greater than wild-type strains. While the SOS response may be induced in these cells, it does not contribute to the mutational load. Inactivating mismatch repair in a proofreading-deficient strain increases the mutation rate another 1.5-fold. DNA polymerase has a bias for converting G:C to A:T base pairs, but proofreading reduces the impact of these mutations, helping to maintain the genomic G:C content. These findings give an unprecedented view of how polymerase and error-correction pathways work together to maintain E. coli' s low mutation rate of 1 per thousand generations. Copyright © 2018, Genetics.

Single-Color Digital PCR Provides High-Performance Detection of Cancer Mutations from Circulating DNA.

PubMed

Wood-Bouwens, Christina; Lau, Billy T; Handy, Christine M; Lee, HoJoon; Ji, Hanlee P

2017-09-01

We describe a single-color digital PCR assay that detects and quantifies cancer mutations directly from circulating DNA collected from the plasma of cancer patients. This approach relies on a double-stranded DNA intercalator dye and paired allele-specific DNA primer sets to determine an absolute count of both the mutation and wild-type-bearing DNA molecules present in the sample. The cell-free DNA assay uses an input of 1 ng of nonamplified DNA, approximately 300 genome equivalents, and has a molecular limit of detection of three mutation DNA genome-equivalent molecules per assay reaction. When using more genome equivalents as input, we demonstrated a sensitivity of 0.10% for detecting the BRAF V600E and KRAS G12D mutations. We developed several mutation assays specific to the cancer driver mutations of patients' tumors and detected these same mutations directly from the nonamplified, circulating cell-free DNA. This rapid and high-performance digital PCR assay can be configured to detect specific cancer mutations unique to an individual cancer, making it a potentially valuable method for patient-specific longitudinal monitoring. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Human mitochondrial DNA: roles of inherited and somatic mutations

PubMed Central

Schon, Eric A.; DiMauro, Salvatore; Hirano, Michio

2014-01-01

Mutations in the human mitochondrial genome are known to cause an array of diverse disorders, most of which are maternally inherited, and all of which are associated with defects in oxidative energy metabolism. It is now emerging that somatic mutations in mitochondrial DNA (mtDNA) are also linked to other complex traits, including neurodegenerative diseases, ageing and cancer. Here we discuss insights into the roles of mtDNA mutations in a wide variety of diseases, highlighting the interesting genetic characteristics of the mitochondrial genome and challenges in studying its contribution to pathogenesis. PMID:23154810

Basics of DNA biosensors and cancer diagnosis.

PubMed

Sohrabi, Nasrin; Valizadeh, Alireza; Farkhani, Samad Mussa; Akbarzadeh, Abolfazl

2016-01-01

The human genome is exposed to mutations during the life cycle because of many types of changes in the DNA. Viruses, radiation, transposons, mutagenic chemicals, or any errors that happen during DNA replication or the meiotic process in the cell, may cause the mutation. Many mutations have no effect on phenotype or health, while some mutations cause crucial diseases such as cancer or cardiac diseases; therefore, a better understanding of the effects of mutation on phenotype is a very important part of genetic studies. Biosensors based on DNA, RNA, and peptide nucleic acids are the most sensitive tools, due to a strong pairing of lined up nucleotide strands between bases in their complementary parts. These methods can provide information to assist clinicians in making successful treatment decisions and increase the patient survival rate. In this review, we discuss DNA biosensors based on peptide nucleic acids that have an important role in cancer diagnosis.

Somatic Mosaicism: Implications for Disease and Transmission Genetics

PubMed Central

Campbell, Ian M.; Shaw, Chad A.; Stankiewicz, Pawel; Lupski, James R.

2015-01-01

Nearly all of the genetic material among cells within an organism is identical. However, single nucleotide variants (SNVs), indels, copy number variants (CNVs), and other structural variants (SVs) continually accumulate as cells divide during development. This process results in an organism composed of countless cells, each with its own unique personal genome. Thus, every human is undoubtedly mosaic. Mosaic mutations can go unnoticed, underlie genetic disease or normal human variation, and may be transmitted to the next generation as constitutional variants. Here, we review the influence of the developmental timing of mutations, the mechanisms by which they arise, methods for detecting mosaic variants, and the risk of passing these mutations on to the next generation. PMID:25910407

Whole-genome sequencing identifies genomic heterogeneity at a nucleotide and chromosomal level in bladder cancer

PubMed Central

Morrison, Carl D.; Liu, Pengyuan; Woloszynska-Read, Anna; Zhang, Jianmin; Luo, Wei; Qin, Maochun; Bshara, Wiam; Conroy, Jeffrey M.; Sabatini, Linda; Vedell, Peter; Xiong, Donghai; Liu, Song; Wang, Jianmin; Shen, He; Li, Yinwei; Omilian, Angela R.; Hill, Annette; Head, Karen; Guru, Khurshid; Kunnev, Dimiter; Leach, Robert; Eng, Kevin H.; Darlak, Christopher; Hoeflich, Christopher; Veeranki, Srividya; Glenn, Sean; You, Ming; Pruitt, Steven C.; Johnson, Candace S.; Trump, Donald L.

2014-01-01

Using complete genome analysis, we sequenced five bladder tumors accrued from patients with muscle-invasive transitional cell carcinoma of the urinary bladder (TCC-UB) and identified a spectrum of genomic aberrations. In three tumors, complex genotype changes were noted. All three had tumor protein p53 mutations and a relatively large number of single-nucleotide variants (SNVs; average of 11.2 per megabase), structural variants (SVs; average of 46), or both. This group was best characterized by chromothripsis and the presence of subclonal populations of neoplastic cells or intratumoral mutational heterogeneity. Here, we provide evidence that the process of chromothripsis in TCC-UB is mediated by nonhomologous end-joining using kilobase, rather than megabase, fragments of DNA, which we refer to as “stitchers,” to repair this process. We postulate that a potential unifying theme among tumors with the more complex genotype group is a defective replication–licensing complex. A second group (two bladder tumors) had no chromothripsis, and a simpler genotype, WT tumor protein p53, had relatively few SNVs (average of 5.9 per megabase) and only a single SV. There was no evidence of a subclonal population of neoplastic cells. In this group, we used a preclinical model of bladder carcinoma cell lines to study a unique SV (translocation and amplification) of the gene glutamate receptor ionotropic N-methyl D-aspertate as a potential new therapeutic target in bladder cancer. PMID:24469795

Efficient CRISPR/Cas9-mediated Targeted Mutagenesis in Populus in the First Generation

PubMed Central

Fan, Di; Liu, Tingting; Li, Chaofeng; Jiao, Bo; Li, Shuang; Hou, Yishu; Luo, Keming

2015-01-01

Recently, RNA-guided genome editing using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system has been applied to edit the plant genome in several herbaceous plant species. However, it remains unknown whether this system can be used for genome editing in woody plants. In this study, we describe the genome editing and targeted gene mutation in a woody species, Populus tomentosa Carr. via the CRISPR/Cas9 system. Four guide RNAs (gRNAs) were designed to target with distinct poplar genomic sites of the phytoene desaturase gene 8 (PtoPDS) which are followed by the protospacer-adjacent motif (PAM). After Agrobacterium-mediated transformation, obvious albino phenotype was observed in transgenic poplar plants. By analyzing the RNA-guided genome-editing events, 30 out of 59 PCR clones were homozygous mutants, 2 out of 59 were heterozygous mutants and the mutation efficiency at these target sites was estimated to be 51.7%. Our data demonstrate that the Cas9/sgRNA system can be exploited to precisely edit genomic sequence and effectively create knockout mutations in woody plants. PMID:26193631

Genomic analysis and clinical management of adolescent cutaneous melanoma.

PubMed

Rabbie, Roy; Rashid, Mamunur; Arance, Ana M; Sánchez, Marcelo; Tell-Marti, Gemma; Potrony, Miriam; Conill, Carles; van Doorn, Remco; Dentro, Stefan; Gruis, Nelleke A; Corrie, Pippa; Iyer, Vivek; Robles-Espinoza, Carla Daniela; Puig-Butille, Joan A; Puig, Susana; Adams, David J

2017-05-01

Melanoma in young children is rare; however, its incidence in adolescents and young adults is rising. We describe the clinical course of a 15-year-old female diagnosed with AJCC stage IB non-ulcerated primary melanoma, who died from metastatic disease 4 years after diagnosis despite three lines of modern systemic therapy. We also present the complete genomic profile of her tumour and compare this to a further series of 13 adolescent melanomas and 275 adult cutaneous melanomas. A somatic BRAF V 600E mutation and a high mutational load equivalent to that found in adult melanoma and composed primarily of C>T mutations were observed. A germline genomic analysis alongside a series of 23 children and adolescents with melanoma revealed no mutations in known germline melanoma-predisposing genes. Adolescent melanomas appear to have genomes that are as complex as those arising in adulthood and their clinical course can, as with adults, be unpredictable. © 2017 The Authors. Pigment Cell & Melanoma Research published by John Wiley & Sons Ltd.

Germline Mutations and Polymorphisms in the Origins of Cancers in Women

PubMed Central

Hirshfield, Kim M.; Rebbeck, Timothy R.; Levine, Arnold J.

2010-01-01

Several female malignancies including breast, ovarian, and endometrial cancers can be characterized based on known somatic and germline mutations. Initiation and propagation of tumors reflect underlying genomic alterations such as mutations, polymorphisms, and copy number variations found in genes of multiple cellular pathways. The contributions of any single genetic variation or mutation in a population depend on its frequency and penetrance as well as tissue-specific functionality. Genome wide association studies, fluorescence in situ hybridization, comparative genomic hybridization, and candidate gene studies have enumerated genetic contributors to cancers in women. These include p53, BRCA1, BRCA2, STK11, PTEN, CHEK2, ATM, BRIP1, PALB2, FGFR2, TGFB1, MDM2, MDM4 as well as several other chromosomal loci. Based on the heterogeneity within a specific tumor type, a combination of genomic alterations defines the cancer subtype, biologic behavior, and in some cases, response to therapeutics. Consideration of tumor heterogeneity is therefore important in the critical analysis of gene associations in cancer. PMID:20111735

An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes

PubMed Central

Racher, Hilary; Phelps, Ian G.; Toedt, Grischa; Kennedy, Julie; Wunderlich, Kirsten A.; Sorusch, Nasrin; Abdelhamed, Zakia A.; Natarajan, Subaashini; Herridge, Warren; van Reeuwijk, Jeroen; Horn, Nicola; Boldt, Karsten; Parry, David A.; Letteboer, Stef J.F.; Roosing, Susanne; Adams, Matthew; Bell, Sandra M.; Bond, Jacquelyn; Higgins, Julie; Morrison, Ewan E.; Tomlinson, Darren C.; Slaats, Gisela G.; van Dam, Teunis J. P.; Huang, Lijia; Kessler, Kristin; Giessl, Andreas; Logan, Clare V.; Boyle, Evan A.; Shendure, Jay; Anazi, Shamsa; Aldahmesh, Mohammed; Al Hazzaa, Selwa; Hegele, Robert A.; Ober, Carole; Frosk, Patrick; Mhanni, Aizeddin A.; Chodirker, Bernard N.; Chudley, Albert E.; Lamont, Ryan; Bernier, Francois P.; Beaulieu, Chandree L.; Gordon, Paul; Pon, Richard T.; Donahue, Clem; Barkovich, A. James; Wolf, Louis; Toomes, Carmel; Thiel, Christian T.; Boycott, Kym M.; McKibbin, Martin; Inglehearn, Chris F.; Stewart, Fiona; Omran, Heymut; Huynen, Martijn A.; Sergouniotis, Panagiotis I.; Alkuraya, Fowzan S.; Parboosingh, Jillian S.; Innes, A Micheil; Willoughby, Colin E.; Giles, Rachel H.; Webster, Andrew R.; Ueffing, Marius; Blacque, Oliver; Gleeson, Joseph G.; Wolfrum, Uwe; Beales, Philip L.; Gibson, Toby

2015-01-01

Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin-proteasome system, 12 G-protein-coupled receptors, and three pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localise to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1/CEP90 and C21orf2/LRRC76 as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2-variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease. PMID:26167768

The infectivities of turnip yellow mosaic virus genomes with altered tRNA mimicry are not dependent on compensating mutations in the viral replication protein.

PubMed

Filichkin, S A; Bransom, K L; Goodwin, J B; Dreher, T W

2000-09-01

Five highly infectious turnip yellow mosaic virus (TYMV) genomes with sequence changes in their 3'-terminal regions that result in altered aminoacylation and eEF1A binding have been studied. These genomes were derived from cloned parental RNAs of low infectivity by sequential passaging in plants. Three of these genomes that are incapable of aminoacylation have been reported previously (J. B. Goodwin, J. M. Skuzeski, and T. W. Dreher, Virology 230:113-124, 1997). We now demonstrate by subcloning the 3' untranslated regions into wild-type TYMV RNA that the high infectivities and replication rates of these genomes compared to their progenitors are mostly due to a small number of mutations acquired in the 3' tRNA-like structure during passaging. Mutations in other parts of the genome, including the replication protein coding region, are not required for high infectivity but probably do play a role in optimizing viral amplification and spread in plants. Two other TYMV RNA variants of suboptimal infectivities, one that accepts methionine instead of the usual valine and one that interacts less tightly with eEF1A, were sequentially passaged to produce highly infectious genomes. The improved infectivities of these RNAs were not associated with increased replication in protoplasts, and no mutations were acquired in their 3' tRNA-like structures. Complete sequencing of one genome identified two mutations that result in amino acid changes in the movement protein gene, suggesting that improved infectivity may be a function of improved viral dissemination in plants. Our results show that the wild-type TYMV replication proteins are able to amplify genomes with 3' termini of variable sequence and tRNA mimicry. These and previous results have led to a model in which the binding of eEF1A to the 3' end to antagonize minus-strand initiation is a major role of the tRNA-like structure.

Exploration of sequence space as the basis of viral RNA genome segmentation.

PubMed

Moreno, Elena; Ojosnegros, Samuel; García-Arriaza, Juan; Escarmís, Cristina; Domingo, Esteban; Perales, Celia

2014-05-06

The mechanisms of viral RNA genome segmentation are unknown. On extensive passage of foot-and-mouth disease virus in baby hamster kidney-21 cells, the virus accumulated multiple point mutations and underwent a transition akin to genome segmentation. The standard single RNA genome molecule was replaced by genomes harboring internal in-frame deletions affecting the L- or capsid-coding region. These genomes were infectious and killed cells by complementation. Here we show that the point mutations in the nonstructural protein-coding region (P2, P3) that accumulated in the standard genome before segmentation increased the relative fitness of the segmented version relative to the standard genome. Fitness increase was documented by intracellular expression of virus-coded proteins and infectious progeny production by RNAs with the internal deletions placed in the sequence context of the parental and evolved genome. The complementation activity involved several viral proteins, one of them being the leader proteinase L. Thus, a history of genetic drift with accumulation of point mutations was needed to allow a major variation in the structure of a viral genome. Thus, exploration of sequence space by a viral genome (in this case an unsegmented RNA) can reach a point of the space in which a totally different genome structure (in this case, a segmented RNA) is favored over the form that performed the exploration.

Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia

PubMed Central

Puente, Xose S.; Pinyol, Magda; Quesada, Víctor; Conde, Laura; Ordóñez, Gonzalo R.; Villamor, Neus; Escaramis, Georgia; Jares, Pedro; Beà, Sílvia; González-Díaz, Marcos; Bassaganyas, Laia; Baumann, Tycho; Juan, Manel; López-Guerra, Mónica; Colomer, Dolors; Tubío, José M. C.; López, Cristina; Navarro, Alba; Tornador, Cristian; Aymerich, Marta; Rozman, María; Hernández, Jesús M.; Puente, Diana A.; Freije, José M. P.; Velasco, Gloria; Gutiérrez-Fernández, Ana; Costa, Dolors; Carrió, Anna; Guijarro, Sara; Enjuanes, Anna; Hernández, Lluís; Yagüe, Jordi; Nicolás, Pilar; Romeo-Casabona, Carlos M.; Himmelbauer, Heinz; Castillo, Ester; Dohm, Juliane C.; de Sanjosé, Silvia; Piris, Miguel A.; de Alava, Enrique; Miguel, Jesús San; Royo, Romina; Gelpí, Josep L.; Torrents, David; Orozco, Modesto; Pisano, David G.; Valencia, Alfonso; Guigó, Roderic; Bayés, Mónica; Heath, Simon; Gut, Marta; Klatt, Peter; Marshall, John; Raine, Keiran; Stebbings, Lucy A.; Futreal, P. Andrew; Stratton, Michael R.; Campbell, Peter J.; Gut, Ivo; López-Guillermo, Armando; Estivill, Xavier; Montserrat, Emili; López-Otín, Carlos; Campo, Elías

2012-01-01

Chronic lymphocytic leukaemia (CLL), the most frequent leukaemia in adults in Western countries, is a heterogeneous disease with variable clinical presentation and evolution1,2. Two major molecular subtypes can be distinguished, characterized respectively by a high or low number of somatic hypermutations in the variable region of immunoglobulin genes3,4. The molecular changes leading to the pathogenesis of the disease are still poorly understood. Here we performed whole-genome sequencing of four cases of CLL and identified 46 somatic mutations that potentially affect gene function. Further analysis of these mutations in 363 patients with CLL identified four genes that are recurrently mutated: notch 1 (NOTCH1), exportin 1 (XPO1), myeloid differentiation primary response gene 88 (MYD88) and kelch-like 6 (KLHL6). Mutations in MYD88 and KLHL6 are predominant in cases of CLL with mutated immunoglobulin genes, whereas NOTCH1 and XPO1 mutations are mainly detected in patients with unmutated immunoglobulins. The patterns of somatic mutation, supported by functional and clinical analyses, strongly indicate that the recurrent NOTCH1, MYD88 and XPO1 mutations are oncogenic changes that contribute to the clinical evolution of the disease. To our knowledge, this is the first comprehensive analysis of CLL combining whole-genome sequencing with clinical characteristics and clinical outcomes. It highlights the usefulness of this approach for the identification of clinically relevant mutations in cancer. PMID:21642962

A Novel Workflow to Enrich and Isolate Patient-Matched EpCAMhigh and EpCAMlow/negative CTCs Enables the Comparative Characterization of the PIK3CA Status in Metastatic Breast Cancer

PubMed Central

Lampignano, Rita; Yang, Liwen; Neumann, Martin H. D.; Franken, André; Fehm, Tanja; Niederacher, Dieter; Neubauer, Hans

2017-01-01

Circulating tumor cells (CTCs), potential precursors of most epithelial solid tumors, are mainly enriched by epithelial cell adhesion molecule (EpCAM)-dependent technologies. Hence, these approaches may overlook mesenchymal CTCs, considered highly malignant. Our aim was to establish a workflow to enrich and isolate patient-matched EpCAMhigh and EpCAMlow/negative CTCs within the same blood samples, and to investigate the phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutational status within single CTCs. We sequentially processed metastatic breast cancer (MBC) blood samples via CellSearch® (EpCAM-based) and via Parsortix™ (size-based) systems. After enrichment, cells captured in Parsortix™ cassettes were stained in situ for nuclei, cytokeratins, EpCAM and CD45. Afterwards, sorted cells were isolated via CellCelector™ micromanipulator and their genomes were amplified. Lastly, PIK3CA mutational status was analyzed by combining an amplicon-based approach with Sanger sequencing. In 54% of patients′ blood samples both EpCAMhigh and EpCAMlow/negative cells were identified and successfully isolated. High genomic integrity was observed in 8% of amplified genomes of EpCAMlow/negative cells vs. 28% of EpCAMhigh cells suggesting an increased apoptosis in the first CTC-subpopulation. Furthermore, PIK3CA hotspot mutations were detected in both EpCAMhigh and EpCAMlow/negative CTCs. Our workflow is suitable for single CTC analysis, permitting—for the first time—assessment of the heterogeneity of PIK3CA mutational status within patient-matched EpCAMhigh and EpCAMlow/negative CTCs. PMID:28858218

A Neutrality Test for Detecting Selection on DNA Methylation Using Single Methylation Polymorphism Frequency Spectrum

PubMed Central

Wang, Jun; Fan, Chuanzhu

2015-01-01

Inheritable epigenetic mutations (epimutations) can contribute to transmittable phenotypic variation. Thus, epimutations can be subject to natural selection and impact the fitness and evolution of organisms. Based on the framework of the modified Tajima’s D test for DNA mutations, we developed a neutrality test with the statistic “Dm” to detect selection forces on DNA methylation mutations using single methylation polymorphisms. With computer simulation and empirical data analysis, we compared the Dm test with the original and modified Tajima’s D tests and demonstrated that the Dm test is suitable for detecting selection on epimutations and outperforms original/modified Tajima’s D tests. Due to the higher resetting rate of epimutations, the interpretation of Dm on epimutations and Tajima’s D test on DNA mutations could be different in inferring natural selection. Analyses using simulated and empirical genome-wide polymorphism data suggested that genes under genetic and epigenetic selections behaved differently. We applied the Dm test to recently originated Arabidopsis and human genes, and showed that newly evolved genes contain higher level of rare epialleles, suggesting that epimutation may play a role in origination and evolution of genes and genomes. Overall, we demonstrate the utility of the Dm test to detect whether the loci are under selection regarding DNA methylation. Our analytical metrics and methodology could contribute to our understanding of evolutionary processes of genes and genomes in the field of epigenetics. The Perl script for the “Dm” test is available at http://fanlab.wayne.edu/ (last accessed December 18, 2014). PMID:25539727

Cell identity regulators link development and stress responses in the Arabidopsis root.

PubMed

Iyer-Pascuzzi, Anjali S; Jackson, Terry; Cui, Hongchang; Petricka, Jalean J; Busch, Wolfgang; Tsukagoshi, Hironaka; Benfey, Philip N

2011-10-18

Stress responses in plants are tightly coordinated with developmental processes, but interaction of these pathways is poorly understood. We used genome-wide assays at high spatiotemporal resolution to understand the processes that link development and stress in the Arabidopsis root. Our meta-analysis finds little evidence for a universal stress response. However, common stress responses appear to exist with many showing cell type specificity. Common stress responses may be mediated by cell identity regulators because mutations in these genes resulted in altered responses to stress. Evidence for a direct role for cell identity regulators came from genome-wide binding profiling of the key regulator SCARECROW, which showed binding to regulatory regions of stress-responsive genes. Coexpression in response to stress was used to identify genes involved in specific developmental processes. These results reveal surprising linkages between stress and development at cellular resolution, and show the power of multiple genome-wide data sets to elucidate biological processes. Copyright © 2011 Elsevier Inc. All rights reserved.

Experimental evolution and the dynamics of genomic mutation rate modifiers.

PubMed

Raynes, Y; Sniegowski, P D

2014-11-01

Because genes that affect mutation rates are themselves subject to mutation, mutation rates can be influenced by natural selection and other evolutionary forces. The population genetics of mutation rate modifier alleles has been a subject of theoretical interest for many decades. Here, we review experimental contributions to our understanding of mutation rate modifier dynamics. Numerous evolution experiments have shown that mutator alleles (modifiers that elevate the genomic mutation rate) can readily rise to high frequencies via genetic hitchhiking in non-recombining microbial populations. Whereas these results certainly provide an explanatory framework for observations of sporadically high mutation rates in pathogenic microbes and in cancer lineages, it is nonetheless true that most natural populations have very low mutation rates. This raises the interesting question of how mutator hitchhiking is suppressed or its phenotypic effect reversed in natural populations. Very little experimental work has addressed this question; with this in mind, we identify some promising areas for future experimental investigation.

Atlantic salmon populations reveal adaptive divergence of immune related genes - a duplicated genome under selection.

PubMed

Kjærner-Semb, Erik; Ayllon, Fernando; Furmanek, Tomasz; Wennevik, Vidar; Dahle, Geir; Niemelä, Eero; Ozerov, Mikhail; Vähä, Juha-Pekka; Glover, Kevin A; Rubin, Carl J; Wargelius, Anna; Edvardsen, Rolf B

2016-08-11

Populations of Atlantic salmon display highly significant genetic differences with unresolved molecular basis. These differences may result from separate postglacial colonization patterns, diversifying natural selection and adaptation, or a combination. Adaptation could be influenced or even facilitated by the recent whole genome duplication in the salmonid lineage which resulted in a partly tetraploid species with duplicated genes and regions. In order to elucidate the genes and genomic regions underlying the genetic differences, we conducted a genome wide association study using whole genome resequencing data from eight populations from Northern and Southern Norway. From a total of ~4.5 million sequencing-derived SNPs, more than 10 % showed significant differentiation between populations from these two regions and ten selective sweeps on chromosomes 5, 10, 11, 13-15, 21, 24 and 25 were identified. These comprised 59 genes, of which 15 had one or more differentiated missense mutation. Our analysis showed that most sweeps have paralogous regions in the partially tetraploid genome, each lacking the high number of significant SNPs found in the sweeps. The most significant sweep was found on Chr 25 and carried several missense mutations in the antiviral mx genes, suggesting that these populations have experienced differing viral pressures. Interestingly the second most significant sweep, found on Chr 5, contains two genes involved in the NF-KB pathway (nkap and nkrf), which is also a known pathogen target that controls a large number of processes in animals. Our results show that natural selection acting on immune related genes has contributed to genetic divergence between salmon populations in Norway. The differences between populations may have been facilitated by the plasticity of the salmon genome. The observed signatures of selection in duplicated genomic regions suggest that the recently duplicated genome has provided raw material for evolutionary adaptation.

Mutational landscape of a chemically-induced mouse model of liver cancer.

PubMed

Connor, Frances; Rayner, Tim F; Aitken, Sarah J; Feig, Christine; Lukk, Margus; Santoyo-Lopez, Javier; Odom, Duncan T

2018-06-26

Carcinogen-induced mouse models of liver cancer are used extensively to study pathogenesis of the disease and have a critical role in validating candidate therapeutics. These models can recapitulate molecular and histological features of human disease. However, it is not known if the genomic alterations driving these mouse tumour genomes are comparable to those found in human tumours. Here, we provide a detailed genomic characterisation of tumours from a commonly used mouse model of hepatocellular carcinoma (HCC). We analysed whole exome sequences of liver tumours arising in mice exposed to diethylnitrosamine (DEN). DEN-initiated tumours had a high, uniform number of somatic single nucleotide variants (SNVs), with few insertions, deletions or copy number alterations, consistent with the known genotoxic action of DEN. Exposure of hepatocytes to DEN left a reproducible mutational imprint in resulting tumour exomes which we could computationally reconstruct using six known COSMIC mutational signatures. The tumours carried a high diversity of low-incidence, non-synonymous point mutations in many oncogenes and tumour suppressors, reflecting the stochastic introduction of SNVs into the hepatocyte genome by the carcinogen. We identified four recurrently mutated genes that were putative oncogenic drivers of HCC in this model. Every neoplasm carried activating hotspot mutations either in codon 61 of Hras, in codon 584 of Braf or in codon 254 of Egfr. Truncating mutations of Apc occurred in 21% of neoplasms, which were exclusively carcinomas supporting a role for deregulation of Wnt/β-catenin signalling in cancer progression. Our study provides detailed insight into the mutational landscape of tumours arising in a commonly-used carcinogen model of HCC, facilitating the future use of this model to understand the human disease. Mouse models are widely used to study the biology of cancer and to test potential therapies. Here, we have described the mutational landscape of tumours arising in a carcinogen-induced mouse model of liver cancer. Since cancer is a disease caused by genomic alterations, information about the patterns and types of mutations in the tumours in this mouse model should facilitate its use to study human liver cancer. Copyright © 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

MC-GenomeKey: a multicloud system for the detection and annotation of genomic variants.

