Sample records for human mutation rates

  1. 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.

  2. 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.

  3. 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.

  4. Variation in genome-wide mutation rates within and between human families.

    PubMed

    Conrad, Donald F; Keebler, Jonathan E M; DePristo, Mark A; Lindsay, Sarah J; Zhang, Yujun; Casals, Ferran; Idaghdour, Youssef; Hartl, Chris L; Torroja, Carlos; Garimella, Kiran V; Zilversmit, Martine; Cartwright, Reed; Rouleau, Guy A; Daly, Mark; Stone, Eric A; Hurles, Matthew E; Awadalla, Philip

    2011-06-12

    J.B.S. Haldane proposed in 1947 that the male germline may be more mutagenic than the female germline. Diverse studies have supported Haldane's contention of a higher average mutation rate in the male germline in a variety of mammals, including humans. Here we present, to our knowledge, the first direct comparative analysis of male and female germline mutation rates from the complete genome sequences of two parent-offspring trios. Through extensive validation, we identified 49 and 35 germline de novo mutations (DNMs) in two trio offspring, as well as 1,586 non-germline DNMs arising either somatically or in the cell lines from which the DNA was derived. Most strikingly, in one family, we observed that 92% of germline DNMs were from the paternal germline, whereas, in contrast, in the other family, 64% of DNMs were from the maternal germline. These observations suggest considerable variation in mutation rates within and between families.

  5. Mutation Rate Variation is a Primary Determinant of the Distribution of Allele Frequencies in Humans

    PubMed Central

    Pritchard, Jonathan K.

    2016-01-01

    The site frequency spectrum (SFS) has long been used to study demographic history and natural selection. Here, we extend this summary by examining the SFS conditional on the alleles found at the same site in other species. We refer to this extension as the “phylogenetically-conditioned SFS” or cSFS. Using recent large-sample data from the Exome Aggregation Consortium (ExAC), combined with primate genome sequences, we find that human variants that occurred independently in closely related primate lineages are at higher frequencies in humans than variants with parallel substitutions in more distant primates. We show that this effect is largely due to sites with elevated mutation rates causing significant departures from the widely-used infinite sites mutation model. Our analysis also suggests substantial variation in mutation rates even among mutations involving the same nucleotide changes. In summary, we show that variable mutation rates are key determinants of the SFS in humans. PMID:27977673

  6. Leveraging Distant Relatedness to Quantify Human Mutation and Gene-Conversion Rates

    PubMed Central

    Palamara, Pier Francesco; Francioli, Laurent C.; Wilton, Peter R.; Genovese, Giulio; Gusev, Alexander; Finucane, Hilary K.; Sankararaman, Sriram; Sunyaev, Shamil R.; de Bakker, Paul I.W.; Wakeley, John; Pe’er, Itsik; Price, Alkes L.

    2015-01-01

    The rate at which human genomes mutate is a central biological parameter that has many implications for our ability to understand demographic and evolutionary phenomena. We present a method for inferring mutation and gene-conversion rates by using the number of sequence differences observed in identical-by-descent (IBD) segments together with a reconstructed model of recent population-size history. This approach is robust to, and can quantify, the presence of substantial genotyping error, as validated in coalescent simulations. We applied the method to 498 trio-phased sequenced Dutch individuals and inferred a point mutation rate of 1.66 × 10−8 per base per generation and a rate of 1.26 × 10−9 for <20 bp indels. By quantifying how estimates varied as a function of allele frequency, we inferred the probability that a site is involved in non-crossover gene conversion as 5.99 × 10−6. We found that recombination does not have observable mutagenic effects after gene conversion is accounted for and that local gene-conversion rates reflect recombination rates. We detected a strong enrichment of recent deleterious variation among mismatching variants found within IBD regions and observed summary statistics of local sharing of IBD segments to closely match previously proposed metrics of background selection; however, we found no significant effects of selection on our mutation-rate estimates. We detected no evidence of strong variation of mutation rates in a number of genomic annotations obtained from several recent studies. Our analysis suggests that a mutation-rate estimate higher than that reported by recent pedigree-based studies should be adopted in the context of DNA-based demographic reconstruction. PMID:26581902

  7. Variation of mutational burden in healthy human tissues suggests non-random strand segregation and allows measuring somatic mutation rates.

    PubMed

    Werner, Benjamin; Sottoriva, Andrea

    2018-06-01

    The immortal strand hypothesis poses that stem cells could produce differentiated progeny while conserving the original template strand, thus avoiding accumulating somatic mutations. However, quantitating the extent of non-random DNA strand segregation in human stem cells remains difficult in vivo. Here we show that the change of the mean and variance of the mutational burden with age in healthy human tissues allows estimating strand segregation probabilities and somatic mutation rates. We analysed deep sequencing data from healthy human colon, small intestine, liver, skin and brain. We found highly effective non-random DNA strand segregation in all adult tissues (mean strand segregation probability: 0.98, standard error bounds (0.97,0.99)). In contrast, non-random strand segregation efficiency is reduced to 0.87 (0.78,0.88) in neural tissue during early development, suggesting stem cell pool expansions due to symmetric self-renewal. Healthy somatic mutation rates differed across tissue types, ranging from 3.5 × 10-9/bp/division in small intestine to 1.6 × 10-7/bp/division in skin.

  8. Distinct contributions of replication and transcription to mutation rate variation of human genomes.

    PubMed

    Cui, Peng; Ding, Feng; Lin, Qiang; Zhang, Lingfang; Li, Ang; Zhang, Zhang; Hu, Songnian; Yu, Jun

    2012-02-01

    Here, we evaluate the contribution of two major biological processes--DNA replication and transcription--to mutation rate variation in human genomes. Based on analysis of the public human tissue transcriptomics data, high-resolution replicating map of Hela cells and dbSNP data, we present significant correlations between expression breadth, replication time in local regions and SNP density. SNP density of tissue-specific (TS) genes is significantly higher than that of housekeeping (HK) genes. TS genes tend to locate in late-replicating genomic regions and genes in such regions have a higher SNP density compared to those in early-replication regions. In addition, SNP density is found to be positively correlated with expression level among HK genes. We conclude that the process of DNA replication generates stronger mutational pressure than transcription-associated biological processes do, resulting in an increase of mutation rate in TS genes while having weaker effects on HK genes. In contrast, transcription-associated processes are mainly responsible for the accumulation of mutations in highly-expressed HK genes. Copyright © 2012 Beijing Genomics Institute. Published by Elsevier Ltd. All rights reserved.

  9. Clock-like mutational processes in human somatic cells

    PubMed Central

    Alexandrov, Ludmil B.; Jones, Philip H.; Wedge, David C.; Sale, Julian E.; Campbell, Peter J.; Nik-Zainal, Serena; Stratton, Michael R.

    2016-01-01

    During the course of a lifetime somatic cells acquire mutations. Different mutational processes may contribute to the mutations accumulated in a cell, with each imprinting a mutational signature on the cell’s genome. Some processes generate mutations throughout life at a constant rate in all individuals and the number of mutations in a cell attributable to these processes will be proportional to the chronological age of the person. Using mutations from 10,250 cancer genomes across 36 cancer types, we investigated clock-like mutational processes that have been operating in normal human cells. Two mutational signatures show clock-like properties. Both exhibit different mutation rates in different tissues. However, their mutation rates are not correlated indicating that the underlying processes are subject to different biological influences. For one signature, the rate of cell division may influence its mutation rate. This study provides the first survey of clock-like mutational processes operative in human somatic cells. PMID:26551669

  10. Clock-like mutational processes in human somatic cells

    DOE PAGES

    Alexandrov, Ludmil B.; Jones, Philip H.; Wedge, David C.; ...

    2015-11-09

    During the course of a lifetime, somatic cells acquire mutations. Different mutational processes may contribute to the mutations accumulated in a cell, with each imprinting a mutational signature on the cell's genome. Some processes generate mutations throughout life at a constant rate in all individuals, and the number of mutations in a cell attributable to these processes will be proportional to the chronological age of the person. Using mutations from 10,250 cancer genomes across 36 cancer types, we investigated clock-like mutational processes that have been operating in normal human cells. Two mutational signatures show clock-like properties. Both exhibit different mutationmore » rates in different tissues. However, their mutation rates are not correlated, indicating that the underlying processes are subject to different biological influences. For one signature, the rate of cell division may influence its mutation rate. This paper provides the first survey of clock-like mutational processes operating in human somatic cells.« less

  11. Human Germline Mutation and the Erratic Evolutionary Clock

    PubMed Central

    Przeworski, Molly

    2016-01-01

    Our understanding of the chronology of human evolution relies on the “molecular clock” provided by the steady accumulation of substitutions on an evolutionary lineage. Recent analyses of human pedigrees have called this understanding into question by revealing unexpectedly low germline mutation rates, which imply that substitutions accrue more slowly than previously believed. Translating mutation rates estimated from pedigrees into substitution rates is not as straightforward as it may seem, however. We dissect the steps involved, emphasizing that dating evolutionary events requires not “a mutation rate” but a precise characterization of how mutations accumulate in development in males and females—knowledge that remains elusive. PMID:27760127

  12. Rapid evolution of the human mutation spectrum

    PubMed Central

    Harris, Kelley; Pritchard, Jonathan K

    2017-01-01

    DNA is a remarkably precise medium for copying and storing biological information. This high fidelity results from the action of hundreds of genes involved in replication, proofreading, and damage repair. Evolutionary theory suggests that in such a system, selection has limited ability to remove genetic variants that change mutation rates by small amounts or in specific sequence contexts. Consistent with this, using SNV variation as a proxy for mutational input, we report here that mutational spectra differ substantially among species, human continental groups and even some closely related populations. Close examination of one signal, an increased TCC→TTC mutation rate in Europeans, indicates a burst of mutations from about 15,000 to 2000 years ago, perhaps due to the appearance, drift, and ultimate elimination of a genetic modifier of mutation rate. Our results suggest that mutation rates can evolve markedly over short evolutionary timescales and suggest the possibility of mapping mutational modifiers. DOI: http://dx.doi.org/10.7554/eLife.24284.001 PMID:28440220

  13. 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

  14. 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

  15. High mutation rates limit evolutionary adaptation in Escherichia coli

    PubMed Central

    Wagner, Andreas

    2018-01-01

    Mutation is fundamental to evolution, because it generates the genetic variation on which selection can act. In nature, genetic changes often increase the mutation rate in systems that range from viruses and bacteria to human tumors. Such an increase promotes the accumulation of frequent deleterious or neutral alleles, but it can also increase the chances that a population acquires rare beneficial alleles. Here, we study how up to 100-fold increases in Escherichia coli’s genomic mutation rate affect adaptive evolution. To do so, we evolved multiple replicate populations of asexual E. coli strains engineered to have four different mutation rates for 3000 generations in the laboratory. We measured the ability of evolved populations to grow in their original environment and in more than 90 novel chemical environments. In addition, we subjected the populations to whole genome population sequencing. Although populations with higher mutation rates accumulated greater genetic diversity, this diversity conveyed benefits only for modestly increased mutation rates, where populations adapted faster and also thrived better than their ancestors in some novel environments. In contrast, some populations at the highest mutation rates showed reduced adaptation during evolution, and failed to thrive in all of the 90 alternative environments. In addition, they experienced a dramatic decrease in mutation rate. Our work demonstrates that the mutation rate changes the global balance between deleterious and beneficial mutational effects on fitness. In contrast to most theoretical models, our experiments suggest that this tipping point already occurs at the modest mutation rates that are found in the wild. PMID:29702649

  16. Influence of Electron–Holes on DNA Sequence-Specific Mutation Rates

    PubMed Central

    Suárez-Villagrán, Martha Y; Azevedo, Ricardo B R; Miller, John H

    2018-01-01

    Abstract Biases in mutation rate can influence molecular evolution, yielding rates of evolution that vary widely in different parts of the genome and even among neighboring nucleotides. Here, we explore one possible mechanism of influence on sequence-specific mutation rates, the electron–hole, which can localize and potentially trigger a replication mismatch. A hole is a mobile site of positive charge created during one-electron oxidation by, for example, radiation, contact with a mutagenic agent, or oxidative stress. Its quantum wavelike properties cause it to localize at various sites with probabilities that vary widely, by orders of magnitude, and depend strongly on the local sequence. We find significant correlations between hole probabilities and mutation rates within base triplets, observed in published mutation accumulation experiments on four species of bacteria. We have also computed hole probability spectra for hypervariable segment I of the human mtDNA control region, which contains several mutational hotspots, and for heptanucleotides in noncoding regions of the human genome, whose polymorphism levels have recently been reported. We observe significant correlations between hole probabilities, and context-specific mutation and substitution rates. The correlation with hole probability cannot be explained entirely by CpG methylation in the heptanucleotide data. Peaks in hole probability tend to coincide with mutational hotspots, even in mtDNA where CpG methylation is rare. Our results suggest that hole-enhanced mutational mechanisms, such as oxidation-stabilized tautomerization and base deamination, contribute to molecular evolution. PMID:29617801

  17. A compositional segmentation of the human mitochondrial genome is related to heterogeneities in the guanine mutation rate

    PubMed Central

    Samuels, David C.; Boys, Richard J.; Henderson, Daniel A.; Chinnery, Patrick F.

    2003-01-01

    We applied a hidden Markov model segmentation method to the human mitochondrial genome to identify patterns in the sequence, to compare these patterns to the gene structure of mtDNA and to see whether these patterns reveal additional characteristics important for our understanding of genome evolution, structure and function. Our analysis identified three segmentation categories based upon the sequence transition probabilities. Category 2 segments corresponded to the tRNA and rRNA genes, with a greater strand-symmetry in these segments. Category 1 and 3 segments covered the protein- coding genes and almost all of the non-coding D-loop. Compared to category 1, the mtDNA segments assigned to category 3 had much lower guanine abundance. A comparison to two independent databases of mitochondrial mutations and polymorphisms showed that the high substitution rate of guanine in human mtDNA is largest in the category 3 segments. Analysis of synonymous mutations showed the same pattern. This suggests that this heterogeneity in the mutation rate is partly independent of respiratory chain function and is a direct property of the genome sequence itself. This has important implications for our understanding of mtDNA evolution and its use as a ‘molecular clock’ to determine the rate of population and species divergence. PMID:14530452

  18. Extensive Variation in the Mutation Rate Between and Within Human Genes Associated with Mendelian Disease.

    PubMed

    Smith, Thomas; Ho, Gladys; Christodoulou, John; Price, Elizabeth Ann; Onadim, Zerrin; Gauthier-Villars, Marion; Dehainault, Catherine; Houdayer, Claude; Parfait, Beatrice; van Minkelen, Rick; Lohman, Dietmar; Eyre-Walker, Adam

    2016-05-01

    We have investigated whether the mutation rate varies between genes and sites using de novo mutations (DNMs) from three genes associated with Mendelian diseases (RB1, NF1, and MECP2). We show that the relative frequency of mutations at CpG dinucleotides relative to non-CpG sites varies between genes and relative to the genomic average. In particular we show that the rate of transition mutation at CpG sites relative to the rate of non-CpG transversion is substantially higher in our disease genes than amongst DNMs in general; the rate of CpG transition can be several hundred-fold greater than the rate of non-CpG transversion. We also show that the mutation rate varies significantly between sites of a particular mutational type, such as non-CpG transversion, within a gene. We estimate that for all categories of sites, except CpG transitions, there is at least a 30-fold difference in the mutation rate between the 10% of sites with the highest and lowest mutation rates. However, our best estimate is that the mutation rate varies by several hundred-fold variation. We suggest that the presence of hypermutable sites may be one reason certain genes are associated with disease. © 2016 WILEY PERIODICALS, INC.

  19. Mutation rate evolution in replicator dynamics.

    PubMed

    Allen, Benjamin; Rosenbloom, Daniel I Scholes

    2012-11-01

    The mutation rate of an organism is itself evolvable. In stable environments, if faithful replication is costless, theory predicts that mutation rates will evolve to zero. However, positive mutation rates can evolve in novel or fluctuating environments, as analytical and empirical studies have shown. Previous work on this question has focused on environments that fluctuate independently of the evolving population. Here we consider fluctuations that arise from frequency-dependent selection in the evolving population itself. We investigate how the dynamics of competing traits can induce selective pressure on the rates of mutation between these traits. To address this question, we introduce a theoretical framework combining replicator dynamics and adaptive dynamics. We suppose that changes in mutation rates are rare, compared to changes in the traits under direct selection, so that the expected evolutionary trajectories of mutation rates can be obtained from analysis of pairwise competition between strains of different rates. Depending on the nature of frequency-dependent trait dynamics, we demonstrate three possible outcomes of this competition. First, if trait frequencies are at a mutation-selection equilibrium, lower mutation rates can displace higher ones. Second, if trait dynamics converge to a heteroclinic cycle-arising, for example, from "rock-paper-scissors" interactions-mutator strains succeed against non-mutators. Third, in cases where selection alone maintains all traits at positive frequencies, zero and nonzero mutation rates can coexist indefinitely. Our second result suggests that relatively high mutation rates may be observed for traits subject to cyclical frequency-dependent dynamics.

  20. Direct estimate of the spontaneous germ line mutation rate in African green monkeys.

    PubMed

    Pfeifer, Susanne P

    2017-12-01

    Here, I provide the first direct estimate of the spontaneous mutation rate in an Old World monkey, using a seven individual, three-generation pedigree of African green monkeys. Eight de novo mutations were identified within ∼1.5 Gbp of accessible genome, corresponding to an estimated point mutation rate of 0.94 × 10 -8 per site per generation, suggesting an effective population size of ∼12000 for the species. This estimation represents a significant improvement in our knowledge of the population genetics of the African green monkey, one of the most important nonhuman primate models in biomedical research. Furthermore, by comparing mutation rates in Old World monkeys with the only other direct estimates in primates to date-humans and chimpanzees-it is possible to uniquely address how mutation rates have evolved over longer time scales. While the estimated spontaneous mutation rate for African green monkeys is slightly lower than the rate of 1.2 × 10 -8 per base pair per generation reported in chimpanzees, it is similar to the lower range of rates of 0.96 × 10 -8 -1.28 × 10 -8 per base pair per generation recently estimated from whole genome pedigrees in humans. This result suggests a long-term constraint on mutation rate that is quite different from similar evidence pertaining to recombination rate evolution in primates. © 2017 The Author(s). Evolution © 2017 The Society for the Study of Evolution.

  1. Determining Mutation Rates in Bacterial Populations

    PubMed Central

    Rosche, William A.; Foster, Patricia L.

    2010-01-01

    When properly determined, spontaneous mutation rates are a more accurate and biologically meaningful reflection of the underlying mutagenic mechanism than are mutation frequencies. Because bacteria grow exponentially and mutations arise stochastically, methods to estimate mutation rates depend on theoretical models that describe the distribution of mutant numbers among parallel cultures, as in the original Luria-Delbrück fluctuation analysis. An accurate determination of mutation rate depends on understanding the strengths and limitations of these methods, and how to design fluctuation assays to optimize a given method. In this paper we describe a number of methods to estimate mutation rates, give brief accounts of their derivations, and discuss how they behave under various experimental conditions. PMID:10610800

  2. 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

  3. 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.

  4. 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

  5. Methods for Determining Spontaneous Mutation Rates

    PubMed Central

    Foster, Patricia L.

    2007-01-01

    Spontaneous mutations arise as a result of cellular processes that act upon or damage DNA. Accurate determination of spontaneous mutation rates can contribute to our understanding of these processes and the enzymatic pathways that deal with them. The methods that are used to calculate mutation rates are based on the model for the expansion of mutant clones originally described by Luria and Delbrück and extended by Lea and Coulson. The accurate determination of mutation rates depends on understanding the strengths and limitations of these methods and how to optimize a fluctuation assay for a given method. This chapter describes the proper design of a fluctuation assay, several of the methods used to calculate mutation rates, and ways to evaluate the results statistically. PMID:16793403

  6. Mutation rate estimation for 15 autosomal STR loci in a large population from Mainland China.

    PubMed

    Zhao, Zhuo; Zhang, Jie; Wang, Hua; Liu, Zhi-Peng; Liu, Ming; Zhang, Yuan; Sun, Li; Zhang, Hui

    2015-09-01

    STR, short tandem repeats, are well known as a type of powerful genetic marker and widely used in studying human population genetics. Compared with the conventional genetic markers, the mutation rate of STR is higher. Additionally, the mutations of STR loci do not lead to genetic inconsistencies between the genotypes of parents and children; therefore, the analysis of STR mutation is more suited to assess the population mutation. In this study, we focused on 15 autosomal STR loci. DNA samples from a total of 42,416 unrelated healthy individuals (19,037 trios) from the population of Mainland China collected between Jan 2012 and May 2014 were successfully investigated. In our study, the allele frequencies, paternal mutation rates, maternal mutation rates and average mutation rates were detected. Furthermore, we also investigated the relationship between paternal ages, maternal ages, area, the time of pregnancy and average mutation rate. We found that the paternal mutation rate was higher than the maternal mutation rate and the paternal, maternal, and average mutation rates had a positive correlation with paternal age, maternal age and the time of pregnancy respectively. Additionally, the average mutation rate of coastal areas was higher than that of inland areas.

  7. Mutations, mutation rates, and evolution at the hypervariable VNTR loci of Yersinia pestis.

    PubMed

    Vogler, Amy J; Keys, Christine E; Allender, Christopher; Bailey, Ira; Girard, Jessica; Pearson, Talima; Smith, Kimothy L; Wagner, David M; Keim, Paul

    2007-03-01

    VNTRs are able to discriminate among closely related isolates of recently emerged clonal pathogens, including Yersinia pestis the etiologic agent of plague, because of their great diversity. Diversity is driven largely by mutation but little is known about VNTR mutation rates, factors affecting mutation rates, or the mutational mechanisms. The molecular epidemiological utility of VNTRs will be greatly enhanced when this foundational knowledge is available. Here, we measure mutation rates for 43 VNTR loci in Y. pestis using an in vitro generated population encompassing approximately 96,000 generations. We estimate the combined 43-locus rate and individual rates for 14 loci. A comparison of Y. pestis and Escherichia coli O157:H7 VNTR mutation rates and products revealed a similar relationship between diversity and mutation rate in these two species. Likewise, the relationship between repeat copy number and mutation rate is nearly identical between these species, suggesting a generalized relationship that may be applicable to other species. The single- versus multiple-repeat mutation ratios and the insertion versus deletion mutation ratios were also similar, providing support for a general model for the mutations associated with VNTRs. Finally, we use two small sets of Y. pestis isolates to show how this general model and our estimated mutation rates can be used to compare alternate phylogenies, and to evaluate the significance of genotype matches, near-matches, and mismatches found in empirical comparisons with a reference database.

  8. 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.

  9. The study of human mutation rates

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

    Neel, J.V.

    1992-01-01

    We will describe recent developments regarding the question of induced mutations in the survivors of the atomic bombings of Hiroshima and Nagasaki. As part of that work we, describe some developments with respect to the Amerindian blood samples collected under DoE sponsorship between 1964 and 1982. Then developments regarding the application of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) to the study of genetic variation and mutation affecting protein characteristics. In particular, we will report on the identification and isolation of genes of especial interest as reflected in the behavior of the proteins which they encode.

  10. 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

  11. Resolving rates of mutation in the brain using single-neuron genomics

    PubMed Central

    Evrony, Gilad D; Lee, Eunjung; Park, Peter J; Walsh, Christopher A

    2016-01-01

    Whether somatic mutations contribute functional diversity to brain cells is a long-standing question. Single-neuron genomics enables direct measurement of somatic mutation rates in human brain and promises to answer this question. A recent study (Upton et al., 2015) reported high rates of somatic LINE-1 element (L1) retrotransposition in the hippocampus and cerebral cortex that would have major implications for normal brain function, and suggested that these events preferentially impact genes important for neuronal function. We identify aspects of the single-cell sequencing approach, bioinformatic analysis, and validation methods that led to thousands of artifacts being interpreted as somatic mutation events. Our reanalysis supports a mutation frequency of approximately 0.2 events per cell, which is about fifty-fold lower than reported, confirming that L1 elements mobilize in some human neurons but indicating that L1 mosaicism is not ubiquitous. Through consideration of the challenges identified, we provide a foundation and framework for designing single-cell genomics studies. DOI: http://dx.doi.org/10.7554/eLife.12966.001 PMID:26901440

  12. Anaerobically Grown Escherichia coli Has an Enhanced Mutation Rate and Distinct Mutational Spectra

    PubMed Central

    Shewaramani, Sonal; Finn, Thomas J.; Kassen, Rees; Rainey, Paul B.

    2017-01-01

    Oxidative stress is a major cause of mutation but little is known about how growth in the absence of oxygen impacts the rate and spectrum of mutations. We employed long-term mutation accumulation experiments to directly measure the rates and spectra of spontaneous mutation events in Escherichia coli populations propagated under aerobic and anaerobic conditions. To detect mutations, whole genome sequencing was coupled with methods of analysis sufficient to identify a broad range of mutational classes, including structural variants (SVs) generated by movement of repetitive elements. The anaerobically grown populations displayed a mutation rate nearly twice that of the aerobic populations, showed distinct asymmetric mutational strand biases, and greater insertion element activity. Consistent with mutation rate and spectra observations, genes for transposition and recombination repair associated with SVs were up-regulated during anaerobic growth. Together, these results define differences in mutational spectra affecting the evolution of facultative anaerobes. PMID:28103245

  13. 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

  14. A resolution of the mutation load paradox in humans.

    PubMed

    Lesecque, Yann; Keightley, Peter D; Eyre-Walker, Adam

    2012-08-01

    Current information on the rate of mutation and the fraction of sites in the genome that are subject to selection suggests that each human has received, on average, at least two new harmful mutations from its parents. These mutations were subsequently removed by natural selection through reduced survival or fertility. It has been argued that the mutation load, the proportional reduction in population mean fitness relative to the fitness of an idealized mutation-free individual, allows a theoretical prediction of the proportion of individuals in the population that fail to reproduce as a consequence of these harmful mutations. Application of this theory to humans implies that at least 88% of individuals should fail to reproduce and that each female would need to have more than 16 offspring to maintain population size. This prediction is clearly at odds with the low reproductive excess of human populations. Here, we derive expressions for the fraction of individuals that fail to reproduce as a consequence of recurrent deleterious mutation () for a model in which selection occurs via differences in relative fitness, such as would occur through competition between individuals. We show that is much smaller than the value predicted by comparing fitness to that of a mutation-free genotype. Under the relative fitness model, we show that depends jointly on U and the selective effects of new deleterious mutations and that a species could tolerate 10's or even 100's of new deleterious mutations per genome each generation.

  15. 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

  16. 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

  17. Nucleotide selectivity defect and mutator phenotype conferred by a colon cancer-associated DNA polymerase δ mutation in human cells

    PubMed Central

    Mertz, Tony M.; Baranovskiy, Andrey G.; Wang, Jing; Tahirov, Tahir H.; Shcherbakova, Polina V.

    2017-01-01

    Mutations in the POLD1 and POLE genes encoding DNA polymerases δ (Polδ) and ε (Polε) cause hereditary colorectal cancer (CRC) and have been found in many sporadic colorectal and endometrial tumors. Much attention has been focused on POLE exonuclease domain mutations, which occur frequently in hypermutated DNA mismatch repair (MMR)-proficient tumors and appear to be responsible for the bulk of genomic instability in these tumors. In contrast, somatic POLD1 mutations are seen less frequently and typically occur in MMR-deficient tumors. Their functional significance is often unclear. Here we demonstrate that expression of the cancer-associated POLD1-R689W allele is strongly mutagenic in human cells. The mutation rate increased synergistically when the POLD1-R689W expression was combined with a MMR defect, indicating that the mutator effect of POLD1-R689W results from a high rate of replication errors. Purified human Polδ-R689W has normal exonuclease activity, but the nucleotide selectivity of the enzyme is severely impaired, providing a mechanistic explanation for the increased mutation rate in the POLD1-R689W-expressing cells. The vast majority of mutations induced by the POLD1-R689W are GC→TA transversions and GC→AT transitions, with transversions showing a strong strand bias and a remarkable preference for polypurine/polypyrimidine sequences. The mutational specificity of the Polδ variant matches that of the hypermutated CRC cell line, HCT15, in which this variant was first identified. The results provide compelling evidence for the pathogenic role of the POLD1-R689W mutation in the development of the human tumor and emphasize the need to experimentally determine the significance of Polδ variants present in sporadic tumors. PMID:28368425

  18. 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

  19. Parent-progeny sequencing indicates higher mutation rates in heterozygotes.

    PubMed

    Yang, Sihai; Wang, Long; Huang, Ju; Zhang, Xiaohui; Yuan, Yang; Chen, Jian-Qun; Hurst, Laurence D; Tian, Dacheng

    2015-07-23

    Mutation rates vary within genomes, but the causes of this remain unclear. As many prior inferences rely on methods that assume an absence of selection, potentially leading to artefactual results, we call mutation events directly using a parent-offspring sequencing strategy focusing on Arabidopsis and using rice and honey bee for replication. Here we show that mutation rates are higher in heterozygotes and in proximity to crossover events. A correlation between recombination rate and intraspecific diversity is in part owing to a higher mutation rate in domains of high recombination/diversity. Implicating diversity per se as a cause, we find an ∼3.5-fold higher mutation rate in heterozygotes than in homozygotes, with mutations occurring in closer proximity to heterozygous sites than expected by chance. In a genome that is a patchwork of heterozygous and homozygous domains, mutations occur disproportionately more often in the heterozygous domains. If segregating mutations predispose to a higher local mutation rate, clusters of genes dominantly under purifying selection (more commonly homozygous) and under balancing selection (more commonly heterozygous), might have low and high mutation rates, respectively. Our results are consistent with this, there being a ten times higher mutation rate in pathogen resistance genes, expected to be under positive or balancing selection. Consequently, we do not necessarily need to evoke extremely weak selection on the mutation rate to explain why mutational hot and cold spots might correspond to regions under positive/balancing and purifying selection, respectively.

  20. Exome-wide Sequencing Shows Low Mutation Rates and Identifies Novel Mutated Genes in Seminomas.

    PubMed

    Cutcutache, Ioana; Suzuki, Yuka; Tan, Iain Beehuat; Ramgopal, Subhashini; Zhang, Shenli; Ramnarayanan, Kalpana; Gan, Anna; Lee, Heng Hong; Tay, Su Ting; Ooi, Aikseng; Ong, Choon Kiat; Bolthouse, Jonathan T; Lane, Brian R; Anema, John G; Kahnoski, Richard J; Tan, Patrick; Teh, Bin Tean; Rozen, Steven G

    2015-07-01

    Testicular germ cell tumors are the most common cancer diagnosed in young men, and seminomas are the most common type of these cancers. There have been no exome-wide examinations of genes mutated in seminomas or of overall rates of nonsilent somatic mutations in these tumors. The objective was to analyze somatic mutations in seminomas to determine which genes are affected and to determine rates of nonsilent mutations. Eight seminomas and matched normal samples were surgically obtained from eight patients. DNA was extracted from tissue samples and exome sequenced on massively parallel Illumina DNA sequencers. Single-nucleotide polymorphism chip-based copy number analysis was also performed to assess copy number alterations. The DNA sequencing read data were analyzed to detect somatic mutations including single-nucleotide substitutions and short insertions and deletions. The detected mutations were validated by independent sequencing and further checked for subclonality. The rate of nonsynonymous somatic mutations averaged 0.31 mutations/Mb. We detected nonsilent somatic mutations in 96 genes that were not previously known to be mutated in seminomas, of which some may be driver mutations. Many of the mutations appear to have been present in subclonal populations. In addition, two genes, KIT and KRAS, were affected in two tumors each with mutations that were previously observed in other cancers and are presumably oncogenic. Our study, the first report on exome sequencing of seminomas, detected somatic mutations in 96 new genes, several of which may be targetable drivers. Furthermore, our results show that seminoma mutation rates are five times higher than previously thought, but are nevertheless low compared to other common cancers. Similar low rates are seen in other cancers that also have excellent rates of remission achieved with chemotherapy. We examined the DNA sequences of seminomas, the most common type of testicular germ cell cancer. Our study identified 96

  1. 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.

  2. Male mutation rates and the cost of sex for females

    NASA Astrophysics Data System (ADS)

    Redfield, Rosemary J.

    1994-05-01

    ALTHOUGH we do not know why sex evolved, the twofold cost of meiosis for females provides a standard against which postulated benefits of sex can be evaluated1. The most reliable benefit is sex's ability to reduce the impact of deleterious mutations2,3. But deleterious mutations may themselves generate a large and previously overlooked female-specific cost of sex. DNA sequence comparisons have confirmed Haldane's suggestion that most mutations arise in the male germ line4,5; recent estimates of α, the ratio of male to female mutation rates, are ten, six and two in humans, primates and rodents, respectively6-8. Consequently, male gametes may give progeny more mutations than the associated sexual recombination eliminates. Here I describe computer simulations showing that the cost of male mutations can easily exceed the benefits of recombination, causing females to produce fitter progeny by parthenogenesis than by mating. The persistence of sexual reproduction by females thus becomes even more problematic.

  3. 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.

  4. 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

  5. Strong effects of ionizing radiation from Chernobyl on mutation rates.

    PubMed

    Møller, Anders Pape; Mousseau, Timothy A

    2015-02-10

    In this paper we use a meta-analysis to examine the relationship between radiation and mutation rates in Chernobyl across 45 published studies, covering 30 species. Overall effect size of radiation on mutation rates estimated as Pearson's product-moment correlation coefficient was very large (E = 0.67; 95% confidence intervals (CI) 0.59 to 0.73), accounting for 44.3% of the total variance in an unstructured random-effects model. Fail-safe calculations reflecting the number of unpublished null results needed to eliminate this average effect size showed the extreme robustness of this finding (Rosenberg's method: 4135 at p = 0.05). Indirect tests did not provide any evidence of publication bias. The effect of radiation on mutations varied among taxa, with plants showing a larger effect than animals. Humans were shown to have intermediate sensitivity of mutations to radiation compared to other species. Effect size did not decrease over time, providing no evidence for an improvement in environmental conditions. The surprisingly high mean effect size suggests a strong impact of radioactive contamination on individual fitness in current and future generations, with potentially significant population-level consequences, even beyond the area contaminated with radioactive material.

  6. Strong effects of ionizing radiation from Chernobyl on mutation rates

    NASA Astrophysics Data System (ADS)

    Møller, Anders Pape; Mousseau, Timothy A.

    2015-02-01

    In this paper we use a meta-analysis to examine the relationship between radiation and mutation rates in Chernobyl across 45 published studies, covering 30 species. Overall effect size of radiation on mutation rates estimated as Pearson's product-moment correlation coefficient was very large (E = 0.67; 95% confidence intervals (CI) 0.59 to 0.73), accounting for 44.3% of the total variance in an unstructured random-effects model. Fail-safe calculations reflecting the number of unpublished null results needed to eliminate this average effect size showed the extreme robustness of this finding (Rosenberg's method: 4135 at p = 0.05). Indirect tests did not provide any evidence of publication bias. The effect of radiation on mutations varied among taxa, with plants showing a larger effect than animals. Humans were shown to have intermediate sensitivity of mutations to radiation compared to other species. Effect size did not decrease over time, providing no evidence for an improvement in environmental conditions. The surprisingly high mean effect size suggests a strong impact of radioactive contamination on individual fitness in current and future generations, with potentially significant population-level consequences, even beyond the area contaminated with radioactive material.

  7. Elevated germline mutation rate in teenage fathers

    PubMed Central

    Forster, Peter; Hohoff, Carsten; Dunkelmann, Bettina; Schürenkamp, Marianne; Pfeiffer, Heidi; Neuhuber, Franz; Brinkmann, Bernd

    2015-01-01

    Men age and die, while cells in their germline are programmed to be immortal. To elucidate how germ cells maintain viable DNA despite increasing parental age, we analysed DNA from 24 097 parents and their children, from Europe, the Middle East and Africa. We chose repetitive microsatellite DNA that mutates (unlike point mutations) only as a result of cellular replication, providing us with a natural ‘cell-cycle counter’. We observe, as expected, that the overall mutation rate for fathers is seven times higher than for mothers. Also as expected, mothers have a low and lifelong constant DNA mutation rate. Surprisingly, however, we discover that (i) teenage fathers already set out from a much higher mutation rate than teenage mothers (potentially equivalent to 77–196 male germline cell divisions by puberty); and (ii) ageing men maintain sperm DNA quality similar to that of teenagers, presumably by using fresh batches of stem cells known as ‘A-dark spermatogonia’. PMID:25694621

  8. Elevated germline mutation rate in teenage fathers.

    PubMed

    Forster, Peter; Hohoff, Carsten; Dunkelmann, Bettina; Schürenkamp, Marianne; Pfeiffer, Heidi; Neuhuber, Franz; Brinkmann, Bernd

    2015-03-22

    Men age and die, while cells in their germline are programmed to be immortal. To elucidate how germ cells maintain viable DNA despite increasing parental age, we analysed DNA from 24 097 parents and their children, from Europe, the Middle East and Africa. We chose repetitive microsatellite DNA that mutates (unlike point mutations) only as a result of cellular replication, providing us with a natural 'cell-cycle counter'. We observe, as expected, that the overall mutation rate for fathers is seven times higher than for mothers. Also as expected, mothers have a low and lifelong constant DNA mutation rate. Surprisingly, however, we discover that (i) teenage fathers already set out from a much higher mutation rate than teenage mothers (potentially equivalent to 77-196 male germline cell divisions by puberty); and (ii) ageing men maintain sperm DNA quality similar to that of teenagers, presumably by using fresh batches of stem cells known as 'A-dark spermatogonia'.

  9. The antiretrovirus drug 3'-azido-3'-deoxythymidine increases the retrovirus mutation rate.

    PubMed Central

    Julias, J G; Kim, T; Arnold, G; Pathak, V K

    1997-01-01

    It was previously observed that the nucleoside analog 5-azacytidine increased the spleen necrosis virus (SNV) mutation rate 13-fold in one cycle of retrovirus replication (V. K. Pathak and H. M. Temin, J. Virol. 66:3093-3100, 1992). Based on this observation, we hypothesized that nucleoside analogs used as antiviral drugs may also increase retrovirus mutation rates. We sought to determine if 3'-azido-3'-deoxythymidine (AZT), the primary treatment for human immunodeficiency virus type 1 (HIV-1) infection, increases the retrovirus mutation rate. Two assays were used to determine the effects of AZT on retrovirus mutation rates. The strategy of the first assay involved measuring the in vivo rate of inactivation of the lacZ gene in one replication cycle of SNV- and murine leukemia virus-based retroviral vectors. We observed 7- and 10-fold increases in the SNV mutant frequency following treatment of target cells with 0.1 and 0.5 microM AZT, respectively. The murine leukemia virus mutant frequency increased two- and threefold following treatment of target cells with 0.5 and 1.0 microM AZT, respectively. The second assay used an SNV-based shuttle vector containing the lacZ alpha gene. Proviruses were recovered as plasmids in Escherichia coli, and the rate of inactivation of lacZ alpha was measured. The results indicated that treatment of target cells increased the overall mutation rate two- to threefold. DNA sequence analysis of mutant proviruses indicated that AZT increased both the deletion and substitution rates. These results suggest that AZT treatment of HIV-1 infection may increase the degree of viral variation and alter virus evolution or pathogenesis. PMID:9151812

  10. 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.

  11. Frequency-dependent selection can lead to evolution of high mutation rates.

    PubMed

    Rosenbloom, Daniel I S; Allen, Benjamin

    2014-05-01

    Theoretical and experimental studies have shown that high mutation rates can be advantageous, especially in novel or fluctuating environments. Here we examine how frequency-dependent competition may lead to fluctuations in trait frequencies that exert upward selective pressure on mutation rates. We use a mathematical model to show that cyclical trait dynamics generated by "rock-paper-scissors" competition can cause the mutation rate in a population to converge to a high evolutionarily stable mutation rate, reflecting a trade-off between generating novelty and reproducing past success. Introducing recombination lowers the evolutionarily stable mutation rate but allows stable coexistence between mutation rates above and below the evolutionarily stable rate. Even considering strong mutational load and ignoring the costs of faithful replication, evolution favors positive mutation rates if the selective advantage of prevailing in competition exceeds the ratio of recombining to nonrecombining offspring. We discuss a number of genomic mechanisms that may meet our theoretical requirements for the adaptive evolution of mutation. Overall, our results suggest that local mutation rates may be higher on genes influencing cyclical competition and that global mutation rates in asexual species may be higher in populations subject to strong cyclical competition.

  12. Monotonicity of fitness landscapes and mutation rate control.

    PubMed

    Belavkin, Roman V; Channon, Alastair; Aston, Elizabeth; Aston, John; Krašovec, Rok; Knight, Christopher G

    2016-12-01

    A common view in evolutionary biology is that mutation rates are minimised. However, studies in combinatorial optimisation and search have shown a clear advantage of using variable mutation rates as a control parameter to optimise the performance of evolutionary algorithms. Much biological theory in this area is based on Ronald Fisher's work, who used Euclidean geometry to study the relation between mutation size and expected fitness of the offspring in infinite phenotypic spaces. Here we reconsider this theory based on the alternative geometry of discrete and finite spaces of DNA sequences. First, we consider the geometric case of fitness being isomorphic to distance from an optimum, and show how problems of optimal mutation rate control can be solved exactly or approximately depending on additional constraints of the problem. Then we consider the general case of fitness communicating only partial information about the distance. We define weak monotonicity of fitness landscapes and prove that this property holds in all landscapes that are continuous and open at the optimum. This theoretical result motivates our hypothesis that optimal mutation rate functions in such landscapes will increase when fitness decreases in some neighbourhood of an optimum, resembling the control functions derived in the geometric case. We test this hypothesis experimentally by analysing approximately optimal mutation rate control functions in 115 complete landscapes of binding scores between DNA sequences and transcription factors. Our findings support the hypothesis and find that the increase of mutation rate is more rapid in landscapes that are less monotonic (more rugged). We discuss the relevance of these findings to living organisms.

  13. Pseudouridines have context-dependent mutation and stop rates in high-throughput sequencing.

    PubMed

    Zhou, Katherine I; Clark, Wesley C; Pan, David W; Eckwahl, Matthew J; Dai, Qing; Pan, Tao

    2018-05-11

    The abundant RNA modification pseudouridine (Ψ) has been mapped transcriptome-wide by chemically modifying pseudouridines with carbodiimide and detecting the resulting reverse transcription stops in high-throughput sequencing. However, these methods have limited sensitivity and specificity, in part due to the use of reverse transcription stops. We sought to use mutations rather than just stops in sequencing data to identify pseudouridine sites. Here, we identify reverse transcription conditions that allow read-through of carbodiimide-modified pseudouridine (CMC-Ψ), and we show that pseudouridines in carbodiimide-treated human ribosomal RNA have context-dependent mutation and stop rates in high-throughput sequencing libraries prepared under these conditions. Furthermore, accounting for the context-dependence of mutation and stop rates can enhance the detection of pseudouridine sites. Similar approaches could contribute to the sequencing-based detection of many RNA modifications.

  14. Population Heterogeneity in Mutation Rate Increases the Frequency of Higher-Order Mutants and Reduces Long-Term Mutational Load

    PubMed Central

    Alexander, Helen K.; Mayer, Stephanie I.; Bonhoeffer, Sebastian

    2017-01-01

    Abstract Mutation rate is a crucial evolutionary parameter that has typically been treated as a constant in population genetic analyses. However, the propensity to mutate is likely to vary among co-existing individuals within a population, due to genetic polymorphisms, heterogeneous environmental influences, and random physiological fluctuations. We review the evidence for mutation rate heterogeneity and explore its consequences by extending classic population genetic models to allow an arbitrary distribution of mutation rate among individuals, either with or without inheritance. With this general new framework, we rigorously establish the effects of heterogeneity at various evolutionary timescales. In a single generation, variation of mutation rate about the mean increases the probability of producing zero or many simultaneous mutations on a genome. Over multiple generations of mutation and selection, heterogeneity accelerates the appearance of both deleterious and beneficial multi-point mutants. At mutation-selection balance, higher-order mutant frequencies are likewise boosted, while lower-order mutants exhibit subtler effects; nonetheless, population mean fitness is always enhanced. We quantify the dependencies on moments of the mutation rate distribution and selection coefficients, and clarify the role of mutation rate inheritance. While typical methods of estimating mutation rate will recover only the population mean, analyses assuming mutation rate is fixed to this mean could underestimate the potential for multi-locus adaptation, including medically relevant evolution in pathogenic and cancerous populations. We discuss the potential to empirically parameterize mutation rate distributions, which have to date hardly been quantified. PMID:27836985

  15. Somatic mutation detection in human biomonitoring.

    PubMed

    Olsen, L S; Nielsen, L R; Nexø, B A; Wassermann, K

    1996-06-01

    Somatic cell gene mutation arising in vivo may be considered to be a biomarker for genotoxicity. Assays detecting mutations of the haemoglobin and glycophorin A genes in red blood cells and of the hypoxanthine-guanine phosphoribosyltransferase and human leucocyte antigenes in T-lymphocytes are available in humans. This MiniReview describes these assays and their application to studies of individuals exposed to genotoxic agents. Moreover, with the implementation of techniques of molecular biology mutation spectra can now be defined in addition to the quantitation of in vivo mutant frequencies. We describe current screening methods for unknown mutations, including the denaturing gradient gel electrophoresis, single strand conformation polymorphism analysis, heteroduplex analysis, chemical modification techniques and enzymatic cleavage methods. The advantage of mutation detection as a biomarker is that it integrates exposure and sensitivity in one measurement. With the analysis of mutation spectra it may thus be possible to identify the causative genotoxic agent.

  16. Elevated mutation rate during meiosis in Saccharomyces cerevisiae.

    PubMed

    Rattray, Alison; Santoyo, Gustavo; Shafer, Brenda; Strathern, Jeffrey N

    2015-01-01

    Mutations accumulate during all stages of growth, but only germ line mutations contribute to evolution. While meiosis contributes to evolution by reassortment of parental alleles, we show here that the process itself is inherently mutagenic. We have previously shown that the DNA synthesis associated with repair of a double-strand break is about 1000-fold less accurate than S-phase synthesis. Since the process of meiosis involves many programmed DSBs, we reasoned that this repair might also be mutagenic. Indeed, in the early 1960's Magni and Von Borstel observed elevated reversion of recessive alleles during meiosis, and found that the revertants were more likely to be associated with a crossover than non-revertants, a process that they called "the meiotic effect." Here we use a forward mutation reporter (CAN1 HIS3) placed at either a meiotic recombination coldspot or hotspot near the MAT locus on Chromosome III. We find that the increased mutation rate at CAN1 (6 to 21 -fold) correlates with the underlying recombination rate at the locus. Importantly, we show that the elevated mutation rate is fully dependent upon Spo11, the protein that introduces the meiosis specific DSBs. To examine associated recombination we selected for random spores with or without a mutation in CAN1. We find that the mutations isolated this way show an increased association with recombination (crossovers, loss of crossover interference and/or increased gene conversion tracts). Polζ appears to contribute about half of the mutations induced during meiosis, but is not the only source of mutations for the meiotic effect. We see no difference in either the spectrum or distribution of mutations between mitosis and meiosis. The correlation of hotspots with elevated mutagenesis provides a mechanism for organisms to control evolution rates in a gene specific manner.

  17. Impacts of Usher syndrome type IB mutations on human myosin VIIa motor function.

    PubMed

    Watanabe, Shinya; Umeki, Nobuhisa; Ikebe, Reiko; Ikebe, Mitsuo

    2008-09-09

    Usher syndrome (USH) is a human hereditary disorder characterized by profound congenital deafness, retinitis pigmentosa, and vestibular dysfunction. Myosin VIIa has been identified as the responsible gene for USH type 1B, and a number of missense mutations have been identified in the affected families. However, the molecular basis of the dysfunction of USH gene, myosin VIIa, in the affected families is unknown to date. Here we clarified the effects of USH1B mutations on human myosin VIIa motor function for the first time. The missense mutations of USH1B significantly inhibited the actin activation of ATPase activity of myosin VIIa. G25R, R212C, A397D, and E450Q mutations abolished the actin-activated ATPase activity completely. P503L mutation increased the basal ATPase activity for 2-3-fold but reduced the actin-activated ATPase activity to 50% of the wild type. While all of the mutations examined, except for R302H, reduced the affinity for actin and the ATP hydrolysis cycling rate, they did not largely decrease the rate of ADP release from actomyosin, suggesting that the mutations reduce the duty ratio of myosin VIIa. Taken together, the results suggest that the mutations responsible for USH1B cause the complete loss of the actin-activated ATPase activity or the reduction of duty ratio of myosin VIIa.

  18. Impacts of Usher Syndrome Type IB Mutations on Human Myosin VIIa Motor Function†

    PubMed Central

    Watanabe, Shinya; Umeki, Nobuhisa; Ikebe, Reiko; Ikebe, Mitsuo

    2010-01-01

    Usher syndrome (USH) is a human hereditary disorder characterized by profound congenital deafness, retinitis pigmentosa and vestibular dysfunction. Myosin VIIa has been identified as the responsible gene for USH type 1B, and a number of missense mutations have been identified in the affected families. However, the molecular basis of the dysfunction of USH gene, myosin VIIa, in the affected families is unknown to date. Here we clarified the effects of USH1B mutations on human myosin VIIa motor function for the first time. The missense mutations of USH1B significantly inhibited the actin activation of ATPase activity of myosin VIIa. G25R, R212C, A397D and E450Q mutations abolished the actin-activated ATPase activity completely. P503L mutation increased the basal ATPase activity for 2-3 fold, but reduced the actin-activated ATPase activity to 50% of the wild type. While all the mutations examined, except for R302H, reduced the affinity for actin and the ATP hydrolysis cycling rate, they did not largely decrease the rate of ADP release from acto-myosin, suggesting that the mutations reduce the duty ratio of myosin VIIa. Taken together, the results suggest that the mutations responsible for USH1B cause the complete loss of the actin-activated ATPase activity or the reduction of duty ratio of myosin VIIa. PMID:18700726

  19. Different mutations of the human c-mpl gene indicate distinct haematopoietic diseases.

    PubMed

    He, Xin; Chen, Zhigang; Jiang, Yangyan; Qiu, Xi; Zhao, Xiaoying

    2013-01-25

    The human c-mpl gene (MPL) plays an important role in the development of megakaryocytes and platelets as well as the self-renewal of haematopoietic stem cells. However, numerous MPL mutations have been identified in haematopoietic diseases. These mutations alter the normal regulatory mechanisms and lead to autonomous activation or signalling deficiencies. In this review, we summarise 59 different MPL mutations and classify these mutations into four different groups according to the associated diseases and mutation rates. Using this classification, we clearly distinguish four diverse types of MPL mutations and obtain a deep understand of their clinical significance. This will prove to be useful for both disease diagnosis and the design of individual therapy regimens based on the type of MPL mutations.

  20. Different mutations of the human c-mpl gene indicate distinct haematopoietic diseases

    PubMed Central

    2013-01-01

    The human c-mpl gene (MPL) plays an important role in the development of megakaryocytes and platelets as well as the self-renewal of haematopoietic stem cells. However, numerous MPL mutations have been identified in haematopoietic diseases. These mutations alter the normal regulatory mechanisms and lead to autonomous activation or signalling deficiencies. In this review, we summarise 59 different MPL mutations and classify these mutations into four different groups according to the associated diseases and mutation rates. Using this classification, we clearly distinguish four diverse types of MPL mutations and obtain a deep understand of their clinical significance. This will prove to be useful for both disease diagnosis and the design of individual therapy regimens based on the type of MPL mutations. PMID:23351976

  1. Mutational dynamics of the SARS coronavirus in cell culture and human populations isolated in 2003

    PubMed Central

    Vega, Vinsensius B; Ruan, Yijun; Liu, Jianjun; Lee, Wah Heng; Wei, Chia Lin; Se-Thoe, Su Yun; Tang, Kin Fai; Zhang, Tao; Kolatkar, Prasanna R; Ooi, Eng Eong; Ling, Ai Ee; Stanton, Lawrence W; Long, Philip M; Liu, Edison T

    2004-01-01

    Background The SARS coronavirus is the etiologic agent for the epidemic of the Severe Acute Respiratory Syndrome. The recent emergence of this new pathogen, the careful tracing of its transmission patterns, and the ability to propagate in culture allows the exploration of the mutational dynamics of the SARS-CoV in human populations. Methods We sequenced complete SARS-CoV genomes taken from primary human tissues (SIN3408, SIN3725V, SIN3765V), cultured isolates (SIN848, SIN846, SIN842, SIN845, SIN847, SIN849, SIN850, SIN852, SIN3408L), and five consecutive Vero cell passages (SIN2774_P1, SIN2774_P2, SIN2774_P3, SIN2774_P4, SIN2774_P5) arising from SIN2774 isolate. These represented individual patient samples, serial in vitro passages in cell culture, and paired human and cell culture isolates. Employing a refined mutation filtering scheme and constant mutation rate model, the mutation rates were estimated and the possible date of emergence was calculated. Phylogenetic analysis was used to uncover molecular relationships between the isolates. Results Close examination of whole genome sequence of 54 SARS-CoV isolates identified before 14th October 2003, including 22 from patients in Singapore, revealed the mutations engendered during human-to-Vero and Vero-to-human transmission as well as in multiple Vero cell passages in order to refine our analysis of human-to-human transmission. Though co-infection by different quasipecies in individual tissue samples is observed, the in vitro mutation rate of the SARS-CoV in Vero cell passage is negligible. The in vivo mutation rate, however, is consistent with estimates of other RNA viruses at approximately 5.7 × 10-6 nucleotide substitutions per site per day (0.17 mutations per genome per day), or two mutations per human passage (adjusted R-square = 0.4014). Using the immediate Hotel M contact isolates as roots, we observed that the SARS epidemic has generated four major genetic groups that are geographically associated: two

  2. Rates of genomic divergence in humans, chimpanzees and their lice.

    PubMed

    Johnson, Kevin P; Allen, Julie M; Olds, Brett P; Mugisha, Lawrence; Reed, David L; Paige, Ken N; Pittendrigh, Barry R

    2014-02-22

    The rate of DNA mutation and divergence is highly variable across the tree of life. However, the reasons underlying this variation are not well understood. Comparing the rates of genetic changes between hosts and parasite lineages that diverged at the same time is one way to begin to understand differences in genetic mutation and substitution rates. Such studies have indicated that the rate of genetic divergence in parasites is often faster than that of their hosts when comparing single genes. However, the variation in this relative rate of molecular evolution across different genes in the genome is unknown. We compared the rate of DNA sequence divergence between humans, chimpanzees and their ectoparasitic lice for 1534 protein-coding genes across their genomes. The rate of DNA substitution in these orthologous genes was on average 14 times faster for lice than for humans and chimpanzees. In addition, these rates were positively correlated across genes. Because this correlation only occurred for substitutions that changed the amino acid, this pattern is probably produced by similar functional constraints across the same genes in humans, chimpanzees and their ectoparasites.

  3. An evolutionary reduction principle for mutation rates at multiple Loci.

    PubMed

    Altenberg, Lee

    2011-06-01

    A model of mutation rate evolution for multiple loci under arbitrary selection is analyzed. Results are obtained using techniques from Karlin (Evolutionary Biology, vol. 14, pp. 61-204, 1982) that overcome the weak selection constraints needed for tractability in prior studies of multilocus event models.A multivariate form of the reduction principle is found: reduction results at individual loci combine topologically to produce a surface of mutation rate alterations that are neutral for a new modifier allele. New mutation rates survive if and only if they fall below this surface-a generalization of the hyperplane found by Zhivotovsky et al. (Proc. Natl. Acad. Sci. USA 91, 1079-1083, 1994) for a multilocus recombination modifier. Increases in mutation rates at some loci may evolve if compensated for by decreases at other loci. The strength of selection on the modifier scales in proportion to the number of germline cell divisions, and increases with the number of loci affected. Loci that do not make a difference to marginal fitnesses at equilibrium are not subject to the reduction principle, and under fine tuning of mutation rates would be expected to have higher mutation rates than loci in mutation-selection balance.Other results include the nonexistence of 'viability analogous, Hardy-Weinberg' modifier polymorphisms under multiplicative mutation, and the sufficiency of average transmission rates to encapsulate the effect of modifier polymorphisms on the transmission of loci under selection. A conjecture is offered regarding situations, like recombination in the presence of mutation, that exhibit departures from the reduction principle. Constraints for tractability are: tight linkage of all loci, initial fixation at the modifier locus, and mutation distributions comprising transition probabilities of reversible Markov chains.

  4. 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

  5. Rates of genomic divergence in humans, chimpanzees and their lice

    PubMed Central

    Johnson, Kevin P.; Allen, Julie M.; Olds, Brett P.; Mugisha, Lawrence; Reed, David L.; Paige, Ken N.; Pittendrigh, Barry R.

    2014-01-01

    The rate of DNA mutation and divergence is highly variable across the tree of life. However, the reasons underlying this variation are not well understood. Comparing the rates of genetic changes between hosts and parasite lineages that diverged at the same time is one way to begin to understand differences in genetic mutation and substitution rates. Such studies have indicated that the rate of genetic divergence in parasites is often faster than that of their hosts when comparing single genes. However, the variation in this relative rate of molecular evolution across different genes in the genome is unknown. We compared the rate of DNA sequence divergence between humans, chimpanzees and their ectoparasitic lice for 1534 protein-coding genes across their genomes. The rate of DNA substitution in these orthologous genes was on average 14 times faster for lice than for humans and chimpanzees. In addition, these rates were positively correlated across genes. Because this correlation only occurred for substitutions that changed the amino acid, this pattern is probably produced by similar functional constraints across the same genes in humans, chimpanzees and their ectoparasites. PMID:24403325

  6. 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

  7. An in-silico method for identifying aggregation rate enhancer and mitigator mutations in proteins.

    PubMed

    Rawat, Puneet; Kumar, Sandeep; Michael Gromiha, M

    2018-06-24

    Newly synthesized polypeptides must pass stringent quality controls in cells to ensure appropriate folding and function. However, mutations, environmental stresses and aging can reduce efficiencies of these controls, leading to accumulation of protein aggregates, amyloid fibrils and plaques. In-vitro experiments have shown that even single amino acid substitutions can drastically enhance or mitigate protein aggregation kinetics. In this work, we have collected a dataset of 220 unique mutations in 25 proteins and classified them as enhancers or mitigators on the basis of their effect on protein aggregation rate. The data were analyzed via machine learning to identify features capable of distinguishing between aggregation rate enhancers and mitigators. Our initial Support Vector Machine (SVM) model separated such mutations with an overall accuracy of 69%. When local secondary structures at the mutation sites were considered, the accuracies further improved by 13-15%. The machine-learnt features are distinct for each secondary structure class at mutation sites. Protein stability and flexibility changes are important features for mutations in α-helices. β-strand propensity, polarity and charge become important when mutations occur in β-strands and ability to form secondary structure, helical tendency and aggregation propensity are important for mutations lying in coils. These results have been incorporated into a sequence-based algorithm (available at http://www.iitm.ac.in/bioinfo/aggrerate-disc/) capable of predicting whether a mutation will enhance or mitigate a protein's aggregation rate. This algorithm will find several applications towards understanding protein aggregation in human diseases, enable in-silico optimization of biopharmaceuticals and enzymes for improved biophysical attributes and de novo design of bio-nanomaterials. Copyright © 2018. Published by Elsevier B.V.

  8. Evolutionary rescue of a parasite population by mutation rate evolution.

    PubMed

    Greenspoon, Philip B; Mideo, Nicole

    2017-10-01

    The risk of antibiotic resistance evolution in parasites is a major problem for public health. Identifying factors which promote antibiotic resistance evolution is thus a priority in evolutionary medicine. The rate at which new mutations enter the parasite population is one important predictor; however, mutation rate is not necessarily a fixed quantity, as is often assumed, but can itself evolve. Here we explore the possible impacts of mutation rate evolution on the fate of a disease circulating in a host population, which is being treated with drugs, the use of which varies over time. Using an evolutionary rescue framework, we find that mutation rate evolution provides a dramatic increase in the probability that a parasite population survives treatment in only a limited region, while providing little or no advantage in other regions. Both epidemiological features, such as the virulence of infection, and population genetic parameters, such as recombination rate, play important roles in determining the probability of evolutionary rescue and whether mutation rate evolution enhances the probability of evolutionary rescue or not. While efforts to curtail mutation rate evolution in parasites may be worthwhile under some circumstances, our results suggest that this need not always be the case. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. 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

  10. The population genetics of human disease: The case of recessive, lethal mutations

    PubMed Central

    Gao, Ziyue; Baker, Zachary; Diesel, José Francisco; Simons, Yuval B.; Haque, Imran S.; Pickrell, Joseph; Przeworski, Molly

    2017-01-01

    Do the frequencies of disease mutations in human populations reflect a simple balance between mutation and purifying selection? What other factors shape the prevalence of disease mutations? To begin to answer these questions, we focused on one of the simplest cases: recessive mutations that alone cause lethal diseases or complete sterility. To this end, we generated a hand-curated set of 417 Mendelian mutations in 32 genes reported to cause a recessive, lethal Mendelian disease. We then considered analytic models of mutation-selection balance in infinite and finite populations of constant sizes and simulations of purifying selection in a more realistic demographic setting, and tested how well these models fit allele frequencies estimated from 33,370 individuals of European ancestry. In doing so, we distinguished between CpG transitions, which occur at a substantially elevated rate, and three other mutation types. Intriguingly, the observed frequency for CpG transitions is slightly higher than expectation but close, whereas the frequencies observed for the three other mutation types are an order of magnitude higher than expected, with a bigger deviation from expectation seen for less mutable types. This discrepancy is even larger when subtle fitness effects in heterozygotes or lethal compound heterozygotes are taken into account. In principle, higher than expected frequencies of disease mutations could be due to widespread errors in reporting causal variants, compensation by other mutations, or balancing selection. It is unclear why these factors would have a greater impact on disease mutations that occur at lower rates, however. We argue instead that the unexpectedly high frequency of disease mutations and the relationship to the mutation rate likely reflect an ascertainment bias: of all the mutations that cause recessive lethal diseases, those that by chance have reached higher frequencies are more likely to have been identified and thus to have been included in

  11. 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

  12. Holes influence the mutation spectrum of human mitochondrial DNA

    NASA Astrophysics Data System (ADS)

    Villagran, Martha; Miller, John

    Mutations drive evolution and disease, showing highly non-random patterns of variant frequency vs. nucleotide position. We use computational DNA hole spectroscopy [M.Y. Suarez-Villagran & J.H. Miller, Sci. Rep. 5, 13571 (2015)] to reveal sites of enhanced hole probability in selected regions of human mitochondrial DNA. A hole is a mobile site of positive charge created when an electron is removed, for example by radiation or contact with a mutagenic agent. The hole spectra are quantum mechanically computed using a two-stranded tight binding model of DNA. We observe significant correlation between spectra of hole probabilities and of genetic variation frequencies from the MITOMAP database. These results suggest that hole-enhanced mutation mechanisms exert a substantial, perhaps dominant, influence on mutation patterns in DNA. One example is where a trapped hole induces a hydrogen bond shift, known as tautomerization, which then triggers a base-pair mismatch during replication. Our results deepen overall understanding of sequence specific mutation rates, encompassing both hotspots and cold spots, which drive molecular evolution.

  13. 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

  14. Diverse point mutations in the human gene for polymorphic N-acetyltransferase

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

    Vatsis, K.P.; Martell, K.J.; Weber, W.W.

    1991-07-15

    Classification of humans as rapid or slow acetylators is based on hereditary differences in rates of N-acetylation of therapeutic and carcinogenic agents, but N-acetylation of certain arylamine drugs displays no genetic variation. Two highly homologous human genes for N-acetyltransferase NAT1 and NAT2, presumably code for the genetically invariant and variant NAT proteins, respectively. In the present investigation, 1.9-kilobase human genomic EcoRI fragments encoding NAT2 were generated by the polymerase chain reaction with liver and leukocyte DNA from seven subjects phenotyped as homozygous and heterozygous acetylators. Direct sequencing revealed multiple point mutations in the coding region of two distinct NAT2 variants.more » One of these was derived from leukocytes of a slow acetylator and was distinguished by a silent mutation (coden 94) and a separate G {r arrow} A transition (position 590) leading to replacement of Arg-197 by Gln; the mutated guanine was part of a CpG dinucleotide and a Taq I site. The second NAT2 variant originated from liver with low N-acetylation activity. It was characterized by three nucleotide transitions giving rise to a silent mutation (codon 161), accompanied by obliteration of the sole Kpn I site, and two amino acid substitutions. The results show conclusively that the genetically variant NAT is encoded by NAT2.« less

  15. Interpreting the Dependence of Mutation Rates on Age and Time

    PubMed Central

    Gao, Ziyue; Wyman, Minyoung J.; Sella, Guy; Przeworski, Molly

    2016-01-01

    Mutations can originate from the chance misincorporation of nucleotides during DNA replication or from DNA lesions that arise between replication cycles and are not repaired correctly. We introduce a model that relates the source of mutations to their accumulation with cell divisions, providing a framework for understanding how mutation rates depend on sex, age, and cell division rate. We show that the accrual of mutations should track cell divisions not only when mutations are replicative in origin but also when they are non-replicative and repaired efficiently. One implication is that observations from diverse fields that to date have been interpreted as pointing to a replicative origin of most mutations could instead reflect the accumulation of mutations arising from endogenous reactions or exogenous mutagens. We further find that only mutations that arise from inefficiently repaired lesions will accrue according to absolute time; thus, unless life history traits co-vary, the phylogenetic “molecular clock” should not be expected to run steadily across species. PMID:26761240

  16. Determining Y-STR mutation rates in deep-routing genealogies: Identification of haplogroup differences.

    PubMed

    Claerhout, Sofie; Vandenbosch, Michiel; Nivelle, Kelly; Gruyters, Leen; Peeters, Anke; Larmuseau, Maarten H D; Decorte, Ronny

    2018-05-01

    Knowledge of Y-chromosomal short tandem repeat (Y-STR) mutation rates is essential to determine the most recent common ancestor (MRCA) in familial searching or genealogy research. Up to now, locus-specific mutation rates have been extensively examined especially for commercially available forensic Y-STRs, while haplogroup specific mutation rates have not yet been investigated in detail. Through 450 patrilineally related namesakes distributed over 212 deep-rooting genealogies, the individual mutation rates of 42 Y-STR loci were determined, including 27 forensic Y-STR loci from the Yfiler ® Plus kit and 15 additional Y-STR loci (DYS388, DYS426, DYS442, DYS447, DYS454, DYS455, DYS459a/b, DYS549, DYS607, DYS643, DYS724a/b and YCAIIa/b). At least 726 mutations were observed over 148,596 meiosis and individual Y-STR mutation rates varied from 2.83 × 10 -4 to 1.86 × 10 -2 . The mutation rate was significantly correlated with the average allele size, the complexity of the repeat motif sequence and the age of the father. Significant differences in average Y-STR mutations rates were observed when haplogroup 'I & J' (4.03 × 10 -3 mutations/generation) was compared to 'R1b' (5.35 × 10 -3 mutations/generation) and to the overall mutation rate (5.03 × 10 -3 mutations/generation). A difference in allele size distribution was identified as the only cause for these haplogroup specific mutation rates. The haplogroup specific mutation rates were also present within the commercially available Y-STR kits (Yfiler ® , PowerPlex ® Y23 System and Yfiler ® Plus). This observation has consequences for applications where an average Y-STR mutation rate is used, e.g. tMRCA estimations in familial searching and genealogy research. Copyright © 2018 Elsevier B.V. All rights reserved.

  17. Age-Of Dependent Mutation Rate and Weak Children in the Penna Model in Biological Ageing

    NASA Astrophysics Data System (ADS)

    Berntsen, K. Nikolaj

    We investigate the effect of an age-dependent mutation rate in the Penna model of ageing and then we observe that the high mortality for human babies can be reproduced by the model if one assumes babies to be weaker than adults.

  18. Confounders of mutation-rate estimators: selection and phenotypic lag in Thermus thermophilus

    PubMed Central

    Kissling, Grace E.; Grogan, Dennis W.; Drake, John W.