PubMed

Elshazly, Hatem; Souilmi, Yassine; Tonellato, Peter J; Wall, Dennis P; Abouelhoda, Mohamed

2017-01-20

Next Generation Genome sequencing techniques became affordable for massive sequencing efforts devoted to clinical characterization of human diseases. However, the cost of providing cloud-based data analysis of the mounting datasets remains a concerning bottleneck for providing cost-effective clinical services. To address this computational problem, it is important to optimize the variant analysis workflow and the used analysis tools to reduce the overall computational processing time, and concomitantly reduce the processing cost. Furthermore, it is important to capitalize on the use of the recent development in the cloud computing market, which have witnessed more providers competing in terms of products and prices. In this paper, we present a new package called MC-GenomeKey (Multi-Cloud GenomeKey) that efficiently executes the variant analysis workflow for detecting and annotating mutations using cloud resources from different commercial cloud providers. Our package supports Amazon, Google, and Azure clouds, as well as, any other cloud platform based on OpenStack. Our package allows different scenarios of execution with different levels of sophistication, up to the one where a workflow can be executed using a cluster whose nodes come from different clouds. MC-GenomeKey also supports scenarios to exploit the spot instance model of Amazon in combination with the use of other cloud platforms to provide significant cost reduction. To the best of our knowledge, this is the first solution that optimizes the execution of the workflow using computational resources from different cloud providers. MC-GenomeKey provides an efficient multicloud based solution to detect and annotate mutations. The package can run in different commercial cloud platforms, which enables the user to seize the best offers. The package also provides a reliable means to make use of the low-cost spot instance model of Amazon, as it provides an efficient solution to the sudden termination of spot machines as a result of a sudden price increase. The package has a web-interface and it is available for free for academic use.

Genome-wide screen uncovers novel pathways for tRNA processing and nuclear-cytoplasmic dynamics.

PubMed

Wu, Jingyan; Bao, Alicia; Chatterjee, Kunal; Wan, Yao; Hopper, Anita K

2015-12-15

Transfer ribonucleic acids (tRNAs) are essential for protein synthesis. However, key gene products involved in tRNA biogenesis and subcellular movement remain to be discovered. We conducted the first comprehensive unbiased analysis of the role of nearly an entire proteome in tRNA biology and describe 162 novel and 12 previously known Saccharomyces cerevisiae gene products that function in tRNA processing, turnover, and subcellular movement. tRNA nuclear export is of particular interest because it is essential, but the known tRNA exporters (Los1 [exportin-t] and Msn5 [exportin-5]) are unessential. We report that mutations of CRM1 (Exportin-1), MEX67/MTR2 (TAP/p15), and five nucleoporins cause accumulation of unspliced tRNA, a hallmark of defective tRNA nuclear export. CRM1 mutation genetically interacts with los1Δ and causes altered tRNA nuclear-cytoplasmic distribution. The data implicate roles for the protein and mRNA nuclear export machineries in tRNA nuclear export. Mutations of genes encoding actin cytoskeleton components and mitochondrial outer membrane proteins also cause accumulation of unspliced tRNA, likely due to defective splicing on mitochondria. Additional gene products, such as chromatin modification enzymes, have unanticipated effects on pre-tRNA end processing. Thus, this genome-wide screen uncovered putative novel pathways for tRNA nuclear export and extensive links between tRNA biology and other aspects of cell physiology. © 2015 Wu et al.; Published by Cold Spring Harbor Laboratory Press.

Genome-wide screen uncovers novel pathways for tRNA processing and nuclear–cytoplasmic dynamics

PubMed Central

Wu, Jingyan; Bao, Alicia; Chatterjee, Kunal; Wan, Yao; Hopper, Anita K.

2015-01-01

Transfer ribonucleic acids (tRNAs) are essential for protein synthesis. However, key gene products involved in tRNA biogenesis and subcellular movement remain to be discovered. We conducted the first comprehensive unbiased analysis of the role of nearly an entire proteome in tRNA biology and describe 162 novel and 12 previously known Saccharomyces cerevisiae gene products that function in tRNA processing, turnover, and subcellular movement. tRNA nuclear export is of particular interest because it is essential, but the known tRNA exporters (Los1 [exportin-t] and Msn5 [exportin-5]) are unessential. We report that mutations of CRM1 (Exportin-1), MEX67/MTR2 (TAP/p15), and five nucleoporins cause accumulation of unspliced tRNA, a hallmark of defective tRNA nuclear export. CRM1 mutation genetically interacts with los1Δ and causes altered tRNA nuclear–cytoplasmic distribution. The data implicate roles for the protein and mRNA nuclear export machineries in tRNA nuclear export. Mutations of genes encoding actin cytoskeleton components and mitochondrial outer membrane proteins also cause accumulation of unspliced tRNA, likely due to defective splicing on mitochondria. Additional gene products, such as chromatin modification enzymes, have unanticipated effects on pre-tRNA end processing. Thus, this genome-wide screen uncovered putative novel pathways for tRNA nuclear export and extensive links between tRNA biology and other aspects of cell physiology. PMID:26680305

Distance from sub-Saharan Africa predicts mutational load in diverse human genomes.

PubMed

Henn, Brenna M; Botigué, Laura R; Peischl, Stephan; Dupanloup, Isabelle; Lipatov, Mikhail; Maples, Brian K; Martin, Alicia R; Musharoff, Shaila; Cann, Howard; Snyder, Michael P; Excoffier, Laurent; Kidd, Jeffrey M; Bustamante, Carlos D

2016-01-26

The Out-of-Africa (OOA) dispersal ∼ 50,000 y ago is characterized by a series of founder events as modern humans expanded into multiple continents. Population genetics theory predicts an increase of mutational load in populations undergoing serial founder effects during range expansions. To test this hypothesis, we have sequenced full genomes and high-coverage exomes from seven geographically divergent human populations from Namibia, Congo, Algeria, Pakistan, Cambodia, Siberia, and Mexico. We find that individual genomes vary modestly in the overall number of predicted deleterious alleles. We show via spatially explicit simulations that the observed distribution of deleterious allele frequencies is consistent with the OOA dispersal, particularly under a model where deleterious mutations are recessive. We conclude that there is a strong signal of purifying selection at conserved genomic positions within Africa, but that many predicted deleterious mutations have evolved as if they were neutral during the expansion out of Africa. Under a model where selection is inversely related to dominance, we show that OOA populations are likely to have a higher mutation load due to increased allele frequencies of nearly neutral variants that are recessive or partially recessive.

Whole-genome sequencing of an aggressive BRAF wild-type papillary thyroid cancer identified EML4-ALK translocation as a therapeutic target.

PubMed

Demeure, Michael J; Aziz, Meraj; Rosenberg, Richard; Gurley, Steven D; Bussey, Kimberly J; Carpten, John D

2014-06-01

Recent advances in the treatment of cancer have focused on targeting genomic aberrations with selective therapeutic agents. In radioiodine resistant aggressive papillary thyroid cancers, there remain few effective therapeutic options. A 62-year-old man who underwent multiple operations for papillary thyroid cancer and whose metastases progressed despite standard treatments provided tumor tissue. We analyzed tumor and whole blood DNA by whole genome sequencing, achieving 80× or greater coverage over 94 % of the exome and 90 % of the genome. We determined somatic mutations and structural alterations. We found a total of 57 somatic mutations in 55 genes of the cancer genome. There was notably a lack of mutations in NRAS and BRAF, and no RET/PTC rearrangement. There was a mutation in the TRAPP oncogene and a loss of heterozygosity of the p16, p18, and RB1 tumor suppressor genes. The oncogenic driver for this tumor is a translocation involving the genes for anaplastic lymphoma receptor tyrosine kinase (ALK) and echinoderm microtubule associated protein like 4 (EML4). The EML4-ALK translocation has been reported in approximately 5 % of lung cancers, as well as in pediatric neuroblastoma, and is a therapeutic target for crizotinib. This is the first report of the whole genomic sequencing of a papillary thyroid cancer in which we identified an EML4-ALK translocation of a TRAPP oncogene mutation. These findings suggest that this tumor has a more distinct oncogenesis than BRAF mutant papillary thyroid cancer. Whole genome sequencing can elucidate an oncogenic context and expose potential therapeutic vulnerabilities in rare cancers.

The methylenetetrahydrofolate reductase C677T mutation induces cell-specific changes in genomic DNA methylation and uracil misincorporation: A possible molecular basis for the site-specific cancer risk modification

PubMed Central

Sohn, Kyoung-Jin; Jang, Hyeran; Campan, Mihaela; Weisenberger, Daniel J.; Dickhout, Jeffrey; Wang, Yi-Cheng; Cho, Robert C.; Yates, Zoe; Lucock, Mark; Chiang, En-Pei; Austin, Richard C.; Choi, Sang-Woon; Laird, Peter W.; Kim, Young-In

2009-01-01

The C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene is associated with a decreased risk of colon cancer while it may increase the risk of breast cancer. This polymorphism is associated with changes in intracellular folate cofactors, which may affect DNA methylation and synthesis via altered one-carbon transfer reactions. We investigated the effect of this mutation on DNA methylation and uracil misincorporation and its interaction with exogenous folate in further modulating these biomarkers of one-carbon transfer reactions in an in vitro model of the MTHFR 677T mutation in HCT116 colon and MDA-MB-435 breast adenocarcinoma cells. In HCT116 cells, the MTHFR 677T mutation was associated with significantly increased genomic DNA methylation when folate supply was adequate or high; however, in the setting of folate insufficiency, this mutation was associated with significantly decreased genomic DNA methylation. In contrast, in MDA-MB-435 cells, the MTHFR 677T mutation was associated with significantly decreased genomic DNA methylation when folate supply was adequate or high and with no effect when folate supply was low. The MTHFR 677T mutation was associated with a nonsignificant trend toward decreased and increased uracil misincorporation in HCT116 and MDA-MB-435 cells, respectively. Our data demonstrate for the first time a functional consequence of changes in intracellular folate cofactors resulting from the MTHFR 677T mutation in cells derived from the target organs of interest, thus providing a plausible cellular mechanism that may partly explain the site-specific modification of colon and breast cancer risks associated with the MTHFR C677T mutation. PMID:19123462

The Genome Sequencer FLX System--longer reads, more applications, straight forward bioinformatics and more complete data sets.

PubMed

Droege, Marcus; Hill, Brendon

2008-08-31

The Genome Sequencer FLX System (GS FLX), powered by 454 Sequencing, is a next-generation DNA sequencing technology featuring a unique mix of long reads, exceptional accuracy, and ultra-high throughput. It has been proven to be the most versatile of all currently available next-generation sequencing technologies, supporting many high-profile studies in over seven applications categories. GS FLX users have pursued innovative research in de novo sequencing, re-sequencing of whole genomes and target DNA regions, metagenomics, and RNA analysis. 454 Sequencing is a powerful tool for human genetics research, having recently re-sequenced the genome of an individual human, currently re-sequencing the complete human exome and targeted genomic regions using the NimbleGen sequence capture process, and detected low-frequency somatic mutations linked to cancer.

Towards social acceptance of plant breeding by genome editing.

PubMed

Araki, Motoko; Ishii, Tetsuya

2015-03-01

Although genome-editing technologies facilitate efficient plant breeding without introducing a transgene, it is creating indistinct boundaries in the regulation of genetically modified organisms (GMOs). Rapid advances in plant breeding by genome-editing require the establishment of a new global policy for the new biotechnology, while filling the gap between process-based and product-based GMO regulations. In this Opinion article we review recent developments in producing major crops using genome-editing, and we propose a regulatory model that takes into account the various methodologies to achieve genetic modifications as well as the resulting types of mutation. Moreover, we discuss the future integration of genome-editing crops into society, specifically a possible response to the 'Right to Know' movement which demands labeling of food that contains genetically engineered ingredients. Copyright © 2015 Elsevier Ltd. All rights reserved.

DNMT3A and IDH mutations in acute myeloid leukemia and other myeloid malignancies: associations with prognosis and potential treatment strategies

PubMed Central

Im, AP; Sehgal, AR; Carroll, MP; Smith, BD; Tefferi, A; Johnson, DE; Boyiadzis, M

2014-01-01

The development of effective treatment strategies for most forms of acute myeloid leukemia (AML) has languished for the past several decades. There are a number of reasons for this, but key among them is the considerable heterogeneity of this disease and the paucity of molecular markers that can be used to predict clinical outcomes and responsiveness to different therapies. The recent large-scale sequencing of AML genomes is now providing opportunities for patient stratification and personalized approaches to treatment that are based on individual mutational profiles. It is particularly notable that studies by The Cancer Genome Atlas and others have determined that 44% of patients with AML exhibit mutations in genes that regulate methylation of genomic DNA. In particular, frequent mutation has been observed in the genes encoding DNA methyltransferase 3A (DNMT3A), isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2), as well as Tet oncogene family member 2. This review will summarize the incidence of these mutations, their impact on biochemical functions including epigenetic modification of genomic DNA and their potential usefulness as prognostic indicators. Importantly, the presence of DNMT3A, IDH1 or IDH2 mutations may confer sensitivity to novel therapeutic approaches, including the use of demethylating agents. Therefore, the clinical experience with decitabine and azacitidine in the treatment of patients harboring these mutations will be reviewed. Overall, we propose that understanding the role of these mutations in AML biology will lead to more rational therapeutic approaches targeting molecularly defined subtypes of the disease. PMID:24699305

Single-cell paired-end genome sequencing reveals structural variation per cell cycle

PubMed Central

Voet, Thierry; Kumar, Parveen; Van Loo, Peter; Cooke, Susanna L.; Marshall, John; Lin, Meng-Lay; Zamani Esteki, Masoud; Van der Aa, Niels; Mateiu, Ligia; McBride, David J.; Bignell, Graham R.; McLaren, Stuart; Teague, Jon; Butler, Adam; Raine, Keiran; Stebbings, Lucy A.; Quail, Michael A.; D’Hooghe, Thomas; Moreau, Yves; Futreal, P. Andrew; Stratton, Michael R.; Vermeesch, Joris R.; Campbell, Peter J.

2013-01-01

The nature and pace of genome mutation is largely unknown. Because standard methods sequence DNA from populations of cells, the genetic composition of individual cells is lost, de novo mutations in cells are concealed within the bulk signal and per cell cycle mutation rates and mechanisms remain elusive. Although single-cell genome analyses could resolve these problems, such analyses are error-prone because of whole-genome amplification (WGA) artefacts and are limited in the types of DNA mutation that can be discerned. We developed methods for paired-end sequence analysis of single-cell WGA products that enable (i) detecting multiple classes of DNA mutation, (ii) distinguishing DNA copy number changes from allelic WGA-amplification artefacts by the discovery of matching aberrantly mapping read pairs among the surfeit of paired-end WGA and mapping artefacts and (iii) delineating the break points and architecture of structural variants. By applying the methods, we capture DNA copy number changes acquired over one cell cycle in breast cancer cells and in blastomeres derived from a human zygote after in vitro fertilization. Furthermore, we were able to discover and fine-map a heritable inter-chromosomal rearrangement t(1;16)(p36;p12) by sequencing a single blastomere. The methods will expedite applications in basic genome research and provide a stepping stone to novel approaches for clinical genetic diagnosis. PMID:23630320

Phosphorylation at the Homotypic Interface Regulates Nucleoprotein Oligomerization and Assembly of the Influenza Virus Replication Machinery

PubMed Central

Mondal, Arindam; Potts, Gregory K.; Dawson, Anthony R.; Coon, Joshua J.; Mehle, Andrew

2015-01-01

Negative-sense RNA viruses assemble large ribonucleoprotein (RNP) complexes that direct replication and transcription of the viral genome. Influenza virus RNPs contain the polymerase, genomic RNA and multiple copies of nucleoprotein (NP). During RNP assembly, monomeric NP oligomerizes along the length of the genomic RNA. Regulated assembly of the RNP is essential for virus replication, but how NP is maintained as a monomer that subsequently oligomerizes to form RNPs is poorly understood. Here we elucidate a mechanism whereby NP phosphorylation regulates oligomerization. We identified new evolutionarily conserved phosphorylation sites on NP and demonstrated that phosphorylation of NP decreased formation of higher-order complexes. Two phosphorylation sites were located on opposite sides of the NP:NP interface. In both influenza A and B virus, mutating or mimicking phosphorylation at these residues blocked homotypic interactions and drove NP towards a monomeric form. Highlighting the central role of this process during infection, these mutations impaired RNP formation, polymerase activity and virus replication. Thus, dynamic phosphorylation of NP regulates RNP assembly and modulates progression through the viral life cycle. PMID:25867750

CRISPR-Cas9: from Genome Editing to Cancer Research

PubMed Central

Chen, Si; Sun, Heng; Miao, Kai; Deng, Chu-Xia

2016-01-01

Cancer development is a multistep process triggered by innate and acquired mutations, which cause the functional abnormality and determine the initiation and progression of tumorigenesis. Gene editing is a widely used engineering tool for generating mutations that enhance tumorigenesis. The recent developed clustered regularly interspaced short palindromic repeats-CRISPR-associated 9 (CRISPR-Cas9) system renews the genome editing approach into a more convenient and efficient way. By rapidly introducing genetic modifications in cell lines, organs and animals, CRISPR-Cas9 system extends the gene editing into whole genome screening, both in loss-of-function and gain-of-function manners. Meanwhile, the system accelerates the establishment of animal cancer models, promoting in vivo studies for cancer research. Furthermore, CRISPR-Cas9 system is modified into diverse innovative tools for observing the dynamic bioprocesses in cancer studies, such as image tracing for targeted DNA, regulation of transcription activation or repression. Here, we view recent technical advances in the application of CRISPR-Cas9 system in cancer genetics, large-scale cancer driver gene hunting, animal cancer modeling and functional studies. PMID:27994508

What makes up plant genomes: The vanishing line between transposable elements and genes.

PubMed

Zhao, Dongyan; Ferguson, Ann A; Jiang, Ning

2016-02-01

The ultimate source of evolution is mutation. As the largest component in plant genomes, transposable elements (TEs) create numerous types of mutations that cannot be mimicked by other genetic mechanisms. When TEs insert into genomic sequences, they influence the expression of nearby genes as well as genes unlinked to the insertion. TEs can duplicate, mobilize, and recombine normal genes or gene fragments, with the potential to generate new genes or modify the structure of existing genes. TEs also donate their transposase coding regions for cellular functions in a process called TE domestication. Despite the host defense against TE activity, a subset of TEs survived and thrived through discreet selection of transposition activity, target site, element size, and the internal sequence. Finally, TEs have established strategies to reduce the efficacy of host defense system by increasing the cost of silencing TEs. This review discusses the recent progress in the area of plant TEs with a focus on the interaction between TEs and genes. Copyright © 2015 Elsevier B.V. All rights reserved.

Sequence diversity and molecular evolutionary rates between buffalo and cattle.

PubMed

Moaeen-ud-Din, M; Bilal, G

2015-02-01

Identification of genes of importance regarding production traits in buffalo is impaired by a paucity of genomic resources. Choice to fill this gap is to exploit data available for cow. The cross-species application of comparative genomics tools is potential gear to investigate the buffalo genome. However, this is dependent on nucleotide sequences similarity. In this study, gene diversity between buffalo and cattle was determined using 86 gene orthologues. There was approximately 3% difference in all genes in terms of nucleotide diversity and 0.267 ± 0.134 in amino acids, indicating the possibility for successfully using cross-species strategies for genomic studies. There were significantly higher non-synonymous substitutions both in cattle and buffalo; however, there was similar difference in terms of dN- dS (4.414 versus 4.745) in buffalo and cattle, respectively. Higher rate of non-synonymous substitutions at similar level in buffalo and cattle indicated a similar positive selection pressure. Results for relative rate test were assessed with the chi-squared test. There was no significance difference on unique mutations between cattle and buffalo lineages at synonymous sites. However, there was a significance difference on unique mutations for non-synonymous sites, indicating ongoing mutagenic process that generates substitutional mutation at approximately the same rate at silent sites. Moreover, despite of common ancestry, our results indicate a different divergent time among genes of cattle and buffalo. This is the first demonstration that variable rates of molecular evolution may be present within the family Bovidae. © 2014 Blackwell Verlag GmbH.

Global Gene Expression Patterns and Somatic Mutations in Sporadic Intracranial Aneurysms.

PubMed

Li, Zhili; Tan, Haibin; Shi, Yi; Huang, Guangfu; Wang, Zhenyu; Liu, Ling; Yin, Cheng; Wang, Qi

2017-04-01

High-throughput sequencing technologies can expand our understanding of the pathologic basis of intracranial aneurysms (IAs). Our study was aimed to decipher the gene expression signature and genetic factors associated with IAs. We determined the gene expression levels of 3 cases of IAs by RNA sequencing. Bioinformatics analysis was conducted to identify the differentially expressed genes (DEGs) and uncover their biological function. In addition, whole genome sequencing was performed on an additional 6 cases of IAs to detect the potential somatic alterations in DEGs. Compared with the normal arterial tissue, 1709 genes were differentially expressed in IAs arterial tissue. The most significantly up-regulated gene and down-regulated gene, H19 and HIST1H3J, may be essential for tumorigenesis of IAs. Hub protein of IKBKG in protein-protein interaction network was probably involved in the inflammation process in aneurysms. Another 2 hub proteins, ACTB and MKI67IP, as well as up-regulated genes, might be abnormally activated in aneurysms and involved in the pathogenesis of IAs. Further whole genome sequencing and filtering yielded 4 candidate somatic single nucleotide variants including MUC3B, and BLM may be involved in the pathogenesis of IAs. Even though, our results do not support the hypothesis of somatic mutations occurred in the DEGs. Two-dimensional genomic data from transcriptome and whole genome sequencing indicated that no somatic mutations occurred in DEGs. In addition, 3 DEGs (IKBKG, ACTB, and MKI67IP) and 2 mutant genes (MUC3B and BLM) were essential in IAs. Copyright © 2017 Elsevier Inc. All rights reserved.

Nuclear Proximity of Mtr4 with RNA exosome restricts DNA mutational asymmetry

PubMed Central

Lim, Junghyun; Giri, Pankaj Kumar; Kazadi, David; Laffleur, Brice; Zhang, Wanwei; Grinstein, Veronika; Pefanis, Evangelos; Brown, Lewis M.; Ladewig, Erik; Martin, Ophélie; Chen, Yuling; Rabadan, Raul; Boyer, François; Rothschild, Gerson; Cogné, Michel; Pinaud, Eric; Deng, Haiteng; Basu, Uttiya

2017-01-01

SUMMARY The distribution of sense and antisense strand DNA mutations on transcribed duplex DNA contributes to the development of immune and neural systems along with the progression of cancer. Because developmentally matured B cells undergo biologically programmed strand-specific DNA mutagenesis at focal DNA/RNA hybrid structures, they make a convenient system to investigate strand-specific mutagenesis mechanisms. We demonstrate that the sense and antisense strand DNA mutagenesis at the immunoglobulin heavy chain locus and some other regions of the B cell genome depends upon localized RNA processing protein complex formation in the nucleus. Both the physical proximity and coupled activities of RNA helicase Mtr4 (and Senataxin) with the noncoding RNA processing function of RNA exosome determine the strand specific distribution of DNA mutations. Our study suggests that strand-specific DNA mutagenesis-associated mechanisms will play major roles in other undiscovered aspects of organismic development. PMID:28431250

Implementation of Quality Management in Core Service Laboratories

PubMed Central

Creavalle, T.; Haque, K.; Raley, C.; Subleski, M.; Smith, M.W.; Hicks, B.

2010-01-01

CF-28 The Genetics and Genomics group of the Advanced Technology Program of SAIC-Frederick exists to bring innovative genomic expertise, tools and analysis to NCI and the scientific community. The Sequencing Facility (SF) provides next generation short read (Illumina) sequencing capacity to investigators using a streamlined production approach. The Laboratory of Molecular Technology (LMT) offers a wide range of genomics core services including microarray expression analysis, miRNA analysis, array comparative genome hybridization, long read (Roche) next generation sequencing, quantitative real time PCR, transgenic genotyping, Sanger sequencing, and clinical mutation detection services to investigators from across the NIH. As the technology supporting this genomic research becomes more complex, the need for basic quality processes within all aspects of the core service groups becomes critical. The Quality Management group works alongside members of these labs to establish or improve processes supporting operations control (equipment, reagent and materials management), process improvement (reengineering/optimization, automation, acceptance criteria for new technologies and tech transfer), and quality assurance and customer support (controlled documentation/SOPs, training, service deficiencies and continual improvement efforts). Implementation and expansion of quality programs within unregulated environments demonstrates SAIC-Frederick's dedication to providing the highest quality products and services to the NIH community.

Mutations Affecting Expression of the rosy Locus in Drosophila melanogaster

PubMed Central

Lee, Chong Sung; Curtis, Daniel; McCarron, Margaret; Love, Carol; Gray, Mark; Bender, Welcome; Chovnick, Arthur

1987-01-01

The rosy locus in Drosophila melanogaster codes for the enzyme xanthine dehydrogenase (XDH). Previous studies defined a "control element" near the 5' end of the gene, where variant sites affected the amount of rosy mRNA and protein produced. We have determined the DNA sequence of this region from both genomic and cDNA clones, and from the ry+10 underproducer strain. This variant strain had many sequence differences, so that the site of the regulatory change could not be fixed. A mutagenesis was also undertaken to isolate new regulatory mutations. We induced 376 new mutations with 1-ethyl-1-nitrosourea (ENU) and screened them to isolate those that reduced the amount of XDH protein produced, but did not change the properties of the enzyme. Genetic mapping was used to find mutations located near the 5' end of the gene. DNA from each of seven mutants was cloned and sequenced through the 5' region. Mutant base changes were identified in all seven; they appear to affect splicing and translation of the rosy mRNA. In a related study (T. P. Keith et al. 1987), the genomic and cDNA sequences are extended through the 3' end of the gene; the combined sequences define the processing pattern of the rosy transcript and predict the amino acid sequence of XDH. PMID:3036645

Mutation allele burden remains unchanged in chronic myelomonocytic leukaemia responding to hypomethylating agents

DOE PAGES

Merlevede, Jane; Droin, Nathalie; Qin, Tingting; ...

2016-02-24

The cytidine analogues azacytidine and 5-aza-2’-deoxycytidine (decitabine) are commonly used to treat myelodysplastic syndromes, with or without a myeloproliferative component. It remains unclear whether the response to these hypomethylating agents results from a cytotoxic or an epigenetic effect. In this study, we address this question in chronic myelomonocytic leukaemia. We describe a comprehensive analysis of the mutational landscape of these tumours, combining whole-exome and whole-genome sequencing. We identify an average of 14 ± 5 somatic mutations in coding sequences of sorted monocyte DNA and the signatures of three mutational processes. Serial sequencing demonstrates that the response to hypomethylating agents ismore » associated with changes in DNA methylation and gene expression, without any decrease in the mutation allele burden, nor prevention of new genetic alteration occurence. Lastly, our findings indicate that cytosine analogues restore a balanced haematopoiesis without decreasing the size of the mutated clone, arguing for a predominantly epigenetic effect.« less

New observations on maternal age effect on germline de novo mutations.

PubMed

Wong, Wendy S W; Solomon, Benjamin D; Bodian, Dale L; Kothiyal, Prachi; Eley, Greg; Huddleston, Kathi C; Baker, Robin; Thach, Dzung C; Iyer, Ramaswamy K; Vockley, Joseph G; Niederhuber, John E

2016-01-19

Germline mutations are the source of evolution and contribute substantially to many health-related processes. Here we use whole-genome deep sequencing data from 693 parents-offspring trios to examine the de novo point mutations (DNMs) in the offspring. Our estimate for the mutation rate per base pair per generation is 1.05 × 10(-8), well within the range of previous studies. We show that maternal age has a small but significant correlation with the total number of DNMs in the offspring after controlling for paternal age (0.51 additional mutations per year, 95% CI: 0.29, 0.73), which was not detectable in the smaller and younger parental cohorts of earlier studies. Furthermore, while the total number of DNMs increases at a constant rate for paternal age, the contribution from the mother increases at an accelerated rate with age.These observations have implications related to the incidence of de novo mutations relating to maternal age.

Mutation allele burden remains unchanged in chronic myelomonocytic leukaemia responding to hypomethylating agents

PubMed Central

Merlevede, Jane; Droin, Nathalie; Qin, Tingting; Meldi, Kristen; Yoshida, Kenichi; Morabito, Margot; Chautard, Emilie; Auboeuf, Didier; Fenaux, Pierre; Braun, Thorsten; Itzykson, Raphael; de Botton, Stéphane; Quesnel, Bruno; Commes, Thérèse; Jourdan, Eric; Vainchenker, William; Bernard, Olivier; Pata-Merci, Noemie; Solier, Stéphanie; Gayevskiy, Velimir; Dinger, Marcel E.; Cowley, Mark J.; Selimoglu-Buet, Dorothée; Meyer, Vincent; Artiguenave, François; Deleuze, Jean-François; Preudhomme, Claude; Stratton, Michael R.; Alexandrov, Ludmil B.; Padron, Eric; Ogawa, Seishi; Koscielny, Serge; Figueroa, Maria; Solary, Eric

2016-01-01

The cytidine analogues azacytidine and 5-aza-2'-deoxycytidine (decitabine) are commonly used to treat myelodysplastic syndromes, with or without a myeloproliferative component. It remains unclear whether the response to these hypomethylating agents results from a cytotoxic or an epigenetic effect. In this study, we address this question in chronic myelomonocytic leukaemia. We describe a comprehensive analysis of the mutational landscape of these tumours, combining whole-exome and whole-genome sequencing. We identify an average of 14±5 somatic mutations in coding sequences of sorted monocyte DNA and the signatures of three mutational processes. Serial sequencing demonstrates that the response to hypomethylating agents is associated with changes in DNA methylation and gene expression, without any decrease in the mutation allele burden, nor prevention of new genetic alteration occurence. Our findings indicate that cytosine analogues restore a balanced haematopoiesis without decreasing the size of the mutated clone, arguing for a predominantly epigenetic effect. PMID:26908133

Faster-X evolution: Theory and evidence from Drosophila.

PubMed

Charlesworth, Brian; Campos, José L; Jackson, Benjamin C

2018-02-12

A faster rate of adaptive evolution of X-linked genes compared with autosomal genes can be caused by the fixation of recessive or partially recessive advantageous mutations, due to the full expression of X-linked mutations in hemizygous males. Other processes, including recombination rate and mutation rate differences between X chromosomes and autosomes, may also cause faster evolution of X-linked genes. We review population genetics theory concerning the expected relative values of variability and rates of evolution of X-linked and autosomal DNA sequences. The theoretical predictions are compared with data from population genomic studies of several species of Drosophila. We conclude that there is evidence for adaptive faster-X evolution of several classes of functionally significant nucleotides. We also find evidence for potential differences in mutation rates between X-linked and autosomal genes, due to differences in mutational bias towards GC to AT mutations. Many aspects of the data are consistent with the male hemizygosity model, although not all possible confounding factors can be excluded. © 2018 John Wiley & Sons Ltd.

Comparative Genome Analysis Between Aspergillus oryzae Strains Reveals Close Relationship Between Sites of Mutation Localization and Regions of Highly Divergent Genes among Aspergillus Species

PubMed Central

Umemura, Myco; Koike, Hideaki; Yamane, Noriko; Koyama, Yoshinori; Satou, Yuki; Kikuzato, Ikuya; Teruya, Morimi; Tsukahara, Masatoshi; Imada, Yumi; Wachi, Youji; Miwa, Yukino; Yano, Shuichi; Tamano, Koichi; Kawarabayasi, Yutaka; Fujimori, Kazuhiro E.; Machida, Masayuki; Hirano, Takashi

2012-01-01

Aspergillus oryzae has been utilized for over 1000 years in Japan for the production of various traditional foods, and a large number of A. oryzae strains have been isolated and/or selected for the effective fermentation of food ingredients. Characteristics of genetic alterations among the strains used are of particular interest in studies of A. oryzae. Here, we have sequenced the whole genome of an industrial fungal isolate, A. oryzae RIB326, by using a next-generation sequencing system and compared the data with those of A. oryzae RIB40, a wild-type strain sequenced in 2005. The aim of this study was to evaluate the mutation pressure on the non-syntenic blocks (NSBs) of the genome, which were previously identified through comparative genomic analysis of A. oryzae, Aspergillus fumigatus, and Aspergillus nidulans. We found that genes within the NSBs of RIB326 accumulate mutations more frequently than those within the SBs, regardless of their distance from the telomeres or of their expression level. Our findings suggest that the high mutation frequency of NSBs might contribute to maintaining the diversity of the A. oryzae genome. PMID:22912434

Comparative genome analysis between Aspergillus oryzae strains reveals close relationship between sites of mutation localization and regions of highly divergent genes among Aspergillus species.

PubMed

Umemura, Myco; Koike, Hideaki; Yamane, Noriko; Koyama, Yoshinori; Satou, Yuki; Kikuzato, Ikuya; Teruya, Morimi; Tsukahara, Masatoshi; Imada, Yumi; Wachi, Youji; Miwa, Yukino; Yano, Shuichi; Tamano, Koichi; Kawarabayasi, Yutaka; Fujimori, Kazuhiro E; Machida, Masayuki; Hirano, Takashi

2012-10-01

Aspergillus oryzae has been utilized for over 1000 years in Japan for the production of various traditional foods, and a large number of A. oryzae strains have been isolated and/or selected for the effective fermentation of food ingredients. Characteristics of genetic alterations among the strains used are of particular interest in studies of A. oryzae. Here, we have sequenced the whole genome of an industrial fungal isolate, A. oryzae RIB326, by using a next-generation sequencing system and compared the data with those of A. oryzae RIB40, a wild-type strain sequenced in 2005. The aim of this study was to evaluate the mutation pressure on the non-syntenic blocks (NSBs) of the genome, which were previously identified through comparative genomic analysis of A. oryzae, Aspergillus fumigatus, and Aspergillus nidulans. We found that genes within the NSBs of RIB326 accumulate mutations more frequently than those within the SBs, regardless of their distance from the telomeres or of their expression level. Our findings suggest that the high mutation frequency of NSBs might contribute to maintaining the diversity of the A. oryzae genome.

The Genomic Basis of Evolutionary Innovation in Pseudomonas aeruginosa

PubMed Central

Wagner, Andreas; MacLean, R. Craig

2016-01-01

Novel traits play a key role in evolution, but their origins remain poorly understood. Here we address this problem by using experimental evolution to study bacterial innovation in real time. We allowed 380 populations of Pseudomonas aeruginosa to adapt to 95 different carbon sources that challenged bacteria with either evolving novel metabolic traits or optimizing existing traits. Whole genome sequencing of more than 80 clones revealed profound differences in the genetic basis of innovation and optimization. Innovation was associated with the rapid acquisition of mutations in genes involved in transcription and metabolism. Mutations in pre-existing duplicate genes in the P. aeruginosa genome were common during innovation, but not optimization. These duplicate genes may have been acquired by P. aeruginosa due to either spontaneous gene amplification or horizontal gene transfer. High throughput phenotype assays revealed that novelty was associated with increased pleiotropic costs that are likely to constrain innovation. However, mutations in duplicate genes with close homologs in the P. aeruginosa genome were associated with low pleiotropic costs compared to mutations in duplicate genes with distant homologs in the P. aeruginosa genome, suggesting that functional redundancy between duplicates facilitates innovation by buffering pleiotropic costs. PMID:27149698

Mitigating Mitochondrial Genome Erosion Without Recombination.

PubMed

Radzvilavicius, Arunas L; Kokko, Hanna; Christie, Joshua R

2017-11-01

Mitochondria are ATP-producing organelles of bacterial ancestry that played a key role in the origin and early evolution of complex eukaryotic cells. Most modern eukaryotes transmit mitochondrial genes uniparentally, often without recombination among genetically divergent organelles. While this asymmetric inheritance maintains the efficacy of purifying selection at the level of the cell, the absence of recombination could also make the genome susceptible to Muller's ratchet. How mitochondria escape this irreversible defect accumulation is a fundamental unsolved question. Occasional paternal leakage could in principle promote recombination, but it would also compromise the purifying selection benefits of uniparental inheritance. We assess this tradeoff using a stochastic population-genetic model. In the absence of recombination, uniparental inheritance of freely-segregating genomes mitigates mutational erosion, while paternal leakage exacerbates the ratchet effect. Mitochondrial fusion-fission cycles ensure independent genome segregation, improving purifying selection. Paternal leakage provides opportunity for recombination to slow down the mutation accumulation, but always at a cost of increased steady-state mutation load. Our findings indicate that random segregation of mitochondrial genomes under uniparental inheritance can effectively combat the mutational meltdown, and that homologous recombination under paternal leakage might not be needed. Copyright © 2017 by the Genetics Society of America.

Genome-Wide Linkage Analysis to Identify Genetic Modifiers of ALK Mutation Penetrance in Familial Neuroblastoma

PubMed Central

Devoto, Marcella; Specchia, Claudia; Laudenslager, Marci; Longo, Luca; Hakonarson, Hakon; Maris, John; Mossé, Yael

2011-01-01

Background Neuroblastoma (NB) is an important childhood cancer with a strong genetic component related to disease susceptibility. Approximately 1% of NB cases have a positive family history. Following a genome-wide linkage analysis and sequencing of candidate genes in the critical region, we identified ALK as the major familial NB gene. Dominant mutations in ALK are found in more than 50% of familial NB cases. However, in the families used for the linkage study, only about 50% of carriers of ALK mutations are affected by NB. Methods To test whether genetic variation may explain the reduced penetrance of the disease phenotype, we analyzed genome-wide genotype data in ALK mutation-positive families using a model-based linkage approach with different liability classes for carriers and non-carriers of ALK mutations. Results The region with the highest LOD score was located at chromosome 2p23–p24 and included the ALK locus under models of dominant and recessive inheritance. Conclusions This finding suggests that variants in the non-mutated ALK gene or another gene linked to it may affect penetrance of the ALK mutations and risk of developing NB in familial cases. PMID:21734404

Significant associations between driver gene mutations and DNA methylation alterations across many cancer types

PubMed Central

Chen, Yun-Ching; Margolin, Gennady

2017-01-01

Recent evidence shows that mutations in several driver genes can cause aberrant methylation patterns, a hallmark of cancer. In light of these findings, we hypothesized that the landscapes of tumor genomes and epigenomes are tightly interconnected. We measured this relationship using principal component analyses and methylation-mutation associations applied at the nucleotide level and with respect to genome-wide trends. We found that a few mutated driver genes were associated with genome-wide patterns of aberrant hypomethylation or CpG island hypermethylation in specific cancer types. In addition, we identified associations between 737 mutated driver genes and site-specific methylation changes. Moreover, using these mutation-methylation associations, we were able to distinguish between two uterine and two thyroid cancer subtypes. The driver gene mutation–associated methylation differences between the thyroid cancer subtypes were linked to differential gene expression in JAK-STAT signaling, NADPH oxidation, and other cancer-related pathways. These results establish that driver gene mutations are associated with methylation alterations capable of shaping regulatory network functions. In addition, the methodology presented here can be used to subdivide tumors into more homogeneous subsets corresponding to underlying molecular characteristics, which could improve treatment efficacy. PMID:29125844

A shifting mutational landscape in 6 nutritional states: Stress-induced mutagenesis as a series of distinct stress input-mutation output relationships.

PubMed

Maharjan, Ram P; Ferenci, Thomas

2017-06-01

Environmental stresses increase genetic variation in bacteria, plants, and human cancer cells. The linkage between various environments and mutational outcomes has not been systematically investigated, however. Here, we established the influence of nutritional stresses commonly found in the biosphere (carbon, phosphate, nitrogen, oxygen, or iron limitation) on both the rate and spectrum of mutations in Escherichia coli. We found that each limitation was associated with a remarkably distinct mutational profile. Overall mutation rates were not always elevated, and nitrogen, iron, and oxygen limitation resulted in major spectral changes but no net increase in rate. Our results thus suggest that stress-induced mutagenesis is a diverse series of stress input-mutation output linkages that is distinct in every condition. Environment-specific spectra resulted in the differential emergence of traits needing particular mutations in these settings. Mutations requiring transpositions were highest under iron and oxygen limitation, whereas base-pair substitutions and indels were highest under phosphate limitation. The unexpected diversity of input-output effects explains some important phenomena in the mutational biases of evolving genomes. The prevalence of bacterial insertion sequence transpositions in the mammalian gut or in anaerobically stored cultures is due to environmentally determined mutation availability. Likewise, the much-discussed genomic bias towards transition base substitutions in evolving genomes can now be explained as an environment-specific output. Altogether, our conclusion is that environments influence genetic variation as well as selection.

A shifting mutational landscape in 6 nutritional states: Stress-induced mutagenesis as a series of distinct stress input–mutation output relationships

PubMed Central

Maharjan, Ram P.

2017-01-01

Environmental stresses increase genetic variation in bacteria, plants, and human cancer cells. The linkage between various environments and mutational outcomes has not been systematically investigated, however. Here, we established the influence of nutritional stresses commonly found in the biosphere (carbon, phosphate, nitrogen, oxygen, or iron limitation) on both the rate and spectrum of mutations in Escherichia coli. We found that each limitation was associated with a remarkably distinct mutational profile. Overall mutation rates were not always elevated, and nitrogen, iron, and oxygen limitation resulted in major spectral changes but no net increase in rate. Our results thus suggest that stress-induced mutagenesis is a diverse series of stress input–mutation output linkages that is distinct in every condition. Environment-specific spectra resulted in the differential emergence of traits needing particular mutations in these settings. Mutations requiring transpositions were highest under iron and oxygen limitation, whereas base-pair substitutions and indels were highest under phosphate limitation. The unexpected diversity of input–output effects explains some important phenomena in the mutational biases of evolving genomes. The prevalence of bacterial insertion sequence transpositions in the mammalian gut or in anaerobically stored cultures is due to environmentally determined mutation availability. Likewise, the much-discussed genomic bias towards transition base substitutions in evolving genomes can now be explained as an environment-specific output. Altogether, our conclusion is that environments influence genetic variation as well as selection. PMID:28594817

New mutations of DAX-1 genes in two Japanese patients with X-linked congenital adrenal hypoplasia and hypogonadotropic hypogonadism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yanase, Toshihiko; Takayanagi, Ryoichi; Oba, Koichi

Congenital adrenal hypoplasia, an X-linked disorder, is characterized by primary adrenal insufficiency and frequent association with hypogonadotropic hypogonadism. The X-chromosome gene DAX-1 has been most recently identified and shown to be responsible for this disorder. We analyzed the DAX-1 genes of two unrelated Japanese patients with congenital adrenal hypoplasia and hypogonadotropic hypogonadism by using PCR amplification of genomic DNA and its complete exonic sequencing. In a family containing several affected individuals, the proband male patient had a stop codon (TGA) in place of tryptophan (TGG) at amino acid position 171. As expected, his mother was a heterozygous carrier for themore » mutation, whereas his father and unaffected brother did not carry this mutation. In another male patient with noncontributory family history, sequencing revealed a 1-bp (T) deletion at amino acid position 280, leading to a frame shift and, subsequently a premature stop codon at amino acid position 371. The presence of this mutation in the patients` genome was further confirmed by digestion of genomic PCR product with MspI created by this mutation. Family studies using MspI digestion of genomic PCR products revealed that neither parent of this individual carried the mutation. These results clearly indicate that congenital adrenal hypoplasia and hypogonadotropic hypogonadism result from not only inherited but also de novo mutation in the DAX-1 gene. 31 refs., 4 figs., 2 tabs.« less

Targeted sequencing identifies genetic alterations that confer primary resistance to EGFR tyrosine kinase inhibitor (Korean Lung Cancer Consortium).

PubMed

Lim, Sun Min; Kim, Hye Ryun; Cho, Eun Kyung; Min, Young Joo; Ahn, Jin Seok; Ahn, Myung-Ju; Park, Keunchil; Cho, Byoung Chul; Lee, Ji-Hyun; Jeong, Hye Cheol; Kim, Eun Kyung; Kim, Joo-Hang

2016-06-14

Non-small-cell lung cancer (NSCLC) patients with activating epidermal growth factor receptor (EGFR) mutations may exhibit primary resistance to EGFR tyrosine kinase inhibitor (TKI). We aimed to examine genomic alterations associated with de novo resistance to gefitinib in a prospective study of NSCLC patients. One-hundred and fifty two patients with activating EGFR mutations were included in this study and 136 patients' tumor sample were available for targeted sequencing of genomic alterations in 22 genes using the Colon and Lung Cancer panel (Ampliseq, Life Technologies). All 132 patients with EGFR mutation were treated with gefitinib for their treatment of advanced NSCLC. Twenty patients showed primary resistance to EGFR TKI, and were classified as non-responders. A total of 543 somatic single-nucleotide variants (498 missense, 13 nonsense) and 32 frameshift insertions/deletions, with a median of 3 mutations per sample. TP53 was most commonly mutated (47%) and mutations in SMAD4 was also common (19%), as well as DDR2 (16%), PIK3CA (15%), STK11 (14%), and BRAF (7%). Genomic mutations in the PI3K/Akt/mTOR pathway were commonly found in non-responders (45%) compared to responders (27%), and they had significantly shorter progression-free survival and overall survival compared to patients without mutations (2.1 vs. 12.8 months, P=0.04, 15.7 vs. not reached, P<0.001). FGFR 1-3 alterations, KRAS mutations and TP53 mutations were more commonly detected in non-responders compared to responders. Genomic mutations in the PI3K/Akt/mTOR pathway were commonly identified in non-responders and may confer resistance to EGFR TKI. Screening lung adenocarcinoma patients with clinical cancer gene test may aid in selecting out those who show primary resistance to EGFR TKI (NCT01697163).

Clinicopathological analysis of endometrial carcinomas harboring somatic POLE exonuclease domain mutations.

PubMed

Hussein, Yaser R; Weigelt, Britta; Levine, Douglas A; Schoolmeester, J Kenneth; Dao, Linda N; Balzer, Bonnie L; Liles, Georgia; Karlan, Beth; Köbel, Martin; Lee, Cheng-Han; Soslow, Robert A

2015-04-01

The Cancer Genome Atlas described four major genomic groups of endometrial carcinomas, including a POLE ultramutated subtype comprising ∼10% of endometrioid adenocarcinoma, characterized by POLE exonuclease domain mutations, ultrahigh somatic mutation rates, and favorable outcome. Our aim was to examine the morphological and clinicopathological features of ultramutated endometrial carcinomas harboring somatic POLE exonuclease domain mutations. Hematoxylin and eosin slides and pathology reports for 8/17 POLE-mutated endometrial carcinomas described in the Cancer Genome Atlas study were studied; for the remaining cases, virtual whole slide images publicly available at cBioPortal (www.cbioportal.org) were examined. A second cohort of eight POLE mutated endometrial carcinomas from University of Calgary was also studied. Median age was 55 years (range 33-87 years). Nineteen patients presented as stage I, 1 stage II, and 5 stage III. The majority of cases (24 of the 25) demonstrated defining morphological features of endometrioid differentiation. The studied cases were frequently high grade (60%) and rich in tumor-infiltrating lymphocytes and/or peri-tumoral lymphocytes (84%); many tumors showed morphological heterogeneity (52%) and ambiguity (16%). Foci demonstrating severe nuclear atypia led to concern for serous carcinoma in 28% of cases. At the molecular level, the majority of the Cancer Genome Atlas POLE-mutated tumors were microsatellite stable (65%), and TP53 mutations were present in 35% of cases. They also harbored mutations in PTEN (94%), FBXW7 (82%), ARID1A (76%), and PIK3CA (71%). All patients from both cohorts were alive without disease, and none of the patients developed recurrence at the time of follow-up (median 33 months; range 2-102 months). In conclusion, the recognition of ultramutated endometrial carcinomas with POLE exonuclease domain mutation is important given their favorable outcome. Our histopathological review revealed that these tumors are commonly high grade, have obvious lymphocytic infiltrates, and can show ambiguous morphology. As they frequently harbor TP53 mutations, it is important not to misclassify them as serous carcinoma.

High Resolution Melt (HRM) analysis is an efficient tool to genotype EMS mutants in complex crop genomes.

PubMed

Lochlainn, Seosamh Ó; Amoah, Stephen; Graham, Neil S; Alamer, Khalid; Rios, Juan J; Kurup, Smita; Stoute, Andrew; Hammond, John P; Østergaard, Lars; King, Graham J; White, Phillip J; Broadley, Martin R

2011-12-08

Targeted Induced Loci Lesions IN Genomes (TILLING) is increasingly being used to generate and identify mutations in target genes of crop genomes. TILLING populations of several thousand lines have been generated in a number of crop species including Brassica rapa. Genetic analysis of mutants identified by TILLING requires an efficient, high-throughput and cost effective genotyping method to track the mutations through numerous generations. High resolution melt (HRM) analysis has been used in a number of systems to identify single nucleotide polymorphisms (SNPs) and insertion/deletions (IN/DELs) enabling the genotyping of different types of samples. HRM is ideally suited to high-throughput genotyping of multiple TILLING mutants in complex crop genomes. To date it has been used to identify mutants and genotype single mutations. The aim of this study was to determine if HRM can facilitate downstream analysis of multiple mutant lines identified by TILLING in order to characterise allelic series of EMS induced mutations in target genes across a number of generations in complex crop genomes. We demonstrate that HRM can be used to genotype allelic series of mutations in two genes, BraA.CAX1a and BraA.MET1.a in Brassica rapa. We analysed 12 mutations in BraA.CAX1.a and five in BraA.MET1.a over two generations including a back-cross to the wild-type. Using a commercially available HRM kit and the Lightscanner™ system we were able to detect mutations in heterozygous and homozygous states for both genes. Using HRM genotyping on TILLING derived mutants, it is possible to generate an allelic series of mutations within multiple target genes rapidly. Lines suitable for phenotypic analysis can be isolated approximately 8-9 months (3 generations) from receiving M3 seed of Brassica rapa from the RevGenUK TILLING service.

High Resolution Melt (HRM) analysis is an efficient tool to genotype EMS mutants in complex crop genomes

PubMed Central

2011-01-01

Background Targeted Induced Loci Lesions IN Genomes (TILLING) is increasingly being used to generate and identify mutations in target genes of crop genomes. TILLING populations of several thousand lines have been generated in a number of crop species including Brassica rapa. Genetic analysis of mutants identified by TILLING requires an efficient, high-throughput and cost effective genotyping method to track the mutations through numerous generations. High resolution melt (HRM) analysis has been used in a number of systems to identify single nucleotide polymorphisms (SNPs) and insertion/deletions (IN/DELs) enabling the genotyping of different types of samples. HRM is ideally suited to high-throughput genotyping of multiple TILLING mutants in complex crop genomes. To date it has been used to identify mutants and genotype single mutations. The aim of this study was to determine if HRM can facilitate downstream analysis of multiple mutant lines identified by TILLING in order to characterise allelic series of EMS induced mutations in target genes across a number of generations in complex crop genomes. Results We demonstrate that HRM can be used to genotype allelic series of mutations in two genes, BraA.CAX1a and BraA.MET1.a in Brassica rapa. We analysed 12 mutations in BraA.CAX1.a and five in BraA.MET1.a over two generations including a back-cross to the wild-type. Using a commercially available HRM kit and the Lightscanner™ system we were able to detect mutations in heterozygous and homozygous states for both genes. Conclusions Using HRM genotyping on TILLING derived mutants, it is possible to generate an allelic series of mutations within multiple target genes rapidly. Lines suitable for phenotypic analysis can be isolated approximately 8-9 months (3 generations) from receiving M3 seed of Brassica rapa from the RevGenUK TILLING service. PMID:22152063

Spontaneous Transformation of Murine Epithelial Cells Requires the Early Acquisition of Specific Chromosomal Aneuploidies and Genomic Imbalances

PubMed Central

Padilla-Nash, Hesed M.; Hathcock, Karen; McNeil, Nicole E.; Mack, David; Hoeppner, Daniel; Ravin, Rea; Knutsen, Turid; Yonescu, Raluca; Wangsa, Danny; Dorritie, Kathleen; Barenboim, Linda; Hu, Yue; Ried, Thomas

2011-01-01

Human carcinomas are defined by recurrent chromosomal aneuploidies, which result in tissue-specific distribution of genomic imbalances. In order to develop models for these genome mutations and determine their role in tumorigenesis, we generated 45 spontaneously transformed murine cell lines from normal epithelial cells derived from bladder, cervix, colon, kidney, lung, and mammary gland. Phenotypic changes, chromosomal aberrations, centrosome number, and telomerase activity were assayed in control uncultured cells and in three subsequent stages of transformation. Supernumerary centrosomes, bi-nucleate cells, and tetraploidy were observed as early as 48 hr after explantation. In addition, telomerase activity increased throughout progression. Live-cell imaging revealed that failure of cytokinesis, not cell fusion, promoted genome duplication. Spectral karyotyping demonstrated that aneuploidy preceded immortalization, consisting predominantly of whole chromosome losses (4, 9, 12, 13, 16, and Y) and gains (1, 10, 15, and 19). After transformation, focal amplifications of the oncogenes Myc and Mdm2 were frequently detected. Fifty percent of the transformed lines resulted in tumors upon injection into immuno-compromised mice. The phenotypic and genomic alterations observed in spontaneously transformed murine epithelial cells recapitulated the aberration pattern observed during human carcinogenesis. The dominant aberration of these cell lines was the presence of specific chromosomal aneuploidies. We propose that our newly derived cancer models will be useful tools to dissect the sequential steps of genome mutations during malignant transformation, and also to identify cancer-specific genes, signaling pathways, and the role of chromosomal instability in this process. PMID:22161874

Integrating transcriptome and genome re-sequencing data to identify key genes and mutations affecting chicken eggshell qualities.

PubMed

Zhang, Quan; Zhu, Feng; Liu, Long; Zheng, Chuan Wei; Wang, De He; Hou, Zhuo Cheng; Ning, Zhong Hua

2015-01-01

Eggshell damages lead to economic losses in the egg production industry and are a threat to human health. We examined 49-wk-old Rhode Island White hens (Gallus gallus) that laid eggs having shells with significantly different strengths and thicknesses. We used HiSeq 2000 (Illumina) sequencing to characterize the chicken transcriptome and whole genome to identify the key genes and genetic mutations associated with eggshell calcification. We identified a total of 14,234 genes expressed in the chicken uterus, representing 89% of all annotated chicken genes. A total of 889 differentially expressed genes were identified by comparing low eggshell strength (LES) and normal eggshell strength (NES) genomes. The DEGs are enriched in calcification-related processes, including calcium ion transport and calcium signaling pathways as revealed by gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. Some important matrix proteins, such as OC-116, LTF and SPP1, were also expressed differentially between two groups. A total of 3,671,919 single-nucleotide polymorphisms (SNPs) and 508,035 Indels were detected in protein coding genes by whole-genome re-sequencing, including 1775 non-synonymous variations and 19 frame-shift Indels in DEGs. SNPs and Indels found in this study could be further investigated for eggshell traits. This is the first report to integrate the transcriptome and genome re-sequencing to target the genetic variations which decreased the eggshell qualities. These findings further advance our understanding of eggshell calcification in the chicken uterus.

Ecological genomics of natural plant populations: the Israeli perspective.