    2015-01-01

    In a recent description of the rate and character of spontaneous mutation in the hyperthermophilic bacterium Thermus thermophilus, the mutation rate was observed to be substantially lower than seen in several mesophiles. Subsequently, a report appeared indicating that this bacterium maintains an average of about 4.5 genomes per cell. This number of genomes might result in a segregation lag for the expression of a recessive mutation and might therefore lead to an underestimate of the rate of mutation. Here we describe some kinds of problems that may arise when estimating mutation rates and outline ways to adjust the rates accordingly. The emphasis is mainly on differential rates of growth of mutants versus their parents and on various kinds of phenotypic lag. We then apply these methods to the T. thermophilus data and conclude that there is as yet no reliable impact on a previously described rate. PMID:23916418

  19. Sample features associated with success rates in population-based EGFR mutation testing.

    PubMed

    Shiau, Carolyn J; Babwah, Jesse P; da Cunha Santos, Gilda; Sykes, Jenna R; Boerner, Scott L; Geddie, William R; Leighl, Natasha B; Wei, Cuihong; Kamel-Reid, Suzanne; Hwang, David M; Tsao, Ming-Sound

    2014-07-01

    Epidermal growth factor receptor (EGFR) mutation testing has become critical in the treatment of patients with advanced non-small-cell lung cancer. This study involves a large cohort and epidemiologically unselected series of EGFR mutation testing for patients with nonsquamous non-small-cell lung cancer in a North American population to determine sample-related factors that influence success in clinical EGFR testing. Data from consecutive cases of Canadian province-wide testing at a centralized diagnostic laboratory for a 24-month period were reviewed. Samples were tested for exon-19 deletion and exon-21 L858R mutations using a validated polymerase chain reaction method with 1% to 5% detection sensitivity. From 2651 samples submitted, 2404 samples were tested with 2293 samples eligible for analysis (1780 histology and 513 cytology specimens). The overall test-failure rate was 5.4% with overall mutation rate of 20.6%. No significant differences in the failure rate, mutation rate, or mutation type were found between histology and cytology samples. Although tumor cellularity was significantly associated with test-success or mutation rates in histology and cytology specimens, respectively, mutations could be detected in all specimen types. Significant rates of EGFR mutation were detected in cases with thyroid transcription factor (TTF)-1-negative immunohistochemistry (6.7%) and mucinous component (9.0%). EGFR mutation testing should be attempted in any specimen, whether histologic or cytologic. Samples should not be excluded from testing based on TTF-1 status or histologic features. Pathologists should report the amount of available tumor for testing. However, suboptimal samples with a negative EGFR mutation result should be considered for repeat testing with an alternate sample.

  20. An alternative derivation of the stationary distribution of the multivariate neutral Wright-Fisher model for low mutation rates with a view to mutation rate estimation from site frequency data.

    PubMed

    Schrempf, Dominik; Hobolth, Asger

    2017-04-01

    Recently, Burden and Tang (2016) provided an analytical expression for the stationary distribution of the multivariate neutral Wright-Fisher model with low mutation rates. In this paper we present a simple, alternative derivation that illustrates the approximation. Our proof is based on the discrete multivariate boundary mutation model which has three key ingredients. First, the decoupled Moran model is used to describe genetic drift. Second, low mutation rates are assumed by limiting mutations to monomorphic states. Third, the mutation rate matrix is separated into a time-reversible part and a flux part, as suggested by Burden and Tang (2016). An application of our result to data from several great apes reveals that the assumption of stationarity may be inadequate or that other evolutionary forces like selection or biased gene conversion are acting. Furthermore we find that the model with a reversible mutation rate matrix provides a reasonably good fit to the data compared to the one with a non-reversible mutation rate matrix. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  1. The application of a linear algebra to the analysis of mutation rates.

    PubMed

    Jones, M E; Thomas, S M; Clarke, K

    1999-07-07

    Cells and bacteria growing in culture are subject to mutation, and as this mutation is the ultimate substrate for selection and evolution, the factors controlling the mutation rate are of some interest. The mutational event is not observed directly, but is inferred from the phenotype of the original mutant or of its descendants; the rate of mutation is inferred from the number of such mutant phenotypes. Such inference presumes a knowledge of the probability distribution for the size of a clone arising from a single mutation. We develop a mathematical formulation that assists in the design and analysis of experiments which investigate mutation rates and mutant clone size distribution, and we use it to analyse data for which the classical Luria-Delbrück clone-size distribution must be rejected. Copyright 1999 Academic Press.

  2. Mitochondrial Mutation Rate, Spectrum and Heteroplasmy in Caenorhabditis elegans Spontaneous Mutation Accumulation Lines of Differing Population Size.

    PubMed

    Konrad, Anke; Thompson, Owen; Waterston, Robert H; Moerman, Donald G; Keightley, Peter D; Bergthorsson, Ulfar; Katju, Vaishali

    2017-06-01

    Mitochondrial genomes of metazoans, given their elevated rates of evolution, have served as pivotal markers for phylogeographic studies and recent phylogenetic events. In order to determine the dynamics of spontaneous mitochondrial mutations in small populations in the absence and presence of selection, we evolved mutation accumulation (MA) lines of Caenorhabditis elegans in parallel over 409 consecutive generations at three varying population sizes of N = 1, 10, and 100 hermaphrodites. The N =1 populations should have a minimal influence of natural selection to provide the spontaneous mutation rate and the expected rate of neutral evolution, whereas larger population sizes should experience increasing intensity of selection. New mutations were identified by Illumina paired-end sequencing of 86 mtDNA genomes across 35 experimental lines and compared with published genomes of natural isolates. The spontaneous mitochondrial mutation rate was estimated at 1.05 × 10-7/site/generation. A strong G/C→A/T mutational bias was observed in both the MA lines and the natural isolates. This suggests that the low G + C content at synonymous sites is the product of mutation bias rather than selection as previously proposed. The mitochondrial effective population size per worm generation was estimated to be 62. Although it was previously concluded that heteroplasmy was rare in C. elegans, the vast majority of mutations in this study were heteroplasmic despite an experimental regime exceeding 400 generations. The frequencies of frameshift and nonsynonymous mutations were negatively correlated with population size, which suggests their deleterious effects on fitness and a potent role for selection in their eradication. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. 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

  4. Renal Aplasia in Humans Is Associated with RET Mutations

    PubMed Central

    Skinner, Michael A.; Safford, Shawn D.; Reeves, Justin G.; Jackson, Margaret E.; Freemerman, Alex J.

    2008-01-01

    In animal models, kidney formation is known to be controlled by the proteins RET, GDNF, and GFRA1; however, no human studies to date have shown an association between abnormal kidney development and mutation of these genes. We hypothesized that stillborn fetuses with congenital renal agenesis or severe dysplasia would possess mutations in RET, GDNF, or GFRA1. We assayed for mutations in these genes in 33 stillborn fetuses that had bilateral or unilateral renal agenesis (29 subjects) or severe congenital renal dysplasia (4 subjects). Mutations in RET were found in 7 of 19 fetuses with bilateral renal agenesis (37%) and 2 of 10 fetuses (20%) with unilateral agenesis. In two fetuses, there were two different RET mutations found, and a total of ten different sequence variations were identified. We also investigated whether these mutations affected RET activation; in each case, RET phosphorylation was either absent or constitutively activated. A GNDF mutation was identified in only one fetus with unilateral agenesis; this subject also had two RET mutations. No GFRA1 mutations were seen in any fetuses. These data suggest that in humans, mutations in RET and GDNF may contribute significantly to abnormal kidney development. PMID:18252215

  5. Mutations in Prickle Orthologs Cause Seizures in Flies, Mice, and Humans

    PubMed Central

    Tao, Hirotaka; Manak, J. Robert; Sowers, Levi; Mei, Xue; Kiyonari, Hiroshi; Abe, Takaya; Dahdaleh, Nader S.; Yang, Tian; Wu, Shu; Chen, Shan; Fox, Mark H.; Gurnett, Christina; Montine, Thomas; Bird, Thomas; Shaffer, Lisa G.; Rosenfeld, Jill A.; McConnell, Juliann; Madan-Khetarpal, Suneeta; Berry-Kravis, Elizabeth; Griesbach, Hilary; Saneto, Russell P.; Scott, Matthew P.; Antic, Dragana; Reed, Jordan; Boland, Riley; Ehaideb, Salleh N.; El-Shanti, Hatem; Mahajan, Vinit B.; Ferguson, Polly J.; Axelrod, Jeffrey D.; Lehesjoki, Anna-Elina; Fritzsch, Bernd; Slusarski, Diane C.; Wemmie, John; Ueno, Naoto; Bassuk, Alexander G.

    2011-01-01

    Epilepsy is heritable, yet few causative gene mutations have been identified, and thus far no human epilepsy gene mutations have been found to produce seizures in invertebrates. Here we show that mutations in prickle genes are associated with seizures in humans, mice, and flies. We identified human epilepsy patients with heterozygous mutations in either PRICKLE1 or PRICKLE2. In overexpression assays in zebrafish, prickle mutations resulted in aberrant prickle function. A seizure phenotype was present in the Prickle1-null mutant mouse, two Prickle1 point mutant (missense and nonsense) mice, and a Prickle2-null mutant mouse. Drosophila with prickle mutations displayed seizures that were responsive to anti-epileptic medication, and homozygous mutant embryos showed neuronal defects. These results suggest that prickle mutations have caused seizures throughout evolution. PMID:21276947

  6. Complete-proteome mapping of human influenza A adaptive mutations: implications for human transmissibility of zoonotic strains.

    PubMed

    Miotto, Olivo; Heiny, A T; Albrecht, Randy; García-Sastre, Adolfo; Tan, Tin Wee; August, J Thomas; Brusic, Vladimir

    2010-02-03

    There is widespread concern that H5N1 avian influenza A viruses will emerge as a pandemic threat, if they become capable of human-to-human (H2H) transmission. Avian strains lack this capability, which suggests that it requires important adaptive mutations. We performed a large-scale comparative analysis of proteins from avian and human strains, to produce a catalogue of mutations associated with H2H transmissibility, and to detect their presence in avian isolates. We constructed a dataset of influenza A protein sequences from 92,343 public database records. Human and avian sequence subsets were compared, using a method based on mutual information, to identify characteristic sites where human isolates present conserved mutations. The resulting catalogue comprises 68 characteristic sites in eight internal proteins. Subtype variability prevented the identification of adaptive mutations in the hemagglutinin and neuraminidase proteins. The high number of sites in the ribonucleoprotein complex suggests interdependence between mutations in multiple proteins. Characteristic sites are often clustered within known functional regions, suggesting their functional roles in cellular processes. By isolating and concatenating characteristic site residues, we defined adaptation signatures, which summarize the adaptive potential of specific isolates. Most adaptive mutations emerged within three decades after the 1918 pandemic, and have remained remarkably stable thereafter. Two lineages with stable internal protein constellations have circulated among humans without reassorting. On the contrary, H5N1 avian and swine viruses reassort frequently, causing both gains and losses of adaptive mutations. Human host adaptation appears to be complex and systemic, involving nearly all influenza proteins. Adaptation signatures suggest that the ability of H5N1 strains to infect humans is related to the presence of an unusually high number of adaptive mutations. However, these mutations appear

  7. Elevated mutation rates in the germ line of first- and second-generation offspring of irradiated male mice

    PubMed Central

    Barber, Ruth; Plumb, Mark A.; Boulton, Emma; Roux, Isabelle; Dubrova, Yuri E.

    2002-01-01

    Mutation rates at two expanded simple tandem repeat loci were studied in the germ line of first- and second-generation offspring of inbred male CBA/H, C57BL/6, and BALB/c mice exposed to either high linear energy transfer fission neutrons or low linear energy transfer x-rays. Paternal CBA/H exposure to either x-rays or fission neutrons resulted in increased mutation rates in the germ line of two subsequent generations. Comparable transgenerational effects were observed also in neutron-irradiated C57BL/6 and x-irradiated BALB/c mice. The levels of spontaneous mutation rates and radiation-induced transgenerational instability varied between strains (BALB/c>CBA/H>C57BL/6). Pre- and postmeiotic paternal exposure resulted in similar increases in mutation rate in the germ line of both generations of CBA/H mice, which together with our previous results suggests that radiation-induced expanded simple tandem repeat instability is manifested in diploid cells after fertilization. The remarkable finding that radiation-induced germ-line instability persists for at least two generations raises important issues of risk evaluation in humans. PMID:11997464

  8. MUTATIONS INDUCED BY URBAN AIR AND DRINKING WATER: DO THEY LEAVE A MUTATIONAL SIGNATURE IN HUMAN TUMORS?

    EPA Science Inventory

    Mutations Induced by Urban Air and Drinking Water: Do They Leave a Mutational Signature in Human Tumors?

    Mutation spectra of complex environmental mixtures have been determined thus far only in Salmonella. We have determined mutation spectra for the particulate organics ...

  9. 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.

  10. The study of human mutation rates. Progress report, 1989--1992

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

    Neel, J.V.

    1992-12-01

    We will describe recent developments regarding the question of induced mutations in the survivors of the atomic bombings of Hiroshima and Nagasaki. As part of that work we, describe some developments with respect to the Amerindian blood samples collected under DoE sponsorship between 1964 and 1982. Then developments regarding the application of two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) to the study of genetic variation and mutation affecting protein characteristics. In particular, we will report on the identification and isolation of genes of especial interest as reflected in the behavior of the proteins which they encode.

  11. Hybridization alters spontaneous mutation rates in a parent-of-origin-dependent fashion in Arabidopsis.

    PubMed

    Bashir, Tufail; Sailer, Christian; Gerber, Florian; Loganathan, Nitin; Bhoopalan, Hemadev; Eichenberger, Christof; Grossniklaus, Ueli; Baskar, Ramamurthy

    2014-05-01

    Over 70 years ago, increased spontaneous mutation rates were observed in Drosophila spp. hybrids, but the genetic basis of this phenomenon is not well understood. The model plant Arabidopsis (Arabidopsis thaliana) offers unique opportunities to study the types of mutations induced upon hybridization and the frequency of their occurrence. Understanding the mutational effects of hybridization is important, as many crop plants are grown as hybrids. Besides, hybridization is important for speciation and its effects on genome integrity could be critical, as chromosomal rearrangements can lead to reproductive isolation. We examined the rates of hybridization-induced point and frameshift mutations as well as homologous recombination events in intraspecific Arabidopsis hybrids using a set of transgenic mutation detector lines that carry mutated or truncated versions of a reporter gene. We found that hybridization alters the frequency of different kinds of mutations. In general, Columbia (Col)×Cape Verde Islands and Col×C24 hybrid progeny had decreased T→G and T→A transversion rates but an increased C→T transition rate. Significant changes in frameshift mutation rates were also observed in some hybrids. In Col×C24 hybrids, there is a trend for increased homologous recombination rates, except for the hybrids from one line, while in Col×Cape Verde Islands hybrids, this rate is decreased. The overall genetic distance of the parents had no influence on mutation rates in the progeny, as closely related accessions on occasion displayed higher mutation rates than accessions that are separated farther apart. However, reciprocal hybrids had significantly different mutation rates, suggesting parent-of-origin-dependent effects on the mutation frequency.

  12. cis-Regulatory Mutations Are a Genetic Cause of Human Limb Malformations

    PubMed Central

    VanderMeer, Julia E.; Ahituv, Nadav

    2011-01-01

    The underlying mutations that cause human limb malformations are often difficult to determine, particularly for limb malformations that occur as isolated traits. Evidence from a variety of studies shows that cis-regulatory mutations, specifically in enhancers, can lead to some of these isolated limb malformations. Here, we provide a review of human limb malformations that have been shown to be caused by enhancer mutations and propose that cis-regulatory mutations will continue to be identified as the cause of additional human malformations as our understanding of regulatory sequences improves. PMID:21509892

  13. Extensive de novo mutation rate variation between individuals and across the genome of Chlamydomonas reinhardtii

    PubMed Central

    Ness, Rob W.; Morgan, Andrew D.; Vasanthakrishnan, Radhakrishnan B.; Colegrave, Nick; Keightley, Peter D.

    2015-01-01

    Describing the process of spontaneous mutation is fundamental for understanding the genetic basis of disease, the threat posed by declining population size in conservation biology, and much of evolutionary biology. Directly studying spontaneous mutation has been difficult, however, because new mutations are rare. Mutation accumulation (MA) experiments overcome this by allowing mutations to build up over many generations in the near absence of natural selection. Here, we sequenced the genomes of 85 MA lines derived from six genetically diverse strains of the green alga Chlamydomonas reinhardtii. We identified 6843 new mutations, more than any other study of spontaneous mutation. We observed sevenfold variation in the mutation rate among strains and that mutator genotypes arose, increasing the mutation rate approximately eightfold in some replicates. We also found evidence for fine-scale heterogeneity in the mutation rate, with certain sequence motifs mutating at much higher rates, and clusters of multiple mutations occurring at closely linked sites. There was little evidence, however, for mutation rate heterogeneity between chromosomes or over large genomic regions of 200 kbp. We generated a predictive model of the mutability of sites based on their genomic properties, including local GC content, gene expression level, and local sequence context. Our model accurately predicted the average mutation rate and natural levels of genetic diversity of sites across the genome. Notably, trinucleotides vary 17-fold in rate between the most and least mutable sites. Our results uncover a rich heterogeneity in the process of spontaneous mutation both among individuals and across the genome. PMID:26260971

  14. Distribution of fixed beneficial mutations and the rate of adaptation in asexual populations

    PubMed Central

    Good, Benjamin H.; Rouzine, Igor M.; Balick, Daniel J.; Hallatschek, Oskar; Desai, Michael M.

    2012-01-01

    When large asexual populations adapt, competition between simultaneously segregating mutations slows the rate of adaptation and restricts the set of mutations that eventually fix. This phenomenon of interference arises from competition between mutations of different strengths as well as competition between mutations that arise on different fitness backgrounds. Previous work has explored each of these effects in isolation, but the way they combine to influence the dynamics of adaptation remains largely unknown. Here, we describe a theoretical model to treat both aspects of interference in large populations. We calculate the rate of adaptation and the distribution of fixed mutational effects accumulated by the population. We focus particular attention on the case when the effects of beneficial mutations are exponentially distributed, as well as on a more general class of exponential-like distributions. In both cases, we show that the rate of adaptation and the influence of genetic background on the fixation of new mutants is equivalent to an effective model with a single selection coefficient and rescaled mutation rate, and we explicitly calculate these effective parameters. We find that the effective selection coefficient exactly coincides with the most common fixed mutational effect. This equivalence leads to an intuitive picture of the relative importance of different types of interference effects, which can shift dramatically as a function of the population size, mutation rate, and the underlying distribution of fitness effects. PMID:22371564

  15. Mutation and Evolutionary Rates in Adélie Penguins from the Antarctic

    PubMed Central

    Millar, Craig D.; Dodd, Andrew; Anderson, Jennifer; Gibb, Gillian C.; Ritchie, Peter A.; Baroni, Carlo; Woodhams, Michael D.; Hendy, Michael D.; Lambert, David M.

    2008-01-01

    Precise estimations of molecular rates are fundamental to our understanding of the processes of evolution. In principle, mutation and evolutionary rates for neutral regions of the same species are expected to be equal. However, a number of recent studies have shown that mutation rates estimated from pedigree material are much faster than evolutionary rates measured over longer time periods. To resolve this apparent contradiction, we have examined the hypervariable region (HVR I) of the mitochondrial genome using families of Adélie penguins (Pygoscelis adeliae) from the Antarctic. We sequenced 344 bps of the HVR I from penguins comprising 508 families with 915 chicks, together with both their parents. All of the 62 germline heteroplasmies that we detected in mothers were also detected in their offspring, consistent with maternal inheritance. These data give an estimated mutation rate (μ) of 0.55 mutations/site/Myrs (HPD 95% confidence interval of 0.29–0.88 mutations/site/Myrs) after accounting for the persistence of these heteroplasmies and the sensitivity of current detection methods. In comparison, the rate of evolution (k) of the same HVR I region, determined using DNA sequences from 162 known age sub-fossil bones spanning a 37,000-year period, was 0.86 substitutions/site/Myrs (HPD 95% confidence interval of 0.53 and 1.17). Importantly, the latter rate is not statistically different from our estimate of the mutation rate. These results are in contrast to the view that molecular rates are time dependent. PMID:18833304

  16. A Cryptochrome 2 mutation yields advanced sleep phase in humans.

    PubMed

    Hirano, Arisa; Shi, Guangsen; Jones, Christopher R; Lipzen, Anna; Pennacchio, Len A; Xu, Ying; Hallows, William C; McMahon, Thomas; Yamazaki, Maya; Ptáček, Louis J; Fu, Ying-Hui

    2016-08-16

    Familial Advanced Sleep Phase (FASP) is a heritable human sleep phenotype characterized by very early sleep and wake times. We identified a missense mutation in the human Cryptochrome 2 (CRY2) gene that co-segregates with FASP in one family. The mutation leads to replacement of an alanine residue at position 260 with a threonine (A260T). In mice, the CRY2 mutation causes a shortened circadian period and reduced phase-shift to early-night light pulse associated with phase-advanced behavioral rhythms in the light-dark cycle. The A260T mutation is located in the phosphate loop of the flavin adenine dinucleotide (FAD) binding domain of CRY2. The mutation alters the conformation of CRY2, increasing its accessibility and affinity for FBXL3 (an E3 ubiquitin ligase), thus promoting its degradation. These results demonstrate that CRY2 stability controlled by FBXL3 plays a key role in the regulation of human sleep wake behavior.

  17. High mitochondrial mutation rates estimated from deep-rooting Costa Rican pedigrees

    PubMed Central

    Madrigal, Lorena; Melendez-Obando, Mauricio; Villegas-Palma, Ramon; Barrantes, Ramiro; Raventos, Henrieta; Pereira, Reynaldo; Luiselli, Donata; Pettener, Davide; Barbujani, Guido

    2012-01-01

    Estimates of mutation rates for the noncoding hypervariable Region I (HVR-I) of mitochondrial DNA (mtDNA) vary widely, depending on whether they are inferred from phylogenies (assuming that molecular evolution is clock-like) or directly from pedigrees. All pedigree-based studies so far were conducted on populations of European origin. In this paper we analyzed 19 deep-rooting pedigrees in a population of mixed origin in Costa Rica. We calculated two estimates of the HVR-I mutation rate, one considering all apparent mutations, and one disregarding changes at sites known to be mutational hot spots and eliminating genealogy branches which might be suspected to include errors, or unrecognized adoptions along the female lines. At the end of this procedure, we still observed a mutation rate equal to 1.24 × 10−6, per site per year, i.e., at least three-fold as high as estimates derived from phylogenies. Our results confirm that mutation rates observed in pedigrees are much higher than estimated assuming a neutral model of long-term HVRI evolution. We argue that, until the cause of these discrepancies will be fully understood, both lower estimates (i.e., those derived from phylogenetic comparisons) and higher, direct estimates such as those obtained in this study, should be considered when modeling evolutionary and demographic processes. PMID:22460349

  18. Spontaneous mutation rate is a plastic trait associated with population density across domains of life.

    PubMed

    Krašovec, Rok; Richards, Huw; Gifford, Danna R; Hatcher, Charlie; Faulkner, Katy J; Belavkin, Roman V; Channon, Alastair; Aston, Elizabeth; McBain, Andrew J; Knight, Christopher G

    2017-08-01

    Rates of random, spontaneous mutation can vary plastically, dependent upon the environment. Such plasticity affects evolutionary trajectories and may be adaptive. We recently identified an inverse plastic association between mutation rate and population density at 1 locus in 1 species of bacterium. It is unknown how widespread this association is, whether it varies among organisms, and what molecular mechanisms of mutagenesis or repair are required for this mutation-rate plasticity. Here, we address all 3 questions. We identify a strong negative association between mutation rate and population density across 70 years of published literature, comprising hundreds of mutation rates estimated using phenotypic markers of mutation (fluctuation tests) from all domains of life and viruses. We test this relationship experimentally, determining that there is indeed density-associated mutation-rate plasticity (DAMP) at multiple loci in both eukaryotes and bacteria, with up to 23-fold lower mutation rates at higher population densities. We find that the degree of plasticity varies, even among closely related organisms. Nonetheless, in each domain tested, DAMP requires proteins scavenging the mutagenic oxidised nucleotide 8-oxo-dGTP. This implies that phenotypic markers give a more precise view of mutation rate than previously believed: having accounted for other known factors affecting mutation rate, controlling for population density can reduce variation in mutation-rate estimates by 93%. Widespread DAMP, which we manipulate genetically in disparate organisms, also provides a novel trait to use in the fight against the evolution of antimicrobial resistance. Such a prevalent environmental association and conserved mechanism suggest that mutation has varied plastically with population density since the early origins of life.

  19. Spontaneous mutation rate is a plastic trait associated with population density across domains of life

    PubMed Central

    Gifford, Danna R.; Hatcher, Charlie; Faulkner, Katy J.; Belavkin, Roman V.; Channon, Alastair; Aston, Elizabeth; McBain, Andrew J.

    2017-01-01

    Rates of random, spontaneous mutation can vary plastically, dependent upon the environment. Such plasticity affects evolutionary trajectories and may be adaptive. We recently identified an inverse plastic association between mutation rate and population density at 1 locus in 1 species of bacterium. It is unknown how widespread this association is, whether it varies among organisms, and what molecular mechanisms of mutagenesis or repair are required for this mutation-rate plasticity. Here, we address all 3 questions. We identify a strong negative association between mutation rate and population density across 70 years of published literature, comprising hundreds of mutation rates estimated using phenotypic markers of mutation (fluctuation tests) from all domains of life and viruses. We test this relationship experimentally, determining that there is indeed density-associated mutation-rate plasticity (DAMP) at multiple loci in both eukaryotes and bacteria, with up to 23-fold lower mutation rates at higher population densities. We find that the degree of plasticity varies, even among closely related organisms. Nonetheless, in each domain tested, DAMP requires proteins scavenging the mutagenic oxidised nucleotide 8-oxo-dGTP. This implies that phenotypic markers give a more precise view of mutation rate than previously believed: having accounted for other known factors affecting mutation rate, controlling for population density can reduce variation in mutation-rate estimates by 93%. Widespread DAMP, which we manipulate genetically in disparate organisms, also provides a novel trait to use in the fight against the evolution of antimicrobial resistance. Such a prevalent environmental association and conserved mechanism suggest that mutation has varied plastically with population density since the early origins of life. PMID:28837573

  20. The allele-frequency spectrum in a decoupled Moran model with mutation, drift, and directional selection, assuming small mutation rates.

    PubMed

    Vogl, Claus; Clemente, Florian

    2012-05-01

    We analyze a decoupled Moran model with haploid population size N, a biallelic locus under mutation and drift with scaled forward and backward mutation rates θ(1)=μ(1)N and θ(0)=μ(0)N, and directional selection with scaled strength γ=sN. With small scaled mutation rates θ(0) and θ(1), which is appropriate for single nucleotide polymorphism data in highly recombining regions, we derive a simple approximate equilibrium distribution for polymorphic alleles with a constant of proportionality. We also put forth an even simpler model, where all mutations originate from monomorphic states. Using this model we derive the sojourn times, conditional on the ancestral and fixed allele, and under equilibrium the distributions of fixed and polymorphic alleles and fixation rates. Furthermore, we also derive the distribution of small samples in the diffusion limit and provide convenient recurrence relations for calculating this distribution. This enables us to give formulas analogous to the Ewens-Watterson estimator of θ for biased mutation rates and selection. We apply this theory to a polymorphism dataset of fourfold degenerate sites in Drosophila melanogaster. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. Rate of de novo mutations and the importance of father's age to disease risk.

    PubMed

    Kong, Augustine; Frigge, Michael L; Masson, Gisli; Besenbacher, Soren; Sulem, Patrick; Magnusson, Gisli; Gudjonsson, Sigurjon A; Sigurdsson, Asgeir; Jonasdottir, Aslaug; Jonasdottir, Adalbjorg; Wong, Wendy S W; Sigurdsson, Gunnar; Walters, G Bragi; Steinberg, Stacy; Helgason, Hannes; Thorleifsson, Gudmar; Gudbjartsson, Daniel F; Helgason, Agnar; Magnusson, Olafur Th; Thorsteinsdottir, Unnur; Stefansson, Kari

    2012-08-23

    Mutations generate sequence diversity and provide a substrate for selection. The rate of de novo mutations is therefore of major importance to evolution. Here we conduct a study of genome-wide mutation rates by sequencing the entire genomes of 78 Icelandic parent-offspring trios at high coverage. We show that in our samples, with an average father's age of 29.7, the average de novo mutation rate is 1.20 × 10(-8) per nucleotide per generation. Most notably, the diversity in mutation rate of single nucleotide polymorphisms is dominated by the age of the father at conception of the child. The effect is an increase of about two mutations per year. An exponential model estimates paternal mutations doubling every 16.5 years. After accounting for random Poisson variation, father's age is estimated to explain nearly all of the remaining variation in the de novo mutation counts. These observations shed light on the importance of the father's age on the risk of diseases such as schizophrenia and autism.

  2. Adaptive tuning of mutation rates allows fast response to lethal stress in Escherichia coli

    PubMed Central

    Swings, Toon; Van den Bergh, Bram; Wuyts, Sander; Oeyen, Eline; Voordeckers, Karin; Verstrepen, Kevin J; Fauvart, Maarten; Verstraeten, Natalie; Michiels, Jan

    2017-01-01

    While specific mutations allow organisms to adapt to stressful environments, most changes in an organism's DNA negatively impact fitness. The mutation rate is therefore strictly regulated and often considered a slowly-evolving parameter. In contrast, we demonstrate an unexpected flexibility in cellular mutation rates as a response to changes in selective pressure. We show that hypermutation independently evolves when different Escherichia coli cultures adapt to high ethanol stress. Furthermore, hypermutator states are transitory and repeatedly alternate with decreases in mutation rate. Specifically, population mutation rates rise when cells experience higher stress and decline again once cells are adapted. Interestingly, we identified cellular mortality as the major force driving the quick evolution of mutation rates. Together, these findings show how organisms balance robustness and evolvability and help explain the prevalence of hypermutation in various settings, ranging from emergence of antibiotic resistance in microbes to cancer relapses upon chemotherapy. DOI: http://dx.doi.org/10.7554/eLife.22939.001 PMID:28460660

  3. Death and population dynamics affect mutation rate estimates and evolvability under stress in bacteria

    PubMed Central

    Bonhoeffer, Sebastian

    2018-01-01

    The stress-induced mutagenesis hypothesis postulates that in response to stress, bacteria increase their genome-wide mutation rate, in turn increasing the chances that a descendant is able to better withstand the stress. This has implications for antibiotic treatment: exposure to subinhibitory doses of antibiotics has been reported to increase bacterial mutation rates and thus probably the rate at which resistance mutations appear and lead to treatment failure. More generally, the hypothesis posits that stress increases evolvability (the ability of a population to generate adaptive genetic diversity) and thus accelerates evolution. Measuring mutation rates under stress, however, is problematic, because existing methods assume there is no death. Yet subinhibitory stress levels may induce a substantial death rate. Death events need to be compensated by extra replication to reach a given population size, thus providing more opportunities to acquire mutations. We show that ignoring death leads to a systematic overestimation of mutation rates under stress. We developed a system based on plasmid segregation that allows us to measure death and division rates simultaneously in bacterial populations. Using this system, we found that a substantial death rate occurs at the tested subinhibitory concentrations previously reported to increase mutation rate. Taking this death rate into account lowers and sometimes removes the signal for stress-induced mutagenesis. Moreover, even when antibiotics increase mutation rate, we show that subinhibitory treatments do not increase genetic diversity and evolvability, again because of effects of the antibiotics on population dynamics. We conclude that antibiotic-induced mutagenesis is overestimated because of death and that understanding evolvability under stress requires accounting for the effects of stress on population dynamics as much as on mutation rate. Our goal here is dual: we show that population dynamics and, in particular, the

  4. Death and population dynamics affect mutation rate estimates and evolvability under stress in bacteria.

    PubMed

    Frenoy, Antoine; Bonhoeffer, Sebastian

    2018-05-01

    The stress-induced mutagenesis hypothesis postulates that in response to stress, bacteria increase their genome-wide mutation rate, in turn increasing the chances that a descendant is able to better withstand the stress. This has implications for antibiotic treatment: exposure to subinhibitory doses of antibiotics has been reported to increase bacterial mutation rates and thus probably the rate at which resistance mutations appear and lead to treatment failure. More generally, the hypothesis posits that stress increases evolvability (the ability of a population to generate adaptive genetic diversity) and thus accelerates evolution. Measuring mutation rates under stress, however, is problematic, because existing methods assume there is no death. Yet subinhibitory stress levels may induce a substantial death rate. Death events need to be compensated by extra replication to reach a given population size, thus providing more opportunities to acquire mutations. We show that ignoring death leads to a systematic overestimation of mutation rates under stress. We developed a system based on plasmid segregation that allows us to measure death and division rates simultaneously in bacterial populations. Using this system, we found that a substantial death rate occurs at the tested subinhibitory concentrations previously reported to increase mutation rate. Taking this death rate into account lowers and sometimes removes the signal for stress-induced mutagenesis. Moreover, even when antibiotics increase mutation rate, we show that subinhibitory treatments do not increase genetic diversity and evolvability, again because of effects of the antibiotics on population dynamics. We conclude that antibiotic-induced mutagenesis is overestimated because of death and that understanding evolvability under stress requires accounting for the effects of stress on population dynamics as much as on mutation rate. Our goal here is dual: we show that population dynamics and, in particular, the

  5. Does smoking alter the mutation profile of human papillomavirus-driven head and neck cancers?

    PubMed

    Mirghani, Haitham; Lacroix, Ludovic; Rossoni, Caroline; Sun, Roger; Aupérin, Anne; Casiraghi, Odile; Villepelet, Aude; Lacave, Roger; Faucher, Gladwys; Marty, Virginie; Ferté, Charles; Soria, Jean Charles; Even, Caroline

    2018-05-01

    Human papillomavirus (HPV)-driven oropharyngeal cancer (OPC) patients are characterised by a better prognosis than their HPV-negative counterparts. However, this significant survival advantage is not homogeneous and among HPV-positive patients those with a smoking history have a significantly increased risk of oncologic failure. The reason why tobacco consumption impacts negatively the prognosis is still elusive. Tobacco might induce additional genetic alterations leading to a more aggressive phenotype. The purpose of this study was to characterise the mutational profile of HPV-positive OPCs by smoking status. We hypothesise a higher frequency of mutations affecting smokers. Targeted next-generation sequencing of 39 genes that are recurrently mutated in head and neck cancers (HNCs) caused by tobacco/alcohol consumption was performed in 62 HPV-driven OPC cases including smokers and non-smokers. The study population included 37 (60%) non-smokers and 25 (40%) smokers. Twenty (32%) patients had no mutation, 14 (23%) had 1 mutation and 28 (45%) had 2 or more mutations. The most commonly mutated genes regardless of tobacco consumption were PIK3CA (19%), MLL2 (19%), TP53 (8%), FAT 1 (15%), FBXW7 (16%), NOTCH1 (10%) and FGFR3 (10%). Mutation rate was not significantly different in smokers compared with non-smokers even when analyses focused on heavy smokers (>20 pack-years vs. <20 pack-years). Similarly, there was no significant difference in mutations patterns according to tobacco consumption. In HPV-positive patients, smoking does not increase the mutation rate of genes that are recurrently mutated in traditional HNC. Additional studies are warranted to further describe the molecular landscape of HPV-driven OPC according to tobacco consumption. Copyright © 2018 Elsevier Ltd. All rights reserved.

  6. Induced mutations in mice and genetic risk assessment in humans

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

    Selby, P.B.

    1980-01-01

    In studies on mice, in contrast to studies on humans, it is possible to perform carefully controlled experiments with the exposures one desires. The necessity for having separate mammalian tests for looking at the induction of gene mutations and small deficiencies, and at the induction of chromosomal aberrations, is obvious. Mutagens can differ as to which of these types of damage they are more likely to cause. The reason for focusing attention on the mouse in a discussion of hazard from induced gene mutations and small deficiencies is the existence of techniques in this mammal for readily studying the inductionmore » of such genetic effects. Many mutations at the molecular level cause no apparent changes at the gene-product level and many mutations that cause changes at the gene-product level cause no detectable phenotypic changes in heterozygotes. Many dominant mutations that change the phenotype cause no serious handicap. For these reasons, risk estimation for important chemicals must rely heavily on studies on the induction of those germinal mutations in mammals that are easily related to human dominant disorders, such as skeletal and cataract mutations. Molecular or enzyme studies cannot provide definitive answers about risk. The specific-locus method should help greatly in assessing the genetic risks to humans from chemicals. The new sensitive-indicator method should complement it in providing a tool for attacking the question of what treatments induce gene mutations and small deficiencies and for approximating first-generation damage to the skeleton. (ERB)« less

  7. Radiation-quality dependent cellular response in mutation induction in normal human cells.

    PubMed

    Suzuki, Masao; Tsuruoka, Chizuru; Uchihori, Yukio; Kitamura, Hisashi; Liu, Cui Hua

    2009-09-01

    We studied cellular responses in normal human fibroblasts induced with low-dose (rate) or low-fluence irradiations of different radiation types, such as gamma rays, neutrons and high linear energy transfer (LET) heavy ions. The cells were pretreated with low-dose (rate) or low-fluence irradiations (approximately 1 mGy/7-8 h) of 137Cs gamma rays, 241Am-Be neutrons, helium, carbon and iron ions before irradiations with an X-ray challenging dose (1.5 Gy). Helium (LET = 2.3 keV/microm), carbon (LET = 13.3 keV/microm) and iron (LET = 200 keV/microm) ions were produced by the Heavy Ion Medical Accelerator in Chiba (HIMAC), Japan. No difference in cell-killing effect, measured by a colony forming assay, was observed among the pretreatment with different radiation types. In mutation induction, which was detected in the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus to measure 6-thioguanine resistant clones, there was no difference in mutation frequency induced by the X-ray challenging dose between unpretreated and gamma-ray pretreated cells. In the case of the pretreatment of heavy ions, X-ray-induced mutation was around 1.8 times higher in helium-ion pretreated and 4.0 times higher in carbon-ion pretreated cells than in unpretreated cells (X-ray challenging dose alone). However, the mutation frequency in cells pretreated with iron ions was the same level as either unpretreated or gamma-ray pretreated cells. In contrast, it was reduced at 0.15 times in cells pretreated with neutrons when compared to unpretreated cells. The results show that cellular responses caused by the influence of hprt mutation induced in cells pretreated with low-dose-rate or low-fluence irradiations of different radiation types were radiation-quality dependent manner.

  8. 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.

  9. Rates of Spontaneous Mutation in Bacteriophage T4 Are Independent of Host Fidelity Determinants

    PubMed Central

    Santos, M. E.; Drake, J. W.

    1994-01-01

    Bacteriophage T4 encodes most of the genes whose products are required for its DNA metabolism, and host (Escherichia coli) genes can only infrequently complement mutationally inactivated T4 genes. We screened the following host mutator mutations for effects on spontaneous mutation rates in T4: mutT (destruction of aberrant dGTPs), polA, polB and polC (DNA polymerases), dnaQ (exonucleolytic proofreading), mutH, mutS, mutL and uvrD (methyl-directed DNA mismatch repair), mutM and mutY (excision repair of oxygen-damaged DNA), mutA (function unknown), and topB and osmZ (affecting DNA topology). None increased T4 spontaneous mutation rates within a resolving power of about twofold (nor did optA, which is not a mutator but overexpresses a host dGTPase). Previous screens in T4 have revealed strong mutator mutations only in the gene encoding the viral DNA polymerase and proofreading 3'-exonuclease, plus weak mutators in several polymerase accessory proteins or determinants of dNTP pool sizes. T4 maintains a spontaneous mutation rate per base pair about 30-fold greater than that of its host. Thus, the joint high fidelity of insertion by T4 DNA polymerase and proofreading by its associated 3'-exonuclease appear to determine the T4 spontaneous mutation rate, whereas the host requires numerous additional systems to achieve high replication fidelity. PMID:7851754

  10. 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

  11. Rates of spontaneous mutation in an archaeon from geothermal environments.

    PubMed Central

    Jacobs, K L; Grogan, D W

    1997-01-01

    To estimate the efficacy of mechanisms which may prevent or repair thermal damage to DNA in thermophilic archaea, a quantitative assay of forward mutation at extremely high temperature was developed for Sulfolobus acidocaldarius, based on the selection of pyrimidine-requiring mutants resistant to 5-fluoro-orotic acid. Maximum-likelihood analysis of spontaneous mutant distributions in wild-type cultures yielded maximal estimates of (2.8 +/- 0.7) x 10(-7) and (1.5 +/- 0.6) x 10(-7) mutational events per cell per division cycle for the pyrE and pyrF loci, respectively. To our knowledge, these results provide the first accurate measurement of the genetic fidelity maintained by archaea that populate geothermal environments. The measured rates of forward mutation at the pyrE and pyrF loci in S. acidocaldarius are close to corresponding rates reported for protein-encoding genes of Escherichia coli. The normal rate of spontaneous mutation in E. coli at 37 degrees C is known to require the functioning of several enzyme systems that repair spontaneous damage in DNA. Our results provide indirect evidence that S. acidocaldarius has cellular mechanisms, as yet unidentified, which effectively compensate for the higher chemical instability of DNA at the temperatures and pHs that prevail within growing Sulfolobus cells. PMID:9150227

  12. Mutation analysis of the Smad3 gene in human osteoarthritis.

    PubMed

    Yao, Jun-Yan; Wang, Yan; An, Jing; Mao, Chun-Ming; Hou, Ning; Lv, Ya-Xin; Wang, You-Liang; Cui, Fang; Huang, Min; Yang, Xiao

    2003-09-01

    Osteoarthritis (OA) is the most common joint disease worldwide. Recent studies have shown that targeted disruption of Smad3 in mouse results in OA. To reveal the possible association between the Smad3 gene mutation and human OA, we employed polymerase chain reaction-single strand conformation polymorphism and sequencing to screen mutations in all nine exons of the Smad3 gene in 32 patients with knee OA and 50 patients with only bone fracture. A missense mutation of the Smad3 gene was found in one patient. The single base mutation located in the linker region of the SMAD3 protein was A --> T change in the position 2 of codon 197 and resulted in an asparagine to isoleucine amino-acid substitution. The expressions of matrix metalloproteinase 2 (MMP-2) and MMP-9 in sera of the patient carrying the mutation were higher than other OA patients and controls. This is the first report showing that the Smad3 gene mutations could be associated with the pathogenesis of human OA.

  13. 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

  14. Mutation rates at 42 Y chromosomal short tandem repeats in Chinese Han population in Eastern China.

    PubMed

    Wu, Weiwei; Ren, Wenyan; Hao, Honglei; Nan, Hailun; He, Xin; Liu, Qiuling; Lu, Dejian

    2018-01-31

    Mutation analysis of 42 Y chromosomal short tandem repeats (Y-STRs) loci was performed using a sample of 1160 father-son pairs from the Chinese Han population in Eastern China. The results showed that the average mutation rate across the 42 Y-STR loci was 0.0041 (95% CI 0.0036-0.0047) per locus per generation. The locus-specific mutation rates varied from 0.000 to 0.0190. No mutation was found at DYS388, DYS437, DYS448, DYS531, and GATA_H4. DYS627, DYS570, DYS576, and DYS449 could be classified as rapidly mutating Y-STRs, with mutation rates higher than 1.0 × 10 -2 . DYS458, DYS630, and DYS518 were moderately mutating Y-STRs, with mutation rates ranging from 8 × 10 -3 to 1 × 10 -2 . Although the characteristics of the Y-STR mutations were consistent with those in previous studies, mutation rate differences between our data and previous published data were found at some rapidly mutating Y-STRs. The single-copy loci located on the short arm of the Y chromosome (Yp) showed relatively higher mutation rates more frequently than the multi-copy loci. These results will not only extend the data for Y-STR mutations but also be important for kinship analysis, paternal lineage identification, and family relationship reconstruction in forensic Y-STR analysis.

  15. Retroviral mutation rates and A-to-G hypermutations during different stages of retroviral replication.

    PubMed Central

    Kim, T; Mudry, R A; Rexrode, C A; Pathak, V K

    1996-01-01

    Retroviruses mutate at a high rate in vivo during viral replication. Mutations may occur during proviral transcription by RNA polymerase II, during minus-strand DNA synthesis (RNA template) by viral reverse transcriptase, or during plus-strand DNA synthesis (DNA template) by reverse transcriptase. To determine the contributions of different stages of replication to the retroviral mutation rates, we developed a spleen necrosis virus-based in vivo system to selectively identify mutations occurring during the early stage (RNA transcription plus minus-strand synthesis) and the late stage (plus-strand synthesis plus DNA repair). A lacZalpha reporter gene was inserted into the long terminal repeat (LTR) of a spleen necrosis virus shuttle vector, and proviruses were recovered from infected cells as plasmids containing either one or both LTRs. Plasmids containing both LTRs generated a mutant phenotype only if the lacZalpha genes in both LTRs were mutated, which is most likely to occur during the early stage. Mutant phenotypes were identified from plasmids containing one LTR regardless of the stage at which the mutations occurred. Thus, mutant frequencies obtained after recovery of plasmids containing both LTRs or one LTR provided early-stage and total mutation rates, respectively. Analysis of 56,409 proviruses suggested that the retroviral mutation rates during the early and late stages of replication were equal or within twofold of each other. In addition, two mutants with A-to-G hypermutations were discovered, suggesting a role for mammalian double-stranded RNA adenosine deaminase enzyme in retroviral mutations. These experiments provide a system to selectively identify mutations in the early stage of retroviral replication and to provide upper and lower limits to the in vivo mutation rates during minus-strand and plus-strand synthesis, respectively. PMID:8892879

  16. Can robots patch-clamp as well as humans? Characterization of a novel sodium channel mutation

    PubMed Central

    Estacion, M; Choi, J S; Eastman, E M; Lin, Z; Li, Y; Tyrrell, L; Yang, Y; Dib-Hajj, S D; Waxman, S G

    2010-01-01

    Ion channel missense mutations cause disorders of excitability by changing channel biophysical properties. As an increasing number of new naturally occurring mutations have been identified, and the number of other mutations produced by molecular approaches such as in situ mutagenesis has increased, the need for functional analysis by patch-clamp has become rate limiting. Here we compare a patch-clamp robot using planar-chip technology with human patch-clamp in a functional assessment of a previously undescribed Nav1.7 sodium channel mutation, S211P, which causes erythromelalgia. This robotic patch-clamp device can increase throughput (the number of cells analysed per day) by 3- to 10-fold. Both modes of analysis show that the mutation hyperpolarizes activation voltage dependence (−8 mV by manual profiling, −11 mV by robotic profiling), alters steady-state fast inactivation so that it requires an additional Boltzmann function for a second fraction of total current (∼20% manual, ∼40% robotic), and enhances slow inactivation (hyperpolarizing shift −15 mV by human, −13 mV robotic). Manual patch-clamping demonstrated slower deactivation and enhanced (∼2-fold) ramp response for the mutant channel while robotic recording did not, possibly due to increased temperature and reduced signal-to-noise ratio on the robotic platform. If robotic profiling is used to screen ion channel mutations, we recommend that each measurement or protocol be validated by initial comparison to manual recording. With this caveat, we suggest that, if results are interpreted cautiously, robotic patch-clamp can be used with supervision and subsequent confirmation from human physiologists to facilitate the initial profiling of a variety of electrophysiological parameters of ion channel mutations. PMID:20123784

  17. Critical Mutation Rate Has an Exponential Dependence on Population Size in Haploid and Diploid Populations

    PubMed Central

    Aston, Elizabeth; Channon, Alastair; Day, Charles; Knight, Christopher G.

    2013-01-01

    Understanding the effect of population size on the key parameters of evolution is particularly important for populations nearing extinction. There are evolutionary pressures to evolve sequences that are both fit and robust. At high mutation rates, individuals with greater mutational robustness can outcompete those with higher fitness. This is survival-of-the-flattest, and has been observed in digital organisms, theoretically, in simulated RNA evolution, and in RNA viruses. We introduce an algorithmic method capable of determining the relationship between population size, the critical mutation rate at which individuals with greater robustness to mutation are favoured over individuals with greater fitness, and the error threshold. Verification for this method is provided against analytical models for the error threshold. We show that the critical mutation rate for increasing haploid population sizes can be approximated by an exponential function, with much lower mutation rates tolerated by small populations. This is in contrast to previous studies which identified that critical mutation rate was independent of population size. The algorithm is extended to diploid populations in a system modelled on the biological process of meiosis. The results confirm that the relationship remains exponential, but show that both the critical mutation rate and error threshold are lower for diploids, rather than higher as might have been expected. Analyzing the transition from critical mutation rate to error threshold provides an improved definition of critical mutation rate. Natural populations with their numbers in decline can be expected to lose genetic material in line with the exponential model, accelerating and potentially irreversibly advancing their decline, and this could potentially affect extinction, recovery and population management strategy. The effect of population size is particularly strong in small populations with 100 individuals or less; the exponential model has

  18. The Rate of Beneficial Mutations Surfing on the Wave of a Range Expansion

    PubMed Central

    Lehe, Rémi; Hallatschek, Oskar; Peliti, Luca

    2012-01-01

    Many theoretical and experimental studies suggest that range expansions can have severe consequences for the gene pool of the expanding population. Due to strongly enhanced genetic drift at the advancing frontier, neutral and weakly deleterious mutations can reach large frequencies in the newly colonized regions, as if they were surfing the front of the range expansion. These findings raise the question of how frequently beneficial mutations successfully surf at shifting range margins, thereby promoting adaptation towards a range-expansion phenotype. Here, we use individual-based simulations to study the surfing statistics of recurrent beneficial mutations on wave-like range expansions in linear habitats. We show that the rate of surfing depends on two strongly antagonistic factors, the probability of surfing given the spatial location of a novel mutation and the rate of occurrence of mutations at that location. The surfing probability strongly increases towards the tip of the wave. Novel mutations are unlikely to surf unless they enjoy a spatial head start compared to the bulk of the population. The needed head start is shown to be proportional to the inverse fitness of the mutant type, and only weakly dependent on the carrying capacity. The precise location dependence of surfing probabilities is derived from the non-extinction probability of a branching process within a moving field of growth rates. The second factor is the mutation occurrence which strongly decreases towards the tip of the wave. Thus, most successful mutations arise at an intermediate position in the front of the wave. We present an analytic theory for the tradeoff between these factors that allows to predict how frequently substitutions by beneficial mutations occur at invasion fronts. We find that small amounts of genetic drift increase the fixation rate of beneficial mutations at the advancing front, and thus could be important for adaptation during species invasions. PMID:22479175

  19. Computational analysis of the human sinus node action potential: model development and effects of mutations

    PubMed Central

    Fabbri, Alan; Fantini, Matteo; Wilders, Ronald

    2017-01-01

    Key points We constructed a comprehensive mathematical model of the spontaneous electrical activity of a human sinoatrial node (SAN) pacemaker cell, starting from the recent Severi–DiFrancesco model of rabbit SAN cells.Our model is based on electrophysiological data from isolated human SAN pacemaker cells and closely matches the action potentials and calcium transient that were recorded experimentally.Simulated ion channelopathies explain the clinically observed changes in heart rate in corresponding mutation carriers, providing an independent qualitative validation of the model.The model shows that the modulatory role of the ‘funny current’ (I f) in the pacing rate of human SAN pacemaker cells is highly similar to that of rabbit SAN cells, despite its considerably lower amplitude.The model may prove useful in the design of experiments and the development of heart‐rate modulating drugs. Abstract The sinoatrial node (SAN) is the normal pacemaker of the mammalian heart.  Over several decades, a large amount of data on the ionic mechanisms underlying the spontaneous electrical activity of SAN pacemaker cells has been obtained, mostly in experiments on single cells isolated from rabbit SAN. This wealth of data has allowed the development of mathematical models of the electrical activity of rabbit SAN pacemaker cells. The present study aimed to construct a comprehensive model of the electrical activity of a human SAN pacemaker cell using recently obtained electrophysiological data from human SAN pacemaker cells.  We based our model on the recent Severi–DiFrancesco model of a rabbit SAN pacemaker cell. The action potential and calcium transient of the resulting model are close to the experimentally recorded values. The model has a much smaller ‘funny current’ (I f) than do rabbit cells, although its modulatory role is highly similar. Changes in pacing rate upon the implementation of mutations associated with sinus node dysfunction agree with the clinical

  20. Law-medicine interfacing: patenting of human genes and mutations.

    PubMed

    Fialho, Arsenio M; Chakrabarty, Ananda M

    2011-08-01

    Mutations, Single Nucleotide Polymorphisms (SNPs), deletions and genetic rearrangements in specific genes in the human genome account for not only our physical characteristics and behavior, but can lead to many in-born and acquired diseases. Such changes in the genome can also predispose people to cancers, as well as significantly affect the metabolism and efficacy of many drugs, resulting in some cases in acute toxicity to the drug. The testing of the presence of such genetic mutations and rearrangements is of great practical and commercial value, leading many of these genes and their mutations/deletions and genetic rearrangements to be patented. A recent decision by a judge in the Federal District Court in the Southern District of New York, has created major uncertainties, based on the revocation of BRCA1 and BRCA2 gene patents, in the eligibility of all human and presumably other gene patents. This article argues that while patents on BRCA1 and BRCA2 genes could be challenged based on a lack of utility, the patenting of the mutations and genetic rearrangements is of great importance to further development and commercialization of genetic tests that can save human lives and prevent suffering, and should be allowed.

  1. The effects of a deleterious mutation load on patterns of influenza A/H3N2's antigenic evolution in humans

    PubMed Central

    Koelle, Katia; Rasmussen, David A

    2015-01-01

    Recent phylogenetic analyses indicate that RNA virus populations carry a significant deleterious mutation load. This mutation load has the potential to shape patterns of adaptive evolution via genetic linkage to beneficial mutations. Here, we examine the effect of deleterious mutations on patterns of influenza A subtype H3N2's antigenic evolution in humans. By first analyzing simple models of influenza that incorporate a mutation load, we show that deleterious mutations, as expected, act to slow the virus's rate of antigenic evolution, while making it more punctuated in nature. These models further predict three distinct molecular pathways by which antigenic cluster transitions occur, and we find phylogenetic patterns consistent with each of these pathways in influenza virus sequences. Simulations of a more complex phylodynamic model further indicate that antigenic mutations act in concert with deleterious mutations to reproduce influenza's spindly hemagglutinin phylogeny, co-circulation of antigenic variants, and high annual attack rates. DOI: http://dx.doi.org/10.7554/eLife.07361.001 PMID:26371556

  2. Mutation-profile-based methods for understanding selection forces in cancer somatic mutations: a comparative analysis.

    PubMed

    Zhou, Zhan; Zou, Yangyun; Liu, Gangbiao; Zhou, Jingqi; Wu, Jingcheng; Zhao, Shimin; Su, Zhixi; Gu, Xun

    2017-08-29

    Human genes exhibit different effects on fitness in cancer and normal cells. Here, we present an evolutionary approach to measure the selection pressure on human genes, using the well-known ratio of the nonsynonymous to synonymous substitution rate in both cancer genomes ( C N / C S ) and normal populations ( p N / p S ). A new mutation-profile-based method that adopts sample-specific mutation rate profiles instead of conventional substitution models was developed. We found that cancer-specific selection pressure is quite different from the selection pressure at the species and population levels. Both the relaxation of purifying selection on passenger mutations and the positive selection of driver mutations may contribute to the increased C N / C S values of human genes in cancer genomes compared with the p N / p S values in human populations. The C N / C S values also contribute to the improved classification of cancer genes and a better understanding of the onco-functionalization of cancer genes during oncogenesis. The use of our computational pipeline to identify cancer-specific positively and negatively selected genes may provide useful information for understanding the evolution of cancers and identifying possible targets for therapeutic intervention.

  3. 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

  4. Bottleneck Effect on Evolutionary Rate in the Nearly Neutral Mutation Model

    PubMed Central

    Araki, H.; Tachida, H.

    1997-01-01

    Variances of evolutionary rates among lineages in some proteins are larger than those expected from simple Poisson processes. This phenomenon is called overdispersion of the molecular clock. If population size N is constant, the overdispersion is observed only in a limited range of 2Nσ under the nearly neutral mutation model, where σ represents the standard deviation of selection coefficients of new mutants. In this paper, we investigated effects of changing population size on the evolutionary rate by computer simulations assuming the nearly neutral mutation model. The size was changed cyclically between two numbers, N(1) and N(2) (N(1) > N(2)), in the simulations. The overdispersion is observed if 2N(2)σ is less than two and the state of reduced size (bottleneck state) continues for more than ~0.1/u generations, where u is the mutation rate. The overdispersion results mainly because the average fitnesses of only a portion of populations go down when the population size is reduced and only in these populations subsequent advantageous substitutions occur after the population size becomes large. Since the fitness reduction after the bottleneck is stochastic, acceleration of the evolutionary rate does not necessarily occur uniformly among loci. From these results, we argue that the nearly neutral mutation model is a candidate mechanism to explain the overdispersed molecular clock. PMID:9335622

  5. Atrial arrhythmogenicity of KCNJ2 mutations in short QT syndrome: Insights from virtual human atria

    PubMed Central

    El Harchi, Aziza; Hancox, Jules C.

    2017-01-01

    Gain-of-function mutations in KCNJ2-encoded Kir2.1 channels underlie variant 3 (SQT3) of the short QT syndrome, which is associated with atrial fibrillation (AF). Using biophysically-detailed human atria computer models, this study investigated the mechanistic link between SQT3 mutations and atrial arrhythmogenesis, and potential ion channel targets for treatment of SQT3. A contemporary model of the human atrial action potential (AP) was modified to recapitulate functional changes in IK1 due to heterozygous and homozygous forms of the D172N and E299V Kir2.1 mutations. Wild-type (WT) and mutant formulations were incorporated into multi-scale homogeneous and heterogeneous tissue models. Effects of mutations on AP duration (APD), conduction velocity (CV), effective refractory period (ERP), tissue excitation threshold and their rate-dependence, as well as the wavelength of re-entry (WL) were quantified. The D172N and E299V Kir2.1 mutations produced distinct effects on IK1 and APD shortening. Both mutations decreased WL for re-entry through a reduction in ERP and CV. Stability of re-entrant excitation waves in 2D and 3D tissue models was mediated by changes to tissue excitability and dispersion of APD in mutation conditions. Combined block of IK1 and IKr was effective in terminating re-entry associated with heterozygous D172N conditions, whereas IKr block alone may be a safer alternative for the E299V mutation. Combined inhibition of IKr and IKur produced a synergistic anti-arrhythmic effect in both forms of SQT3. In conclusion, this study provides mechanistic insights into atrial proarrhythmia with SQT3 Kir2.1 mutations and highlights possible pharmacological strategies for management of SQT3-linked AF. PMID:28609477

  6. Thailand mutation and variation database (ThaiMUT).

    PubMed

    Ruangrit, Uttapong; Srikummool, Metawee; Assawamakin, Anunchai; Ngamphiw, Chumpol; Chuechote, Suparat; Thaiprasarnsup, Vilasinee; Agavatpanitch, Gallissara; Pasomsab, Ekawat; Yenchitsomanus, Pa-Thai; Mahasirimongkol, Surakameth; Chantratita, Wasun; Palittapongarnpim, Prasit; Uyyanonvara, Bunyarit; Limwongse, Chanin; Tongsima, Sissades

    2008-08-01

    With the completion of the human genome project, novel sequencing and genotyping technologies had been utilized to detect mutations. Such mutations have continually been produced at exponential rate by researchers in various communities. Based on the population's mutation spectra, occurrences of Mendelian diseases are different across ethnic groups. A proportion of Mendelian diseases can be observed in some countries at higher rates than others. Recognizing the importance of mutation effects in Thailand, we established a National and Ethnic Mutation Database (NEMDB) for Thai people. This database, named Thailand Mutation and Variation database (ThaiMUT), offers a web-based access to genetic mutation and variation information in Thai population. This NEMDB initiative is an important informatics tool for both research and clinical purposes to retrieve and deposit human variation data. The mutation data cataloged in ThaiMUT database were derived from journal articles available in PubMed and local publications. In addition to collected mutation data, ThaiMUT also records genetic polymorphisms located in drug related genes. ThaiMUT could then provide useful information for clinical mutation screening services for Mendelian diseases and pharmacogenomic researches. ThaiMUT can be publicly accessed from http://gi.biotec.or.th/thaimut.

  7. Three mutations switch H7N9 influenza to human-type receptor specificity.

    PubMed

    de Vries, Robert P; Peng, Wenjie; Grant, Oliver C; Thompson, Andrew J; Zhu, Xueyong; Bouwman, Kim M; de la Pena, Alba T Torrents; van Breemen, Marielle J; Ambepitiya Wickramasinghe, Iresha N; de Haan, Cornelis A M; Yu, Wenli; McBride, Ryan; Sanders, Rogier W; Woods, Robert J; Verheije, Monique H; Wilson, Ian A; Paulson, James C

    2017-06-01

    The avian H7N9 influenza outbreak in 2013 resulted from an unprecedented incidence of influenza transmission to humans from infected poultry. The majority of human H7N9 isolates contained a hemagglutinin (HA) mutation (Q226L) that has previously been associated with a switch in receptor specificity from avian-type (NeuAcα2-3Gal) to human-type (NeuAcα2-6Gal), as documented for the avian progenitors of the 1957 (H2N2) and 1968 (H3N2) human influenza pandemic viruses. While this raised concern that the H7N9 virus was adapting to humans, the mutation was not sufficient to switch the receptor specificity of H7N9, and has not resulted in sustained transmission in humans. To determine if the H7 HA was capable of acquiring human-type receptor specificity, we conducted mutation analyses. Remarkably, three amino acid mutations conferred a switch in specificity for human-type receptors that resembled the specificity of the 2009 human H1 pandemic virus, and promoted binding to human trachea epithelial cells.

  8. Three mutations switch H7N9 influenza to human-type receptor specificity

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

    de Vries, Robert P.; Peng, Wenjie; Grant, Oliver C.

    The avian H7N9 influenza outbreak in 2013 resulted from an unprecedented incidence of influenza transmission to humans from infected poultry. The majority of human H7N9 isolates contained a hemagglutinin (HA) mutation (Q226L) that has previously been associated with a switch in receptor specificity from avian-type (NeuAcα2-3Gal) to human-type (NeuAcα2-6Gal), as documented for the avian progenitors of the 1957 (H2N2) and 1968 (H3N2) human influenza pandemic viruses. While this raised concern that the H7N9 virus was adapting to humans, the mutation was not sufficient to switch the receptor specificity of H7N9, and has not resulted in sustained transmission in humans. Tomore » determine if the H7 HA was capable of acquiring human-type receptor specificity, we conducted mutation analyses. Remarkably, three amino acid mutations conferred a switch in specificity for human-type receptors that resembled the specificity of the 2009 human H1 pandemic virus, and promoted binding to human trachea epithelial cells.« less

  9. The Effect of Coexistence of a Pair of Mutated Oncogenes on the Survival Rate of Invasive Breast Carcinoma Patients

    NASA Astrophysics Data System (ADS)

    Nair, D. R.

    2017-12-01

    The purpose of this project was to determine the effect of two mutated oncogenes on the survival rate from invasive breast carcinoma when in comparison to the mutation of a single oncogene on the survival rate. An oncogene is defined as a gene, that when mutated, can lead to cancer. The two oncogenes used in this project were human epidermal growth factor receptor 2 (HER2) and c-myc (MYC). HER2 and MYC are both oncogenes that contribute to the formation of cancer. HER2 proteins are receptors on breast cells, and when the HER2 gene is mutated, there is an overexpression of HER2 protein on the breast cell. This makes the breast cells proliferate uncontrollably. MYC is a gene that codes for a transcription factor that plays a role in cell cycle progression. The overexpression of MYC also leads to the proliferation of cells. I hypothesized that if there is a mutation in both the MYC and HER2 genes, then the survival rate of invasive breast carcinoma patients will be lower compared to patients with the mutations of only MYC or HER2. To test this hypothesis, we conducted individual gene searches in CBioPortal for HER2 in the datasets from the studies titled TCGA Nature 2012, TCGA Cell 2015, and TCGA Provisional. We conducted individual gene searches in CBioPortal for MYC in the same datasets. The survival rate data was then exported and analyzed for patients with mutations of either HER2 or MYC and with mutations of both genes. To determine the cases that had both HER2 and MYC mutations, we found the overlapping cases in both HER2 and MYC groups for all three datasets. We calculated the median of the survival data for cases where either HER2 or MYC was mutated and cases where both MYC and HER2 were mutated. From the first dataset, the median of MYC data was 95.53, HER2 data was 95.83, and both HER2 and MYC data was 91.24. In the second dataset, the median of MYC data was 92.17 , HER2 data was 93.5, and both HER2 and MYC data was 87.95 . In the third dataset, the median

  10. DRUMS: a human disease related unique gene mutation search engine.

    PubMed

    Li, Zuofeng; Liu, Xingnan; Wen, Jingran; Xu, Ye; Zhao, Xin; Li, Xuan; Liu, Lei; Zhang, Xiaoyan

    2011-10-01

    With the completion of the human genome project and the development of new methods for gene variant detection, the integration of mutation data and its phenotypic consequences has become more important than ever. Among all available resources, locus-specific databases (LSDBs) curate one or more specific genes' mutation data along with high-quality phenotypes. Although some genotype-phenotype data from LSDB have been integrated into central databases little effort has been made to integrate all these data by a search engine approach. In this work, we have developed disease related unique gene mutation search engine (DRUMS), a search engine for human disease related unique gene mutation as a convenient tool for biologists or physicians to retrieve gene variant and related phenotype information. Gene variant and phenotype information were stored in a gene-centred relational database. Moreover, the relationships between mutations and diseases were indexed by the uniform resource identifier from LSDB, or another central database. By querying DRUMS, users can access the most popular mutation databases under one interface. DRUMS could be treated as a domain specific search engine. By using web crawling, indexing, and searching technologies, it provides a competitively efficient interface for searching and retrieving mutation data and their relationships to diseases. The present system is freely accessible at http://www.scbit.org/glif/new/drums/index.html. © 2011 Wiley-Liss, Inc.

  11. Predicting protein folding rate change upon point mutation using residue-level coevolutionary information.

    PubMed

    Mallik, Saurav; Das, Smita; Kundu, Sudip

    2016-01-01

    Change in folding kinetics of globular proteins upon point mutation is crucial to a wide spectrum of biological research, such as protein misfolding, toxicity, and aggregations. Here we seek to address whether residue-level coevolutionary information of globular proteins can be informative to folding rate changes upon point mutations. Generating residue-level coevolutionary networks of globular proteins, we analyze three parameters: relative coevolution order (rCEO), network density (ND), and characteristic path length (CPL). A point mutation is considered to be equivalent to a node deletion of this network and respective percentage changes in rCEO, ND, CPL are found linearly correlated (0.84, 0.73, and -0.61, respectively) with experimental folding rate changes. The three parameters predict the folding rate change upon a point mutation with 0.031, 0.045, and 0.059 standard errors, respectively. © 2015 Wiley Periodicals, Inc.

  12. Somatic deleterious mutation rate in a woody plant: estimation from phenotypic data

    PubMed Central

    Bobiwash, K; Schultz, S T; Schoen, D J

    2013-01-01

    We conducted controlled crosses in populations of the long-lived clonal shrub, Vaccinium angustifolium (lowbush blueberry) to estimate inbreeding depression and mutation parameters associated with somatic deleterious mutation. Inbreeding depression level was high, with many plants failing to set fruit after self-pollination. We also compared fruit set from autogamous pollinations (pollen collected from within the same inflorescence) with fruit set from geitonogamous pollinations (pollen collected from the same plant but from inflorescences separated by several meters of branch growth). The difference between geitonogamous versus autogamous fitness within single plants is referred to as ‘autogamy depression' (AD). AD can be caused by somatic deleterious mutation. AD was significantly different from zero for fruit set. We developed a maximum-likelihood procedure to estimate somatic mutation parameters from AD, and applied it to geitonogamous and autogamous fruit set data from this experiment. We infer that, on average, approximately three sublethal, partially dominant somatic mutations exist within the crowns of the plants studied. We conclude that somatic mutation in this woody plant results in an overall genomic deleterious mutation rate that exceeds the rate measured to date for annual plants. Some implications of this result for evolutionary biology and agriculture are discussed. PMID:23778990

  13. 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

  14. Mutations in TUBB8 and Human Oocyte Meiotic Arrest

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

    Feng, Ruizhi; Sang, Qing; Kuang, Yanping

    BACKGROUND: We present that human reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to the arrest of human oocyte maturation are unknown. METHODS: We sequenced the exomes of five members of a four-generation family, three of whom had infertility due to oocyte meiosis I arrest. We performed Sanger sequencing of a candidate gene, TUBB8, in DNA samples from these members, additional family members, and members of 23 other affected families. The expression of TUBB8 and all other β-tubulin isotypes was assessed in human oocytes, earlymore » embryos, sperm cells, and several somatic tissues by means of a quantitative reverse- transcriptase-polymerase-chain-reaction assay. We evaluated the effect of the TUBB8 mutations on the assembly of the heterodimer consisting of one α-tubulin polypeptide and one β-tubulin polypeptide (α/β-tubulin heterodimer) in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes. RESULTSL: We identified seven mutations in the primate-specific gene TUBB8 that were responsible for oocyte meiosis I arrest in 7 of the 24 families. TUBB8 expression is unique to oocytes and the early embryo, in which this gene accounts for almost all the expressed β-tubulin. The mutations affect chaperone-dependent folding and assembly of the α/β-tubulin heterodimer, disrupt microtubule behavior on expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle-assembly defects and maturation arrest on expression in mouse and human oocytes. CONCLUSIONS: Lastly, TUBB8 mutations have dominant-negative effects that disrupt microtubule behavior and oocyte meiotic spindle assembly and maturation, causing female infertility.« less

  15. Mutations in TUBB8 and Human Oocyte Meiotic Arrest

    DOE PAGES

    Feng, Ruizhi; Sang, Qing; Kuang, Yanping; ...