PubMed

Nevo, Eviatar

2009-01-01

The genomic era revolutionized evolutionary population biology. The ecological genomics of the wild progenitors of wheat and barley reviewed here was central in the research program of the Institute of Evolution, University of Haifa, since 1975 ( http://evolution.haifa.ac.il ). We explored the following questions: (1) How much of the genomic and phenomic diversity of wild progenitors of cultivars (wild emmer wheat, Triticum dicoccoides, the progenitor of most wheat, plus wild relatives of the Aegilops species; wild barley, Hordeum spontaneum, the progenitor of cultivated barley; wild oat, Avena sterilis, the progenitor of cultivated oats; and wild lettuce species, Lactuca, the progenitor and relatives of cultivated lettuce) are adaptive and processed by natural selection at both coding and noncoding genomic regions? (2) What is the origin and evolution of genomic adaptation and speciation processes and their regulation by mutation, recombination, and transposons under spatiotemporal variables and stressful macrogeographic and microgeographic environments? (3) How much genetic resources are harbored in the wild progenitors for crop improvement? We advanced ecological genetics into ecological genomics and analyzed (regionally across Israel and the entire Near East Fertile Crescent and locally at microsites, focusing on the "Evolution Canyon" model) hundreds of populations and thousands of genotypes for protein (allozyme) and deoxyribonucleic acid (DNA) (coding and noncoding) diversity, partly combined with phenotypic diversity. The environmental stresses analyzed included abiotic (climatic and microclimatic, edaphic) and biotic (pathogens, demographic) stresses. Recently, we introduced genetic maps, cloning, and transformation of candidate genes. Our results indicate abundant genotypic and phenotypic diversity in natural plant populations. The organization and evolution of molecular and organismal diversity in plant populations, at all genomic regions and geographical scales, are nonrandom and are positively correlated with, and partly predictable by, abiotic and biotic environmental heterogeneity and stress. Biodiversity evolution, even in small isolated populations, is primarily driven by natural selection including diversifying, balancing, cyclical, and purifying selection regimes interacting with, but, ultimately, overriding the effects of mutation, migration, and stochasticity. The progenitors of cultivated plants harbor rich genetic resources and are the best hope for crop improvement by both classical and modern biotechnological methods. Future studies should focus on the interplay between structural and functional genome organization focusing on gene regulation.

Genome-wide analysis reveals signatures of selection for important traits in domestic sheep from different ecoregions.

PubMed

Liu, Zhaohua; Ji, Zhibin; Wang, Guizhi; Chao, Tianle; Hou, Lei; Wang, Jianmin

2016-11-03

Throughout a long period of adaptation and selection, sheep have thrived in a diverse range of ecological environments. Mongolian sheep is the common ancestor of the Chinese short fat-tailed sheep. Migration to different ecoregions leads to changes in selection pressures and results in microevolution. Mongolian sheep and its subspecies differ in a number of important traits, especially reproductive traits. Genome-wide intraspecific variation is required to dissect the genetic basis of these traits. This research resequenced 3 short fat-tailed sheep breeds with a 43.2-fold coverage of the sheep genome. We report more than 17 million single nucleotide polymorphisms and 2.9 million indels and identify 143 genomic regions with reduced pooled heterozygosity or increased genetic distance to each other breed that represent likely targets for selection during the migration. These regions harbor genes related to developmental processes, cellular processes, multicellular organismal processes, biological regulation, metabolic processes, reproduction, localization, growth and various components of the stress responses. Furthermore, we examined the haplotype diversity of 3 genomic regions involved in reproduction and found significant differences in TSHR and PRL gene regions among 8 sheep breeds. Our results provide useful genomic information for identifying genes or causal mutations associated with important economic traits in sheep and for understanding the genetic basis of adaptation to different ecological environments.

University of Texas MD Anderson Cancer Center: High-Throughput Screening Identifying Driving Mutations in Endometrial Cancer | Office of Cancer Genomics

Cancer.gov

Recent advances in next-generation sequencing technology have enabled the unprecedented characterization of a full spectrum of somatic alterations in cancer genomes. Given the large numbers of somatic mutations typically detected by this approach, a key challenge in the downstream analysis is to distinguish “drivers” that functionally contribute to tumorigenesis from “passengers” that occur as the consequence of genomic instability.

Going, going, gone: predicting the fate of genomic insertions in plant RNA viruses.

PubMed

Willemsen, Anouk; Carrasco, José L; Elena, Santiago F; Zwart, Mark P

2018-05-10

Horizontal gene transfer is common among viruses, while they also have highly compact genomes and tend to lose artificial genomic insertions rapidly. Understanding the stability of genomic insertions in viral genomes is therefore relevant for explaining and predicting their evolutionary patterns. Here, we revisit a large body of experimental research on a plant RNA virus, tobacco etch potyvirus (TEV), to identify the patterns underlying the stability of a range of homologous and heterologous insertions in the viral genome. We obtained a wide range of estimates for the recombination rate-the rate at which deletions removing the insertion occur-and these appeared to be independent of the type of insertion and its location. Of the factors we considered, recombination rate was the best predictor of insertion stability, although we could not identify the specific sequence characteristics that would help predict insertion instability. We also considered experimentally the possibility that functional insertions lead to higher mutational robustness through increased redundancy. However, our observations suggest that both functional and non-functional increases in genome size decreased the mutational robustness. Our results therefore demonstrate the importance of recombination rates for predicting the long-term stability and evolution of viral RNA genomes and suggest that there are unexpected drawbacks to increases in genome size for mutational robustness.

Insights into the Evolution of Mitochondrial Genome Size from Complete Sequences of Citrullus lanatus and Cucurbita pepo (Cucurbitaceae)

PubMed Central

Alverson, Andrew J.; Wei, XiaoXin; Rice, Danny W.; Stern, David B.; Barry, Kerrie; Palmer, Jeffrey D.

2010-01-01

The mitochondrial genomes of seed plants are unusually large and vary in size by at least an order of magnitude. Much of this variation occurs within a single family, the Cucurbitaceae, whose genomes range from an estimated 390 to 2,900 kb in size. We sequenced the mitochondrial genomes of Citrullus lanatus (watermelon: 379,236 nt) and Cucurbita pepo (zucchini: 982,833 nt)—the two smallest characterized cucurbit mitochondrial genomes—and determined their RNA editing content. The relatively compact Citrullus mitochondrial genome actually contains more and longer genes and introns, longer segmental duplications, and more discernibly nuclear-derived DNA. The large size of the Cucurbita mitochondrial genome reflects the accumulation of unprecedented amounts of both chloroplast sequences (>113 kb) and short repeated sequences (>370 kb). A low mutation rate has been hypothesized to underlie increases in both genome size and RNA editing frequency in plant mitochondria. However, despite its much larger genome, Cucurbita has a significantly higher synonymous substitution rate (and presumably mutation rate) than Citrullus but comparable levels of RNA editing. The evolution of mutation rate, genome size, and RNA editing are apparently decoupled in Cucurbitaceae, reflecting either simple stochastic variation or governance by different factors. PMID:20118192

Sequenced sorghum mutant library- an efficient platform for discovery of causal gene mutations

USDA-ARS?s Scientific Manuscript database

Ethyl methanesulfonate (EMS) efficiently generates high-density mutations in genomes. We applied whole-genome sequencing to 256 phenotyped mutant lines of sorghum (Sorghum bicolor L. Moench) to 16x coverage. Comparisons with the reference sequence revealed >1.8 million canonical EMS-induced G/C to A...

Optimizing complex phenotypes through model-guided multiplex genome engineering

DOE PAGES

Kuznetsov, Gleb; Goodman, Daniel B.; Filsinger, Gabriel T.; ...

2017-05-25

Here, we present a method for identifying genomic modifications that optimize a complex phenotype through multiplex genome engineering and predictive modeling. We apply our method to identify six single nucleotide mutations that recover 59% of the fitness defect exhibited by the 63-codon E. coli strain C321.ΔA. By introducing targeted combinations of changes in multiplex we generate rich genotypic and phenotypic diversity and characterize clones using whole-genome sequencing and doubling time measurements. Regularized multivariate linear regression accurately quantifies individual allelic effects and overcomes bias from hitchhiking mutations and context-dependence of genome editing efficiency that would confound other strategies.

Optimizing complex phenotypes through model-guided multiplex genome engineering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuznetsov, Gleb; Goodman, Daniel B.; Filsinger, Gabriel T.

Here, we present a method for identifying genomic modifications that optimize a complex phenotype through multiplex genome engineering and predictive modeling. We apply our method to identify six single nucleotide mutations that recover 59% of the fitness defect exhibited by the 63-codon E. coli strain C321.ΔA. By introducing targeted combinations of changes in multiplex we generate rich genotypic and phenotypic diversity and characterize clones using whole-genome sequencing and doubling time measurements. Regularized multivariate linear regression accurately quantifies individual allelic effects and overcomes bias from hitchhiking mutations and context-dependence of genome editing efficiency that would confound other strategies.

Targeted Analysis of Whole Genome Sequence Data to Diagnose Genetic Cardiomyopathy

DOE PAGES

Golbus, Jessica R.; Puckelwartz, Megan J.; Dellefave-Castillo, Lisa; ...

2014-09-01

Background—Cardiomyopathy is highly heritable but genetically diverse. At present, genetic testing for cardiomyopathy uses targeted sequencing to simultaneously assess the coding regions of more than 50 genes. New genes are routinely added to panels to improve the diagnostic yield. With the anticipated $1000 genome, it is expected that genetic testing will shift towards comprehensive genome sequencing accompanied by targeted gene analysis. Therefore, we assessed the reliability of whole genome sequencing and targeted analysis to identify cardiomyopathy variants in 11 subjects with cardiomyopathy. Methods and Results—Whole genome sequencing with an average of 37× coverage was combined with targeted analysis focused onmore » 204 genes linked to cardiomyopathy. Genetic variants were scored using multiple prediction algorithms combined with frequency data from public databases. This pipeline yielded 1-14 potentially pathogenic variants per individual. Variants were further analyzed using clinical criteria and/or segregation analysis. Three of three previously identified primary mutations were detected by this analysis. In six subjects for whom the primary mutation was previously unknown, we identified mutations that segregated with disease, had clinical correlates, and/or had additional pathological correlation to provide evidence for causality. For two subjects with previously known primary mutations, we identified additional variants that may act as modifiers of disease severity. In total, we identified the likely pathological mutation in 9 of 11 (82%) subjects. We conclude that these pilot data demonstrate that ~30-40× coverage whole genome sequencing combined with targeted analysis is feasible and sensitive to identify rare variants in cardiomyopathy-associated genes.« less

Localization of causal locus in the genome of the brown macroalga Ectocarpus: NGS-based mapping and positional cloning approaches

PubMed Central

Billoud, Bernard; Jouanno, Émilie; Nehr, Zofia; Carton, Baptiste; Rolland, Élodie; Chenivesse, Sabine; Charrier, Bénédicte

2015-01-01

Mutagenesis is the only process by which unpredicted biological gene function can be identified. Despite that several macroalgal developmental mutants have been generated, their causal mutation was never identified, because experimental conditions were not gathered at that time. Today, progresses in macroalgal genomics and judicious choices of suitable genetic models make mutated gene identification possible. This article presents a comparative study of two methods aiming at identifying a genetic locus in the brown alga Ectocarpus siliculosus: positional cloning and Next-Generation Sequencing (NGS)-based mapping. Once necessary preliminary experimental tools were gathered, we tested both analyses on an Ectocarpus morphogenetic mutant. We show how a narrower localization results from the combination of the two methods. Advantages and drawbacks of these two approaches as well as potential transfer to other macroalgae are discussed. PMID:25745426

Rates of spontaneous mutation among RNA viruses.

PubMed Central

Drake, J W

1993-01-01

Simple methods are presented to estimate rates of spontaneous mutation from mutant frequencies and population parameters in RNA viruses. Published mutant frequencies yield a wide range of mutation rates per genome per replication, mainly because mutational targets have usually been small and, thus, poor samples of the mutability of the average base. Nevertheless, there is a clear central tendency for lytic RNA viruses (bacteriophage Q beta, poliomyelitis, vesicular stomatitis, and influenza A) to display rates of spontaneous mutation of approximately 1 per genome per replication. This rate is some 300-fold higher than previously reported for DNA-based microbes. Lytic RNA viruses thus mutate at a rate close to the maximum value compatible with viability. Retroviruses (spleen necrosis, murine leukemia, Rous sarcoma), however, mutate at an average rate about an order of magnitude lower than lytic RNA viruses. PMID:8387212

Activation of the ALT pathway for telomere maintenance can affect other sequences in the human genome.

PubMed

Jeyapalan, Jennie N; Varley, Helen; Foxon, Jenny L; Pollock, Raphael E; Jeffreys, Alec J; Henson, Jeremy D; Reddel, Roger R; Royle, Nicola J

2005-07-01

Immortal human cells maintain telomere length by the expression of telomerase or through the alternative lengthening of telomeres (ALT). The ALT mechanism involves a recombination-like process that allows the rapid elongation of shortened telomeres. However, it is not known whether activation of the ALT pathway affects other sequences in the genome. To address this we have investigated, in ALT-expressing cell lines and tumours, the stability of tandem repeat sequences known to mutate via homologous recombination in the human germline. We have shown extraordinary somatic instability in the human minisatellite MS32 (D1S8) in ALT-expressing (ALT+) but not in normal or telomerase-expressing cell lines. The MS32 mutation frequency varied across 15 ALT+ cell lines and was on average 55-fold greater than in ALT- cell lines. The MS32 minisatellite was also highly unstable in three of eight ALT+ soft tissue sarcomas, indicating that somatic destabilization occurs in vivo. The MS32 mutation rates estimated for two ALT+ cell lines were similar to that seen in the germline. However, the internal structures of ALT and germline mutant alleles are very different, indicating differences in the underlying mutation mechanisms. Five other hypervariable minisatellites did not show elevated instability in ALT-expressing cell lines, indicating that minisatellite destabilization is not universal. The elevation of MS32 instability upon activation of the ALT pathway and telomere length maintenance suggests there is overlap between the underlying processes that may be tractable through analysis of the D1S8 locus.

Advances in computational approaches for prioritizing driver mutations and significantly mutated genes in cancer genomes.

PubMed

Cheng, Feixiong; Zhao, Junfei; Zhao, Zhongming

2016-07-01

Cancer is often driven by the accumulation of genetic alterations, including single nucleotide variants, small insertions or deletions, gene fusions, copy-number variations, and large chromosomal rearrangements. Recent advances in next-generation sequencing technologies have helped investigators generate massive amounts of cancer genomic data and catalog somatic mutations in both common and rare cancer types. So far, the somatic mutation landscapes and signatures of >10 major cancer types have been reported; however, pinpointing driver mutations and cancer genes from millions of available cancer somatic mutations remains a monumental challenge. To tackle this important task, many methods and computational tools have been developed during the past several years and, thus, a review of its advances is urgently needed. Here, we first summarize the main features of these methods and tools for whole-exome, whole-genome and whole-transcriptome sequencing data. Then, we discuss major challenges like tumor intra-heterogeneity, tumor sample saturation and functionality of synonymous mutations in cancer, all of which may result in false-positive discoveries. Finally, we highlight new directions in studying regulatory roles of noncoding somatic mutations and quantitatively measuring circulating tumor DNA in cancer. This review may help investigators find an appropriate tool for detecting potential driver or actionable mutations in rapidly emerging precision cancer medicine. © The Author 2015. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

Tumor Genomic Profiling in Breast Cancer Patients Using Targeted Massively Parallel Sequencing

DTIC Science & Technology

2016-03-01

recently, we identified several novel alterations in in ER+ breast tumors, including translocations in ESR1 , the gene that encodes the estrogen receptor...modified our bait design to include genomic coordinates across select introns in ESR1 . In addition, two papers from the Broad Institute published in...with PIK3CA mutations, 23% with ESR1 ligand-binding domain mutations, 9% with ERBB2 mutations, 9% with FGFR1/2 amplifications, and 1% with

Dosage Mutator Genes in Saccharomyces cerevisiae: A Novel Mutator Mode-of-Action of the Mph1 DNA Helicase.

PubMed

Ang, J Sidney; Duffy, Supipi; Segovia, Romulo; Stirling, Peter C; Hieter, Philip

2016-11-01

Mutations that cause genome instability are considered important predisposing events that contribute to initiation and progression of cancer. Genome instability arises either due to defects in genes that cause an increased mutation rate (mutator phenotype), or defects in genes that cause chromosome instability (CIN). To extend the catalog of genome instability genes, we systematically explored the effects of gene overexpression on mutation rate, using a forward-mutation screen in budding yeast. We screened ∼5100 plasmids, each overexpressing a unique single gene, and characterized the five strongest mutators, MPH1 (mutator phenotype 1), RRM3, UBP12, PIF1, and DNA2 We show that, for MPH1, the yeast homolog of Fanconi Anemia complementation group M (FANCM), the overexpression mutator phenotype is distinct from that of mph1Δ. Moreover, while four of our top hits encode DNA helicases, the overexpression of 48 other DNA helicases did not cause a mutator phenotype, suggesting this is not a general property of helicases. For Mph1 overexpression, helicase activity was not required for the mutator phenotype; in contrast Mph1 DEAH-box function was required for hypermutation. Mutagenesis by MPH1 overexpression was independent of translesion synthesis (TLS), but was suppressed by overexpression of RAD27, a conserved flap endonuclease. We propose that binding of DNA flap structures by excess Mph1 may block Rad27 action, creating a mutator phenotype that phenocopies rad27Δ. We believe this represents a novel mutator mode-of-action and opens up new prospects to understand how upregulation of DNA repair proteins may contribute to mutagenesis. Copyright © 2016 by the Genetics Society of America.

A deletion mutation in the 5' part of the pol gene of Moloney murine leukemia virus blocks proteolytic processing of the gag and pol polyproteins.

PubMed Central

Crawford, S; Goff, S P

1985-01-01

Deletion mutations in the 5' part of the pol gene of Moloney murine leukemia virus were generated by restriction enzyme site-directed mutagenesis of cloned proviral DNA. DNA sequence analysis indicated that one such deletion was localized entirely within the 5' part of the pol gene, did not affect the region encoding reverse transcriptase, and preserved the translational reading frame downstream of the mutation. The major viral precursor polyproteins (Pr65gag, Pr200gag-pol, and gPr80env) were synthesized at wild-type levels in cell lines carrying the mutant genome. These cell lines assembled and released wild-type levels of virion particles into the medium. Cleavage of both Pr65gag and Pr200gag-pol precursors to the mature proteins was completely blocked in the mutant virions. Surprisingly, these virions contained high levels of active reverse transcriptase; examination of the endogenous reverse transcription products synthesized by the mutant virions revealed normal amounts of minus-strand strong-stop DNA, indicating that the RNA genome was packaged and that reverse transcription in detergent-permeabilized virions was not significantly impaired. Processing of gPr80env to gP70env and P15E was not affected by the mutation, but cleavage of P15E to P12E was not observed. The mutant particles were poorly infectious; analysis indicated that infection was blocked at an early stage. The data are consistent with the idea that the 5' part of the pol gene encodes a protease directly responsible for processing Pr65gag, and possibly Pr200gag-pol, to the structural virion proteins. It appears that cleavage of the gag gene product is not required for budding and release of virions and that complete processing of the pol gene product to the mature form of reverse transcriptase is not required for its functional activation. Images PMID:3882995

Identification of a recently active Prunus-specific non-autonomous Mutator element with considerable genome shaping force.

PubMed

Halász, Júlia; Kodad, Ossama; Hegedűs, Attila

2014-07-01

Miniature inverted-repeat transposable elements (MITEs) are known to contribute to the evolution of plants, but only limited information is available for MITEs in the Prunus genome. We identified a MITE that has been named Falling Stones, FaSt. All structural features (349-bp size, 82-bp terminal inverted repeats and 9-bp target site duplications) are consistent with this MITE being a putative member of the Mutator transposase superfamily. FaSt showed a preferential accumulation in the short AT-rich segments of the euchromatin region of the peach genome. DNA sequencing and pollination experiments have been performed to confirm that the nested insertion of FaSt into the S-haplotype-specific F-box gene of apricot resulted in the breakdown of self-incompatibility (SI). A bioinformatics-based survey of the known Rosaceae and other genomes and a newly designed polymerase chain reaction (PCR) assay verified the Prunoideae-specific occurrence of FaSt elements. Phylogenetic analysis suggested a recent activity of FaSt in the Prunus genome. The occurrence of a nested insertion in the apricot genome further supports the recent activity of FaSt in response to abiotic stress conditions. This study reports on a presumably active non-autonomous Mutator element in Prunus that exhibits a major indirect genome shaping force through inducing loss-of-function mutation in the SI locus. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Mutation rates among RNA viruses

PubMed Central

Drake, John W.; Holland, John J.

1999-01-01

The rate of spontaneous mutation is a key parameter in modeling the genetic structure and evolution of populations. The impact of the accumulated load of mutations and the consequences of increasing the mutation rate are important in assessing the genetic health of populations. Mutation frequencies are among the more directly measurable population parameters, although the information needed to convert them into mutation rates is often lacking. A previous analysis of mutation rates in RNA viruses (specifically in riboviruses rather than retroviruses) was constrained by the quality and quantity of available measurements and by the lack of a specific theoretical framework for converting mutation frequencies into mutation rates in this group of organisms. Here, we describe a simple relation between ribovirus mutation frequencies and mutation rates, apply it to the best (albeit far from satisfactory) available data, and observe a central value for the mutation rate per genome per replication of μg ≈ 0.76. (The rate per round of cell infection is twice this value or about 1.5.) This value is so large, and ribovirus genomes are so informationally dense, that even a modest increase extinguishes the population. PMID:10570172

Rare beneficial mutations can halt Muller's ratchet

NASA Astrophysics Data System (ADS)

Balick, Daniel; Goyal, Sidhartha; Jerison, Elizabeth; Neher, Richard; Shraiman, Boris; Desai, Michael

2012-02-01

In viral, bacterial, and other asexual populations, the vast majority of non-neutral mutations are deleterious. This motivates the application of models without beneficial mutations. Here we show that the presence of surprisingly few compensatory mutations halts fitness decay in these models. Production of deleterious mutations is balanced by purifying selection, stabilizing the fitness distribution. However, stochastic vanishing of fitness classes can lead to slow fitness decay (i.e. Muller's ratchet). For weakly deleterious mutations, production overwhelms purification, rapidly decreasing population fitness. We show that when beneficial mutations are introduced, a stable steady state emerges in the form of a dynamic mutation-selection balance. We argue this state is generic for all mutation rates and population sizes, and is reached as an end state as genomes become saturated by either beneficial or deleterious mutations. Assuming all mutations have the same magnitude selective effect, we calculate the fraction of beneficial mutations necessary to maintain the dynamic balance. This may explain the unexpected maintenance of asexual genomes, as in mitochondria, in the presence of selection. This will affect in the statistics of genetic diversity in these populations.

Comprehensive identification of mutations induced by heavy-ion beam irradiation in Arabidopsis thaliana.

PubMed

Hirano, Tomonari; Kazama, Yusuke; Ishii, Kotaro; Ohbu, Sumie; Shirakawa, Yuki; Abe, Tomoko

2015-04-01

Heavy-ion beams are widely used for mutation breeding and molecular biology. Although the mutagenic effects of heavy-ion beam irradiation have been characterized by sequence analysis of some restricted chromosomal regions or loci, there have been no evaluations at the whole-genome level or of the detailed genomic rearrangements in the mutant genomes. In this study, using array comparative genomic hybridization (array-CGH) and resequencing, we comprehensively characterized the mutations in Arabidopsis thaliana genomes irradiated with Ar or Fe ions. We subsequently used this information to investigate the mutagenic effects of the heavy-ion beams. Array-CGH demonstrated that the average number of deleted areas per genome were 1.9 and 3.7 following Ar-ion and Fe-ion irradiation, respectively, with deletion sizes ranging from 149 to 602,180 bp; 81% of the deletions were accompanied by genomic rearrangements. To provide a further detailed analysis, the genomes of the mutants induced by Ar-ion beam irradiation were resequenced, and total mutations, including base substitutions, duplications, in/dels, inversions, and translocations, were detected using three algorithms. All three resequenced mutants had genomic rearrangements. Of the 22 DNA fragments that contributed to the rearrangements, 19 fragments were responsible for the intrachromosomal rearrangements, and multiple rearrangements were formed in the localized regions of the chromosomes. The interchromosomal rearrangements were detected in the multiply rearranged regions. These results indicate that the heavy-ion beams led to clustered DNA damage in the chromosome, and that they have great potential to induce complicated intrachromosomal rearrangements. Heavy-ion beams will prove useful as unique mutagens for plant breeding and the establishment of mutant lines. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Cross-species comparison of aCGH data from mouse and human BRCA1- and BRCA2-mutated breast cancers

PubMed Central

2010-01-01

Background Genomic gains and losses are a result of genomic instability in many types of cancers. BRCA1- and BRCA2-mutated breast cancers are associated with increased amounts of chromosomal aberrations, presumably due their functions in genome repair. Some of these genomic aberrations may harbor genes whose absence or overexpression may give rise to cellular growth advantage. So far, it has not been easy to identify the driver genes underlying gains and losses. A powerful approach to identify these driver genes could be a cross-species comparison of array comparative genomic hybridization (aCGH) data from cognate mouse and human tumors. Orthologous regions of mouse and human tumors that are commonly gained or lost might represent essential genomic regions selected for gain or loss during tumor development. Methods To identify genomic regions that are associated with BRCA1- and BRCA2-mutated breast cancers we compared aCGH data from 130 mouse Brca1Δ/Δ;p53Δ/Δ, Brca2Δ/Δ;p53Δ/Δ and p53Δ/Δ mammary tumor groups with 103 human BRCA1-mutated, BRCA2-mutated and non-hereditary breast cancers. Results Our genome-wide cross-species analysis yielded a complete collection of loci and genes that are commonly gained or lost in mouse and human breast cancer. Principal common CNAs were the well known MYC-associated gain and RB1/INTS6-associated loss that occurred in all mouse and human tumor groups, and the AURKA-associated gain occurred in BRCA2-related tumors from both species. However, there were also important differences between tumor profiles of both species, such as the prominent gain on chromosome 10 in mouse Brca2Δ/Δ;p53Δ/Δ tumors and the PIK3CA associated 3q gain in human BRCA1-mutated tumors, which occurred in tumors from one species but not in tumors from the other species. This disparity in recurrent aberrations in mouse and human tumors might be due to differences in tumor cell type or genomic organization between both species. Conclusions The selection of the oncogenome during mouse and human breast tumor development is markedly different, apart from the MYC gain and RB1-associated loss. These differences should be kept in mind when using mouse models for preclinical studies. PMID:20735817

Identification of novel mutational drivers reveals oncogene dependencies in multiple myeloma.

PubMed

Walker, Brian A; Mavrommatis, Konstantinos; Wardell, Christopher P; Ashby, T Cody; Bauer, Michael; Davies, Faith E; Rosenthal, Adam; Wang, Hongwei; Qu, Pingping; Hoering, Antje; Samur, Mehmet; Towfic, Fadi; Ortiz, Maria; Flynt, Erin; Yu, Zhinuan; Yang, Zhihong; Rozelle, Dan; Obenauer, John; Trotter, Matthew; Auclair, Daniel; Keats, Jonathan; Bolli, Niccolo; Fulciniti, Mariateresa; Szalat, Raphael; Moreau, Philippe; Durie, Brian; Stewart, A Keith; Goldschmidt, Hartmut; Raab, Marc S; Einsele, Hermann; Sonneveld, Pieter; San Miguel, Jesus; Lonial, Sagar; Jackson, Graham H; Anderson, Kenneth C; Avet-Loiseau, Herve; Munshi, Nikhil; Thakurta, Anjan; Morgan, Gareth J

2018-06-08

Understanding the profile of oncogene and tumor suppressor gene mutations with their interactions and impact on the prognosis of multiple myeloma (MM) can improve the definition of disease subsets and identify pathways important in disease pathobiology. Using integrated genomics of 1,273 newly diagnosed patients with multiple myeloma we identify 63 driver genes, some of which are novel including IDH1 , IDH2 , HUWE1 , KLHL6 , and PTPN11 Oncogene mutations are significantly more clonal than tumor suppressor mutations, indicating they may exert a bigger selective pressure. Patients with more mutations in driver genes are associated with a worse outcome, as are those with identified mechanisms of genomic instability. Oncogenic dependencies were identified between mutations in driver genes, common regions of copy number change, and primary translocation and hyperdiploidy events. These dependencies included associations with t(4;14) and mutations in FGFR3 , DIS3 and PRKD2 ; t(11;14) with mutations in CCND1 and IRF4 ; t(14;16) with mutations in MAF , BRAF , DIS3 and ATM ; and hyperdiploidy with gain 11q, mutations in FAM46C and MYC rearrangements. These associations indicate that the genomic landscape of myeloma is pre-determined by the primary events upon which further dependencies are built, giving rise to a non-random accumulation of genetic hits. Understanding these dependencies may elucidate potential evolutionary patterns and lead to better treatment regimens. Copyright © 2018 American Society of Hematology.

Restriction digest screening facilitates efficient detection of site-directed mutations introduced by CRISPR in C. albicans UME6.

PubMed

Evans, Ben A; Smith, Olivia L; Pickerill, Ethan S; York, Mary K; Buenconsejo, Kristen J P; Chambers, Antonio E; Bernstein, Douglas A

2018-01-01

Introduction of point mutations to a gene of interest is a powerful tool when determining protein function. CRISPR-mediated genome editing allows for more efficient transfer of a desired mutation into a wide range of model organisms. Traditionally, PCR amplification and DNA sequencing is used to determine if isolates contain the intended mutation. However, mutation efficiency is highly variable, potentially making sequencing costly and time consuming. To more efficiently screen for correct transformants, we have identified restriction enzymes sites that encode for two identical amino acids or one or two stop codons. We used CRISPR to introduce these restriction sites directly upstream of the Candida albicans UME6 Zn 2+ -binding domain, a known regulator of C. albicans filamentation. While repair templates coding for different restriction sites were not equally successful at introducing mutations, restriction digest screening enabled us to rapidly identify isolates with the intended mutation in a cost-efficient manner. In addition, mutated isolates have clear defects in filamentation and virulence compared to wild type C. albicans . Our data suggest restriction digestion screening efficiently identifies point mutations introduced by CRISPR and streamlines the process of identifying residues important for a phenotype of interest.

Discovery of Genomic Breakpoints Affecting Breast Cancer Progression and Prognosis

DTIC Science & Technology

2009-10-01

157 genomic breakpoints in MCF-7 cells that could be confirmed by PCR across breakpoint joins as likely somatic mutations . A total of 79 genes are...SUPPLEMENTARY NOTES 14. ABSTRACT 157 genomic breakpoints could be confirmed as likely somatic mutations . We focused on breakpoints predicted to lead to...enrichment for breakpoints containing genes (50.3% vs 77.4%), and for fusion-containing breakpoints (6.4% vs 16.1%). Also, all chimeric mRNA products are

Unbiased Combinatorial Genomic Approaches to Identify Alternative Therapeutic Targets within the TSC Signaling Network

DTIC Science & Technology

2015-09-01

assessed the specificity of mutation in Drosophila S2R+ cells. We generated a quantitative mutation reporter vector in which an sgRNA target sequence ...phosphatases (563 genes) in the Drosophila genome (Figure 4). 65 samples that displayed synthetic lethality (15 genes) or synthetic increases in viability...targeting all kinases and phosphatases (563 genes) in the Drosophila genome . . Identified three hits (mRNA-Cap, Pitslre and CycT) that scored as

University of Texas MD Anderson Cancer Center (UT-MDACC): High-Throughput Screening Identifying Driving Mutations in Endometrial Cancer | Office of Cancer Genomics

Cancer.gov

Recent advances in next-generation sequencing technology have enabled the unprecedented characterization of a full spectrum of somatic alterations in cancer genomes. Given the large numbers of somatic mutations typically detected by this approach, a key challenge in the downstream analysis is to distinguish “drivers” that functionally contribute to tumorigenesis from “passengers” that occur as the consequence of genomic instability.

Novel genomic findings in multiple myeloma identified through routine diagnostic sequencing.

PubMed

Ryland, Georgina L; Jones, Kate; Chin, Melody; Markham, John; Aydogan, Elle; Kankanige, Yamuna; Caruso, Marisa; Guinto, Jerick; Dickinson, Michael; Prince, H Miles; Yong, Kwee; Blombery, Piers

2018-05-14

Multiple myeloma is a genomically complex haematological malignancy with many genomic alterations recognised as important in diagnosis, prognosis and therapeutic decision making. Here, we provide a summary of genomic findings identified through routine diagnostic next-generation sequencing at our centre. A cohort of 86 patients with multiple myeloma underwent diagnostic sequencing using a custom hybridisation-based panel targeting 104 genes. Sequence variants, genome-wide copy number changes and structural rearrangements were detected using an inhouse-developed bioinformatics pipeline. At least one mutation was found in 69 (80%) patients. Frequently mutated genes included TP53 (36%), KRAS (22.1%), NRAS (15.1%), FAM46C/DIS3 (8.1%) and TET2/FGFR3 (5.8%), including multiple mutations not previously described in myeloma. Importantly we observed TP53 mutations in the absence of a 17 p deletion in 8% of the cohort, highlighting the need for sequencing-based assessment in addition to cytogenetics to identify these high-risk patients. Multiple novel copy number changes and immunoglobulin heavy chain translocations are also discussed. Our results demonstrate that many clinically relevant genomic findings remain in multiple myeloma which have not yet been identified through large-scale sequencing efforts, and provide important mechanistic insights into plasma cell pathobiology. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Mitochondrial DNA mutations and cognition: a case-series report.

PubMed

Inczedy-Farkas, Gabriella; Trampush, Joey W; Perczel Forintos, Dora; Beech, Danielle; Andrejkovics, Monika; Varga, Zsofia; Remenyi, Viktoria; Bereznai, Benjamin; Gal, Aniko; Molnar, Maria Judit

2014-06-01

Mutations in the mitochondrial genome can impair normal metabolic function in the central nervous system (CNS) where cellular energy demand is high. Primary mitochondrial DNA (mtDNA) mutations have been linked to several mitochondrial disorders that have comorbid psychiatric, neurologic, and cognitive sequelae. Here, we present a series of cases with primary mtDNA mutations who were genotyped and evaluated across a common neuropsychological battery. Nineteen patients with mtDNA mutations were genotyped and clinically and cognitively evaluated. Pronounced deficits in nonverbal/visuoperceptual reasoning, verbal recall, semantic word generativity, and processing speed were evident and consistent with a "mitochondrial dementia" that has been posited. However, variation in cognitive performance was noteworthy, suggesting that the phenotypic landscape of cognition linked to primary mtDNA mutations is heterogeneous. Our patients with mtDNA mutations evidenced cognitive deficits quite similar to those commonly seen in Alzheimer's disease and could have clinical relevance to the evaluation of dementia. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Genome-wide mapping of mutations at single-nucleotide resolution for protein, metabolic and genome engineering.

PubMed

Garst, Andrew D; Bassalo, Marcelo C; Pines, Gur; Lynch, Sean A; Halweg-Edwards, Andrea L; Liu, Rongming; Liang, Liya; Wang, Zhiwen; Zeitoun, Ramsey; Alexander, William G; Gill, Ryan T

2017-01-01

Improvements in DNA synthesis and sequencing have underpinned comprehensive assessment of gene function in bacteria and eukaryotes. Genome-wide analyses require high-throughput methods to generate mutations and analyze their phenotypes, but approaches to date have been unable to efficiently link the effects of mutations in coding regions or promoter elements in a highly parallel fashion. We report that CRISPR-Cas9 gene editing in combination with massively parallel oligomer synthesis can enable trackable editing on a genome-wide scale. Our method, CRISPR-enabled trackable genome engineering (CREATE), links each guide RNA to homologous repair cassettes that both edit loci and function as barcodes to track genotype-phenotype relationships. We apply CREATE to site saturation mutagenesis for protein engineering, reconstruction of adaptive laboratory evolution experiments, and identification of stress tolerance and antibiotic resistance genes in bacteria. We provide preliminary evidence that CREATE will work in yeast. We also provide a webtool to design multiplex CREATE libraries.

Elucidating the genomic architecture of Asian EGFR-mutant lung adenocarcinoma through multi-region exome sequencing.

PubMed

Nahar, Rahul; Zhai, Weiwei; Zhang, Tong; Takano, Angela; Khng, Alexis J; Lee, Yin Yeng; Liu, Xingliang; Lim, Chong Hee; Koh, Tina P T; Aung, Zaw Win; Lim, Tony Kiat Hon; Veeravalli, Lavanya; Yuan, Ju; Teo, Audrey S M; Chan, Cheryl X; Poh, Huay Mei; Chua, Ivan M L; Liew, Audrey Ann; Lau, Dawn Ping Xi; Kwang, Xue Lin; Toh, Chee Keong; Lim, Wan-Teck; Lim, Bing; Tam, Wai Leong; Tan, Eng-Huat; Hillmer, Axel M; Tan, Daniel S W

2018-01-15

EGFR-mutant lung adenocarcinomas (LUAD) display diverse clinical trajectories and are characterized by rapid but short-lived responses to EGFR tyrosine kinase inhibitors (TKIs). Through sequencing of 79 spatially distinct regions from 16 early stage tumors, we show that despite low mutation burdens, EGFR-mutant Asian LUADs unexpectedly exhibit a complex genomic landscape with frequent and early whole-genome doubling, aneuploidy, and high clonal diversity. Multiple truncal alterations, including TP53 mutations and loss of CDKN2A and RB1, converge on cell cycle dysregulation, with late sector-specific high-amplitude amplifications and deletions that potentially beget drug resistant clones. We highlight the association between genomic architecture and clinical phenotypes, such as co-occurring truncal drivers and primary TKI resistance. Through comparative analysis with published smoking-related LUAD, we postulate that the high intra-tumor heterogeneity observed in Asian EGFR-mutant LUAD may be contributed by an early dominant driver, genomic instability, and low background mutation rates.

Genomic catastrophes frequently arise in esophageal adenocarcinoma and drive tumorigenesis

PubMed Central

Patch, Ann-Marie; Bailey, Peter; Newell, Felicity; Holmes, Oliver; Fink, J. Lynn; Quinn, Michael C.J.; Tang, Yue Hang; Lampe, Guy; Quek, Kelly; Loffler, Kelly A.; Manning, Suzanne; Idrisoglu, Senel; Miller, David; Xu, Qinying; Waddell, Nick; Wilson, Peter J.; Bruxner, Timothy J.C.; Christ, Angelika N.; Harliwong, Ivon; Nourse, Craig; Nourbakhsh, Ehsan; Anderson, Matthew; Kazakoff, Stephen; Leonard, Conrad; Wood, Scott; Simpson, Peter T.; Reid, Lynne E.; Krause, Lutz; Hussey, Damian J.; Watson, David I.; Lord, Reginald V.; Nancarrow, Derek; Phillips, Wayne A.; Gotley, David; Smithers, B. Mark; Whiteman, David C.; Hayward, Nicholas K.; Campbell, Peter J.; Pearson, John V.; Grimmond, Sean M.; Barbour, Andrew P.

2015-01-01

Oesophageal adenocarcinoma (EAC) incidence is rapidly increasing in Western countries. A better understanding of EAC underpins efforts to improve early detection and treatment outcomes. While large EAC exome sequencing efforts to date have found recurrent loss-of-function mutations, oncogenic driving events have been underrepresented. Here we use a combination of whole-genome sequencing (WGS) and single-nucleotide polymorphism-array profiling to show that genomic catastrophes are frequent in EAC, with almost a third (32%, n = 40/123) undergoing chromothriptic events. WGS of 22 EAC cases show that catastrophes may lead to oncogene amplification through chromothripsis-derived double-minute chromosome formation (MYC and MDM2) or breakage-fusion-bridge (KRAS, MDM2 and RFC3). Telomere shortening is more prominent in EACs bearing localized complex rearrangements. Mutational signature analysis also confirms that extreme genomic instability in EAC can be driven by somatic BRCA2 mutations. These findings suggest that genomic catastrophes have a significant role in the malignant transformation of EAC. PMID:25351503

NetNorM: Capturing cancer-relevant information in somatic exome mutation data with gene networks for cancer stratification and prognosis.

PubMed

Le Morvan, Marine; Zinovyev, Andrei; Vert, Jean-Philippe

2017-06-01

Genome-wide somatic mutation profiles of tumours can now be assessed efficiently and promise to move precision medicine forward. Statistical analysis of mutation profiles is however challenging due to the low frequency of most mutations, the varying mutation rates across tumours, and the presence of a majority of passenger events that hide the contribution of driver events. Here we propose a method, NetNorM, to represent whole-exome somatic mutation data in a form that enhances cancer-relevant information using a gene network as background knowledge. We evaluate its relevance for two tasks: survival prediction and unsupervised patient stratification. Using data from 8 cancer types from The Cancer Genome Atlas (TCGA), we show that it improves over the raw binary mutation data and network diffusion for these two tasks. In doing so, we also provide a thorough assessment of somatic mutations prognostic power which has been overlooked by previous studies because of the sparse and binary nature of mutations.

NetNorM: Capturing cancer-relevant information in somatic exome mutation data with gene networks for cancer stratification and prognosis

PubMed Central

2017-01-01

Genome-wide somatic mutation profiles of tumours can now be assessed efficiently and promise to move precision medicine forward. Statistical analysis of mutation profiles is however challenging due to the low frequency of most mutations, the varying mutation rates across tumours, and the presence of a majority of passenger events that hide the contribution of driver events. Here we propose a method, NetNorM, to represent whole-exome somatic mutation data in a form that enhances cancer-relevant information using a gene network as background knowledge. We evaluate its relevance for two tasks: survival prediction and unsupervised patient stratification. Using data from 8 cancer types from The Cancer Genome Atlas (TCGA), we show that it improves over the raw binary mutation data and network diffusion for these two tasks. In doing so, we also provide a thorough assessment of somatic mutations prognostic power which has been overlooked by previous studies because of the sparse and binary nature of mutations. PMID:28650955

The Association Between Molecular Markers in Colorectal Sessile Serrated Polyps and Colorectal Cancer Risk

DTIC Science & Technology

2017-08-01

mouse and human colon epithelium; Aim 2.) Perform genome editing using CRISPR /Cas9 on immortalized human colon epithelial cells to introduce CRC...relevant gene mutations; Aim 3.) Use CRISPR /Cas9 genome editing in colon organoid cultures to introduce CRC relevant gene mutations into primary colon cells

Mapping a Mutation in "Caenorhabditis elegans" Using a Polymerase Chain Reaction-Based Approach

ERIC Educational Resources Information Center

Myers, Edith M.

2014-01-01

Many single nucleotide polymorphisms (SNPs) have been identified within the "Caenorhabditis elegans" genome. SNPs present in the genomes of two isogenic "C. elegans" strains have been routinely used as a tool in forward genetics to map a mutation to a particular chromosome. This article describes a laboratory exercise in which…

Truncation- and motif-based pan-cancer analysis reveals tumor-suppressing kinases.

PubMed

Hudson, Andrew M; Stephenson, Natalie L; Li, Cynthia; Trotter, Eleanor; Fletcher, Adam J; Katona, Gitta; Bieniasz-Krzywiec, Patrycja; Howell, Matthew; Wirth, Chris; Furney, Simon; Miller, Crispin J; Brognard, John

2018-04-17

A major challenge in cancer genomics is identifying "driver" mutations from the many neutral "passenger" mutations within a given tumor. To identify driver mutations that would otherwise be lost within mutational noise, we filtered genomic data by motifs that are critical for kinase activity. In the first step of our screen, we used data from the Cancer Cell Line Encyclopedia and The Cancer Genome Atlas to identify kinases with truncation mutations occurring within or before the kinase domain. The top 30 tumor-suppressing kinases were aligned, and hotspots for loss-of-function (LOF) mutations were identified on the basis of amino acid conservation and mutational frequency. The functional consequences of new LOF mutations were biochemically validated, and the top 15 hotspot LOF residues were used in a pan-cancer analysis to define the tumor-suppressing kinome. A ranked list revealed MAP2K7, an essential mediator of the c-Jun N-terminal kinase (JNK) pathway, as a candidate tumor suppressor in gastric cancer, despite its mutational frequency falling within the mutational noise for this cancer type. The majority of mutations in MAP2K7 abolished its catalytic activity, and reactivation of the JNK pathway in gastric cancer cells harboring LOF mutations in MAP2K7 or the downstream kinase JNK suppressed clonogenicity and growth in soft agar, demonstrating the functional relevance of inactivating the JNK pathway in gastric cancer. Together, our data highlight a broadly applicable strategy to identify functional cancer driver mutations and define the JNK pathway as tumor-suppressive in gastric cancer. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Novel recurrently mutated genes and a prognostic mutation signature in colorectal cancer.

PubMed

Yu, Jun; Wu, William K K; Li, Xiangchun; He, Jun; Li, Xiao-Xing; Ng, Simon S M; Yu, Chang; Gao, Zhibo; Yang, Jie; Li, Miao; Wang, Qiaoxiu; Liang, Qiaoyi; Pan, Yi; Tong, Joanna H; To, Ka F; Wong, Nathalie; Zhang, Ning; Chen, Jie; Lu, Youyong; Lai, Paul B S; Chan, Francis K L; Li, Yingrui; Kung, Hsiang-Fu; Yang, Huanming; Wang, Jun; Sung, Joseph J Y

2015-04-01

Characterisation of colorectal cancer (CRC) genomes by next-generation sequencing has led to the discovery of novel recurrently mutated genes. Nevertheless, genomic data has not yet been used for CRC prognostication. To identify recurrent somatic mutations with prognostic significance in patients with CRC. Exome sequencing was performed to identify somatic mutations in tumour tissues of 22 patients with CRC, followed by validation of 187 recurrent and pathway-related genes using targeted capture sequencing in additional 160 cases. Seven significantly mutated genes, including four reported (APC, TP53, KRAS and SMAD4) and three novel recurrently mutated genes (CDH10, FAT4 and DOCK2), exhibited high mutation prevalence (6-14% for novel cancer genes) and higher-than-expected number of non-silent mutations in our CRC cohort. For prognostication, a five-gene-signature (CDH10, COL6A3, SMAD4, TMEM132D, VCAN) was devised, in which mutation(s) in one or more of these genes was significantly associated with better overall survival independent of tumor-node-metastasis (TNM) staging. The median survival time was 80.4 months in the mutant group versus 42.4 months in the wild type group (p=0.0051). The prognostic significance of this signature was successfully verified using the data set from the Cancer Genome Atlas study. The application of next-generation sequencing has led to the identification of three novel significantly mutated genes in CRC and a mutation signature that predicts survival outcomes for stratifying patients with CRC independent of TNM staging. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Pediatric-type nodal follicular lymphoma: a biologically distinct lymphoma with frequent MAPK pathway mutations

PubMed Central

Schafernak, Kristian T.; Geyer, Julia T.; Kovach, Alexandra E.; Ghandi, Mahmoud; Gratzinger, Dita; Roth, Christine G.; Paxton, Christian N.; Kim, Sunhee; Namgyal, Chungdak; Morin, Ryan; Morgan, Elizabeth A.; Neuberg, Donna S.; South, Sarah T.; Harris, Marian H.; Hasserjian, Robert P.; Hochberg, Ephraim P.; Garraway, Levi A.; Harris, Nancy Lee; Weinstock, David M.

2016-01-01

Pediatric-type nodal follicular lymphoma (PTNFL) is a variant of follicular lymphoma (FL) characterized by limited-stage presentation and invariably benign behavior despite often high-grade histological appearance. It is important to distinguish PTNFL from typical FL in order to avoid unnecessary treatment; however, this distinction relies solely on clinical and pathological criteria, which may be variably applied. To define the genetic landscape of PTNFL, we performed copy number analysis and exome and/or targeted sequencing of 26 PTNFLs (16 pediatric and 10 adult). The most commonly mutated gene in PTNFL was MAP2K1, encoding MEK1, with a mutation frequency of 43%. All MAP2K1 mutations were activating missense mutations localized to exons 2 and 3, which encode negative regulatory and catalytic domains, respectively. Missense mutations in MAPK1 (2/22) and RRAS (1/22) were identified in cases that lacked MAP2K1 mutations. The second most commonly mutated gene in PTNFL was TNFRSF14, with a mutation frequency of 29%, similar to that seen in limited-stage typical FL (P = .35). PTNFL was otherwise genomically bland and specifically lacked recurrent mutations in epigenetic modifiers (eg, CREBBP, KMT2D). Copy number aberrations affected a mean of only 0.5% of PTNFL genomes, compared with 10% of limited-stage typical FL genomes (P < .02). Importantly, the mutational profiles of PTNFLs in children and adults were highly similar. Together, these findings define PTNFL as a biologically and clinically distinct indolent lymphoma of children and adults characterized by a high prevalence of MAPK pathway mutations and a near absence of mutations in epigenetic modifiers. PMID:27325104

Pediatric-type nodal follicular lymphoma: a biologically distinct lymphoma with frequent MAPK pathway mutations.

PubMed

Louissaint, Abner; Schafernak, Kristian T; Geyer, Julia T; Kovach, Alexandra E; Ghandi, Mahmoud; Gratzinger, Dita; Roth, Christine G; Paxton, Christian N; Kim, Sunhee; Namgyal, Chungdak; Morin, Ryan; Morgan, Elizabeth A; Neuberg, Donna S; South, Sarah T; Harris, Marian H; Hasserjian, Robert P; Hochberg, Ephraim P; Garraway, Levi A; Harris, Nancy Lee; Weinstock, David M

2016-08-25

Pediatric-type nodal follicular lymphoma (PTNFL) is a variant of follicular lymphoma (FL) characterized by limited-stage presentation and invariably benign behavior despite often high-grade histological appearance. It is important to distinguish PTNFL from typical FL in order to avoid unnecessary treatment; however, this distinction relies solely on clinical and pathological criteria, which may be variably applied. To define the genetic landscape of PTNFL, we performed copy number analysis and exome and/or targeted sequencing of 26 PTNFLs (16 pediatric and 10 adult). The most commonly mutated gene in PTNFL was MAP2K1, encoding MEK1, with a mutation frequency of 43%. All MAP2K1 mutations were activating missense mutations localized to exons 2 and 3, which encode negative regulatory and catalytic domains, respectively. Missense mutations in MAPK1 (2/22) and RRAS (1/22) were identified in cases that lacked MAP2K1 mutations. The second most commonly mutated gene in PTNFL was TNFRSF14, with a mutation frequency of 29%, similar to that seen in limited-stage typical FL (P = .35). PTNFL was otherwise genomically bland and specifically lacked recurrent mutations in epigenetic modifiers (eg, CREBBP, KMT2D). Copy number aberrations affected a mean of only 0.5% of PTNFL genomes, compared with 10% of limited-stage typical FL genomes (P < .02). Importantly, the mutational profiles of PTNFLs in children and adults were highly similar. Together, these findings define PTNFL as a biologically and clinically distinct indolent lymphoma of children and adults characterized by a high prevalence of MAPK pathway mutations and a near absence of mutations in epigenetic modifiers. © 2016 by The American Society of Hematology.

An Unbiased Genome-Wide View of the Mutation Rate and Spectrum of the Endosymbiotic Bacterium Teredinibacter turnerae.

PubMed

Senra, Marcus V X; Sung, Way; Ackerman, Matthew; Miller, Samuel F; Lynch, Michael; Soares, Carlos Augusto G

2018-03-01

Mutations contribute to genetic variation in all living systems. Thus, precise estimates of mutation rates and spectra across a diversity of organisms are required for a full comprehension of evolution. Here, a mutation-accumulation (MA) assay was carried out on the endosymbiotic bacterium Teredinibacter turnerae. After ∼3,025 generations, base-pair substitutions (BPSs) and insertion-deletion (indel) events were characterized by whole-genome sequencing analysis of 47 independent MA lines, yielding a BPS rate of 1.14 × 10-9 per site per generation and indel rate of 1.55 × 10-10 events per site per generation, which are among the highest within free-living and facultative intracellular bacteria. As in other endosymbionts, a significant bias of BPSs toward A/T and an excess of deletion mutations over insertion mutations are observed for these MA lines. However, even with a deletion bias, the genome remains relatively large (∼5.2 Mb) for an endosymbiotic bacterium. The estimate of the effective population size (Ne) in T. turnerae is quite high and comparable to free-living bacteria (∼4.5 × 107), suggesting that the heavy bottlenecking associated with many endosymbiotic relationships is not prevalent during the life of this endosymbiont. The efficiency of selection scales with increasing Ne and such strong selection may have been operating against the deletion bias, preventing genome erosion. The observed mutation rate in this endosymbiont is of the same order of magnitude of those with similar Ne, consistent with the idea that population size is a primary determinant of mutation-rate evolution within endosymbionts, and that not all endosymbionts have low Ne.

Detection of somatic mutations in the mitochondrial DNA control region D-loop in brain tumors: The first report in Malaysian patients.

PubMed

Mohamed Yusoff, Abdul Aziz; Mohd Nasir, Khairol Naaim; Haris, Khalilah; Mohd Khair, Siti Zulaikha Nashwa; Abdul Ghani, Abdul Rahman Izaini; Idris, Zamzuri; Abdullah, Jafri Malin

2017-11-01

Although the role of nuclear-encoded gene alterations has been well documented in brain tumor development, the involvement of the mitochondrial genome in brain tumorigenesis has not yet been fully elucidated and remains controversial. The present study aimed to identify mutations in the mitochondrial DNA (mtDNA) control region D-loop in patients with brain tumors in Malaysia. A mutation analysis was performed in which DNA was extracted from paired tumor tissue and blood samples obtained from 49 patients with brain tumors. The D-loop region DNA was amplified using the PCR technique, and genetic data from DNA sequencing analyses were compared with the published revised Cambridge sequence to identify somatic mutations. Among the 49 brain tumor tissue samples evaluated, 25 cases (51%) had somatic mutations of the mtDNA D-loop, with a total of 48 mutations. Novel mutations that had not previously been identified in the D-loop region (176 A-deletion, 476 C>A, 566 C>A and 16405 A-deletion) were also classified. No significant associations between the D-loop mutation status and the clinicopathological parameters were observed. To the best of our knowledge, the current study presents the first evidence of alterations in the mtDNA D-loop regions in the brain tumors of Malaysian patients. These results may provide an overview and data regarding the incidence of mitochondrial genome alterations in Malaysian patients with brain tumors. In addition to nuclear genome aberrations, these specific mitochondrial genome alterations may also be considered as potential cancer biomarkers for the diagnosis and staging of brain cancers.

Bioinformatics analysis and genetic diversity of the poliovirus.

PubMed

Liu, Yanhan; Ma, Tengfei; Liu, Jianzhu; Zhao, Xiaona; Cheng, Ziqiang; Guo, Huijun; Wang, Shujing; Xu, Ruixue

2014-12-01

Poliomyelitis, a disease which can manifest as muscle paralysis, is caused by the poliovirus, which is a human enterovirus and member of the family Picornaviridae that usually transmits by the faecal-oral route. The viruses of the OPV (oral poliovirus attenuated-live vaccine) strains can mutate in the human intestine during replication and some of these mutations can lead to the recovery of serious neurovirulence. Informatics research of the poliovirus genome can be used to explain further the characteristics of this virus. In this study, sequences from 100 poliovirus isolates were acquired from GenBank. To determine the evolutionary relationship between the strains, we compared and analysed the sequences of the complete poliovirus genome and the VP1 region. The reconstructed phylogenetic trees for the complete sequences and the VP1 sequences were both divided into two branches, indicating that the genetic relationships of the whole poliovirus genome and the VP1 sequences are very similar. This branching indicates that the virulence and pathogenicity of poliomyelitis may be associated with the VP1 region. Sequence alignment of the VP1 region revealed numerous mutation sites in which mutation rates of >30 % were detected. In a group of strains recorded in the USA, mutation sites and mutation types were the same and this may be associated with their distribution in the evolutionary tree and their genetic relationship. In conclusion, the genetic evolutionary relationships of poliovirus isolate sequences are determined to a great extent by the VP1 protein, and poliovirus strains located on the same branch of the phylogenetic tree contain the same mutation spots and mutation types. Hence, the genetic characteristics of the VP1 region in the poliovirus genome should be analysed to identify the transmission route of poliovirus and provide the basis of viral immunity development. © 2014 The Authors.