    2016-01-21

    BACKGROUND: We present that human reproduction depends on the fusion of a mature oocyte with a sperm cell to form a fertilized egg. The genetic events that lead to the arrest of human oocyte maturation are unknown. METHODS: We sequenced the exomes of five members of a four-generation family, three of whom had infertility due to oocyte meiosis I arrest. We performed Sanger sequencing of a candidate gene, TUBB8, in DNA samples from these members, additional family members, and members of 23 other affected families. The expression of TUBB8 and all other β-tubulin isotypes was assessed in human oocytes, earlymore » embryos, sperm cells, and several somatic tissues by means of a quantitative reverse- transcriptase-polymerase-chain-reaction assay. We evaluated the effect of the TUBB8 mutations on the assembly of the heterodimer consisting of one α-tubulin polypeptide and one β-tubulin polypeptide (α/β-tubulin heterodimer) in vitro, on microtubule architecture in HeLa cells, on microtubule dynamics in yeast cells, and on spindle assembly in mouse and human oocytes. RESULTSL: We identified seven mutations in the primate-specific gene TUBB8 that were responsible for oocyte meiosis I arrest in 7 of the 24 families. TUBB8 expression is unique to oocytes and the early embryo, in which this gene accounts for almost all the expressed β-tubulin. The mutations affect chaperone-dependent folding and assembly of the α/β-tubulin heterodimer, disrupt microtubule behavior on expression in cultured cells, alter microtubule dynamics in vivo, and cause catastrophic spindle-assembly defects and maturation arrest on expression in mouse and human oocytes. CONCLUSIONS: Lastly, TUBB8 mutations have dominant-negative effects that disrupt microtubule behavior and oocyte meiotic spindle assembly and maturation, causing female infertility.« less

  16. Human knockouts and phenotypic analysis in a cohort with a high rate of consanguinity.

    PubMed

    Saleheen, Danish; Natarajan, Pradeep; Armean, Irina M; Zhao, Wei; Rasheed, Asif; Khetarpal, Sumeet A; Won, Hong-Hee; Karczewski, Konrad J; O'Donnell-Luria, Anne H; Samocha, Kaitlin E; Weisburd, Benjamin; Gupta, Namrata; Zaidi, Mozzam; Samuel, Maria; Imran, Atif; Abbas, Shahid; Majeed, Faisal; Ishaq, Madiha; Akhtar, Saba; Trindade, Kevin; Mucksavage, Megan; Qamar, Nadeem; Zaman, Khan Shah; Yaqoob, Zia; Saghir, Tahir; Rizvi, Syed Nadeem Hasan; Memon, Anis; Hayyat Mallick, Nadeem; Ishaq, Mohammad; Rasheed, Syed Zahed; Memon, Fazal-Ur-Rehman; Mahmood, Khalid; Ahmed, Naveeduddin; Do, Ron; Krauss, Ronald M; MacArthur, Daniel G; Gabriel, Stacey; Lander, Eric S; Daly, Mark J; Frossard, Philippe; Danesh, John; Rader, Daniel J; Kathiresan, Sekar

    2017-04-12

    A major goal of biomedicine is to understand the function of every gene in the human genome. Loss-of-function mutations can disrupt both copies of a given gene in humans and phenotypic analysis of such 'human knockouts' can provide insight into gene function. Consanguineous unions are more likely to result in offspring carrying homozygous loss-of-function mutations. In Pakistan, consanguinity rates are notably high. Here we sequence the protein-coding regions of 10,503 adult participants in the Pakistan Risk of Myocardial Infarction Study (PROMIS), designed to understand the determinants of cardiometabolic diseases in individuals from South Asia. We identified individuals carrying homozygous predicted loss-of-function (pLoF) mutations, and performed phenotypic analysis involving more than 200 biochemical and disease traits. We enumerated 49,138 rare (<1% minor allele frequency) pLoF mutations. These pLoF mutations are estimated to knock out 1,317 genes, each in at least one participant. Homozygosity for pLoF mutations at PLA2G7 was associated with absent enzymatic activity of soluble lipoprotein-associated phospholipase A2; at CYP2F1, with higher plasma interleukin-8 concentrations; at TREH, with lower concentrations of apoB-containing lipoprotein subfractions; at either A3GALT2 or NRG4, with markedly reduced plasma insulin C-peptide concentrations; and at SLC9A3R1, with mediators of calcium and phosphate signalling. Heterozygous deficiency of APOC3 has been shown to protect against coronary heart disease; we identified APOC3 homozygous pLoF carriers in our cohort. We recruited these human knockouts and challenged them with an oral fat load. Compared with family members lacking the mutation, individuals with APOC3 knocked out displayed marked blunting of the usual post-prandial rise in plasma triglycerides. Overall, these observations provide a roadmap for a 'human knockout project', a systematic effort to understand the phenotypic consequences of complete

  17. Low Base-Substitution Mutation Rate in the Germline Genome of the Ciliate Tetrahymena thermophila

    DTIC Science & Technology

    2016-09-15

    generations of mutation accumulation (MA). We applied an existing mutation-calling pipeline and developed a new probabilistic mutation detection approach...noise introduced by mismapped reads. We used both our new method and an existing mutation-calling pipeline (Sung, Tucker, et al. 2012) to analyse the...and larger MA experiments will be required to confidently estimate the mutational spectrum of a species with such a low mutation rate. Materials and

  18. 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

  19. TP53 mutations, expression and interaction networks in human cancers

    PubMed Central

    Wang, Xiaosheng; Sun, Qingrong

    2017-01-01

    Although the associations of p53 dysfunction, p53 interaction networks and oncogenesis have been widely explored, a systematic analysis of TP53 mutations and its related interaction networks in various types of human cancers is lacking. Our study explored the associations of TP53 mutations, gene expression, clinical outcomes, and TP53 interaction networks across 33 cancer types using data from The Cancer Genome Atlas (TCGA). We show that TP53 is the most frequently mutated gene in a number of cancers, and its mutations appear to be early events in cancer initiation. We identified genes potentially repressed by p53, and genes whose expression correlates significantly with TP53 expression. These gene products may be especially important nodes in p53 interaction networks in human cancers. This study shows that while TP53-truncating mutations often result in decreased TP53 expression, other non-truncating TP53 mutations result in increased TP53 expression in some cancers. Survival analyses in a number of cancers show that patients with TP53 mutations are more likely to have worse prognoses than TP53-wildtype patients, and that elevated TP53 expression often leads to poor clinical outcomes. We identified a set of candidate synthetic lethal (SL) genes for TP53, and validated some of these SL interactions using data from the Cancer Cell Line Project. These predicted SL genes are promising candidates for experimental validation and the development of personalized therapeutics for patients with TP53-mutated cancers. PMID:27880943

  20. TP53 mutations, expression and interaction networks in human cancers.

    PubMed

    Wang, Xiaosheng; Sun, Qingrong

    2017-01-03

    Although the associations of p53 dysfunction, p53 interaction networks and oncogenesis have been widely explored, a systematic analysis of TP53 mutations and its related interaction networks in various types of human cancers is lacking. Our study explored the associations of TP53 mutations, gene expression, clinical outcomes, and TP53 interaction networks across 33 cancer types using data from The Cancer Genome Atlas (TCGA). We show that TP53 is the most frequently mutated gene in a number of cancers, and its mutations appear to be early events in cancer initiation. We identified genes potentially repressed by p53, and genes whose expression correlates significantly with TP53 expression. These gene products may be especially important nodes in p53 interaction networks in human cancers. This study shows that while TP53-truncating mutations often result in decreased TP53 expression, other non-truncating TP53 mutations result in increased TP53 expression in some cancers. Survival analyses in a number of cancers show that patients with TP53 mutations are more likely to have worse prognoses than TP53-wildtype patients, and that elevated TP53 expression often leads to poor clinical outcomes. We identified a set of candidate synthetic lethal (SL) genes for TP53, and validated some of these SL interactions using data from the Cancer Cell Line Project. These predicted SL genes are promising candidates for experimental validation and the development of personalized therapeutics for patients with TP53-mutated cancers.

  1. Functional and Genomic Features of Human Genes Mutated in Neuropsychiatric Disorders.

    PubMed

    Forero, Diego A; Prada, Carlos F; Perry, George

    2016-01-01

    In recent years, a large number of studies around the world have led to the identification of causal genes for hereditary types of common and rare neurological and psychiatric disorders. To explore the functional and genomic features of known human genes mutated in neuropsychiatric disorders. A systematic search was used to develop a comprehensive catalog of genes mutated in neuropsychiatric disorders (NPD). Functional enrichment and protein-protein interaction analyses were carried out. A false discovery rate approach was used for correction for multiple testing. We found several functional categories that are enriched among NPD genes, such as gene ontologies, protein domains, tissue expression, signaling pathways and regulation by brain-expressed miRNAs and transcription factors. Sixty six of those NPD genes are known to be druggable. Several topographic parameters of protein-protein interaction networks and the degree of conservation between orthologous genes were identified as significant among NPD genes. These results represent one of the first analyses of enrichment of functional categories of genes known to harbor mutations for NPD. These findings could be useful for a future creation of computational tools for prioritization of novel candidate genes for NPD.

  2. Functional and Genomic Features of Human Genes Mutated in Neuropsychiatric Disorders

    PubMed Central

    Forero, Diego A.; Prada, Carlos F.; Perry, George

    2016-01-01

    Background: In recent years, a large number of studies around the world have led to the identification of causal genes for hereditary types of common and rare neurological and psychiatric disorders. Objective: To explore the functional and genomic features of known human genes mutated in neuropsychiatric disorders. Methods: A systematic search was used to develop a comprehensive catalog of genes mutated in neuropsychiatric disorders (NPD). Functional enrichment and protein-protein interaction analyses were carried out. A false discovery rate approach was used for correction for multiple testing. Results: We found several functional categories that are enriched among NPD genes, such as gene ontologies, protein domains, tissue expression, signaling pathways and regulation by brain-expressed miRNAs and transcription factors. Sixty six of those NPD genes are known to be druggable. Several topographic parameters of protein-protein interaction networks and the degree of conservation between orthologous genes were identified as significant among NPD genes. Conclusion: These results represent one of the first analyses of enrichment of functional categories of genes known to harbor mutations for NPD. These findings could be useful for a future creation of computational tools for prioritization of novel candidate genes for NPD. PMID:27990183

  3. 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.

  4. No evidence of radiation effect on mutation rates at hypervariable minisatellite loci in the germ cells of atomic bomb survivors.

    PubMed

    Kodaira, Mieko; Izumi, Shizue; Takahashi, Norio; Nakamura, Nori

    2004-10-01

    Human minisatellites consist of tandem arrays of short repeat sequences, and some are highly polymorphic in numbers of repeats among individuals. Since these loci mutate much more frequently than coding sequences, they make attractive markers for screening populations for genetic effects of mutagenic agents. Here we report the results of our analysis of mutations at eight hypervariable minisatellite loci in the offspring (61 from exposed families in 60 of which only one parent was exposed, and 58 from unexposed parents) of atomic bomb survivors with mean doses of >1 Sv. We found 44 mutations in paternal alleles and eight mutations in maternal alleles with no indication that the high doses of acutely applied radiation had caused significant genetic effects. Our finding contrasts with those of some other studies in which much lower radiation doses, applied chronically, caused significantly increased mutation rates. Possible reasons for this discrepancy are discussed.

  5. Dynamically correlated mutations drive human Influenza A evolution.

    PubMed

    Tria, F; Pompei, S; Loreto, V

    2013-01-01

    Human Influenza A virus undergoes recurrent changes in the hemagglutinin (HA) surface protein, primarily involved in the human antibody recognition. Relevant antigenic changes, enabling the virus to evade host immune response, have been recognized to occur in parallel to multiple mutations at antigenic sites in HA. Yet, the role of correlated mutations (epistasis) in driving the molecular evolution of the virus still represents a challenging puzzle. Further, though circulation at a global geographic level is key for the survival of Influenza A, its role in shaping the viral phylodynamics remains largely unexplored. Here we show, through a sequence based epidemiological model, that epistatic effects between amino acids substitutions, coupled with a reservoir that mimics worldwide circulating viruses, are key determinants that drive human Influenza A evolution. Our approach explains all the up-to-date observations characterizing the evolution of H3N2 subtype, including phylogenetic properties, nucleotide fixation patterns, and composition of antigenic clusters.

  6. Mutations in the human GlyT2 gene define a presynaptic component of human startle disease

    PubMed Central

    Rees, Mark I.; Harvey, Kirsten; Pearce, Brian R.; Chung, Seo-Kyung; Duguid, Ian C.; Thomas, Philip; Beatty, Sarah; Graham, Gail E.; Armstrong, Linlea; Shiang, Rita; Abbott, Kim J.; Zuberi, Sameer M.; Stephenson, John B.P.; Owen, Michael J.; Tijssen, Marina A.J.; van den Maagdenberg, Arn M.J.M.; Smart, Trevor G.; Supplisson, Stéphane; Harvey, Robert J.

    2011-01-01

    Hyperekplexia is a human neurological disorder characterized by an excessive startle response and is typically caused by missense and nonsense mutations in the gene encoding the inhibitory glycine receptor (GlyR) α1 subunit (GLRA1)1-3. Genetic heterogeneity has been confirmed in isolated sporadic cases with mutations in other postsynaptic glycinergic proteins including the GlyR β subunit (GLRB)4, gephyrin (GPHN)5 and RhoGEF collybistin (ARHGEF9)6. However, many sporadic patients diagnosed with hyperekplexia do not carry mutations in these genes2-7. Here we reveal that missense, nonsense and frameshift mutations in the presynaptic glycine transporter 2 (GlyT2) gene (SLC6A5)8 also cause hyperekplexia. Patients harbouring mutations in SLC6A5 presented with hypertonia, an exaggerated startle response to tactile or acoustic stimuli, and life-threatening neonatal apnoea episodes. GlyT2 mutations result in defective subcellular localisation and/or decreased glycine uptake, with selected mutations affecting predicted glycine and Na+ binding sites. Our results demonstrate that SLC6A5 is a major gene for hyperekplexia and define the first neurological disorder linked to mutations in a Na+/Cl−-dependent transporter for a classical fast neurotransmitter. By analogy, we suggest that in other human disorders where defects in postsynaptic receptors have been identified, similar symptoms could result from defects in the cognate presynaptic neurotransmitter transporter. PMID:16751771

  7. A protein domain-centric approach for the comparative analysis of human and yeast phenotypically relevant mutations

    PubMed Central

    2013-01-01

    Background The body of disease mutations with known phenotypic relevance continues to increase and is expected to do so even faster with the advent of new experimental techniques such as whole-genome sequencing coupled with disease association studies. However, genomic association studies are limited by the molecular complexity of the phenotype being studied and the population size needed to have adequate statistical power. One way to circumvent this problem, which is critical for the study of rare diseases, is to study the molecular patterns emerging from functional studies of existing disease mutations. Current gene-centric analyses to study mutations in coding regions are limited by their inability to account for the functional modularity of the protein. Previous studies of the functional patterns of known human disease mutations have shown a significant tendency to cluster at protein domain positions, namely position-based domain hotspots of disease mutations. However, the limited number of known disease mutations remains the main factor hindering the advancement of mutation studies at a functional level. In this paper, we address this problem by incorporating mutations known to be disruptive of phenotypes in other species. Focusing on two evolutionarily distant organisms, human and yeast, we describe the first inter-species analysis of mutations of phenotypic relevance at the protein domain level. Results The results of this analysis reveal that phenotypic mutations from yeast cluster at specific positions on protein domains, a characteristic previously revealed to be displayed by human disease mutations. We found over one hundred domain hotspots in yeast with approximately 50% in the exact same domain position as known human disease mutations. Conclusions We describe an analysis using protein domains as a framework for transferring functional information by studying domain hotspots in human and yeast and relating phenotypic changes in yeast to diseases in

  8. Exonuclease mutations in DNA polymerase epsilon reveal replication strand specific mutation patterns and human origins of replication

    PubMed Central

    Shinbrot, Eve; Henninger, Erin E.; Weinhold, Nils; Covington, Kyle R.; Göksenin, A. Yasemin; Schultz, Nikolaus; Chao, Hsu; Doddapaneni, HarshaVardhan; Muzny, Donna M.; Gibbs, Richard A.; Sander, Chris; Pursell, Zachary F.

    2014-01-01

    Tumors with somatic mutations in the proofreading exonuclease domain of DNA polymerase epsilon (POLE-exo*) exhibit a novel mutator phenotype, with markedly elevated TCT→TAT and TCG→TTG mutations and overall mutation frequencies often exceeding 100 mutations/Mb. Here, we identify POLE-exo* tumors in numerous cancers and classify them into two groups, A and B, according to their mutational properties. Group A mutants are found only in POLE, whereas Group B mutants are found in POLE and POLD1 and appear to be nonfunctional. In Group A, cell-free polymerase assays confirm that mutations in the exonuclease domain result in high mutation frequencies with a preference for C→A mutation. We describe the patterns of amino acid substitutions caused by POLE-exo* and compare them to other tumor types. The nucleotide preference of POLE-exo* leads to increased frequencies of recurrent nonsense mutations in key tumor suppressors such as TP53, ATM, and PIK3R1. We further demonstrate that strand-specific mutation patterns arise from some of these POLE-exo* mutants during genome duplication. This is the first direct proof of leading strand-specific replication by human POLE, which has only been demonstrated in yeast so far. Taken together, the extremely high mutation frequency and strand specificity of mutations provide a unique identifier of eukaryotic origins of replication. PMID:25228659

  9. High mutation detection rate in TCOF1 among Treacher Collins syndrome patients reveals clustering of mutations and 16 novel pathogenic changes.

    PubMed

    Splendore, A; Silva, E O; Alonso, L G; Richieri-Costa, A; Alonso, N; Rosa, A; Carakushanky, G; Cavalcanti, D P; Brunoni, D; Passos-Bueno, M R

    2000-10-01

    Twenty-eight families with a clinical diagnosis of Treacher Collins syndrome were screened for mutations in the 25 coding exons of TCOF1 and their adjacent splice junctions through SSCP and direct sequencing. Pathogenic mutations were detected in 26 patients, yielding the highest detection rate reported so far for this disease (93%) and bringing the number of known disease-causing mutations from 35 to 51. This is the first report to describe clustering of pathogenic mutations. Thirteen novel polymorphic alterations were characterized, confirming previous reports that TCOF1 has an unusually high rate of single-nucleotide polymorphisms (SNPs) within its coding region. We suggest a possible different mechanism leading to TCS or genetic heterogeneity for this condition, as we identified two families with no apparent pathogenic mutation in the gene. Furthermore, our data confirm the absence of genotype-phenotype correlation and reinforce that the apparent anticipation often observed in TCS families is due to ascertainment bias. Copyright 2000 Wiley-Liss, Inc.

  10. 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.

  11. Mutation rates for 20 STR loci in a population from São Paulo state, Southeast, Brazil.

    PubMed

    Martinez, Juliana; Braganholi, Danilo Faustino; Ambrósio, Isabela Brunelli; Polverari, Fernanda Silva; Cicarelli, Regina Maria Barretto

    2017-11-01

    Short tandem repeats (STRs) are genetic markers largely employed in forensic analysis and paternity investigation cases. When an inconsistency between the parent and child is considered as a possible mutation, the mutation rate should be incorporated into paternity index calculations to give a robust result and to reduce the chance of misinterpretation. The aim of this study was to estimate the mutation rates of 20 autosomal STRs loci used for paternity tests. In these loci we analysed 29,831 parent-child allelic transfers from 929 duo or trio paternity tests carried out during 2012?2016 from São Paulo State, Brazil. We identified 35 mutations in 16 loci, and they were more frequent in the paternal germline compared to the maternal germline. The loci with the highest rate were vWA and FGA and the ones with the lowest rate were PENTA E, PENTA D, D21S11, D7S820 and D6S1043. We did not identified any mutation in D2S1338, TH01, TPOX and D16S539 loci. All mutations consisted of losses or gains of one repeat unit. Mutation rates found in the São Paulo population have peculiarities, which justifies the use of regional databases in laboratories.

  12. Human knockouts and phenotypic analysis in a cohort with a high rate of consanguinity

    PubMed Central

    Saleheen, Danish; Natarajan, Pradeep; Armean, Irina M.; Zhao, Wei; Rasheed, Asif; Khetarpal, Sumeet; Won, Hong-Hee; Karczewski, Konrad J.; O’Donnell-Luria, Anne H.; Samocha, Kaitlin E.; Weisburd, Benjamin; Gupta, Namrata; Zaidi, Mozzam; Samuel, Maria; Imran, Atif; Abbas, Shahid; Majeed, Faisal; Ishaq, Madiha; Akhtar, Saba; Trindade, Kevin; Mucksavage, Megan; Qamar, Nadeem; Zaman, Khan Shah; Yaqoob, Zia; Saghir, Tahir; Rizvi, Syed Nadeem Hasan; Memon, Anis; Mallick, Nadeem Hayyat; Ishaq, Mohammad; Rasheed, Syed Zahed; Memon, Fazal-ur-Rehman; Mahmood, Khalid; Ahmed, Naveeduddin; Do, Ron; Krauss, Ronald M.; MacArthur, Daniel G.; Gabriel, Stacey; Lander, Eric S.; Daly, Mark J.; Frossard, Philippe; Danesh, John; Rader, Daniel J.; Kathiresan, Sekar

    2017-01-01

    A major goal of biomedicine is to understand the function of every gene in the human genome.1 Loss-of-function (LoF) mutations can disrupt both copies of a given gene in humans and phenotypic analysis of such ‘human knockouts’ can provide insight into gene function. Consanguineous unions are more likely to result in offspring who carry LoF mutations in a homozygous state. In Pakistan, consanguinity rates are notably high.2 Here, we sequenced the protein-coding regions of 10,503 adult participants in the Pakistan Risk of Myocardial Infarction Study (PROMIS) designed to understand the determinants of cardiometabolic diseases in South Asians.3 We identified individuals carrying predicted LoF (pLoF) mutations in the homozygous state, and performed phenotypic analysis involving >200 biochemical and disease traits. We enumerated 49,138 rare (<1 % minor allele frequency) pLoF mutations. These pLoF mutations are predicted to knock out 1,317 genes in at least one participant. Homozygosity for pLoF mutations at PLAG27 was associated with absent enzymatic activity of soluble lipoprotein-associated phospholipase A2; at CYP2F1, with higher plasma interleukin-8 concentrations; at TREH, with lower concentrations of apoB-containing lipoprotein subfractions; at either A3GALT2 or NRG4, with markedly reduced plasma insulin C-peptide concentrations; and at SLC9A3R1, with mediators of calcium and phosphate signaling. Finally, APOC3 is a gene which retards clearance of plasma triglyceride-rich lipoproteins and where heterozygous deficiency confers protection against coronary heart disease.4,5 In Pakistan, we now observe APOC3 homozygous pLoF carriers; we recalled these knockout humans and challenged with an oral fat load. Compared with wild-type family members, APOC3 knockouts displayed marked blunting of the usual post-prandial rise in plasma triglycerides. Overall, these observations provide a roadmap for a ‘human knockout project’, a systematic effort to understand the

  13. Male Mutation Bias Is the Main Force Shaping Chromosomal Substitution Rates in Monotreme Mammals

    PubMed Central

    Link, Vivian; Aguilar-Gómez, Diana; Ramírez-Suástegui, Ciro; Hurst, Laurence D.

    2017-01-01

    Abstract In many species, spermatogenesis involves more cell divisions than oogenesis, and the male germline, therefore, accumulates more DNA replication errors, a phenomenon known as male mutation bias. The extent of male mutation bias (α) is estimated by comparing substitution rates of the X, Y, and autosomal chromosomes, as these chromosomes spend different proportions of their time in the germlines of the two sexes. Male mutation bias has been characterized in placental and marsupial mammals as well as birds, but analyses in monotremes failed to detect any such bias. Monotremes are an ancient lineage of egg-laying mammals with distinct biological properties, which include unique germline features. Here, we sought to assess the presence and potential characteristics of male mutation bias in platypus and the short-beaked echidna based on substitution rate analyses of X, Y, and autosomes. We established the presence of moderate male mutation bias in monotremes, corresponding to an α value of 2.12–3.69. Given that it has been unclear what proportion of the variation in substitution rates on the different chromosomal classes is really due to differential number of replications, we analyzed the influence of other confounding forces (selection, replication-timing, etc.) and found that male mutation bias is the main force explaining the between-chromosome classes differences in substitution rates. Finally, we estimated the proportion of variation at the gene level in substitution rates that is owing to replication effects and found that this phenomenon can explain >68% of these variations in monotremes, and in control species, rodents, and primates. PMID:28922870

  14. 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.

  15. Mitochondrial DNA mutations in single human blood cells.

    PubMed

    Yao, Yong-Gang; Kajigaya, Sachiko; Young, Neal S

    2015-09-01

    Determination mitochondrial DNA (mtDNA) sequences from extremely small amounts of DNA extracted from tissue of limited amounts and/or degraded samples is frequently employed in medical, forensic, and anthropologic studies. Polymerase chain reaction (PCR) amplification followed by DNA cloning is a routine method, especially to examine heteroplasmy of mtDNA mutations. In this review, we compare the mtDNA mutation patterns detected by three different sequencing strategies. Cloning and sequencing methods that are based on PCR amplification of DNA extracted from either single cells or pooled cells yield a high frequency of mutations, partly due to the artifacts introduced by PCR and/or the DNA cloning process. Direct sequencing of PCR product which has been amplified from DNA in individual cells is able to detect the low levels of mtDNA mutations present within a cell. We further summarize the findings in our recent studies that utilized this single cell method to assay mtDNA mutation patterns in different human blood cells. Our data show that many somatic mutations observed in the end-stage differentiated cells are found in hematopoietic stem cells (HSCs) and progenitors within the CD34(+) cell compartment. Accumulation of mtDNA variations in the individual CD34+ cells is affected by both aging and family genetic background. Granulocytes harbor higher numbers of mutations compared with the other cells, such as CD34(+) cells and lymphocytes. Serial assessment of mtDNA mutations in a population of single CD34(+) cells obtained from the same donor over time suggests stability of some somatic mutations. CD34(+) cell clones from a donor marked by specific mtDNA somatic mutations can be found in the recipient after transplantation. The significance of these findings is discussed in terms of the lineage tracing of HSCs, aging effect on accumulation of mtDNA mutations and the usage of mtDNA sequence in forensic identification. Copyright © 2015 Elsevier B.V. All rights

  16. Contributions of intrinsic mutation rate and selfish selection to levels of de novo HRAS mutations in the paternal germline

    PubMed Central

    Giannoulatou, Eleni; McVean, Gilean; Taylor, Indira B.; McGowan, Simon J.; Maher, Geoffrey J.; Iqbal, Zamin; Pfeifer, Susanne P.; Turner, Isaac; Burkitt Wright, Emma M. M.; Shorto, Jennifer; Itani, Aysha; Turner, Karen; Gregory, Lorna; Buck, David; Rajpert-De Meyts, Ewa; Looijenga, Leendert H. J.; Kerr, Bronwyn; Wilkie, Andrew O. M.; Goriely, Anne

    2013-01-01

    The RAS proto-oncogene Harvey rat sarcoma viral oncogene homolog (HRAS) encodes a small GTPase that transduces signals from cell surface receptors to intracellular effectors to control cellular behavior. Although somatic HRAS mutations have been described in many cancers, germline mutations cause Costello syndrome (CS), a congenital disorder associated with predisposition to malignancy. Based on the epidemiology of CS and the occurrence of HRAS mutations in spermatocytic seminoma, we proposed that activating HRAS mutations become enriched in sperm through a process akin to tumorigenesis, termed selfish spermatogonial selection. To test this hypothesis, we quantified the levels, in blood and sperm samples, of HRAS mutations at the p.G12 codon and compared the results to changes at the p.A11 codon, at which activating mutations do not occur. The data strongly support the role of selection in determining HRAS mutation levels in sperm, and hence the occurrence of CS, but we also found differences from the mutation pattern in tumorigenesis. First, the relative prevalence of mutations in sperm correlates weakly with their in vitro activating properties and occurrence in cancers. Second, specific tandem base substitutions (predominantly GC>TT/AA) occur in sperm but not in cancers; genomewide analysis showed that this same mutation is also overrepresented in constitutional pathogenic and polymorphic variants, suggesting a heightened vulnerability to these mutations in the germline. We developed a statistical model to show how both intrinsic mutation rate and selfish selection contribute to the mutational burden borne by the paternal germline. PMID:24259709

  17. Contributions of intrinsic mutation rate and selfish selection to levels of de novo HRAS mutations in the paternal germline.

    PubMed

    Giannoulatou, Eleni; McVean, Gilean; Taylor, Indira B; McGowan, Simon J; Maher, Geoffrey J; Iqbal, Zamin; Pfeifer, Susanne P; Turner, Isaac; Burkitt Wright, Emma M M; Shorto, Jennifer; Itani, Aysha; Turner, Karen; Gregory, Lorna; Buck, David; Rajpert-De Meyts, Ewa; Looijenga, Leendert H J; Kerr, Bronwyn; Wilkie, Andrew O M; Goriely, Anne

    2013-12-10

    The RAS proto-oncogene Harvey rat sarcoma viral oncogene homolog (HRAS) encodes a small GTPase that transduces signals from cell surface receptors to intracellular effectors to control cellular behavior. Although somatic HRAS mutations have been described in many cancers, germline mutations cause Costello syndrome (CS), a congenital disorder associated with predisposition to malignancy. Based on the epidemiology of CS and the occurrence of HRAS mutations in spermatocytic seminoma, we proposed that activating HRAS mutations become enriched in sperm through a process akin to tumorigenesis, termed selfish spermatogonial selection. To test this hypothesis, we quantified the levels, in blood and sperm samples, of HRAS mutations at the p.G12 codon and compared the results to changes at the p.A11 codon, at which activating mutations do not occur. The data strongly support the role of selection in determining HRAS mutation levels in sperm, and hence the occurrence of CS, but we also found differences from the mutation pattern in tumorigenesis. First, the relative prevalence of mutations in sperm correlates weakly with their in vitro activating properties and occurrence in cancers. Second, specific tandem base substitutions (predominantly GC>TT/AA) occur in sperm but not in cancers; genomewide analysis showed that this same mutation is also overrepresented in constitutional pathogenic and polymorphic variants, suggesting a heightened vulnerability to these mutations in the germline. We developed a statistical model to show how both intrinsic mutation rate and selfish selection contribute to the mutational burden borne by the paternal germline.

  18. Characterizing Changes in the Rate of Protein-Protein Dissociation upon Interface Mutation Using Hotspot Energy and Organization

    PubMed Central

    Agius, Rudi; Torchala, Mieczyslaw; Moal, Iain H.; Fernández-Recio, Juan; Bates, Paul A.

    2013-01-01

    Predicting the effects of mutations on the kinetic rate constants of protein-protein interactions is central to both the modeling of complex diseases and the design of effective peptide drug inhibitors. However, while most studies have concentrated on the determination of association rate constants, dissociation rates have received less attention. In this work we take a novel approach by relating the changes in dissociation rates upon mutation to the energetics and architecture of hotspots and hotregions, by performing alanine scans pre- and post-mutation. From these scans, we design a set of descriptors that capture the change in hotspot energy and distribution. The method is benchmarked on 713 kinetically characterized mutations from the SKEMPI database. Our investigations show that, with the use of hotspot descriptors, energies from single-point alanine mutations may be used for the estimation of off-rate mutations to any residue type and also multi-point mutations. A number of machine learning models are built from a combination of molecular and hotspot descriptors, with the best models achieving a Pearson's Correlation Coefficient of 0.79 with experimental off-rates and a Matthew's Correlation Coefficient of 0.6 in the detection of rare stabilizing mutations. Using specialized feature selection models we identify descriptors that are highly specific and, conversely, broadly important to predicting the effects of different classes of mutations, interface regions and complexes. Our results also indicate that the distribution of the critical stability regions across protein-protein interfaces is a function of complex size more strongly than interface area. In addition, mutations at the rim are critical for the stability of small complexes, but consistently harder to characterize. The relationship between hotregion size and the dissociation rate is also investigated and, using hotspot descriptors which model cooperative effects within hotregions, we show how the

  19. Solving the mystery of human sleep schedules one mutation at a time.

    PubMed

    Hallows, William C; Ptáček, Louis J; Fu, Ying-Hui

    2013-01-01

    Sleep behavior remains one of the most enigmatic areas of life. The unanswered questions range from "why do we sleep?" to "how we can improve sleep in today's society?" Identification of mutations responsible for altered circadian regulation of human sleep lead to unique opportunities for probing these territories. In this review, we summarize causative circadian mutations found from familial genetic studies to date. We also describe how these mutations mechanistically affect circadian function and lead to altered sleep behaviors, including shifted or shortening of sleep patterns. In addition, we discuss how the investigation of mutations can not only expand our understanding of the molecular mechanisms regulating the circadian clock and sleep duration, but also bridge the pathways between clock/sleep and other human physiological conditions and ailments such as metabolic regulation and migraine headaches.

  20. Third trimester fetal heart rate predicts phenotype and mutation burden in the type 1 long QT syndrome.

    PubMed

    Winbo, Annika; Fosdal, Inger; Lindh, Maria; Diamant, Ulla-Britt; Persson, Johan; Wettrell, Göran; Rydberg, Annika

    2015-08-01

    Early diagnosis and risk stratification is of clinical importance in the long QT syndrome (LQTS), however, little genotype-specific data are available regarding fetal LQTS. We investigate third trimester fetal heart rate, routinely recorded within public maternal health care, as a possible marker for LQT1 genotype and phenotype. This retrospective study includes 184 fetuses from 2 LQT1 founder populations segregating p.Y111C and p.R518X (74 noncarriers and 110 KCNQ1 mutation carriers, whereof 13 double mutation carriers). Pedigree-based measured genotype analysis revealed significant associations between fetal heart rate, genotype, and phenotype; mean third trimester prelabor fetal heart rates obtained from obstetric records (gestational week 29-41) were lower per added mutation (no mutation, 143±5 beats per minute; single mutation, 134±8 beats per minute; double mutations, 111±6 beats per minute; P<0.0001), and lower in symptomatic versus asymptomatic mutation carriers (122±10 versus 137±9 beats per minute; P<0.0001). Strong correlations between fetal heart rate and neonatal heart rate (r=0.700; P<0.001), and postnatal QTc (r=-0.762; P<0.001) were found. In a multivariable model, fetal genotype explained the majority of variance in fetal heart rate (-10 beats per minute per added mutation; P<1.0×10(-23)). Arrhythmia symptoms and intrauterine β-blocker exposure each predicted -7 beats per minute, P<0.0001. In this study including 184 fetuses from 2 LQT1 founder populations, third trimester fetal heart rate discriminated between fetal genotypes and correlated with severity of postnatal cardiac phenotype. This finding strengthens the role of fetal heart rate in the early detection and risk stratification of LQTS, particularly for fetuses with double mutations, at high risk of early life-threatening arrhythmias. © 2015 American Heart Association, Inc.

  1. Enhanced Human-Type Receptor Binding by Ferret-Transmissible H5N1 with a K193T Mutation.

    PubMed

    Peng, Wenjie; Bouwman, Kim M; McBride, Ryan; Grant, Oliver C; Woods, Robert J; Verheije, Monique H; Paulson, James C; de Vries, Robert P

    2018-05-15

    All human influenza pandemics have originated from avian influenza viruses. Although multiple changes are needed for an avian virus to be able to transmit between humans, binding to human-type receptors is essential. Several research groups have reported mutations in H5N1 viruses that exhibit specificity for human-type receptors and promote respiratory droplet transmission between ferrets. Upon detailed analysis, we have found that these mutants exhibit significant differences in fine receptor specificity compared to human H1N1 and H3N2 and retain avian-type receptor binding. We have recently shown that human influenza viruses preferentially bind to α2-6-sialylated branched N-linked glycans, where the sialic acids on each branch can bind to receptor sites on two protomers of the same hemagglutinin (HA) trimer. In this binding mode, the glycan projects over the 190 helix at the top of the receptor-binding pocket, which in H5N1 would create a stearic clash with lysine at position 193. Thus, we hypothesized that a K193T mutation would improve binding to branched N-linked receptors. Indeed, the addition of the K193T mutation to the H5 HA of a respiratory-droplet-transmissible virus dramatically improves both binding to human trachea epithelial cells and specificity for extended α2-6-sialylated N-linked glycans recognized by human influenza viruses. IMPORTANCE Infections by avian H5N1 viruses are associated with a high mortality rate in several species, including humans. Fortunately, H5N1 viruses do not transmit between humans because they do not bind to human-type receptors. In 2012, three seminal papers have shown how these viruses can be engineered to transmit between ferrets, the human model for influenza virus infection. Receptor binding, among others, was changed, and the viruses now bind to human-type receptors. Receptor specificity was still markedly different compared to that of human influenza viruses. Here we report an additional mutation in ferret

  2. Male Mutation Bias Is the Main Force Shaping Chromosomal Substitution Rates in Monotreme Mammals.

    PubMed

    Link, Vivian; Aguilar-Gómez, Diana; Ramírez-Suástegui, Ciro; Hurst, Laurence D; Cortez, Diego

    2017-09-01

    In many species, spermatogenesis involves more cell divisions than oogenesis, and the male germline, therefore, accumulates more DNA replication errors, a phenomenon known as male mutation bias. The extent of male mutation bias (α) is estimated by comparing substitution rates of the X, Y, and autosomal chromosomes, as these chromosomes spend different proportions of their time in the germlines of the two sexes. Male mutation bias has been characterized in placental and marsupial mammals as well as birds, but analyses in monotremes failed to detect any such bias. Monotremes are an ancient lineage of egg-laying mammals with distinct biological properties, which include unique germline features. Here, we sought to assess the presence and potential characteristics of male mutation bias in platypus and the short-beaked echidna based on substitution rate analyses of X, Y, and autosomes. We established the presence of moderate male mutation bias in monotremes, corresponding to an α value of 2.12-3.69. Given that it has been unclear what proportion of the variation in substitution rates on the different chromosomal classes is really due to differential number of replications, we analyzed the influence of other confounding forces (selection, replication-timing, etc.) and found that male mutation bias is the main force explaining the between-chromosome classes differences in substitution rates. Finally, we estimated the proportion of variation at the gene level in substitution rates that is owing to replication effects and found that this phenomenon can explain >68% of these variations in monotremes, and in control species, rodents, and primates. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  3. Genetic mutations in human rectal cancers detected by targeted sequencing.

    PubMed

    Bai, Jun; Gao, Jinglong; Mao, Zhijun; Wang, Jianhua; Li, Jianhui; Li, Wensheng; Lei, Yu; Li, Shuaishuai; Wu, Zhuo; Tang, Chuanning; Jones, Lindsey; Ye, Hua; Lou, Feng; Liu, Zhiyuan; Dong, Zhishou; Guo, Baishuai; Huang, Xue F; Chen, Si-Yi; Zhang, Enke

    2015-10-01

    Colorectal cancer (CRC) is widespread with significant mortality. Both inherited and sporadic mutations in various signaling pathways influence the development and progression of the cancer. Identifying genetic mutations in CRC is important for optimal patient treatment and many approaches currently exist to uncover these mutations, including next-generation sequencing (NGS) and commercially available kits. In the present study, we used a semiconductor-based targeted DNA-sequencing approach to sequence and identify genetic mutations in 91 human rectal cancer samples. Analysis revealed frequent mutations in KRAS (58.2%), TP53 (28.6%), APC (16.5%), FBXW7 (9.9%) and PIK3CA (9.9%), and additional mutations in BRAF, CTNNB1, ERBB2 and SMAD4 were also detected at lesser frequencies. Thirty-eight samples (41.8%) also contained two or more mutations, with common combination mutations occurring between KRAS and TP53 (42.1%), and KRAS and APC (31.6%). DNA sequencing for individual cancers is of clinical importance for targeted drug therapy and the advantages of such targeted gene sequencing over other NGS platforms or commercially available kits in sensitivity, cost and time effectiveness may aid clinicians in treating CRC patients in the near future.

  4. Different rates of spontaneous mutation of chloroplastic and nuclear viroids as determined by high-fidelity ultra-deep sequencing.

    PubMed

    López-Carrasco, Amparo; Ballesteros, Cristina; Sentandreu, Vicente; Delgado, Sonia; Gago-Zachert, Selma; Flores, Ricardo; Sanjuán, Rafael

    2017-09-01

    Mutation rates vary by orders of magnitude across biological systems, being higher for simpler genomes. The simplest known genomes correspond to viroids, subviral plant replicons constituted by circular non-coding RNAs of few hundred bases. Previous work has revealed an extremely high mutation rate for chrysanthemum chlorotic mottle viroid, a chloroplast-replicating viroid. However, whether this is a general feature of viroids remains unclear. Here, we have used high-fidelity ultra-deep sequencing to determine the mutation rate in a common host (eggplant) of two viroids, each representative of one family: the chloroplastic eggplant latent viroid (ELVd, Avsunviroidae) and the nuclear potato spindle tuber viroid (PSTVd, Pospiviroidae). This revealed higher mutation frequencies in ELVd than in PSTVd, as well as marked differences in the types of mutations produced. Rates of spontaneous mutation, quantified in vivo using the lethal mutation method, ranged from 1/1000 to 1/800 for ELVd and from 1/7000 to 1/3800 for PSTVd depending on sequencing run. These results suggest that extremely high mutability is a common feature of chloroplastic viroids, whereas the mutation rates of PSTVd and potentially other nuclear viroids appear significantly lower and closer to those of some RNA viruses.

  5. Fungal Infection Increases the Rate of Somatic Mutation in Scots Pine (Pinus sylvestris L.).

    PubMed

    Ranade, Sonali Sachin; Ganea, Laura-Stefana; Razzak, Abdur M; García Gil, M R

    2015-01-01

    Somatic mutations are transmitted during mitosis in developing somatic tissue. Somatic cells bearing the mutations can develop into reproductive (germ) cells and the somatic mutations are then passed on to the next generation of plants. Somatic mutations are a source of variation essential to evolve new defense strategies and adapt to the environment. Stem rust disease in Scots pine has a negative effect on wood quality, and thus adversely affects the economy. It is caused by the 2 most destructive fungal species in Scandinavia: Peridermium pini and Cronartium flaccidum. We studied nuclear genome stability in Scots pine under biotic stress (fungus-infected, 22 trees) compared to a control population (plantation, 20 trees). Stability was assessed as accumulation of new somatic mutations in 10 microsatellite loci selected for genotyping. Microsatellites are widely used as molecular markers in population genetics studies of plants, and are particularly used for detection of somatic mutations as their rate of mutation is of a much higher magnitude when compared with other DNA markers. We report double the rate of somatic mutation per locus in the fungus-infected trees (4.8×10(-3) mutations per locus), as compared to the controls (2.0×10(-3) mutations per locus) when individual samples were analyzed at 10 different microsatellite markers. Pearson's chi-squared test indicated a significant effect of the fungal infection which increased the number of mutations in the fungus-infected trees (χ(2) = 12.9883, df = 1, P = 0.0003134). © The American Genetic Association 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  6. Increase of the spontaneous mutation rate in a long-term experiment with Drosophila melanogaster.

    PubMed

    Avila, Victoria; Chavarrías, David; Sánchez, Enrique; Manrique, Antonio; López-Fanjul, Carlos; García-Dorado, Aurora

    2006-05-01

    In a previous experiment, the effect of 255 generations of mutation accumulation (MA) on the second chromosome viability of Drosophila melanogaster was studied using 200 full-sib MA1 lines and a large C1 control, both derived from a genetically homogeneous base population. At generation 265, one of those MA1 lines was expanded to start 150 new full-sib MA2 lines and a new C2 large control. After 46 generations, the rate of decline in mean viability in MA2 was approximately 2.5 times that estimated in MA1, while the average degree of dominance of mutations was small and nonsignificant by generation 40 and moderate by generation 80. In parallel, the inbreeding depression rate for viability and the amount of additive variance for two bristle traits in C2 were 2-3 times larger than those in C1. The results are consistent with a mutation rate in the line from which MA2 and C2 were derived about 2.5 times larger than that in MA1. The mean viability of C2 remained roughly similar to that of C1, but the rate of MA2 line extinction increased progressively, leading to mutational collapse, which can be ascribed to accelerated mutation and/or synergy after important deleterious accumulation.

  7. Increase of the Spontaneous Mutation Rate in a Long-Term Experiment With Drosophila melanogaster

    PubMed Central

    Ávila, Victoria; Chavarrías, David; Sánchez, Enrique; Manrique, Antonio; López-Fanjul, Carlos; García-Dorado, Aurora

    2006-01-01

    In a previous experiment, the effect of 255 generations of mutation accumulation (MA) on the second chromosome viability of Drosophila melanogaster was studied using 200 full-sib MA1 lines and a large C1 control, both derived from a genetically homogeneous base population. At generation 265, one of those MA1 lines was expanded to start 150 new full-sib MA2 lines and a new C2 large control. After 46 generations, the rate of decline in mean viability in MA2 was ∼2.5 times that estimated in MA1, while the average degree of dominance of mutations was small and nonsignificant by generation 40 and moderate by generation 80. In parallel, the inbreeding depression rate for viability and the amount of additive variance for two bristle traits in C2 were 2–3 times larger than those in C1. The results are consistent with a mutation rate in the line from which MA2 and C2 were derived about 2.5 times larger than that in MA1. The mean viability of C2 remained roughly similar to that of C1, but the rate of MA2 line extinction increased progressively, leading to mutational collapse, which can be ascribed to accelerated mutation and/or synergy after important deleterious accumulation. PMID:16547099

  8. MutHTP: Mutations in Human Transmembrane Proteins.

    PubMed

    A, Kulandaisamy; S, Binny Priya; R, Sakthivel; Tarnovskaya, Svetlana; Bizin, Ilya; Hönigschmid, Peter; Frishman, Dmitrij; Gromiha, M Michael

    2018-02-01

    We have developed a novel database, MutHTP, which contains information on 183395 disease-associated and 17827 neutral mutations in human transmembrane proteins. For each mutation site MutHTP provides a description of its location with respect to the membrane protein topology, structural environment (if available) and functional features. Comprehensive visualization, search, display and download options are available. The database is publicly available at http://www.iitm.ac.in/bioinfo/MutHTP/. The website is implemented using HTML, PHP and javascript and supports recent versions of all major browsers, such as Firefox, Chrome and Opera. gromiha@iitm.ac.in. Supplementary data are available at Bioinformatics online. © The Author (2018). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  9. 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

  10. Germline stem cell competition, mutation hot spots, genetic disorders and older dads

    PubMed Central

    Arnheim, Norman; Calabrese, Peter

    2016-01-01

    Some de novo human mutations arise at frequencies far exceeding the genome average mutation rate. Examples are the common mutations at one or a few sites in the genes causing achondroplasia, Noonan syndrome, multiple endocrine neoplasia 2B and Apert syndrome. These mutations are recurrent, provide a gain of function, are paternally derived and are more likely transmitted as the father ages. Recent experiments tested whether the high mutation frequencies are due to an elevated mutation rate per cell division, as expected, or an advantage of the mutant spermatogonial stem cells over wild-type stem cells. The evidence, which includes the surprising discovery of testis mutation clusters, rejects the former model but not the latter. We propose how the mutations might alter spermatogonial stem cell function and discuss how germline selection contributes to the paternal age effect, the human mutational load and adaptive evolution. PMID:27070266

  11. Pathogenicity of a Human Laminin β2 Mutation Revealed in Models of Alport Syndrome.

    PubMed

    Funk, Steven D; Bayer, Raymond H; Malone, Andrew F; McKee, Karen K; Yurchenco, Peter D; Miner, Jeffrey H

    2018-03-01

    Pierson syndrome is a congenital nephrotic syndrome with eye and neurologic defects caused by mutations in laminin β 2 ( LAMB2 ), a major component of the glomerular basement membrane (GBM). Pathogenic missense mutations in human LAMB2 cluster in or near the laminin amino-terminal (LN) domain, a domain required for extracellular polymerization of laminin trimers and basement membrane scaffolding. Here, we investigated an LN domain missense mutation, LAMB2-S80R, which was discovered in a patient with Pierson syndrome and unusually late onset of proteinuria. Biochemical data indicated that this mutation impairs laminin polymerization, which we hypothesized to be the cause of the patient's nephrotic syndrome. Testing this hypothesis in genetically altered mice showed that the corresponding amino acid change (LAMB2-S83R) alone is not pathogenic. However, expression of LAMB2-S83R significantly increased the rate of progression to kidney failure in a Col4a3 -/- mouse model of autosomal recessive Alport syndrome and increased proteinuria in Col4a5 +/- females that exhibit a mild form of X-linked Alport syndrome due to mosaic deposition of collagen α 3 α 4 α 5(IV) in the GBM. Collectively, these data show the pathogenicity of LAMB2-S80R and provide the first evidence of genetic modification of Alport phenotypes by variation in another GBM component. This finding could help explain the wide range of Alport syndrome onset and severity observed in patients with Alport syndrome, even for family members who share the same COL4 mutation. Our results also show the complexities of using model organisms to investigate genetic variants suspected of being pathogenic in humans. Copyright © 2018 by the American Society of Nephrology.

  12. 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

  13. A novel twelve class fluctuation test reveals higher than expected mutation rates for influenza A viruses

    PubMed Central

    Pauly, Matthew D; Procario, Megan C; Lauring, Adam S

    2017-01-01

    Influenza virus’ low replicative fidelity contributes to its capacity for rapid evolution. Clonal sequencing and fluctuation tests have suggested that the influenza virus mutation rate is 2.7 × 10–6 - 3.0 × 10–5 substitutions per nucleotide per strand copied (s/n/r). However, sequencing assays are biased toward mutations with minimal fitness impacts and fluctuation tests typically investigate only a subset of all possible single nucleotide mutations. We developed a fluctuation test based on reversion to fluorescence in a set of virally encoded mutant green fluorescent proteins, which allowed us to measure the rates of selectively neutral mutations representative of the twelve different mutation types. We measured an overall mutation rate of 1.8 × 10–4 s/n/r for PR8 (H1N1) and 2.5 × 10–4 s/n/r for Hong Kong 2014 (H3N2) and a transitional bias of 2.7–3.6. Our data suggest that each replicated genome will have an average of 2–3 mutations and highlight the importance of mutational load in influenza virus evolution. DOI: http://dx.doi.org/10.7554/eLife.26437.001 PMID:28598328

  14. Functional modules, mutational load and human genetic disease

    PubMed Central

    Zaghloul, Norann A.; Katsanis, Nicholas

    2013-01-01

    The ability to generate a massive amount of sequencing and genotyping data is transforming the study of human genetic disorders. Driven by such innovation, it is likely that whole exome and whole-genome resequencing will replace regionally focused approaches for gene discovery and clinical testing in the next few years. However, this opportunity brings a significant interpretative challenge to assigning function and phenotypic variance to common and rare alleles. Understanding the effect of individual mutations in the context of the remaining genomic variation represents a major challenge to our interpretation of disease. Here, we discuss the challenges of assigning mutation functionality and, drawing from the examples of ciliopathies as well as cohesinopathies and channelopathies, discuss possibilities for the functional modularization of the human genome. Functional modularization in addition to the development of physiologically-relevant assays to test allele functionality will accelerate our understanding of disease architecture and enable the use of genome-wide sequence data for disease diagnosis and phenotypic prediction in individuals. PMID:20226561

  15. A site specific model and analysis of the neutral somatic mutation rate in whole-genome cancer data.

    PubMed

    Bertl, Johanna; Guo, Qianyun; Juul, Malene; Besenbacher, Søren; Nielsen, Morten Muhlig; Hornshøj, Henrik; Pedersen, Jakob Skou; Hobolth, Asger

    2018-04-19

    Detailed modelling of the neutral mutational process in cancer cells is crucial for identifying driver mutations and understanding the mutational mechanisms that act during cancer development. The neutral mutational process is very complex: whole-genome analyses have revealed that the mutation rate differs between cancer types, between patients and along the genome depending on the genetic and epigenetic context. Therefore, methods that predict the number of different types of mutations in regions or specific genomic elements must consider local genomic explanatory variables. A major drawback of most methods is the need to average the explanatory variables across the entire region or genomic element. This procedure is particularly problematic if the explanatory variable varies dramatically in the element under consideration. To take into account the fine scale of the explanatory variables, we model the probabilities of different types of mutations for each position in the genome by multinomial logistic regression. We analyse 505 cancer genomes from 14 different cancer types and compare the performance in predicting mutation rate for both regional based models and site-specific models. We show that for 1000 randomly selected genomic positions, the site-specific model predicts the mutation rate much better than regional based models. We use a forward selection procedure to identify the most important explanatory variables. The procedure identifies site-specific conservation (phyloP), replication timing, and expression level as the best predictors for the mutation rate. Finally, our model confirms and quantifies certain well-known mutational signatures. We find that our site-specific multinomial regression model outperforms the regional based models. The possibility of including genomic variables on different scales and patient specific variables makes it a versatile framework for studying different mutational mechanisms. Our model can serve as the neutral null model

  16. A novel mouse model of Niemann-Pick type C disease carrying a D1005G-Npc1 mutation comparable to commonly observed human mutations.

    PubMed

    Maue, Robert A; Burgess, Robert W; Wang, Bing; Wooley, Christine M; Seburn, Kevin L; Vanier, Marie T; Rogers, Maximillian A; Chang, Catherine C; Chang, Ta-Yuan; Harris, Brent T; Graber, David J; Penatti, Carlos A A; Porter, Donna M; Szwergold, Benjamin S; Henderson, Leslie P; Totenhagen, John W; Trouard, Theodore P; Borbon, Ivan A; Erickson, Robert P

    2012-02-15

    We have identified a point mutation in Npc1 that creates a novel mouse model (Npc1(nmf164)) of Niemann-Pick type C1 (NPC) disease: a single nucleotide change (A to G at cDNA bp 3163) that results in an aspartate to glycine change at position 1005 (D1005G). This change is in the cysteine-rich luminal loop of the NPC1 protein and is highly similar to commonly occurring human mutations. Genetic and molecular biological analyses, including sequencing the Npc1(spm) allele and identifying a truncating mutation, confirm that the mutation in Npc1(nmf164) mice is distinct from those in other existing mouse models of NPC disease (Npc1(nih), Npc1(spm)). Analyses of lifespan, body and spleen weight, gait and other motor activities, as well as acoustic startle responses all reveal a more slowly developing phenotype in Npc1(nmf164) mutant mice than in mice with the null mutations (Npc1(nih), Npc1(spm)). Although Npc1 mRNA levels appear relatively normal, Npc1(nmf164) brain and liver display dramatic reductions in Npc1 protein, as well as abnormal cholesterol metabolism and altered glycolipid expression. Furthermore, histological analyses of liver, spleen, hippocampus, cortex and cerebellum reveal abnormal cholesterol accumulation, glial activation and Purkinje cell loss at a slower rate than in the Npc1(nih) mouse model. Magnetic resonance imaging studies also reveal significantly less demyelination/dysmyelination than in the null alleles. Thus, although prior mouse models may correspond to the severe infantile onset forms of NPC disease, Npc1(nmf164) mice offer many advantages as a model for the late-onset, more slowly progressing forms of NPC disease that comprise the large majority of human cases.

  17. A novel mouse model of Niemann–Pick type C disease carrying a D1005G-Npc1 mutation comparable to commonly observed human mutations

    PubMed Central

    Maue, Robert A.; Burgess, Robert W.; Wang, Bing; Wooley, Christine M.; Seburn, Kevin L.; Vanier, Marie T.; Rogers, Maximillian A.; Chang, Catherine C.; Chang, Ta-Yuan; Harris, Brent T.; Graber, David J.; Penatti, Carlos A.A.; Porter, Donna M.; Szwergold, Benjamin S.; Henderson, Leslie P.; Totenhagen, John W.; Trouard, Theodore P.; Borbon, Ivan A.; Erickson, Robert P.

    2012-01-01

    We have identified a point mutation in Npc1 that creates a novel mouse model (Npc1nmf164) of Niemann–Pick type C1 (NPC) disease: a single nucleotide change (A to G at cDNA bp 3163) that results in an aspartate to glycine change at position 1005 (D1005G). This change is in the cysteine-rich luminal loop of the NPC1 protein and is highly similar to commonly occurring human mutations. Genetic and molecular biological analyses, including sequencing the Npc1spm allele and identifying a truncating mutation, confirm that the mutation in Npc1nmf164 mice is distinct from those in other existing mouse models of NPC disease (Npc1nih, Npc1spm). Analyses of lifespan, body and spleen weight, gait and other motor activities, as well as acoustic startle responses all reveal a more slowly developing phenotype in Npc1nmf164 mutant mice than in mice with the null mutations (Npc1nih, Npc1spm). Although Npc1 mRNA levels appear relatively normal, Npc1nmf164 brain and liver display dramatic reductions in Npc1 protein, as well as abnormal cholesterol metabolism and altered glycolipid expression. Furthermore, histological analyses of liver, spleen, hippocampus, cortex and cerebellum reveal abnormal cholesterol accumulation, glial activation and Purkinje cell loss at a slower rate than in the Npc1nih mouse model. Magnetic resonance imaging studies also reveal significantly less demyelination/dysmyelination than in the null alleles. Thus, although prior mouse models may correspond to the severe infantile onset forms of NPC disease, Npc1nmf164 mice offer many advantages as a model for the late-onset, more slowly progressing forms of NPC disease that comprise the large majority of human cases. PMID:22048958

  18. 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.

  19. Genetic mutation analysis of human gastric adenocarcinomas using ion torrent sequencing platform.

    PubMed

    Xu, Zhi; Huo, Xinying; Ye, Hua; Tang, Chuanning; Nandakumar, Vijayalakshmi; Lou, Feng; Zhang, Dandan; Dong, Haichao; Sun, Hong; Jiang, Shouwen; Zhang, Guangchun; Liu, Zhiyuan; Dong, Zhishou; Guo, Baishuai; He, Yan; Yan, Chaowei; Wang, Lu; Su, Ziyi; Li, Yangyang; Gu, Dongying; Zhang, Xiaojing; Wu, Xiaomin; Wei, Xiaowei; Hong, Lingzhi; Zhang, Yangmei; Yang, Jinsong; Gong, Yonglin; Tang, Cuiju; Jones, Lindsey; Huang, Xue F; Chen, Si-Yi; Chen, Jinfei

    2014-01-01

    Gastric cancer is the one of the major causes of cancer-related death, especially in Asia. Gastric adenocarcinoma, the most common type of gastric cancer, is heterogeneous and its incidence and cause varies widely with geographical regions, gender, ethnicity, and diet. Since unique mutations have been observed in individual human cancer samples, identification and characterization of the molecular alterations underlying individual gastric adenocarcinomas is a critical step for developing more effective, personalized therapies. Until recently, identifying genetic mutations on an individual basis by DNA sequencing remained a daunting task. Recent advances in new next-generation DNA sequencing technologies, such as the semiconductor-based Ion Torrent sequencing platform, makes DNA sequencing cheaper, faster, and more reliable. In this study, we aim to identify genetic mutations in the genes which are targeted by drugs in clinical use or are under development in individual human gastric adenocarcinoma samples using Ion Torrent sequencing. We sequenced 737 loci from 45 cancer-related genes in 238 human gastric adenocarcinoma samples using the Ion Torrent Ampliseq Cancer Panel. The sequencing analysis revealed a high occurrence of mutations along the TP53 locus (9.7%) in our sample set. Thus, this study indicates the utility of a cost and time efficient tool such as Ion Torrent sequencing to screen cancer mutations for the development of personalized cancer therapy.

  20. An approximate stationary solution for multi-allele neutral diffusion with low mutation rates.

    PubMed

    Burden, Conrad J; Tang, Yurong

    2016-12-01

    We address the problem of determining the stationary distribution of the multi-allelic, neutral-evolution Wright-Fisher model in the diffusion limit. A full solution to this problem for an arbitrary K×K mutation rate matrix involves solving for the stationary solution of a forward Kolmogorov equation over a (K-1)-dimensional simplex, and remains intractable. In most practical situations mutations rates are slow on the scale of the diffusion limit and the solution is heavily concentrated on the corners and edges of the simplex. In this paper we present a practical approximate solution for slow mutation rates in the form of a set of line densities along the edges of the simplex. The method of solution relies on parameterising the general non-reversible rate matrix as the sum of a reversible part and a set of (K-1)(K-2)/2 independent terms corresponding to fluxes of probability along closed paths around faces of the simplex. The solution is potentially a first step in estimating non-reversible evolutionary rate matrices from observed allele frequency spectra. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations

    PubMed Central

    Kamitaki, Nolan; Mitchell, Jana; Avior, Yishai; Mello, Curtis; Kashin, Seva; Mekhoubad, Shila; Ilic, Dusko; Charlton, Maura; Saphier, Genevieve; Handsaker, Robert E.; Genovese, Giulio; Bar, Shiran; Benvenisty, Nissim; McCarroll, Steven A.; Eggan, Kevin

    2017-01-01

    Human pluripotent stem cells (hPSCs) can self-renew indefinitely, making them an attractive source for regenerative therapies. This expansion potential has been linked with acquisition of large copy number variants (CNVs) that provide mutant cells with a growth advantage in culture1–3. However, the nature, extent, and functional impact of other acquired genome sequence mutations in cultured hPSCs is not known. Here, we sequenced the protein-coding genes (exomes) of 140 independent human embryonic stem cell (hESC) lines, including 26 lines prepared for potential clinical use4. We then applied computational strategies for identifying mutations present in a subset of cells5. Though such mosaic mutations were generally rare, we identified five unrelated hESC lines that carried six mutations in the TP53 gene that encodes the tumor suppressor P53. Notably, the TP53 mutations we observed are dominant negative and are the mutations most commonly seen in human cancers. We used droplet digital PCR to demonstrate that the TP53 mutant allelic fraction increased with passage number under standard culture conditions, suggesting that P53 mutation confers selective advantage. When we then mined published RNA sequencing data from 117 hPSC lines, we observed another nine TP53 mutations, all resulting in coding changes in the DNA binding domain of P53. Strikingly, in three lines, the allelic fraction exceeded 50%, suggesting additional selective advantage resulting from loss of heterozygosity at the TP53 locus. As the acquisition and favored expansion of cancer-associated mutations in hPSCs may go unnoticed during most applications, we suggest that careful genetic characterization of hPSCs and their differentiated derivatives should be carried out prior to clinical use. PMID:28445466

  2. Different rates of spontaneous mutation of chloroplastic and nuclear viroids as determined by high-fidelity ultra-deep sequencing

    PubMed Central

    Ballesteros, Cristina; Sentandreu, Vicente; Gago-Zachert, Selma

    2017-01-01

    Mutation rates vary by orders of magnitude across biological systems, being higher for simpler genomes. The simplest known genomes correspond to viroids, subviral plant replicons constituted by circular non-coding RNAs of few hundred bases. Previous work has revealed an extremely high mutation rate for chrysanthemum chlorotic mottle viroid, a chloroplast-replicating viroid. However, whether this is a general feature of viroids remains unclear. Here, we have used high-fidelity ultra-deep sequencing to determine the mutation rate in a common host (eggplant) of two viroids, each representative of one family: the chloroplastic eggplant latent viroid (ELVd, Avsunviroidae) and the nuclear potato spindle tuber viroid (PSTVd, Pospiviroidae). This revealed higher mutation frequencies in ELVd than in PSTVd, as well as marked differences in the types of mutations produced. Rates of spontaneous mutation, quantified in vivo using the lethal mutation method, ranged from 1/1000 to 1/800 for ELVd and from 1/7000 to 1/3800 for PSTVd depending on sequencing run. These results suggest that extremely high mutability is a common feature of chloroplastic viroids, whereas the mutation rates of PSTVd and potentially other nuclear viroids appear significantly lower and closer to those of some RNA viruses. PMID:28910391

  3. Quantitative evaluation of DNA damage and mutation rate by atmospheric and room-temperature plasma (ARTP) and conventional mutagenesis.

    PubMed

    Zhang, Xue; Zhang, Chong; Zhou, Qian-Qian; Zhang, Xiao-Fei; Wang, Li-Yan; Chang, Hai-Bo; Li, He-Ping; Oda, Yoshimitsu; Xing, Xin-Hui

    2015-07-01

    DNA damage is the dominant source of mutation, which is the driving force of evolution. Therefore, it is important to quantitatively analyze the DNA damage caused by different mutagenesis methods, the subsequent mutation rates, and their relationship. Atmospheric and room temperature plasma (ARTP) mutagenesis has been used for the mutation breeding of more than 40 microorganisms. However, ARTP mutagenesis has not been quantitatively compared with conventional mutation methods. In this study, the umu test using a flow-cytometric analysis was developed to quantify the DNA damage in individual viable cells using Salmonella typhimurium NM2009 as the model strain and to determine the mutation rate. The newly developed method was used to evaluate four different mutagenesis systems: a new ARTP tool, ultraviolet radiation, 4-nitroquinoline-1-oxide (4-NQO), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis. The mutation rate was proportional to the corresponding SOS response induced by DNA damage. ARTP caused greater DNA damage to individual living cells than the other conventional mutagenesis methods, and the mutation rate was also higher. By quantitatively comparing the DNA damage and consequent mutation rate after different types of mutagenesis, we have shown that ARTP is a potentially powerful mutagenesis tool with which to improve the characteristics of microbial cell factories.

  4. Ultraviolet A within Sunlight Induces Mutations in the Epidermal Basal Layer of Engineered Human Skin

    PubMed Central

    Huang, Xiao Xuan; Bernerd, Françoise; Halliday, Gary Mark

    2009-01-01

    The ultraviolet B (UVB) waveband within sunlight is an important carcinogen; however, UVA is also likely to be involved. By ascribing mutations to being either UVB or UVA induced, we have previously shown that human skin cancers contain similar numbers of UVB- and UVA-induced mutations, and, importantly, the UVA mutations were at the base of the epidermis of the tumors. To determine whether these mutations occurred in response to UV, we exposed engineered human skin (EHS) to UVA, UVB, or a mixture that resembled sunlight, and then detected mutations by both denaturing high-performance liquid chromatography and DNA sequencing. EHS resembles human skin, modeling differential waveband penetration to the basal, dividing keratinocytes. We administered only four low doses of UV exposure. Both UVA and UVB induced p53 mutations in irradiated EHS, suggesting that sunlight doses that are achievable during normal daily activities are mutagenic. UVA- but not UVB-induced mutations predominated in the basal epidermis that contains dividing keratinocytes and are thought to give rise to skin tumors. These studies indicate that both UVA and UVB at physiological doses are mutagenic to keratinocytes in EHS. PMID:19264911

  5. Elevated heart rate triggers action potential alternans and sudden death. translational study of a homozygous KCNH2 mutation.

    PubMed

    Schweigmann, Ulrich; Biliczki, Peter; Ramirez, Rafael J; Marschall, Christoph; Takac, Ina; Brandes, Ralf P; Kotzot, Dieter; Girmatsion, Zenawit; Hohnloser, Stefan H; Ehrlich, Joachim R

    2014-01-01

    Long QT syndrome (LQTS) leads to arrhythmic events and increased risk for sudden cardiac death (SCD). Homozygous KCNH2 mutations underlying LQTS-2 have previously been termed "human HERG knockout" and typically express severe phenotypes. We studied genotype-phenotype correlations of an LQTS type 2 mutation identified in the homozygous index patient from a consanguineous Turkish family after his brother died suddenly during febrile illness. Clinical work-up, DNA sequencing, mutagenesis, cell culture, patch-clamp, in silico mathematical modelling, protein biochemistry, confocal microscopy were performed. Genetic analysis revealed a homozygous C-terminal KCNH2 mutation (p.R835Q) in the index patient (QTc ∼506 ms with notched T waves). Parents were I° cousins - both heterozygous for the mutation and clinically unremarkable (QTc ∼447 ms, father and ∼396 ms, mother). Heterologous expression of KCNH2-R835Q showed mildly reduced current amplitudes. Biophysical properties of ionic currents were also only nominally changed with slight acceleration of deactivation and more negative V50 in R835Q-currents. Protein biochemistry and confocal microscopy revealed similar expression patterns and trafficking of WT and R835Q, even at elevated temperature. In silico analysis demonstrated mildly prolonged ventricular action potential duration (APD) compared to WT at a cycle length of 1000 ms. At a cycle length of 350 ms M-cell APD remained stable in WT, but displayed APD alternans in R835Q. Kv11.1 channels affected by the C-terminal R835Q mutation display mildly modified biophysical properties, but leads to M-cell APD alternans with elevated heart rate and could precipitate SCD under specific clinical circumstances associated with high heart rates.