Scalable Open Science Approach for Mutation Calling of Tumor Exomes Using Multiple Genomic Pipelines.

PubMed

Ellrott, Kyle; Bailey, Matthew H; Saksena, Gordon; Covington, Kyle R; Kandoth, Cyriac; Stewart, Chip; Hess, Julian; Ma, Singer; Chiotti, Kami E; McLellan, Michael; Sofia, Heidi J; Hutter, Carolyn; Getz, Gad; Wheeler, David; Ding, Li

2018-03-28

The Cancer Genome Atlas (TCGA) cancer genomics dataset includes over 10,000 tumor-normal exome pairs across 33 different cancer types, in total >400 TB of raw data files requiring analysis. Here we describe the Multi-Center Mutation Calling in Multiple Cancers project, our effort to generate a comprehensive encyclopedia of somatic mutation calls for the TCGA data to enable robust cross-tumor-type analyses. Our approach accounts for variance and batch effects introduced by the rapid advancement of DNA extraction, hybridization-capture, sequencing, and analysis methods over time. We present best practices for applying an ensemble of seven mutation-calling algorithms with scoring and artifact filtering. The dataset created by this analysis includes 3.5 million somatic variants and forms the basis for PanCan Atlas papers. The results have been made available to the research community along with the methods used to generate them. This project is the result of collaboration from a number of institutes and demonstrates how team science drives extremely large genomics projects. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Recombination Is Responsible for the Increased Recovery of Drug-Resistant Mutants with Hypermutated Genomes in Resting Yeast Diploids Expressing APOBEC Deaminases

PubMed Central

Lada, Artem G.; Stepchenkova, Elena I.; Zhuk, Anna S.; Kliver, Sergei F.; Rogozin, Igor B.; Polev, Dmitrii E.; Dhar, Alok; Pavlov, Youri I.

2017-01-01

DNA editing deaminases (APOBECs) are implicated in generation of mutations in somatic cells during tumorigenesis. APOBEC-dependent mutagenesis is thought to occur during transient exposure of unprotected single-stranded DNA. Mutations frequently occur in clusters (kataegis). We investigated mechanisms of mutant generation in growing and resting diploid yeast expressing APOBEC from sea lamprey, PmCDA1, whose kataegistic effect was previously shown to be associated with transcription. We have found that the frequency of canavanine-resistant mutants kept raising after growth cessation, while the profile of transcription remained unchanged. Surprisingly, the overall number of mutations in the genomes did not elevate in resting cells. Thus, mutations were accumulated during vigorous growth stage with both intense replication and transcription. We found that the elevated recovery of can1 mutant clones in non-growing cells is the result of loss of heterozygosity (LOH) leading to clusters of homozygous mutations in the chromosomal regions distal to the reporter gene. We confirmed that recombination frequency in resting cells was elevated by orders of magnitude, suggesting that cells were transiently committed to meiotic levels of recombination, a process referred to in yeast genetics as return-to-growth. In its extreme, on day 6 of starvation, a few mutant clones were haploid, likely resulting from completed meiosis. Distribution of mutations along chromosomes indicated that PmCDA1 was active during ongoing recombination events and sometimes produced characteristic kataegis near initial breakpoints. AID and APOBEC1 behaved similar to PmCDA1. We conclude that replication, transcription, and mitotic recombination contribute to the recovered APOBEC-induced mutations in resting diploids. The mechanism is relevant to the initial stages of oncogenic transformation in terminally differentiated cells, when recombination may lead to the LOH exposing recessive mutations induced by APOBECs in cell’s history and to acquisition of new mutations near original break. PMID:29312434

Recombination Is Responsible for the Increased Recovery of Drug-Resistant Mutants with Hypermutated Genomes in Resting Yeast Diploids Expressing APOBEC Deaminases.

PubMed

Lada, Artem G; Stepchenkova, Elena I; Zhuk, Anna S; Kliver, Sergei F; Rogozin, Igor B; Polev, Dmitrii E; Dhar, Alok; Pavlov, Youri I

2017-01-01

DNA editing deaminases (APOBECs) are implicated in generation of mutations in somatic cells during tumorigenesis. APOBEC-dependent mutagenesis is thought to occur during transient exposure of unprotected single-stranded DNA. Mutations frequently occur in clusters ( kataegis ). We investigated mechanisms of mutant generation in growing and resting diploid yeast expressing APOBEC from sea lamprey, PmCDA1, whose kataegistic effect was previously shown to be associated with transcription. We have found that the frequency of canavanine-resistant mutants kept raising after growth cessation, while the profile of transcription remained unchanged. Surprisingly, the overall number of mutations in the genomes did not elevate in resting cells. Thus, mutations were accumulated during vigorous growth stage with both intense replication and transcription. We found that the elevated recovery of can1 mutant clones in non-growing cells is the result of loss of heterozygosity (LOH) leading to clusters of homozygous mutations in the chromosomal regions distal to the reporter gene. We confirmed that recombination frequency in resting cells was elevated by orders of magnitude, suggesting that cells were transiently committed to meiotic levels of recombination, a process referred to in yeast genetics as return-to-growth. In its extreme, on day 6 of starvation, a few mutant clones were haploid, likely resulting from completed meiosis. Distribution of mutations along chromosomes indicated that PmCDA1 was active during ongoing recombination events and sometimes produced characteristic kataegis near initial breakpoints. AID and APOBEC1 behaved similar to PmCDA1. We conclude that replication, transcription, and mitotic recombination contribute to the recovered APOBEC-induced mutations in resting diploids. The mechanism is relevant to the initial stages of oncogenic transformation in terminally differentiated cells, when recombination may lead to the LOH exposing recessive mutations induced by APOBECs in cell's history and to acquisition of new mutations near original break.

Genetic transformation of Neurospora tetrasperma, demonstration of repeat-induced point mutation (RIP) in self-crosses and a screen for recessive RIP-defective mutants.

PubMed Central

Bhat, Ashwin; Tamuli, Ranjan; Kasbekar, Durgadas P

2004-01-01

The pseudohomothallic fungus Neurospora tetrasperma is naturally resistant to the antibiotic hygromycin. We discovered that mutation of its erg-3 (sterol C-14 reductase) gene confers a hygromycin-sensitive phenotype that can be used to select transformants on hygromycin medium by complementation with the N. crassa erg-3+ and bacterial hph genes. Cotransformation of hph with PCR-amplified DNA of other genes enabled us to construct strains duplicated for the amplified DNA. Using transformation we constructed self-fertile strains that were homoallelic for an ectopic erg-3+ transgene and a mutant erg-3 allele at the endogenous locus. Self-crosses of these strains yielded erg-3 mutant ascospores that produced colonies with the characteristic morphology on Vogel's sorbose agar described previously for erg-3 mutants of N. crassa. The mutants were generated by repeat-induced point mutation (RIP), a genome defense process that causes numerous G:C to A:T mutations in duplicated DNA sequences. Homozygosity for novel recessive RIP-deficient mutations was signaled by self-crosses of erg-3-duplication strains that fail to produce erg-3 mutant progeny. Using this assay we isolated a UV-induced mutant with a putative partial RIP defect. RIP-induced mutants were isolated in rid-1 and sad-1, which are essential genes, respectively, for RIP and another genome defense mechanism called meiotic silencing by unpaired DNA. PMID:15280231

Relapsed neuroblastomas show frequent RAS-MAPK pathway mutations | Office of Cancer Genomics

Cancer.gov

The majority of patients with neuroblastoma have tumors that initially respond to chemotherapy, but a large proportion will experience therapy-resistant relapses. The molecular basis of this aggressive phenotype is unknown. Whole-genome sequencing of 23 paired diagnostic and relapse neuroblastomas showed clonal evolution from the diagnostic tumor, with a median of 29 somatic mutations unique to the relapse sample. Eighteen of the 23 relapse tumors (78%) showed mutations predicted to activate the RAS-MAPK pathway.

The molecular landscape of pediatric acute myeloid leukemia reveals recurrent structural alterations and age-specific mutational interactions | Office of Cancer Genomics

Cancer.gov

We present the molecular landscape of pediatric acute myeloid leukemia (AML) and characterize nearly 1,000 participants in Children’s Oncology Group (COG) AML trials. The COG–National Cancer Institute (NCI) TARGET AML initiative assessed cases by whole-genome, targeted DNA, mRNA and microRNA sequencing and CpG methylation profiling. Validated DNA variants corresponded to diverse, infrequent mutations, with fewer than 40 genes mutated in >2% of cases.

Regulation of AID, the B-cell genome mutator

PubMed Central

Keim, Celia; Kazadi, David; Rothschild, Gerson; Basu, Uttiya

2013-01-01

The mechanisms by which B cells somatically engineer their genomes to generate the vast diversity of antibodies required to challenge the nearly infinite number of antigens that immune systems encounter are of tremendous clinical and academic interest. The DNA cytidine deaminase activation-induced deaminase (AID) catalyzes two of these mechanisms: class switch recombination (CSR) and somatic hypermutation (SHM). Recent discoveries indicate a significant promiscuous targeting of this B-cell mutator enzyme genome-wide. Here we discuss the various regulatory elements that control AID activity and prevent AID from inducing genomic instability and thereby initiating oncogenesis. PMID:23307864

Mutations in DONSON disrupt replication fork stability and cause microcephalic dwarfism

PubMed Central

Reynolds, John J; Bicknell, Louise S; Carroll, Paula; Higgs, Martin R; Shaheen, Ranad; Murray, Jennie E; Papadopoulos, Dimitrios K; Leitch, Andrea; Murina, Olga; Tarnauskaitė, Žygimantė; Wessel, Sarah R; Zlatanou, Anastasia; Vernet, Audrey; von Kriegsheim, Alex; Mottram, Rachel MA; Logan, Clare V; Bye, Hannah; Li, Yun; Brean, Alexander; Maddirevula, Sateesh; Challis, Rachel C; Skouloudaki, Kassiani; Almoisheer, Agaadir; Alsaif, Hessa S; Amar, Ariella; Prescott, Natalie J; Bober, Michael B; Duker, Angela; Faqeih, Eissa; Seidahmed, Mohammed Zain; Al Tala, Saeed; Alswaid, Abdulrahman; Ahmed, Saleem; Al-Aama, Jumana Yousuf; Altmüller, Janine; Al Balwi, Mohammed; Brady, Angela F; Chessa, Luciana; Cox, Helen; Fischetto, Rita; Heller, Raoul; Henderson, Bertram D; Hobson, Emma; Nürnberg, Peter; Percin, E Ferda; Peron, Angela; Spaccini, Luigina; Quigley, Alan J; Thakur, Seema; Wise, Carol A; Yoon, Grace; Alnemer, Maha; Tomancak, Pavel; Yigit, Gökhan; Taylor, A Malcolm R; Reijns, Martin AM; Simpson, Michael A; Cortez, David; Alkuraya, Fowzan S; Mathew, Christopher G; Jackson, Andrew P; Stewart, Grant S

2017-01-01

To ensure efficient genome duplication, cells have evolved numerous factors that promote unperturbed DNA replication, and protect, repair and restart damaged forks. Here we identify DONSON as a novel fork protection factor, and report biallelic DONSON mutations in 29 individuals with microcephalic dwarfism. We demonstrate that DONSON is a replisome component that stabilises forks during genome replication. Loss of DONSON leads to severe replication-associated DNA damage arising from nucleolytic cleavage of stalled replication forks. Furthermore, ATR-dependent signalling in response to replication stress is impaired in DONSON-deficient cells, resulting in decreased checkpoint activity, and potentiating chromosomal instability. Hypomorphic mutations substantially reduce DONSON protein levels and impair fork stability in patient cells, consistent with defective DNA replication underlying the disease phenotype. In summary, we identify mutations in DONSON as a common cause of microcephalic dwarfism, and establish DONSON as a critical replication fork protein required for mammalian DNA replication and genome stability. PMID:28191891

Initial genome sequencing and analysis of multiple myeloma

PubMed Central

Chapman, Michael A.; Lawrence, Michael S.; Keats, Jonathan J.; Cibulskis, Kristian; Sougnez, Carrie; Schinzel, Anna C.; Harview, Christina L.; Brunet, Jean-Philippe; Ahmann, Gregory J.; Adli, Mazhar; Anderson, Kenneth C.; Ardlie, Kristin G.; Auclair, Daniel; Baker, Angela; Bergsagel, P. Leif; Bernstein, Bradley E.; Drier, Yotam; Fonseca, Rafael; Gabriel, Stacey B.; Hofmeister, Craig C.; Jagannath, Sundar; Jakubowiak, Andrzej J.; Krishnan, Amrita; Levy, Joan; Liefeld, Ted; Lonial, Sagar; Mahan, Scott; Mfuko, Bunmi; Monti, Stefano; Perkins, Louise M.; Onofrio, Robb; Pugh, Trevor J.; Vincent Rajkumar, S.; Ramos, Alex H.; Siegel, David S.; Sivachenko, Andrey; Trudel, Suzanne; Vij, Ravi; Voet, Douglas; Winckler, Wendy; Zimmerman, Todd; Carpten, John; Trent, Jeff; Hahn, William C.; Garraway, Levi A.; Meyerson, Matthew; Lander, Eric S.; Getz, Gad; Golub, Todd R.

2013-01-01

Multiple myeloma is an incurable malignancy of plasma cells, and its pathogenesis is poorly understood. Here we report the massively parallel sequencing of 38 tumor genomes and their comparison to matched normal DNAs. Several new and unexpected oncogenic mechanisms were suggested by the pattern of somatic mutation across the dataset. These include the mutation of genes involved in protein translation (seen in nearly half of the patients), genes involved in histone methylation, and genes involved in blood coagulation. In addition, a broader than anticipated role of NF-κB signaling was suggested by mutations in 11 members of the NF-κB pathway. Of potential immediate clinical relevance, activating mutations of the kinase BRAF were observed in 4% of patients, suggesting the evaluation of BRAF inhibitors in multiple myeloma clinical trials. These results indicate that cancer genome sequencing of large collections of samples will yield new insights into cancer not anticipated by existing knowledge. PMID:21430775

Diagnosis of mitochondrial disorders by concomitant next-generation sequencing of the exome and mitochondrial genome

PubMed Central

Dinwiddie, Darrell L.; Smith, Laurie D.; Miller, Neil A.; Atherton, Andrea M.; Farrow, Emily G.; Strenk, Meghan E.; Soden, Sarah E.; Saunders, Carol J.; Kingsmore, Stephen F.

2015-01-01

Mitochondrial diseases are notoriously difficult to diagnose due to extreme locus and allelic heterogeneity, with both nuclear and mitochondrial genomes potentially liable. Using exome sequencing we demonstrate the ability to rapidly and cost effectively evaluate both the nuclear and mitochondrial genomes to obtain a molecular diagnosis for four patients with three distinct mitochondrial disorders. One patient was found to have Leigh syndrome due to a mutation in MT-ATP6, two affected siblings were discovered to be compound heterozygous for mutations in the NDUFV1 gene, which causes mitochondrial complex I deficiency, and one patient was found to have coenzyme Q10 deficiency due to compound heterozygous mutations in COQ2. In all cases conventional diagnostic testing failed to identify a molecular diagnosis. We suggest that additional studies should be conducted to evaluate exome sequencing as a primary diagnostic test for mitochondrial diseases, including those due to mtDNA mutations. PMID:23631824

DOE Office of Scientific and Technical Information (OSTI.GOV)

Golbus, Jessica R.; Puckelwartz, Megan J.; Dellefave-Castillo, Lisa

Background—Cardiomyopathy is highly heritable but genetically diverse. At present, genetic testing for cardiomyopathy uses targeted sequencing to simultaneously assess the coding regions of more than 50 genes. New genes are routinely added to panels to improve the diagnostic yield. With the anticipated $1000 genome, it is expected that genetic testing will shift towards comprehensive genome sequencing accompanied by targeted gene analysis. Therefore, we assessed the reliability of whole genome sequencing and targeted analysis to identify cardiomyopathy variants in 11 subjects with cardiomyopathy. Methods and Results—Whole genome sequencing with an average of 37× coverage was combined with targeted analysis focused onmore » 204 genes linked to cardiomyopathy. Genetic variants were scored using multiple prediction algorithms combined with frequency data from public databases. This pipeline yielded 1-14 potentially pathogenic variants per individual. Variants were further analyzed using clinical criteria and/or segregation analysis. Three of three previously identified primary mutations were detected by this analysis. In six subjects for whom the primary mutation was previously unknown, we identified mutations that segregated with disease, had clinical correlates, and/or had additional pathological correlation to provide evidence for causality. For two subjects with previously known primary mutations, we identified additional variants that may act as modifiers of disease severity. In total, we identified the likely pathological mutation in 9 of 11 (82%) subjects. We conclude that these pilot data demonstrate that ~30-40× coverage whole genome sequencing combined with targeted analysis is feasible and sensitive to identify rare variants in cardiomyopathy-associated genes.« less

Comprehensive genomic analysis of rhabdomyosarcoma reveals a landscape of alterations affecting a common genetic axis in fusion-positive and fusion-negative tumors.

PubMed

Shern, Jack F; Chen, Li; Chmielecki, Juliann; Wei, Jun S; Patidar, Rajesh; Rosenberg, Mara; Ambrogio, Lauren; Auclair, Daniel; Wang, Jianjun; Song, Young K; Tolman, Catherine; Hurd, Laura; Liao, Hongling; Zhang, Shile; Bogen, Dominik; Brohl, Andrew S; Sindiri, Sivasish; Catchpoole, Daniel; Badgett, Thomas; Getz, Gad; Mora, Jaume; Anderson, James R; Skapek, Stephen X; Barr, Frederic G; Meyerson, Matthew; Hawkins, Douglas S; Khan, Javed

2014-02-01

Despite gains in survival, outcomes for patients with metastatic or recurrent rhabdomyosarcoma remain dismal. In a collaboration between the National Cancer Institute, Children's Oncology Group, and Broad Institute, we performed whole-genome, whole-exome, and transcriptome sequencing to characterize the landscape of somatic alterations in 147 tumor/normal pairs. Two genotypes are evident in rhabdomyosarcoma tumors: those characterized by the PAX3 or PAX7 fusion and those that lack these fusions but harbor mutations in key signaling pathways. The overall burden of somatic mutations in rhabdomyosarcoma is relatively low, especially in tumors that harbor a PAX3/7 gene fusion. In addition to previously reported mutations in NRAS, KRAS, HRAS, FGFR4, PIK3CA, and CTNNB1, we found novel recurrent mutations in FBXW7 and BCOR, providing potential new avenues for therapeutic intervention. Furthermore, alteration of the receptor tyrosine kinase/RAS/PIK3CA axis affects 93% of cases, providing a framework for genomics-directed therapies that might improve outcomes for patients with rhabdomyosarcoma. This is the most comprehensive genomic analysis of rhabdomyosarcoma to date. Despite a relatively low mutation rate, multiple genes were recurrently altered, including NRAS, KRAS, HRAS, FGFR4, PIK3CA, CTNNB1, FBXW7, and BCOR. In addition, a majority of rhabdomyosarcoma tumors alter the receptor tyrosine kinase/RAS/PIK3CA axis, providing an opportunity for genomics-guided intervention. 2014 AACR

RNA-Mediated Epigenetic Programming of Genome Rearrangements

PubMed Central

Nowacki, Mariusz; Shetty, Keerthi; Landweber, Laura F.

2012-01-01

RNA, normally thought of as a conduit in gene expression, has a novel mode of action in ciliated protozoa. Maternal RNA templates provide both an organizing guide for DNA rearrangements and a template that can transport somatic mutations to the next generation. This opportunity for RNA-mediated genome rearrangement and DNA repair is profound in the ciliate Oxytricha, which deletes 95% of its germline genome during development in a process that severely fragments its chromosomes and then sorts and reorders the hundreds of thousands of pieces remaining. Oxytricha’s somatic nuclear genome is therefore an epigenome formed through RNA templates and signals arising from the previous generation. Furthermore, this mechanism of RNA-mediated epigenetic inheritance can function across multiple generations, and the discovery of maternal template RNA molecules has revealed new biological roles for RNA and has hinted at the power of RNA molecules to sculpt genomic information in cells. PMID:21801022

Whole-genome sequencing of a malignant granular cell tumor with metabolic response to pazopanib

PubMed Central

Wei, Lei; Liu, Song; Conroy, Jeffrey; Wang, Jianmin; Papanicolau-Sengos, Antonios; Glenn, Sean T.; Murakami, Mitsuko; Liu, Lu; Hu, Qiang; Conroy, Jacob; Miles, Kiersten Marie; Nowak, David E.; Liu, Biao; Qin, Maochun; Bshara, Wiam; Omilian, Angela R.; Head, Karen; Bianchi, Michael; Burgher, Blake; Darlak, Christopher; Kane, John; Merzianu, Mihai; Cheney, Richard; Fabiano, Andrew; Salerno, Kilian; Talati, Chetasi; Khushalani, Nikhil I.; Trump, Donald L.; Johnson, Candace S.; Morrison, Carl D.

2015-01-01

Granular cell tumors are an uncommon soft tissue neoplasm. Malignant granular cell tumors comprise <2% of all granular cell tumors, are associated with aggressive behavior and poor clinical outcome, and are poorly understood in terms of tumor etiology and systematic treatment. Because of its rarity, the genetic basis of malignant granular cell tumor remains unknown. We performed whole-genome sequencing of one malignant granular cell tumor with metabolic response to pazopanib. This tumor exhibited a very low mutation rate and an overall stable genome with local complex rearrangements. The mutation signature was dominated by C>T transitions, particularly when immediately preceded by a 5′ G. A loss-of-function mutation was detected in a newly recognized tumor suppressor candidate, BRD7. No mutations were found in known targets of pazopanib. However, we identified a receptor tyrosine kinase pathway mutation in GFRA2 that warrants further evaluation. To the best of our knowledge, this is only the second reported case of a malignant granular cell tumor exhibiting a response to pazopanib, and the first whole-genome sequencing of this uncommon tumor type. The findings provide insight into the genetic basis of malignant granular cell tumors and identify potential targets for further investigation. PMID:27148567

DNA and RNA editing of retrotransposons accelerate mammalian genome evolution.

PubMed

Knisbacher, Binyamin A; Levanon, Erez Y

2015-04-01

Genome evolution is commonly viewed as a gradual process that is driven by random mutations that accumulate over time. However, DNA- and RNA-editing enzymes have been identified that can accelerate evolution by actively modifying the genomically encoded information. The apolipoprotein B mRNA editing enzymes, catalytic polypeptide-like (APOBECs) are potent restriction factors that can inhibit retroelements by cytosine-to-uridine editing of retroelement DNA after reverse transcription. In some cases, a retroelement may successfully integrate into the genome despite being hypermutated. Such events introduce unique sequences into the genome and are thus a source of genomic innovation. adenosine deaminases that act on RNA (ADARs) catalyze adenosine-to-inosine editing in double-stranded RNA, commonly formed by oppositely oriented retroelements. The RNA editing confers plasticity to the transcriptome by generating many transcript variants from a single genomic locus. If the editing produces a beneficial variant, the genome may maintain the locus that produces the RNA-edited transcript for its novel function. Here, we discuss how these two powerful editing mechanisms, which both target inserted retroelements, facilitate expedited genome evolution. © 2015 New York Academy of Sciences.

Interpreting short tandem repeat variations in humans using mutational constraint

PubMed Central

Gymrek, Melissa; Willems, Thomas; Reich, David; Erlich, Yaniv

2017-01-01

Identifying regions of the genome that are depleted of mutations can reveal potentially deleterious variants. Short tandem repeats (STRs), also known as microsatellites, are among the largest contributors of de novo mutations in humans. However, per-locus studies of STR mutations have been limited to highly ascertained panels of several dozen loci. Here, we harnessed bioinformatics tools and a novel analytical framework to estimate mutation parameters for each STR in the human genome by correlating STR genotypes with local sequence heterozygosity. We applied our method to obtain robust estimates of the impact of local sequence features on mutation parameters and used this to create a framework for measuring constraint at STRs by comparing observed vs. expected mutation rates. Constraint scores identified known pathogenic variants with early onset effects. Our metric will provide a valuable tool for prioritizing pathogenic STRs in medical genetics studies. PMID:28892063

Generation and Repair of AID-initiated DNA Lesions in B Lymphocytes

PubMed Central

Chen, Zhangguo; Wang, Jing H.

2014-01-01

Activation-induced deaminase (AID) initiates the secondary antibody diversification process in B lymphocytes. In mammalian B cells, this process includes somatic hypermutation (SHM) and class switch recombination (CSR), both of which require AID. AID induces U:G mismatch lesions in DNA that are subsequently converted into point mutations or DNA double stranded breaks during SHM/CSR. In a physiological context, AID targets immunoglobulin (Ig) loci to mediate SHM/CSR. However, recent studies reveal genome-wide access of AID to numerous non-Ig loci. Thus, AID poses a threat to the genome of B cells if AID-initiated DNA lesions cannot be properly repaired. In this review, we focus on the molecular mechanisms that regulate the specificity of AID targeting and the repair pathways responsible for processing AID-initiated DNA lesions. PMID:24748462

Replicative age induces mitotic recombination in the ribosomal RNA gene cluster of Saccharomyces cerevisiae.

PubMed

Lindstrom, Derek L; Leverich, Christina K; Henderson, Kiersten A; Gottschling, Daniel E

2011-03-01

Somatic mutations contribute to the development of age-associated disease. In earlier work, we found that, at high frequency, aging Saccharomyces cerevisiae diploid cells produce daughters without mitochondrial DNA, leading to loss of respiration competence and increased loss of heterozygosity (LOH) in the nuclear genome. Here we used the recently developed Mother Enrichment Program to ask whether aging cells that maintain the ability to produce respiration-competent daughters also experience increased genomic instability. We discovered that this population exhibits a distinct genomic instability phenotype that primarily affects the repeated ribosomal RNA gene array (rDNA array). As diploid cells passed their median replicative life span, recombination rates between rDNA arrays on homologous chromosomes progressively increased, resulting in mutational events that generated LOH at >300 contiguous open reading frames on the right arm of chromosome XII. We show that, while these recombination events were dependent on the replication fork block protein Fob1, the aging process that underlies this phenotype is Fob1-independent. Furthermore, we provide evidence that this aging process is not driven by mechanisms that modulate rDNA recombination in young cells, including loss of cohesion within the rDNA array or loss of Sir2 function. Instead, we suggest that the age-associated increase in rDNA recombination is a response to increasing DNA replication stress generated in aging cells.

Mutation Scanning in Wheat by Exon Capture and Next-Generation Sequencing.