  6. Fast maximum likelihood estimation of mutation rates using a birth-death process.

    PubMed

    Wu, Xiaowei; Zhu, Hongxiao

    2015-02-07

    Since fluctuation analysis was first introduced by Luria and Delbrück in 1943, it has been widely used to make inference about spontaneous mutation rates in cultured cells. Under certain model assumptions, the probability distribution of the number of mutants that appear in a fluctuation experiment can be derived explicitly, which provides the basis of mutation rate estimation. It has been shown that, among various existing estimators, the maximum likelihood estimator usually demonstrates some desirable properties such as consistency and lower mean squared error. However, its application in real experimental data is often hindered by slow computation of likelihood due to the recursive form of the mutant-count distribution. We propose a fast maximum likelihood estimator of mutation rates, MLE-BD, based on a birth-death process model with non-differential growth assumption. Simulation studies demonstrate that, compared with the conventional maximum likelihood estimator derived from the Luria-Delbrück distribution, MLE-BD achieves substantial improvement on computational speed and is applicable to arbitrarily large number of mutants. In addition, it still retains good accuracy on point estimation. Published by Elsevier Ltd.

  7. 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

  8. 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

  9. Prediction of phenotypes of missense mutations in human proteins from biological assemblies.

    PubMed

    Wei, Qiong; Xu, Qifang; Dunbrack, Roland L

    2013-02-01

    Single nucleotide polymorphisms (SNPs) are the most frequent variation in the human genome. Nonsynonymous SNPs that lead to missense mutations can be neutral or deleterious, and several computational methods have been presented that predict the phenotype of human missense mutations. These methods use sequence-based and structure-based features in various combinations, relying on different statistical distributions of these features for deleterious and neutral mutations. One structure-based feature that has not been studied significantly is the accessible surface area within biologically relevant oligomeric assemblies. These assemblies are different from the crystallographic asymmetric unit for more than half of X-ray crystal structures. We find that mutations in the core of proteins or in the interfaces in biological assemblies are significantly more likely to be disease-associated than those on the surface of the biological assemblies. For structures with more than one protein in the biological assembly (whether the same sequence or different), we find the accessible surface area from biological assemblies provides a statistically significant improvement in prediction over the accessible surface area of monomers from protein crystal structures (P = 6e-5). When adding this information to sequence-based features such as the difference between wildtype and mutant position-specific profile scores, the improvement from biological assemblies is statistically significant but much smaller (P = 0.018). Combining this information with sequence-based features in a support vector machine leads to 82% accuracy on a balanced dataset of 50% disease-associated mutations from SwissVar and 50% neutral mutations from human/primate sequence differences in orthologous proteins. Copyright © 2012 Wiley Periodicals, Inc.

  10. The rate and potential relevance of new mutations in a colonizing plant lineage

    PubMed Central

    Schuenemann, Verena J.; Reiter, Ella; Setzer, Claudia; Slovak, Radka; Brachi, Benjamin; Hagmann, Jörg; Grimm, Dominik G.; Chen, Jiahui; Ness, Rob W.

    2018-01-01

    By following the evolution of populations that are initially genetically homogeneous, much can be learned about core biological principles. For example, it allows for detailed studies of the rate of emergence of de novo mutations and their change in frequency due to drift and selection. Unfortunately, in multicellular organisms with generation times of months or years, it is difficult to set up and carry out such experiments over many generations. An alternative is provided by “natural evolution experiments” that started from colonizations or invasions of new habitats by selfing lineages. With limited or missing gene flow from other lineages, new mutations and their effects can be easily detected. North America has been colonized in historic times by the plant Arabidopsis thaliana, and although multiple intercrossing lineages are found today, many of the individuals belong to a single lineage, HPG1. To determine in this lineage the rate of substitutions—the subset of mutations that survived natural selection and drift–, we have sequenced genomes from plants collected between 1863 and 2006. We identified 73 modern and 27 herbarium specimens that belonged to HPG1. Using the estimated substitution rate, we infer that the last common HPG1 ancestor lived in the early 17th century, when it was most likely introduced by chance from Europe. Mutations in coding regions are depleted in frequency compared to those in other portions of the genome, consistent with purifying selection. Nevertheless, a handful of mutations is found at high frequency in present-day populations. We link these to detectable phenotypic variance in traits of known ecological importance, life history and growth, which could reflect their adaptive value. Our work showcases how, by applying genomics methods to a combination of modern and historic samples from colonizing lineages, we can directly study new mutations and their potential evolutionary relevance. PMID:29432421

  11. Perturbed length-dependent activation in human hypertrophic cardiomyopathy with missense sarcomeric gene mutations.

    PubMed

    Sequeira, Vasco; Wijnker, Paul J M; Nijenkamp, Louise L A M; Kuster, Diederik W D; Najafi, Aref; Witjas-Paalberends, E Rosalie; Regan, Jessica A; Boontje, Nicky; Ten Cate, Folkert J; Germans, Tjeerd; Carrier, Lucie; Sadayappan, Sakthivel; van Slegtenhorst, Marjon A; Zaremba, Ruud; Foster, D Brian; Murphy, Anne M; Poggesi, Corrado; Dos Remedios, Cris; Stienen, Ger J M; Ho, Carolyn Y; Michels, Michelle; van der Velden, Jolanda

    2013-05-24

    High-myofilament Ca(2+) sensitivity has been proposed as a trigger of disease pathogenesis in familial hypertrophic cardiomyopathy (HCM) on the basis of in vitro and transgenic mice studies. However, myofilament Ca(2+) sensitivity depends on protein phosphorylation and muscle length, and at present, data in humans are scarce. To investigate whether high myofilament Ca(2+) sensitivity and perturbed length-dependent activation are characteristics for human HCM with mutations in thick and thin filament proteins. Cardiac samples from patients with HCM harboring mutations in genes encoding thick (MYH7, MYBPC3) and thin (TNNT2, TNNI3, TPM1) filament proteins were compared with sarcomere mutation-negative HCM and nonfailing donors. Cardiomyocyte force measurements showed higher myofilament Ca(2+) sensitivity in all HCM samples and low phosphorylation of protein kinase A (PKA) targets compared with donors. After exogenous PKA treatment, myofilament Ca(2+) sensitivity was similar (MYBPC3mut, TPM1mut, sarcomere mutation-negative HCM), higher (MYH7mut, TNNT2mut), or even significantly lower (TNNI3mut) compared with donors. Length-dependent activation was significantly smaller in all HCM than in donor samples. PKA treatment increased phosphorylation of PKA-targets in HCM myocardium and normalized length-dependent activation to donor values in sarcomere mutation-negative HCM and HCM with truncating MYBPC3 mutations but not in HCM with missense mutations. Replacement of mutant by wild-type troponin in TNNT2mut and TNNI3mut corrected length-dependent activation to donor values. High-myofilament Ca(2+) sensitivity is a common characteristic of human HCM and partly reflects hypophosphorylation of PKA targets compared with donors. Length-dependent sarcomere activation is perturbed by missense mutations, possibly via posttranslational modifications other than PKA hypophosphorylation or altered protein-protein interactions, and represents a common pathomechanism in HCM.

  12. Prediction of change in protein unfolding rates upon point mutations in two state proteins.

    PubMed

    Chaudhary, Priyashree; Naganathan, Athi N; Gromiha, M Michael

    2016-09-01

    Studies on protein unfolding rates are limited and challenging due to the complexity of unfolding mechanism and the larger dynamic range of the experimental data. Though attempts have been made to predict unfolding rates using protein sequence-structure information there is no available method for predicting the unfolding rates of proteins upon specific point mutations. In this work, we have systematically analyzed a set of 790 single mutants and developed a robust method for predicting protein unfolding rates upon mutations (Δlnku) in two-state proteins by combining amino acid properties and knowledge-based classification of mutants with multiple linear regression technique. We obtain a mean absolute error (MAE) of 0.79/s and a Pearson correlation coefficient (PCC) of 0.71 between predicted unfolding rates and experimental observations using jack-knife test. We have developed a web server for predicting protein unfolding rates upon mutation and it is freely available at https://www.iitm.ac.in/bioinfo/proteinunfolding/unfoldingrace.html. Prominent features that determine unfolding kinetics as well as plausible reasons for the observed outliers are also discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. A homozygous missense mutation in human KLOTHO causes severe tumoral calcinosis

    PubMed Central

    Ichikawa, Shoji; Imel, Erik A.; Kreiter, Mary L.; Yu, Xijie; Mackenzie, Donald S.; Sorenson, Andrea H.; Goetz, Regina; Mohammadi, Moosa; White, Kenneth E.; Econs, Michael J.

    2007-01-01

    Familial tumoral calcinosis is characterized by ectopic calcifications and hyperphosphatemia due to inactivating mutations in FGF23 or UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3). Herein we report a homozygous missense mutation (H193R) in the KLOTHO (KL) gene of a 13-year-old girl who presented with severe tumoral calcinosis with dural and carotid artery calcifications. This patient exhibited defects in mineral ion homeostasis with marked hyperphosphatemia and hypercalcemia as well as elevated serum levels of parathyroid hormone and FGF23. Mapping of H193R mutation onto the crystal structure of myrosinase, a plant homolog of KL, revealed that this histidine residue was at the base of the deep catalytic cleft and mutation of this histidine to arginine should destabilize the putative glycosidase domain (KL1) of KL, thereby attenuating production of membrane-bound and secreted KL. Indeed, compared with wild-type KL, expression and secretion of H193R KL were markedly reduced in vitro, resulting in diminished ability of FGF23 to signal via its cognate FGF receptors. Taken together, our findings provide what we believe to be the first evidence that loss-of-function mutations in human KL impair FGF23 bioactivity, underscoring the essential role of KL in FGF23-mediated phosphate and vitamin D homeostasis in humans. PMID:17710231

  14. Experimental Estimation of Mutation Rates in a Wheat Population With a Gene Genealogy Approach

    PubMed Central

    Raquin, Anne-Laure; Depaulis, Frantz; Lambert, Amaury; Galic, Nathalie; Brabant, Philippe; Goldringer, Isabelle

    2008-01-01

    Microsatellite markers are extensively used to evaluate genetic diversity in natural or experimental evolving populations. Their high degree of polymorphism reflects their high mutation rates. Estimates of the mutation rates are therefore necessary when characterizing diversity in populations. As a complement to the classical experimental designs, we propose to use experimental populations, where the initial state is entirely known and some intermediate states have been thoroughly surveyed, thus providing a short timescale estimation together with a large number of cumulated meioses. In this article, we derived four original gene genealogy-based methods to assess mutation rates with limited bias due to relevant model assumptions incorporating the initial state, the number of new alleles, and the genetic effective population size. We studied the evolution of genetic diversity at 21 microsatellite markers, after 15 generations in an experimental wheat population. Compared to the parents, 23 new alleles were found in generation 15 at 9 of the 21 loci studied. We provide evidence that they arose by mutation. Corresponding estimates of the mutation rates ranged from 0 to 4.97 × 10−3 per generation (i.e., year). Sequences of several alleles revealed that length polymorphism was only due to variation in the core of the microsatellite. Among different microsatellite characteristics, both the motif repeat number and an independent estimation of the Nei diversity were correlated with the novel diversity. Despite a reduced genetic effective size, global diversity at microsatellite markers increased in this population, suggesting that microsatellite diversity should be used with caution as an indicator in biodiversity conservation issues. PMID:18689900

  15. Experimental estimation of mutation rates in a wheat population with a gene genealogy approach.

    PubMed

    Raquin, Anne-Laure; Depaulis, Frantz; Lambert, Amaury; Galic, Nathalie; Brabant, Philippe; Goldringer, Isabelle

    2008-08-01

    Microsatellite markers are extensively used to evaluate genetic diversity in natural or experimental evolving populations. Their high degree of polymorphism reflects their high mutation rates. Estimates of the mutation rates are therefore necessary when characterizing diversity in populations. As a complement to the classical experimental designs, we propose to use experimental populations, where the initial state is entirely known and some intermediate states have been thoroughly surveyed, thus providing a short timescale estimation together with a large number of cumulated meioses. In this article, we derived four original gene genealogy-based methods to assess mutation rates with limited bias due to relevant model assumptions incorporating the initial state, the number of new alleles, and the genetic effective population size. We studied the evolution of genetic diversity at 21 microsatellite markers, after 15 generations in an experimental wheat population. Compared to the parents, 23 new alleles were found in generation 15 at 9 of the 21 loci studied. We provide evidence that they arose by mutation. Corresponding estimates of the mutation rates ranged from 0 to 4.97 x 10(-3) per generation (i.e., year). Sequences of several alleles revealed that length polymorphism was only due to variation in the core of the microsatellite. Among different microsatellite characteristics, both the motif repeat number and an independent estimation of the Nei diversity were correlated with the novel diversity. Despite a reduced genetic effective size, global diversity at microsatellite markers increased in this population, suggesting that microsatellite diversity should be used with caution as an indicator in biodiversity conservation issues.

  16. Clinical and pathological characterization of HER2 mutations in human breast cancer: a systematic review of the literature.

    PubMed

    Petrelli, Fausto; Tomasello, Gianluca; Barni, Sandro; Lonati, Veronica; Passalacqua, Rodolfo; Ghidini, Michele

    2017-11-01

    HER2 gene is a member of the epidermal growth factor receptor (EGFR) family. Across different malignancies, aberrations of HER2 gene commonly correspond to gain-of-function alterations leading to increased receptor signaling. We have reviewed the literature currently available on HER2 mutations in human breast cancer (BC) evaluating type and frequency of such mutations. The primary objective was to determine the frequency and the number of patients with HER2-mut in the series analyzed. The secondary objectives were to assess characteristics of mutated cases (ER and HER2 status and stage of disease, type of mutations, and finally the clinical outcome if reported). We retrieved 31 published papers, and the pooled rate of HER2 mutations across 12,905 BC patients was calculated. Overall, the frequency of HER2 mutations was 2.7% with most involving the intracellular domain. About 4% of patients were finally mutated. The predictive role was not described. Only 30% of these patients were simultaneously HER2 positive and 63% were ER positive. We have found that the prevalence of HER2 mutations is about 3%. These genic alterations are independently associated with HER2 amplification status, occurring in both ER-positive/HER2-negative diseases or HER2-enriched cancers. Ongoing trials are investigating small molecules tyrosine kinase inhibitors in patients harboring these mutations.

  17. Biallelic mutations in IRF8 impair human NK cell maturation and function

    PubMed Central

    Mace, Emily M.; Gunesch, Justin T.; Chinn, Ivan K.; Angelo, Laura S.; Maisuria, Sheetal; Keller, Michael D.; Togi, Sumihito; Watkin, Levi B.; LaRosa, David F.; Jhangiani, Shalini N.; Muzny, Donna M.; Stray-Pedersen, Asbjørg; Coban Akdemir, Zeynep; Smith, Jansen B.; Hernández-Sanabria, Mayra; Le, Duy T.; Hogg, Graham D.; Cao, Tram N.; Freud, Aharon G.; Szymanski, Eva P.; Collin, Matthew; Cant, Andrew J.; Gibbs, Richard A.; Holland, Steven M.; Caligiuri, Michael A.; Ozato, Keiko; Paust, Silke; Doody, Gina M.; Lupski, James R.; Orange, Jordan S.

    2016-01-01

    Human NK cell deficiencies are rare yet result in severe and often fatal disease, particularly as a result of viral susceptibility. NK cells develop from hematopoietic stem cells, and few monogenic errors that specifically interrupt NK cell development have been reported. Here we have described biallelic mutations in IRF8, which encodes an interferon regulatory factor, as a cause of familial NK cell deficiency that results in fatal and severe viral disease. Compound heterozygous or homozygous mutations in IRF8 in 3 unrelated families resulted in a paucity of mature CD56dim NK cells and an increase in the frequency of the immature CD56bright NK cells, and this impairment in terminal maturation was also observed in Irf8–/–, but not Irf8+/–, mice. We then determined that impaired maturation was NK cell intrinsic, and gene expression analysis of human NK cell developmental subsets showed that multiple genes were dysregulated by IRF8 mutation. The phenotype was accompanied by deficient NK cell function and was stable over time. Together, these data indicate that human NK cells require IRF8 for development and functional maturation and that dysregulation of this function results in severe human disease, thereby emphasizing a critical role for NK cells in human antiviral defense. PMID:27893462

  18. Biallelic mutations in IRF8 impair human NK cell maturation and function.

    PubMed

    Mace, Emily M; Bigley, Venetia; Gunesch, Justin T; Chinn, Ivan K; Angelo, Laura S; Care, Matthew A; Maisuria, Sheetal; Keller, Michael D; Togi, Sumihito; Watkin, Levi B; LaRosa, David F; Jhangiani, Shalini N; Muzny, Donna M; Stray-Pedersen, Asbjørg; Coban Akdemir, Zeynep; Smith, Jansen B; Hernández-Sanabria, Mayra; Le, Duy T; Hogg, Graham D; Cao, Tram N; Freud, Aharon G; Szymanski, Eva P; Savic, Sinisa; Collin, Matthew; Cant, Andrew J; Gibbs, Richard A; Holland, Steven M; Caligiuri, Michael A; Ozato, Keiko; Paust, Silke; Doody, Gina M; Lupski, James R; Orange, Jordan S

    2017-01-03

    Human NK cell deficiencies are rare yet result in severe and often fatal disease, particularly as a result of viral susceptibility. NK cells develop from hematopoietic stem cells, and few monogenic errors that specifically interrupt NK cell development have been reported. Here we have described biallelic mutations in IRF8, which encodes an interferon regulatory factor, as a cause of familial NK cell deficiency that results in fatal and severe viral disease. Compound heterozygous or homozygous mutations in IRF8 in 3 unrelated families resulted in a paucity of mature CD56dim NK cells and an increase in the frequency of the immature CD56bright NK cells, and this impairment in terminal maturation was also observed in Irf8-/-, but not Irf8+/-, mice. We then determined that impaired maturation was NK cell intrinsic, and gene expression analysis of human NK cell developmental subsets showed that multiple genes were dysregulated by IRF8 mutation. The phenotype was accompanied by deficient NK cell function and was stable over time. Together, these data indicate that human NK cells require IRF8 for development and functional maturation and that dysregulation of this function results in severe human disease, thereby emphasizing a critical role for NK cells in human antiviral defense.

  19. Testing computational prediction of missense mutation phenotypes: Functional characterization of 204 mutations of human cystathionine beta synthase

    PubMed Central

    Wei, Qiong; Wang, Liqun; Wang, Qiang; Kruger, Warren D.; Dunbrack, Roland L.

    2010-01-01

    Predicting the phenotypes of missense mutations uncovered by large-scale sequencing projects is an important goal in computational biology. High-confidence predictions can be an aid in focusing experimental and association studies on those mutations most likely to be associated with causative relationships between mutation and disease. As an aid in developing these methods further, we have derived a set of random mutations of the enzymatic domains of human cystathionine beta synthase. This enzyme is a dimeric protein that catalyzes the condensation of serine and homocysteine to produce cystathionine. Yeast missing this enzyme cannot grow on medium lacking a source of cysteine, while transfection of functional human CBS into yeast strains missing endogenous enzyme can successfully complement for the missing gene. We used PCR mutagenesis with error-prone Taq polymerase to produce 948 colonies, and compared cell growth in the presence or absence of a cysteine source as a measure of CBS function. We were able to infer the phenotypes of 204 single-site mutants, 79 of them deleterious and 125 neutral. This set was used to test the accuracy of six publicly available prediction methods for phenotype prediction of missense mutations: SIFT, PolyPhen, PMut, SNPs3D, PhD-SNP, and nsSNPAnalyzer. The top methods are PolyPhen, SIFT, and nsSNPAnalyzer, which have similar performance. Using kernel discriminant functions, we found that the difference in position-specific scoring matrix values is more predictive than the wild-type PSSM score alone, and that the relative surface area in the biologically relevant complex is more predictive than that of the monomeric proteins. PMID:20455263

  20. Hypomorphic PCNA mutation underlies a human DNA repair disorder

    PubMed Central

    Baple, Emma L.; Chambers, Helen; Cross, Harold E.; Fawcett, Heather; Nakazawa, Yuka; Chioza, Barry A.; Harlalka, Gaurav V.; Mansour, Sahar; Sreekantan-Nair, Ajith; Patton, Michael A.; Muggenthaler, Martina; Rich, Phillip; Wagner, Karin; Coblentz, Roselyn; Stein, Constance K.; Last, James I.; Taylor, A. Malcolm R.; Jackson, Andrew P.; Ogi, Tomoo; Lehmann, Alan R.; Green, Catherine M.; Crosby, Andrew H.

    2014-01-01

    Numerous human disorders, including Cockayne syndrome, UV-sensitive syndrome, xeroderma pigmentosum, and trichothiodystrophy, result from the mutation of genes encoding molecules important for nucleotide excision repair. Here, we describe a syndrome in which the cardinal clinical features include short stature, hearing loss, premature aging, telangiectasia, neurodegeneration, and photosensitivity, resulting from a homozygous missense (p.Ser228Ile) sequence alteration of the proliferating cell nuclear antigen (PCNA). PCNA is a highly conserved sliding clamp protein essential for DNA replication and repair. Due to this fundamental role, mutations in PCNA that profoundly impair protein function would be incompatible with life. Interestingly, while the p.Ser228Ile alteration appeared to have no effect on protein levels or DNA replication, patient cells exhibited marked abnormalities in response to UV irradiation, displaying substantial reductions in both UV survival and RNA synthesis recovery. The p.Ser228Ile change also profoundly altered PCNA’s interaction with Flap endonuclease 1 and DNA Ligase 1, DNA metabolism enzymes. Together, our findings detail a mutation of PCNA in humans associated with a neurodegenerative phenotype, displaying clinical and molecular features common to other DNA repair disorders, which we showed to be attributable to a hypomorphic amino acid alteration. PMID:24911150

  1. Functional modules, mutational load and human genetic disease.

    PubMed

    Zaghloul, Norann A; Katsanis, Nicholas

    2010-04-01

    The ability to generate a massive amount of sequencing and genotyping data is transforming the study of human genetic disorders. Driven by such innovation, it is likely that whole exome and whole-genome resequencing will replace regionally focused approaches for gene discovery and clinical testing in the next few years. However, this opportunity brings a significant interpretative challenge to assigning function and phenotypic variance to common and rare alleles. Understanding the effect of individual mutations in the context of the remaining genomic variation represents a major challenge to our interpretation of disease. Here, we discuss the challenges of assigning mutation functionality and, drawing from the examples of ciliopathies as well as cohesinopathies and channelopathies, discuss possibilities for the functional modularization of the human genome. Functional modularization in addition to the development of physiologically relevant assays to test allele functionality will accelerate our understanding of disease architecture and enable the use of genome-wide sequence data for disease diagnosis and phenotypic prediction in individuals. Copyright 2010 Elsevier Ltd. All rights reserved.

  2. Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase that increases the production rate of D-tagatose.

    PubMed

    Kim, H-J; Kim, J-H; Oh, H-J; Oh, D-K

    2006-07-01

    Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase used to increase the production rate of D-tagatose. A mutated gene was obtained by an error-prone polymerase chain reaction using L-arabinose isomerase gene from G. stearothermophilus as a template and the gene was expressed in Escherichia coli. The expressed mutated L-arabinose isomerase exhibited the change of three amino acids (Met322-->Val, Ser393-->Thr, and Val408-->Ala), compared with the wild-type enzyme and was then purified to homogeneity. The mutated enzyme had a maximum galactose isomerization activity at pH 8.0, 65 degrees C, and 1.0 mM Co2+, while the wild-type enzyme had a maximum activity at pH 8.0, 60 degrees C, and 1.0-mM Mn2+. The mutated L-arabinose isomerase exhibited increases in D-galactose isomerization activity, optimum temperature, catalytic efficiency (kcat/Km) for D-galactose, and the production rate of D-tagatose from D-galactose. The mutated L-arabinose isomerase from G. stearothermophilus is valuable for the commercial production of D-tagatose. This work contributes knowledge on the characterization of a mutated L-arabinose isomerase, and allows an increased production rate for D-tagatose from D-galactose using the mutated enzyme.

  3. Mutations to PB2 and NP proteins of an avian influenza virus combine to confer efficient growth in primary human respiratory cells.

    PubMed

    Danzy, Shamika; Studdard, Lydia R; Manicassamy, Balaji; Solorzano, Alicia; Marshall, Nicolle; García-Sastre, Adolfo; Steel, John; Lowen, Anice C

    2014-11-01

    Influenza pandemics occur when influenza A viruses (IAV) adapted to other host species enter humans and spread through the population. Pandemics are relatively rare due to host restriction of IAV: strains adapted to nonhuman species do not readily infect, replicate in, or transmit among humans. IAV can overcome host restriction through reassortment or adaptive evolution, and these are mechanisms by which pandemic strains arise in nature. To identify mutations that facilitate growth of avian IAV in humans, we have adapted influenza A/duck/Alberta/35/1976 (H1N1) (dk/AB/76) virus to a high-growth phenotype in differentiated human tracheo-bronchial epithelial (HTBE) cells. Following 10 serial passages of three independent lineages, the bulk populations showed similar growth in HTBE cells to that of a human seasonal virus. The coding changes present in six clonal isolates were determined. The majority of changes were located in the polymerase complex and nucleoprotein (NP), and all isolates carried mutations in the PB2 627 domain and regions of NP thought to interact with PB2. Using reverse genetics, the impact on growth and polymerase activity of individual and paired mutations in PB2 and NP was evaluated. The results indicate that coupling of the mammalian-adaptive mutation PB2 E627K or Q591K to selected mutations in NP further augments the growth of the corresponding viruses. In addition, minimal combinations of three (PB2 Q236H, E627K, and NP N309K) or two (PB2 Q591K and NP S50G) mutations were sufficient to recapitulate the efficient growth in HTBE cells of dk/AB/76 viruses isolated after 10 passages in this substrate. Influenza A viruses adapted to birds do not typically grow well in humans. However, as has been seen recently with H5N1 and H7N9 subtype viruses, productive and virulent infection of humans with avian influenza viruses can occur. The ability of avian influenza viruses to adapt to new host species is a consequence of their high mutation rate that

  4. Mutations to PB2 and NP Proteins of an Avian Influenza Virus Combine To Confer Efficient Growth in Primary Human Respiratory Cells

    PubMed Central

    Danzy, Shamika; Studdard, Lydia R.; Manicassamy, Balaji; Solorzano, Alicia; Marshall, Nicolle; García-Sastre, Adolfo; Steel, John

    2014-01-01

    mutation rate that supports their zoonotic potential. Understanding of the adaptation of avian viruses to mammals strengthens public health efforts aimed at controlling influenza. In particular, it is critical to know how readily and through mutation to which functional components avian influenza viruses gain the ability to grow efficiently in humans. Our data show that as few as three mutations, in the PB2 and NP proteins, support robust growth of a low-pathogenic, H1N1 duck isolate in primary human respiratory cells. PMID:25210184

  5. Mutations in SULT2B1 Cause Autosomal-Recessive Congenital Ichthyosis in Humans.

    PubMed

    Heinz, Lisa; Kim, Gwang-Jin; Marrakchi, Slaheddine; Christiansen, Julie; Turki, Hamida; Rauschendorf, Marc-Alexander; Lathrop, Mark; Hausser, Ingrid; Zimmer, Andreas D; Fischer, Judith

    2017-06-01

    Ichthyoses are a clinically and genetically heterogeneous group of genodermatoses associated with abnormal scaling of the skin over the whole body. Mutations in nine genes are known to cause non-syndromic forms of autosomal-recessive congenital ichthyosis (ARCI). However, not all genetic causes for ARCI have been discovered to date. Using whole-exome sequencing (WES) and multigene panel screening, we identified 6 ARCI-affected individuals from three unrelated families with mutations in Sulfotransferase family 2B member 1 (SULT2B1), showing their causative association with ARCI. Cytosolic sulfotransferases form a large family of enzymes that are involved in the synthesis and metabolism of several steroids in humans. We identified four distinct mutations including missense, nonsense, and splice site mutations. We demonstrated the loss of SULT2B1 expression at RNA and protein levels in keratinocytes from individuals with ARCI by functional analyses. Furthermore, we succeeded in reconstructing the morphologic skin alterations in a 3D organotypic tissue culture model with SULT2B1-deficient keratinocytes and fibroblasts. By thin layer chromatography (TLC) of extracts from these organotypic cultures, we could show the absence of cholesterol sulfate, the metabolite of SULT2B1, and an increased level of cholesterol, indicating a disturbed cholesterol metabolism of the skin upon loss-of-function mutation in SULT2B1. In conclusion, our study reveals an essential role for SULT2B1 in the proper development of healthy human skin. Mutation in SULT2B1 leads to an ARCI phenotype via increased proliferation of human keratinocytes, thickening of epithelial layers, and altered epidermal cholesterol metabolism. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  6. Mutation mechanisms that underlie turnover of a human telomere-adjacent segmental duplication containing an unstable minisatellite.

    PubMed

    Hills, Mark; Jeyapalan, Jennie N; Foxon, Jennifer L; Royle, Nicola J

    2007-04-01

    Subterminal regions, juxtaposed to telomeres on human chromosomes, contain a high density of segmental duplications, but relatively little is known about the evolutionary processes that underlie sequence turnover in these regions. We have characterized a segmental duplication adjacent to the Xp/Yp telomere, each copy containing a hypervariable array of the DXYS14 minisatellite. Both DXYS14 repeat arrays mutate at a high rate (0.3 and 0.2% per gamete) but linkage disequilibrium analysis across 27 SNPs and a direct crossover assay show that recombination during meiosis is suppressed. Therefore instability at DXYS14a and b is dominated by intra-allelic processes or possibly conversion limited to the repeat arrays. Furthermore some chromosomes (14%) carry only one copy of the duplicon, including one DXYS14 repeat array that is also highly mutable (1.2% per gamete). To explain these and other observations, we propose there is another low-rate mutation process that causes copy number change in part or all of the duplicon.

  7. Identification of T-cell Receptors Targeting KRAS-mutated Human Tumors

    PubMed Central

    Wang, Qiong J.; Yu, Zhiya; Griffith, Kayla; Hanada, Ken-ichi; Restifo, Nicholas P.; Yang, James C.

    2015-01-01

    KRAS is one of the most frequently mutated proto-oncogenes in human cancers. The dominant oncogenic mutations of KRAS are single amino acid substitutions at codon 12, in particular G12D and G12V present in 60–70% of pancreatic cancers and 20–30% of colorectal cancers. The consistency, frequency, and tumor specificity of these “neo-antigens” make them attractive therapeutic targets. Recent data associates T cells that target mutated antigens with clinical immunotherapy responses in patients with metastatic melanoma, lung cancer, or cholangiocarcinoma. Using HLA-peptide prediction algorithms, we noted that HLA-A*11:01 could potentially present mutated KRAS variants. By immunizing HLA-A*11:01 transgenic mice, we generated murine T cells and subsequently isolated T-cell receptors (TCRs) highly reactive to the mutated KRAS variants G12V and G12D. Peripheral blood lymphocytes (PBLs) transduced with these TCRs could recognize multiple HLA-A*11:01+ tumor lines bearing the appropriate KRAS mutations. In a xenograft model of large established tumor, adoptive transfer of these transduced PBLs reactive with an HLA-A*11:01, G12D-mutated pancreatic cell line could significantly reduce its growth in NSG mice (P = 0.002). The success of adoptive transfer of TCR-engineered T cells against melanoma and other cancers support clinical trials with these T cells that recognize mutated KRAS in patients with a variety of common cancer types. PMID:26701267

  8. Comparison of Detection Rate and Mutational Pattern of Drug-Resistant Mutations Between a Large Cohort of Genotype B and Genotype C Hepatitis B Virus-Infected Patients in North China.

    PubMed

    Li, Xiaodong; Liu, Yan; Xin, Shaojie; Ji, Dong; You, Shaoli; Hu, Jinhua; Zhao, Jun; Wu, Jingjing; Liao, Hao; Zhang, Xin-Xin; Xu, Dongping

    2017-06-01

    The study aimed to investigate the association of prevalent genotypes in China (HBV/C and HBV/B) with HBV drug-resistant mutations. A total of 13,847 nucleos(t)ide analogue (NA)-treated patients with chronic HBV infection from North China were enrolled. HBV genotypes and resistant mutations were determined by direct sequencing and confirmed by clonal sequencing if necessary. HBV/B, HBV/C, and HBV/D occupied 14.3%, 84.9%, and 0.8% across the study population, respectively. NA usage had no significant difference between HBV/B- and HBV/C-infected patients. Lamivudine-resistant mutations were more frequently detected in HBV/C-infected patients, compared with HBV/B-infected patients (31.67% vs. 25.26%, p < 0.01). Adefovir- and entecavir-resistant mutation detection rates were similar, but the mutational pattern was different between the two genotypes. For adefovir-resistant mutations, HBV/C-infected patients had a higher detection rate of rtA181 V (HBV/C 5.29% vs. HBV/B 1.36%, p < 0.01) and a lower detection rate of rtN236T (2.70% vs. 6.54%, p < 0.01). For entecavir-resistant mutations, HBV/C-infected patients had a higher detection rate of rtM204 V/I+T184 substitution or S202G/C (3.66% vs. 2.16%, p < 0.01) and a lower detection rate of rtM204 V/I+M250 V/I/L substitution (0.67% vs. 1.46%, p < 0.01). Multidrug-resistant mutations (defined as coexistence of mutation to nucleoside and nucleotide analogues) were detected in 104 patients. HBV/C-infected patients had a higher detection rate of multidrug-resistant mutation than HBV/B-infected patients (0.83% vs. 0.35%, p < 0.05). The study for the first time clarified that HBV/C-infected patients had a higher risk to develop multidrug-resistant mutations, compared with HBV/B-infected patients; and HBV/C- and HBV/B-infected patients had different inclinations in the ETV-resistant mutational pattern.

  9. 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

  10. Structure-based assessment of disease-related mutations in human voltage-gated sodium channels.

    PubMed

    Huang, Weiyun; Liu, Minhao; Yan, S Frank; Yan, Nieng

    2017-06-01

    Voltage-gated sodium (Na v ) channels are essential for the rapid upstroke of action potentials and the propagation of electrical signals in nerves and muscles. Defects of Na v channels are associated with a variety of channelopathies. More than 1000 disease-related mutations have been identified in Na v channels, with Na v 1.1 and Na v 1.5 each harboring more than 400 mutations. Na v channels represent major targets for a wide array of neurotoxins and drugs. Atomic structures of Na v channels are required to understand their function and disease mechanisms. The recently determined atomic structure of the rabbit voltage-gated calcium (Ca v ) channel Ca v 1.1 provides a template for homology-based structural modeling of the evolutionarily related Na v channels. In this Resource article, we summarized all the reported disease-related mutations in human Na v channels, generated a homologous model of human Na v 1.7, and structurally mapped disease-associated mutations. Before the determination of structures of human Na v channels, the analysis presented here serves as the base framework for mechanistic investigation of Na v channelopathies and for potential structure-based drug discovery.

  11. The Effective Mutation Rate at Y Chromosome Short Tandem Repeats, with Application to Human Population-Divergence Time

    PubMed Central

    Zhivotovsky, Lev A.; Underhill, Peter A.; Cinnioğlu, Cengiz; Kayser, Manfred; Morar, Bharti; Kivisild, Toomas; Scozzari, Rosaria; Cruciani, Fulvio; Destro-Bisol, Giovanni; Spedini, Gabriella; Chambers, Geoffrey K.; Herrera, Rene J.; Yong, Kiau Kiun; Gresham, David; Tournev, Ivailo; Feldman, Marcus W.; Kalaydjieva, Luba

    2004-01-01

    We estimate an effective mutation rate at an average Y chromosome short-tandem repeat locus as 6.9×10-4 per 25 years, with a standard deviation across loci of 5.7×10-4, using data on microsatellite variation within Y chromosome haplogroups defined by unique-event polymorphisms in populations with documented short-term histories, as well as comparative data on worldwide populations at both the Y chromosome and various autosomal loci. This value is used to estimate the times of the African Bantu expansion, the divergence of Polynesian populations (the Maoris, Cook Islanders, and Samoans), and the origin of Gypsy populations from Bulgaria. PMID:14691732

  12. Analysis of human MutS homolog 2 missense mutations in patients with colorectal cancer.

    PubMed

    Zhang, Xiaomei; Chen, Senqing; Yu, Jun; Zhang, Yuanying; Lv, Min; Zhu, Ming

    2018-05-01

    Germline mutations of DNA mismatch repair gene human MutS homolog 2 ( hMSH2 ) are associated with hereditary nonpolyposis colorectal cancer (HNPCC). A total of one-third of these mutations are missense mutations. Several hMSH2 missense mutations have been identified in patients in East Asia, although their function has not been evaluated. In the present study, the role of ten hMSH2 missense mutations in the pathogenesis of colorectal cancer was examined. The hMSH2/hMSH6 protein interaction system was established using yeast two-hybrid screening. Next, the missense mutations were analyzed for their ability to affect the protein interaction of hMSH2 with its partner hMSH6. Additionally, the Sorting Intolerant from Tolerant tool was applied to predict the effects of different amino acid substitutions. The results demonstrated that certain hMSH2 mutations (L173R and C199R) caused a significant functional change in the human hMutSα complex and were identified to be pathological mutations. The Y408C, D603Y, P696L and S703Y mutations partially affected interaction and partly affected the function of hMSH2. The remaining four variants, T8M, I169V, A370T and Q419K, may be non-functional polymorphisms or could affect protein function through other molecular mechanisms. The present study evaluated the functional consequences of previously unknown missense mutations in hMSH2 , and may contribute to improved clinical diagnosis and mutation screening of HNPCC.

  13. Identification of a Novel GJA8 (Cx50) Point Mutation Causes Human Dominant Congenital Cataracts

    NASA Astrophysics Data System (ADS)

    Ge, Xiang-Lian; Zhang, Yilan; Wu, Yaming; Lv, Jineng; Zhang, Wei; Jin, Zi-Bing; Qu, Jia; Gu, Feng

    2014-02-01

    Hereditary cataracts are clinically and genetically heterogeneous lens diseases that cause a significant proportion of visual impairment and blindness in children. Human cataracts have been linked with mutations in two genes, GJA3 and GJA8, respectively. To identify the causative mutation in a family with hereditary cataracts, family members were screened for mutations by PCR for both genes. Sequencing the coding regions of GJA8, coding for connexin 50, revealed a C > A transversion at nucleotide 264, which caused p.P88T mutation. To dissect the molecular consequences of this mutation, plasmids carrying wild-type and mutant mouse ORFs of Gja8 were generated and ectopically expressed in HEK293 cells and human lens epithelial cells, respectively. The recombinant proteins were assessed by confocal microscopy and Western blotting. The results demonstrate that the molecular consequences of the p.P88T mutation in GJA8 include changes in connexin 50 protein localization patterns, accumulation of mutant protein, and increased cell growth.

  14. 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.

  15. Mastocytosis in mice expressing human Kit receptor with the activating Asp816Val mutation

    PubMed Central

    Zappulla, Jacques P.; Dubreuil, Patrice; Desbois, Sabine; Létard, Sébastien; Hamouda, Nadine Ben; Daëron, Marc; Delsol, Georges; Arock, Michel; Liblau, Roland S.

    2005-01-01

    Mastocytosis is a rare neoplastic disease characterized by a pathologic accumulation of tissue mast cells (MCs). Mastocytosis is often associated with a somatic point mutation in the Kit protooncogene leading to an Asp/Val substitution at position 816 in the kinase domain of this receptor. The contribution of this mutation to mastocytosis development remains unclear. In addition, the clinical heterogeneity presented by mastocytosis patients carrying the same mutation is unexplained. We report that a disease with striking similarities to human mastocytosis develops spontaneously in transgenic mice expressing the human Asp816Val mutant Kit protooncogene specifically in MCs. This disease is characterized by clinical signs ranging from a localized and indolent MC hyperplasia to an invasive MC tumor. In addition, bone marrow–derived MCs from transgenic animals can be maintained in culture for >24 mo and acquire growth factor independency for proliferation. These results demonstrate a causal link in vivo between the Asp816Val Kit mutation and MC neoplasia and suggest a basis for the clinical heterogeneity of human mastocytosis. PMID:16352739

  16. Mutation rates at the glycophorin A and HPRT loci in uranium miners exposed to radon progeny.

    PubMed Central

    Shanahan, E M; Peterson, D; Roxby, D; Quintana, J; Morely, A A; Woodward, A

    1996-01-01

    OBJECTIVES--To find whether a relation exists between estimated levels of exposure to radon and its progeny and mutations in hypoxanthine phosphoribosyl transferase (HPRT) and glycophorin A in a cohort of former uranium miners. METHODS--A cohort study involving a sample of miners from the Radium Hill uranium mine in South Australia, which operated from 1952 to 1961. Radiation exposures underground at Radium Hill were estimated from historical radon gas measures with a job exposure matrix. Workers from the mine who worked exclusively above ground according to mine records were selected as controls. In 1991-2 miners were interviewed and blood taken for measurement of somatic mutations. Mutation rates for HPRT and glycophorin A were estimated with standard assay techniques. RESULTS--Homozygous mutations of glycophorin A were increased in underground miners (P = 0.0027) and the mutation rate tended to rise with increasing exposure with the exception of the highest exposure (> 10 working level months). However, there was no association between place of work and either the hemizygous mutations of glycophorin A or the HPRT mutation. CONCLUSIONS--There may be an association between glycophorin A mutations and previous occupational exposure to ionising radiation. However, not enough is known at present to use these assays as biomarkers for historical exposure in underground mining cohorts. PMID:8704866

  17. Detecting Mutations in the Mycobacterium tuberculosis Pyrazinamidase Gene pncA to Improve Infection Control and Decrease Drug Resistance Rates in Human Immunodeficiency Virus Coinfection.

    PubMed

    Dudley, Matthew Z; Sheen, Patricia; Gilman, Robert H; Ticona, Eduardo; Friedland, Jon S; Kirwan, Daniela E; Caviedes, Luz; Rodriguez, Richard; Cabrera, Lilia Z; Coronel, Jorge; Grandjean, Louis; Moore, David A J; Evans, Carlton A; Huaroto, Luz; Chávez-Pérez, Víctor; Zimic, Mirko

    2016-12-07

    Hospital infection control measures are crucial to tuberculosis (TB) control strategies within settings caring for human immunodeficiency virus (HIV)-positive patients, as these patients are at heightened risk of developing TB. Pyrazinamide (PZA) is a potent drug that effectively sterilizes persistent Mycobacterium tuberculosis bacilli. However, PZA resistance associated with mutations in the nicotinamidase/pyrazinamidase coding gene, pncA, is increasing. A total of 794 patient isolates obtained from four sites in Lima, Peru, underwent spoligotyping and drug resistance testing. In one of these sites, the HIV unit of Hospital Dos de Mayo (HDM), an isolation ward for HIV/TB coinfected patients opened during the study as an infection control intervention: circulating genotypes and drug resistance pre- and postintervention were compared. All other sites cared for HIV-negative outpatients: genotypes and drug resistance rates from these sites were compared with those from HDM. HDM patients showed high concordance between multidrug resistance, PZA resistance according to the Wayne method, the two most common genotypes (spoligotype international type [SIT] 42 of the Latino American-Mediterranean (LAM)-9 clade and SIT 53 of the T1 clade), and the two most common pncA mutations (G145A and A403C). These associations were absent among community isolates. The infection control intervention was associated with 58-92% reductions in TB caused by SIT 42 or SIT 53 genotypes (odds ratio [OR] = 0.420, P = 0.003); multidrug-resistant TB (OR = 0.349, P < 0.001); and PZA-resistant TB (OR = 0.076, P < 0.001). In conclusion, pncA mutation typing, with resistance testing and spoligotyping, was useful in identifying a nosocomial TB outbreak and demonstrating its resolution after implementation of infection control measures. © The American Society of Tropical Medicine and Hygiene.

  18. Detecting Mutations in the Mycobacterium tuberculosis Pyrazinamidase Gene pncA to Improve Infection Control and Decrease Drug Resistance Rates in Human Immunodeficiency Virus Coinfection

    PubMed Central

    Dudley, Matthew Z.; Sheen, Patricia; Gilman, Robert H.; Ticona, Eduardo; Friedland, Jon S.; Kirwan, Daniela E.; Caviedes, Luz; Rodriguez, Richard; Cabrera, Lilia Z.; Coronel, Jorge; Grandjean, Louis; Moore, David A. J.; Evans, Carlton A.; Huaroto, Luz; Chávez-Pérez, Víctor; Zimic, Mirko

    2016-01-01

    Hospital infection control measures are crucial to tuberculosis (TB) control strategies within settings caring for human immunodeficiency virus (HIV)–positive patients, as these patients are at heightened risk of developing TB. Pyrazinamide (PZA) is a potent drug that effectively sterilizes persistent Mycobacterium tuberculosis bacilli. However, PZA resistance associated with mutations in the nicotinamidase/pyrazinamidase coding gene, pncA, is increasing. A total of 794 patient isolates obtained from four sites in Lima, Peru, underwent spoligotyping and drug resistance testing. In one of these sites, the HIV unit of Hospital Dos de Mayo (HDM), an isolation ward for HIV/TB coinfected patients opened during the study as an infection control intervention: circulating genotypes and drug resistance pre- and postintervention were compared. All other sites cared for HIV-negative outpatients: genotypes and drug resistance rates from these sites were compared with those from HDM. HDM patients showed high concordance between multidrug resistance, PZA resistance according to the Wayne method, the two most common genotypes (spoligotype international type [SIT] 42 of the Latino American-Mediterranean (LAM)-9 clade and SIT 53 of the T1 clade), and the two most common pncA mutations (G145A and A403C). These associations were absent among community isolates. The infection control intervention was associated with 58–92% reductions in TB caused by SIT 42 or SIT 53 genotypes (odds ratio [OR] = 0.420, P = 0.003); multidrug-resistant TB (OR = 0.349, P < 0.001); and PZA-resistant TB (OR = 0.076, P < 0.001). In conclusion, pncA mutation typing, with resistance testing and spoligotyping, was useful in identifying a nosocomial TB outbreak and demonstrating its resolution after implementation of infection control measures. PMID:27928075

  19. A bacterial model for expression of mutations in the human ornithine transcarbamylase (OTC) gene

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

    Tuchman, M.; McCann, M.T.; Qureshi, A.A.

    1994-09-01

    OTC is a mitochondrial enzyme catalyzing the formation of citrulline from carbamyl phosphate and ornithine. X-linked deficiency of OTC is the most prevalent genetic defect of ureagenesis. Mutations and polymorphisms in the OTC gene identified in deficient patients have indicated the occurrence of many family-specific, unique alleles. Due to the low frequency of recurrent mutations, distinguishing between deleterious mutations and polymorphisms is difficult. Using a human OTC gene containing plasmid driven by a tac promoter, we have devised a simple and efficient method for expressing mutations in the mature human OTC enzyme. To demonstrate this method, PCR engineered site-directed mutagenesismore » was employed to generated cDNA fragments which contained either the R151Q or R277W known mutations found in patients with neonatal and late-onset OTC deficiency, respectively. The normal allele for each mutation was also generated by an identical PCR procedure. Digestion with Bgl II- and Sty I-generated mutant and normal replacement cassettes containing the respective mutant and wild type sequences. Upon transformation of JM109 E.coli cells, OTC enzymatic activity was measured at log and stationary phases of growth using a radiochromatographic method. The R141Q mutation abolished enzymatic activity (<0.02% of normal), whereas the R277W mutation expressed partial activity (2.3% of normal). In addition, a PCR-generated mutation, A280V, resulted in 73% loss of catalytic activity. This OTC expression system is clinically applicable for distinguishing between mutations and polymorphisms, and it can be used to investigate the effects of mutations on various domains of the OTC gene.« less

  20. Genetic characterization of an adapted pandemic 2009 H1N1 influenza virus that reveals improved replication rates in human lung epithelial cells

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

    Wörmann, Xenia; Lesch, Markus; Steinbeis Innovation gGmbH, Center for Systems Biomedicine, Falkensee

    The 2009 influenza pandemic originated from a swine-origin H1N1 virus, which, although less pathogenic than anticipated, may acquire additional virulence-associated mutations in the future. To estimate the potential risk, we sequentially passaged the isolate A/Hamburg/04/2009 in A549 human lung epithelial cells. After passage 6, we observed a 100-fold increased replication rate. High-throughput sequencing of viral gene segments identified five dominant mutations, whose contribution to the enhanced growth was analyzed by reverse genetics. The increased replication rate was pinpointed to two mutations within the hemagglutinin (HA) gene segment (HA{sub 1} D130E, HA{sub 2} I91L), near the receptor binding site and themore » stem domain. The adapted virus also replicated more efficiently in mice in vivo. Enhanced replication rate correlated with increased fusion pH of the HA protein and a decrease in receptor affinity. Our data might be relevant for surveillance of pre-pandemic strains and development of high titer cell culture strains for vaccine production. - Highlights: • We observed a spontaneous mutation of a 2009-pandemic H1N1 influenza virus in vitro. • The adaptation led to a 100-fold rise in replication rate in human A549 cells. • Adaptation was caused by two mutations in the HA gene segment. • Adaptation correlates with increased fusion pH and decreased receptor affinity.« less

  1. 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.

  2. LET and ion-species dependence for mutation induction and mutation spectrum on hprt locus in normal human fibroblasts.

    PubMed

    Tsuruoka, Chizuru; Suzuki, Masao; Fujitaka, Kazunobu

    2004-11-01

    We have been studying LET and ion species dependence of RBE in mutation frequency and mutation spectrum of deletion pattern of exons in hprt locus. Normal human skin fibroblasts were irradiated with heavy-ion beams, such as carbon- (290 MeV/u and 135 MeV/u), neon- (230 MeV/u and 400 MeV/u), silicon- (490 MeV/u) and iron- (500 MeV/u) ion beams, generated by Heavy Ion Medical Accelerator in Chiba (HIMAC) at national Institute of Radiological Sciences (NIRS). Mutation induction in hprt locus was detected to measure 6-thioguanine resistant colonies and deletion spectrum of exons was analyzed by multiplex PCR. The LET-RBE curves of mutation induction for carbon- and neon-ion beams showed a peak around 75 keV/micrometers and 155 keV/micrometers, respectively. On the other hand, there observed no clear peak for silicon-ion beams. The deletion spectrum of exons was different in induced mutants among different ion species. These results suggested that quantitative and qualitative difference in mutation occurred when using different ion species even if similar LET values.

  3. The presence of highly disruptive 16S rRNA mutations in clinical samples indicates a wider role for mutations of the mitochondrial ribosome in human disease

    PubMed Central

    Elson, Joanna L.; Smith, Paul M.; Greaves, Laura C.; Lightowlers, Robert N.; Chrzanowska-Lightowlers, Zofia M.A.; Taylor, Robert W.; Vila-Sanjurjo, Antón

    2015-01-01

    Mitochondrial DNA mutations are well recognized as an important cause of disease, with over two hundred variants in the protein encoding and mt-tRNA genes associated with human disorders. In contrast, the two genes encoding the mitochondrial rRNAs (mt-rRNAs) have been studied in far less detail. This is because establishing the pathogenicity of mt-rRNA mutations is a major diagnostic challenge. Only two disease causing mutations have been identified at these loci, both mapping to the small subunit (SSU). On the large subunit (LSU), however, the evidence for the presence of pathogenic LSU mt-rRNA changes is particularly sparse. We have previously expanded the list of deleterious SSU mt-rRNA mutations by identifying highly disruptive base changes capable of blocking the activity of the mitoribosomal SSU. To do this, we used a new methodology named heterologous inferential analysis (HIA). The recent arrival of near-atomic-resolution structures of the human mitoribosomal LSU, has enhanced the power of our approach by permitting the analysis of the corresponding sites of mutation within their natural structural context. Here, we have used these tools to determine whether LSU mt-rRNA mutations found in the context of human disease and/or ageing could disrupt the function of the mitoribosomal LSU. Our results clearly show that, much like the for SSU mt-rRNA, LSU mt-rRNAs mutations capable of compromising the function of the mitoribosomal LSU are indeed present in clinical samples. Thus, our work constitutes an important contribution to an emerging view of the mitoribosome as an important element in human health. PMID:26349026

  4. Immune dysregulation in human subjects with heterozygous germline mutations in CTLA4

    PubMed Central

    Deenick, Elissa K.; Niemela, Julie E.; Avery, Danielle T.; Schickel, Jean-Nicolas; Tran, Dat Q.; Stoddard, Jennifer; Zhang, Yu; Frucht, David M.; Dumitriu, Bogdan; Scheinberg, Phillip; Folio, Les R.; Frein, Cathleen A.; Price, Susan; Koh, Christopher; Heller, Theo; Seroogy, Christine M.; Huttenlocher, Anna; Rao, V. Koneti; Su, Helen C.; Kleiner, David; Notarangelo, Luigi D.; Rampertaap, Yajesh; Olivier, Kenneth N.; McElwee, Joshua; Hughes, Jason; Pittaluga, Stefania; Oliveira, Joao B.; Meffre, Eric; Fleisher, Thomas A.; Holland, Steven M.; Lenardo, Michael J.; Tangye, Stuart G.; Uzel, Gulbu

    2015-01-01

    Cytotoxic T lymphocyte antigen–4 (CTLA-4) is an inhibitory receptor found on immune cells. The consequences of mutations in CTLA4 in humans are unknown. We identified germline heterozygous mutations in CTLA4 in subjects with severe immune dysregulation from four unrelated families. Whereas Ctla4 heterozygous mice have no obvious phenotype, human CTLA4 haploinsufficiency caused dysregulation of FoxP3+ regulatory T (Treg) cells, hyperactivation of effector T cells, and lymphocytic infiltration of target organs. Patients also exhibited progressive loss of circulating B cells, associated with an increase of predominantly autoreactive CD21lo B cells and accumulation of B cells in nonlymphoid organs. Inherited human CTLA4 haploinsufficiency demonstrates a critical quantitative role for CTLA-4 in governing T and B lymphocyte homeostasis. PMID:25213377

  5. [Evaluation of performance of five bioinformatics software for the prediction of missense mutations].

    PubMed

    Chen, Qianting; Dai, Congling; Zhang, Qianjun; Du, Juan; Li, Wen

    2016-10-01

    To study the prediction performance evaluation with five kinds of bioinformatics software (SIFT, PolyPhen2, MutationTaster, Provean, MutationAssessor). From own database for genetic mutations collected over the past five years, Chinese literature database, Human Gene Mutation Database, and dbSNP, 121 missense mutations confirmed by functional studies, and 121 missense mutations suspected to be pathogenic by pedigree analysis were used as positive gold standard, while 242 missense mutations with minor allele frequency (MAF)>5% in dominant hereditary diseases were used as negative gold standard. The selected mutations were predicted with the five software. Based on the results, the performance of the five software was evaluated for their sensitivity, specificity, positive predict value, false positive rate, negative predict value, false negative rate, false discovery rate, accuracy, and receiver operating characteristic curve (ROC). In terms of sensitivity, negative predictive value and false negative rate, the rank was MutationTaster, PolyPhen2, Provean, SIFT, and MutationAssessor. For specificity and false positive rate, the rank was MutationTaster, Provean, MutationAssessor, SIFT, and PolyPhen2. For positive predict value and false discovery rate, the rank was MutationTaster, Provean, MutationAssessor, PolyPhen2, and SIFT. For area under the ROC curve (AUC) and accuracy, the rank was MutationTaster, Provean, PolyPhen2, MutationAssessor, and SIFT. The prediction performance of software may be different when using different parameters. Among the five software, MutationTaster has the best prediction performance.

  6. Mutations in α-Tubulin Cause Abnormal Neuronal Migration in Mice and Lissencephaly in Humans

    PubMed Central

    Keays, David A.; Tian, Guoling; Poirier, Karine; Huang, Guo-Jen; Siebold, Christian; Cleak, James; Oliver, Peter L.; Fray, Martin; Harvey, Robert J.; Molnár, Zoltán; Piñon, Maria C.; Dear, Neil; Valdar, William; Brown, Steve D.M.; Davies, Kay E.; Rawlins, J. Nicholas P.; Cowan, Nicholas J.; Nolan, Patrick; Chelly, Jamel; Flint, Jonathan

    2007-01-01

    Summary The development of the mammalian brain is dependent on extensive neuronal migration. Mutations in mice and humans that affect neuronal migration result in abnormal lamination of brain structures with associated behavioral deficits. Here, we report the identification of a hyperactive N-ethyl-N-nitrosourea (ENU)-induced mouse mutant with abnormalities in the laminar architecture of the hippocampus and cortex, accompanied by impaired neuronal migration. We show that the causative mutation lies in the guanosine triphosphate (GTP) binding pocket of α-1 tubulin (Tuba1) and affects tubulin heterodimer formation. Phenotypic similarity with existing mouse models of lissencephaly led us to screen a cohort of patients with developmental brain anomalies. We identified two patients with de novo mutations in TUBA3, the human homolog of Tuba1. This study demonstrates the utility of ENU mutagenesis in the mouse as a means to discover the basis of human neurodevelopmental disorders. PMID:17218254

  7. Many private mutations originate from the first few divisions of a human colorectal adenoma.

    PubMed

    Kang, Haeyoun; Salomon, Matthew P; Sottoriva, Andrea; Zhao, Junsong; Toy, Morgan; Press, Michael F; Curtis, Christina; Marjoram, Paul; Siegmund, Kimberly; Shibata, Darryl

    2015-11-01

    Intratumoural mutational heterogeneity (ITH) or the presence of different private mutations in different parts of the same tumour is commonly observed in human tumours. The mechanisms generating such ITH are uncertain. Here we find that ITH can be remarkably well structured by measuring point mutations, chromosome copy numbers, and DNA passenger methylation from opposite sides and individual glands of a 6 cm human colorectal adenoma. ITH was present between tumour sides and individual glands, but the private mutations were side-specific and subdivided the adenoma into two major subclones. Furthermore, ITH disappeared within individual glands because the glands were clonal populations composed of cells with identical mutant genotypes. Despite mutation clonality, the glands were relatively old, diverse populations when their individual cells were compared for passenger methylation and by FISH. These observations can be organized into an expanding star-like ancestral tree with co-clonal expansion, where many private mutations and multiple related clones arise during the first few divisions. As a consequence, most detectable mutational ITH in the final tumour originates from the first few divisions. Much of the early history of a tumour, especially the first few divisions, may be embedded within the detectable ITH of tumour genomes. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  8. Foxp2 mutations impair auditory-motor association learning.

    PubMed

    Kurt, Simone; Fisher, Simon E; Ehret, Günter

    2012-01-01

    Heterozygous mutations of the human FOXP2 transcription factor gene cause the best-described examples of monogenic speech and language disorders. Acquisition of proficient spoken language involves auditory-guided vocal learning, a specialized form of sensory-motor association learning. The impact of etiological Foxp2 mutations on learning of auditory-motor associations in mammals has not been determined yet. Here, we directly assess this type of learning using a newly developed conditioned avoidance paradigm in a shuttle-box for mice. We show striking deficits in mice heterozygous for either of two different Foxp2 mutations previously implicated in human speech disorders. Both mutations cause delays in acquiring new motor skills. The magnitude of impairments in association learning, however, depends on the nature of the mutation. Mice with a missense mutation in the DNA-binding domain are able to learn, but at a much slower rate than wild type animals, while mice carrying an early nonsense mutation learn very little. These results are consistent with expression of Foxp2 in distributed circuits of the cortex, striatum and cerebellum that are known to play key roles in acquisition of motor skills and sensory-motor association learning, and suggest differing in vivo effects for distinct variants of the Foxp2 protein. Given the importance of such networks for the acquisition of human spoken language, and the fact that similar mutations in human FOXP2 cause problems with speech development, this work opens up a new perspective on the use of mouse models for understanding pathways underlying speech and language disorders.

  9. 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.

  10. Non-coding cancer driver candidates identified with a sample- and position-specific model of the somatic mutation rate

    PubMed Central

    Juul, Malene; Bertl, Johanna; Guo, Qianyun; Nielsen, Morten Muhlig; Świtnicki, Michał; Hornshøj, Henrik; Madsen, Tobias; Hobolth, Asger; Pedersen, Jakob Skou

    2017-01-01

    Non-coding mutations may drive cancer development. Statistical detection of non-coding driver regions is challenged by a varying mutation rate and uncertainty of functional impact. Here, we develop a statistically founded non-coding driver-detection method, ncdDetect, which includes sample-specific mutational signatures, long-range mutation rate variation, and position-specific impact measures. Using ncdDetect, we screened non-coding regulatory regions of protein-coding genes across a pan-cancer set of whole-genomes (n = 505), which top-ranked known drivers and identified new candidates. For individual candidates, presence of non-coding mutations associates with altered expression or decreased patient survival across an independent pan-cancer sample set (n = 5454). This includes an antigen-presenting gene (CD1A), where 5’UTR mutations correlate significantly with decreased survival in melanoma. Additionally, mutations in a base-excision-repair gene (SMUG1) correlate with a C-to-T mutational-signature. Overall, we find that a rich model of mutational heterogeneity facilitates non-coding driver identification and integrative analysis points to candidates of potential clinical relevance. DOI: http://dx.doi.org/10.7554/eLife.21778.001 PMID:28362259

  11. Population data and mutation rates of 20 autosomal STR loci in a Chinese Han population from Yunnan Province, Southwest China.

    PubMed

    Zhang, Xiufeng; Liu, Linlin; Xie, Runfang; Wang, Guiyi; Shi, Yuan; Gu, Tao; Hu, Liping; Nie, Shengjie

    2018-07-01

    The genetic polymorphisms of 20 autosomal short tandem repeat (STR) loci included in the PowerPlex® 21 kit were evaluated from 2068 unrelated, healthy individuals from the Chinese Han population of Yunnan Province in southwest China. All of the loci reached Hardy-Weinberg equilibrium. These loci were examined to determine allele frequencies and forensic statistical parameters. The genetic relationships among the Yunnan Han and other Chinese populations were also estimated. The combined discrimination power and probability of excluding paternity of the 20 STR loci were 0.99999999999999999999999126 and 0.999999975, respectively. In addition, mutation rates from 4363 parentage cases (2215 trios and 2148 duos) were investigated in this study. A total of 164 mutations were observed in 6578 meioses from the 20 loci. The highest mutation rate was observed in D12S391 (0.30%), and the lowest mutation rates were observed in D13S317 (0.03%) and TPOX (0.03%). The average mutation rate for the 20 loci was estimated to be 1.246 × 10 -3 per meiosis. The mutations were primarily single-step and paternal mutations.

  12. The role of the prokineticin 2 pathway in human reproduction: evidence from the study of human and murine gene mutations.

    PubMed

    Martin, Cecilia; Balasubramanian, Ravikumar; Dwyer, Andrew A; Au, Margaret G; Sidis, Yisrael; Kaiser, Ursula B; Seminara, Stephanie B; Pitteloud, Nelly; Zhou, Qun-Yong; Crowley, William F

    2011-04-01

    A widely dispersed network of hypothalamic GnRH neurons controls the reproductive axis in mammals. Genetic investigation of the human disease model of isolated GnRH deficiency has revealed several key genes crucial for GnRH neuronal ontogeny and GnRH secretion. Among these genes, prokineticin 2 (PROK2), and PROK2 receptor (PROKR2) have recently emerged as critical regulators of reproduction in both mice and humans. Both prok2- and prokr2-deficient mice recapitulate the human Kallmann syndrome phenotype. Additionally, PROK2 and PROKR2 mutations are seen in humans with Kallmann syndrome, thus implicating this pathway in GnRH neuronal migration. However, PROK2/PROKR2 mutations are also seen in normosmic GnRH deficiency, suggesting a role for the prokineticin signaling system in GnRH biology that is beyond neuronal migration. This observation is particularly surprising because mature GnRH neurons do not express PROKR2. Moreover, mutations in both PROK2 and PROKR2 are predominantly detected in the heterozygous state with incomplete penetrance or variable expressivity frequently seen within and across pedigrees. In some of these pedigrees, a "second hit" or oligogenicity has been documented. Besides reproduction, a pleiotropic physiological role for PROK2 is now recognized, including regulation of pain perception, circadian rhythms, hematopoiesis, and immune response. Therefore, further detailed clinical studies of patients with PROK2/PROKR2 mutations will help to map the broader biological role of the PROK2/PROKR2 pathway and identify other interacting genes/proteins that mediate its molecular effects in humans.

  13. The Role of the Prokineticin 2 Pathway in Human Reproduction: Evidence from the Study of Human and Murine Gene Mutations

    PubMed Central

    Martin, Cecilia; Balasubramanian, Ravikumar; Dwyer, Andrew A.; Au, Margaret G.; Sidis, Yisrael; Kaiser, Ursula B.; Seminara, Stephanie B.; Pitteloud, Nelly; Zhou, Qun-Yong

    2011-01-01

    A widely dispersed network of hypothalamic GnRH neurons controls the reproductive axis in mammals. Genetic investigation of the human disease model of isolated GnRH deficiency has revealed several key genes crucial for GnRH neuronal ontogeny and GnRH secretion. Among these genes, prokineticin 2 (PROK2), and PROK2 receptor (PROKR2) have recently emerged as critical regulators of reproduction in both mice and humans. Both prok2- and prokr2-deficient mice recapitulate the human Kallmann syndrome phenotype. Additionally, PROK2 and PROKR2 mutations are seen in humans with Kallmann syndrome, thus implicating this pathway in GnRH neuronal migration. However, PROK2/PROKR2 mutations are also seen in normosmic GnRH deficiency, suggesting a role for the prokineticin signaling system in GnRH biology that is beyond neuronal migration. This observation is particularly surprising because mature GnRH neurons do not express PROKR2. Moreover, mutations in both PROK2 and PROKR2 are predominantly detected in the heterozygous state with incomplete penetrance or variable expressivity frequently seen within and across pedigrees. In some of these pedigrees, a “second hit” or oligogenicity has been documented. Besides reproduction, a pleiotropic physiological role for PROK2 is now recognized, including regulation of pain perception, circadian rhythms, hematopoiesis, and immune response. Therefore, further detailed clinical studies of patients with PROK2/PROKR2 mutations will help to map the broader biological role of the PROK2/PROKR2 pathway and identify other interacting genes/proteins that mediate its molecular effects in humans. PMID:21037178

  14. Changing rates and patterns of drug resistance mutations in antiretroviral-experienced HIV-infected patients.

    PubMed

    de Mendoza, Carmen; Garrido, Carolina; Corral, Angelica; Ramírez-Olivencia, German; Jiménez-Nacher, Inmaculada; Zahonero, Natalia; Gonzalez-Lahoz, Juan; Soriano, Vincent

    2007-07-01

    Surveillance of drug resistance mutations in antiretroviral-experienced HIV(+) patients may provide useful information regarding options available for rescue interventions. All resistance tests performed from 1999 to 2005 on antiretroviral-experienced individuals at one reference laboratory in Madrid were examined. Only mutations associated with drug resistance recorded at the September 2006 IAS-USA list were considered. A total of 2137 specimens were analyzed. Overall, 71.1% showed resistance mutations to at least one drug class, 56.1% to at least two, and 21% to all three drug families. Resistance mutations were 65% for NRTI, 44.4% for NNRTI, and 42.5% for PI. Mutations T215Y/F, M184V, and M41L were the most frequent for NRTI. Their rate significantly declined since 1999. K65R significantly increased since 1999 (0.8%) to 2003 (7.3%) but declined up to 3.3% in 2005. For NNRTI, K103N significantly increased from 21.8% in 1999 to 29.5% in 2005 (p < 0.01). The most frequent PI resistance mutations were L90M (24.3%), V82X (19.9%), M46I/L (19.5%), and I54V (17.1%). The presence of five or more was 58.8% in 1999 but declined to 22.2% in 2005. The rate of drug resistance mutations causing NRTI and PI resistance has steadily declined in antiretroviral-experienced patients since 1999. The availability of a large number and/or more convenient NRTI as well as the wide use of ritonavir-boosted PI could explain these observations. However, broad PI cross-resistance was seen in nearly 25% of antiretroviral-experienced patients in 2005. Therefore, there is a still need for new antiretrovirals with different resistance profiles.

  15. Missense Mutations in the N-Terminal Domain of Human Phenylalanine Hydroxylase Interfere with Binding of Regulatory Phenylalanine

    PubMed Central

    Gjetting, Torben; Petersen, Marie; Guldberg, Per; Güttler, Flemming

    2001-01-01

    Hyperphenylalaninemia due to a deficiency of phenylalanine hydroxylase (PAH) is an autosomal recessive disorder caused by >400 mutations in the PAH gene. Recent work has suggested that the majority of PAH missense mutations impair enzyme activity by causing increased protein instability and aggregation. In this study, we describe an alternative mechanism by which some PAH mutations may render PAH defective. Database searches were used to identify regions in the N-terminal domain of PAH with homology to the regulatory domain of prephenate dehydratase (PDH), the rate-limiting enzyme in the bacterial phenylalanine biosynthesis pathway. Naturally occurring N-terminal PAH mutations are distributed in a nonrandom pattern and cluster within residues 46–48 (GAL) and 65–69 (IESRP), two motifs highly conserved in PDH. To examine whether N-terminal PAH mutations affect the ability of PAH to bind phenylalanine at the regulatory domain, wild-type and five mutant (G46S, A47V, T63P/H64N, I65T, and R68S) forms of the N-terminal domain (residues 2–120) of human PAH were expressed as fusion proteins in Escherichia coli. Binding studies showed that the wild-type form of this domain specifically binds phenylalanine, whereas all mutations abolished or significantly reduced this phenylalanine-binding capacity. Our data suggest that impairment of phenylalanine-mediated activation of PAH may be an important disease-causing mechanism of some N-terminal PAH mutations, which may explain some well-documented genotype-phenotype discrepancies in PAH deficiency. PMID:11326337

  16. Muver, a computational framework for accurately calling accumulated mutations.

    PubMed

    Burkholder, Adam B; Lujan, Scott A; Lavender, Christopher A; Grimm, Sara A; Kunkel, Thomas A; Fargo, David C

    2018-05-09

    Identification of mutations from next-generation sequencing data typically requires a balance between sensitivity and accuracy. This is particularly true of DNA insertions and deletions (indels), that can impart significant phenotypic consequences on cells but are harder to call than substitution mutations from whole genome mutation accumulation experiments. To overcome these difficulties, we present muver, a computational framework that integrates established bioinformatics tools with novel analytical methods to generate mutation calls with the extremely low false positive rates and high sensitivity required for accurate mutation rate determination and comparison. Muver uses statistical comparison of ancestral and descendant allelic frequencies to identify variant loci and assigns genotypes with models that include per-sample assessments of sequencing errors by mutation type and repeat context. Muver identifies maximally parsimonious mutation pathways that connect these genotypes, differentiating potential allelic conversion events and delineating ambiguities in mutation location, type, and size. Benchmarking with a human gold standard father-son pair demonstrates muver's sensitivity and low false positive rates. In DNA mismatch repair (MMR) deficient Saccharomyces cerevisiae, muver detects multi-base deletions in homopolymers longer than the replicative polymerase footprint at rates greater than predicted for sequential single-base deletions, implying a novel multi-repeat-unit slippage mechanism. Benchmarking results demonstrate the high accuracy and sensitivity achieved with muver, particularly for indels, relative to available tools. Applied to an MMR-deficient Saccharomyces cerevisiae system, muver mutation calls facilitate mechanistic insights into DNA replication fidelity.

  17. Application of Digital PCR in Detecting Human Diseases Associated Gene Mutation.

    PubMed

    Tong, Yu; Shen, Shizhen; Jiang, Hui; Chen, Zhi

    2017-01-01

    Gene mutation has been considered a research hotspot, and the rapid development of biomedicine has enabled significant advances in the evaluation of gene mutations. The advent of digital polymerase chain reaction (dPCR) elevates the detection of gene mutations to unprecedented levels of precision, especially in cancer-associated genes. dPCR has been utilized in the detection of tumor markers in cell-free DNA (cfDNA) samples from patients with different types of cancer in samples such as plasma, cerebrospinal fluid, urine and sputum, which confers significant value for dPCR in both clinical applications and basic research. Moreover, dPCR is extensively used in detecting pathogen mutations related to typical features of infectious diseases (e.g., drug resistance) and mutation status of heteroplasmic mitochondrial DNA, which determines the manifestation and progression of mtDNA-related diseases, as well as allows for the prenatal diagnosis of monogenic diseases and the assessment of the genome editing effects. Compared with real-time PCR (qPCR) and sequencing, the higher sensitivity and accuracy of dPCR indicates a great advantage in the detection of rare mutation. As a new technique, dPCR has some limitations, such as the necessity of highly allele-specific probes and a large sample volume. In this review, we summarize the application of dPCR in the detection of human disease-associated gene mutations. © 2017 The Author(s). Published by S. Karger AG, Basel.

  18. 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

  19. 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

  20. Heterogeneity of Clonal Expansion and Maturation-Linked Mutation Acquisition in Hematopoietic Progenitors in Human Acute Myeloid Leukemia

    PubMed Central

    Walter, Roland B.; Laszlo, George S.; Lionberger, Jack M.; Pollard, Jessica A.; Harrington, Kimberly H.; Gudgeon, Chelsea J.; Othus, Megan; Rafii, Shahin; Meshinchi, Soheil; Appelbaum, Frederick R.; Bernstein, Irwin D.

    2014-01-01

    Recent technological advances led to an appreciation of the genetic complexity of human acute myeloid leukemia (AML) but underlying progenitor cells remain poorly understood because their rarity precludes direct study. We developed a co-culture method integrating hypoxia, aryl hydrocarbon receptor inhibition, and micro-environmental support via human endothelial cells to isolate these cells. X-chromosome inactivation studies of the least mature precursors derived following prolonged culture of CD34+/CD33− cells revealed polyclonal growth in highly curable AMLs, suggesting mutations necessary for clonal expansion were acquired in more mature progenitors. Consistently, in core-binding factor (CBF) leukemias with known complementing mutations, immature precursors derived following prolonged culture of CD34+/CD33− cells harbored neither mutation or the CBF mutation alone, whereas more mature precursors often carried both mutations. These results were in contrast to those with leukemias with poor prognosis that showed clonal dominance in the least mature precursors. These data indicate heterogeneity among progenitors in human AML that may have prognostic and therapeutic implications. PMID:24721792

  1. Associations between mutations and a VNTR in the human phenylalanine hydroxylase gene

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

    Goltsov, A.A.; Eisensmith, R.C.; Woo, S.L.C.

    1992-09-01

    The HindIII RFLP in the human phenylalanine hydroxylase (PAH) gene is caused by the presence of an AT-rich (70%) minisatellite region. This region contains various multiples of 30-bp tandem repeats and is located 3 kb downstream of the final exon of the gene. PCR-mediated amplification of this region from haplotyped PAH chromosomes indicates that the previously reported 4.0-kb HindIII allele contains three of these repeats, while the 4.4-kb HindIII allele contains 12 of these repeats. The 4.2-kb HindIII fragment can contain six, seven, eight, or nine copies of this repeat. These variations permit more detailed analysis of mutant haplotypes 1,more » 5, 6, and, possibly, others. Kindred analysis in phenylketonuria families demonstrates Mendelian segregation of these VNTR alleles, as well as associations between theses alleles and certain PAH mutations. The R261Q mutation, associated with haplotype 1, is associated almost exclusively with an allele containing eight repeats; the R408W mutation, when occurring on a haplotype 1 background, may also be associated with the eight-repeat VNTR allele. Other PAH mutations associated with haplotype 1, R252W and P281L, do not appear to segregate with specific VNTR alleles. The IVS-10 mutation, when associated with haplotype 6, is associated exclusively with an allele containing seven repeats. The combined use of this VNTR system and the existing RFLP haplotype system will increase the performance of prenatal diagnostic tests based on haplotype analysis. In addition, this VNTR may prove useful in studies concerning the origins and distributions of PAH mutations in different human populations. 32 refs., 3 figs., 3 tabs.« less

  2. 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.

  3. 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.

  4. Ionotropic GABA and Glutamate Receptor Mutations and Human Neurologic Diseases.

    PubMed

    Yuan, Hongjie; Low, Chian-Ming; Moody, Olivia A; Jenkins, Andrew; Traynelis, Stephen F

    2015-07-01

    The advent of whole exome/genome sequencing and the technology-driven reduction in the cost of next-generation sequencing as well as the introduction of diagnostic-targeted sequencing chips have resulted in an unprecedented volume of data directly linking patient genomic variability to disorders of the brain. This information has the potential to transform our understanding of neurologic disorders by improving diagnoses, illuminating the molecular heterogeneity underlying diseases, and identifying new targets for therapeutic treatment. There is a strong history of mutations in GABA receptor genes being involved in neurologic diseases, particularly the epilepsies. In addition, a substantial number of variants and mutations have been found in GABA receptor genes in patients with autism, schizophrenia, and addiction, suggesting potential links between the GABA receptors and these conditions. A new and unexpected outcome from sequencing efforts has been the surprising number of mutations found in glutamate receptor subunits, with the GRIN2A gene encoding the GluN2A N-methyl-d-aspartate receptor subunit being most often affected. These mutations are associated with multiple neurologic conditions, for which seizure disorders comprise the largest group. The GluN2A subunit appears to be a locus for epilepsy, which holds important therapeutic implications. Virtually all α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor mutations, most of which occur within GRIA3, are from patients with intellectual disabilities, suggesting a link to this condition. Similarly, the most common phenotype for kainate receptor variants is intellectual disability. Herein, we summarize the current understanding of disease-associated mutations in ionotropic GABA and glutamate receptor families, and discuss implications regarding the identification of human mutations and treatment of neurologic diseases. Copyright © 2015 by The American Society for Pharmacology and Experimental

  5. Ionotropic GABA and Glutamate Receptor Mutations and Human Neurologic Diseases

    PubMed Central

    Yuan, Hongjie; Low, Chian-Ming; Moody, Olivia A.; Jenkins, Andrew

    2015-01-01

    The advent of whole exome/genome sequencing and the technology-driven reduction in the cost of next-generation sequencing as well as the introduction of diagnostic-targeted sequencing chips have resulted in an unprecedented volume of data directly linking patient genomic variability to disorders of the brain. This information has the potential to transform our understanding of neurologic disorders by improving diagnoses, illuminating the molecular heterogeneity underlying diseases, and identifying new targets for therapeutic treatment. There is a strong history of mutations in GABA receptor genes being involved in neurologic diseases, particularly the epilepsies. In addition, a substantial number of variants and mutations have been found in GABA receptor genes in patients with autism, schizophrenia, and addiction, suggesting potential links between the GABA receptors and these conditions. A new and unexpected outcome from sequencing efforts has been the surprising number of mutations found in glutamate receptor subunits, with the GRIN2A gene encoding the GluN2A N-methyl-d-aspartate receptor subunit being most often affected. These mutations are associated with multiple neurologic conditions, for which seizure disorders comprise the largest group. The GluN2A subunit appears to be a locus for epilepsy, which holds important therapeutic implications. Virtually all α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor mutations, most of which occur within GRIA3, are from patients with intellectual disabilities, suggesting a link to this condition. Similarly, the most common phenotype for kainate receptor variants is intellectual disability. Herein, we summarize the current understanding of disease-associated mutations in ionotropic GABA and glutamate receptor families, and discuss implications regarding the identification of human mutations and treatment of neurologic diseases. PMID:25904555

  6. De novo Uroplakin IIIa heterozygous mutations cause human renal adysplasia leading to severe kidney failure.

    PubMed

    Jenkins, Dagan; Bitner-Glindzicz, Maria; Malcolm, Sue; Hu, Chih-Chi A; Allison, Jennifer; Winyard, Paul J D; Gullett, Ambrose M; Thomas, David F M; Belk, Rachel A; Feather, Sally A; Sun, Tung-Tien; Woolf, Adrian S

    2005-07-01

    Human renal adysplasia usually occurs sporadically, and bilateral disease is the most common cause of childhood end-stage renal failure, a condition that is lethal without intervention using dialysis or transplantation. De novo heterozygous mutations in Uroplakin IIIa (UPIIIa) are reported in four of 17 children with kidney failure caused by renal adysplasia in the absence of an overt urinary tract obstruction. One girl and one boy in unrelated kindreds had a missense mutation at a CpG dinucleotide in the cytoplasmic domain of UPIIIa (Pro273Leu), both of whom had severe vesicoureteric reflux, and the girl had persistent cloaca; two other patients had de novo mutations in the 3' UTR (963 T-->G; 1003 T-->C), and they had renal adysplasia in the absence of any other anomaly. The mutations were absent in all sets of parents and in siblings, none of whom had radiologic evidence of renal adysplasia, and mutations were absent in two panels of 192 ethnically matched control chromosomes. UPIIIa was expressed in nascent urothelia in ureter and renal pelvis of human embryos, and it is suggested that perturbed urothelial differentiation may generate human kidney malformations, perhaps by altering differentiation of adjacent smooth muscle cells such that the metanephros is exposed to a functional obstruction of urine flow. With advances in renal replacement therapy, children with renal failure, who would otherwise have died, are surviving to adulthood. Therefore, although the mechanisms of action of the UPIIIa mutations have yet to be determined, these findings have important implications regarding genetic counseling of affected individuals who reach reproductive age.

  7. Analysis and implications of mutational variation.

    PubMed

    Keightley, Peter D; Halligan, Daniel L

    2009-06-01

    Variation from new mutations is important for several questions in quantitative genetics. Key parameters are the genomic mutation rate and the distribution of effects of mutations (DEM), which determine the amount of new quantitative variation that arises per generation from mutation (V(M)). Here, we review methods and empirical results concerning mutation accumulation (MA) experiments that have shed light on properties of mutations affecting quantitative traits. Surprisingly, most data on fitness traits from laboratory assays of MA lines indicate that the DEM is platykurtic in form (i.e., substantially less leptokurtic than an exponential distribution), and imply that most variation is produced by mutations of moderate to large effect. This finding contrasts with results from MA or mutagenesis experiments in which mutational changes to the DNA can be assayed directly, which imply that the vast majority of mutations have very small phenotypic effects, and that the distribution has a leptokurtic form. We compare these findings with recent approaches that attempt to infer the DEM for fitness based on comparing the frequency spectra of segregating nucleotide polymorphisms at putatively neutral and selected sites in population samples. When applied to data for humans and Drosophila, these analyses also indicate that the DEM is strongly leptokurtic. However, by combining the resultant estimates of parameters of the DEM with estimates of the mutation rate per nucleotide, the predicted V(M) for fitness is only a tiny fraction of V(M) observed in MA experiments. This discrepancy can be explained if we postulate that a few deleterious mutations of large effect contribute most of the mutational variation observed in MA experiments and that such mutations segregate at very low frequencies in natural populations, and effectively are never seen in population samples.

  8. Racial disparities in patient survival and tumor mutation burden, and the association between tumor mutation burden and cancer incidence rate.

    PubMed

    Zhang, Wensheng; Edwards, Andrea; Flemington, Erik K; Zhang, Kun

    2017-10-20

    The causes underlying racial disparities in cancer are multifactorial. In addition to socioeconomic issues, biological factors may contribute to these inequities, especially in disease incidence and patient survival. To date, there have been few studies that relate the disparities in these aspects to genetic aberrations. In this work, we studied the impacts of race on the patient survival and tumor mutation burden using the data released by the Cancer Genome Atlas (TCGA). The potential relationship between mutation burden and disease incidence is further inferred by an integrative analysis of TCGA data and the data from the Surveillance, Epidemiology, and End Results (SEER) Program. The results show that disparities are present (p < 0.05) in patient survival of five cancers, such as head and neck squamous cell carcinoma. The numbers of tumor driver mutations are differentiated (p < 0.05) over the racial groups in five cancers, such as lung adenocarcinoma. By treating a specific cancer type and a racial group as an "experimental unit", driver mutation numbers demonstrate a significant (r = 0.46, p < 0.002) positive correlation with cancer incidence rates, especially when the five cancers with mutational disparities are exclusively focused (r = 0.88, p < 0.00002). These results enrich our understanding of racial disparities in cancer and carcinogenic process.

  9. Improving solubility and refolding efficiency of human V(H)s by a novel mutational approach.

    PubMed

    Tanha, Jamshid; Nguyen, Thanh-Dung; Ng, Andy; Ryan, Shannon; Ni, Feng; Mackenzie, Roger

    2006-11-01

    The antibody V(H) domains of camelids tend to be soluble and to resist aggregation, in contrast to human V(H) domains. For immunotherapy, attempts have therefore been made to improve the properties of human V(H)s by camelization of a small set of framework residues. Here, we have identified through sequence comparison of well-folded llama V(H) domains an alternative set of residues (not typically camelid) for mutation. Thus, the solubility and thermal refolding efficiency of a typical human V(H), derived from the human antibody BT32/A6, were improved by introduction of two mutations in framework region (FR) 1 and 4 to generate BT32/A6.L1. Three more mutations in FR3 of BT32/A6.L1 further improved the thermal refolding efficiency while retaining solubility and cooperative melting profiles. To demonstrate practical utility, BT32/A6.L1 was used to construct a phage display library from which were isolated human V(H)s with good antigen binding activity and solubility. The engineered human V(H) domains described here may be useful for immunotherapy, due to their expected low immunogenicity, and in applications involving transient high temperatures, due to their efficient refolding after thermal denaturation.

  10. Parkin Mutations Reduce the Complexity of Neuronal Processes in iPSC-derived Human Neurons

    PubMed Central

    Ren, Yong; Jiang, Houbo; Hu, Zhixing; Fan, Kevin; Wang, Jun; Janoschka, Stephen; Wang, Xiaomin; Ge, Shaoyu; Feng, Jian

    2015-01-01

    Parkinson’s disease (PD) is characterized by the degeneration of nigral dopaminergic (DA) neurons and non-DA neurons in many parts of the brain. Mutations of parkin, an E3 ubiquitin ligase that strongly binds to microtubules, are the most frequent cause of recessively inherited Parkinson’s disease. The lack of robust PD phenotype in parkin knockout mice suggests a unique vulnerability of human neurons to parkin mutations. Here, we show that the complexity of neuronal processes as measured by total neurite length, number of terminals, number of branch points and Sholl analysis, was greatly reduced in induced pluripotent stem cell (iPSC)-derived TH+ or TH− neurons from PD patients with parkin mutations. Consistent with these, microtubule stability was significantly decreased by parkin mutations in iPSC-derived neurons. Overexpression of parkin, but not its PD-linked mutant nor GFP, restored the complexity of neuronal processes and the stability of microtubules. Consistent with these, the microtubule-depolymerizing agent colchicine mimicked the effect of parkin mutations by decreasing neurite length and complexity in control neurons while the microtubule-stabilizing drug taxol mimicked the effect of parkin overexpression by enhancing the morphology of parkin-deficient neurons. The results suggest that parkin maintains the morphological complexity of human neurons by stabilizing microtubules. PMID:25332110

  11. Human germline hedgehog pathway mutations predispose to fatty liver.

    PubMed

    Guillen-Sacoto, Maria J; Martinez, Ariel F; Abe, Yu; Kruszka, Paul; Weiss, Karin; Everson, Joshua L; Bataller, Ramon; Kleiner, David E; Ward, Jerrold M; Sulik, Kathleen K; Lipinski, Robert J; Solomon, Benjamin D; Muenke, Maximilian

    2017-10-01

    Non-alcoholic fatty liver disease (NAFLD) is the most common form of liver disease. Activation of hedgehog (Hh) signaling has been implicated in the progression of NAFLD and proposed as a therapeutic target; however, the effects of Hh signaling inhibition have not been studied in humans with germline mutations that affect this pathway. Patients with holoprosencephaly (HPE), a disorder associated with germline mutations disrupting Sonic hedgehog (SHH) signaling, were clinically evaluated for NAFLD. A combined mouse model of Hh signaling attenuation (Gli2 heterozygous null: Gli2 +/- ) and diet-induced NAFLD was used to examine aspects of NAFLD and hepatic gene expression profiles, including molecular markers of hepatic fibrosis and inflammation. Patients with HPE had a higher prevalence of liver steatosis compared to the general population, independent of obesity. Exposure of Gli2 +/- mice to fatty liver-inducing diets resulted in increased liver steatosis compared to wild-type mice. Similar to humans, this effect was independent of obesity in the mutant mice and was associated with decreased expression of pro-fibrotic and pro-inflammatory genes, and increased expression of PPARγ, a potent anti-fibrogenic and anti-inflammatory regulator. Interestingly, tumor suppressors p53 and p16INK4 were found to be downregulated in the Gli2 +/- mice exposed to a high-fat diet. Our results indicate that germline mutations disrupting Hh signaling promotes liver steatosis, independent of obesity, with reduced fibrosis. While Hh signaling inhibition has been associated with a better NAFLD prognosis, further studies are required to evaluate the long-term effects of mutations affecting this pathway. Lay summary: Non-alcoholic fatty liver disease (NAFLD) is characterized by excess fat deposition in the liver predominantly due to high calorie intake and a sedentary lifestyle. NAFLD progression is usually accompanied by activation of the Sonic hedgehog (SHH) pathway leading to fibrous

  12. 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

  13. Integrin Alpha 8 Recessive Mutations Are Responsible for Bilateral Renal Agenesis in Humans

    PubMed Central

    Humbert, Camille; Silbermann, Flora; Morar, Bharti; Parisot, Mélanie; Zarhrate, Mohammed; Masson, Cécile; Tores, Frédéric; Blanchet, Patricia; Perez, Marie-José; Petrov, Yuliya; Khau Van Kien, Philippe; Roume, Joelle; Leroy, Brigitte; Gribouval, Olivier; Kalaydjieva, Luba; Heidet, Laurence; Salomon, Rémi; Antignac, Corinne; Benmerah, Alexandre; Saunier, Sophie; Jeanpierre, Cécile

    2014-01-01

    Renal hypodysplasia (RHD) is a heterogeneous condition encompassing a spectrum of kidney development defects including renal agenesis, hypoplasia, and (cystic) dysplasia. Heterozygous mutations of several genes have been identified as genetic causes of RHD with various severity. However, these genes and mutations are not associated with bilateral renal agenesis, except for RET mutations, which could be involved in a few cases. The pathophysiological mechanisms leading to total absence of kidney development thus remain largely elusive. By using a whole-exome sequencing approach in families with several fetuses with bilateral renal agenesis, we identified recessive mutations in the integrin α8-encoding gene ITGA8 in two families. Itga8 homozygous knockout in mice is known to result in absence of kidney development. We provide evidence of a damaging effect of the human ITGA8 mutations. These results demonstrate that mutations of ITGA8 are a genetic cause of bilateral renal agenesis and that, at least in some cases, bilateral renal agenesis is an autosomal-recessive disease. PMID:24439109

  14. Analysis of APC mutation in human ameloblastoma and clinical significance.

    PubMed

    Li, Ning; Liu, Bing; Sui, Chengguang; Jiang, Youhong

    2016-01-01

    As a highly conserved signaling pathway, Wnt/β-catenin signal transduction pathway plays an important role in many processes. Either in the occurrence or development of tumor, activation of this pathway takes an important place. APC inhibits Wnt/β-catenin pathway to regulate cell proliferation and differentiation. This study aimed to investigate the function of cancer suppressor gene. PCR amplification and sequencing method was used to analyze APC mutations of human clinical specimens. The pathological specimens were collected for PCR and clear electrophoretic bands were obtained after electrophoresis. The gene sequence obtained after purification and sequencing analysis was compared with the known APC gene sequence (NM_000038.5). Base mutations at APC 1543 (T → C), APC-4564 (G → A), APC-5353 (T → G), APC-5550 (T → A) and APC-5969 (G → A) locus existed in 22 (27.5 %), 12 (15 %), 5 (6.25 %), 13 (16.25 %) and 12 patients (15 %), respectively. Gene mutations existed in ameloblastoma, and the mutation loci were 1543 locus (T → C), 4564 locus (G → A), 5353 locus (T → G), 5550 locus (T → A) and 5969 locus (G → A) 15 %, respectively. APC mutation plays a certain role in monitoring the tumor malignant degree as it may indicate the transition process of ameloblastoma malignant phenotype.

  15. Parallel Evolution of High-Level Aminoglycoside Resistance in Escherichia coli Under Low and High Mutation Supply Rates.

    PubMed

    Ibacache-Quiroga, Claudia; Oliveros, Juan C; Couce, Alejandro; Blázquez, Jesus

    2018-01-01

    Antibiotic resistance is a major concern in public health worldwide, thus there is much interest in characterizing the mutational pathways through which susceptible bacteria evolve resistance. Here we use experimental evolution to explore the mutational pathways toward aminoglycoside resistance, using gentamicin as a model, under low and high mutation supply rates. Our results show that both normo and hypermutable strains of Escherichia coli are able to develop resistance to drug dosages > 1,000-fold higher than the minimal inhibitory concentration for their ancestors. Interestingly, such level of resistance was often associated with changes in susceptibility to other antibiotics, most prominently with increased resistance to fosfomycin. Whole-genome sequencing revealed that all resistant derivatives presented diverse mutations in five common genetic elements: fhuA, fusA and the atpIBEFHAGDC, cyoABCDE , and potABCD operons. Despite the large number of mutations acquired, hypermutable strains did not pay, apparently, fitness cost. In contrast to recent studies, we found that the mutation supply rate mainly affected the speed (tempo) but not the pattern (mode) of evolution: both backgrounds acquired the mutations in the same order, although the hypermutator strain did it faster. This observation is compatible with the adaptive landscape for high-level gentamicin resistance being relatively smooth, with few local maxima; which might be a common feature among antibiotics for which resistance involves multiple loci.

  16. Parallel Evolution of High-Level Aminoglycoside Resistance in Escherichia coli Under Low and High Mutation Supply Rates

    PubMed Central

    Ibacache-Quiroga, Claudia; Oliveros, Juan C.; Couce, Alejandro; Blázquez, Jesus

    2018-01-01

    Antibiotic resistance is a major concern in public health worldwide, thus there is much interest in characterizing the mutational pathways through which susceptible bacteria evolve resistance. Here we use experimental evolution to explore the mutational pathways toward aminoglycoside resistance, using gentamicin as a model, under low and high mutation supply rates. Our results show that both normo and hypermutable strains of Escherichia coli are able to develop resistance to drug dosages > 1,000-fold higher than the minimal inhibitory concentration for their ancestors. Interestingly, such level of resistance was often associated with changes in susceptibility to other antibiotics, most prominently with increased resistance to fosfomycin. Whole-genome sequencing revealed that all resistant derivatives presented diverse mutations in five common genetic elements: fhuA, fusA and the atpIBEFHAGDC, cyoABCDE, and potABCD operons. Despite the large number of mutations acquired, hypermutable strains did not pay, apparently, fitness cost. In contrast to recent studies, we found that the mutation supply rate mainly affected the speed (tempo) but not the pattern (mode) of evolution: both backgrounds acquired the mutations in the same order, although the hypermutator strain did it faster. This observation is compatible with the adaptive landscape for high-level gentamicin resistance being relatively smooth, with few local maxima; which might be a common feature among antibiotics for which resistance involves multiple loci. PMID:29615988

  17. Aku, a mutation of the mouse homologous to human alkaptonuria, maps to chromosome 16

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

    Montagutelli, X.; Lalouette, A.; Guenet, J.L.

    1994-01-01

    Alkaptonuria is a human hereditary metabolic disease characterized by a very high urinary excretion of homogentisic acid, an intermediary product in the metabolism of tyrosine, in association with ochronosis and arthritis. This disease is due to a deficiency in the enzyme homogentisic acid oxidase and is inherited as an autosomal recessive condition. The authors have found a new recessive mutation (aku) in the mouse that is homologous to human alkaptonuria, during a mutagenesis program with ethylnitrosourea. Affected mice show high levels of urinary homogentisic acid without signs of ochronosis or arthritis. This mutation has been mapped to Chr 16 closemore » to the D16Mit4 locus, in a region of synteny with human 3q. 22 refs., 1 fig., 1 tab.« less

  18. Human TUBB3 mutations perturb microtubule dynamics, kinesin interactions, and axon guidance

    PubMed Central

    Tischfield, Max A.; Baris, Hagit N.; Wu, Chen; Rudolph, Guenther; Van Maldergem, Lionel; He, Wei; Chan, Wai-Man; Andrews, Caroline; Demer, Joseph L.; Robertson, Richard L.; Mackey, David A.; Ruddle, Jonathan B.; Bird, Thomas D.; Gottlob, Irene; Pieh, Christina; Traboulsi, Elias I.; Pomeroy, Scott L.; Hunter, David G.; Soul, Janet S.; Newlin, Anna; Sabol, Louise J.; Doherty, Edward J.; de Uzcátegui, Clara E.; de Uzcátegui, Nicolas; Collins, Mary Louise Z.; Sener, Emin C.; Wabbels, Bettina; Hellebrand, Heide; Meitinger, Thomas; de Berardinis, Teresa; Magli, Adriano; Schiavi, Costantino; Pastore-Trossello, Marco; Koc, Feray; Wong, Agnes M.; Levin, Alex V.; Geraghty, Michael T.; Descartes, Maria; Flaherty, Maree; Jamieson, Robyn V.; Møller, H. U.; Meuthen, Ingo; Callen, David F.; Kerwin, Janet; Lindsay, Susan; Meindl, Alfons; Gupta, Mohan L.; Pellman, David; Engle, Elizabeth C.

    2011-01-01

    We report that eight heterozygous missense mutations in TUBB3, encoding the neuron-specific β-tubulin isotype III, result in a spectrum of human nervous system disorders we now call the TUBB3 syndromes. Each mutation causes the ocular motility disorder CFEOM3, whereas some also result in intellectual and behavioral impairments, facial paralysis, and/or later-onset axonal sensorimotor polyneuropathy. Neuroimaging reveals a spectrum of abnormalities including hypoplasia of oculomotor nerves, and dysgenesis of the corpus callosum, anterior commissure, and corticospinal tracts. A knock-in disease mouse model reveals axon guidance defects without evidence of cortical cell migration abnormalities. We show the disease-associated mutations can impair tubulin heterodimer formation in vitro, although folded mutant heterodimers can still polymerize into microtubules. Modeling each mutation in yeast tubulin demonstrates that all alter dynamic instability whereas a subset disrupts the interaction of microtubules with kinesin motors. These findings demonstrate normal TUBB3 is required for axon guidance and maintenance in mammals. PMID:20074521

  19. Exome Sequencing Identifies Truncating Mutations in Human SERPINF1 in Autosomal-Recessive Osteogenesis Imperfecta

    PubMed Central

    Becker, Jutta; Semler, Oliver; Gilissen, Christian; Li, Yun; Bolz, Hanno Jörn; Giunta, Cecilia; Bergmann, Carsten; Rohrbach, Marianne; Koerber, Friederike; Zimmermann, Katharina; de Vries, Petra; Wirth, Brunhilde; Schoenau, Eckhard; Wollnik, Bernd; Veltman, Joris A.; Hoischen, Alexander; Netzer, Christian

    2011-01-01

    Osteogenesis imperfecta (OI) is a heterogeneous genetic disorder characterized by bone fragility and susceptibility to fractures after minimal trauma. After mutations in all known OI genes had been excluded by Sanger sequencing, we applied next-generation sequencing to analyze the exome of a single individual who has a severe form of the disease and whose parents are second cousins. A total of 26,922 variations from the human reference genome sequence were subjected to several filtering steps. In addition, we extracted the genotypes of all dbSNP130-annotated SNPs from the exome sequencing data and used these 299,494 genotypes as markers for the genome-wide identification of homozygous regions. A single homozygous truncating mutation, affecting SERPINF1 on chromosome 17p13.3, that was embedded into a homozygous stretch of 2.99 Mb remained. The mutation was also homozygous in the affected brother of the index patient. Subsequently, we identified homozygosity for two different truncating SERPINF1 mutations in two unrelated patients with OI and parental consanguinity. All four individuals with SERPINF1 mutations have severe OI. Fractures of long bones and severe vertebral compression fractures with resulting deformities were observed as early as the first year of life in these individuals. Collagen analyses with cultured dermal fibroblasts displayed no evidence for impaired collagen folding, posttranslational modification, or secretion. SERPINF1 encodes pigment epithelium-derived factor (PEDF), a secreted glycoprotein of the serpin superfamily. PEDF is a multifunctional protein and one of the strongest inhibitors of angiogenesis currently known in humans. Our data provide genetic evidence for PEDF involvement in human bone homeostasis. PMID:21353196

  20. SelTarbase, a database of human mononucleotide-microsatellite mutations and their potential impact to tumorigenesis and immunology

    PubMed Central

    Woerner, Stefan M.; Yuan, Yan P.; Benner, Axel; Korff, Sebastian; von Knebel Doeberitz, Magnus; Bork, Peer

    2010-01-01

    About 15% of human colorectal cancers and, at varying degrees, other tumor entities as well as nearly all tumors related to Lynch syndrome are hallmarked by microsatellite instability (MSI) as a result of a defective mismatch repair system. The functional impact of resulting mutations depends on their genomic localization. Alterations within coding mononucleotide repeat tracts (MNRs) can lead to protein truncation and formation of neopeptides, whereas alterations within untranslated MNRs can alter transcription level or transcript stability. These mutations may provide selective advantage or disadvantage to affected cells. They may further concern the biology of microsatellite unstable cells, e.g. by generating immunogenic peptides induced by frameshifts mutations. The Selective Targets database (http://www.seltarbase.org) is a curated database of a growing number of public MNR mutation data in microsatellite unstable human tumors. Regression calculations for various MSI–H tumor entities indicating statistically deviant mutation frequencies predict TGFBR2, BAX, ACVR2A and others that are shown or highly suspected to be involved in MSI tumorigenesis. Many useful tools for further analyzing genomic DNA, derived wild-type and mutated cDNAs and peptides are integrated. A comprehensive database of all human coding, untranslated, non-coding RNA- and intronic MNRs (MNR_ensembl) is also included. Herewith, SelTarbase presents as a plenty instrument for MSI-carcinogenesis-related research, diagnostics and therapy. PMID:19820113

  1. Recessive mutations in ELOVL4 cause ichthyosis, intellectual disability, and spastic quadriplegia.

    PubMed

    Aldahmesh, Mohammed A; Mohamed, Jawahir Y; Alkuraya, Hisham S; Verma, Ishwar C; Puri, Ratna D; Alaiya, Ayodele A; Rizzo, William B; Alkuraya, Fowzan S

    2011-12-09

    Very-long-chain fatty acids (VLCFAs) play important roles in membrane structure and cellular signaling, and their contribution to human health is increasingly recognized. Fatty acid elongases catalyze the first and rate-limiting step in VLCFA synthesis. Heterozygous mutations in ELOVL4, the gene encoding one of the elongases, are known to cause macular degeneration in humans and retinal abnormalities in mice. However, biallelic ELOVL4 mutations have not been observed in humans, and murine models with homozygous mutations die within hours of birth as a result of a defective epidermal water barrier. Here, we report on two human individuals with recessive ELOVL4 mutations revealed by a combination of autozygome analysis and exome sequencing. These individuals exhibit clinical features of ichthyosis, seizures, mental retardation, and spasticity-a constellation that resembles Sjögren-Larsson syndrome (SLS) but presents a more severe neurologic phenotype. Our findings identify recessive mutations in ELOVL4 as the cause of a neuro-ichthyotic disease and emphasize the importance of VLCFA synthesis in brain and cutaneous development. Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  2. A novel human autoimmune syndrome caused by combined hypomorphic and activating mutations in ZAP-70

    PubMed Central

    Chan, Alice Y.; Punwani, Divya; Kadlecek, Theresa A.; Cowan, Morton J.; Olson, Jean L.; Mathes, Erin F.; Sunderam, Uma; Man Fu, Shu; Srinivasan, Rajgopal; Kuriyan, John; Brenner, Steven E.; Weiss, Arthur

    2016-01-01

    A brother and sister developed a previously undescribed constellation of autoimmune manifestations within their first year of life, with uncontrollable bullous pemphigoid, colitis, and proteinuria. The boy had hemophilia due to a factor VIII autoantibody and nephrotic syndrome. Both children required allogeneic hematopoietic cell transplantation (HCT), which resolved their autoimmunity. The early onset, severity, and distinctive findings suggested a single gene disorder underlying the phenotype. Whole-exome sequencing performed on five family members revealed the affected siblings to be compound heterozygous for two unique missense mutations in the 70-kD T cell receptor ζ-chain associated protein (ZAP-70). Healthy relatives were heterozygous mutation carriers. Although pre-HCT patient T cells were not available, mutation effects were determined using transfected cell lines and peripheral blood from carriers and controls. Mutation R192W in the C-SH2 domain exhibited reduced binding to phosphorylated ζ-chain, whereas mutation R360P in the N lobe of the catalytic domain disrupted an autoinhibitory mechanism, producing a weakly hyperactive ZAP-70 protein. Although human ZAP-70 deficiency can have dysregulated T cells, and autoreactive mouse thymocytes with weak Zap-70 signaling can escape tolerance, our patients’ combination of hypomorphic and activating mutations suggested a new disease mechanism and produced previously undescribed human ZAP-70–associated autoimmune disease. PMID:26783323

  3. 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

  4. The CDC Hemophilia B mutation project mutation list: a new online resource.

    PubMed

    Li, Tengguo; Miller, Connie H; Payne, Amanda B; Craig Hooper, W

    2013-11-01

    Hemophilia B (HB) is caused by mutations in the human gene F9. The mutation type plays a pivotal role in genetic counseling and prediction of inhibitor development. To help the HB community understand the molecular etiology of HB, we have developed a listing of all F9 mutations that are reported to cause HB based on the literature and existing databases. The Centers for Disease Control and Prevention (CDC) Hemophilia B Mutation Project (CHBMP) mutation list is compiled in an easily accessible format of Microsoft Excel and contains 1083 unique mutations that are reported to cause HB. Each mutation is identified using Human Genome Variation Society (HGVS) nomenclature standards. The mutation types and the predicted changes in amino acids, if applicable, are also provided. Related information including the location of mutation, severity of HB, the presence of inhibitor, and original publication reference are listed as well. Therefore, our mutation list provides an easily accessible resource for genetic counselors and HB researchers to predict inhibitors. The CHBMP mutation list is freely accessible at http://www.cdc.gov/hemophiliamutations.

  5. Distinct pattern of TP53 mutations in human immunodeficiency virus-related head and neck squamous cell carcinoma.

    PubMed

    Gleber-Netto, Frederico O; Zhao, Mei; Trivedi, Sanchit; Wang, Jiping; Jasser, Samar; McDowell, Christina; Kadara, Humam; Zhang, Jiexin; Wang, Jing; William, William N; Lee, J Jack; Nguyen, Minh Ly; Pai, Sara I; Walline, Heather M; Shin, Dong M; Ferris, Robert L; Carey, Thomas E; Myers, Jeffrey N; Pickering, Curtis R

    2018-01-01

    Human immunodeficiency virus-infected individuals (HIVIIs) have a higher incidence of head and neck squamous cell carcinoma (HNSCC), and clinical and histopathological differences have been observed in their tumors in comparison with those of HNSCC patients without a human immunodeficiency virus (HIV) infection. The reasons for these differences are not clear, and molecular differences between HIV-related HNSCC and non-HIV-related HNSCC may exist. This study compared the mutational patterns of HIV-related HNSCC and non-HIV-related HNSCC. The DNA of 20 samples of HIV-related HNSCCs and 32 samples of non-HIV-related HNSCCs was sequenced. DNA libraries covering exons of 18 genes frequently mutated in HNSCC (AJUBA, CASP8, CCND1, CDKN2A, EGFR, FAT1, FBXW7, HLA-A, HRAS, KEAP1, NFE2L2, NOTCH1, NOTCH2, NSD1, PIK3CA, TGFBR2, TP53, and TP63) were prepared and sequenced on an Ion Personal Genome Machine sequencer. DNA sequencing data were analyzed with Ion Reporter software. The human papillomavirus (HPV) status of the tumor samples was assessed with in situ hybridization, the MassARRAY HPV multiplex polymerase chain reaction assay, and p16 immunostaining. Mutation calls were compared among the studied groups. HIV-related HNSCC revealed a distinct pattern of mutations in comparison with non-HIV-related HNSCC. TP53 mutation frequencies were significantly lower in HIV-related HNSCC. Mutations in HIV+ patients tended to be TpC>T nucleotide changes for all mutated genes but especially for TP53. HNSCC in HIVIIs presents a distinct pattern of genetic mutations, particularly in the TP53 gene. HIV-related HNSCC may have a distinct biology, and an effect of the HIV virus on the pathogenesis of these tumors should not be ruled out. Cancer 2018;124:84-94. © 2017 American Cancer Society. © 2017 American Cancer Society.

  6. Evidence for a high mutation rate at rapidly evolving yeast centromeres.

    PubMed

    Bensasson, Douda

    2011-07-18

    Although their role in cell division is essential, centromeres evolve rapidly in animals, plants and yeasts. Unlike the complex centromeres of plants and aminals, the point centromeres of Saccharomcyes yeasts can be readily sequenced to distinguish amongst the possible explanations for fast centromere evolution. Using DNA sequences of all 16 centromeres from 34 strains of Saccharomyces cerevisiae and population genomic data from Saccharomyces paradoxus, I show that centromeres in both species evolve 3 times more rapidly even than selectively unconstrained DNA. Exceptionally high levels of polymorphism seen in multiple yeast populations suggest that rapid centromere evolution does not result from the repeated selective sweeps expected under meiotic drive. I further show that there is little evidence for crossing-over or gene conversion within centromeres, although there is clear evidence for recombination in their immediate vicinity. Finally I show that the mutation spectrum at centromeres is consistent with the pattern of spontaneous mutation elsewhere in the genome. These results indicate that rapid centromere evolution is a common phenomenon in yeast species. Furthermore, these results suggest that rapid centromere evolution does not result from the mutagenic effect of gene conversion, but from a generalised increase in the mutation rate, perhaps arising from the unusual chromatin structure at centromeres in yeast and other eukaryotes.

  7. Functional studies of RYR1 mutations in the skeletal muscle ryanodine receptor using human RYR1 complementary DNA.

    PubMed

    Sato, Keisaku; Pollock, Neil; Stowell, Kathryn M

    2010-06-01

    Malignant hyperthermia is associated with mutations within the gene encoding the skeletal muscle ryanodine receptor, the calcium channel that releases Ca from sarcoplasmic reticulum stores triggering muscle contraction, and other metabolic activities. More than 200 variants have been identified in the ryanodine receptor, but only some of these have been shown to functionally affect the calcium channel. To implement genetic testing for malignant hyperthermia, variants must be shown to alter the function of the channel. A number of different ex vivo methods can be used to demonstrate functionality, as long as cells from human patients can be obtained and cultured from at least two unrelated families. Because malignant hyperthermia is an uncommon disorder and many variants seem to be private, including the newly identified H4833Y mutation, these approaches are limited. The authors cloned the human skeletal muscle ryanodine receptor complementary DNA and expressed both normal and mutated forms in HEK-293 cells and carried out functional analysis using ryanodine binding assays in the presence of a specific agonist, 4-chloro-m-cresol, and the antagonist Mg. Transiently expressed human ryanodine receptor proteins colocalized with an endoplasmic reticulum marker in HEK-293 cells. Ryanodine binding assays confirmed that mutations causing malignant hyperthermia resulted in a hypersensitive channel, while those causing central core disease resulted in a hyposensitive channel. The functional assays validate recombinant human skeletal muscle ryanodine receptor for analysis of variants and add an additional mutation (H4833Y) to the repertoire of mutations that can be used for the genetic diagnosis of malignant hyperthermia.

  8. Insights into the Mutation-Induced HHH Syndrome from Modeling Human Mitochondrial Ornithine Transporter-1

    PubMed Central

    Wang, Jing-Fang; Chou, Kuo-Chen

    2012-01-01

    Human mitochondrial ornithine transporter-1 is reported in coupling with the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, which is a rare autosomal recessive disorder. For in-depth understanding of the molecular mechanism of the disease, it is crucially important to acquire the 3D structure of human mitochondrial ornithine transporter-1. Since no such structure is available in the current protein structure database, we have developed it via computational approaches based on the recent NMR structure of human mitochondrial uncoupling protein (Berardi MJ, Chou JJ, et al. Nature 2011, 476:109–113). Subsequently, we docked the ligand L-ornithine into the computational structure to search for the favorable binding mode. It was observed that the binding interaction for the most favorable binding mode is featured by six remarkable hydrogen bonds between the receptor and ligand, and that the most favorable binding mode shared the same ligand-binding site with most of the homologous mitochondrial carriers from different organisms, implying that the ligand-binding sites are quite conservative in the mitochondrial carriers family although their sequences similarity is very low with 20% or so. Moreover, according to our structural analysis, the relationship between the disease-causing mutations of human mitochondrial ornithine transporter-1 and the HHH syndrome can be classified into the following three categories: (i) the mutation occurs in the pseudo-repeat regions so as to change the region of the protein closer to the mitochondrial matrix; (ii) the mutation is directly affecting the substrate binding pocket so as to reduce the substrate binding affinity; (iii) the mutation is located in the structural region closer to the intermembrane space that can significantly break the salt bridge networks of the protein. These findings may provide useful insights for in-depth understanding of the molecular mechanism of the HHH syndrome and developing effective

  9. Somatic mutations of GUCY2F, EPHA3, and NTRK3 in human cancers.

    PubMed

    Wood, Laura D; Calhoun, Eric S; Silliman, Natalie; Ptak, Janine; Szabo, Steve; Powell, Steve M; Riggins, Gregory J; Wang, Tian-Li; Yan, Hai; Gazdar, Adi; Kern, Scott E; Pennacchio, Len; Kinzler, Kenneth W; Vogelstein, Bert; Velculescu, Victor E

    2006-10-01

    Tyrosine kinases are major regulators of signal transduction cascades involved in cellular proliferation and have important roles in tumorigenesis. We have recently analyzed the tyrosine kinase gene family for alterations in human colorectal cancers and identified somatic mutations in seven members of this gene family. In this study we have used high-throughput sequencing approaches to further evaluate this subset of genes for genetic alterations in other human tumors. We identified somatic mutations in GUCY2F, EPHA3, and NTRK3 in breast, lung, and pancreatic cancers. Our results implicate these tyrosine kinase genes in the pathogenesis of other tumor types and suggest that they may be useful targets for diagnostic and therapeutic intervention in selected patients.

  10. Systematic discovery of mutation-specific synthetic lethals by mining pan-cancer human primary tumor data

    NASA Astrophysics Data System (ADS)

    Sinha, Subarna; Thomas, Daniel; Chan, Steven; Gao, Yang; Brunen, Diede; Torabi, Damoun; Reinisch, Andreas; Hernandez, David; Chan, Andy; Rankin, Erinn B.; Bernards, Rene; Majeti, Ravindra; Dill, David L.

    2017-05-01

    Two genes are synthetically lethal (SL) when defects in both are lethal to a cell but a single defect is non-lethal. SL partners of cancer mutations are of great interest as pharmacological targets; however, identifying them by cell line-based methods is challenging. Here we develop MiSL (Mining Synthetic Lethals), an algorithm that mines pan-cancer human primary tumour data to identify mutation-specific SL partners for specific cancers. We apply MiSL to 12 different cancers and predict 145,891 SL partners for 3,120 mutations, including known mutation-specific SL partners. Comparisons with functional screens show that MiSL predictions are enriched for SLs in multiple cancers. We extensively validate a SL interaction identified by MiSL between the IDH1 mutation and ACACA in leukaemia using gene targeting and patient-derived xenografts. Furthermore, we apply MiSL to pinpoint genetic biomarkers for drug sensitivity. These results demonstrate that MiSL can accelerate precision oncology by identifying mutation-specific targets and biomarkers.

  11. G protein-coupled receptor mutations and human genetic disease.

    PubMed

    Thompson, Miles D; Hendy, Geoffrey N; Percy, Maire E; Bichet, Daniel G; Cole, David E C

    2014-01-01

    Genetic variations in G protein-coupled receptor genes (GPCRs) disrupt GPCR function in a wide variety of human genetic diseases. In vitro strategies and animal models have been used to identify the molecular pathologies underlying naturally occurring GPCR mutations. Inactive, overactive, or constitutively active receptors have been identified that result in pathology. These receptor variants may alter ligand binding, G protein coupling, receptor desensitization and receptor recycling. Receptor systems discussed include rhodopsin, thyrotropin, parathyroid hormone, melanocortin, follicle-stimulating hormone (FSH), luteinizing hormone, gonadotropin-releasing hormone (GNRHR), adrenocorticotropic hormone, vasopressin, endothelin-β, purinergic, and the G protein associated with asthma (GPRA or neuropeptide S receptor 1 (NPSR1)). The role of activating and inactivating calcium-sensing receptor (CaSR) mutations is discussed in detail with respect to familial hypocalciuric hypercalcemia (FHH) and autosomal dominant hypocalemia (ADH). The CASR mutations have been associated with epilepsy. Diseases caused by the genetic disruption of GPCR functions are discussed in the context of their potential to be selectively targeted by drugs that rescue altered receptors. Examples of drugs developed as a result of targeting GPCRs mutated in disease include: calcimimetics and calcilytics, therapeutics targeting melanocortin receptors in obesity, interventions that alter GNRHR loss from the cell surface in idiopathic hypogonadotropic hypogonadism and novel drugs that might rescue the P2RY12 receptor congenital bleeding phenotype. De-orphanization projects have identified novel disease-associated receptors, such as NPSR1 and GPR35. The identification of variants in these receptors provides genetic reagents useful in drug screens. Discussion of the variety of GPCRs that are disrupted in monogenic Mendelian disorders provides the basis for examining the significance of common

  12. Somatic mutations of the histone H3K27 demethylase, UTX, in human cancer

    PubMed Central

    van Haaften, Gijs; Dalgliesh, Gillian L; Davies, Helen; Chen, Lina; Bignell, Graham; Greenman, Chris; Edkins, Sarah; Hardy, Claire; O’Meara, Sarah; Teague, Jon; Butler, Adam; Hinton, Jonathan; Latimer, Calli; Andrews, Jenny; Barthorpe, Syd; Beare, Dave; Buck, Gemma; Campbell, Peter J; Cole, Jennifer; Dunmore, Rebecca; Forbes, Simon; Jia, Mingming; Jones, David; Kok, Chai Yin; Leroy, Catherine; Lin, Meng-Lay; McBride, David J; Maddison, Mark; Maquire, Simon; McLay, Kirsten; Menzies, Andrew; Mironenko, Tatiana; Lee, Mulderrig; Mudie, Laura; Pleasance, Erin; Shepherd, Rebecca; Smith, Raffaella; Stebbings, Lucy; Stephens, Philip; Tang, Gurpreet; Tarpey, Patrick S; Turner, Rachel; Turrell, Kelly; Varian, Jennifer; West, Sofie; Widaa, Sara; Wray, Paul; Collins, V Peter; Ichimura, Koichi; Law, Simon; Wong, John; Yuen, Siu Tsan; Leung, Suet Yi; Tonon, Giovanni; DePinho, Ronald A; Tai, Yu-Tzu; Anderson, Kenneth C; Kahnoski, Richard J.; Massie, Aaron; Khoo, Sok Kean; Teh, Bin Tean; Stratton, Michael R; Futreal, P Andrew

    2010-01-01

    Somatically acquired epigenetic changes are present in many cancers. Epigenetic regulation is maintained via post-translational modifications of core histones. Here, we describe inactivating somatic mutations in the histone lysine demethylase, UTX, pointing to histone H3 lysine methylation deregulation in multiple tumour types. UTX reintroduction into cancer cells with inactivating UTX mutations resulted in slowing of proliferation and marked transcriptional changes. These data identify UTX as a new human cancer gene. PMID:19330029

  13. JAK2V617F somatic mutation in the general population: myeloproliferative neoplasm development and progression rate

    PubMed Central

    Nielsen, Camilla; Bojesen, Stig E.; Nordestgaard, Børge G.; Kofoed, Klaus F.; Birgens, Henrik S.

    2014-01-01

    Clinical significance of the JAK2V617F mutation in patients with a myeloproliferative neoplasm has been the target of intensive research in recent years. However, there is considerably uncertainty about prognosis in JAK2V617F positive individuals without overt signs of myeloproliferative disease. In this study, we tested the hypothesis that increased JAK2V617F somatic mutation burden is associated with myeloproliferative neoplasm progression rate in the general population. Among 49,488 individuals from the Copenhagen General Population Study, 63 (0.1%) tested positive for the JAK2V617F mutation in the time period 2003–2008. Of these, 48 were available for re-examination in 2012. Level of JAK2V617F mutation burden was associated with myeloproliferative neoplasm progression rate, consistent with a biological continuum of increasing JAK2V617F mutation burden across increasing severity of myeloproliferative neoplasm from no disease (n=8 at re-examination) through essential thrombocythemia (n=20) and polycythemia vera (n=13) to primary myelofibrosis (n=7). Among those diagnosed with a myeloproliferative neoplasm only at re-examination in 2012, in the preceding years JAK2V617F mutation burden increased by 0.55% per year, erythrocyte volume fraction increased by 1.19% per year, and erythrocyte mean corpuscular volume increased by 1.25% per year, while there was no change in platelet count or erythropoietin levels. Furthermore, we established a JAK2V617F mutation burden cut-off point of 2% indicative of disease versus no disease; however, individuals with a mutation burden below 2% may suffer from a latent form of myeloproliferative disease revealed by a slightly larger spleen and/or slightly higher lactic acid dehydrogenase concentration compared to controls. Of all 63 JAK2V617F positive individuals, 48 were eventually diagnosed with a myeloproliferative neoplasm. PMID:24907356

  14. Mutational Spectrum of MYO15A and the Molecular Mechanisms of DFNB3 Human Deafness

    PubMed Central

    Rehman, Atteeq U.; Bird, Jonathan E.; Faridi, Rabia; Shahzad, Mohsin; Shah, Sujay; Lee, Kwanghyuk; Khan, Shaheen N.; Imtiaz, Ayesha; Ahmed, Zubair M.; Riazuddin, Saima; Santos-Cortez, Regie Lyn P.; Ahmad, Wasim; Leal, Suzanne M.; Riazuddin, Sheikh; Friedman, Thomas B.

    2016-01-01

    Deafness in humans is a common neurosensory disorder and is genetically heterogeneous. Across diverse ethnic groups, mutations of MYO15A at the DFNB3 locus appear to be the third or fourth most common cause of autosomal recessive, nonsyndromic deafness. In 49 of the 67 exons of MYO15A, there are currently 192 recessive mutations identified, including 14 novel mutations reported here. These mutations are distributed uniformly across MYO15A with one enigmatic exception; the alternatively spliced giant exon 2, encoding 1,233 residues, has 17 truncating mutations but no convincing deafness-causing missense mutations. MYO15A encodes three distinct isoform classes, one of which is 395 kDa (3,530 residues), the largest member of the myosin superfamily of molecular motors. Studies of Myo15 mouse models that recapitulate DFNB3 revealed two different pathogenic mechanisms of hearing loss. In the inner ear, myosin 15 is necessary both for the development and the long-term maintenance of stereocilia, mechanosensory sound-transducing organelles that extend from the apical surface of hair cells. The goal of this Mutation Update is to provide a comprehensive review of mutations and functions of MYO15A. PMID:27375115

  15. Development of an in vitro PIG-A gene mutation assay in human cells

    PubMed Central

    Rees, Benjamin J; Tate, Matthew; Lynch, Anthony M; Thornton, Catherine A; Jenkins, Gareth J; Walmsley, Richard M; Johnson, George E

    2017-01-01

    Abstract Mutagens can be carcinogens, and traditionally, they have been identified in vitro using the Salmonella ‘Ames’ reverse mutation assay. However, prokaryotic DNA packaging, replication and repair systems are mechanistically very different to those in the humans we inevitably seek to protect. Therefore, for many years, mammalian cell line genotoxicity assays that can detect eukaryotic mutagens as well as clastogens and aneugens have been used. The apparent lack of specificity in these largely rodent systems, due partly to their mutant p53 status, has contributed to the use of animal studies to resolve data conflicts. Recently, silencing mutations at the PIG-A locus have been demonstrated to prevent glycophosphatidylinositol (GPI) anchor synthesis and consequentially result in loss of GPI-anchored proteins from the cell’s extracellular surface. The successful exploitation of this mutant phenotype in animal studies has triggered interest in the development of an analogous in vitro PIG-A mutation screening assay. This article describes the development of a robust assay design using metabolically active human cells. The assay includes viability and cell membrane integrity assessment and conforms to the future ideas of the 21st-century toxicology testing. PMID:28057708

  16. Benzo[a]pyrene, Aflatoxine B1 and Acetaldehyde Mutational Patterns in TP53 Gene Using a Functional Assay: Relevance to Human Cancer Aetiology

    PubMed Central

    Paget, Vincent; Lechevrel, Mathilde; André, Véronique; Le Goff, Jérémie; Pottier, Didier; Billet, Sylvain; Garçon, Guillaume; Shirali, Pirouz; Sichel, François

    2012-01-01

    Mutations in the TP53 gene are the most common alterations in human tumours. TP53 mutational patterns have sometimes been linked to carcinogen exposure. In hepatocellular carcinoma, a specific G>T transversion on codon 249 is classically described as a fingerprint of aflatoxin B1 exposure. Likewise G>T transversions in codons 157 and 158 have been related to tobacco exposure in human lung cancers. However, controversies remain about the interpretation of TP53 mutational pattern in tumours as the fingerprint of genotoxin exposure. By using a functional assay, the Functional Analysis of Separated Alleles in Yeast (FASAY), the present study depicts the mutational pattern of TP53 in normal human fibroblasts after in vitro exposure to well-known carcinogens: benzo[a]pyrene, aflatoxin B1 and acetaldehyde. These in vitro patterns of mutations were then compared to those found in human tumours by using the IARC database of TP53 mutations. The results show that the TP53 mutational patterns found in human tumours can be only partly ascribed to genotoxin exposure. A complex interplay between the functional impact of the mutations on p53 phenotype and the cancer natural history may affect these patterns. However, our results strongly support that genotoxins exposure plays a major role in the aetiology of the considered cancers. PMID:22319594

  17. Gamma radiation at a human relevant low dose rate is genotoxic in mice

    NASA Astrophysics Data System (ADS)

    Graupner, Anne; Eide, Dag M.; Instanes, Christine; Andersen, Jill M.; Brede, Dag A.; Dertinger, Stephen D.; Lind, Ole C.; Brandt-Kjelsen, Anicke; Bjerke, Hans; Salbu, Brit; Oughton, Deborah; Brunborg, Gunnar; Olsen, Ann K.

    2016-09-01

    Even today, 70 years after Hiroshima and accidents like in Chernobyl and Fukushima, we still have limited knowledge about the health effects of low dose rate (LDR) radiation. Despite their human relevance after occupational and accidental exposure, only few animal studies on the genotoxic effects of chronic LDR radiation have been performed. Selenium (Se) is involved in oxidative stress defence, protecting DNA and other biomolecules from reactive oxygen species (ROS). It is hypothesised that Se deficiency, as it occurs in several parts of the world, may aggravate harmful effects of ROS-inducing stressors such as ionising radiation. We performed a study in the newly established LDR-facility Figaro on the combined effects of Se deprivation and LDR γ exposure in DNA repair knockout mice (Ogg1-/-) and control animals (Ogg1+/-). Genotoxic effects were seen after continuous radiation (1.4 mGy/h) for 45 days. Chromosomal damage (micronucleus), phenotypic mutations (Pig-a gene mutation of RBCCD24-) and DNA lesions (single strand breaks/alkali labile sites) were significantly increased in blood cells of irradiated animals, covering three types of genotoxic activity. This study demonstrates that chronic LDR γ radiation is genotoxic in an exposure scenario realistic for humans, supporting the hypothesis that even LDR γ radiation may induce cancer.

  18. Can a few non‐coding mutations make a human brain?

    PubMed Central

    Franchini, Lucía F.

    2015-01-01

    The recent finding that the human version of a neurodevelopmental enhancer of the Wnt receptor Frizzled 8 (FZD8) gene alters neural progenitor cell cycle timing and brain size is a step forward to understanding human brain evolution. The human brain is distinctive in terms of its cognitive abilities as well as its susceptibility to neurological disease. Identifying which of the millions of genomic changes that occurred during human evolution led to these and other uniquely human traits is extremely challenging. Recent studies have demonstrated that many of the fastest evolving regions of the human genome function as gene regulatory enhancers during embryonic development and that the human‐specific mutations in them might alter expression patterns. However, elucidating molecular and cellular effects of sequence or expression pattern changes is a major obstacle to discovering the genetic bases of the evolution of our species. There is much work to do before human‐specific genetic and genomic changes are linked to complex human traits. Also watch the Video Abstract. PMID:26350501

  19. A canine model of Alzheimer's disease generated by overexpressing a mutated human amyloid precursor protein.

    PubMed

    Lee, Geun-Shik; Jeong, Yeon Woo; Kim, Joung Joo; Park, Sun Woo; Ko, Kyeong Hee; Kang, Mina; Kim, Yu Kyung; Jung, Eui-Man; Moon, Changjong; Hyun, Sang Hwan; Hwang, Kyu-Chan; Kim, Nam-Hyung; Shin, Taeyoung; Jeung, Eui-Bae; Hwang, Woo Suk

    2014-04-01

    Canines are considered the most authentic model for studying multifactorial human diseases, as these animals typically share a common environment with man. Somatic cell nuclear transfer (SCNT) technology along with genetic engineering of nuclear donor cells provides a unique opportunity for examining human diseases using transgenic canines. In the present study, we generated transgenic canines that overexpressed the human amyloid precursor protein (APP) gene containing well-characterized familial Alzheimer's disease (AD) mutations. We successfully obtained five out of six live puppies by SCNT. This was confirmed by observing the expression of green fluorescence protein in the body as a visual transgenic marker and the overexpression of the mutated APP gene in the brain. The transgenic canines developed AD-like symptoms, such as enlarged ventricles, an atrophied hippocampus, and β-amyloid plaques in the brain. Thus, the transgenic canines we created can serve as a novel animal model for studying human AD.

  20. Exome-wide Mutation Profile in Benzo[a]pyrene-derived Post-stasis and Immortal Human Mammary Epithelial Cells

    PubMed Central

    Severson, Paul L.; Vrba, Lukas; Stampfer, Martha R.; Futscher, Bernard W.

    2014-01-01

    Genetic mutations are known to drive cancer progression and certain tumors have mutation signatures that reflect exposures to environmental carcinogens. Benzo[a]pyrene (BaP) has a known mutation signature and has proven capable of inducing changes to DNA sequence that drives normal pre-stasis human mammary epithelial cells (HMEC) past a first tumor suppressor barrier (stasis) and towards immortality. We analyzed normal, pre-stasis HMEC, three independent BaP-derived post-stasis HMEC strains (184Aa, 184Be, 184Ce) and two of their immortal derivatives(184A1 and 184BE1) by whole exome sequencing. The independent post-stasis strains exhibited between 93 and 233 BaP-induced mutations in exons. Seventy percent of the mutations were C:G>A:T transversions, consistent with the known mutation spectrum of BaP. Mutations predicted to impact protein function occurred in several known and putative cancer drivers including p16, PLCG1, MED12, TAF1 in 184Aa; PIK3CG, HSP90AB1, WHSC1L1, LCP1 in 184Be and FANCA, LPP in 184Ce. Biological processes that typically harbor cancer driver mutations such as cell cycle, regulation of cell death and proliferation, RNA processing, chromatin modification and DNA repair were found to have mutations predicted to impact function in each of the post-stasis strains. Spontaneously immortalized HMEC lines derived from two of the BaP-derived post-stasis strains shared greater than 95% of their BaP-induced mutations with their precursor cells. These immortal HMEC had 10 or fewer additional point mutations relative to their post-stasis precursors, but acquired chromosomal anomalies during immortalization that arose independent of BaP. The results of this study indicate that acute exposures of HMEC to high dose BaP recapitulate mutation patterns of human tumors and can induce mutations in a number of cancer driver genes. PMID:25435355

  1. Exome-wide mutation profile in benzo[a]pyrene-derived post-stasis and immortal human mammary epithelial cells.

    PubMed

    Severson, Paul L; Vrba, Lukas; Stampfer, Martha R; Futscher, Bernard W

    2014-12-01

    Genetic mutations are known to drive cancer progression and certain tumors have mutation signatures that reflect exposures to environmental carcinogens. Benzo[a]pyrene (BaP) has a known mutation signature and has proven capable of inducing changes to DNA sequence that drives normal pre-stasis human mammary epithelial cells (HMEC) past a first tumor suppressor barrier (stasis) and toward immortality. We analyzed normal, pre-stasis HMEC, three independent BaP-derived post-stasis HMEC strains (184Aa, 184Be, 184Ce) and two of their immortal derivatives(184A1 and 184BE1) by whole exome sequencing. The independent post-stasis strains exhibited between 93 and 233 BaP-induced mutations in exons. Seventy percent of the mutations were C:G>A:T transversions, consistent with the known mutation spectrum of BaP. Mutations predicted to impact protein function occurred in several known and putative cancer drivers including p16, PLCG1, MED12, TAF1 in 184Aa; PIK3CG, HSP90AB1, WHSC1L1, LCP1 in 184Be and FANCA, LPP in 184Ce. Biological processes that typically harbor cancer driver mutations such as cell cycle, regulation of cell death and proliferation, RNA processing, chromatin modification and DNA repair were found to have mutations predicted to impact function in each of the post-stasis strains. Spontaneously immortalized HMEC lines derived from two of the BaP-derived post-stasis strains shared greater than 95% of their BaP-induced mutations with their precursor cells. These immortal HMEC had 10 or fewer additional point mutations relative to their post-stasis precursors, but acquired chromosomal anomalies during immortalization that arose independent of BaP. The results of this study indicate that acute exposures of HMEC to high dose BaP recapitulate mutation patterns of human tumors and can induce mutations in a number of cancer driver genes. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Evolution Models with Conditional Mutation Rates: Strange Plateaus in Population Distribution

    NASA Astrophysics Data System (ADS)

    Saakian, David B.

    2017-08-01

    Cancer is related to clonal evolution with a strongly nonlinear, collective behavior. Here we investigate a slightly advanced version of the popular Crow-Kimura evolution model, suggested recently, by simply assuming a conditional mutation rate. We investigated the steady-state solution and found a highly intriguing plateau in the distribution. There are selective and nonselective phases, with a rather narrow plateau in the distribution at the peak in the first phase, and a wide plateau for many Hamming classes (a collection of genomes with the same number of mutations from the reference genome) in the second phase. We analytically solved the steady state distribution in the selective and nonselective phases, calculating the widths of the plateaus. Numerically, we also found an intermediate phase with several plateaus in the steady-state distribution, related to large finite-genome-length corrections. We assume that the newly observed phenomena should exist in other versions of evolution dynamics when the parameters of the model are conditioned to the population distribution.

  3. Molecular and biochemical characterization of a unique mutation in CCS, the human copper chaperone to superoxide dismutase.

    PubMed

    Huppke, Peter; Brendel, Cornelia; Korenke, Georg Christoph; Marquardt, Iris; Donsante, Anthony; Yi, Ling; Hicks, Julia D; Steinbach, Peter J; Wilson, Callum; Elpeleg, Orly; Møller, Lisbeth Birk; Christodoulou, John; Kaler, Stephen G; Gärtner, Jutta

    2012-08-01

    Copper (Cu) is a trace metal that readily gains and donates electrons, a property that renders it desirable as an enzyme cofactor but dangerous as a source of free radicals. To regulate cellular Cu metabolism, an elaborate system of chaperones and transporters has evolved, although no human Cu chaperone mutations have been described to date. We describe a child from a consanguineous family who inherited homozygous mutations in the SLC33A1, encoding an acetyl CoA transporter, and in CCS, encoding the Cu chaperone for superoxide dismutase. The CCS mutation, p.Arg163Trp, predicts substitution of a highly conserved arginine residue at position 163, with tryptophan in domain II of CCS, which interacts directly with superoxide dismutase 1 (SOD1). Biochemical analyses of the patient's fibroblasts, mammalian cell transfections, immunoprecipitation assays, and Lys7Δ (CCS homolog) yeast complementation support the pathogenicity of the mutation. Expression of CCS was reduced and binding of CCS to SOD1 impaired. As a result, this mutation causes reduced SOD1 activity and may impair other mechanisms important for normal Cu homeostasis. CCS-Arg163Trp represents the primary example of a human mutation in a gene coding for a Cu chaperone. © 2012 Wiley Periodicals, Inc.