PubMed

King, Robert; Bird, Nicholas; Ramirez-Gonzalez, Ricardo; Coghill, Jane A; Patil, Archana; Hassani-Pak, Keywan; Uauy, Cristobal; Phillips, Andrew L

2015-01-01

Targeted Induced Local Lesions in Genomes (TILLING) is a reverse genetics approach to identify novel sequence variation in genomes, with the aims of investigating gene function and/or developing useful alleles for breeding. Despite recent advances in wheat genomics, most current TILLING methods are low to medium in throughput, being based on PCR amplification of the target genes. We performed a pilot-scale evaluation of TILLING in wheat by next-generation sequencing through exon capture. An oligonucleotide-based enrichment array covering ~2 Mbp of wheat coding sequence was used to carry out exon capture and sequencing on three mutagenised lines of wheat containing previously-identified mutations in the TaGA20ox1 homoeologous genes. After testing different mapping algorithms and settings, candidate SNPs were identified by mapping to the IWGSC wheat Chromosome Survey Sequences. Where sequence data for all three homoeologues were found in the reference, mutant calls were unambiguous; however, where the reference lacked one or two of the homoeologues, captured reads from these genes were mis-mapped to other homoeologues, resulting either in dilution of the variant allele frequency or assignment of mutations to the wrong homoeologue. Competitive PCR assays were used to validate the putative SNPs and estimate cut-off levels for SNP filtering. At least 464 high-confidence SNPs were detected across the three mutagenized lines, including the three known alleles in TaGA20ox1, indicating a mutation rate of ~35 SNPs per Mb, similar to that estimated by PCR-based TILLING. This demonstrates the feasibility of using exon capture for genome re-sequencing as a method of mutation detection in polyploid wheat, but accurate mutation calling will require an improved genomic reference with more comprehensive coverage of homoeologues.

Dissecting Vancomycin-Intermediate Resistance in Staphylococcus aureus Using Genome-Wide Association

PubMed Central

Alam, Md Tauqeer; Petit, Robert A.; Crispell, Emily K.; Thornton, Timothy A.; Conneely, Karen N.; Jiang, Yunxuan; Satola, Sarah W.; Read, Timothy D.

2014-01-01

Vancomycin-intermediate Staphylococcus aureus (VISA) is currently defined as having minimal inhibitory concentration (MIC) of 4–8 µg/ml. VISA evolves through changes in multiple genetic loci with at least 16 candidate genes identified in clinical and in vitro-selected VISA strains. We report a whole-genome comparative analysis of 49 vancomycin-sensitive S. aureus and 26 VISA strains. Resistance to vancomycin was determined by broth microdilution, Etest, and population analysis profile-area under the curve (PAP-AUC). Genome-wide association studies (GWAS) of 55,977 single-nucleotide polymorphisms identified in one or more strains found one highly significant association (P = 8.78E-08) between a nonsynonymous mutation at codon 481 (H481) of the rpoB gene and increased vancomycin MIC. Additionally, we used a database of public S. aureus genome sequences to identify rare mutations in candidate genes associated with VISA. On the basis of these data, we proposed a preliminary model called ECM+RMCG for the VISA phenotype as a benchmark for future efforts. The model predicted VISA based on the presence of a rare mutation in a set of candidate genes (walKR, vraSR, graSR, and agrA) and/or three previously experimentally verified mutations (including the rpoB H481 locus) with an accuracy of 81% and a sensitivity of 73%. Further, the level of resistance measured by both Etest and PAP-AUC regressed positively with the number of mutations present in a strain. This study demonstrated 1) the power of GWAS for identifying common genetic variants associated with antibiotic resistance in bacteria and 2) that rare mutations in candidate gene, identified using large genomic data sets, can also be associated with resistance phenotypes. PMID:24787619

[Advances in genome editing technologies for treating muscular dystrophy.

PubMed

Makita, Yukimasa; Hozumi, Hiroyuki; Hotta, Akitsu

Recent advances in genome editing technologies have opened the possibility for treating genetic diseases, such as Duchenne muscular dystrophy(DMD), by correcting the causing gene mutations in dystrophin gene. In fact, there are several reports that demonstrated the restoration of the mutated dystrophin gene in DMD patient-derived iPS cell or functional recovery of forelimb grip strength in DMD model mice. For future clinical applications, there are several aspects that need to be taken into consideration:efficient delivery of the genome editing components, risk of off-target mutagenesis and immunogenicity against genome editing enzyme. In this review, we summarize the current status and future prospective of the research in applying genome editing technologies to DMD.

RAG-mediated recombination is the predominant driver of oncogenic rearrangement in ETV6-RUNX1 acute lymphoblastic leukemia

PubMed Central

Papaemmanuil, Elli; Rapado, Inmaculada; Li, Yilong; Potter, Nicola E; Wedge, David C; Tubio, Jose; Alexandrov, Ludmil B; Van Loo, Peter; Cooke, Susanna L; Marshall, John; Martincorena, Inigo; Hinton, Jonathan; Gundem, Gunes; van Delft, Frederik W; Nik-Zainal, Serena; Jones, David R; Ramakrishna, Manasa; Titley, Ian; Stebbings, Lucy; Leroy, Catherine; Menzies, Andrew; Gamble, John; Robinson, Ben; Mudie, Laura; Raine, Keiran; O’Meara, Sarah; Teague, Jon W; Butler, Adam P; Cazzaniga, Giovanni; Biondi, Andrea; Zuna, Jan; Kempski, Helena; Muschen, Markus; Ford, Anthony M; Stratton, Michael R; Greaves, Mel; Campbell, Peter J

2014-01-01

The ETV6-RUNX1 fusion gene, found in 25% of childhood acute lymphoblastic leukemia (ALL), is acquired in utero but requires additional somatic mutations for overt leukemia. We used exome and low-coverage whole-genome sequencing to characterize secondary events associated with leukemic transformation. RAG-mediated deletions emerge as the dominant mutational process, characterized by recombination signal sequence motifs near the breakpoints; incorporation of non-templated sequence at the junction; ~30-fold enrichment at promoters and enhancers of genes actively transcribed in B-cell development and an unexpectedly high ratio of recurrent to non-recurrent structural variants. Single cell tracking shows that this mechanism is active throughout leukemic evolution with evidence of localized clustering and re-iterated deletions. Integration of point mutation and rearrangement data identifies ATF7IP and MGA as two new tumor suppressor genes in ALL. Thus, a remarkably parsimonious mutational process transforms ETV6-RUNX1 lymphoblasts, targeting the promoters, enhancers and first exons of genes that normally regulate B-cell differentiation. PMID:24413735

Broad Detection of Alterations Predicted to Confer Lack of Benefit From EGFR Antibodies or Sensitivity to Targeted Therapy in Advanced Colorectal Cancer.

PubMed

Rankin, Andrew; Klempner, Samuel J; Erlich, Rachel; Sun, James X; Grothey, Axel; Fakih, Marwan; George, Thomas J; Lee, Jeeyun; Ross, Jeffrey S; Stephens, Philip J; Miller, Vincent A; Ali, Siraj M; Schrock, Alexa B

2016-09-28

A KRAS mutation represented the first genomic biomarker to predict lack of benefit from anti-epidermal growth factor receptor (EGFR) antibody therapy in advanced colorectal cancer (CRC). Expanded RAS testing has further refined the treatment approach, but understanding of genomic alterations underlying primary and acquired resistance is limited and further study is needed. We prospectively analyzed 4,422 clinical samples from patients with advanced CRC, using hybrid-capture based comprehensive genomic profiling (CGP) at the request of the individual treating physicians. Comparison with prior molecular testing results, when available, was performed to assess concordance. We identified a RAS/RAF pathway mutation or amplification in 62% of cases, including samples harboring KRAS mutations outside of the codon 12/13 hotspot region in 6.4% of cases. Among cases with KRAS non-codon 12/13 alterations for which prior test results were available, 79 of 90 (88%) were not identified by focused testing. Of 1,644 RAS/RAF wild-type cases analyzed by CGP, 31% harbored a genomic alteration (GA) associated with resistance to anti-EGFR therapy in advanced CRC including mutations in PIK3CA, PTEN, EGFR, and ERBB2. We also identified other targetable GA, including novel kinase fusions, receptor tyrosine kinase amplification, activating point mutations, as well as microsatellite instability. Extended genomic profiling reliably detects alterations associated with lack of benefit to anti-EGFR therapy in advanced CRC, while simultaneously identifying alterations potentially important in guiding treatment. The use of CGP during the course of clinical care allows for the refined selection of appropriate targeted therapies and clinical trials, increasing the chance of clinical benefit and avoiding therapeutic futility. Comprehensive genomic profiling (CGP) detects diverse genomic alterations associated with lack of benefit to anti-epidermal growth factor receptor therapy in advanced colorectal cancer (CRC), as well as targetable alterations in many other genes. This includes detection of a broad spectrum of activating KRAS alterations frequently missed by focused molecular hotspot testing, as well as other RAS/RAF pathway alterations, mutations shown to disrupt antibody binding, RTK activating point mutations, amplifications, and rearrangements, and activating alterations in downstream effectors including PI3K and MEK1. The use of CGP in clinical practice is critical to guide appropriate selection of targeted therapies for patients with advanced CRC. ©AlphaMed Press.

Quantitative genome re-sequencing defines multiple mutations conferring chloroquine resistance in rodent malaria

PubMed Central

2012-01-01

Background Drug resistance in the malaria parasite Plasmodium falciparum severely compromises the treatment and control of malaria. A knowledge of the critical mutations conferring resistance to particular drugs is important in understanding modes of drug action and mechanisms of resistances. They are required to design better therapies and limit drug resistance. A mutation in the gene (pfcrt) encoding a membrane transporter has been identified as a principal determinant of chloroquine resistance in P. falciparum, but we lack a full account of higher level chloroquine resistance. Furthermore, the determinants of resistance in the other major human malaria parasite, P. vivax, are not known. To address these questions, we investigated the genetic basis of chloroquine resistance in an isogenic lineage of rodent malaria parasite P. chabaudi in which high level resistance to chloroquine has been progressively selected under laboratory conditions. Results Loci containing the critical genes were mapped by Linkage Group Selection, using a genetic cross between the high-level chloroquine-resistant mutant and a genetically distinct sensitive strain. A novel high-resolution quantitative whole-genome re-sequencing approach was used to reveal three regions of selection on chr11, chr03 and chr02 that appear progressively at increasing drug doses on three chromosomes. Whole-genome sequencing of the chloroquine-resistant parent identified just four point mutations in different genes on these chromosomes. Three mutations are located at the foci of the selection valleys and are therefore predicted to confer different levels of chloroquine resistance. The critical mutation conferring the first level of chloroquine resistance is found in aat1, a putative aminoacid transporter. Conclusions Quantitative trait loci conferring selectable phenotypes, such as drug resistance, can be mapped directly using progressive genome-wide linkage group selection. Quantitative genome-wide short-read genome resequencing can be used to reveal these signatures of drug selection at high resolution. The identities of three genes (and mutations within them) conferring different levels of chloroquine resistance generate insights regarding the genetic architecture and mechanisms of resistance to chloroquine and other drugs. Importantly, their orthologues may now be evaluated for critical or accessory roles in chloroquine resistance in human malarias P. vivax and P. falciparum. PMID:22435897

The Infectivities of Turnip Yellow Mosaic Virus Genomes with Altered tRNA Mimicry Are Not Dependent on Compensating Mutations in the Viral Replication Protein†

PubMed Central

Filichkin, Sergei A.; Bransom, Kay L.; Goodwin, Joel B.; Dreher, Theo W.

2000-01-01

Five highly infectious turnip yellow mosaic virus (TYMV) genomes with sequence changes in their 3′-terminal regions that result in altered aminoacylation and eEF1A binding have been studied. These genomes were derived from cloned parental RNAs of low infectivity by sequential passaging in plants. Three of these genomes that are incapable of aminoacylation have been reported previously (J. B. Goodwin, J. M. Skuzeski, and T. W. Dreher, Virology 230:113–124, 1997). We now demonstrate by subcloning the 3′ untranslated regions into wild-type TYMV RNA that the high infectivities and replication rates of these genomes compared to their progenitors are mostly due to a small number of mutations acquired in the 3′ tRNA-like structure during passaging. Mutations in other parts of the genome, including the replication protein coding region, are not required for high infectivity but probably do play a role in optimizing viral amplification and spread in plants. Two other TYMV RNA variants of suboptimal infectivities, one that accepts methionine instead of the usual valine and one that interacts less tightly with eEF1A, were sequentially passaged to produce highly infectious genomes. The improved infectivities of these RNAs were not associated with increased replication in protoplasts, and no mutations were acquired in their 3′ tRNA-like structures. Complete sequencing of one genome identified two mutations that result in amino acid changes in the movement protein gene, suggesting that improved infectivity may be a function of improved viral dissemination in plants. Our results show that the wild-type TYMV replication proteins are able to amplify genomes with 3′ termini of variable sequence and tRNA mimicry. These and previous results have led to a model in which the binding of eEF1A to the 3′ end to antagonize minus-strand initiation is a major role of the tRNA-like structure. PMID:10954536

A Streamlined Protocol for Molecular Testing of the DMD Gene within a Diagnostic Laboratory: A Combination of Array Comparative Genomic Hybridization and Bidirectional Sequence Analysis

PubMed Central

Marquis-Nicholson, Renate; Lai, Daniel; Love, Jennifer M.; Love, Donald R.

2013-01-01

Purpose. The aim of this study was to develop a streamlined mutation screening protocol for the DMD gene in order to confirm a clinical diagnosis of Duchenne or Becker muscular dystrophy in affected males and to clarify the carrier status of female family members. Methods. Sequence analysis and array comparative genomic hybridization (aCGH) were used to identify mutations in the dystrophin DMD gene. We analysed genomic DNA from six individuals with a range of previously characterised mutations and from eight individuals who had not previously undergone any form of molecular analysis. Results. We successfully identified the known mutations in all six patients. A molecular diagnosis was also made in three of the four patients with a clinical diagnosis who had not undergone prior genetic screening, and testing for familial mutations was successfully completed for the remaining four patients. Conclusion. The mutation screening protocol described here meets best practice guidelines for molecular testing of the DMD gene in a diagnostic laboratory. The aCGH method is a superior alternative to more conventional assays such as multiplex ligation-dependent probe amplification (MLPA). The combination of aCGH and sequence analysis will detect mutations in 98% of patients with the Duchenne or Becker muscular dystrophy. PMID:23476807

The Sequences of 1504 Mutants in the Model Rice Variety Kitaake Facilitate Rapid Functional Genomic Studies

PubMed Central

Pham, Nikki T.; Wei, Tong; Schackwitz, Wendy S.; Lipzen, Anna M.; Duong, Phat Q.; Jones, Kyle C.; Ruan, Deling; Bauer, Diane; Peng, Yi; Schmutz, Jeremy

2017-01-01

The availability of a whole-genome sequenced mutant population and the cataloging of mutations of each line at a single-nucleotide resolution facilitate functional genomic analysis. To this end, we generated and sequenced a fast-neutron-induced mutant population in the model rice cultivar Kitaake (Oryza sativa ssp japonica), which completes its life cycle in 9 weeks. We sequenced 1504 mutant lines at 45-fold coverage and identified 91,513 mutations affecting 32,307 genes, i.e., 58% of all rice genes. We detected an average of 61 mutations per line. Mutation types include single-base substitutions, deletions, insertions, inversions, translocations, and tandem duplications. We observed a high proportion of loss-of-function mutations. We identified an inversion affecting a single gene as the causative mutation for the short-grain phenotype in one mutant line. This result reveals the usefulness of the resource for efficient, cost-effective identification of genes conferring specific phenotypes. To facilitate public access to this genetic resource, we established an open access database called KitBase that provides access to sequence data and seed stocks. This population complements other available mutant collections and gene-editing technologies. This work demonstrates how inexpensive next-generation sequencing can be applied to generate a high-density catalog of mutations. PMID:28576844

Development of a genome editing technique using the CRISPR/Cas9 system in the industrial filamentous fungus Aspergillus oryzae.

PubMed

Katayama, Takuya; Tanaka, Yuki; Okabe, Tomoya; Nakamura, Hidetoshi; Fujii, Wataru; Kitamoto, Katsuhiko; Maruyama, Jun-Ichi

2016-04-01

To develop a genome editing method using the CRISPR/Cas9 system in Aspergillus oryzae, the industrial filamentous fungus used in Japanese traditional fermentation and for the production of enzymes and heterologous proteins. To develop the CRISPR/Cas9 system as a genome editing technique for A. oryzae, we constructed plasmids expressing the gene encoding Cas9 nuclease and single guide RNAs for the mutagenesis of target genes. We introduced these into an A. oryzae strain and obtained transformants containing mutations within each target gene that exhibited expected phenotypes. The mutational rates ranged from 10 to 20 %, and 1 bp deletions or insertions were the most commonly induced mutations. We developed a functional and versatile genome editing method using the CRISPR/Cas9 system in A. oryzae. This technique will contribute to the use of efficient targeted mutagenesis in many A. oryzae industrial strains.

RNA-guided genome editing for target gene mutations in wheat.

PubMed

Upadhyay, Santosh Kumar; Kumar, Jitesh; Alok, Anshu; Tuli, Rakesh

2013-12-09

The clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) system has been used as an efficient tool for genome editing. We report the application of CRISPR-Cas-mediated genome editing to wheat (Triticum aestivum), the most important food crop plant with a very large and complex genome. The mutations were targeted in the inositol oxygenase (inox) and phytoene desaturase (pds) genes using cell suspension culture of wheat and in the pds gene in leaves of Nicotiana benthamiana. The expression of chimeric guide RNAs (cgRNA) targeting single and multiple sites resulted in indel mutations in all the tested samples. The expression of Cas9 or sgRNA alone did not cause any mutation. The expression of duplex cgRNA with Cas9 targeting two sites in the same gene resulted in deletion of DNA fragment between the targeted sequences. Multiplexing the cgRNA could target two genes at one time. Target specificity analysis of cgRNA showed that mismatches at the 3' end of the target site abolished the cleavage activity completely. The mismatches at the 5' end reduced cleavage, suggesting that the off target effects can be abolished in vivo by selecting target sites with unique sequences at 3' end. This approach provides a powerful method for genome engineering in plants.

Precision medicine driven by cancer systems biology.

PubMed

Filipp, Fabian V

2017-03-01

Molecular insights from genome and systems biology are influencing how cancer is diagnosed and treated. We critically evaluate big data challenges in precision medicine. The melanoma research community has identified distinct subtypes involving chronic sun-induced damage and the mitogen-activated protein kinase driver pathway. In addition, despite low mutation burden, non-genomic mitogen-activated protein kinase melanoma drivers are found in membrane receptors, metabolism, or epigenetic signaling with the ability to bypass central mitogen-activated protein kinase molecules and activating a similar program of mitogenic effectors. Mutation hotspots, structural modeling, UV signature, and genomic as well as non-genomic mechanisms of disease initiation and progression are taken into consideration to identify resistance mutations and novel drug targets. A comprehensive precision medicine profile of a malignant melanoma patient illustrates future rational drug targeting strategies. Network analysis emphasizes an important role of epigenetic and metabolic master regulators in oncogenesis. Co-occurrence of driver mutations in signaling, metabolic, and epigenetic factors highlights how cumulative alterations of our genomes and epigenomes progressively lead to uncontrolled cell proliferation. Precision insights have the ability to identify independent molecular pathways suitable for drug targeting. Synergistic treatment combinations of orthogonal modalities including immunotherapy, mitogen-activated protein kinase inhibitors, epigenetic inhibitors, and metabolic inhibitors have the potential to overcome immune evasion, side effects, and drug resistance.

Comprehensive genomic profiling reveals inactivating SMARCA4 mutations and low tumor mutational burden in small cell carcinoma of the ovary, hypercalcemic-type.

PubMed

Lin, Douglas I; Chudnovsky, Yakov; Duggan, Bridget; Zajchowski, Deborah; Greenbowe, Joel; Ross, Jeffrey S; Gay, Laurie M; Ali, Siraj M; Elvin, Julia A

2017-12-01

Small cell carcinoma of the ovary, hypercalcemic-type (SCCOHT) is a rare, extremely aggressive neoplasm that usually occurs in young women and is characterized by deleterious germline or somatic SMARCA4 mutations. We performed comprehensive genomic profiling (CGP) to potentially identify additional clinically and pathophysiologically relevant genomic alterations in SCCOHT. CGP assessment of all classes of coding alterations in up to 406 genes commonly altered in cancer and intronic regions for up to 31 genes commonly rearranged in cancer was performed on 18 SCCOHT cases (16 exhibiting classic morphology and 2 cases exhibiting exclusive a large cell variant morphology). In addition, a retrospective database search for clinically advanced ovarian tumors with genomic profiles similar to SCCOHT yielded 3 additional cases originally diagnosed as non-SCCOHT. CGP revealed inactivating SMARCA4 alterations and low tumor mutational burden (TMB) (<6mutations/Mb) in 94% (15/16) of SCCOHT with classic morphology. In contrast, both (2/2) cases exhibiting only large cell variant morphology were hypermutated (TMB scores of 90 and 360mut/Mb) and were wildtype for SMARCA4. In our retrospective search, an index ovarian cancer patient harboring inactivating SMARCA4 alterations, initially diagnosed as endometrioid carcinoma, was re-classified as SCCOHT and responded to an SCCOHT chemotherapy regimen. The vast majority of SCCOHT demonstrate genomic SMARCA4 loss with only rare co-occurring alterations. Our data support a role for CGP in the diagnosis and management of SCCOHT and of other lesions with overlapping histological and clinical features, since identifying the former by genomic profile suggests benefit from an appropriate regimen and treatment decisions, as illustrated by an index patient. Copyright © 2017 Elsevier Inc. All rights reserved.

A single nucleotide mutation in Nppc is associated with a long bone abnormality in lbab mice.

PubMed

Jiao, Yan; Yan, Jian; Jiao, Feng; Yang, Hongbin; Donahue, Leah Rae; Li, Xinmin; Roe, Bruce A; Stuart, John; Gu, Weikuan

2007-04-17

The long bone abnormality (lbab) mouse is a new autosomal recessive mutant characterized by overall smaller body size with proportionate dwarfing of all organs and shorter long bones. Previous linkage analysis has located the lbab mutation on chromosome 1 between the markers D1Mit9 and D1Mit488. A genome-based positional approach was used to identify a mutation associated with lbab disease. A total of 122 genes and expressed sequence tags at the lbab region were screened for possible mutation by using genomic DNA from lbabl/lbab, lbab/+, and +/+ B6 mice and high throughput temperature gradient capillary electrophoresis. A sequence difference was identified in one of the amplicons of gene Nppc between lbab/lbab and +/+ mice. One-step reverse transcriptase polymerase chain reaction was performed to validate the difference of Nppc in different types of mice at the mRNA level. The mutation of Nppc was unique in lbab/lbab mice among multiple mouse inbred strains. The mutation of Nppc is co-segregated with lbab disease in 200 progenies produced from heterozygous lbab/+ parents. A single nucleotide mutation of Nppc is associated with dwarfism in lbab/lbab mice. Current genome information and technology allow us to efficiently identify single nucleotide mutations from roughly mapped disease loci. The lbab mouse is a useful model for hereditary human achondroplasia.

A single nucleotide mutation in Nppc is associated with a long bone abnormality in lbab mice

PubMed Central

Jiao, Yan; Yan, Jian; Jiao, Feng; Yang, HongBin; Donahue, Leah Rae; Li, Xinmin; Roe, Bruce A; Stuart, John; Gu, Weikuan

2007-01-01

Background The long bone abnormality (lbab) mouse is a new autosomal recessive mutant characterized by overall smaller body size with proportionate dwarfing of all organs and shorter long bones. Previous linkage analysis has located the lbab mutation on chromosome 1 between the markers D1Mit9 and D1Mit488. Results A genome-based positional approach was used to identify a mutation associated with lbab disease. A total of 122 genes and expressed sequence tags at the lbab region were screened for possible mutation by using genomic DNA from lbabl/lbab, lbab/+, and +/+ B6 mice and high throughput temperature gradient capillary electrophoresis. A sequence difference was identified in one of the amplicons of gene Nppc between lbab/lbab and +/+ mice. One-step reverse transcriptase polymerase chain reaction was performed to validate the difference of Nppc in different types of mice at the mRNA level. The mutation of Nppc was unique in lbab/lbab mice among multiple mouse inbred strains. The mutation of Nppc is co-segregated with lbab disease in 200 progenies produced from heterozygous lbab/+ parents. Conclusion A single nucleotide mutation of Nppc is associated with dwarfism in lbab/lbab mice. Current genome information and technology allow us to efficiently identify single nucleotide mutations from roughly mapped disease loci. The lbab mouse is a useful model for hereditary human achondroplasia. PMID:17439653

Whole-genome resequencing of Escherichia coli K-12 MG1655 undergoing short-term laboratory evolution in lactate minimal media reveals flexible selection of adaptive mutations

PubMed Central

2009-01-01

Background Short-term laboratory evolution of bacteria followed by genomic sequencing provides insight into the mechanism of adaptive evolution, such as the number of mutations needed for adaptation, genotype-phenotype relationships, and the reproducibility of adaptive outcomes. Results In the present study, we describe the genome sequencing of 11 endpoints of Escherichia coli that underwent 60-day laboratory adaptive evolution under growth rate selection pressure in lactate minimal media. Two to eight mutations were identified per endpoint. Generally, each endpoint acquired mutations to different genes. The most notable exception was an 82 base-pair deletion in the rph-pyrE operon that appeared in 7 of the 11 adapted strains. This mutation conferred an approximately 15% increase to the growth rate when experimentally introduced to the wild-type background and resulted in an approximately 30% increase to growth rate when introduced to a background already harboring two adaptive mutations. Additionally, most endpoints had a mutation in a regulatory gene (crp or relA, for example) or the RNA polymerase. Conclusions The 82 base-pair deletion found in the rph-pyrE operon of many endpoints may function to relieve a pyrimidine biosynthesis defect present in MG1655. In contrast, a variety of regulators acquire mutations in the different endpoints, suggesting flexibility in overcoming regulatory challenges in the adaptation. PMID:19849850

Using Drosophila melanogaster as a Model for Genotoxic Chemical Mutational Studies with a New Program, SnpSift

PubMed Central

Cingolani, Pablo; Patel, Viral M.; Coon, Melissa; Nguyen, Tung; Land, Susan J.; Ruden, Douglas M.; Lu, Xiangyi

2012-01-01

This paper describes a new program SnpSift for filtering differential DNA sequence variants between two or more experimental genomes after genotoxic chemical exposure. Here, we illustrate how SnpSift can be used to identify candidate phenotype-relevant variants including single nucleotide polymorphisms, multiple nucleotide polymorphisms, insertions, and deletions (InDels) in mutant strains isolated from genome-wide chemical mutagenesis of Drosophila melanogaster. First, the genomes of two independently isolated mutant fly strains that are allelic for a novel recessive male-sterile locus generated by genotoxic chemical exposure were sequenced using the Illumina next-generation DNA sequencer to obtain 20- to 29-fold coverage of the euchromatic sequences. The sequencing reads were processed and variants were called using standard bioinformatic tools. Next, SnpEff was used to annotate all sequence variants and their potential mutational effects on associated genes. Then, SnpSift was used to filter and select differential variants that potentially disrupt a common gene in the two allelic mutant strains. The potential causative DNA lesions were partially validated by capillary sequencing of polymerase chain reaction-amplified DNA in the genetic interval as defined by meiotic mapping and deletions that remove defined regions of the chromosome. Of the five candidate genes located in the genetic interval, the Pka-like gene CG12069 was found to carry a separate pre-mature stop codon mutation in each of the two allelic mutants whereas the other four candidate genes within the interval have wild-type sequences. The Pka-like gene is therefore a strong candidate gene for the male-sterile locus. These results demonstrate that combining SnpEff and SnpSift can expedite the identification of candidate phenotype-causative mutations in chemically mutagenized Drosophila strains. This technique can also be used to characterize the variety of mutations generated by genotoxic chemicals. PMID:22435069

Whole Genome Sequencing of Mycobacterium africanum Strains from Mali Provides Insights into the Mechanisms of Geographic Restriction

PubMed Central

Maiga, Mamoudou; Abeel, Thomas; Shea, Terrance; Desjardins, Christopher A.; Diarra, Bassirou; Baya, Bocar; Sanogo, Moumine; Diallo, Souleymane; Earl, Ashlee M.; Bishai, William R.