  4. Molecular and biochemical characterization of a unique mutation in CCS, the human copper chaperone to superoxide dismutase

    PubMed Central

    Huppke, Peter; Brendel, Cornelia; Korenke, Georg Christoph; Marquardt, Iris; Donsante, Anthony; Yi, Ling; Hicks, Julia D.; Steinbach, Peter J.; Wilson, Callum; Elpeleg, Orly; Møller, Lisbeth Birk; Christodoulou, John; Kaler, Stephen G.; Gärtner, Jutta

    2012-01-01

    Copper is a trace metal that readily gains and donates electrons, a property that renders it desirable as an enzyme cofactor but dangerous as a source of free radicals. To regulate cellular copper metabolism, an elaborate system of chaperones and transporters has evolved, although no human copper chaperone mutations have been described to date. We describe a child from a consanguineous family who inherited a homozygous mutations in the SLC33A1, encoding an acetyl CoA transporter, and in CCS, encoding the copper chaperone for superoxide dismutase. The CCS mutation, p.Arg163Trp, predicts substitution of a highly conserved arginine residue at position 163 with tryptophan in domain II of CCS, which interacts directly with SOD1. Biochemical analyses of the patient’s fibroblasts, mammalian cell transfections, immunoprecipitation assays, and Lys7Δ (CCS homolog) yeast complementation support the pathogenicity of the mutation. Expression of CCS was reduced and binding of CCS to SOD1 impaired. As a result this mutation causes reduced SOD1 activity and may impair other mechanisms important for normal copper homeostasis. CCS-Arg163Trp represents the primary example of a human mutation in a gene coding for a copper chaperone. PMID:22508683

  5. Evidence for a high mutation rate at rapidly evolving yeast centromeres

    PubMed Central

    2011-01-01

    Background Although their role in cell division is essential, centromeres evolve rapidly in animals, plants and yeasts. Unlike the complex centromeres of plants and aminals, the point centromeres of Saccharomcyes yeasts can be readily sequenced to distinguish amongst the possible explanations for fast centromere evolution. Results Using DNA sequences of all 16 centromeres from 34 strains of Saccharomyces cerevisiae and population genomic data from Saccharomyces paradoxus, I show that centromeres in both species evolve 3 times more rapidly even than selectively unconstrained DNA. Exceptionally high levels of polymorphism seen in multiple yeast populations suggest that rapid centromere evolution does not result from the repeated selective sweeps expected under meiotic drive. I further show that there is little evidence for crossing-over or gene conversion within centromeres, although there is clear evidence for recombination in their immediate vicinity. Finally I show that the mutation spectrum at centromeres is consistent with the pattern of spontaneous mutation elsewhere in the genome. Conclusions These results indicate that rapid centromere evolution is a common phenomenon in yeast species. Furthermore, these results suggest that rapid centromere evolution does not result from the mutagenic effect of gene conversion, but from a generalised increase in the mutation rate, perhaps arising from the unusual chromatin structure at centromeres in yeast and other eukaryotes. PMID:21767380

  6. Mutation spectrum of Chinese patients with Bartter syndrome.

    PubMed

    Han, Yue; Lin, Yi; Sun, Qing; Wang, Shujuan; Gao, Yanxia; Shao, Leping

    2017-11-24

    Bartter syndrome (BS) has been rarely reported in Chinese population except for a few case reports. This investigation was aimed to analyze the mutations of the causal genes in sixteen Chinese patients with BS, and review their followup and treatment. Identify mutations by the next generation sequencing and the multiplex ligation-dependent probe amplification (MLPA). Clinical characteristics and biochemical findings at the first presentation as well as follow-up were reviewed. 15 different CLCNKB gene mutations were identified in fourteen patients with BS, including 11 novel ones. A novel missense mutation and a novel small deletion were found from SLC12A1 gene. A novel gross deletion was found in CLCNKA gene. A recurrent missense mutation was identified from BSND gene. We found that the whole gene deletion mutation of CLCNKB gene was the most frequent mutation (32%), and the rate of gross deletion was up to 50 percent in this group of Chinese patients. The present study has found 19 mutations, including 14 novel ones, which would enrich the human gene mutation database (HGMD) and provide valuable references to the genetic counseling and diagnosis of the Chinese population.

  7. Mutation spectrum of Chinese patients with Bartter syndrome

    PubMed Central

    Han, Yue; Lin, Yi; Sun, Qing; Wang, Shujuan; Gao, Yanxia; Shao, Leping

    2017-01-01

    Objective Bartter syndrome (BS) has been rarely reported in Chinese population except for a few case reports. This investigation was aimed to analyze the mutations of the causal genes in sixteen Chinese patients with BS, and review their followup and treatment. Methods Identify mutations by the next generation sequencing and the multiplex ligation-dependent probe amplification (MLPA). Clinical characteristics and biochemical findings at the first presentation as well as follow-up were reviewed. Results 15 different CLCNKB gene mutations were identified in fourteen patients with BS, including 11 novel ones. A novel missense mutation and a novel small deletion were found from SLC12A1 gene. A novel gross deletion was found in CLCNKA gene. A recurrent missense mutation was identified from BSND gene. We found that the whole gene deletion mutation of CLCNKB gene was the most frequent mutation (32%), and the rate of gross deletion was up to 50 percent in this group of Chinese patients. Conclusion The present study has found 19 mutations, including 14 novel ones, which would enrich the human gene mutation database (HGMD) and provide valuable references to the genetic counseling and diagnosis of the Chinese population. PMID:29254190

  8. [Genetic mutation databases: stakes and perspectives for orphan genetic diseases].

    PubMed

    Humbertclaude, V; Tuffery-Giraud, S; Bareil, C; Thèze, C; Paulet, D; Desmet, F-O; Hamroun, D; Baux, D; Girardet, A; Collod-Béroud, G; Khau Van Kien, P; Roux, A-F; des Georges, M; Béroud, C; Claustres, M

    2010-10-01

    New technologies, which constantly become available for mutation detection and gene analysis, have contributed to an exponential rate of discovery of disease genes and variation in the human genome. The task of collecting and documenting this enormous amount of data in genetic databases represents a major challenge for the future of biological and medical science. The Locus Specific Databases (LSDBs) are so far the most efficient mutation databases. This review presents the main types of databases available for the analysis of mutations responsible for genetic disorders, as well as open perspectives for new therapeutic research or challenges for future medicine. Accurate and exhaustive collection of variations in human genomes will be crucial for research and personalized delivery of healthcare. Copyright © 2009 Elsevier Masson SAS. All rights reserved.

  9. Structure of human POFUT1, its requirement in ligand-independent oncogenic Notch signaling, and functional effects of Dowling-Degos mutations

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

    McMillan, Brian J.; Zimmerman, Brandon; Egan, Emily D.

    Protein O-fucosyltransferase-1 (POFUT1), which transfers fucose residues to acceptor sites on serine and threonine residues of epidermal growth factor-like repeats of recipient proteins, is essential for Notch signal transduction in mammals. Here, we examine the consequences of POFUT1 loss on the oncogenic signaling associated with certain leukemia-associated mutations of human Notch1, report the structures of human POFUT1 in free and GDP-fucose bound states, and assess the effects of Dowling-Degos mutations on human POFUT1 function. CRISPR-mediated knockout of POFUT1 in U2OS cells suppresses both normal Notch1 signaling, and the ligand-independent signaling associated with leukemogenic mutations of Notch1. Normal and oncogenic signalingmore » are rescued by wild-type POFUT1 but rescue is impaired by an active-site R240A mutation. The overall structure of the human enzyme closely resembles that of the Caenorhabditis elegans protein, with an overall backbone RMSD of 0.93 Å, despite primary sequence identity of only 39% in the mature protein. GDP-fucose binding to the human enzyme induces limited backbone conformational movement, though the side chains of R43 and D244 reorient to make direct contact with the fucose moiety in the complex. The reported Dowling-Degos mutations of POFUT1, except for M262T, fail to rescue Notch1 signaling efficiently in the CRISPR-engineered POFUT1 -/- background. Together, these studies identify POFUT1 as a potential target for cancers driven by Notch1 mutations and provide a structural roadmap for its inhibition.« less

  10. Mapping Interaction Sites on Human Chemokine Receptors by Deep Mutational Scanning.

    PubMed

    Heredia, Jeremiah D; Park, Jihye; Brubaker, Riley J; Szymanski, Steven K; Gill, Kevin S; Procko, Erik

    2018-06-01

    Chemokine receptors CXCR4 and CCR5 regulate WBC trafficking and are engaged by the HIV-1 envelope glycoprotein gp120 during infection. We combine a selection of human CXCR4 and CCR5 libraries comprising nearly all of ∼7000 single amino acid substitutions with deep sequencing to define sequence-activity landscapes for surface expression and ligand interactions. After consideration of sequence constraints for surface expression, known interaction sites with HIV-1-blocking Abs were appropriately identified as conserved residues following library sorting for Ab binding, validating the use of deep mutational scanning to map functional interaction sites in G protein-coupled receptors. Chemokine CXCL12 was found to interact with residues extending asymmetrically into the CXCR4 ligand-binding cavity, similar to the binding surface of CXCR4 recognized by an antagonistic viral chemokine previously observed crystallographically. CXCR4 mutations distal from the chemokine binding site were identified that enhance chemokine recognition. This included disruptive mutations in the G protein-coupling site that diminished calcium mobilization, as well as conservative mutations to a membrane-exposed site (CXCR4 residues H79 2.45 and W161 4.50 ) that increased ligand binding without loss of signaling. Compared with CXCR4-CXCL12 interactions, CCR5 residues conserved for gp120 (HIV-1 BaL strain) interactions map to a more expansive surface, mimicking how the cognate chemokine CCL5 makes contacts across the entire CCR5 binding cavity. Acidic substitutions in the CCR5 N terminus and extracellular loops enhanced gp120 binding. This study demonstrates how comprehensive mutational scanning can define functional interaction sites on receptors, and novel mutations that enhance receptor activities can be found simultaneously. Copyright © 2018 by The American Association of Immunologists, Inc.

  11. Altered heme catabolism by heme oxygenase-1 caused by mutations in human NADPH cytochrome P450 reductase

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

    Pandey, Amit V., E-mail: amit@pandeylab.org; Flueck, Christa E.; Mullis, Primus E.

    2010-09-24

    Research highlights: {yields} Mutations in POR identified from patients lead to reduced HO-1 activities. {yields} POR mutation Y181D affecting FMN binding results in total loss of HO-1 activity. {yields} POR mutations A287P, C569Y and V608F, lost 50-70% activity. {yields} Mutations in FAD binding domain, R457H, Y459H and V492E lost all HO-1 activity. {yields} POR polymorphisms P228L, R316W, G413S, A503V and G504R have normal activity. -- Abstract: Human heme oxygenase-1 (HO-1) carries out heme catabolism supported by electrons supplied from the NADPH through NADPH P450 reductase (POR, CPR). Previously we have shown that mutations in human POR cause a rare formmore » of congenital adrenal hyperplasia. In this study, we have evaluated the effects of mutations in POR on HO-1 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified HO-1 to measure heme degradation in a coupled assay using biliverdin reductase. Here we show that mutations in POR found in patients may reduce HO-1 activity, potentially influencing heme catabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had total loss of HO-1 activity, while POR mutations A287P, C569Y and V608F lost 50-70% activity. The POR variants P228L, R316W and G413S, A503V and G504R identified as polymorphs had close to WT activity. Loss of HO-1 activity may result in increased oxidative neurotoxicity, anemia, growth retardation and iron deposition. Further examination of patients affected with POR deficiency will be required to assess the metabolic effects of reduced HO-1 activity in affected individuals.« less

  12. Published sequences do not support transfer of oseltamivir resistance mutations from avian to human influenza A virus strains.

    PubMed

    Norberg, Peter; Lindh, Magnus; Olofsson, Sigvard

    2015-03-28

    Tamiflu (oseltamivir phosphate ester, OE) is a widely used antiviral active against influenza A virus. Its active metabolite, oseltamivir carboxylate (OC), is chemically stable and secreted into wastewater treatment plants. OC contamination of natural habitats of waterfowl might induce OC resistance in influenza viruses persistently infecting waterfowl, and lead to transfer of OC-resistance from avian to human influenza. The aim of this study was to evaluate whether such has occurred. A genomics approach including phylogenetic analysis and probability calculations for homologous recombination was applied on altogether 19,755 neuraminidase (N1 and N2) genes from virus sampled in humans and birds, with and without resistance mutations. No evidence for transfer of OE resistance mutations from avian to human N genes was obtained, and events suggesting recombination between human and avian influenza virus variants could not be traced in the sequence material studied. The results indicate that resistance in influenza viruses infecting humans is due to the selection pressure posed by the global OE administration in humans rather than transfer from avian influenza A virus strains carrying mutations induced by environmental exposure to OC.

  13. Computational analysis of histidine mutations on the structural stability of human tyrosinases leading to albinism insurgence.

    PubMed

    Hassan, Mubashir; Abbas, Qamar; Raza, Hussain; Moustafa, Ahmed A; Seo, Sung-Yum

    2017-07-25

    Misfolding and structural alteration in proteins lead to serious malfunctions and cause various diseases in humans. Mutations at the active binding site in tyrosinase impair structural stability and cause lethal albinism by abolishing copper binding. To evaluate the histidine mutational effect, all mutated structures were built using homology modelling. The protein sequence was retrieved from the UniProt database, and 3D models of original and mutated human tyrosinase sequences were predicted by changing the residual positions within the target sequence separately. Structural and mutational analyses were performed to interpret the significance of mutated residues (N 180 , R 202 , Q 202 , R 211 , Y 363 , R 367 , Y 367 and D 390 ) at the active binding site of tyrosinases. CSpritz analysis depicted that 23.25% residues actively participate in the instability of tyrosinase. The accuracy of predicted models was confirmed through online servers ProSA-web, ERRAT score and VERIFY 3D values. The theoretical pI and GRAVY generated results also showed the accuracy of the predicted models. The CCA negative correlation results depicted that the replacement of mutated residues at His within the active binding site disturbs the structural stability of tyrosinases. The predicted CCA scores of Tyr 367 (-0.079) and Q/R 202 (0.032) revealed that both mutations have more potential to disturb the structural stability. MD simulation analyses of all predicted models justified that Gln 202 , Arg 202 , Tyr 367 and D 390 replacement made the protein structures more susceptible to destabilization. Mutational results showed that the replacement of His with Q/R 202 and Y/R 363 has a lethal effect and may cause melanin associated diseases such as OCA1. Taken together, our computational analysis depicts that the mutated residues such as Q/R 202 and Y/R 363 actively participate in instability and misfolding of tyrosinases, which may govern OCA1 through disturbing the melanin biosynthetic pathway.

  14. T antigen mutations are a human tumor-specific signature for Merkel cell polyomavirus

    PubMed Central

    Shuda, Masahiro; Feng, Huichen; Kwun, Hyun Jin; Rosen, Steven T.; Gjoerup, Ole; Moore, Patrick S.; Chang, Yuan

    2008-01-01

    Merkel cell polyomavirus (MCV) is a virus discovered in our laboratory at the University of Pittsburgh that is monoclonally integrated into the genome of ≈80% of human Merkel cell carcinomas (MCCs). Transcript mapping was performed to show that MCV expresses transcripts in MCCs similar to large T (LT), small T (ST), and 17kT transcripts of SV40. Nine MCC tumor-derived LT genomic sequences have been examined, and all were found to harbor mutations prematurely truncating the MCV LT helicase. In contrast, four presumed episomal viruses from nontumor sources did not possess this T antigen signature mutation. Using coimmunoprecipitation and origin replication assays, we show that tumor-derived virus mutations do not affect retinoblastoma tumor suppressor protein (Rb) binding by LT but do eliminate viral DNA replication capacity. Identification of an MCC cell line (MKL-1) having monoclonal MCV integration and the signature LT mutation allowed us to functionally test both tumor-derived and wild type (WT) T antigens. Only WT LT expression activates replication of integrated MCV DNA in MKL-1 cells. Our findings suggest that MCV-positive MCC tumor cells undergo selection for LT mutations to prevent autoactivation of integrated virus replication that would be detrimental to cell survival. Because these mutations render the virus replication-incompetent, MCV is not a “passenger virus” that secondarily infects MCC tumors. PMID:18812503

  15. Exome-wide mutation profile in benzo[a]pyrene-derived post-stasis and immortal human mammary epithelial cells

    DOE PAGES

    Severson, Paul L.; Vrba, Lukas; Stampfer, Martha R.; ...

    2014-11-04

    Genetic mutations are known to drive cancer progression and certain tumors have mutation signatures that reflect exposures to environmental carcinogens. Benzo[a]pyrene (BaP) has a known mutation signature and has proven capable of inducing changes to DNA sequence that drives normal pre-stasis human mammary epithelial cells (HMEC) past a first tumor suppressor barrier (stasis) and toward immortality. We analyzed normal, pre-stasis HMEC, three independent BaP-derived post-stasis HMEC strains (184Aa, 184Be, 184Ce) and two of their immortal derivatives(184A1 and 184BE1) by whole exome sequencing. The independent post-stasis strains exhibited between 93 and 233 BaP-induced mutations in exons. Seventy percent of the mutationsmore » were C:G>A:T transversions, consistent with the known mutation spectrum of BaP. Mutations predicted to impact protein function occurred in several known and putative cancer drivers including p16, PLCG1, MED12, TAF1 in 184Aa; PIK3CG, HSP90AB1, WHSC1L1, LCP1 in 184Be and FANCA, LPP in 184Ce. Biological processes that typically harbor cancer driver mutations such as cell cycle, regulation of cell death and proliferation, RNA processing, chromatin modification and DNA repair were found to have mutations predicted to impact function in each of the post-stasis strains. Spontaneously immortalized HMEC lines derived from two of the BaP-derived post-stasis strains shared greater than 95% of their BaP-induced mutations with their precursor cells. These immortal HMEC had 10 or fewer additional point mutations relative to their post-stasis precursors, but acquired chromosomal anomalies during immortalization that arose independent of BaP. In conclusion, the results of this study indicate that acute exposures of HMEC to high dose BaP recapitulate mutation patterns of human tumors and can induce mutations in a number of cancer driver genes.« less

  16. Mutational Spectrum of MYO15A and the Molecular Mechanisms of DFNB3 Human Deafness.

    PubMed

    Rehman, Atteeq U; Bird, Jonathan E; Faridi, Rabia; Shahzad, Mohsin; Shah, Sujay; Lee, Kwanghyuk; Khan, Shaheen N; Imtiaz, Ayesha; Ahmed, Zubair M; Riazuddin, Saima; Santos-Cortez, Regie Lyn P; Ahmad, Wasim; Leal, Suzanne M; Riazuddin, Sheikh; Friedman, Thomas B

    2016-10-01

    Deafness in humans is a common neurosensory disorder and is genetically heterogeneous. Across diverse ethnic groups, mutations of MYO15A at the DFNB3 locus appear to be the third or fourth most common cause of autosomal-recessive, nonsyndromic deafness. In 49 of the 67 exons of MYO15A, there are currently 192 recessive mutations identified, including 14 novel mutations reported here. These mutations are distributed uniformly across MYO15A with one enigmatic exception; the alternatively spliced giant exon 2, encoding 1,233 residues, has 17 truncating mutations but no convincing deafness-causing missense mutations. MYO15A encodes three distinct isoform classes, one of which is 395 kDa (3,530 residues), the largest member of the myosin superfamily of molecular motors. Studies of Myo15 mouse models that recapitulate DFNB3 revealed two different pathogenic mechanisms of hearing loss. In the inner ear, myosin 15 is necessary both for the development and the long-term maintenance of stereocilia, mechanosensory sound-transducing organelles that extend from the apical surface of hair cells. The goal of this Mutation Update is to provide a comprehensive review of mutations and functions of MYO15A. © 2016 WILEY PERIODICALS, INC.

  17. Germline mitochondrial DNA mutations aggravate ageing and can impair brain development.

    PubMed

    Ross, Jaime M; Stewart, James B; Hagström, Erik; Brené, Stefan; Mourier, Arnaud; Coppotelli, Giuseppe; Freyer, Christoph; Lagouge, Marie; Hoffer, Barry J; Olson, Lars; Larsson, Nils-Göran

    2013-09-19

    Ageing is due to an accumulation of various types of damage, and mitochondrial dysfunction has long been considered to be important in this process. There is substantial sequence variation in mammalian mitochondrial DNA (mtDNA), and the high mutation rate is counteracted by different mechanisms that decrease maternal transmission of mutated mtDNA. Despite these protective mechanisms, it is becoming increasingly clear that low-level mtDNA heteroplasmy is quite common and often inherited in humans. We designed a series of mouse mutants to investigate the extent to which inherited mtDNA mutations can contribute to ageing. Here we report that maternally transmitted mtDNA mutations can induce mild ageing phenotypes in mice with a wild-type nuclear genome. Furthermore, maternally transmitted mtDNA mutations lead to anticipation of reduced fertility in mice that are heterozygous for the mtDNA mutator allele (PolgA(wt/mut)) and aggravate premature ageing phenotypes in mtDNA mutator mice (PolgA(mut/mut)). Unexpectedly, a combination of maternally transmitted and somatic mtDNA mutations also leads to stochastic brain malformations. Our findings show that a pre-existing mutation load will not only allow somatic mutagenesis to create a critically high total mtDNA mutation load sooner but will also increase clonal expansion of mtDNA mutations to enhance the normally occurring mosaic respiratory chain deficiency in ageing tissues. Our findings suggest that maternally transmitted mtDNA mutations may have a similar role in aggravating aspects of normal human ageing.

  18. Visualizing the origins of selfish de novo mutations in individual seminiferous tubules of human testes

    PubMed Central

    Maher, Geoffrey J.; McGowan, Simon J.; Giannoulatou, Eleni; Verrill, Clare; Goriely, Anne; Wilkie, Andrew O. M.

    2016-01-01

    De novo point mutations arise predominantly in the male germline and increase in frequency with age, but it has not previously been possible to locate specific, identifiable mutations directly within the seminiferous tubules of human testes. Using microdissection of tubules exhibiting altered expression of the spermatogonial markers MAGEA4, FGFR3, and phospho-AKT, whole genome amplification, and DNA sequencing, we establish an in situ strategy for discovery and analysis of pathogenic de novo mutations. In 14 testes from men aged 39–90 y, we identified 11 distinct gain-of-function mutations in five genes (fibroblast growth factor receptors FGFR2 and FGFR3, tyrosine phosphatase PTPN11, and RAS oncogene homologs HRAS and KRAS) from 16 of 22 tubules analyzed; all mutations have known associations with severe diseases, ranging from congenital or perinatal lethal disorders to somatically acquired cancers. These results support proposed selfish selection of spermatogonial mutations affecting growth factor receptor-RAS signaling, highlight its prevalence in older men, and enable direct visualization of the microscopic anatomy of elongated mutant clones. PMID:26858415

  19. Visualizing the origins of selfish de novo mutations in individual seminiferous tubules of human testes.

    PubMed

    Maher, Geoffrey J; McGowan, Simon J; Giannoulatou, Eleni; Verrill, Clare; Goriely, Anne; Wilkie, Andrew O M

    2016-03-01

    De novo point mutations arise predominantly in the male germline and increase in frequency with age, but it has not previously been possible to locate specific, identifiable mutations directly within the seminiferous tubules of human testes. Using microdissection of tubules exhibiting altered expression of the spermatogonial markers MAGEA4, FGFR3, and phospho-AKT, whole genome amplification, and DNA sequencing, we establish an in situ strategy for discovery and analysis of pathogenic de novo mutations. In 14 testes from men aged 39-90 y, we identified 11 distinct gain-of-function mutations in five genes (fibroblast growth factor receptors FGFR2 and FGFR3, tyrosine phosphatase PTPN11, and RAS oncogene homologs HRAS and KRAS) from 16 of 22 tubules analyzed; all mutations have known associations with severe diseases, ranging from congenital or perinatal lethal disorders to somatically acquired cancers. These results support proposed selfish selection of spermatogonial mutations affecting growth factor receptor-RAS signaling, highlight its prevalence in older men, and enable direct visualization of the microscopic anatomy of elongated mutant clones.

  20. Is High Resolution Melting Analysis (HRMA) Accurate for Detection of Human Disease-Associated Mutations? A Meta Analysis

    PubMed Central

    Ma, Feng-Li; Jiang, Bo; Song, Xiao-Xiao; Xu, An-Gao

    2011-01-01

    Background High Resolution Melting Analysis (HRMA) is becoming the preferred method for mutation detection. However, its accuracy in the individual clinical diagnostic setting is variable. To assess the diagnostic accuracy of HRMA for human mutations in comparison to DNA sequencing in different routine clinical settings, we have conducted a meta-analysis of published reports. Methodology/Principal Findings Out of 195 publications obtained from the initial search criteria, thirty-four studies assessing the accuracy of HRMA were included in the meta-analysis. We found that HRMA was a highly sensitive test for detecting disease-associated mutations in humans. Overall, the summary sensitivity was 97.5% (95% confidence interval (CI): 96.8–98.5; I2 = 27.0%). Subgroup analysis showed even higher sensitivity for non-HR-1 instruments (sensitivity 98.7% (95%CI: 97.7–99.3; I2 = 0.0%)) and an eligible sample size subgroup (sensitivity 99.3% (95%CI: 98.1–99.8; I2 = 0.0%)). HRMA specificity showed considerable heterogeneity between studies. Sensitivity of the techniques was influenced by sample size and instrument type but by not sample source or dye type. Conclusions/Significance These findings show that HRMA is a highly sensitive, simple and low-cost test to detect human disease-associated mutations, especially for samples with mutations of low incidence. The burden on DNA sequencing could be significantly reduced by the implementation of HRMA, but it should be recognized that its sensitivity varies according to the number of samples with/without mutations, and positive results require DNA sequencing for confirmation. PMID:22194806

  1. Multiple point mutations in a shuttle vector propagated in human cells: evidence for an error-prone DNA polymerase activity.

    PubMed

    Seidman, M M; Bredberg, A; Seetharam, S; Kraemer, K H

    1987-07-01

    Mutagenesis was studied at the DNA-sequence level in human fibroblast and lymphoid cells by use of a shuttle vector plasmid, pZ189, containing a suppressor tRNA marker gene. In a series of experiments, 62 plasmids were recovered that had two to six base substitutions in the 160-base-pair marker gene. Approximately 20-30% of the mutant plasmids that were recovered after passing ultraviolet-treated pZ189 through a repair-proficient human fibroblast line contained these multiple mutations. In contrast, passage of ultraviolet-treated pZ189 through an excision-repair-deficient (xeroderma pigmentosum) line yielded only 2% multiple base substitution mutants. Introducing a single-strand nick in otherwise unmodified pZ189 adjacent to the marker, followed by passage through the xeroderma pigmentosum cells, resulted in about 66% multiple base substitution mutants. The multiple mutations were found in a 160-base-pair region containing the marker gene but were rarely found in an adjacent 170-base-pair region. Passing ultraviolet-treated or nicked pZ189 through a repair-proficient human B-cell line also yielded multiple base substitution mutations in 20-33% of the mutant plasmids. An explanation for these multiple mutations is that they were generated by an error-prone polymerase while filling gaps. These mutations share many of the properties displayed by mutations in the immunoglobulin hypervariable regions.

  2. Heart-specific expression of laminopathic mutations in transgenic zebrafish.

    PubMed

    Verma, Ajay D; Parnaik, Veena K

    2017-07-01

    Lamins are key determinants of nuclear organization and function in the metazoan nucleus. Mutations in human lamin A cause a spectrum of genetic diseases that affect cardiac muscle and skeletal muscle as well as other tissues. A few laminopathies have been modeled using the mouse. As zebrafish is a well established model for the study of cardiac development and disease, we have investigated the effects of heart-specific lamin A mutations in transgenic zebrafish. We have developed transgenic lines of zebrafish expressing conserved lamin A mutations that cause cardiac dysfunction in humans. Expression of zlamin A mutations Q291P and M368K in the heart was driven by the zebrafish cardiac troponin T2 promoter. Homozygous mutant embryos displayed nuclear abnormalities in cardiomyocyte nuclei. Expression analysis showed the upregulation of genes involved in heart regeneration in transgenic mutant embryos and a cell proliferation marker was increased in adult heart tissue. At the physiological level, there was deviation of up to 20% from normal heart rate in transgenic embryos expressing mutant lamins. Adult homozygous zebrafish were fertile and did not show signs of early mortality. Our results suggest that transgenic zebrafish models of heart-specific laminopathies show cardiac regeneration and moderate deviations in heart rate during embryonic development. © 2017 International Federation for Cell Biology.

  3. Characterization of mouse and human GTP cyclohydrolase I genes. Mutations in patients with GTP cyclohydrolase I deficiency.

    PubMed

    Ichinose, H; Ohye, T; Matsuda, Y; Hori, T; Blau, N; Burlina, A; Rouse, B; Matalon, R; Fujita, K; Nagatsu, T

    1995-04-28

    GTP cyclohydrolase I is the first and rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin in mammals. Previously, we reported three species of human GTP cyclohydrolase I cDNA in a human liver cDNA library (Togari, A., Ichinose, H., Matsumoto, S., Fujita, K., and Nagatsu, T. (1992) Biochem. Biophys. Res. Commun. 187, 359-365). Furthermore, very recently, we found that the GTP cyclohydrolase I gene is causative for hereditary progressive dystonia with marked diurnal fluctuation, also known as DOPA-responsive dystonia (Ichinose, H., Ohye, T., Takahashi, E., Seki, N., Hori, T., Segawa, M., Nomura, Y., Endo, K., Tanaka, H., Tsuji, S., Fujita, K., and Nagatsu, T. (1994) Nature Genetics 8, 236-242). To clarify the mechanisms that regulate transcription of the GTP cyclohydrolase I gene and to generate multiple species of mRNA, we isolated genomic DNA clones for the human and mouse GTP cyclohydrolase I genes. Structural analysis of the isolated clones revealed that the GTP cyclohydrolase I gene is encoded by a single copy gene and is composed of six exons spanning approximately 30 kilobases. We sequenced all exon/intron boundaries of the human and mouse genes. Structural analysis also demonstrated that the heterogeneity of GTP cyclohydrolase I mRNA is caused by an alternative usage of the splicing acceptor site at the sixth exon. The transcription start site of the mouse GTP cyclohydrolase I gene and the 5'-flanking sequences of the mouse and human genes were determined. We performed regional mapping of the mouse gene by fluorescence in situ hybridization, and the mouse GTP cyclohydrolase I gene was assigned to region C2-3 of mouse chromosome 14. We identified missense mutations in patients with GTP cyclohydrolase I deficiency and expressed mutated enzymes in Escherichia coli to confirm alterations in the enzyme activity.

  4. Accumulation of Human-Adapting Mutations during Circulation of A(H1N1)pdm09 Influenza Virus in Humans in the United Kingdom

    PubMed Central

    Elderfield, Ruth A.; Watson, Simon J.; Godlee, Alexandra; Adamson, Walt E.; Thompson, Catherine I.; Dunning, Jake; Fernandez-Alonso, Mirian; Blumenkrantz, Deena; Hussell, Tracy; Zambon, Maria; Openshaw, Peter; Kellam, Paul

    2014-01-01

    ABSTRACT The influenza pandemic that emerged in 2009 provided an unprecedented opportunity to study adaptation of a virus recently acquired from an animal source during human transmission. In the United Kingdom, the novel virus spread in three temporally distinct waves between 2009 and 2011. Phylogenetic analysis of complete viral genomes showed that mutations accumulated over time. Second- and third-wave viruses replicated more rapidly in human airway epithelial (HAE) cells than did the first-wave virus. In infected mice, weight loss varied between viral isolates from the same wave but showed no distinct pattern with wave and did not correlate with viral load in the mouse lungs or severity of disease in the human donor. However, second- and third-wave viruses induced less alpha interferon in the infected mouse lungs. NS1 protein, an interferon antagonist, had accumulated several mutations in second- and third-wave viruses. Recombinant viruses with the third-wave NS gene induced less interferon in human cells, but this alone did not account for increased virus fitness in HAE cells. Mutations in HA and NA genes in third-wave viruses caused increased binding to α-2,6-sialic acid and enhanced infectivity in human mucus. A recombinant virus with these two segments replicated more efficiently in HAE cells. A mutation in PA (N321K) enhanced polymerase activity of third-wave viruses and also provided a replicative advantage in HAE cells. Therefore, multiple mutations allowed incremental changes in viral fitness, which together may have contributed to the apparent increase in severity of A(H1N1)pdm09 influenza virus during successive waves. IMPORTANCE Although most people infected with the 2009 pandemic influenza virus had mild or unapparent symptoms, some suffered severe and devastating disease. The reasons for this variability were unknown, but the numbers of severe cases increased during successive waves of human infection in the United Kingdom. To determine the causes

  5. Accumulation of human-adapting mutations during circulation of A(H1N1)pdm09 influenza virus in humans in the United Kingdom.

    PubMed

    Elderfield, Ruth A; Watson, Simon J; Godlee, Alexandra; Adamson, Walt E; Thompson, Catherine I; Dunning, Jake; Fernandez-Alonso, Mirian; Blumenkrantz, Deena; Hussell, Tracy; Zambon, Maria; Openshaw, Peter; Kellam, Paul; Barclay, Wendy S

    2014-11-01

    The influenza pandemic that emerged in 2009 provided an unprecedented opportunity to study adaptation of a virus recently acquired from an animal source during human transmission. In the United Kingdom, the novel virus spread in three temporally distinct waves between 2009 and 2011. Phylogenetic analysis of complete viral genomes showed that mutations accumulated over time. Second- and third-wave viruses replicated more rapidly in human airway epithelial (HAE) cells than did the first-wave virus. In infected mice, weight loss varied between viral isolates from the same wave but showed no distinct pattern with wave and did not correlate with viral load in the mouse lungs or severity of disease in the human donor. However, second- and third-wave viruses induced less alpha interferon in the infected mouse lungs. NS1 protein, an interferon antagonist, had accumulated several mutations in second- and third-wave viruses. Recombinant viruses with the third-wave NS gene induced less interferon in human cells, but this alone did not account for increased virus fitness in HAE cells. Mutations in HA and NA genes in third-wave viruses caused increased binding to α-2,6-sialic acid and enhanced infectivity in human mucus. A recombinant virus with these two segments replicated more efficiently in HAE cells. A mutation in PA (N321K) enhanced polymerase activity of third-wave viruses and also provided a replicative advantage in HAE cells. Therefore, multiple mutations allowed incremental changes in viral fitness, which together may have contributed to the apparent increase in severity of A(H1N1)pdm09 influenza virus during successive waves. Although most people infected with the 2009 pandemic influenza virus had mild or unapparent symptoms, some suffered severe and devastating disease. The reasons for this variability were unknown, but the numbers of severe cases increased during successive waves of human infection in the United Kingdom. To determine the causes of this variation

  6. Bi-allelic Mutations in PKD1L1 Are Associated with Laterality Defects in Humans.

    PubMed

    Vetrini, Francesco; D'Alessandro, Lisa C A; Akdemir, Zeynep C; Braxton, Alicia; Azamian, Mahshid S; Eldomery, Mohammad K; Miller, Kathryn; Kois, Chelsea; Sack, Virginia; Shur, Natasha; Rijhsinghani, Asha; Chandarana, Jignesh; Ding, Yan; Holtzman, Judy; Jhangiani, Shalini N; Muzny, Donna M; Gibbs, Richard A; Eng, Christine M; Hanchard, Neil A; Harel, Tamar; Rosenfeld, Jill A; Belmont, John W; Lupski, James R; Yang, Yaping

    2016-10-06

    Disruption of the establishment of left-right (L-R) asymmetry leads to situs anomalies ranging from situs inversus totalis (SIT) to situs ambiguus (heterotaxy). The genetic causes of laterality defects in humans are highly heterogeneous. Via whole-exome sequencing (WES), we identified homozygous mutations in PKD1L1 from three affected individuals in two unrelated families. PKD1L1 encodes a polycystin-1-like protein and its loss of function is known to cause laterality defects in mouse and medaka fish models. Family 1 had one fetus and one deceased child with heterotaxy and complex congenital heart malformations. WES identified a homozygous splicing mutation, c.6473+2_6473+3delTG, which disrupts the invariant splice donor site in intron 42, in both affected individuals. In the second family, a homozygous c.5072G>C (p.Cys1691Ser) missense mutation was detected in an individual with SIT and congenital heart disease. The p.Cys1691Ser substitution affects a highly conserved cysteine residue and is predicted by molecular modeling to disrupt a disulfide bridge essential for the proper folding of the G protein-coupled receptor proteolytic site (GPS) motif. Damaging effects associated with substitutions of this conserved cysteine residue in the GPS motif have also been reported in other genes, namely GPR56, BAI3, and PKD1 in human and lat-1 in C. elegans, further supporting the likely pathogenicity of p.Cys1691Ser in PKD1L1. The identification of bi-allelic PKD1L1 mutations recapitulates previous findings regarding phenotypic consequences of loss of function of the orthologous genes in mice and medaka fish and further expands our understanding of genetic contributions to laterality defects in humans. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  7. Loss-of-function mutation in GATA4 causes anomalies of human testicular development

    PubMed Central

    Lourenço, Diana; Brauner, Raja; Rybczyńska, Magda; Nihoul-Fékété, Claire; McElreavey, Ken; Bashamboo, Anu

    2011-01-01

    Approximately 1 of every 250 newborns has some abnormality of genital and/or gonadal development. However, a specific molecular cause is identified in only 20% of these cases of disorder of sex development (DSD). We identified a family of French origin presenting with 46,XY DSD and congenital heart disease. Sequencing of the ORF of GATA4 identified a heterozygous missense mutation (p.Gly221Arg) in the conserved N-terminal zinc finger of GATA4. This mutation was not observed in 450 ancestry-matched control individuals. The mutation compromised the ability of the protein to bind to and transactivate the anti-Müllerian hormone (AMH) promoter. The mutation does not interfere with the direct protein–protein interaction, but it disrupts synergistic activation of the AMH promoter by GATA4 and NR5A1. The p.Gly221Arg mutant protein also failed to bind to a known protein partner FOG2 that is essential for gonad formation. Our data demonstrate the key role of GATA4 in human testicular development. PMID:21220346

  8. Recombination or mutational hot spots in human mtDNA?

    PubMed

    Innan, Hideki; Nordborg, Magnus

    2002-07-01

    Awadalla, Eyre-Walker, and Maynard Smith (1999) recently argued that there might be recombination in human mitochondrial DNA (mtDNA). Their claim was based on their observation of decaying linkage disequilibrium (LD) as a function of physical distance. Their study was much criticized, and follow-up studies have failed to find any evidence for recombination. We argue that the criticisms levied, even if correct, could not possibly explain the findings of Awadalla, Eyre-Walker, and Maynard Smith (1999). Nonetheless, the test proposed by Awadalla, Eyre-Walker, and Maynard Smith (1999 ) is not robust because recombination is not the only explanation for decay of LD. We show that such a pattern can be caused by mutational hot spots as well. However, a closer look at the data suggests that the pattern observed was not caused by mutational hot spots but rather by chance. Thus, there appears to be no evidence for recombination in the mtDNA polymorphism data. In conclusion, we discuss the possibility of detecting recombination in mtDNA and the implications of its existence.

  9. Origin of Somatic Mutations in β-Catenin versus Adenomatous Polyposis Coli in Colon Cancer: Random Mutagenesis in Animal Models versus Nonrandom Mutagenesis in Humans.

    PubMed

    Yang, Da; Zhang, Min; Gold, Barry

    2017-07-17

    Wnt signaling is compromised early in the development of human colorectal cancer (CRC) due to truncating nonsense mutations in adenomatous polyposis coli (APC). CRC induced by chemical carcinogens, such as heterocyclic aromatic amines and azoxymethane, in mice also involves dysregulation of Wnt signaling but via activating missense mutations in the β-catenin oncogene despite the fact that genetically modified mice harboring an inactive APC allele efficiently develop CRC. In contrast, activating mutations in β-catenin are rarely observed in human CRC. Dysregulation of the Wnt signaling pathway by the two distinct mechanisms reveals insights into the etiology of human CRC. On the basis of calculations related to DNA adduct levels produced in mouse CRC models using mutagens, and the number of stem cells in the mouse colon, we show that two nonsense mutations required for biallelic disruption of APC are statistically unlikely to produce CRC in experiments using small numbers of mice. We calculate that an activating mutation in one allele near the critical GSK3β phosphorylation site on β-catenin is >10 5 -times more likely to produce CRC by random mutagenesis due to chemicals than inactivating two alleles in APC, yet it does not occur in humans. Therefore, the mutagenesis mechanism in human CRC cannot be random. We explain that nonsense APC mutations predominate in human CRC because of deamination at 5-methylcytosine at CGA and CAG codons, coupled with the number of human colonic stem cells and lifespan. Our analyses, including a comparison of mutation type and age at CRC diagnosis in U.S. and Chinese patients, also indicate that APC mutations in CRC are not due to environmental mutagens that randomly damage DNA.

  10. An interactive mutation database for human coagulation factor IX provides novel insights into the phenotypes and genetics of hemophilia B.

    PubMed

    Rallapalli, P M; Kemball-Cook, G; Tuddenham, E G; Gomez, K; Perkins, S J

    2013-07-01

    Factor IX (FIX) is important in the coagulation cascade, being activated to FIXa on cleavage. Defects in the human F9 gene frequently lead to hemophilia B. To assess 1113 unique F9 mutations corresponding to 3721 patient entries in a new and up-to-date interactive web database alongside the FIXa protein structure. The mutations database was built using MySQL and structural analyses were based on a homology model for the human FIXa structure based on closely-related crystal structures. Mutations have been found in 336 (73%) out of 461 residues in FIX. There were 812 unique point mutations, 182 deletions, 54 polymorphisms, 39 insertions and 26 others that together comprise a total of 1113 unique variants. The 64 unique mild severity mutations in the mature protein with known circulating protein phenotypes include 15 (23%) quantitative type I mutations and 41 (64%) predominantly qualitative type II mutations. Inhibitors were described in 59 reports (1.6%) corresponding to 25 unique mutations. The interactive database provides insights into mechanisms of hemophilia B. Type II mutations are deduced to disrupt predominantly those structural regions involved with functional interactions. The interactive features of the database will assist in making judgments about patient management. © 2013 International Society on Thrombosis and Haemostasis.

  11. Somatic mutations of the histone H3K27 demethylase gene UTX in human cancer.

    PubMed

    van Haaften, Gijs; Dalgliesh, Gillian L; Davies, Helen; Chen, Lina; Bignell, Graham; Greenman, Chris; Edkins, Sarah; Hardy, Claire; O'Meara, Sarah; Teague, Jon; Butler, Adam; Hinton, Jonathan; Latimer, Calli; Andrews, Jenny; Barthorpe, Syd; Beare, Dave; Buck, Gemma; Campbell, Peter J; Cole, Jennifer; Forbes, Simon; Jia, Mingming; Jones, David; Kok, Chai Yin; Leroy, Catherine; Lin, Meng-Lay; McBride, David J; Maddison, Mark; Maquire, Simon; McLay, Kirsten; Menzies, Andrew; Mironenko, Tatiana; Mulderrig, Lee; Mudie, Laura; Pleasance, Erin; Shepherd, Rebecca; Smith, Raffaella; Stebbings, Lucy; Stephens, Philip; Tang, Gurpreet; Tarpey, Patrick S; Turner, Rachel; Turrell, Kelly; Varian, Jennifer; West, Sofie; Widaa, Sara; Wray, Paul; Collins, V Peter; Ichimura, Koichi; Law, Simon; Wong, John; Yuen, Siu Tsan; Leung, Suet Yi; Tonon, Giovanni; DePinho, Ronald A; Tai, Yu-Tzu; Anderson, Kenneth C; Kahnoski, Richard J; Massie, Aaron; Khoo, Sok Kean; Teh, Bin Tean; Stratton, Michael R; Futreal, P Andrew

    2009-05-01

    Somatically acquired epigenetic changes are present in many cancers. Epigenetic regulation is maintained via post-translational modifications of core histones. Here, we describe inactivating somatic mutations in the histone lysine demethylase gene UTX, pointing to histone H3 lysine methylation deregulation in multiple tumor types. UTX reintroduction into cancer cells with inactivating UTX mutations resulted in slowing of proliferation and marked transcriptional changes. These data identify UTX as a new human cancer gene.

  12. Liquid biopsy for detection of actionable oncogenic mutations in human cancers and electric field induced release and measurement liquid biopsy (eLB).

    PubMed

    Tu, Michael; Chia, David; Wei, Fang; Wong, David

    2016-01-21

    Oncogenic activations by mutations in key cancer genes such as EGFR and KRAS are frequently associated with human cancers. Molecular targeting of specific oncogenic mutations in human cancer is a major therapeutic inroad for anti-cancer drug therapy. In addition, progressive developments of oncogene mutations lead to drug resistance. Therefore, the ability to detect and continuously monitor key actionable oncogenic mutations is important to guide the use of targeted molecular therapies to improve long-term clinical outcomes in cancer patients. Current oncogenic mutation detection is based on direct sampling of cancer tissue by surgical resection or biopsy. Oncogenic mutations were recently shown to be detectable in circulating bodily fluids of cancer patients. This field of investigation, termed liquid biopsy, permits a less invasive means of assessing the oncogenic mutation profile of a patient. This paper will review the analytical strategies used to assess oncogenic mutations from biofluid samples. Clinical applications will also be discussed.

  13. Liquid Biopsy for Detection of Actionable Oncogenic Mutations in Human Cancers and Electric Field Induced Release and Measurement Liquid Biopsy (eLB)

    PubMed Central

    Tu, Michael; Chia, David; Wei, Fang; Wong, David

    2015-01-01

    Oncogenic activations by mutations in key cancer genes such as EGFR and KRAS are frequently associated with human cancers. Molecular targeting of specific oncogenic mutations in human cancer is a major therapeutic inroad for anti-cancer drug therapy. In addition, progressive developments of oncogene mutations lead to drug resistance. Therefore, the ability to detect and continuously monitor key actionable oncogenic mutations is important to guide the use of targeted molecular therapies to improve long-term clinical outcomes in cancer patients. Current oncogenic mutation detection is based on direct sampling of cancer tissue by surgical resection or biopsy. Oncogenic mutations were recently shown to be detectable in circulating bodily fluids of cancer patients. This field of investigation, termed liquid biopsy, permits a less invasive means of assessing the oncogenic mutation profile of a patient. This paper will review the analytical strategies used to assess oncogenic mutations from biofluid samples. Clinical applications will also be discussed. PMID:26645892

  14. Structure of the human sulfhydryl oxidase augmenter of liver regeneration and characterization of a human mutation causing an autosomal recessive myopathy†, ‡

    PubMed Central

    Daithankar, Vidyadhar N.; Schaefer, Stephanie A.; Dong, Ming; Bahnson, Brian J.; Thorpe, Colin

    2010-01-01

    The sulfhydryl oxidase augmenter of liver regeneration (ALR) binds FAD in a helix-rich domain that presents a CxxC disulfide proximal to the isoalloxazine ring of the flavin. Head-to-tail interchain disulfide bonds link subunits within the homodimer of both the short, cytokine-like, form of ALR (sfALR), and a longer form (lfALR) which resides in the mitochondrial intermembrane space (IMS). lfALR has an 80-residue N-terminal extension with an additional CxxC motif required for the reoxidation of reduced Mia40 during oxidative protein folding within the IMS. Recently Di Fonzo et al. (Di Fonzo, A., Ronchi, D., Lodi, T., Fassone, E., Tigano, M., Lamperti, C., Corti, S., Bordoni, A., Fortunato, F., Nizzardo, M., Napoli, L., Donadoni, C., Salani, S., Saladino, F., Moggio, M., Bresolin, N., Ferrero, I., and Comi, G. P. (2009) Am. J. Hum. Genet. 84, 594–604) described an R194H mutation of human ALR that led to cataract, progressive muscle hypotonia, and hearing loss in three children. The current work presents a structural and enzymological characterization of the human R194H mutant in lf- and sfALR. A crystal structure of human sfALR was determined by molecular replacement using the rat sfALR structure. R194 is located at the subunit interface of sfALR, close to the intersubunit disulfide bridges. The R194 guanidino moiety participates in three H-bonds: two main-chain carbonyl oxygen atoms (from R194 itself, and from C95 of the intersubunit disulfide of the other protomer) and with the 2' OH of the FAD ribose. The R194H mutation has minimal effect on the enzyme activity using model and physiological substrates of short and long ALR forms. However the mutation adversely affects the stability of both ALR forms: e.g. by decreasing the melting temperature by about 10 °C, by increasing the rate of dissociation of FAD from the holoenzyme by about 45-fold, and by strongly enhancing the susceptibility of sfALR to partial proteolysis and to reduction of its intersubunit

  15. The mutational landscape of MYCN, Lin28b and ALK F1174L driven murine neuroblastoma mimics human disease.

    PubMed

    De Wilde, Bram; Beckers, Anneleen; Lindner, Sven; Kristina, Althoff; De Preter, Katleen; Depuydt, Pauline; Mestdagh, Pieter; Sante, Tom; Lefever, Steve; Hertwig, Falk; Peng, Zhiyu; Shi, Le-Ming; Lee, Sangkyun; Vandermarliere, Elien; Martens, Lennart; Menten, Björn; Schramm, Alexander; Fischer, Matthias; Schulte, Johannes; Vandesompele, Jo; Speleman, Frank

    2018-02-02

    Genetically engineered mouse models have proven to be essential tools for unraveling fundamental aspects of cancer biology and for testing novel therapeutic strategies. To optimally serve these goals, it is essential that the mouse model faithfully recapitulates the human disease. Recently, novel mouse models for neuroblastoma have been developed. Here, we report on the further genomic characterization through exome sequencing and DNA copy number analysis of four of the currently available murine neuroblastoma model systems ( ALK, Th- MYCN, Dbh- MYCN and Lin28b ). The murine tumors revealed a low number of genomic alterations - in keeping with human neuroblastoma - and a positive correlation of the number of genetic lesions with the time to onset of tumor formation was observed. Gene copy number alterations are the hallmark of both murine and human disease and frequently affect syntenic genomic regions. Despite low mutational load, the genes mutated in murine disease were found to be enriched for genes mutated in human disease. Taken together, our study further supports the validity of the tested mouse models for mechanistic and preclinical studies of human neuroblastoma.

  16. Diphtheria toxin resistance in human lymphocytes and lymphoblasts in the in vivo somatic cell mutation test

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

    Tomkins, D.J.; Wei, L.; Laurie, K.E.

    1985-01-01

    It has been shown that circulating peripheral blood lymphocytes can be used for the enumeration of 6-thioguanine-resistant cells that presumably arise by mutation in vivo. This somatic cell mutation test has been studied in lymphocytes from human populations exposed to known mutagens and/or carcinogens. The sensitivity of the test could be further enhanced by including other gene markers, since there is evidence for locus-specific differences in response to mutagens. Resistance to diphtheria toxin (Dip/sup r/) seemed like a potential marker to incorporate into the test because the mutation acts codominantly, can readily be selected in human diploid fibroblasts and Chinesemore » hamster cells with no evidence for cell density or cross-feeding effects, and can be assayed for in nondividing cells by measuring protein synthesis inhibition. Blood samples were collected from seven individuals, and fresh, cryopreserved, or Epstein-Barr virus (EBV)-transformed lymphocytes were tested for continued DNA synthesis (TH-thymidine, autoradiography) or protein synthesis (TVS-methionine, scintillation counting). Both fresh and cryopreserved lymphocytes, stimulated to divide with phytohemagglutinin (PHA), continued to synthesize DNA in the presence of high doses of diphtheria toxin (DT). Similarly, both dividing (PHA-stimulated) and nondividing fresh lymphocytes carried on significant levels of protein synthesis even 68 hr after exposure to 100 flocculating units (LF)/ml DT. The results suggest that human T and B lymphocytes may not be as sensitive to DT protein synthesis inhibition as human fibroblast and Chinese hamster cells. For this reason, Dip/sup r/ may not be a suitable marker for the somatic cell mutation test.« less

  17. Dihydrofolate-Reductase Mutations in Plasmodium knowlesi Appear Unrelated to Selective Drug Pressure from Putative Human-To-Human Transmission in Sabah, Malaysia.

    PubMed

    Grigg, Matthew J; Barber, Bridget E; Marfurt, Jutta; Imwong, Mallika; William, Timothy; Bird, Elspeth; Piera, Kim A; Aziz, Ammar; Boonyuen, Usa; Drakeley, Christopher J; Cox, Jonathan; White, Nicholas J; Cheng, Qin; Yeo, Tsin W; Auburn, Sarah; Anstey, Nicholas M

    2016-01-01

    Malaria caused by zoonotic Plasmodium knowlesi is an emerging threat in Eastern Malaysia. Despite demonstrated vector competency, it is unknown whether human-to-human (H-H) transmission is occurring naturally. We sought evidence of drug selection pressure from the antimalarial sulfadoxine-pyrimethamine (SP) as a potential marker of H-H transmission. The P. knowlesi dihdyrofolate-reductase (pkdhfr) gene was sequenced from 449 P. knowlesi malaria cases from Sabah (Malaysian Borneo) and genotypes evaluated for association with clinical and epidemiological factors. Homology modelling using the pvdhfr template was used to assess the effect of pkdhfr mutations on the pyrimethamine binding pocket. Fourteen non-synonymous mutations were detected, with the most common being at codon T91P (10.2%) and R34L (10.0%), resulting in 21 different genotypes, including the wild-type, 14 single mutants, and six double mutants. One third of the P. knowlesi infections were with pkdhfr mutants; 145 (32%) patients had single mutants and 14 (3%) had double-mutants. In contrast, among the 47 P. falciparum isolates sequenced, three pfdhfr genotypes were found, with the double mutant 108N+59R being fixed and the triple mutants 108N+59R+51I and 108N+59R+164L occurring with frequencies of 4% and 8%, respectively. Two non-random spatio-temporal clusters were identified with pkdhfr genotypes. There was no association between pkdhfr mutations and hyperparasitaemia or malaria severity, both hypothesized to be indicators of H-H transmission. The orthologous loci associated with resistance in P. falciparum were not mutated in pkdhfr. Subsequent homology modelling of pkdhfr revealed gene loci 13, 53, 120, and 173 as being critical for pyrimethamine binding, however, there were no mutations at these sites among the 449 P. knowlesi isolates. Although moderate diversity was observed in pkdhfr in Sabah, there was no evidence this reflected selective antifolate drug pressure in humans.

  18. piRNA-mediated transposon regulation and the germ-line mutation rate in Drosophila melanogaster males.

    PubMed

    Simmons, Michael J; Peterson, Mark P; Thorp, Michael W; Buschette, Jared T; DiPrima, Stephanie N; Harter, Christine L; Skolnick, Matthew J

    2015-03-01

    Transposons, especially retrotransposons, are abundant in the genome of Drosophila melanogaster. These mobile elements are regulated by small RNAs that interact with the Piwi family of proteins-the piwi-interacting or piRNAs. The Piwi proteins are encoded by the genes argonaute3 (ago3), aubergine (aub), and piwi. Heterochromatin Protein 1 (HP1), a chromatin-organizing protein encoded by the Suppressor of variegation 205 [Su(var)205] gene, also plays a role in this regulation. To assess the mutational impact of weakening the system for transposon regulation, we measured the frequency of recessive X-linked lethal mutations occurring in the germ lines of males from stocks that were heterozygous for mutant alleles of the ago3, aub, piwi, or Su(var)205 genes. These mutant alleles are expected to deplete the wild-type proteins encoded by these genes by as much as 50%. The mutant alleles of piwi and Su(var)205 significantly increased the X-linked lethal mutation frequency, whereas the mutant alleles of ago3 did not. An increased mutation frequency was also observed in males from one of two mutant aub stocks, but this increase may not have been due to the aub mutant. The increased mutation frequency caused by depleting Piwi or HP1suggests that chromatin-organizing proteins play important roles in minimizing the germ-line mutation rate, possibly by stabilizing the structure of the heterochromatin in which many transposons are situated. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Dramatic effect of single-base mutation on the conformational dynamics of human telomeric G-quadruplex

    PubMed Central

    Lee, Ja Yil; Kim, D. S.

    2009-01-01

    Guanine-rich DNA sequences can form G-quadruplexes. These four-stranded structures are known to form in several genomic regions and to influence certain biological activities. Sometimes, the instability of G-quadruplexes causes the abnormal biological processes. Mutation is a culprit for the destabilization of G-quadruplexes, but the details of mutated G-quadruplexes are poorly understood. In this article, we investigated the conformational dynamics of single-base mutated human telomeric G-quadruplexes in the presence of K+ with single-molecule FRET spectroscopy. We observed that the replacement of single guanine by thymine in a G-track induces various folded structures, i.e. structural polymorphism. Moreover, direct observation of their dynamics revealed that a single-base mutation causes fast unfolding of folded states under physiological conditions. Furthermore, we found that the degree of destabilization varies according to mutation positions. When the central guanine of a G-track is replaced, the G-quadruplexes unfold quickly at any K+ concentrations and temperature. Meanwhile, outer-quartet mutated G-quadruplexes have heterogeneous dynamics at intermediate K+ concentrations and longstanding folded states at high K+ concentrations. Several factors such as base-stacking interaction and K+ coordination are responsible for the different dynamics according to the mutation position. PMID:19359361

  20. Gene mutations and increased levels of p53 protein in human squamous cell carcinomas and their cell lines.

    PubMed Central

    Burns, J. E.; Baird, M. C.; Clark, L. J.; Burns, P. A.; Edington, K.; Chapman, C.; Mitchell, R.; Robertson, G.; Soutar, D.; Parkinson, E. K.

    1993-01-01

    Using immunocytochemical and Western blotting techniques we have demonstrated the presence of abnormally high levels of p53 protein in 8/24 (33%) of human squamous cell carcinomas (SCC) and 9/18 (50%) of SCC cell lines. There was a correlation between the immunocytochemical results obtained with eight SCC samples and their corresponding cell lines. Direct sequencing of PCR-amplified, reverse transcribed, p53 mRNA confirmed the expression of point mutations in six of the positive cell lines and detected in-frame deletions in two others. We also detected two stop mutations and three out-of-frame deletions in five lines which did not express elevated levels of p53 protein. Several of the mutations found in SCC of the tongue (3/7) were in a region (codons 144-166) previously identified as being a p53 mutational hot spot in non-small cell lung tumours (Mitsudomi et al., 1992). In 11/13 cases only the mutant alleles were expressed suggesting loss or reduced expression of the wild type alleles in these cases. Six of the mutations were also detected in the SCCs from which the lines were derived, strongly suggesting that the mutations occurred, and were selected, in vivo. The 12th mutation GTG-->GGG (valine-->glycine) at codon 216 was expressed in line SCC-12 clone B along with an apparently normal p53 allele and is to our knowledge a novel mutation. Line BICR-19 also expressed a normal p53 allele in addition to one where exon 10 was deleted. Additionally 15 of the SCC lines (including all of those which did not show elevated p53 protein levels) were screened for the presence of human papillomavirus types 16 and 18 and were found to be negative. These results are discussed in relation to the pathogenesis of SCC and the immortalisation of human keratinocytes in vitro. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:8390283

  1. Infrequent detectable somatic mutations of the RET and glial cell line-derived neurotrophic factor (GDNF) genes in human pituitary adenomas.

    PubMed

    Yoshimoto, K; Tanaka, C; Moritani, M; Shimizu, E; Yamaoka, T; Yamada, S; Sano, T; Itakura, M

    1999-02-01

    RET is a receptor tyrosine kinase expressed in neuroendocrine cells and tumors. RET is activated by a ligand complex comprising glial cell line-derived neurotrophic factor (GDNF) and GDNF receptor-alpha (GDNFR-alpha). Activating mutations of the RET proto-oncogene were found in multiple endocrine neoplasia (MEN) 2 and in sporadic medullary thyroid carcinoma and pheochromocytoma of neuroendocrine origin. Mutations of the RET proto-oncogene and the glial cell line-derived neurotrophic factor (GDNF) gene were examined in human pituitary tumors. No mutations of the RET proto-oncogene including the cysteine-rich region or codon 768 and 918 in the tyrosine kinase domain were detected in 172 human pituitary adenomas either by polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) or by PCR-restriction fragment length polymorphism (RFLP). Further, somatic mutations of the GDNF gene in 33 human pituitary adenomas were not detected by PCR-SSCP. One polymorphism of the GDNF gene at codon 145 of TGC or TGT was observed in a prolactinoma. The RET proto-oncogene message was detected in a normal human pituitary gland or 4 of 4 human pituitary adenomas with reverse transcription (RT)-PCR, and in rodent pituitary tumor cell lines with Western blotting. The expression of GDNF gene was detected in 1 of 4 human somatotroph adenomas, 1 of 2 corticotroph adenomas, and 2 of 6 rodent pituitary tumor cell lines with RT-PCR. Based on these, it is concluded that somatic mutations of the RET proto-oncogene or the GDNF gene do not appear to play a major role in the pituitary tumorigenesis in examined tumors.

  2. Mutations in PIK3CA are infrequent in neuroblastoma

    PubMed Central

    Dam, Vincent; Morgan, Brian T; Mazanek, Pavel; Hogarty, Michael D

    2006-01-01

    Background Neuroblastoma is a frequently lethal pediatric cancer in which MYCN genomic amplification is highly correlated with aggressive disease. Deregulated MYC genes require co-operative lesions to foster tumourigenesis and both direct and indirect evidence support activated Ras signaling for this purpose in many cancers. Yet Ras genes and Braf, while often activated in cancer cells, are infrequent targets for activation in neuroblastoma. Recently, the Ras effector PIK3CA was shown to be activated in diverse human cancers. We therefore assessed PIK3CA for mutation in human neuroblastomas, as well as in neuroblastomas arising in transgenic mice with MYCN overexpressed in neural-crest tissues. In this murine model we additionally surveyed for Ras family and Braf mutations as these have not been previously reported. Methods Sixty-nine human neuroblastomas (42 primary tumors and 27 cell lines) were sequenced for PIK3CA activating mutations within the C2, helical and kinase domain "hot spots" where 80% of mutations cluster. Constitutional DNA was sequenced in cases with confirmed alterations to assess for germline or somatic acquisition. Additionally, Ras family members (Hras1, Kras2 and Nras) and the downstream effectors Pik3ca and Braf, were sequenced from twenty-five neuroblastomas arising in neuroblastoma-prone transgenic mice. Results We identified mutations in the PIK3CA gene in 2 of 69 human neuroblastomas (2.9%). Neither mutation (R524M and E982D) has been studied to date for effects on lipid kinase activity. Though both occurred in tumors with MYCN amplification the overall rate of PIK3CA mutations in MYCN amplified and single-copy tumors did not differ appreciably (2 of 31 versus 0 of 38, respectively). Further, no activating mutations were identified in a survey of Ras signal transduction genes (including Hras1, Kras2, Nras, Pik3ca, or Braf genes) in twenty-five neuroblastic tumors arising in the MYCN-initiated transgenic mouse model. Conclusion These data

  3. Natural mismatch repair mutations mediate phenotypic diversity and drug resistance in Cryptococcus deuterogattii.

    PubMed

    Billmyre, R Blake; Clancey, Shelly Applen; Heitman, Joseph

    2017-09-26

    Pathogenic microbes confront an evolutionary conflict between the pressure to maintain genome stability and the need to adapt to mounting external stresses. Bacteria often respond with elevated mutation rates, but little evidence exists of stable eukaryotic hypermutators in nature. Whole genome resequencing of the human fungal pathogen Cryptococcus deuterogattii identified an outbreak lineage characterized by a nonsense mutation in the mismatch repair component MSH2. This defect results in a moderate mutation rate increase in typical genes, and a larger increase in genes containing homopolymer runs. This allows facile inactivation of genes with coding homopolymer runs including FRR1 , which encodes the target of the immunosuppresive antifungal drugs FK506 and rapamycin. Our study identifies a eukaryotic hypermutator lineage spread over two continents and suggests that pathogenic eukaryotic microbes may experience similar selection pressures on mutation rate as bacterial pathogens, particularly during long periods of clonal growth or while expanding into new environments.

  4. An MRPS12 mutation modifies aminoglycoside sensitivity caused by 12S rRNA mutations

    PubMed Central

    Emperador, Sonia; Pacheu-Grau, David; Bayona-Bafaluy, M. Pilar; Garrido-Pérez, Nuria; Martín-Navarro, Antonio; López-Pérez, Manuel J.; Montoya, Julio; Ruiz-Pesini, Eduardo

    2015-01-01

    Several homoplasmic pathologic mutations in mitochondrial DNA, such as those causing Leber hereditary optic neuropathy or non-syndromic hearing loss, show incomplete penetrance. Therefore, other elements must modify their pathogenicity. Discovery of these modifying factors is not an easy task because in multifactorial diseases conventional genetic approaches may not always be informative. Here, we have taken an evolutionary approach to unmask putative modifying factors for a particular homoplasmic pathologic mutation causing aminoglycoside-induced and non-syndromic hearing loss, the m.1494C>T transition in the mitochondrial DNA. The mutation is located in the decoding site of the mitochondrial ribosomal RNA. We first looked at mammalian species that had fixed the human pathologic mutation. These mutations are called compensated pathogenic deviations because an organism carrying one must also have another that suppresses the deleterious effect of the first. We found that species from the primate family Cercopithecidae (old world monkeys) harbor the m.1494T allele even if their auditory function is normal. In humans the m.1494T allele increases the susceptibility to aminoglycosides. However, in primary fibroblasts from a Cercopithecidae species, aminoglycosides do not impair cell growth, respiratory complex IV activity and quantity or the mitochondrial protein synthesis. Interestingly, this species also carries a fixed mutation in the mitochondrial ribosomal protein S12. We show that the expression of this variant in a human m.1494T cell line reduces its susceptibility to aminoglycosides. Because several mutations in this human protein have been described, they may possibly explain the absence of pathologic phenotype in some pedigree members with the most frequent pathologic mutations in mitochondrial ribosomal RNA. PMID:25642242

  5. Molecular consequences of the R453C hypertrophic cardiomyopathy mutation on human β-cardiac myosin motor function.

    PubMed

    Sommese, Ruth F; Sung, Jongmin; Nag, Suman; Sutton, Shirley; Deacon, John C; Choe, Elizabeth; Leinwand, Leslie A; Ruppel, Kathleen; Spudich, James A

    2013-07-30

    Cardiovascular disorders are the leading cause of morbidity and mortality in the developed world, and hypertrophic cardiomyopathy (HCM) is among the most frequently occurring inherited cardiac disorders. HCM is caused by mutations in the genes encoding the fundamental force-generating machinery of the cardiac muscle, including β-cardiac myosin. Here, we present a biomechanical analysis of the HCM-causing mutation, R453C, in the context of human β-cardiac myosin. We found that this mutation causes a ∼30% decrease in the maximum ATPase of the human β-cardiac subfragment 1, the motor domain of myosin, and a similar percent decrease in the in vitro velocity. The major change in the R453C human β-cardiac subfragment 1 is a 50% increase in the intrinsic force of the motor compared with wild type, with no appreciable change in the stroke size, as observed with a dual-beam optical trap. These results predict that the overall force of the ensemble of myosin molecules in the muscle should be higher in the R453C mutant compared with wild type. Loaded in vitro motility assay confirms that the net force in the ensemble is indeed increased. Overall, this study suggests that the R453C mutation should result in a hypercontractile state in the heart muscle.

  6. Mutations involving the SRY-related gene SOX8 are associated with a spectrum of human reproductive anomalies.

    PubMed

    Portnoi, Marie-France; Dumargne, Marie-Charlotte; Rojo, Sandra; Witchel, Selma F; Duncan, Andrew J; Eozenou, Caroline; Bignon-Topalovic, Joelle; Yatsenko, Svetlana A; Rajkovic, Aleksandar; Reyes-Mugica, Miguel; Almstrup, Kristian; Fusee, Leila; Srivastava, Yogesh; Chantot-Bastaraud, Sandra; Hyon, Capucine; Louis-Sylvestre, Christine; Validire, Pierre; de Malleray Pichard, Caroline; Ravel, Celia; Christin-Maitre, Sophie; Brauner, Raja; Rossetti, Raffaella; Persani, Luca; Charreau, Eduardo H; Dain, Liliana; Chiauzzi, Violeta A; Mazen, Inas; Rouba, Hassan; Schluth-Bolard, Caroline; MacGowan, Stuart; McLean, W H Irwin; Patin, Etienne; Rajpert-De Meyts, Ewa; Jauch, Ralf; Achermann, John C; Siffroi, Jean-Pierre; McElreavey, Ken; Bashamboo, Anu

    2018-04-01

    SOX8 is an HMG-box transcription factor closely related to SRY and SOX9. Deletion of the gene encoding Sox8 in mice causes reproductive dysfunction but the role of SOX8 in humans is unknown. Here, we show that SOX8 is expressed in the somatic cells of the early developing gonad in the human and influences human sex determination. We identified two individuals with 46, XY disorders/differences in sex development (DSD) and chromosomal rearrangements encompassing the SOX8 locus and a third individual with 46, XY DSD and a missense mutation in the HMG-box of SOX8. In vitro functional assays indicate that this mutation alters the biological activity of the protein. As an emerging body of evidence suggests that DSDs and infertility can have common etiologies, we also analysed SOX8 in a cohort of infertile men (n = 274) and two independent cohorts of women with primary ovarian insufficiency (POI; n = 153 and n = 104). SOX8 mutations were found at increased frequency in oligozoospermic men (3.5%; P < 0.05) and POI (5.06%; P = 4.5 × 10-5) as compared with fertile/normospermic control populations (0.74%). The mutant proteins identified altered SOX8 biological activity as compared with the wild-type protein. These data demonstrate that SOX8 plays an important role in human reproduction and SOX8 mutations contribute to a spectrum of phenotypes including 46, XY DSD, male infertility and 46, XX POI.

  7. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9.

    PubMed

    Paquet, Dominik; Kwart, Dylan; Chen, Antonia; Sproul, Andrew; Jacob, Samson; Teo, Shaun; Olsen, Kimberly Moore; Gregg, Andrew; Noggle, Scott; Tessier-Lavigne, Marc

    2016-05-05

    The bacterial CRISPR/Cas9 system allows sequence-specific gene editing in many organisms and holds promise as a tool to generate models of human diseases, for example, in human pluripotent stem cells. CRISPR/Cas9 introduces targeted double-stranded breaks (DSBs) with high efficiency, which are typically repaired by non-homologous end-joining (NHEJ) resulting in nonspecific insertions, deletions or other mutations (indels). DSBs may also be repaired by homology-directed repair (HDR) using a DNA repair template, such as an introduced single-stranded oligo DNA nucleotide (ssODN), allowing knock-in of specific mutations. Although CRISPR/Cas9 is used extensively to engineer gene knockouts through NHEJ, editing by HDR remains inefficient and can be corrupted by additional indels, preventing its widespread use for modelling genetic disorders through introducing disease-associated mutations. Furthermore, targeted mutational knock-in at single alleles to model diseases caused by heterozygous mutations has not been reported. Here we describe a CRISPR/Cas9-based genome-editing framework that allows selective introduction of mono- and bi-allelic sequence changes with high efficiency and accuracy. We show that HDR accuracy is increased dramatically by incorporating silent CRISPR/Cas-blocking mutations along with pathogenic mutations, and establish a method termed 'CORRECT' for scarless genome editing. By characterizing and exploiting a stereotyped inverse relationship between a mutation's incorporation rate and its distance to the DSB, we achieve predictable control of zygosity. Homozygous introduction requires a guide RNA targeting close to the intended mutation, whereas heterozygous introduction can be accomplished by distance-dependent suboptimal mutation incorporation or by use of mixed repair templates. Using this approach, we generated human induced pluripotent stem cells with heterozygous and homozygous dominant early onset Alzheimer's disease-causing mutations in

  8. High mutation rates explain low population genetic divergence at copy-number-variable loci in Homo sapiens.

    PubMed

    Hu, Xin-Sheng; Yeh, Francis C; Hu, Yang; Deng, Li-Ting; Ennos, Richard A; Chen, Xiaoyang

    2017-02-22

    Copy-number-variable (CNV) loci differ from single nucleotide polymorphic (SNP) sites in size, mutation rate, and mechanisms of maintenance in natural populations. It is therefore hypothesized that population genetic divergence at CNV loci will differ from that found at SNP sites. Here, we test this hypothesis by analysing 856 CNV loci from the genomes of 1184 healthy individuals from 11 HapMap populations with a wide range of ancestry. The results show that population genetic divergence at the CNV loci is generally more than three times lower than at genome-wide SNP sites. Populations generally exhibit very small genetic divergence (G st  = 0.05 ± 0.049). The smallest divergence is among African populations (G st  = 0.0081 ± 0.0025), with increased divergence among non-African populations (G st  = 0.0217 ± 0.0109) and then among African and non-African populations (G st  = 0.0324 ± 0.0064). Genetic diversity is high in African populations (~0.13), low in Asian populations (~0.11), and intermediate in the remaining 11 populations. Few significant linkage disequilibria (LDs) occur between the genome-wide CNV loci. Patterns of gametic and zygotic LDs indicate the absence of epistasis among CNV loci. Mutation rate is about twice as large as the migration rate in the non-African populations, suggesting that the high mutation rates play dominant roles in producing the low population genetic divergence at CNV loci.

  9. The observed human sperm mutation frequency cannot explain the achondroplasia paternal age effect

    PubMed Central

    Tiemann-Boege, Irene; Navidi, William; Grewal, Raji; Cohn, Dan; Eskenazi, Brenda; Wyrobek, Andrew J.; Arnheim, Norman

    2002-01-01

    The lifelong spermatogonial stem cell divisions unique to male germ cell production are thought to contribute to a higher mutation frequency in males. The fact that certain de novo human genetic conditions (e.g., achondroplasia) increase in incidence with the age of the father is consistent with this idea. Although it is assumed that the paternal age effect is the result of an increasing frequency of mutant sperm as a man grows older, no direct molecular measurement of the germ-line mutation frequency has been made to confirm this hypothesis. Using sperm DNA from donors of different ages, we determined the frequency of the nucleotide substitution in the fibroblast growth factor receptor 3 (FGFR3) gene that causes achondroplasia. Surprisingly, the magnitude of the increase in mutation frequency with age appears insufficient to explain why older fathers have a greater chance of having a child with this condition. A number of alternatives may explain this discrepancy, including selection for sperm that carry the mutation or an age-dependent increase in premutagenic lesions that remain unrepaired in sperm and are inefficiently detected by the PCR assay. PMID:12397172

  10. A novel 'splice site' HCN4 Gene mutation, c.1737+1 G>T, causes familial bradycardia, reduced heart rate response, impaired chronotropic competence and increased short-term heart rate variability.

    PubMed

    Hategan, Lidia; Csányi, Beáta; Ördög, Balázs; Kákonyi, Kornél; Tringer, Annamária; Kiss, Orsolya; Orosz, Andrea; Sághy, László; Nagy, István; Hegedűs, Zoltán; Rudas, László; Széll, Márta; Varró, András; Forster, Tamás; Sepp, Róbert

    2017-08-15

    The most important molecular determinant of heart rate regulation in sino-atrial pacemaker cells includes hyperpolarization-activated, cyclic nucleotide-gated ion channels, the major isoform of which is encoded by the HCN4 gene. Mutations affecting the HCN4 gene are associated primarily with sick sinus syndrome. A novel c.1737+1 G>T 'splice-site' HCN4 mutation was identified in a large family with familial bradycardia which co-segregated with the disease providing a two-point LOD score of 4.87. Twelve out of the 22 investigated family members [4 males, 8 females average age 36 (SD 6) years] were considered as clinically affected (heart rate<60/min on resting ECG). Minimum [36 (SD 7) vs. 47 (SD 5) bpm, p=0.0087) and average heart rates [62 (SD 8) vs. 73 (SD 8) bpm, p=0.0168) were significantly lower in carriers on 24-hour Holter recordings. Under maximum exercise test carriers achieved significantly lower heart rates than non-carrier family members, and percent heart rate reserve and percent corrected heart rate reserve were significantly lower in carriers. Applying rigorous criteria for chronotropic incompetence a higher number of carriers exhibited chronotropic incompetence. Parameters, characterizing short-term variability of heart rate (i.e. rMSSD and pNN50%) were increased in carrier family members, even after normalization for heart rate, in the 24-hour ECG recordings with the same relative increase in 5-minute recordings. The identified novel 'splice site' HCN4 gene mutation, c.1737+1 G>T, causes familial bradycardia and leads to reduced heart rate response, impaired chronotropic competence and increased short-term heart rate variability in the mutation carriers. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Mutations in Human Tubulin Proximal to the Kinesin-Binding Site Alter Dynamic Instability at Microtubule Plus- and Minus-Ends

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

    Ti, Shih-Chieh; Pamula, Melissa C.; Howes, Stuart C.

    The assembly of microtubule-based cellular structures depends on regulated tubulin polymerization and directional transport. In this research, we have purified and characterized tubulin heterodimers that have human β-tubulin isotype III (TUBB3), as well as heterodimers with one of two β-tubulin mutations (D417H or R262H). Both point mutations are proximal to the kinesin-binding site and have been linked to an ocular motility disorder in humans. Compared to wild-type, microtubules with these mutations have decreased catastrophe frequencies and increased average lifetimes of plus- and minus-end-stabilizing caps. Importantly, the D417H mutation does not alter microtubule lattice structure or Mal3 binding to growing filaments.more » Instead, this mutation reduces the affinity of tubulin for TOG domains and colchicine, suggesting that the distribution of tubulin heterodimer conformations is changed. Together, our findings reveal how residues on the surface of microtubules, distal from the GTP-hydrolysis site and inter-subunit contacts, can alter polymerization dynamics at the plus- and minus-ends of microtubules.« less

  12. Introducing the human Leigh syndrome mutation T9176G into Saccharomyces cerevisiae mitochondrial DNA leads to severe defects in the incorporation of Atp6p into the ATP synthase and in the mitochondrial morphology.

    PubMed

    Kucharczyk, Roza; Salin, Bénédicte; di Rago, J-P

    2009-08-01

    The Leigh syndrome is a severe neurological disorder that has been associated with mutations affecting the mitochondrial energy transducing system. One of these mutations, T9176G, has been localized in the mitochondrial ATP6 gene encoding the Atp6p (or a) subunit of the ATP synthase. This mutation converts a highly conserved leucine residue into arginine within a presumed trans-membrane alpha-helical segment, at position 217 of Atp6p. The T9176G mutation was previously shown to severely reduce the rate of mitochondrial ATP production in cultured human cells containing high loads of this mutation. However, the underlying mechanism responsible for the impaired ATP production is still unknown. To better understand how T9176G affects the ATP synthase, we have created and analyzed the properties of a yeast strain bearing an equivalent of this mutation. We show that incorporation of Atp6p within the ATP synthase was almost completely prevented in the modified yeast. Based on previous partial biochemical characterization of human T9176G cells, it is likely that this mutation similarly affects the human ATP synthase instead of causing a block in the rotary mechanism of this enzyme as it had been suggested. Interestingly, the T9176G yeast exhibits important anomalies in mitochondrial morphology, an observation which indicates that the pathogenicity of T9176G may not be limited to a bioenergetic deficiency.

  13. Calculation of Heavy Ion Inactivation and Mutation Rates in Radial Dose Model of Track Structure

    NASA Technical Reports Server (NTRS)

    Cucinotta, Francis A.; Wilson, John W.; Shavers, Mark R.; Katz, Robert

    1997-01-01

    In the track structure model, the inactivation cross section is found by summing an inactivation probability over all impact parameters from the ion to the sensitive sites within the cell nucleus. The inactivation probability is evaluated by using the dose response of the system to gamma rays and the radial dose of the ions and may be equal to unity at small impact parameters. We apply the track structure model to recent data with heavy ion beams irradiating biological samples of E. Coli, B. Subtilis spores, and Chinese hamster (V79) cells. Heavy ions have observed cross sections for inactivation that approach and sometimes exceed the geometric size of the cell nucleus. We show how the effects of inactivation may be taken into account in the evaluation of the mutation cross sections in the track structure model through correlation of sites for gene mutation and cell inactivation. The model is fit to available data for HPRT (hypoxanthine guanine phosphoribosyl transferase) mutations in V79 cells, and good agreement is found. Calculations show the high probability for mutation by relativistic ions due to the radial extension of ions track from delta rays. The effects of inactivation on mutation rates make it very unlikely that a single parameter such as LET (linear energy transfer) can be used to specify radiation quality for heavy ion bombardment.

  14. Running on empty: does mitochondrial DNA mutation limit replicative lifespan in yeast?: Mutations that increase the division rate of cells lacking mitochondrial DNA also extend replicative lifespan in Saccharomyces cerevisiae.

    PubMed

    Dunn, Cory D

    2011-10-01

    Mitochondrial DNA (mtDNA) mutations escalate with increasing age in higher organisms. However, it has so far been difficult to experimentally determine whether mtDNA mutation merely correlates with age or directly limits lifespan. A recent study shows that budding yeast can also lose functional mtDNA late in life. Interestingly, independent studies of replicative lifespan (RLS) and of mtDNA-deficient cells show that the same mutations can increase both RLS and the division rate of yeast lacking the mitochondrial genome. These exciting, parallel findings imply a potential causal relationship between mtDNA mutation and replicative senescence. Furthermore, these results suggest more efficient methods for discovering genes that determine lifespan. Copyright © 2011 WILEY Periodicals, Inc.

  15. Single-Cell Analysis of Human Pancreas Reveals Transcriptional Signatures of Aging and Somatic Mutation Patterns.

    PubMed

    Enge, Martin; Arda, H Efsun; Mignardi, Marco; Beausang, John; Bottino, Rita; Kim, Seung K; Quake, Stephen R

    2017-10-05

    As organisms age, cells accumulate genetic and epigenetic errors that eventually lead to impaired organ function or catastrophic transformation such as cancer. Because aging reflects a stochastic process of increasing disorder, cells in an organ will be individually affected in different ways, thus rendering bulk analyses of postmitotic adult cells difficult to interpret. Here, we directly measure the effects of aging in human tissue by performing single-cell transcriptome analysis of 2,544 human pancreas cells from eight donors spanning six decades of life. We find that islet endocrine cells from older donors display increased levels of transcriptional noise and potential fate drift. By determining the mutational history of individual cells, we uncover a novel mutational signature in healthy aging endocrine cells. Our results demonstrate the feasibility of using single-cell RNA sequencing (RNA-seq) data from primary cells to derive insights into genetic and transcriptional processes that operate on aging human tissue. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Mutational analysis of the human nucleotide excision repair gene ERCC1.

    PubMed Central

    Sijbers, A M; van der Spek, P J; Odijk, H; van den Berg, J; van Duin, M; Westerveld, A; Jaspers, N G; Bootsma, D; Hoeijmakers, J H

    1996-01-01

    The human DNA repair protein ERCC1 resides in a complex together with the ERCC4, ERCC11 and XP-F correcting activities, thought to perform the 5' strand incision during nucleotide excision repair (NER). Its yeast counterpart, RAD1-RAD10, has an additional engagement in a mitotic recombination pathway, probably required for repair of DNA cross-links. Mutational analysis revealed that the poorly conserved N-terminal 91 amino acids of ERCC1 are dispensable for both repair functions, in contrast to a deletion of only four residues from the C-terminus. A database search revealed a strongly conserved motif in this C-terminus sharing sequence homology with many DNA break processing proteins, indicating that this part is primarily required for the presumed structure-specific endonuclease activity of ERCC1. Most missense mutations in the central region give rise to an unstable protein (complex). Accordingly, we found that free ERCC1 is very rapidly degraded, suggesting that protein-protein interactions provide stability. Survival experiments show that the removal of cross-links requires less ERCC1 than UV repair. This suggests that the ERCC1-dependent step in cross-link repair occurs outside the context of NER and provides an explanation for the phenotype of the human repair syndrome xeroderma pigmentosum group F. PMID:8811092

  17. Two novel mutations in the glycine-rich region of human PAX6 gene: Implications for an association of cataracts and anosmia with aniridia

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

    Martha, A.; Ferrel, R.E.; Hittner, H.M.

    1994-09-01

    Aniridia (iris hyplasia) is a autosomal dominant congenital disorder of the eye. Mutations in the human aniridia (PAX6) gene have now been identified in many patients from various ethnic groups. In the present study we describe new mutations in this gene. Out of four mutations found, three were novel mutations; the fourth one is identical to the previously reported mutations (C{yields}T transition at nt 240). The three novel mutations analyzed were in the glycine-rich region (two) and in the proline/serine/threonine-rich (PST) region (one). Previously no mutations were reported for the glycine-rich region in humans. One of the mutations found inmore » this region is associated with cataracts in an aniridia family. The other splice mutation found in the PST domain is associated with anosmia (lack of sensation to smell) in a sporadic aniridia case. Two of the mutations presented here, one in the glycine-rich region and the other in the PST domain, were not detected by SSCR. These mutations could be detected by using MDE gel and heteroduplex information. All mutations found in the present study are similar in that 32 of 33 PAX6 mutations result in protein truncation and haploinsufficiency.« less

  18. [Characterization of genetic alterations in primary human melanomas carrying BRAF or NRAS mutation].

    PubMed

    Lázár, Viktória

    2013-06-01

    Human malignant melanoma is one of the most aggressive forms of skin cancer with an exceptionally bad prognosis. Melanoma often displays constitutively activated MAPK pathway through BRAF or NRAS mutations. It is also known that these mutations are almost never simultaneously present and that they appear at early stages and preserved throughout tumor progression, although it is proved that these alterations alone are insufficient to cause tumor progression. Therefore the first aim of our study was to evaluate those distinct genetic alterations which can properly differentiate the three important molecular subtypes of primary melanomas with a) BRAF, b) NRAS mutation and c) WT (wild type for both loci). High-resolution array comparative genomic hybridization (array CGH) was used to assess genome-wide analysis of DNA copy number alterations. Primary melanomas with BRAF mutation more frequently exhibited losses on 10q23-10q26 and gains on chromosome 7 and 1q23-1q25 compared to melanomas with NRAS mutation. Loss on the 11q23-11q25 sequence was found mainly in conjunction with NRAS mutation. Based on these results, we proved the existence of marked differences in the genetic pattern of the BRAF and NRAS mutated melanoma subgroups, which might suggest that these mutations contribute to the development of malignant melanoma in conjunction with distinct cooperating oncogenic events. In general, it is an interesting phenomenon suggesting that these mutations provide probably the "guiding force" for these tumors and it also suggests that there are alternative genetic pathways to melanoma. These additional oncogenic events which are associated with BRAF or NRAS mutations can provide rational additional targets for a combination therapy with kinase inhibitors. In this study we also investigated the specific dynamic activities among different signalling pathways highlighting the frequent alterations of genes involved in the signalling interactions between the MAPK-JAK pathways

  19. Mutational scanning of the human serotonin transporter reveals fast translocating serotonin transporter mutants.

    PubMed

    Kristensen, Anders S; Larsen, Mads B; Johnsen, Laust B; Wiborg, Ove

    2004-03-01

    The serotonin transporter (SERT) belongs to a family of sodium-chloride-dependent transporters responsible for uptake of amino acids and biogenic amines from the extracellular space. SERT represents a major pharmacological target in the treatment of several clinical conditions, including depression and anxiety. In the present study we have undertaken a mutational scanning of human SERT in order to identify residues that are responsible for individual differences among related monoamine transporters. One mutant, G100A, was inactive in transport. However, ligand binding affinity was similar to wild-type, suggesting that G100A amongst different possible SERT conformations is restrained to a binding conformation. We suggest that the main role of glycine-100 is to confer structural flexibility during substrate translocation. For the two single mutants, T178A and F263C, uptake rates and K(m) values were both several-fold higher than wild-type while binding affinities and inhibitory potencies decreased considerably for several drugs. Ion dependency increased and only at hyperosmotic concentrations were K(m) values partly restored. For the double mutant, T178A/F263C, shifts in uptake kinetics and ligand affinities, as well as ion dependencies, were drastic. Effects were synergistic compared to the corresponding single mutants. In conclusion, we suggest that mutating threonine-178 to an alanine and phenylalanine-263 to a cysteine mainly alter the overall uptake kinetics of SERT by affecting the conformational equilibrium of different transporter conformations.

  20. Mutations in FOXC2 in humans (lymphoedema distichiasis syndrome) cause lymphatic dysfunction on dependency.

    PubMed

    Mellor, Russell H; Tate, Naomi; Stanton, Anthony W B; Hubert, Charlotte; Mäkinen, Taija; Smith, Alberto; Burnand, Kevin G; Jeffery, Steve; Levick, J Rodney; Mortimer, Peter S

    2011-01-01

    Human lymphoedema distichiasis syndrome (LDS) results from germline mutations in transcription factor FOXC2. In a mouse model, lack of lymphatic and venous valves is observed plus abnormal smooth muscle cell recruitment to initial lymphatics. We investigated the mechanism of lymphoedema in humans with FOXC2 mutations, specifically the effect of gravitational forces on dermal lymphatic function. We performed (1) quantitative fluorescence microlymphangiography (FML) on the skin of the forearm (non-swollen region) at heart level, and the foot (swollen region) below heart level (dependent) and then at heart level, and (2) immunohistochemical staining of microlymphatics in forearm and foot skin biopsies, using antibodies to podoplanin, LYVE-1 and smooth muscle actin. FML revealed a marked reduction in fluid uptake by initial lymphatics in the LDS foot during dependency, yet normal uptake (similar to controls) in the same foot at heart level and in LDS forearms. In control subjects, dependency did not impair initial lymphatic filling. Immunohistochemical microlymphatic density in forearm and foot did not differ between LDS and controls. FOXC2 mutations cause a functional failure of dermal initial lymphatics during gravitational stress (dependency), but not hypoplasia. The results reveal a pathophysiological mechanism contributing to swelling in LDS. Copyright © 2011 S. Karger AG, Basel.

  1. Estimating Exceptionally Rare Germline and Somatic Mutation Frequencies via Next Generation Sequencing

    PubMed Central

    Yoon, Song-Ro; Arnheim, Norman; Calabrese, Peter

    2016-01-01

    We used targeted next generation deep-sequencing (Safe Sequencing System) to measure ultra-rare de novo mutation frequencies in the human male germline by attaching a unique identifier code to each target DNA molecule. Segments from three different human genes (FGFR3, MECP2 and PTPN11) were studied. Regardless of the gene segment, the particular testis donor or the 73 different testis pieces used, the frequencies for any one of the six different mutation types were consistent. Averaging over the C>T/G>A and G>T/C>A mutation types the background mutation frequency was 2.6x10-5 per base pair, while for the four other mutation types the average background frequency was lower at 1.5x10-6 per base pair. These rates far exceed the well documented human genome average frequency per base pair (~10−8) suggesting a non-biological explanation for our data. By computational modeling and a new experimental procedure to distinguish between pre-mutagenic lesion base mismatches and a fully mutated base pair in the original DNA molecule, we argue that most of the base-dependent variation in background frequency is due to a mixture of deamination and oxidation during the first two PCR cycles. Finally, we looked at a previously studied disease mutation in the PTPN11 gene and could easily distinguish true mutations from the SSS background. We also discuss the limits and possibilities of this and other methods to measure exceptionally rare mutation frequencies, and we present calculations for other scientists seeking to design their own such experiments. PMID:27341568

  2. Air pollution and human fertility rates.

    PubMed

    Nieuwenhuijsen, Mark J; Basagaña, Xavier; Dadvand, Payam; Martinez, David; Cirach, Marta; Beelen, Rob; Jacquemin, Bénédicte

    2014-09-01

    Some reports have suggested effects of air pollution on semen quality and success rates of in vitro fertilization (IVF) in humans and lower fertility rates in mice. However, no studies have evaluated the impact of air pollution on human fertility rates. We assessed the association between traffic related air pollution and fertility rates in humans in Barcelona, Spain (2011-2012). We hypothesized that higher air pollution levels would be associated with lower fertility rates. We calculated the general fertility rate which is the number of live births per 1000 women between the ages of 15 and 44 years per census tract. We used land use regression (LUR) modeling to estimate the air pollution concentrations (particulate matter, NO2/NOx) per census tract. We used Besag-York-Mollié models to quantify the relationship between air pollution and fertility rates with adjustment for a number of potential confounders such as maternal age and area level socio-economic status. We found a statistically significant reduction of fertility rates with an increase in traffic related air pollution levels, particularly for the coarse fraction of particulate matter (IRR=0.87 95% CI 0.82, 0.94 per IQR). This is the first study in humans to show an association between reduced fertility rates and higher traffic related air pollution levels. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Frequency of SMARCB1 mutations in familial and sporadic schwannomatosis.

    PubMed

    Smith, Miriam J; Wallace, Andrew J; Bowers, Naomi L; Rustad, Cecilie F; Woods, C Geoff; Leschziner, Guy D; Ferner, Rosalie E; Evans, D Gareth R

    2012-05-01

    Mutations of the SMARCB1 gene have been implicated in several human tumour predisposing syndromes. They have recently been identified as an underlying cause of the tumour suppressor syndrome schwannomatosis. There is a much higher rate of mutation detection in familial disease than in sporadic disease. We have carried out extensive genetic testing on a cohort of familial and sporadic patients who fulfilled clinical diagnostic criteria for schwannomatosis. In our current cohort, we identified novel mutations within the SMARCB1 gene and detected several mutations that have been previously identified in other schwannomatosis cohorts. Of the schwannomatosis screens reported to date, including our current dataset, SMARCB1 mutations have been found in 45 % of familial probands and 7 % of sporadic patients. The exon 1 mutation, c.41C >A, and the 3' untranslated region mutation, c.*82C >T, are the most common changes reported in schwannomatosis disease so far, indicating mutation hotspots at both 5' and 3' portions of the gene. SMARCB1 mutations are found in a significant proportion of schwannomatosis patients, but there remains the possibility that further causative genes remain to be found.

  4. Low dose or low dose rate ionizing radiation-induced health effect in the human.

    PubMed

    Tang, Feng Ru; Loganovsky, Konstantin

    2018-06-05

    The extensive literature review on human epidemiological studies suggests that low dose ionizing radiation (LDIR) (≤100 mSv) or low dose rate ionizing radiation (LDRIR) (<6mSv/H) exposure could induce either negative or positive health effects. These changes may depend on genetic background, age (prenatal day for embryo), sex, nature of radiation exposure, i.e., acute or chronic irradiation, radiation sources (such as atomic bomb attack, fallout from nuclear weapon test, nuclear power plant accidents, 60 Co-contaminated building, space radiation, high background radiation, medical examinations or procedures) and radionuclide components and human epidemiological experimental designs. Epidemiological and clinical studies show that LDIR or LDRIR exposure may induce cancer, congenital abnormalities, cardiovascular and cerebrovascular diseases, cognitive and other neuropsychiatric disorders, cataracts and other eye and somatic pathology (endocrine, bronchopulmonary, digestive, etc). LDIR or LDRIR exposure may also reduce mutation and cancer mortality rates. So far, the mechanisms of LDIR- or LDRIR -induced health effect are poorly understood. Further extensive studies are still needed to clarify under what circumstances, LDIR or LDRIR exposure may induce positive or negative effects, which may facilitate development of new therapeutic approaches to prevent or treat the radiation-induced human diseases or enhance radiation-induced positive health effect. Copyright © 2018 Elsevier Ltd. All rights reserved.

  5. Gain-of-Function R225W Mutation in Human AMPKγ3 Causing Increased Glycogen and Decreased Triglyceride in Skeletal Muscle

    PubMed Central

    Ahituv, Nadav; Chaudhry, Shehla N.; Schackwitz, Wendy S.; Dent, Robert; Pennacchio, Len A.

    2007-01-01

    Background AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme that is evolutionarily conserved from yeast to mammals and functions to maintain cellular and whole body energy homeostasis. Studies in experimental animals demonstrate that activation of AMPK in skeletal muscle protects against insulin resistance, type 2 diabetes and obesity. The regulatory γ3 subunit of AMPK is expressed exclusively in skeletal muscle; however, its importance in controlling overall AMPK activity is unknown. While evidence is emerging that gamma subunit mutations interfere specifically with AMP activation, there remains some controversy regarding the impact of gamma subunit mutations [1]–[3]. Here we report the first gain-of-function mutation in the muscle-specific regulatory γ3 subunit in humans. Methods and Findings We sequenced the exons and splice junctions of the AMPK γ3 gene (PRKAG3) in 761 obese and 759 lean individuals, identifying 87 sequence variants including a novel R225W mutation in subjects from two unrelated families. The γ3 R225W mutation is homologous in location to the γ2R302Q mutation in patients with Wolf-Parkinson-White syndrome and to the γ3R225Q mutation originally linked to an increase in muscle glycogen content in purebred Hampshire Rendement Napole (RN-) pigs. We demonstrate in differentiated muscle satellite cells obtained from the vastus lateralis of R225W carriers that the mutation is associated with an approximate doubling of both basal and AMP-activated AMPK activities. Moreover, subjects bearing the R225W mutation exhibit a ∼90% increase of skeletal muscle glycogen content and a ∼30% decrease in intramuscular triglyceride (IMTG). Conclusions We have identified for the first time a mutation in the skeletal muscle-specific regulatory γ3 subunit of AMPK in humans. The γ3R225W mutation has significant functional effects as demonstrated by increases in basal and AMP-activated AMPK activities, increased muscle glycogen and decreased

  6. Characterization of an MPS I-H Knock-In Mouse that Carries a Nonsense Mutation Analogous to the Human IDUA-W402X Mutation

    PubMed Central

    Wang, Dan; Shukla, Charu; Liu, Xiaoli; Schoeb, Trenton R.; Clarke, Lorne A.; Bedwell, David M.; Keeling, Kim M.

    2009-01-01

    Here we report the characterization of a knock-in mouse model for the autosomal recessive disorder mucopolysaccharidosis type I-Hurler (MPS I-H), also known as Hurler syndrome. MPS I-H is the most severe form of α-L-iduronidase deficiency. α-L-iduronidase (encoded by the IDUA gene) is a lysosomal enzyme that participates in the degradation of dermatan sulfate and heparan sulfate. Using gene replacement methodology, a nucleotide change was introduced into the mouse Idua locus that resulted in a nonsense mutation at codon W392. The Idua-W392X mutation is analogous to the human IDUA-W402X mutation commonly found in MPS I-H patients. We found that the phenotype in homozygous Idua-W392X mice closely correlated with the human MPS I-H disease. Homozygous W392X mice showed no detectable α-L-iduronidase activity. We observed a defect in GAG degradation as evidenced by an increase in sulfated GAGs excreted in the urine and stored in multiple tissues. Histology and electron microscopy also revealed evidence of GAG storage in all tissues examined. Additional assessment revealed bone abnormalities and altered metabolism within the Idua-W392X mouse. This new mouse will provide an important tool to investigate therapeutic approaches for MPS I-H that cannot be addressed using current MPS I-H animal models. PMID:19751987

  7. Determination and Therapeutic Exploitation of Ebola Virus Spontaneous Mutation Frequency.

    PubMed

    Alfson, Kendra J; Worwa, Gabriella; Carrion, Ricardo; Griffiths, Anthony

    2015-12-16

    Ebola virus (EBOV) is an RNA virus that can cause hemorrhagic fever with high fatality rates, and there are no approved vaccines or therapies. Typically, RNA viruses have high spontaneous mutation rates, which permit rapid adaptation to selection pressures and have other important biological consequences. However, it is unknown if filoviruses exhibit high mutation frequencies. Ultradeep sequencing and a recombinant EBOV that carries the gene encoding green fluorescent protein were used to determine the spontaneous mutation frequency of EBOV. The effects of the guanosine analogue ribavirin during EBOV infections were also assessed. Ultradeep sequencing revealed that the mutation frequency for EBOV was high and similar to those of other RNA viruses. Interestingly, significant genetic diversity was not observed in viable viruses, implying that changes were not well tolerated. We hypothesized that this could be exploited therapeutically. In vitro, the presence of ribavirin increased the error rate, and the 50% inhibitory concentration (IC50) was 27 μM. In a mouse model of ribavirin therapy given pre-EBOV exposure, ribavirin treatment corresponded with a significant delay in time to death and up to 75% survival. In mouse and monkey models of therapy given post-EBOV exposure, ribavirin treatment also delayed the time to death and increased survival. These results demonstrate that EBOV has a spontaneous mutation frequency similar to those of other RNA viruses. These data also suggest a potential for therapeutic use of ribavirin for human EBOV infections. Ebola virus (EBOV) causes a severe hemorrhagic disease with high case fatality rates; there are no approved vaccines or therapies. We determined the spontaneous mutation frequency of EBOV, which is relevant to understanding the potential for the virus to adapt. The frequency was similar to those of other RNA viruses. Significant genetic diversity was not observed in viable viruses, implying that changes were not well

  8. The Iranian Human Mutation Gene Bank: a data and sample resource for worldwide collaborative genetics research.

    PubMed

    Najmabadi, Hossein; Neishabury, Maryam; Sahebjam, Farhad; Kahrizi, Kimia; Shafaghati, Yousef; Nikzat, Nushin; Jalalvand, Maryam; Aminy, Farahnaz; Hashemi, Susan Bany; Moghimi, Babak; Noorian, Ali Reza; Jannati, Ali; Mohammadi, Mehrdad; Javan, Khalil

    2003-02-01

    As Human Genome Project exploration continues, the necessity of having a broader spectrum of genomic DNA material from different nationalities to study various aspects of hereditary disease becomes more obvious. The existence of high genetic polymorphism within and between different communities in the world makes it necessary for the gene hunters to investigate many different populations. Iran, a large country with close to 66 million people, is a land of different nationalities, tribes, and religions that offers a highly heterogeneous gene pool to the genetics researcher. The purity of many different races in this country has been highly conserved by geographical borders and by an ancient culture that has always encouraged intrafamilial marriages. All these have created a population that is remarkably heterogeneous yet high in consanguinity rate. During the last five years of investigation we have established a DNA bank, the Iranian Human Mutation Gene Bank (www.IHMGB.com), which contains all genetic diseases studied in Iran that have the Mendelian mode of inheritance. Some of the samples are assigned to common or novel mutations and others belong to patients with clinical profiles associated with particular genetic diseases but undefined mutation. This bank stores samples of DNA from the patient and his/her first-degree relatives together with a comprehensive pedigree and clinical profile for each sample. To facilitate collaboration with other scientists around the world with the same interests, we decided to present our experimental projects online. This DNA bank provides opportunities for us to collaborate with scientists outside Iran. It offers a sample resource to research scientists around the world, at no charge, for the purpose of investigating the various aspects of genetic disorders from prenatal diagnosis to gene structure and function. It is strongly stressed that no commercial benefit is involved in the establishment of this DNA bank and the DNA

  9. Efficient Generation of Gene-Modified Pigs Harboring Precise Orthologous Human Mutation via CRISPR/Cas9-Induced Homology-Directed Repair in Zygotes.

    PubMed

    Zhou, Xiaoyang; Wang, Lulu; Du, Yinan; Xie, Fei; Li, Liang; Liu, Yu; Liu, Chuanhong; Wang, Shiqiang; Zhang, Shibing; Huang, Xingxu; Wang, Yong; Wei, Hong

    2016-01-01

    Precise genetic mutation of model animals is highly valuable for functional investigation of human mutations. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9)-induced homology-directed repair (HDR) is usually used for precise genetic mutation, being limited by the relatively low efficiency compared with that of non-homologous end joining (NHEJ). Although inhibition of NHEJ was shown to enhance HDR-derived mutation, in this work, without inhibition of NHEJ, we first generated gene-modified pigs harboring precise orthologous human mutation (Sox10 c.A325>T) via CRISPR/Cas9-induced HDR in zygotes using single-strand oligo DNA (ssODN) as template with an efficiency as high as 80%, indicating that pig zygotes exhibited high activities of HDR relative to NHEJ and were highly amendable to genetic mutation via CIRSPR/Cas9-induced HDR. Besides, we found a higher concentration of ssODN remarkably reduced HDR-derived mutation in pig zygotes, suggesting a possible balance for optimal HDR-derived mutation in zygotes between the excessive accessibility to HDR templates and the activities of HDR relative to NHEJ which appeared to be negatively correlated to ssODN concentration. In addition, the HDR-derived mutation, as well as those from NHEJ, extensively integrated into various tissues including gonad of founder pig without detected off-targeting, suggesting CRISPR/Cas9-induced HDR in zygotes is a reliable approach for precise genetic mutation in pigs. © 2015 WILEY PERIODICALS, INC.

  10. The carrier rate and mutation spectrum of genes associated with hearing loss in South China hearing female population of childbearing age

    PubMed Central

    2013-01-01

    Background Given that hearing loss occurs in 1 to 3 of 1,000 live births and approximately 90 to 95 percent of them are born into hearing families, it is of importance and necessity to get better understanding about the carrier rate and mutation spectrum of genes associated with hearing impairment in the general population. Methods 7,263 unrelated women of childbearing age with normal hearing and without family history of hearing loss were tested with allele-specific PCR-based universal array. Further genetic testing were provided to the spouses of the screened carriers. For those couples at risk, multiple choices were provided, including prenatal diagnosis. Results Among the 7,263 normal hearing participants, 303 subjects carried pathogenic mutations included in the screening chip, which made the carrier rate 4.17%. Of the 303 screened carriers, 282 harbored heterozygous mutated genes associated with autosomal recessive hearing loss, and 95 spouses took further genetic tests. 8 out of the 9 couples harbored deafness-causing mutations in the same gene received prenatal diagnosis. Conclusions Given that nearly 90 to 95 percent of deaf and hard-of-hearing babies are born into hearing families, better understanding about the carrier rate and mutation spectrum of genes associated with hearing impairment in the female population of childbearing age may be of importance in carrier screening and genetic counseling. PMID:23718755

  11. The carrier rate and mutation spectrum of genes associated with hearing loss in South China hearing female population of childbearing age.

    PubMed

    Yin, Aihua; Liu, Chang; Zhang, Yan; Wu, Jing; Mai, Mingqin; Ding, Hongke; Yang, Jiexia; Zhang, Xiaozhuang

    2013-05-29

    Given that hearing loss occurs in 1 to 3 of 1,000 live births and approximately 90 to 95 percent of them are born into hearing families, it is of importance and necessity to get better understanding about the carrier rate and mutation spectrum of genes associated with hearing impairment in the general population. 7,263 unrelated women of childbearing age with normal hearing and without family history of hearing loss were tested with allele-specific PCR-based universal array. Further genetic testing were provided to the spouses of the screened carriers. For those couples at risk, multiple choices were provided, including prenatal diagnosis. Among the 7,263 normal hearing participants, 303 subjects carried pathogenic mutations included in the screening chip, which made the carrier rate 4.17%. Of the 303 screened carriers, 282 harbored heterozygous mutated genes associated with autosomal recessive hearing loss, and 95 spouses took further genetic tests. 8 out of the 9 couples harbored deafness-causing mutations in the same gene received prenatal diagnosis. Given that nearly 90 to 95 percent of deaf and hard-of-hearing babies are born into hearing families, better understanding about the carrier rate and mutation spectrum of genes associated with hearing impairment in the female population of childbearing age may be of importance in carrier screening and genetic counseling.

  12. Effects of hypertrophic and dilated cardiomyopathy mutations on power output by human β-cardiac myosin.

    PubMed

    Spudich, James A; Aksel, Tural; Bartholomew, Sadie R; Nag, Suman; Kawana, Masataka; Yu, Elizabeth Choe; Sarkar, Saswata S; Sung, Jongmin; Sommese, Ruth F; Sutton, Shirley; Cho, Carol; Adhikari, Arjun S; Taylor, Rebecca; Liu, Chao; Trivedi, Darshan; Ruppel, Kathleen M

    2016-01-01

    Hypertrophic cardiomyopathy is the most frequently occurring inherited cardiovascular disease, with a prevalence of more than one in 500 individuals worldwide. Genetically acquired dilated cardiomyopathy is a related disease that is less prevalent. Both are caused by mutations in the genes encoding the fundamental force-generating protein machinery of the cardiac muscle sarcomere, including human β-cardiac myosin, the motor protein that powers ventricular contraction. Despite numerous studies, most performed with non-human or non-cardiac myosin, there is no clear consensus about the mechanism of action of these mutations on the function of human β-cardiac myosin. We are using a recombinantly expressed human β-cardiac myosin motor domain along with conventional and new methodologies to characterize the forces and velocities of the mutant myosins compared with wild type. Our studies are extending beyond myosin interactions with pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin, the roles of regulatory light chain phosphorylation on the functions of the system, and the possible roles of myosin binding protein-C and titin, important regulatory components of both cardiac and skeletal muscles. © 2016. Published by The Company of Biologists Ltd.

  13. Mutation detection in the human HSP70B′ gene by denaturing high-performance liquid chromatography

    PubMed Central

    Hecker, Karl H.; Asea, Alexzander; Kobayashi, Kaoru; Green, Stacy; Tang, Dan; Calderwood, Stuart K.

    2000-01-01

    Variances, particularly single nucleotide polymorphisms (SNP), in the genomic sequence of individuals are the primary key to understanding gene function as it relates to differences in the susceptibility to disease, environmental influences, and therapy. In this report, the HSP70B′ gene is the target sequence for mutation detection in biopsy samples from human prostate cancer patients undergoing combined hyperthermia and radiation therapy at the Dana-Farber Cancer Institute, using temperature-modulated heteroduplex analysis (TMHA). The underlying principles of TMHA for mutation detection using DHPLC technology are discussed. The procedures involved in amplicon design for mutation analysis by DHPLC are detailed. The melting behavior of the complete coding sequence of the target gene is characterized using WAVEMAKERTM software. Four overlapping amplicons, which span the complete coding region of the HSP70B′ gene, amenable to mutation detection by DHPLC were identified based on the software-predicted melting profile of the target sequence. TMHA was performed on PCR products of individual amplicons of the HSP70B′ gene on the WAVE® Nucleic Acid Fragment Analysis System. The criteria for mutation calling by comparing wild-type and mutant chromatographic patterns are discussed. PMID:11189446

  14. Mutation detection in the human HSP7OB' gene by denaturing high-performance liquid chromatography.

    PubMed

    Hecker, K H; Asea, A; Kobayashi, K; Green, S; Tang, D; Calderwood, S K

    2000-11-01

    Variances, particularly single nucleotide polymorphisms (SNP), in the genomic sequence of individuals are the primary key to understanding gene function as it relates to differences in the susceptibility to disease, environmental influences, and therapy. In this report, the HSP70B' gene is the target sequence for mutation detection in biopsy samples from human prostate cancer patients undergoing combined hyperthermia and radiation therapy at the Dana-Farber Cancer Institute, using temperature-modulated heteroduplex analysis (TMHA). The underlying principles of TMHA for mutation detection using DHPLC technology are discussed. The procedures involved in amplicon design for mutation analysis by DHPLC are detailed. The melting behavior of the complete coding sequence of the target gene is characterized using WAVEMAKER software. Four overlapping amplicons, which span the complete coding region of the HSP70B' gene, amenable to mutation detection by DHPLC were identified based on the software-predicted melting profile of the target sequence. TMHA was performed on PCR products of individual amplicons of the HSP70B' gene on the WAVE Nucleic Acid Fragment Analysis System. The criteria for mutation calling by comparing wild-type and mutant chromatographic patterns are discussed.

  15. The population genetics of mutations: good, bad and indifferent

    PubMed Central

    Loewe, Laurence; Hill, William G.

    2010-01-01

    Population genetics is fundamental to our understanding of evolution, and mutations are essential raw materials for evolution. In this introduction to more detailed papers that follow, we aim to provide an oversight of the field. We review current knowledge on mutation rates and their harmful and beneficial effects on fitness and then consider theories that predict the fate of individual mutations or the consequences of mutation accumulation for quantitative traits. Many advances in the past built on models that treat the evolution of mutations at each DNA site independently, neglecting linkage of sites on chromosomes and interactions of effects between sites (epistasis). We review work that addresses these limitations, to predict how mutations interfere with each other. An understanding of the population genetics of mutations of individual loci and of traits affected by many loci helps in addressing many fundamental and applied questions: for example, how do organisms adapt to changing environments, how did sex evolve, which DNA sequences are medically important, why do we age, which genetic processes can generate new species or drive endangered species to extinction, and how should policy on levels of potentially harmful mutagens introduced into the environment by humans be determined? PMID:20308090

  16. Differential effects of human L1CAM mutations on complementing guidance and synaptic defects in Drosophila melanogaster.

    PubMed

    Kudumala, Sirisha; Freund, Julie; Hortsch, Michael; Godenschwege, Tanja A

    2013-01-01

    A large number of different pathological L1CAM mutations have been identified that result in a broad spectrum of neurological and non-neurological phenotypes. While many of these mutations have been characterized for their effects on homophilic and heterophilic interactions, as well as expression levels in vitro, there are only few studies on their biological consequences in vivo. The single L1-type CAM gene in Drosophila, neuroglian (nrg), has distinct functions during axon guidance and synapse formation and the phenotypes of nrg mutants can be rescued by the expression of human L1CAM. We previously showed that the highly conserved intracellular FIGQY Ankyrin-binding motif is required for L1CAM-mediated synapse formation, but not for neurite outgrowth or axon guidance of the Drosophila giant fiber (GF) neuron. Here, we use the GF as a model neuron to characterize the pathogenic L120V, Y1070C, C264Y, H210Q, E309K and R184Q extracellular L1CAM missense mutations and a L1CAM protein with a disrupted ezrin-moesin-radixin (ERM) binding site to investigate the signaling requirements for neuronal development. We report that different L1CAM mutations have distinct effects on axon guidance and synapse formation. Furthermore, L1CAM homophilic binding and signaling via the ERM motif is essential for axon guidance in Drosophila. In addition, the human pathological H210Q, R184Q and Y1070C, but not the E309K and L120V L1CAM mutations affect outside-in signaling via the FIGQY Ankyrin binding domain which is required for synapse formation. Thus, the pathological phenotypes observed in humans are likely to be caused by the disruption of signaling required for both, guidance and synaptogenesis.

  17. Differential Effects of Human L1CAM Mutations on Complementing Guidance and Synaptic Defects in Drosophila melanogaster

    PubMed Central

    Kudumala, Sirisha; Freund, Julie; Hortsch, Michael; Godenschwege, Tanja A.

    2013-01-01

    A large number of different pathological L1CAM mutations have been identified that result in a broad spectrum of neurological and non-neurological phenotypes. While many of these mutations have been characterized for their effects on homophilic and heterophilic interactions, as well as expression levels in vitro, there are only few studies on their biological consequences in vivo. The single L1-type CAM gene in Drosophila, neuroglian (nrg), has distinct functions during axon guidance and synapse formation and the phenotypes of nrg mutants can be rescued by the expression of human L1CAM. We previously showed that the highly conserved intracellular FIGQY Ankyrin-binding motif is required for L1CAM-mediated synapse formation, but not for neurite outgrowth or axon guidance of the Drosophila giant fiber (GF) neuron. Here, we use the GF as a model neuron to characterize the pathogenic L120V, Y1070C, C264Y, H210Q, E309K and R184Q extracellular L1CAM missense mutations and a L1CAM protein with a disrupted ezrin–moesin–radixin (ERM) binding site to investigate the signaling requirements for neuronal development. We report that different L1CAM mutations have distinct effects on axon guidance and synapse formation. Furthermore, L1CAM homophilic binding and signaling via the ERM motif is essential for axon guidance in Drosophila. In addition, the human pathological H210Q, R184Q and Y1070C, but not the E309K and L120V L1CAM mutations affect outside-in signaling via the FIGQY Ankyrin binding domain which is required for synapse formation. Thus, the pathological phenotypes observed in humans are likely to be caused by the disruption of signaling required for both, guidance and synaptogenesis. PMID:24155914

  18. 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

  19. Generation and Characterization of a Transgenic Mouse Carrying a Functional Human β-Globin Gene with the IVSI-6 Thalassemia Mutation

    PubMed Central

    Mancini, Irene; Lampronti, Ilaria; Salvatori, Francesca; Fabbri, Enrica; Zuccato, Cristina; Cosenza, Lucia C.; Montagner, Giulia; Borgatti, Monica; Altruda, Fiorella; Fagoonee, Sharmila; Carandina, Gianni; Aiello, Vincenzo; Breda, Laura; Rivella, Stefano; Gambari, Roberto

    2015-01-01

    Mouse models that carry mutations causing thalassemia represent a suitable tool to test in vivo new mutation-specific therapeutic approaches. Transgenic mice carrying the β-globin IVSI-6 mutation (the most frequent in Middle-Eastern regions and recurrent in Italy and Greece) are, at present, not available. We report the production and characterization of a transgenic mouse line (TG-β-IVSI-6) carrying the IVSI-6 thalassemia point mutation within the human β-globin gene. In the TG-β-IVSI-6 mouse (a) the transgenic integration region is located in mouse chromosome 7; (b) the expression of the transgene is tissue specific; (c) as expected, normally spliced human β-globin mRNA is produced, giving rise to β-globin production and formation of a human-mouse tetrameric chimeric hemoglobin mu α-globin2/hu β-globin2 and, more importantly, (d) the aberrant β-globin-IVSI-6 RNAs are present in blood cells. The TG-β-IVSI-6 mouse reproduces the molecular features of IVSI-6 β-thalassemia and might be used as an in vivo model to characterize the effects of antisense oligodeoxynucleotides targeting the cryptic sites responsible for the generation of aberrantly spliced β-globin RNA sequences, caused by the IVSI-6 mutation. These experiments are expected to be crucial for the development of a personalized therapy for β-thalassemia. PMID:26097845

  20. Human immunodeficiency virus type 1 pol gene mutations which cause decreased susceptibility to 2',3'-dideoxycytidine.

    PubMed Central

    Fitzgibbon, J E; Howell, R M; Haberzettl, C A; Sperber, S J; Gocke, D J; Dubin, D T

    1992-01-01

    To investigate whether human immunodeficiency virus type 1 pol gene mutations are selected during prolonged 2',3'-dideoxycytidine (ddC) therapy, we used the polymerase chain reaction to amplify a portion of the reverse transcriptase segment of the pol gene from the peripheral blood mononuclear cell DNA of a patient with AIDS before and after an 80-week course of ddC therapy. The consensus sequence from the second sample contained a unique double mutation (ACT to GAT) in the codon for reverse transcriptase amino acid 69, causing substitution of aspartic acid (Asp) for the wild-type threonine (Thr). A mutation (ACA to ATA) also occurred in the codon for position 165, causing substitution of isoleucine (Ile) for Thr. The GAT (Asp) codon was introduced into the pol gene of a molecular clone of human immunodeficiency virus via site-directed mutagenesis. Following transfection, mutant and wild-type viruses were tested for susceptibility to ddC by a plaque reduction assay. The mutant virus was fivefold less susceptible to ddC than the wild type; cross-resistance to 3'-azido-3'-deoxythymidine or 2'3'-dideoxyinosine was not found. The Ile-165 mutation did not confer additional ddC resistance. The Asp-69 substitution may have contributed to the generation of resistant virus in this patient. Images PMID:1317143

  1. Hereditary cancer genes are highly susceptible to splicing mutations

    PubMed Central

    Soemedi, Rachel; Maguire, Samantha; Murray, Michael F.; Monaghan, Sean F.

    2018-01-01

    Substitutions that disrupt pre-mRNA splicing are a common cause of genetic disease. On average, 13.4% of all hereditary disease alleles are classified as splicing mutations mapping to the canonical 5′ and 3′ splice sites. However, splicing mutations present in exons and deeper intronic positions are vastly underreported. A recent re-analysis of coding mutations in exon 10 of the Lynch Syndrome gene, MLH1, revealed an extremely high rate (77%) of mutations that lead to defective splicing. This finding is confirmed by extending the sampling to five other exons in the MLH1 gene. Further analysis suggests a more general phenomenon of defective splicing driving Lynch Syndrome. Of the 36 mutations tested, 11 disrupted splicing. Furthermore, analyzing past reports suggest that MLH1 mutations in canonical splice sites also occupy a much higher fraction (36%) of total mutations than expected. When performing a comprehensive analysis of splicing mutations in human disease genes, we found that three main causal genes of Lynch Syndrome, MLH1, MSH2, and PMS2, belonged to a class of 86 disease genes which are enriched for splicing mutations. Other cancer genes were also enriched in the 86 susceptible genes. The enrichment of splicing mutations in hereditary cancers strongly argues for additional priority in interpreting clinical sequencing data in relation to cancer and splicing. PMID:29505604

  2. PCSK1 Mutations and Human Endocrinopathies: From Obesity to Gastrointestinal Disorders.

    PubMed

    Stijnen, Pieter; Ramos-Molina, Bruno; O'Rahilly, Stephen; Creemers, John W M

    2016-08-01

    Prohormone convertase 1/3, encoded by the PCSK1 gene, is a serine endoprotease that is involved in the processing of a variety of proneuropeptides and prohormones. Humans who are homozygous or compound heterozygous for loss-of-function mutations in PCSK1 exhibit a variable and pleiotropic syndrome consisting of some or all of the following: obesity, malabsorptive diarrhea, hypogonadotropic hypogonadism, altered thyroid and adrenal function, and impaired regulation of plasma glucose levels in association with elevated circulating proinsulin-to-insulin ratio. Recently, more common variants in the PCSK1 gene have been found to be associated with alterations in body mass index, increased circulating proinsulin levels, and defects in glucose homeostasis. This review provides an overview of the endocrinopathies and other disorders observed in prohormone convertase 1/3-deficient patients, discusses the possible biochemical basis for these manifestations of the disease, and proposes a model whereby certain missense mutations in PCSK1 may result in proteins with a dominant negative action.

  3. Cancer-associated TERT promoter mutations abrogate telomerase silencing

    PubMed Central

    Chiba, Kunitoshi; Johnson, Joshua Z; Vogan, Jacob M; Wagner, Tina; Boyle, John M; Hockemeyer, Dirk

    2015-01-01

    Mutations in the human telomerase reverse transcriptase (TERT) promoter are the most frequent non-coding mutations in cancer, but their molecular mechanism in tumorigenesis has not been established. We used genome editing of human pluripotent stem cells with physiological telomerase expression to elucidate the mechanism by which these mutations contribute to human disease. Surprisingly, telomerase-expressing embryonic stem cells engineered to carry any of the three most frequent TERT promoter mutations showed only a modest increase in TERT transcription with no impact on telomerase activity. However, upon differentiation into somatic cells, which normally silence telomerase, cells with TERT promoter mutations failed to silence TERT expression, resulting in increased telomerase activity and aberrantly long telomeres. Thus, TERT promoter mutations are sufficient to overcome the proliferative barrier imposed by telomere shortening without additional tumor-selected mutations. These data establish that TERT promoter mutations can promote immortalization and tumorigenesis of incipient cancer cells. DOI: http://dx.doi.org/10.7554/eLife.07918.001 PMID:26194807

  4. Hypertrophic cardiomyopathy-linked mutation in troponin T causes myofibrillar disarray and pro-arrhythmic action potential changes in human iPSC cardiomyocytes.

    PubMed

    Wang, Lili; Kim, Kyungsoo; Parikh, Shan; Cadar, Adrian Gabriel; Bersell, Kevin R; He, Huan; Pinto, Jose R; Kryshtal, Dmytro O; Knollmann, Bjorn C

    2018-01-01

    Mutations in cardiac troponin T (TnT) are linked to increased risk of ventricular arrhythmia and sudden death despite causing little to no cardiac hypertrophy. Studies in mice suggest that the hypertrophic cardiomyopathy (HCM)-associated TnT-I79N mutation increases myofilament Ca sensitivity and is arrhythmogenic, but whether findings from mice translate to human cardiomyocyte electrophysiology is not known. To study the effects of the TnT-I79N mutation in human cardiomyocytes. Using CRISPR/Cas9, the TnT-I79N mutation was introduced into human induced pluripotent stem cells (hiPSCs). We then used the matrigel mattress method to generate single rod-shaped cardiomyocytes (CMs) and studied contractility, Ca handling and electrophysiology. Compared to isogenic control hiPSC-CMs, TnT-I79N hiPSC-CMs exhibited sarcomere disorganization, increased systolic function and impaired relaxation. The Ca-dependence of contractility was leftward shifted in mutation containing cardiomyocytes, demonstrating increased myofilament Ca sensitivity. In voltage-clamped hiPSC-CMs, TnT-I79N reduced intracellular Ca transients by enhancing cytosolic Ca buffering. These changes in Ca handling resulted in beat-to-beat instability and triangulation of the cardiac action potential, which are predictors of arrhythmia risk. The myofilament Ca sensitizer EMD57033 produced similar action potential triangulation in control hiPSC-CMs. The TnT-I79N hiPSC-CM model not only reproduces key cellular features of TnT-linked HCM such as myofilament disarray, hypercontractility and diastolic dysfunction, but also suggests that this TnT mutation causes pro-arrhythmic changes of the human ventricular action potential. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Effects of Sublethal Fungicides on Mutation Rates and Genomic Variation in Fungal Plant Pathogen, Sclerotinia sclerotiorum.

    PubMed

    Amaradasa, B Sajeewa; Everhart, Sydney E

    2016-01-01

    Pathogen exposure to sublethal doses of fungicides may result in mutations that may represent an important and largely overlooked mechanism of introducing new genetic variation into strictly clonal populations, including acquisition of fungicide resistance. We tested this hypothesis using the clonal plant pathogen, Sclerotinia sclerotiorum. Nine susceptible isolates were exposed independently to five commercial fungicides with different modes of action: boscalid (respiration inhibitor), iprodione (unclear mode of action), thiophanate methyl (inhibition of microtubulin synthesis) and azoxystrobin and pyraclostrobin (quinone outside inhibitors). Mycelium of each isolate was inoculated onto a fungicide gradient and sub-cultured from the 50-100% inhibition zone for 12 generations and experiment repeated. Mutational changes were assessed for all isolates at six neutral microsatellite (SSR) loci and for a subset of isolates using amplified fragment length polymorphisms (AFLPs). SSR analysis showed 12 of 85 fungicide-exposed isolates had a total of 127 stepwise mutations with 42 insertions and 85 deletions. Most stepwise deletions were in iprodione- and azoxystrobin-exposed isolates (n = 40/85 each). Estimated mutation rates were 1.7 to 60-fold higher for mutated loci compared to that expected under neutral conditions. AFLP genotyping of 33 isolates (16 non-exposed control and 17 fungicide exposed) generated 602 polymorphic alleles. Cluster analysis with principal coordinate analysis (PCoA) and discriminant analysis of principal components (DAPC) identified fungicide-exposed isolates as a distinct group from non-exposed control isolates (PhiPT = 0.15, P = 0.001). Dendrograms based on neighbor-joining also supported allelic variation associated with fungicide-exposure. Fungicide sensitivity of isolates measured throughout both experiments did not show consistent trends. For example, eight isolates exposed to boscalid had higher EC50 values at the end of the experiment, and

  6. Effects of Sublethal Fungicides on Mutation Rates and Genomic Variation in Fungal Plant Pathogen, Sclerotinia sclerotiorum

    PubMed Central

    Amaradasa, B. Sajeewa

    2016-01-01

    Pathogen exposure to sublethal doses of fungicides may result in mutations that may represent an important and largely overlooked mechanism of introducing new genetic variation into strictly clonal populations, including acquisition of fungicide resistance. We tested this hypothesis using the clonal plant pathogen, Sclerotinia sclerotiorum. Nine susceptible isolates were exposed independently to five commercial fungicides with different modes of action: boscalid (respiration inhibitor), iprodione (unclear mode of action), thiophanate methyl (inhibition of microtubulin synthesis) and azoxystrobin and pyraclostrobin (quinone outside inhibitors). Mycelium of each isolate was inoculated onto a fungicide gradient and sub-cultured from the 50–100% inhibition zone for 12 generations and experiment repeated. Mutational changes were assessed for all isolates at six neutral microsatellite (SSR) loci and for a subset of isolates using amplified fragment length polymorphisms (AFLPs). SSR analysis showed 12 of 85 fungicide-exposed isolates had a total of 127 stepwise mutations with 42 insertions and 85 deletions. Most stepwise deletions were in iprodione- and azoxystrobin-exposed isolates (n = 40/85 each). Estimated mutation rates were 1.7 to 60-fold higher for mutated loci compared to that expected under neutral conditions. AFLP genotyping of 33 isolates (16 non-exposed control and 17 fungicide exposed) generated 602 polymorphic alleles. Cluster analysis with principal coordinate analysis (PCoA) and discriminant analysis of principal components (DAPC) identified fungicide-exposed isolates as a distinct group from non-exposed control isolates (PhiPT = 0.15, P = 0.001). Dendrograms based on neighbor-joining also supported allelic variation associated with fungicide-exposure. Fungicide sensitivity of isolates measured throughout both experiments did not show consistent trends. For example, eight isolates exposed to boscalid had higher EC50 values at the end of the experiment

  7. Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase

    PubMed Central

    Kucharczyk, Roza; Ezkurdia, Nahia; Couplan, Elodie; Procaccio, Vincent; Ackerman, Sharon H.; Blondel, Marc; di Rago, Jean-Paul

    2010-01-01

    Summary Several human neurological disorders have been associated with various mutations affecting mitochondrial enzymes involved in cellular ATP production. One of these mutations, T9176C in the mitochondrial DNA (mtDNA), changes a highly conserved leucine residue into proline at position 217 of the mitochondrially encoded Atp6p (or a) subunit of the F1FO-ATP synthase. The consequences of this mutation on the mitochondrial ATP synthase are still poorly defined. To gain insight into the primary pathogenic mechanisms induced by T9176C, we have investigated the consequences of this mutation on the ATP synthase of yeast where Atp6p is also encoded by the mtDNA. In vitro, yeast atp6-T9176C mitochondria showed a 30% decrease in the rate of ATP synthesis. When forcing the F1FO complex to work in the reverse mode, i.e. F1-catalyzed hydrolysis of ATP coupled to proton transport out of the mitochondrial matrix, the mutant showed a normal proton-pumping activity and this activity was fully sensitive to oligomycin, an inhibitor of the ATP synthase proton channel. However, under conditions of maximal ATP hydrolytic activity, using non-osmotically protected mitochondria, the mutant ATPase activity was less efficiently inhibited by oligomycin (60% inhibition versus 85% for the wild type control). BN-PAGE analyses revealed that atp6-T9176C yeast accumulated rather good levels of fully assembled ATP synthase complexes. However, a number of subcomplexes (F1, Atp9p-ring, unassembled α-F1 subunits) could be detected as well, presumably because of a decreased stability of Atp6p within the ATP synthase. Although the oxidative phosphorylation capacity was reduced in atp6-T9176C yeast, the number of ATP molecules synthesized per electron transferred to oxygen was similar compared with wild type yeast. It can therefore be inferred that the coupling efficiency within the ATP synthase was mostly unaffected and that the T9176C mutation did not increase the proton permeability of the

  8. Detection of Ultra-Rare Mitochondrial Mutations in Breast Stem Cells by Duplex Sequencing.

    PubMed

    Ahn, Eun Hyun; Hirohata, Kensen; Kohrn, Brendan F; Fox, Edward J; Chang, Chia-Cheng; Loeb, Lawrence A

    2015-01-01

    Long-lived adult stem cells could accumulate non-repaired DNA damage or mutations that increase the risk of tumor formation. To date, studies on mutations in stem cells have concentrated on clonal (homoplasmic) mutations and have not focused on rarely occurring stochastic mutations that may accumulate during stem cell dormancy. A major challenge in investigating these rare mutations is that conventional next generation sequencing (NGS) methods have high error rates. We have established a new method termed Duplex Sequencing (DS), which detects mutations with unprecedented accuracy. We present a comprehensive analysis of mitochondrial DNA mutations in human breast normal stem cells and non-stem cells using DS. The vast majority of mutations occur at low frequency and are not detectable by NGS. The most prevalent point mutation types are the C>T/G>A and A>G/T>C transitions. The mutations exhibit a strand bias with higher prevalence of G>A, T>C, and A>C mutations on the light strand of the mitochondrial genome. The overall rare mutation frequency is significantly lower in stem cells than in the corresponding non-stem cells. We have identified common and unique non-homoplasmic mutations between non-stem and stem cells that include new mutations which have not been reported previously. Four mutations found within the MT-ND5 gene (m.12684G>A, m.12705C>T, m.13095T>C, m.13105A>G) are present in all groups of stem and non-stem cells. Two mutations (m.8567T>C, m.10547C>G) are found only in non-stem cells. This first genome-wide analysis of mitochondrial DNA mutations may aid in characterizing human breast normal epithelial cells and serve as a reference for cancer stem cell mutation profiles.

  9. Novel monoamine oxidase A knock out mice with human-like spontaneous mutation.

    PubMed

    Scott, Anna L; Bortolato, Marco; Chen, Kevin; Shih, Jean C

    2008-05-07

    A novel line of mutant mice [monoamine oxidase A knockout (MAOA KO)] harboring a spontaneous point nonsense mutation in exon 8 of the MAO A gene was serendipitously identified in a 129/SvEvTac colony. This mutation is analogous to the cause of a rare human disorder, Brunner syndrome, characterized by complete MAO A deficiency and impulsive aggressiveness. Concurrent with previous studies of MAO A KO mice generated by insertional mutagenesis ('Tg8'), MAOA(A863T) KO lack MAO A enzyme activity and display enhanced aggression toward intruder mice. MAOA(A863T) KO, however, exhibited lower locomotor activity in a novel, inescapable open field and similar immobility during tail suspension compared with wild type, observations which differ from reports of Tg8. These findings consolidate evidence linking MAO A to aggression and highlight subtle yet distinctive phenotypical characteristics.

  10. MLH1 mutations differentially affect meiotic functions in Saccharomyces cerevisiae.

    PubMed Central

    Hoffmann, Eva R; Shcherbakova, Polina V; Kunkel, Thomas A; Borts, Rhona H

    2003-01-01

    To test whether missense mutations in the cancer susceptibility gene MLH1 adversely affect meiosis, we examined 14 yeast MLH1 mutations for effects on meiotic DNA transactions and gamete viability in the yeast Saccharomyces cerevisiae. Mutations analogous to those associated with hereditary nonpolyposis colorectal cancer (HNPCC) or those that reduce Mlh1p interactions with ATP or DNA all impair replicative mismatch repair as measured by increased mutation rates. However, their effects on meiotic heteroduplex repair, crossing over, chromosome segregation, and gametogenesis vary from complete loss of meiotic functions to no meiotic defect, and mutants defective in one meiotic process are not necessarily defective in others. DNA binding and ATP binding but not ATP hydrolysis are required for meiotic crossing over. The results reveal clear separation of different Mlh1p functions in mitosis and meiosis, and they suggest that some, but not all, MLH1 mutations may be a source of human infertility. PMID:12618391

  11. Mutations in the dopamine beta-hydroxylase gene are associated with human norepinephrine deficiency

    NASA Technical Reports Server (NTRS)

    Kim, Chun-Hyung; Zabetian, Cyrus P.; Cubells, Joseph F.; Cho, Sonhae; Biaggioni, Italo; Cohen, Bruce M.; Robertson, David; Kim, Kwang-Soo

    2002-01-01

    Norepinephrine (NE), a key neurotransmitter of the central and peripheral nervous systems, is synthesized by dopamine beta-hydroxylase (DBH) that catalyzes oxidation of dopamine (DA) to NE. NE deficiency is a congenital disorder of unknown etiology, in which affected patients suffer profound autonomic failure. Biochemical features of the syndrome include undetectable tissue and circulating levels of NE and epinephrine, elevated levels of DA, and undetectable levels of DBH. Here, we report identification of seven novel variants including four potentially pathogenic mutations in the human DBH gene (OMIM 223360) from analysis of two unrelated patients and their families. Both patients are compound heterozygotes for variants affecting expression of DBH protein. Each carries one copy of a T-->C transversion in the splice donor site of DBH intron 1, creating a premature stop codon. In patient 1, there is a missense mutation in DBH exon 2. Patient 2 carries missense mutations in exons 1 and 6 residing in cis. We propose that NE deficiency is an autosomal recessive disorder resulting from heterogeneous molecular lesions at DBH. Copyright 2002 Wiley-Liss, Inc.

  12. 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

  13. A missense mutation in the vasopressin-neurophysin precursor gene cosegregates with human autosomal dominant neurohypophyseal diabetes insipidus.

    PubMed Central

    Bahnsen, U; Oosting, P; Swaab, D F; Nahke, P; Richter, D; Schmale, H

    1992-01-01

    Familial neurohypophyseal diabetes insipidus in humans is a rare disease transmitted as an autosomal dominant trait. Affected individuals have very low or undetectable levels of circulating vasopressin and suffer from polydipsia and polyuria. An obvious candidate gene for the disease is the vasopressin-neurophysin (AVP-NP) precursor gene on human chromosome 20. The 2 kb gene with three exons encodes a composite precursor protein consisting of the neuropeptide vasopressin and two associated proteins, neurophysin and a glycopeptide. Cloning and nucleotide sequence analysis of both alleles of the AVP-NP gene present in a Dutch ADNDI family reveals a point mutation in one allele of the affected family members. Comparison of the nucleotide sequences shows a G----T transversion within the neurophysin-encoding exon B. This missense mutation converts a highly conserved glycine (Gly17 of neurophysin) to a valine residue. RFLP analysis of six related family members indicates cosegregation of the mutant allele with the DI phenotype. The mutation is not present in 96 chromosomes of an unrelated control group. These data suggest that a single amino acid exchange within a highly conserved domain of the human vasopressin-associated neurophysin is the primary cause of one form of ADNDI. Images PMID:1740104

  14. Rules of co-occurring mutations characterize the antigenic evolution of human influenza A/H3N2, A/H1N1 and B viruses.

    PubMed

    Chen, Haifen; Zhou, Xinrui; Zheng, Jie; Kwoh, Chee-Keong

    2016-12-05

    The human influenza viruses undergo rapid evolution (especially in hemagglutinin (HA), a glycoprotein on the surface of the virus), which enables the virus population to constantly evade the human immune system. Therefore, the vaccine has to be updated every year to stay effective. There is a need to characterize the evolution of influenza viruses for better selection of vaccine candidates and the prediction of pandemic strains. Studies have shown that the influenza hemagglutinin evolution is driven by the simultaneous mutations at antigenic sites. Here, we analyze simultaneous or co-occurring mutations in the HA protein of human influenza A/H3N2, A/H1N1 and B viruses to predict potential mutations, characterizing the antigenic evolution. We obtain the rules of mutation co-occurrence using association rule mining after extracting HA1 sequences and detect co-mutation sites under strong selective pressure. Then we predict the potential drifts with specific mutations of the viruses based on the rules and compare the results with the "observed" mutations in different years. The sites under frequent mutations are in antigenic regions (epitopes) or receptor binding sites. Our study demonstrates the co-occurring site mutations obtained by rule mining can capture the evolution of influenza viruses, and confirms that cooperative interactions among sites of HA1 protein drive the influenza antigenic evolution.