2016-01-01

Background Mycobacterium africanum, made up of lineages 5 and 6 within the Mycobacterium tuberculosis complex (MTC), causes up to half of all tuberculosis cases in West Africa, but is rarely found outside of this region. The reasons for this geographical restriction remain unknown. Possible reasons include a geographically restricted animal reservoir, a unique preference for hosts of West African ethnicity, and an inability to compete with other lineages outside of West Africa. These latter two hypotheses could be caused by loss of fitness or altered interactions with the host immune system. Methodology/Principal Findings We sequenced 92 MTC clinical isolates from Mali, including two lineage 5 and 24 lineage 6 strains. Our genome sequencing assembly, alignment, phylogeny and average nucleotide identity analyses enabled us to identify features that typify lineages 5 and 6 and made clear that these lineages do not constitute a distinct species within the MTC. We found that in Mali, lineage 6 and lineage 4 strains have similar levels of diversity and evolve drug resistance through similar mechanisms. In the process, we identified a putative novel streptomycin resistance mutation. In addition, we found evidence of person-to-person transmission of lineage 6 isolates and showed that lineage 6 is not enriched for mutations in virulence-associated genes. Conclusions This is the largest collection of lineage 5 and 6 whole genome sequences to date, and our assembly and alignment data provide valuable insights into what distinguishes these lineages from other MTC lineages. Lineages 5 and 6 do not appear to be geographically restricted due to an inability to transmit between West African hosts or to an elevated number of mutations in virulence-associated genes. However, lineage-specific mutations, such as mutations in cell wall structure, secretion systems and cofactor biosynthesis, provide alternative mechanisms that may lead to host specificity. PMID:26751217

De novo truncating mutations in ASXL3 are associated with a novel clinical phenotype with similarities to Bohring-Opitz syndrome

PubMed Central

2013-01-01

Background Molecular diagnostics can resolve locus heterogeneity underlying clinical phenotypes that may otherwise be co-assigned as a specific syndrome based on shared clinical features, and can associate phenotypically diverse diseases to a single locus through allelic affinity. Here we describe an apparently novel syndrome, likely caused by de novo truncating mutations in ASXL3, which shares characteristics with Bohring-Opitz syndrome, a disease associated with de novo truncating mutations in ASXL1. Methods We used whole-genome and whole-exome sequencing to interrogate the genomes of four subjects with an undiagnosed syndrome. Results Using genome-wide sequencing, we identified heterozygous, de novo truncating mutations in ASXL3, a transcriptional repressor related to ASXL1, in four unrelated probands. We found that these probands shared similar phenotypes, including severe feeding difficulties, failure to thrive, and neurologic abnormalities with significant developmental delay. Further, they showed less phenotypic overlap with patients who had de novo truncating mutations in ASXL1. Conclusion We have identified truncating mutations in ASXL3 as the likely cause of a novel syndrome with phenotypic overlap with Bohring-Opitz syndrome. PMID:23383720

Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy

PubMed Central

Amoasii, Leonela; Long, Chengzu; Li, Hui; Mireault, Alex A.; Shelton, John M.; Sanchez-Ortiz, Efrain; McAnally, John R.; Bhattacharyya, Samadrita; Schmidt, Florian; Grimm, Dirk; Hauschka, Stephen D.; Bassel-Duby, Rhonda; Olson, Eric N.

2017-01-01

Duchenne muscular dystrophy (DMD) is a severe, progressive muscle disease caused by mutations in the dystrophin gene. The majority of DMD mutations are deletions that prematurely terminate the dystrophin protein. Deletions of exon 50 of the dystrophin gene are among the most common single exon deletions causing DMD. Such mutations can be corrected by skipping exon 51, thereby restoring the dystrophin reading frame. Using clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9), we generated a DMD mouse model by deleting exon 50. These ΔEx50 mice displayed severe muscle dysfunction, which was corrected by systemic delivery of adeno-associated virus encoding CRISPR/Cas9 genome editing components. We optimized the method for dystrophin reading frame correction using a single guide RNA that created reframing mutations and allowed skipping of exon 51. In conjunction with muscle-specific expression of Cas9, this approach restored up to 90% of dystrophin protein expression throughout skeletal muscles and the heart of ΔEx50 mice. This method of permanently bypassing DMD mutations using a single cut in genomic DNA represents a step toward clinical correction of DMD mutations and potentially those of other neuromuscular disorders. PMID:29187645

Recapitulating X-Linked Juvenile Retinoschisis in Mouse Model by Knock-In Patient-Specific Novel Mutation.

PubMed

Chen, Ding; Xu, Tao; Tu, Mengjun; Xu, Jinlin; Zhou, Chenchen; Cheng, Lulu; Yang, Ruqing; Yang, Tanchu; Zheng, Weiwei; He, Xiubin; Deng, Ruzhi; Ge, Xianglian; Li, Jin; Song, Zongming; Zhao, Junzhao; Gu, Feng

2017-01-01

X-linked juvenile retinoschisis (XLRS) is a retinal disease caused by mutations in the gene encoding retinoschisin (RS1), which leads to a significant proportion of visual impairment and blindness. To develop personalized genome editing based gene therapy, knock-in animal disease models that have the exact mutation identified in the patients is extremely crucial, and that the way which genome editing in knock-in animals could be easily transferred to the patients. Here we recruited a family diagnosed with XLRS and identified the causative mutation ( RS1 , p.Y65X), then a knock-in mouse model harboring this disease-causative mutation was generated via TALEN (transcription activator-like effector nucleases). We found that the b-wave amplitude of the ERG of the RS1 -KI mice was significantly decreased. Moreover, we observed that the structure of retina in RS1 -KI mice has become disordered, including the disarray of inner nuclear layer and outer nuclear layer, chaos of outer plexiform layer, decreased inner segments of photoreceptor and the loss of outer segments. The novel knock-in mice ( RS1 -KI) harboring patient-specific mutation will be valuable for development of treatment via genome editing mediated gene correction.

Replication and transmission of mammalian-adapted H9 subtype influenza virus in pigs and quail

USDA-ARS?s Scientific Manuscript database

Influenza A is a major pathogen of birds, swine, and humans. Strains can jump from one species to another in a process that often requires genetic mutation and genome reassortment and results in outbreaks and, potentially, pandemics. H9N2 avian influenza is one of the most predominant influenza subt...

Comparative Genomic Analysis of Two Clonally Related Multidrug Resistant Mycobacterium tuberculosis by Single Molecule Real Time Sequencing.

PubMed

Leung, Kenneth Siu-Sing; Siu, Gilman Kit-Hang; Tam, Kingsley King-Gee; To, Sabrina Wai-Chi; Rajwani, Rahim; Ho, Pak-Leung; Wong, Samson Sai-Yin; Zhao, Wei W; Ma, Oliver Chiu-Kit; Yam, Wing-Cheong

2017-01-01

Background: Multidrug-resistant tuberculosis (MDR-TB) is posing a major threat to global TB control. In this study, we focused on two consecutive MDR-TB isolated from the same patient before and after the initiation of anti-TB treatment. To better understand the genomic characteristics of MDR-TB, Single Molecule Real-Time (SMRT) Sequencing and comparative genomic analyses was performed to identify mutations that contributed to the stepwise development of drug resistance and growth fitness in MDR-TB under in vivo challenge of anti-TB drugs. Result: Both pre-treatment and post-treatment strain demonstrated concordant phenotypic and genotypic susceptibility profiles toward rifampicin, pyrazinamide, streptomycin, fluoroquinolones, aminoglycosides, cycloserine, ethionamide, and para-aminosalicylic acid. However, although both strains carried identical missense mutations at rpoB S531L, inhA C-15T, and embB M306V, MYCOTB Sensititre assay showed that the post-treatment strain had 16-, 8-, and 4-fold elevation in the minimum inhibitory concentrations (MICs) toward rifabutin, isoniazid, and ethambutol respectively. The results have indicated the presence of additional resistant-related mutations governing the stepwise development of MDR-TB. Further comparative genomic analyses have identified three additional polymorphisms between the clinical isolates. These include a single nucleotide deletion at nucleotide position 360 of rv0888 in pre-treatment strain, and a missense mutation at rv3303c ( lpdA) V44I and a 6-bp inframe deletion at codon 67-68 in rv2071c ( cobM) in the post-treatment strain. Multiple sequence alignment showed that these mutations were occurring at highly conserved regions among pathogenic mycobacteria. Using structural-based and sequence-based algorithms, we further predicted that the mutations potentially have deleterious effect on protein function. Conclusion: This is the first study that compared the full genomes of two clonally-related MDR-TB clinical isolates during the course of anti-TB treatment. Our work has demonstrated the robustness of SMRT Sequencing in identifying mutations among MDR-TB clinical isolates. Comparative genome analysis also suggested novel mutations at rv0888, lpdA , and cobM that might explain the difference in antibiotic resistance and growth pattern between the two MDR-TB strains.

Amino acid usage is asymmetrically biased in AT- and GC-rich microbial genomes.

PubMed

Bohlin, Jon; Brynildsrud, Ola; Vesth, Tammi; Skjerve, Eystein; Ussery, David W

2013-01-01

Genomic base composition ranges from less than 25% AT to more than 85% AT in prokaryotes. Since only a small fraction of prokaryotic genomes is not protein coding even a minor change in genomic base composition will induce profound protein changes. We examined how amino acid and codon frequencies were distributed in over 2000 microbial genomes and how these distributions were affected by base compositional changes. In addition, we wanted to know how genome-wide amino acid usage was biased in the different genomes and how changes to base composition and mutations affected this bias. To carry this out, we used a Generalized Additive Mixed-effects Model (GAMM) to explore non-linear associations and strong data dependences in closely related microbes; principal component analysis (PCA) was used to examine genomic amino acid- and codon frequencies, while the concept of relative entropy was used to analyze genomic mutation rates. We found that genomic amino acid frequencies carried a stronger phylogenetic signal than codon frequencies, but that this signal was weak compared to that of genomic %AT. Further, in contrast to codon usage bias (CUB), amino acid usage bias (AAUB) was differently distributed in AT- and GC-rich genomes in the sense that AT-rich genomes did not prefer specific amino acids over others to the same extent as GC-rich genomes. AAUB was also associated with relative entropy; genomes with low AAUB contained more random mutations as a consequence of relaxed purifying selection than genomes with higher AAUB. Genomic base composition has a substantial effect on both amino acid- and codon frequencies in bacterial genomes. While phylogeny influenced amino acid usage more in GC-rich genomes, AT-content was driving amino acid usage in AT-rich genomes. We found the GAMM model to be an excellent tool to analyze the genomic data used in this study.

Amino Acid Usage Is Asymmetrically Biased in AT- and GC-Rich Microbial Genomes

PubMed Central

Bohlin, Jon; Brynildsrud, Ola; Vesth, Tammi; Skjerve, Eystein; Ussery, David W.

2013-01-01

Introduction Genomic base composition ranges from less than 25% AT to more than 85% AT in prokaryotes. Since only a small fraction of prokaryotic genomes is not protein coding even a minor change in genomic base composition will induce profound protein changes. We examined how amino acid and codon frequencies were distributed in over 2000 microbial genomes and how these distributions were affected by base compositional changes. In addition, we wanted to know how genome-wide amino acid usage was biased in the different genomes and how changes to base composition and mutations affected this bias. To carry this out, we used a Generalized Additive Mixed-effects Model (GAMM) to explore non-linear associations and strong data dependences in closely related microbes; principal component analysis (PCA) was used to examine genomic amino acid- and codon frequencies, while the concept of relative entropy was used to analyze genomic mutation rates. Results We found that genomic amino acid frequencies carried a stronger phylogenetic signal than codon frequencies, but that this signal was weak compared to that of genomic %AT. Further, in contrast to codon usage bias (CUB), amino acid usage bias (AAUB) was differently distributed in AT- and GC-rich genomes in the sense that AT-rich genomes did not prefer specific amino acids over others to the same extent as GC-rich genomes. AAUB was also associated with relative entropy; genomes with low AAUB contained more random mutations as a consequence of relaxed purifying selection than genomes with higher AAUB. Conclusion Genomic base composition has a substantial effect on both amino acid- and codon frequencies in bacterial genomes. While phylogeny influenced amino acid usage more in GC-rich genomes, AT-content was driving amino acid usage in AT-rich genomes. We found the GAMM model to be an excellent tool to analyze the genomic data used in this study. PMID:23922837

Chemical and UV Mutagenesis.

PubMed

Bose, Jeffrey L

2016-01-01

The ability to create mutations is an important step towards understanding bacterial physiology and virulence. While targeted approaches are invaluable, the ability to produce genome-wide random mutations can lead to crucial discoveries. Transposon mutagenesis is a useful approach, but many interesting mutations can be missed by these insertions that interrupt coding and noncoding sequences due to the integration of an entire transposon. Chemical mutagenesis and UV-based random mutagenesis are alternate approaches to isolate mutations of interest with the potential of only single nucleotide changes. Once a standard method, difficulty in identifying mutation sites had decreased the popularity of this technique. However, thanks to the recent emergence of economical whole-genome sequencing, this approach to making mutations can once again become a viable option. Therefore, this chapter provides an overview protocol for random mutagenesis using UV light or DNA-damaging chemicals.

ALK F1174V mutation confers sensitivity while ALK I1171 mutation confers resistance to alectinib. The importance of serial biopsy post progression.

PubMed

Ou, Sai-Hong; Milliken, Jeffrey C; Azada, Michele C; Miller, Vincent A; Ali, Siraj M; Klempner, Samuel J

2016-01-01

Many acquired resistant mutations to the anaplastic lymphoma kinase (ALK) gene have been identified during treatment of ALK-rearranged non-small cell lung cancer (NSCLC) patients with crizotinib, ceritinib, and alectinib. These various acquired resistant ALK mutations confer differential sensitivities to various ALK inhibitors and may provide guidance on how to sequence the use of many of the second generation ALK inhibitors. We described a patient who developed an acquired ALK F1174V resistant mutation on progression from crizotinib that responded to alectinib for 18 months but then developed an acquired ALK I1171S mutation to alectinib. Both tumor samples had essentially the same genomic profile by comprehensive genomic profiling otherwise. This is the first patient report that demonstrates ALK F1174V mutation is sensitive to alectinib and further confirms missense acquired ALK I1171 mutation is resistant to alectinib. Sequential tumor re-biopsy for comprehensive genomic profiling (CGP) is important to appreciate the selective pressure during treatment with various ALK inhibitors underpinning the evolution of the disease course of ALK+NSCLC patients while on treatment with the various ALK inhibitors. This approach will likely help inform the optimal sequencing strategy as more ALK inhibitors become available. This case report also validates the importance of developing structurally distinct ALK inhibitors for clinical use to overcome non-cross resistant ALK mutations. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

A MELAS syndrome family harboring two mutations in mitochondrial genome.

PubMed

Choi, Byung-Ok; Hwang, Jung Hee; Kim, Joonki; Cho, Eun Min; Cho, Sun Young; Hwang, Su Jin; Lee, Hyang Woon; Kim, Song Ja; Chung, Ki Wha

2008-06-30

Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is a genetically heterogeneous mitochondrial disorder with variable clinical symptoms. Here, from the sequencing of the entire mitochondrial genome, we report a Korean MELAS family harboring two homoplasmic missense mutations, which were reported 9957T>C (Phe251Leu) transition mutation in the cytochrome c oxidase subunit 3 (COX3) gene and a novel 13849A>C (Asn505His) transversion mutation in the NADH dehydrogenase subunit 5 (ND5) gene. Neither of these mutations was found in 205 normal controls. Both mutations were identified from the proband and his mother, but not his father. The patients showed cataract symptom in addition to MELAS phenotype. We believe that the 9957T>C mutation is pathogenic, however, the 13849A>C mutation is of unclear significance. It is likely that the 13849A>C mutation might function as the secondary mutation which increase the expressivity of overlapping phenotypes of MELAS and cataract. This study also demonstrates the importance of full sequencing of mtDNA for the molecular genetic understanding of mitochondrial disorders.

A MELAS syndrome family harboring two mutations in mitochondrial genome

PubMed Central

Choi, Byung-Ok; Hwang, Jung Hee; Kim, Joonki; Cho, Eun Min; Cho, Sun Young; Hwang, Su Jin; Lee, Hyang Woon; Kim, Song Ja

2008-01-01

Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is a genetically heterogeneous mitochondrial disorder with variable clinical symptoms. Here, from the sequencing of the entire mitochondrial genome, we report a Korean MELAS family harboring two homoplasmic missense mutations, which were reported 9957T > C (Phe251Leu) transition mutation in the cytochrome c oxidase subunit 3 (COX3) gene and a novel 13849A > C (Asn505His) transversion mutation in the NADH dehydrogenase subunit 5 (ND5) gene. Neither of these mutations was found in 205 normal controls. Both mutations were identified from the proband and his mother, but not his father. The patients showed cataract symptom in addition to MELAS phenotype. We believe that the 9957T > C mutation is pathogenic, however, the 13849A > C mutation is of unclear significance. It is likely that the 13849A > C mutation might function as the secondary mutation which increase the expressivity of overlapping phenotypes of MELAS and cataract. This study also demonstrates the importance of full sequencing of mtDNA for the molecular genetic understanding of mitochondrial disorders. PMID:18587274

Fitness change in relation to mutation number in spontaneous mutation accumulation lines of Chlamydomonas reinhardtii

PubMed Central

Kraemer, Susanne A.; Böndel, Katharina B.; Ness, Robert W.; Keightley, Peter D.; Colegrave, Nick

2017-01-01

Abstract Although all genetic variation ultimately stems from mutations, their properties are difficult to study directly. Here, we used multiple mutation accumulation (MA) lines derived from five genetic backgrounds of the green algae Chlamydomonas reinhardtii that have been previously subjected to whole genome sequencing to investigate the relationship between the number of spontaneous mutations and change in fitness from a nonevolved ancestor. MA lines were on average less fit than their ancestors and we detected a significantly negative correlation between the change in fitness and the total number of accumulated mutations in the genome. Likewise, the number of mutations located within coding regions significantly and negatively impacted MA line fitness. We used the fitness data to parameterize a maximum likelihood model to estimate discrete categories of mutational effects, and found that models containing one to two mutational effect categories (one neutral and one deleterious category) fitted the data best. However, the best‐fitting mutational effects models were highly dependent on the genetic background of the ancestral strain. PMID:28884790

'PACLIMS': a component LIM system for high-throughput functional genomic analysis.

PubMed

Donofrio, Nicole; Rajagopalon, Ravi; Brown, Douglas; Diener, Stephen; Windham, Donald; Nolin, Shelly; Floyd, Anna; Mitchell, Thomas; Galadima, Natalia; Tucker, Sara; Orbach, Marc J; Patel, Gayatri; Farman, Mark; Pampanwar, Vishal; Soderlund, Cari; Lee, Yong-Hwan; Dean, Ralph A

2005-04-12

Recent advances in sequencing techniques leading to cost reduction have resulted in the generation of a growing number of sequenced eukaryotic genomes. Computational tools greatly assist in defining open reading frames and assigning tentative annotations. However, gene functions cannot be asserted without biological support through, among other things, mutational analysis. In taking a genome-wide approach to functionally annotate an entire organism, in this application the approximately 11,000 predicted genes in the rice blast fungus (Magnaporthe grisea), an effective platform for tracking and storing both the biological materials created and the data produced across several participating institutions was required. The platform designed, named PACLIMS, was built to support our high throughput pipeline for generating 50,000 random insertion mutants of Magnaporthe grisea. To be a useful tool for materials and data tracking and storage, PACLIMS was designed to be simple to use, modifiable to accommodate refinement of research protocols, and cost-efficient. Data entry into PACLIMS was simplified through the use of barcodes and scanners, thus reducing the potential human error, time constraints, and labor. This platform was designed in concert with our experimental protocol so that it leads the researchers through each step of the process from mutant generation through phenotypic assays, thus ensuring that every mutant produced is handled in an identical manner and all necessary data is captured. Many sequenced eukaryotes have reached the point where computational analyses are no longer sufficient and require biological support for their predicted genes. Consequently, there is an increasing need for platforms that support high throughput genome-wide mutational analyses. While PACLIMS was designed specifically for this project, the source and ideas present in its implementation can be used as a model for other high throughput mutational endeavors.

'PACLIMS': A component LIM system for high-throughput functional genomic analysis

PubMed Central

Donofrio, Nicole; Rajagopalon, Ravi; Brown, Douglas; Diener, Stephen; Windham, Donald; Nolin, Shelly; Floyd, Anna; Mitchell, Thomas; Galadima, Natalia; Tucker, Sara; Orbach, Marc J; Patel, Gayatri; Farman, Mark; Pampanwar, Vishal; Soderlund, Cari; Lee, Yong-Hwan; Dean, Ralph A

2005-01-01

Background Recent advances in sequencing techniques leading to cost reduction have resulted in the generation of a growing number of sequenced eukaryotic genomes. Computational tools greatly assist in defining open reading frames and assigning tentative annotations. However, gene functions cannot be asserted without biological support through, among other things, mutational analysis. In taking a genome-wide approach to functionally annotate an entire organism, in this application the ~11,000 predicted genes in the rice blast fungus (Magnaporthe grisea), an effective platform for tracking and storing both the biological materials created and the data produced across several participating institutions was required. Results The platform designed, named PACLIMS, was built to support our high throughput pipeline for generating 50,000 random insertion mutants of Magnaporthe grisea. To be a useful tool for materials and data tracking and storage, PACLIMS was designed to be simple to use, modifiable to accommodate refinement of research protocols, and cost-efficient. Data entry into PACLIMS was simplified through the use of barcodes and scanners, thus reducing the potential human error, time constraints, and labor. This platform was designed in concert with our experimental protocol so that it leads the researchers through each step of the process from mutant generation through phenotypic assays, thus ensuring that every mutant produced is handled in an identical manner and all necessary data is captured. Conclusion Many sequenced eukaryotes have reached the point where computational analyses are no longer sufficient and require biological support for their predicted genes. Consequently, there is an increasing need for platforms that support high throughput genome-wide mutational analyses. While PACLIMS was designed specifically for this project, the source and ideas present in its implementation can be used as a model for other high throughput mutational endeavors. PMID:15826298

Genetic screens for mutations affecting development of Xenopus tropicalis.

PubMed

Goda, Tadahiro; Abu-Daya, Anita; Carruthers, Samantha; Clark, Matthew D; Stemple, Derek L; Zimmerman, Lyle B

2006-06-01

We present here the results of forward and reverse genetic screens for chemically-induced mutations in Xenopus tropicalis. In our forward genetic screen, we have uncovered 77 candidate phenotypes in diverse organogenesis and differentiation processes. Using a gynogenetic screen design, which minimizes time and husbandry space expenditures, we find that if a phenotype is detected in the gynogenetic F2 of a given F1 female twice, it is highly likely to be a heritable abnormality (29/29 cases). We have also demonstrated the feasibility of reverse genetic approaches for obtaining carriers of mutations in specific genes, and have directly determined an induced mutation rate by sequencing specific exons from a mutagenized population. The Xenopus system, with its well-understood embryology, fate map, and gain-of-function approaches, can now be coupled with efficient loss-of-function genetic strategies for vertebrate functional genomics and developmental genetics.

Exercise-induced mitochondrial p53 repairs mtDNA mutations in mutator mice.

PubMed

Safdar, Adeel; Khrapko, Konstantin; Flynn, James M; Saleem, Ayesha; De Lisio, Michael; Johnston, Adam P W; Kratysberg, Yevgenya; Samjoo, Imtiaz A; Kitaoka, Yu; Ogborn, Daniel I; Little, Jonathan P; Raha, Sandeep; Parise, Gianni; Akhtar, Mahmood; Hettinga, Bart P; Rowe, Glenn C; Arany, Zoltan; Prolla, Tomas A; Tarnopolsky, Mark A

2016-01-01

Human genetic disorders and transgenic mouse models have shown that mitochondrial DNA (mtDNA) mutations and telomere dysfunction instigate the aging process. Epidemiologically, exercise is associated with greater life expectancy and reduced risk of chronic diseases. While the beneficial effects of exercise are well established, the molecular mechanisms instigating these observations remain unclear. Endurance exercise reduces mtDNA mutation burden, alleviates multisystem pathology, and increases lifespan of the mutator mice, with proofreading deficient mitochondrial polymerase gamma (POLG1). We report evidence for a POLG1-independent mtDNA repair pathway mediated by exercise, a surprising notion as POLG1 is canonically considered to be the sole mtDNA repair enzyme. Here, we show that the tumor suppressor protein p53 translocates to mitochondria and facilitates mtDNA mutation repair and mitochondrial biogenesis in response to endurance exercise. Indeed, in mutator mice with muscle-specific deletion of p53, exercise failed to prevent mtDNA mutations, induce mitochondrial biogenesis, preserve mitochondrial morphology, reverse sarcopenia, or mitigate premature mortality. Our data establish a new role for p53 in exercise-mediated maintenance of the mtDNA genome and present mitochondrially targeted p53 as a novel therapeutic modality for diseases of mitochondrial etiology.

Detailed analysis of targeted gene mutations caused by the Platinum-Fungal TALENs in Aspergillus oryzae RIB40 strain and a ligD disruptant.

PubMed

Mizutani, Osamu; Arazoe, Takayuki; Toshida, Kenji; Hayashi, Risa; Ohsato, Shuichi; Sakuma, Tetsushi; Yamamoto, Takashi; Kuwata, Shigeru; Yamada, Osamu

2017-03-01

Transcription activator-like effector nucleases (TALENs), which can generate DNA double-strand breaks at specific sites in the desired genome locus, have been used in many organisms as a tool for genome editing. In Aspergilli, including Aspergillus oryzae, however, the use of TALENs has not been validated. In this study, we performed genome editing of A. oryzae wild-type strain via error of nonhomologous end-joining (NHEJ) repair by transient expression of high-efficiency Platinum-Fungal TALENs (PtFg TALENs). Targeted mutations were observed as various mutation patterns. In particular, approximately half of the PtFg TALEN-mediated deletion mutants had deletions larger than 1 kb in the TALEN-targeting region. We also conducted PtFg TALEN-based genome editing in A. oryzae ligD disruptant (ΔligD) lacking the ligD gene involved in the final step of the NHEJ repair and found that mutations were still obtained as well as wild-type. In this case, the ratio of the large deletions reduced compared to PtFg TALEN-based genome editing in the wild-type. In conclusion, we demonstrate that PtFg TALENs are sufficiently functional to cause genome editing via error of NHEJ in A. oryzae. In addition, we reveal that genome editing using TALENs in A. oryzae tends to cause large deletions at the target region, which were partly suppressed by deletion of ligD. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Detection of c. -32T>G (IVS1-13T>G) mutation of Pompe disease by real-time PCR in dried blood spot specimen.

PubMed

Bobillo Lobato, Joaquin; Sánchez Peral, Blas A; Durán Parejo, Pilar; Jiménez Jiménez, Luis M

2013-03-15

Pompe disease, or acid maltase deficiency, is a genetic muscle disorder caused by mutations in the gene encoding the acid alpha-glucosidase (GAA) enzyme, which is essential for the degradation of glycogen to glucose in lysosomes. The wide clinical variability is resulted from genetic heterogeneity, and many different mutations of the GAA gene have been reported. Some of these mutations are associated with specific phenotypes, such as the c. -32T>G (IVS1-13T>G) mutation seen in late-onset Pompe disease. We used a real-time PCR, after genomic DNA extraction isolated from DBS (dried blood spots) and PCR amplification. Our results successfully detected in controls and patients have been 100% concordant with sequencing results. This assay combines simple sample processing and rapid analysis and it allows to detect the patients with a milder form and slower progression of this disease with a high reliability. Copyright © 2013 Elsevier B.V. All rights reserved.