  15. Seven mutations in the human insulin gene linked to permanent neonatal/infancy-onset diabetes mellitus

    PubMed Central

    Colombo, Carlo; Porzio, Ottavia; Liu, Ming; Massa, Ornella; Vasta, Mario; Salardi, Silvana; Beccaria, Luciano; Monciotti, Carla; Toni, Sonia; Pedersen, Oluf; Hansen, Torben; Federici, Luca; Pesavento, Roberta; Cadario, Francesco; Federici, Giorgio; Ghirri, Paolo; Arvan, Peter; Iafusco, Dario; Barbetti, Fabrizio

    2008-01-01

    Permanent neonatal diabetes mellitus (PNDM) is a rare disorder usually presenting within 6 months of birth. Although several genes have been linked to this disorder, in almost half the cases documented in Italy, the genetic cause remains unknown. Because the Akita mouse bearing a mutation in the Ins2 gene exhibits PNDM associated with pancreatic β cell apoptosis, we sequenced the human insulin gene in PNDM subjects with unidentified mutations. We discovered 7 heterozygous mutations in 10 unrelated probands. In 8 of these patients, insulin secretion was detectable at diabetes onset, but rapidly declined over time. When these mutant proinsulins were expressed in HEK293 cells, we observed defects in insulin protein folding and secretion. In these experiments, expression of the mutant proinsulins was also associated with increased Grp78 protein expression and XBP1 mRNA splicing, 2 markers of endoplasmic reticulum stress, and with increased apoptosis. Similarly transfected INS-1E insulinoma cells had diminished viability compared with those expressing WT proinsulin. In conclusion, we find that mutations in the insulin gene that promote proinsulin misfolding may cause PNDM. PMID:18451997

  16. RTTN Mutations Cause Primary Microcephaly and Primordial Dwarfism in Humans.

    PubMed

    Shamseldin, Hanan; Alazami, Anas M; Manning, Melanie; Hashem, Amal; Caluseiu, Oana; Tabarki, Brahim; Esplin, Edward; Schelley, Susan; Innes, A Micheil; Parboosingh, Jillian S; Lamont, Ryan; Majewski, Jacek; Bernier, Francois P; Alkuraya, Fowzan S

    2015-12-03

    Primary microcephaly is a developmental brain anomaly that results from defective proliferation of neuroprogenitors in the germinal periventricular zone. More than a dozen genes are known to be mutated in autosomal-recessive primary microcephaly in isolation or in association with a more generalized growth deficiency (microcephalic primordial dwarfism), but the genetic heterogeneity is probably more extensive. In a research protocol involving autozygome mapping and exome sequencing, we recruited a multiplex consanguineous family who is affected by severe microcephalic primordial dwarfism and tested negative on clinical exome sequencing. Two candidate autozygous intervals were identified, and the second round of exome sequencing revealed a single intronic variant therein (c.2885+8A>G [p.Ser963(∗)] in RTTN exon 23). RT-PCR confirmed that this change creates a cryptic splice donor and thus causes retention of the intervening 7 bp of the intron and leads to premature truncation. On the basis of this finding, we reanalyzed the exome file of a second consanguineous family affected by a similar phenotype and identified another homozygous change in RTTN as the likely causal mutation. Combined linkage analysis of the two families confirmed that RTTN maps to the only significant linkage peak. Finally, through international collaboration, a Canadian multiplex family affected by microcephalic primordial dwarfism and biallelic mutation of RTTN was identified. Our results expand the phenotype of RTTN-related disorders, hitherto limited to polymicrogyria, to include microcephalic primordial dwarfism with a complex brain phenotype involving simplified gyration. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  17. Sleeping Beauty mutagenesis reveals cooperating mutations and pathways in pancreatic adenocarcinoma

    PubMed Central

    Mann, Karen M.; Ward, Jerrold M.; Yew, Christopher Chin Kuan; Kovochich, Anne; Dawson, David W.; Black, Michael A.; Brett, Benjamin T.; Sheetz, Todd E.; Dupuy, Adam J.; Chang, David K.; Biankin, Andrew V.; Waddell, Nicola; Kassahn, Karin S.; Grimmond, Sean M.; Rust, Alistair G.; Adams, David J.; Jenkins, Nancy A.; Copeland, Neal G.

    2012-01-01

    Pancreatic cancer is one of the most deadly cancers affecting the Western world. Because the disease is highly metastatic and difficult to diagnosis until late stages, the 5-y survival rate is around 5%. The identification of molecular cancer drivers is critical for furthering our understanding of the disease and development of improved diagnostic tools and therapeutics. We have conducted a mutagenic screen using Sleeping Beauty (SB) in mice to identify new candidate cancer genes in pancreatic cancer. By combining SB with an oncogenic Kras allele, we observed highly metastatic pancreatic adenocarcinomas. Using two independent statistical methods to identify loci commonly mutated by SB in these tumors, we identified 681 loci that comprise 543 candidate cancer genes (CCGs); 75 of these CCGs, including Mll3 and Ptk2, have known mutations in human pancreatic cancer. We identified point mutations in human pancreatic patient samples for another 11 CCGs, including Acvr2a and Map2k4. Importantly, 10% of the CCGs are involved in chromatin remodeling, including Arid4b, Kdm6a, and Nsd3, and all SB tumors have at least one mutated gene involved in this process; 20 CCGs, including Ctnnd1, Fbxo11, and Vgll4, are also significantly associated with poor patient survival. SB mutagenesis provides a rich resource of mutations in potential cancer drivers for cross-comparative analyses with ongoing sequencing efforts in human pancreatic adenocarcinoma. PMID:22421440

  18. LET and ion-species dependence for cell killing and mutation induction in normal human fibroblasts.

    PubMed

    Tsuruoka, Chizuru; Suzuki, Masao; Fujitaka, Kazunobu

    2003-10-01

    We have been studying LET and ion species dependence of RBE values in cell killing and mutation induction. Normal human skin fibroblasts were irradiated with heavy-ion beams such as carbon (290 Mev/u and 135 Mev/u), neon (230 Mev/u and 400 Mev/u), silicon (490 Mev/u) and iron (500 Mev/u) ion beams, generated by Heavy Ion Medical Accelerator in Chiba (HIMAC) at National Institute of Radiological Sciences (NIRS). Cell killing effect was detected as reproductive cell death using a colony formation assay. Mutation induction in hprt locus was detected to measure 6-thioguanine resistant colonies. The RBE-LET curves of cell killing and mutation induction were different each ion beam. So, we plotted RBE for cell killing and mutation induction as function of Z*2/beta2 instead of LET. RBE-Z*2/beta2 curves of cell killing indicated that the discrepancy of RBE-LET curves was reconciled each ion species. But RBE-Z*2/beta2 curves of mutation induction didn't corresponded between carbon- and silicon-ion beams. These results suggested that different biological endpoints may be suitable for different physical parameter, which represent the track structure of energy deposition of ion beams.

  19. The effect of a germline mutation in the APC gene on β-catenin in human embryonic stem cells.

    PubMed

    Yedid, Nofar; Kalma, Yael; Malcov, Mira; Amit, Ami; Kariv, Revital; Caspi, Michal; Rosin-Arbesfeld, Rina; Ben-Yosef, Dalit

    2016-12-23

    Most cases of colorectal cancer (CRC) are initiated by inactivation mutations in the APC gene, which is a negative regulator of the Wnt-β-catenin pathway. Patients with familial adenomatous polyposis (FAP) inherit a germline mutation in one APC allele, and loss of the second allele leads to the development of polyps that will turn malignant if not removed. It is not fully understood which molecular mechanisms are activated by APC loss and when the loss of the second APC allele occurs. Two FAP human embryonic stem cell (hESCs) lines were derived from APC mutated embryos following pre-implantation genetic diagnosis (PGD) for FAP. These FAP-hESCs were cultured in vitro and following extended culture: 1) β-catenin expression was analyzed by Western blot analysis; 2) Wnt-β-catenin/TCF-mediated transcription luciferase assay was performed; 3) cellular localization of β-catenin was evaluated by immunoflorecence confocal microscopy; and 4) DNA sequencing of the APC gene was performed. We have established a novel human in-vitro model for studying malignant transformation, using hESCs that carry a germline mutation in the APC gene following PGD for FAP. Extended culturing of FAP1 hESCs led to activation of the Wnt signaling pathway, as demonstrated by enhanced β-catenin/TCF-mediated activity. Additionally, β-catenin showed a distinct perinuclear distribution in most (91 %) of the FAP1 hESCs high passage colonies. DNA sequencing of the whole gene detected several polymorphisms in FAP1 hESCs, however, no somatic mutations were discovered in the APC gene. On the other hand, no changes in β-catenin were detected in the FAP2 hESCs, demonstrating the natural diversity of the human FAP population. Our results describe the establishment of novel hESC lines from FAP patients with a predisposition for cancer mutation. These cells can be maintained in culture for long periods of time and may serve as a platform for studying the initial molecular and cellular changes that occur

  20. DNA transposon activity is associated with increased mutation rates in genes of rice and other grasses

    PubMed Central

    Wicker, Thomas; Yu, Yeisoo; Haberer, Georg; Mayer, Klaus F. X.; Marri, Pradeep Reddy; Rounsley, Steve; Chen, Mingsheng; Zuccolo, Andrea; Panaud, Olivier; Wing, Rod A.; Roffler, Stefan

    2016-01-01

    DNA (class 2) transposons are mobile genetic elements which move within their ‘host' genome through excising and re-inserting elsewhere. Although the rice genome contains tens of thousands of such elements, their actual role in evolution is still unclear. Analysing over 650 transposon polymorphisms in the rice species Oryza sativa and Oryza glaberrima, we find that DNA repair following transposon excisions is associated with an increased number of mutations in the sequences neighbouring the transposon. Indeed, the 3,000 bp flanking the excised transposons can contain over 10 times more mutations than the genome-wide average. Since DNA transposons preferably insert near genes, this is correlated with increases in mutation rates in coding sequences and regulatory regions. Most importantly, we find this phenomenon also in maize, wheat and barley. Thus, these findings suggest that DNA transposon activity is a major evolutionary force in grasses which provide the basis of most food consumed by humankind. PMID:27599761

  1. A Foxp2 Mutation Implicated in Human Speech Deficits Alters Sequencing of Ultrasonic Vocalizations in Adult Male Mice.

    PubMed

    Chabout, Jonathan; Sarkar, Abhra; Patel, Sheel R; Radden, Taylor; Dunson, David B; Fisher, Simon E; Jarvis, Erich D

    2016-01-01

    Development of proficient spoken language skills is disrupted by mutations of the FOXP2 transcription factor. A heterozygous missense mutation in the KE family causes speech apraxia, involving difficulty producing words with complex learned sequences of syllables. Manipulations in songbirds have helped to elucidate the role of this gene in vocal learning, but findings in non-human mammals have been limited or inconclusive. Here, we performed a systematic study of ultrasonic vocalizations (USVs) of adult male mice carrying the KE family mutation. Using novel statistical tools, we found that Foxp2 heterozygous mice did not have detectable changes in USV syllable acoustic structure, but produced shorter sequences and did not shift to more complex syntax in social contexts where wildtype animals did. Heterozygous mice also displayed a shift in the position of their rudimentary laryngeal motor cortex (LMC) layer-5 neurons. Our findings indicate that although mouse USVs are mostly innate, the underlying contributions of FoxP2 to sequencing of vocalizations are conserved with humans.

  2. Post-irradiation somatic mutation and clonal stabilisation time in the human colon.

    PubMed Central

    Campbell, F; Williams, G T; Appleton, M A; Dixon, M F; Harris, M; Williams, E D

    1996-01-01

    BACKGROUND: Colorectal crypts are clonal units in which somatic mutation of marker genes in stem cells leads to crypt restricted phenotypic conversion initially involving part of the crypt, later the whole crypt. Studies in mice show that the time taken for the great majority of mutated crypts to be completely converted, the clonal stabilisation time, is four weeks in the colon and 21 weeks in the ileum. Differences in the clonal stabilisation time between tissues and species are thought to reflect differences in stem cell organisation and crypt kinetics. AIM: To study the clonal stabilisation time in the human colorectum. METHODS: Stem cell mutation can lead to crypt restricted loss of O-acetylation of sialomucins in subjects heterozygous for O-acetyltransferase gene activity. mPAS histochemistry was used to visualise and quantify crypts partially or wholly involved by the mutant phenotype in 21 informative cases who had undergone colectomy up to 34 years after radiotherapy. RESULTS: Radiotherapy was followed by a considerable increase in the discordant crypt frequency that remained significantly increased for many years. The proportion of discordant crypts showing partial involvement was initially high but fell to normal levels about 12 months after irradiation. CONCLUSIONS: Crypts wholly involved by a mutant phenotype are stable and persistent while partially involved crypts are transient. The clonal stabilisation time is approximately one year in the human colon compared with four weeks in the mouse. The most likely reason for this is a difference in the number of stem cells in a crypt stem cell niche, although differences in stem cell cycle time and crypt fission may also contribute. These findings are of relevance to colorectal gene therapy and carcinogenesis in stem cell systems. PMID:8944567

  3. Dynamic evolution of plant mitochondrial genomes: Mobile genes and introns and highly variable mutation rates

    PubMed Central

    Palmer, Jeffrey D.; Adams, Keith L.; Cho, Yangrae; Parkinson, Christopher L.; Qiu, Yin-Long; Song, Keming

    2000-01-01

    We summarize our recent studies showing that angiosperm mitochondrial (mt) genomes have experienced remarkably high rates of gene loss and concomitant transfer to the nucleus and of intron acquisition by horizontal transfer. Moreover, we find substantial lineage-specific variation in rates of these structural mutations and also point mutations. These findings mostly arise from a Southern blot survey of gene and intron distribution in 281 diverse angiosperms. These blots reveal numerous losses of mt ribosomal protein genes but, with one exception, only rare loss of respiratory genes. Some lineages of angiosperms have kept all of their mt ribosomal protein genes whereas others have lost most of them. These many losses appear to reflect remarkably high (and variable) rates of functional transfer of mt ribosomal protein genes to the nucleus in angiosperms. The recent transfer of cox2 to the nucleus in legumes provides both an example of interorganellar gene transfer in action and a starting point for discussion of the roles of mechanistic and selective forces in determining the distribution of genetic labor between organellar and nuclear genomes. Plant mt genomes also acquire sequences by horizontal transfer. A striking example of this is a homing group I intron in the mt cox1 gene. This extraordinarily invasive mobile element has probably been acquired over 1,000 times separately during angiosperm evolution via a recent wave of cross-species horizontal transfers. Finally, whereas all previously examined angiosperm mtDNAs have low rates of synonymous substitutions, mtDNAs of two distantly related angiosperms have highly accelerated substitution rates. PMID:10860957

  4. Frequent mutations in the p53 tumor suppressor gene in human leukemia T-cell lines.

    PubMed Central

    Cheng, J; Haas, M

    1990-01-01

    Human T-cell leukemia and T-cell acute lymphoblastic leukemia cell lines were studied for alterations in the p53 tumor suppressor gene. Southern blot analysis of 10 leukemic T-cell lines revealed no gross genomic deletions or rearrangements. Reverse transcription-polymerase chain reaction analysis of p53 mRNA indicated that all 10 lines produced p53 mRNA of normal size. By direct sequencing of polymerase chain reaction-amplified cDNA, we detected 11 missense and nonsense point mutations in 5 of the 10 leukemic T-cell lines studied. The mutations are primarily located in the evolutionarily highly conserved regions of the p53 gene. One of the five cell lines in which a mutation was detected possesses a homozygous point mutation in both p53 alleles, while the other four cell lines harbor from two to four different point mutations. An allelic study of two of the lines (CEM, A3/Kawa) shows that the two missense mutations found in each line are located on separate alleles, thus both alleles of the p53 gene may have been functionally inactivated by two different point mutations. Since cultured leukemic T-cell lines represent a late, fully tumorigenic stage of leukemic T cells, mutation of both (or more) alleles of the p53 gene may reflect the selection of cells possessing an increasingly tumorigenic phenotype, whether the selection took place in vivo or in vitro. Previously, we have shown that the HSB-2 T-cell acute lymphoblastic leukemia cell line had lost both alleles of the retinoblastoma tumor suppressor gene. Taken together, our data show that at least 6 of 10 leukemic T-cell lines examined may have lost the normal function of a known tumor suppressor gene, suggesting that this class of genes serves a critical role in the generation of fully tumorigenic leukemic T cells. Images PMID:2144611

  5. Novel monoamine oxidase A knock out mice with human-like spontaneous mutation

    PubMed Central

    Scott, Anna L.; Bortolato, Marco; Chen, Kevin; Shih, Jean C.

    2012-01-01

    A novel line of mutant mice [monoamine oxidase A knockout (MAOAA863T KO)] harboring a spontaneous point nonsense mutation in exon 8 of the MAO A gene was serendipitously identified in a 129/SvEvTac colony. This mutation is analogous to the cause of a rare human disorder, Brunner syndrome, characterized by complete MAO A deficiency and impulsive aggressiveness. Concurrent with previous studies of MAO A KO mice generated by insertional mutagenesis (‘Tg8’), MAOAA863T KO lack MAO A enzyme activity and display enhanced aggression toward intruder mice. MAOAA863T KO, however, exhibited lower locomotor activity in a novel, inescapable open field and similar immobility during tail suspension compared with wild type, observations which differ from reports of Tg8. These findings consolidate evidence linking MAO A to aggression and highlight subtle yet distinctive phenotypical characteristics. PMID:18418249

  6. Human NR5A1/SF-1 Mutations Show Decreased Activity on BDNF (Brain-Derived Neurotrophic Factor), an Important Regulator of Energy Balance: Testing Impact of Novel SF-1 Mutations Beyond Steroidogenesis

    PubMed Central

    Malikova, Jana; Camats, Núria; Fernández-Cancio, Mónica; Heath, Karen; González, Isabel; Caimarí, María; del Campo, Miguel; Albisu, Marian; Kolouskova, Stanislava; Audí, Laura; Flück, Christa E.

    2014-01-01

    Context Human NR5A1/SF-1 mutations cause 46,XY disorder of sex development (DSD) with broad phenotypic variability, and rarely cause adrenal insufficiency although SF-1 is an important transcription factor for many genes involved in steroidogenesis. In addition, the Sf-1 knockout mouse develops obesity with age. Obesity might be mediated through Sf-1 regulating activity of brain-derived neurotrophic factor (BDNF), an important regulator of energy balance in the ventromedial hypothalamus. Objective To characterize novel SF-1 gene variants in 4 families, clinical, genetic and functional studies were performed with respect to steroidogenesis and energy balance. Patients 5 patients with 46,XY DSD were found to harbor NR5A1/SF-1 mutations including 2 novel variations. One patient harboring a novel mutation also suffered from adrenal insufficiency. Methods SF-1 mutations were studied in cell systems (HEK293, JEG3) for impact on transcription of genes involved in steroidogenesis (CYP11A1, CYP17A1, HSD3B2) and in energy balance (BDNF). BDNF regulation by SF-1 was studied by promoter assays (JEG3). Results Two novel NR5A1/SF-1 mutations (Glu7Stop, His408Profs*159) were confirmed. Glu7Stop is the 4th reported SF-1 mutation causing DSD and adrenal insufficiency. In vitro studies revealed that transcription of the BDNF gene is regulated by SF-1, and that mutant SF-1 decreased BDNF promoter activation (similar to steroid enzyme promoters). However, clinical data from 16 subjects carrying SF-1 mutations showed normal birth weight and BMI. Conclusions Glu7Stop and His408Profs*159 are novel SF-1 mutations identified in patients with 46,XY DSD and adrenal insufficiency (Glu7Stop). In vitro, SF-1 mutations affect not only steroidogenesis but also transcription of BDNF which is involved in energy balance. However, in contrast to mice, consequences on weight were not found in humans with SF-1 mutations. PMID:25122490

  7. Human NR5A1/SF-1 mutations show decreased activity on BDNF (brain-derived neurotrophic factor), an important regulator of energy balance: testing impact of novel SF-1 mutations beyond steroidogenesis.

    PubMed

    Malikova, Jana; Camats, Núria; Fernández-Cancio, Mónica; Heath, Karen; González, Isabel; Caimarí, María; del Campo, Miguel; Albisu, Marian; Kolouskova, Stanislava; Audí, Laura; Flück, Christa E

    2014-01-01

    Human NR5A1/SF-1 mutations cause 46,XY disorder of sex development (DSD) with broad phenotypic variability, and rarely cause adrenal insufficiency although SF-1 is an important transcription factor for many genes involved in steroidogenesis. In addition, the Sf-1 knockout mouse develops obesity with age. Obesity might be mediated through Sf-1 regulating activity of brain-derived neurotrophic factor (BDNF), an important regulator of energy balance in the ventromedial hypothalamus. To characterize novel SF-1 gene variants in 4 families, clinical, genetic and functional studies were performed with respect to steroidogenesis and energy balance. 5 patients with 46,XY DSD were found to harbor NR5A1/SF-1 mutations including 2 novel variations. One patient harboring a novel mutation also suffered from adrenal insufficiency. SF-1 mutations were studied in cell systems (HEK293, JEG3) for impact on transcription of genes involved in steroidogenesis (CYP11A1, CYP17A1, HSD3B2) and in energy balance (BDNF). BDNF regulation by SF-1 was studied by promoter assays (JEG3). Two novel NR5A1/SF-1 mutations (Glu7Stop, His408Profs*159) were confirmed. Glu7Stop is the 4th reported SF-1 mutation causing DSD and adrenal insufficiency. In vitro studies revealed that transcription of the BDNF gene is regulated by SF-1, and that mutant SF-1 decreased BDNF promoter activation (similar to steroid enzyme promoters). However, clinical data from 16 subjects carrying SF-1 mutations showed normal birth weight and BMI. Glu7Stop and His408Profs*159 are novel SF-1 mutations identified in patients with 46,XY DSD and adrenal insufficiency (Glu7Stop). In vitro, SF-1 mutations affect not only steroidogenesis but also transcription of BDNF which is involved in energy balance. However, in contrast to mice, consequences on weight were not found in humans with SF-1 mutations.

  8. Fly Models of Human Diseases: Drosophila as a Model for Understanding Human Mitochondrial Mutations and Disease.

    PubMed

    Sen, A; Cox, R T

    2017-01-01

    Mitochondrial diseases are a prevalent, heterogeneous class of diseases caused by defects in oxidative phosphorylation, whose severity depends upon particular genetic mutations. These diseases can be difficult to diagnose, and current therapeutics have limited efficacy, primarily treating only symptoms. Because mitochondria play a pivotal role in numerous cellular functions, especially ATP production, their diminished activity has dramatic physiological consequences. While this in and of itself makes treating mitochondrial disease complex, these organelles contain their own DNA, mtDNA, whose products are required for ATP production, in addition to the hundreds of nucleus-encoded proteins. Drosophila offers a tractable whole-animal model to understand the mechanisms underlying loss of mitochondrial function, the subsequent cellular and tissue damage that results, and how these organelles are inherited. Human and Drosophila mtDNAs encode the same set of products, and the homologous nucleus-encoded genes required for mitochondrial function are conserved. In addition, Drosophila contain sufficiently complex organ systems to effectively recapitulate many basic symptoms of mitochondrial diseases, yet are relatively easy and fast to genetically manipulate. There are several Drosophila models for specific mitochondrial diseases, which have been recently reviewed (Foriel, Willems, Smeitink, Schenck, & Beyrath, 2015). In this review, we highlight the conservation between human and Drosophila mtDNA, the present and future techniques for creating mtDNA mutations for further study, and how Drosophila has contributed to our current understanding of mitochondrial inheritance. © 2017 Elsevier Inc. All rights reserved.

  9. Mutations in STAT3 and IL12RB1 impair the development of human IL-17–producing T cells

    PubMed Central

    de Beaucoudrey, Ludovic; Puel, Anne; Filipe-Santos, Orchidée; Cobat, Aurélie; Ghandil, Pegah; Chrabieh, Maya; Feinberg, Jacqueline; von Bernuth, Horst; Samarina, Arina; Jannière, Lucile; Fieschi, Claire; Stéphan, Jean-Louis; Boileau, Catherine; Lyonnet, Stanislas; Jondeau, Guillaume; Cormier-Daire, Valérie; Le Merrer, Martine; Hoarau, Cyrille; Lebranchu, Yvon; Lortholary, Olivier; Chandesris, Marie-Olivia; Tron, François; Gambineri, Eleonora; Bianchi, Lucia; Rodriguez-Gallego, Carlos; Zitnik, Simona E.; Vasconcelos, Julia; Guedes, Margarida; Vitor, Artur Bonito; Marodi, Laszlo; Chapel, Helen; Reid, Brenda; Roifman, Chaim; Nadal, David; Reichenbach, Janine; Caragol, Isabel; Garty, Ben-Zion; Dogu, Figen; Camcioglu, Yildiz; Gülle, Sanyie; Sanal, Ozden; Fischer, Alain; Abel, Laurent; Stockinger, Birgitta; Picard, Capucine; Casanova, Jean-Laurent

    2008-01-01

    The cytokines controlling the development of human interleukin (IL) 17–producing T helper cells in vitro have been difficult to identify. We addressed the question of the development of human IL-17–producing T helper cells in vivo by quantifying the production and secretion of IL-17 by fresh T cells ex vivo, and by T cell blasts expanded in vitro from patients with particular genetic traits affecting transforming growth factor (TGF) β, IL-1, IL-6, or IL-23 responses. Activating mutations in TGFB1, TGFBR1, and TGFBR2 (Camurati-Engelmann disease and Marfan-like syndromes) and loss-of-function mutations in IRAK4 and MYD88 (Mendelian predisposition to pyogenic bacterial infections) had no detectable impact. In contrast, dominant-negative mutations in STAT3 (autosomal-dominant hyperimmunoglobulin E syndrome) and, to a lesser extent, null mutations in IL12B and IL12RB1 (Mendelian susceptibility to mycobacterial diseases) impaired the development of IL-17–producing T cells. These data suggest that IL-12Rβ1– and STAT-3–dependent signals play a key role in the differentiation and/or expansion of human IL-17–producing T cell populations in vivo. PMID:18591412

  10. Mutations in KEOPS-complex genes cause nephrotic syndrome with primary microcephaly.

    PubMed

    Braun, Daniela A; Rao, Jia; Mollet, Geraldine; Schapiro, David; Daugeron, Marie-Claire; Tan, Weizhen; Gribouval, Olivier; Boyer, Olivia; Revy, Patrick; Jobst-Schwan, Tilman; Schmidt, Johanna Magdalena; Lawson, Jennifer A; Schanze, Denny; Ashraf, Shazia; Ullmann, Jeremy F P; Hoogstraten, Charlotte A; Boddaert, Nathalie; Collinet, Bruno; Martin, Gaëlle; Liger, Dominique; Lovric, Svjetlana; Furlano, Monica; Guerrera, I Chiara; Sanchez-Ferras, Oraly; Hu, Jennifer F; Boschat, Anne-Claire; Sanquer, Sylvia; Menten, Björn; Vergult, Sarah; De Rocker, Nina; Airik, Merlin; Hermle, Tobias; Shril, Shirlee; Widmeier, Eugen; Gee, Heon Yung; Choi, Won-Il; Sadowski, Carolin E; Pabst, Werner L; Warejko, Jillian K; Daga, Ankana; Basta, Tamara; Matejas, Verena; Scharmann, Karin; Kienast, Sandra D; Behnam, Babak; Beeson, Brendan; Begtrup, Amber; Bruce, Malcolm; Ch'ng, Gaik-Siew; Lin, Shuan-Pei; Chang, Jui-Hsing; Chen, Chao-Huei; Cho, Megan T; Gaffney, Patrick M; Gipson, Patrick E; Hsu, Chyong-Hsin; Kari, Jameela A; Ke, Yu-Yuan; Kiraly-Borri, Cathy; Lai, Wai-Ming; Lemyre, Emmanuelle; Littlejohn, Rebecca Okashah; Masri, Amira; Moghtaderi, Mastaneh; Nakamura, Kazuyuki; Ozaltin, Fatih; Praet, Marleen; Prasad, Chitra; Prytula, Agnieszka; Roeder, Elizabeth R; Rump, Patrick; Schnur, Rhonda E; Shiihara, Takashi; Sinha, Manish D; Soliman, Neveen A; Soulami, Kenza; Sweetser, David A; Tsai, Wen-Hui; Tsai, Jeng-Daw; Topaloglu, Rezan; Vester, Udo; Viskochil, David H; Vatanavicharn, Nithiwat; Waxler, Jessica L; Wierenga, Klaas J; Wolf, Matthias T F; Wong, Sik-Nin; Leidel, Sebastian A; Truglio, Gessica; Dedon, Peter C; Poduri, Annapurna; Mane, Shrikant; Lifton, Richard P; Bouchard, Maxime; Kannu, Peter; Chitayat, David; Magen, Daniella; Callewaert, Bert; van Tilbeurgh, Herman; Zenker, Martin; Antignac, Corinne; Hildebrandt, Friedhelm

    2017-10-01

    Galloway-Mowat syndrome (GAMOS) is an autosomal-recessive disease characterized by the combination of early-onset nephrotic syndrome (SRNS) and microcephaly with brain anomalies. Here we identified recessive mutations in OSGEP, TP53RK, TPRKB, and LAGE3, genes encoding the four subunits of the KEOPS complex, in 37 individuals from 32 families with GAMOS. CRISPR-Cas9 knockout in zebrafish and mice recapitulated the human phenotype of primary microcephaly and resulted in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibited cell proliferation, which human mutations did not rescue. Furthermore, knockdown of these genes impaired protein translation, caused endoplasmic reticulum stress, activated DNA-damage-response signaling, and ultimately induced apoptosis. Knockdown of OSGEP or TP53RK induced defects in the actin cytoskeleton and decreased the migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identified four new monogenic causes of GAMOS, describe a link between KEOPS function and human disease, and delineate potential pathogenic mechanisms.

  11. Genomic organization and mutational analysis of the human UCP2 gene, a prime candidate gene for human obesity.

    PubMed

    Lentes, K U; Tu, N; Chen, H; Winnikes, U; Reinert, I; Marmann, G; Pirke, K M

    1999-01-01

    Uncoupling proteins (UCPs) are mitochondrial membrane transporters which are involved in dissipating the proton electrochemical gradient thereby releasing stored energy as heat. This implies a major role of UCPs in energy metabolism and thermogenesis which when deregulated are key risk factors for the development of obesity and other eating disorders. Recent studies have shown that the sympathetic nervous system, via norepinephrine (beta-adrenoceptors) and cAMP, as well as thyroid hormones and PPAR gamma ligands seem to be major regulators of UCP expression. From the three different UCPs identified so far by gene cloning UCP1 is expressed exclusively in brown adipocytes while UCP2 is widely expressed. The third analogue, UCP3, is expressed predominantly in human skeletal muscle and was found to exist in a long and a short form. At the amino acid level UCP2 has about 59% homology to UCP1 while UCP3 is 73% identical to UCP2. Both UCP2 and UCP3 were mapped in close proximity (75-150 kb) to regions of human chromosome 11 (11q13) that have been linked to obesity and hyper-insulinaemia. Furthermore, there is strong evidence that UCP2, by virtue of its ubiquitous expression, may be important for determining basal metabolic rate. Based on the published full-length cDNA sequence we have deduced the genomic structure of the human UCP2 (hUCP2) gene by PCR and direct sequence analysis. The hUCP2 gene spans over 8.4 kb distributed on 8 exons. The localization of the exon/intron boundaries within the coding region matches precisely the one found in the human UCP1 gene and is almost conserved in the recently discovered UCP3 gene as well. However, the size of each of the introns in the hUCP2 gene differs from its UCP1 and UCP3 counterparts. It varies from 81 bp (intron 5) to about 3 kb (intron 2). The high degree of homology at the nucleotide level and the conservation of the exon/intron boundaries among the three UCP genes suggests that they may have evolved from a common

  12. Parkin dosage mutations have greater pathogenicity in familial PD than simple sequence mutations

    PubMed Central

    Pankratz, N; Kissell, D K.; Pauciulo, M W.; Halter, C A.; Rudolph, A; Pfeiffer, R F.; Marder, K S.; Foroud, T; Nichols, W C.

    2009-01-01

    Objective: Mutations in both alleles of parkin have been shown to result in Parkinson disease (PD). However, it is unclear whether haploinsufficiency (presence of a mutation in only 1 of the 2 parkin alleles) increases the risk for PD. Methods: We performed comprehensive dosage and sequence analysis of all 12 exons of parkin in a sample of 520 independent patients with familial PD and 263 controls. We evaluated whether presence of a single parkin mutation, either a sequence (point mutation or small insertion/deletion) or dosage (whole exon deletion or duplication) mutation, was found at increased frequency in cases as compared with controls. We then compared the clinical characteristics of cases with 0, 1, or 2 parkin mutations. Results: We identified 55 independent patients with PD with at least 1 parkin mutation and 9 controls with a single sequence mutation. Cases and controls had a similar frequency of single sequence mutations (3.1% vs 3.4%, p = 0.83); however, the cases had a significantly higher rate of dosage mutations (2.6% vs 0%, p = 0.009). Cases with a single dosage mutation were more likely to have an earlier age at onset (50% with onset at ≤45 years) compared with those with no parkin mutations (10%, p = 0.00002); this was not true for cases with only a single sequence mutation (25% with onset at ≤45 years, p = 0.06). Conclusions: Parkin haploinsufficiency, specifically for a dosage mutation rather than a point mutation or small insertion/deletion, is a risk factor for familial PD and may be associated with earlier age at onset. GLOSSARY ADL = Activities of Daily Living; GDS = Geriatric Depression Scale; MLPA = multiplex ligation-dependent probe amplification; MMSE = Mini-Mental State Examination; PD = Parkinson disease; UPDRS = Unified Parkinson’s Disease Rating Scale. PMID:19636047

  13. Single Nucleotide Variations of the Human GR Gene Manifested as Pathologic Mutations or Polymorphisms.

    PubMed

    Kino, Tomoshige

    2018-05-11

    The human genome contains numerous single nucleotide variations (SNVs), and the human GR gene harbors ∼450 of these genetic changes. Among them, extremely rare non-synonymous variants known as pathologic GR gene mutations develop a characteristic pathologic condition, familial/sporadic generalized glucocorticoid resistance syndrome, by replacing the amino acids critical for GR protein structure and functions, whereas others known as pathologic polymorphisms develop mild manifestations recognized mainly at population bases by changing the GR activities slightly. Recent progress on the structural analysis to the GR protein and subsequent computer-based structural simulation revealed details of the molecular defects caused by such pathologic GR gene mutations, including their impact on the receptor interaction to ligands, nuclear receptor coactivators (NCoAs) or DNA glucocorticoid response elements (GREs). Indeed, those found in the GR ligand-binding domain significantly damage protein structure of the ligand-binding pocket and/or the activation function-2 transactivation domain and change their molecular interaction to glucocorticoids or the LxxLL signature motif of NCoAs. Two mutations found in GR DBD also affect interaction of the mutant receptors to GRE DNA by affecting the critical amino acid for the interaction or changing local hydrophobic circumstance. In this review, we discuss recent findings on the structural simulation of the pathologic GR mutants in connection to their functional and clinical impacts along with brief explanation to recent research achievement on the GR polymorphisms.

  14. A novel human pain insensitivity disorder caused by a point mutation in ZFHX2

    PubMed Central

    Habib, Abdella M; Matsuyama, Ayako; Okorokov, Andrei L; Santana-Varela, Sonia; Bras, Jose T; Aloisi, Anna Maria; Emery, Edward C; Bogdanov, Yury D; Follenfant, Maryne; Gossage, Sam J; Gras, Mathilde; Humphrey, Jack; Kolesnikov, Anna; Le Cann, Kim; Li, Shengnan; Minett, Michael S; Pereira, Vanessa; Ponsolles, Clara; Sikandar, Shafaq; Torres, Jesus M; Yamaoka, Kenji; Zhao, Jing; Komine, Yuriko; Yamamori, Tetsuo; Maniatis, Nikolas; Panov, Konstantin I; Houlden, Henry; Ramirez, Juan D; Bennett, David L H; Marsili, Letizia; Bachiocco, Valeria; Wood, John N; Cox, James J

    2018-01-01

    Abstract Chronic pain is a major global public health issue causing a severe impact on both the quality of life for sufferers and the wider economy. Despite the significant clinical burden, little progress has been made in terms of therapeutic development. A unique approach to identifying new human-validated analgesic drug targets is to study rare families with inherited pain insensitivity. Here we have analysed an otherwise normal family where six affected individuals display a pain insensitive phenotype that is characterized by hyposensitivity to noxious heat and painless bone fractures. This autosomal dominant disorder is found in three generations and is not associated with a peripheral neuropathy. A novel point mutation in ZFHX2, encoding a putative transcription factor expressed in small diameter sensory neurons, was identified by whole exome sequencing that segregates with the pain insensitivity. The mutation is predicted to change an evolutionarily highly conserved arginine residue 1913 to a lysine within a homeodomain. Bacterial artificial chromosome (BAC) transgenic mice bearing the orthologous murine p.R1907K mutation, as well as Zfhx2 null mutant mice, have significant deficits in pain sensitivity. Gene expression analyses in dorsal root ganglia from mutant and wild-type mice show altered expression of genes implicated in peripheral pain mechanisms. The ZFHX2 variant and downstream regulated genes associated with a human pain-insensitive phenotype are therefore potential novel targets for the development of new analgesic drugs. PMID:29253101

  15. A human-specific mutation leads to the origin of a novel splice form of neuropsin (KLK8), a gene involved in learning and memory.

    PubMed

    Lu, Zhi-xiang; Peng, Jia; Su, Bing

    2007-10-01

    Neuropsin (kallikrein 8, KLK8) is a secreted-type serine protease preferentially expressed in the central nervous system and involved in learning and memory. Its splicing pattern is different in human and mouse, with the longer form (type II) only expressed in human. Sequence analysis suggested a recent origin of type II during primate evolution. Here we demonstrate that the type II form is absent in nonhuman primates, and is thus a human-specific splice form. With the use of an in vitro splicing assay, we show that a human-specific T to A mutation (c.71-127T>A) triggers the change of splicing pattern, leading to the origin of a novel splice form in the human brain. Using mutation assay, we prove that this mutation is not only necessary but also sufficient for type II expression. Our results demonstrate a molecular mechanism for the creation of novel proteins through alternative splicing in the central nervous system during human evolution. Copyright 2007 Wiley-Liss, Inc.

  16. A novel missense-mutation-related feature extraction scheme for 'driver' mutation identification.

    PubMed

    Tan, Hua; Bao, Jiguang; Zhou, Xiaobo

    2012-11-15

    It becomes widely accepted that human cancer is a disease involving dynamic changes in the genome and that the missense mutations constitute the bulk of human genetic variations. A multitude of computational algorithms, especially the machine learning-based ones, has consequently been proposed to distinguish missense changes that contribute to the cancer progression ('driver' mutation) from those that do not ('passenger' mutation). However, the existing methods have multifaceted shortcomings, in the sense that they either adopt incomplete feature space or depend on protein structural databases which are usually far from integrated. In this article, we investigated multiple aspects of a missense mutation and identified a novel feature space that well distinguishes cancer-associated driver mutations from passenger ones. An index (DX score) was proposed to evaluate the discriminating capability of each feature, and a subset of these features which ranks top was selected to build the SVM classifier. Cross-validation showed that the classifier trained on our selected features significantly outperforms the existing ones both in precision and robustness. We applied our method to several datasets of missense mutations culled from published database and literature and obtained more reasonable results than previous studies. The software is available online at http://www.methodisthealth.com/software and https://sites.google.com/site/drivermutationidentification/. xzhou@tmhs.org. Supplementary data are available at Bioinformatics online.

  17. Mutation and polymorphism analysis of the human homogentisate 1, 2-dioxygenase gene in alkaptonuria patients.

    PubMed Central

    Beltrán-Valero de Bernabé, D; Granadino, B; Chiarelli, I; Porfirio, B; Mayatepek, E; Aquaron, R; Moore, M M; Festen, J J; Sanmartí, R; Peñalva, M A; de Córdoba, S R

    1998-01-01

    Alkaptonuria (AKU), a rare hereditary disorder of phenylalanine and tyrosine catabolism, was the first disease to be interpreted as an inborn error of metabolism. AKU patients are deficient for homogentisate 1,2 dioxygenase (HGO); this deficiency causes homogentisic aciduria, ochronosis, and arthritis. We cloned the human HGO gene and characterized two loss-of-function mutations, P230S and V300G, in the HGO gene in AKU patients. Here we report haplotype and mutational analysis of the HGO gene in 29 novel AKU chromosomes. We identified 12 novel mutations: 8 (E42A, W97G, D153G, S189I, I216T, R225H, F227S, and M368V) missense mutations that result in amino acid substitutions at positions conserved in HGO in different species, 1 (F10fs) frameshift mutation, 2 intronic mutations (IVS9-56G-->A, IVS9-17G-->A), and 1 splice-site mutation (IVS5+1G-->T). We also report characterization of five polymorphic sites in HGO and describe the haplotypic associations of alleles at these sites in normal and AKU chromosomes. One of these sites, HGO-3, is a variable dinucleotide repeat; IVS2+35T/A, IVS5+25T/C, and IVS6+46C/A are intronic sites at which single nucleotide substitutions (dimorphisms) have been detected; and c407T/A is a relatively frequent nucleotide substitution in the coding sequence, exon 4, resulting in an amino acid change (H80Q). These data provide insight into the origin and evolution of the various AKU alleles. PMID:9529363

  18. 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

  19. Spiking of contemporary human template DNA with ancient DNA extracts induces mutations under PCR and generates nonauthentic mitochondrial sequences.

    PubMed

    Pusch, Carsten M; Bachmann, Lutz

    2004-05-01

    Proof of authenticity is the greatest challenge in palaeogenetic research, and many safeguards have become standard routine in laboratories specialized on ancient DNA research. Here we describe an as-yet unknown source of artifacts that will require special attention in the future. We show that ancient DNA extracts on their own can have an inhibitory and mutagenic effect under PCR. We have spiked PCR reactions including known human test DNA with 14 selected ancient DNA extracts from human and nonhuman sources. We find that the ancient DNA extracts inhibit the amplification of large fragments to different degrees, suggesting that the usual control against contaminations, i.e., the absence of long amplifiable fragments, is not sufficient. But even more important, we find that the extracts induce mutations in a nonrandom fashion. We have amplified a 148-bp stretch of the mitochondrial HVRI from contemporary human template DNA in spiked PCR reactions. Subsequent analysis of 547 sequences from cloned amplicons revealed that the vast majority (76.97%) differed from the correct sequence by single nucleotide substitutions and/or indels. In total, 34 positions of a 103-bp alignment are affected, and most mutations occur repeatedly in independent PCR amplifications. Several of the induced mutations occur at positions that have previously been detected in studies of ancient hominid sequences, including the Neandertal sequences. Our data imply that PCR-induced mutations are likely to be an intrinsic and general problem of PCR amplifications of ancient templates. Therefore, ancient DNA sequences should be considered with caution, at least as long as the molecular basis for the extract-induced mutations is not understood.

  20. Defining the pathogenesis of the human Atp12p W94R mutation using a Saccharomyces cerevisiae yeast model.

    PubMed

    Meulemans, Ann; Seneca, Sara; Pribyl, Thomas; Smet, Joel; Alderweirldt, Valerie; Waeytens, Anouk; Lissens, Willy; Van Coster, Rudy; De Meirleir, Linda; di Rago, Jean-Paul; Gatti, Domenico L; Ackerman, Sharon H

    2010-02-05

    Studies in yeast have shown that a deficiency in Atp12p prevents assembly of the extrinsic domain (F(1)) of complex V and renders cells unable to make ATP through oxidative phosphorylation. De Meirleir et al. (De Meirleir, L., Seneca, S., Lissens, W., De Clercq, I., Eyskens, F., Gerlo, E., Smet, J., and Van Coster, R. (2004) J. Med. Genet. 41, 120-124) have reported that a homozygous missense mutation in the gene for human Atp12p (HuAtp12p), which replaces Trp-94 with Arg, was linked to the death of a 14-month-old patient. We have investigated the impact of the pathogenic W94R mutation on Atp12p structure/function. Plasmid-borne wild type human Atp12p rescues the respiratory defect of a yeast ATP12 deletion mutant (Deltaatp12). The W94R mutation alters the protein at the most highly conserved position in the Pfam sequence and renders HuAtp12p insoluble in the background of Deltaatp12. In contrast, the yeast protein harboring the corresponding mutation, ScAtp12p(W103R), is soluble in the background of Deltaatp12 but not in the background of Deltaatp12Deltafmc1, a strain that also lacks Fmc1p. Fmc1p is a yeast mitochondrial protein not found in higher eukaryotes. Tryptophan 94 (human) or 103 (yeast) is located in a positively charged region of Atp12p, and hence its mutation to arginine does not alter significantly the electrostatic properties of the protein. Instead, we provide evidence that the primary effect of the substitution is on the dynamic properties of Atp12p.

  1. Reduced white matter MRI transverse relaxation rate in cognitively normal H63D-HFE human carriers and H67D-HFE mice.

    PubMed

    Meadowcroft, Mark D; Wang, Jianli; Purnell, Carson J; Peters, Douglas G; Eslinger, Paul J; Neely, Elizabeth B; Gill, David J; Vasavada, Megha; Ali-Rahmani, Fatima; Yang, Qing X; Connor, James R

    2016-12-01

    Mutations within the HFE protein gene sequence have been associated with increased risk of developing a number of neurodegenerative disorders. To this effect, an animal model has been created which incorporates the mouse homologue to the human H63D-HFE mutation: the H67D-HFE knock-in mouse. These mice exhibit alterations in iron management proteins, have increased neuronal oxidative stress, and a disruption in cholesterol regulation. However, it remains undetermined how these differences translate to human H63D carriers in regards to white matter (WM) integrity. To this endeavor, MRI transverse relaxation rate (R 2 ) parametrics were employed to test the hypothesis that WM alterations are present in H63D human carriers and are recapitulated in the H67D mice. H63D carriers exhibit widespread reductions in brain R 2 compared to non-carriers within white matter association fibers in the brain. Similar R 2 decreases within white matter tracts were observed in the H67D mouse brain. Additionally, an exacerbation of age-related R 2 decrease is found in the H67D animal model in white matter regions of interest. The decrease in R 2 within white matter tracts of both species is speculated to be multifaceted. The R 2 changes are hypothesized to be due to alterations in axonal biochemical tissue composition. The R 2 changes observed in both the human-H63D and mouse-H67D data suggest that modified white matter myelination is occurring in subjects with HFE mutations, potentially increasing vulnerability to neurodegenerative disorders.

  2. Oncogenically active MYD88 mutations in human lymphoma.

    PubMed

    Ngo, Vu N; Young, Ryan M; Schmitz, Roland; Jhavar, Sameer; Xiao, Wenming; Lim, Kian-Huat; Kohlhammer, Holger; Xu, Weihong; Yang, Yandan; Zhao, Hong; Shaffer, Arthur L; Romesser, Paul; Wright, George; Powell, John; Rosenwald, Andreas; Muller-Hermelink, Hans Konrad; Ott, German; Gascoyne, Randy D; Connors, Joseph M; Rimsza, Lisa M; Campo, Elias; Jaffe, Elaine S; Delabie, Jan; Smeland, Erlend B; Fisher, Richard I; Braziel, Rita M; Tubbs, Raymond R; Cook, J R; Weisenburger, Denny D; Chan, Wing C; Staudt, Louis M

    2011-02-03

    The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) remains the least curable form of this malignancy despite recent advances in therapy. Constitutive nuclear factor (NF)-κB and JAK kinase signalling promotes malignant cell survival in these lymphomas, but the genetic basis for this signalling is incompletely understood. Here we describe the dependence of ABC DLBCLs on MYD88, an adaptor protein that mediates toll and interleukin (IL)-1 receptor signalling, and the discovery of highly recurrent oncogenic mutations affecting MYD88 in ABC DLBCL tumours. RNA interference screening revealed that MYD88 and the associated kinases IRAK1 and IRAK4 are essential for ABC DLBCL survival. High-throughput RNA resequencing uncovered MYD88 mutations in ABC DLBCL lines. Notably, 29% of ABC DLBCL tumours harboured the same amino acid substitution, L265P, in the MYD88 Toll/IL-1 receptor (TIR) domain at an evolutionarily invariant residue in its hydrophobic core. This mutation was rare or absent in other DLBCL subtypes and Burkitt's lymphoma, but was observed in 9% of mucosa-associated lymphoid tissue lymphomas. At a lower frequency, additional mutations were observed in the MYD88 TIR domain, occurring in both the ABC and germinal centre B-cell-like (GCB) DLBCL subtypes. Survival of ABC DLBCL cells bearing the L265P mutation was sustained by the mutant but not the wild-type MYD88 isoform, demonstrating that L265P is a gain-of-function driver mutation. The L265P mutant promoted cell survival by spontaneously assembling a protein complex containing IRAK1 and IRAK4, leading to IRAK4 kinase activity, IRAK1 phosphorylation, NF-κB signalling, JAK kinase activation of STAT3, and secretion of IL-6, IL-10 and interferon-β. Hence, the MYD88 signalling pathway is integral to the pathogenesis of ABC DLBCL, supporting the development of inhibitors of IRAK4 kinase and other components of this pathway for the treatment of tumours bearing oncogenic MYD88 mutations.

  3. Mutational signatures associated with tobacco smoking in human cancer

    DOE PAGES

    Alexandrov, Ludmil B.; Ju, Young Seok; Haase, Kerstin; ...

    2016-11-04

    Tobacco smoking increases the risk of at least 17 classes of cancer. Here, we analyzed somatic mutations and DNA methylation in 5,243 cancers of types for which tobacco smoking confers an elevated risk. Smoking is associated with increased mutation burdens of multiple distinct mutational signatures, which contribute to different extents in different cancers. One of these signatures, mainly found in cancers derived from tissues directly exposed to tobacco smoke, is attributable to misreplication of DNA damage caused by tobacco carcinogens. Others likely reflect indirect activation of DNA edi ting by APOBEC cytidine deaminases and of an endogenous clock-like mutational process.more » Smoking is associated with limited differences in methylation. The results are consistent with the proposition that smoking increases cancer risk by increasing the somatic mutation load, although direct evidence for this mechanism is lacking in some smoking-related cancer types.« less

  4. Mutational signatures associated with tobacco smoking in human cancer

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

    Alexandrov, Ludmil B.; Ju, Young Seok; Haase, Kerstin

    Tobacco smoking increases the risk of at least 17 classes of cancer. Here, we analyzed somatic mutations and DNA methylation in 5,243 cancers of types for which tobacco smoking confers an elevated risk. Smoking is associated with increased mutation burdens of multiple distinct mutational signatures, which contribute to different extents in different cancers. One of these signatures, mainly found in cancers derived from tissues directly exposed to tobacco smoke, is attributable to misreplication of DNA damage caused by tobacco carcinogens. Others likely reflect indirect activation of DNA edi ting by APOBEC cytidine deaminases and of an endogenous clock-like mutational process.more » Smoking is associated with limited differences in methylation. The results are consistent with the proposition that smoking increases cancer risk by increasing the somatic mutation load, although direct evidence for this mechanism is lacking in some smoking-related cancer types.« less

  5. Modeling Autism by SHANK Gene Mutations in Mice

    PubMed Central

    Jiang, Yong-hui; Ehlers, Michael D.

    2013-01-01

    Summary Shank family proteins (Shank1, Shank2, and Shank3) are synaptic scaffolding proteins that organize an extensive protein complex at the postsynaptic density (PSD) of excitatory glutamatergic synapses. Recent human genetic studies indicate that SHANK family genes (SHANK1, SHANK2, and SHANK3) are causative genes for idiopathic autism spectrum disorders (ASD). Neurobiological studies of Shank mutations in mice support a general hypothesis of synaptic dysfunction in the pathophysiology of ASD. However, the molecular diversity of SHANK family gene products, as well as the heterogeneity in human and mouse phenotypes, pose challenges to modeling human SHANK mutations. Here, we review the molecular genetics of SHANK mutations in human ASD and discuss recent findings where such mutations have been modeled in mice. Conserved features of synaptic dysfunction and corresponding behaviors in Shank mouse mutants may help dissect the pathophysiology of ASD, but also highlight divergent phenotypes that arise from different mutations in the same gene. PMID:23583105

  6. Novel nonnucleoside inhibitors that select nucleoside inhibitor resistance mutations in human immunodeficiency virus type 1 reverse transcriptase.

    PubMed

    Zhang, Zhijun; Walker, Michelle; Xu, Wen; Shim, Jae Hoon; Girardet, Jean-Luc; Hamatake, Robert K; Hong, Zhi

    2006-08-01

    Mutations in and around the catalytic site of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) are associated with resistance to nucleoside RT inhibitors (NRTIs), whereas changes in the hydrophobic pocket of the RT are attributed to nonnucleoside RT inhibitor (NNRTI) resistance. In this study, we report a novel series of nonnucleoside inhibitors of HIV-1, exemplified by VRX-329747 and VRX-413638, which inhibit both NNRTI- and NRTI-resistant HIV-1 isolates. Enzymatic studies indicated that these compounds are HIV-1 RT inhibitors. Surprisingly, however, following prolonged (6 months) tissue culture selection, this series of nonnucleoside inhibitors did not select NNRTI-resistant mutations in HIV-1 RT. Rather, four mutations (M41L, A62T/V, V118I, and M184V) known to cause resistance to NRTIs and two additional novel mutations (S68N and G112S) adjacent to the catalytic site of the enzyme were selected. Although the M184V mutation appears to be the initial mutation to establish resistance, this mutation alone confers only a two- to fourfold decrease in susceptibility to VRX-329747 and VRX-413638. At least two additional mutations must accumulate for significant resistance. Moreover, while VRX-329747-selected viruses are resistant to lamivudine and emtricitabine due to the M184V mutation, they remain susceptible to zidovudine, stavudine, dideoxyinosine, abacavir, tenofovir, and efavirenz. These results directly demonstrate that VRX-329747 and VRX-413638 are novel nonnucleoside inhibitors of HIV-1 RT with the potential to augment current therapies.

  7. Novel Nonnucleoside Inhibitors That Select Nucleoside Inhibitor Resistance Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase

    PubMed Central

    Zhang, Zhijun; Walker, Michelle; Xu, Wen; Shim, Jae Hoon; Girardet, Jean-Luc; Hamatake, Robert K.; Hong, Zhi

    2006-01-01

    Mutations in and around the catalytic site of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) are associated with resistance to nucleoside RT inhibitors (NRTIs), whereas changes in the hydrophobic pocket of the RT are attributed to nonnucleoside RT inhibitor (NNRTI) resistance. In this study, we report a novel series of nonnucleoside inhibitors of HIV-1, exemplified by VRX-329747 and VRX-413638, which inhibit both NNRTI- and NRTI-resistant HIV-1 isolates. Enzymatic studies indicated that these compounds are HIV-1 RT inhibitors. Surprisingly, however, following prolonged (6 months) tissue culture selection, this series of nonnucleoside inhibitors did not select NNRTI-resistant mutations in HIV-1 RT. Rather, four mutations (M41L, A62T/V, V118I, and M184V) known to cause resistance to NRTIs and two additional novel mutations (S68N and G112S) adjacent to the catalytic site of the enzyme were selected. Although the M184V mutation appears to be the initial mutation to establish resistance, this mutation alone confers only a two- to fourfold decrease in susceptibility to VRX-329747 and VRX-413638. At least two additional mutations must accumulate for significant resistance. Moreover, while VRX-329747-selected viruses are resistant to lamivudine and emtricitabine due to the M184V mutation, they remain susceptible to zidovudine, stavudine, dideoxyinosine, abacavir, tenofovir, and efavirenz. These results directly demonstrate that VRX-329747 and VRX-413638 are novel nonnucleoside inhibitors of HIV-1 RT with the potential to augment current therapies. PMID:16870771

  8. Population data and mutation rate of nine Y-STRs in a mestizo Mexican population from Guadalajara, Jalisco, México.

    PubMed

    Padilla-Gutiérrez, Jorge Ramón; Valle, Yeminia; Quintero-Ramos, Antonio; Hernández, Guillermo; Rodarte, Katya; Ortiz, Rocío; Olivares, Norma; Rivas, Fernando

    2008-11-01

    Nine Y-STR (DYS19, DYS390, DYS391, DYS392, DYS446, DYS447, DYS448, DYS456 and DYS458) were analyzed in a male sample of 285 unrelated individuals from Guadalajara, Jalisco, México. The haplotype diversity (0.996) and discrimination capacity (0.986) were calculated. A family study of around 200 father/son pairs and among 1828 meiosis showed five mutational events. All mutations were single step. The overall mutation rate estimated across the nine Y-STRs was 2.7 x 10(-3) (95% CI 1.2-6.4 x 10(-3))/locus/meiosis. The results indicate that these nine loci are useful Y-linked markers for forensic applications.

  9. 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

  10. P16 UV mutations in human skin epithelial tumors.

    PubMed

    Soufir, N; Molès, J P; Vilmer, C; Moch, C; Verola, O; Rivet, J; Tesniere, A; Dubertret, L; Basset-Seguin, N

    1999-09-23

    The p16 gene expresses two alternative transcripts (p16alpha and p16beta) involved in tumor suppression via the retinoblastoma (Rb) or p53 pathways. Disruption of these pathways can occur through inactivation of p16 or p53, or activating mutations of cyclin dependant kinase 4 gene (Cdk4). We searched for p16, Cdk4 and p53 gene mutations in 20 squamous cell carcinomas (SSCs), 1 actinic keratosis (AK), and 28 basal cell carcinomas (BCCs), using PCR-SSCP. A deletion and methylation analysis of p16 was also performed. Six different mutations (12%) were detected in exon 2 of p16 (common to p16alpha and p16beta), in five out of 21 squamous lesions (24%) (one AK and four SCCs) and one out of 28 BCCs (3.5%). These included four (66%) ultraviolet (UV)-type mutations (two tandems CC : GG to TT : AA transitions and two C : G to T : A transitions at dipyrimidic site) and two transversions. P53 mutations were present in 18 samples (37%), mostly of UV type. Of these, only two (one BCC and one AK) harboured simultaneously mutations of p16, but with no consequence on p16beta transcript. Our data demonstrate for the first time the presence of p16 UV induced mutations in non melanoma skin cancer, particularly in the most aggressive SCC type, and support that p16 and p53 are involved in two independent pathways in skin carcinogenesis.

  11. Investigating the Consequences of Interference between Multiple CD8+ T Cell Escape Mutations in Early HIV Infection

    PubMed Central

    Garcia, Victor; Feldman, Marcus W.; Regoes, Roland R.

    2016-01-01

    During early human immunodeficiency virus (HIV) infection multiple CD8+ T cell responses are elicited almost simultaneously. These responses exert strong selective pressures on different parts of HIV’s genome, and select for mutations that escape recognition and are thus beneficial to the virus. Some studies reveal that the later these escape mutations emerge, the more slowly they go to fixation. This pattern of escape rate decrease(ERD) can arise by distinct mechanisms. In particular, in large populations with high beneficial mutation rates interference among different escape strains –an effect that can emerge in evolution with asexual reproduction and results in delayed fixation times of beneficial mutations compared to sexual reproduction– could significantly impact the escape rates of mutations. In this paper, we investigated how interference between these concurrent escape mutations affects their escape rates in systems with multiple epitopes, and whether it could be a source of the ERD pattern. To address these issues, we developed a multilocus Wright-Fisher model of HIV dynamics with selection, mutation and recombination, serving as a null-model for interference. We also derived an interference-free null model assuming initial neutral evolution before immune response elicitation. We found that interference between several equally selectively advantageous mutations can generate the observed ERD pattern. We also found that the number of loci, as well as recombination rates substantially affect ERD. These effects can be explained by the underexponential decline of escape rates over time. Lastly, we found that the observed ERD pattern in HIV infected individuals is consistent with both independent, interference-free mutations as well as interference effects. Our results confirm that interference effects should be considered when analyzing HIV escape mutations. The challenge in estimating escape rates and mutation-associated selective coefficients posed by

  12. Erythrocytosis and Pulmonary Hypertension in a Mouse Model of Human HIF2A Gain of Function Mutation*

    PubMed Central

    Tan, Qiulin; Kerestes, Heddy; Percy, Melanie J.; Pietrofesa, Ralph; Chen, Li; Khurana, Tejvir S.; Christofidou-Solomidou, Melpo; Lappin, Terence R. J.; Lee, Frank S.

    2013-01-01

    The central pathway for oxygen-dependent control of red cell mass is the prolyl hydroxylase domain protein (PHD):hypoxia inducible factor (HIF) pathway. PHD site specifically prolyl hydroxylates the transcription factor HIF-α, thereby targeting the latter for degradation. Under hypoxia, this modification is attenuated, allowing stabilized HIF-α to activate target genes, including that for erythropoietin (EPO). Studies employing genetically modified mice point to Hif-2α, one of two main Hif-α isoforms, as being the critical regulator of Epo in the adult mouse. More recently, erythrocytosis patients with heterozygous point mutations in the HIF2A gene have been identified; whether these mutations were polymorphisms unrelated to the phenotype could not be ruled out. In the present report, we characterize a mouse line bearing a G536W missense mutation in the Hif2a gene that corresponds to the first such human mutation identified (G537W). We obtained mice bearing both heterozygous and homozygous mutations at this locus. We find that these mice display, in a mutation dose-dependent manner, erythrocytosis and pulmonary hypertension with a high degree of penetrance. These findings firmly establish missense mutations in HIF-2α as a cause of erythrocytosis, highlight the importance of this HIF-α isoform in erythropoiesis, and point to physiologic consequences of HIF-2α dysregulation. PMID:23640890

  13. Human Cataract Mutations in EPHA2 SAM Domain Alter Receptor Stability and Function

    PubMed Central

    Park, Jeong Eun; Son, Alexander I.; Hua, Rui; Wang, Lianqing; Zhang, Xue; Zhou, Renping

    2012-01-01

    The cellular and molecular mechanisms underlying the pathogenesis of cataracts leading to visual impairment remain poorly understood. In recent studies, several mutations in the cytoplasmic sterile-α-motif (SAM) domain of human EPHA2 on chromosome 1p36 have been associated with hereditary cataracts in several families. Here, we have investigated how these SAM domain mutations affect EPHA2 activity. We showed that the SAM domain mutations dramatically destabilized the EPHA2 protein in a proteasome-dependent pathway, as evidenced by the increase of EPHA2 receptor levels in the presence of the proteasome inhibitor MG132. In addition, the expression of wild-type EPHA2 promoted the migration of the mouse lens epithelial αTN4-1 cells in the absence of ligand stimulation, whereas the mutants exhibited significantly reduced activity. In contrast, stimulation of EPHA2 with its ligand ephrin-A5 eradicates the enhancement of cell migration accompanied by Akt activation. Taken together, our studies suggest that the SAM domain of the EPHA2 protein plays critical roles in enhancing the stability of EPHA2 by modulating the proteasome-dependent process. Furthermore, activation of Akt switches EPHA2 from promoting to inhibiting cell migration upon ephrin-A5 binding. Our results provide the first report of multiple EPHA2 cataract mutations contributing to the destabilization of the receptor and causing the loss of cell migration activity. PMID:22570727

  14. Structure of the human DNA-repair protein RAD52 containing surface mutations.

    PubMed

    Saotome, Mika; Saito, Kengo; Onodera, Keiichi; Kurumizaka, Hitoshi; Kagawa, Wataru

    2016-08-01

    The Rad52 protein is a eukaryotic single-strand DNA-annealing protein that is involved in the homologous recombinational repair of DNA double-strand breaks. The isolated N-terminal half of the human RAD52 protein (RAD52(1-212)) forms an undecameric ring structure with a surface that is mostly positively charged. In the present study, it was found that RAD52(1-212) containing alanine mutations of the charged surface residues (Lys102, Lys133 and Glu202) is highly amenable to crystallization. The structure of the mutant RAD52(1-212) was solved at 2.4 Å resolution. The structure revealed an association between the symmetry-related RAD52(1-212) rings, in which a partially unfolded, C-terminal region of RAD52 extended into the DNA-binding groove of the neighbouring ring in the crystal. The alanine mutations probably reduced the surface entropy of the RAD52(1-212) ring and stabilized the ring-ring association observed in the crystal.

  15. Clinical Significance of Epigenetic Alterations in Human Hepatocellular Carcinoma and Its Association with Genetic Mutations.

    PubMed

    Nishida, Naoshi; Kudo, Masatoshi

    Accumulation of genetic and epigenetic alterations is a hallmark of cancer genomes, including those in hepatocellular carcinoma (HCC). Particularly, in human HCC, epigenetic changes are more frequently observed than genetic changes in a variety of cancer-related genes, suggesting a potential role for epigenetic alterations during hepatocarcinogenesis. Several environmental factors, such as inflammation, obesity, and steatosis, are reported to affect the epigenetic status in hepatocytes, which could play a role in HCC development. In addition, genetic mutations in histone modulators and chromatin regulators would be critical for the acceleration of epigenetic alteration. It is also possible that major genetic mutations of HCC, such as TP53 and CNTTB1 mutations, are associated with the disturbance of epigenetic integrity. For example, specific TP53 mutations frequently induced by aflatoxin B1 exposure might affect histone modifiers and nucleosome remodelers. Generally, epigenetic alteration is reversible, because of which dysregulation of transcription takes place, without affecting protein structure. Therefore, differentiation therapy is one of the potential approaches for HCC with advanced epigenetic alterations. On the other hand, a tumor carrying an accumulation of genetic mutations would result in many abnormal proteins that could be recognized as non-self and could be targets for immune reactions; thus, immune-checkpoint blockers should be effective for HCCs with genetic hypermutation. Although the emergence of genetic and epigenetic alterations could be linked to each other and there could be some crossover or convergence between these cancer pathways, characterization of the mutation spectrum of genetic and epigenetic alterations could influence future HCC treatment. © 2016 S. Karger AG, Basel.

  16. Effects of track structure and cell inactivation on the calculation of heavy ion mutation rates in mammalian cells

    NASA Technical Reports Server (NTRS)

    Cucinotta, F. A.; Wilson, J. W.; Shavers, M. R.; Katz, R.

    1996-01-01

    It has long been suggested that inactivation severely effects the probability of mutation by heavy ions in mammalian cells. Heavy ions have observed cross sections of inactivation that approach and sometimes exceed the geometric size of the cell nucleus in mammalian cells. In the track structure model of Katz the inactivation cross section is found by summing an inactivation probability over all impact parameters from the ion to the sensitive sites within the cell nucleus. The inactivation probability is evaluated using the dose-response of the system to gamma-rays and the radial dose of the ions and may be equal to unity at small impact parameters for some ions. We show how the effects of inactivation may be taken into account in the evaluation of the mutation cross sections from heavy ions in the track structure model through correlation of sites for gene mutation and cell inactivation. The model is fit to available data for HPRT mutations in Chinese hamster cells and good agreement is found. The resulting calculations qualitatively show that mutation cross sections for heavy ions display minima at velocities where inactivation cross sections display maxima. Also, calculations show the high probability of mutation by relativistic heavy ions due to the radial extension of ions track from delta-rays in agreement with the microlesion concept. The effects of inactivation on mutations rates make it very unlikely that a single parameter such as LET or Z*2/beta(2) can be used to specify radiation quality for heavy ion bombardment.

  17. 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

  18. Estimation of pea (Pisum sativum L.) microsatellite mutation rate based on pedigree and single-seed descent analyses.

    PubMed

    Cieslarová, Jaroslava; Hanáček, Pavel; Fialová, Eva; Hýbl, Miroslav; Smýkal, Petr

    2011-11-01

    Microsatellites, or simple sequence repeats (SSRs) are widespread class of repetitive DNA sequences, used in population genetics, genetic diversity and mapping studies. In spite of the SSR utility, the genetic and evolutionary mechanisms are not fully understood. We have investigated three microsatellite loci with different position in the pea (Pisum sativum L.) genome, the A9 locus residing in LTR region of abundant retrotransposon, AD270 as intergenic and AF016458 located in 5'untranslated region of expressed gene. Comparative analysis of a 35 pair samples from seven pea varieties propagated by single-seed descent for ten generations, revealed single 4 bp mutation in 10th generation sample at AD270 locus corresponding to stepwise increase in one additional ATCT repeat unit. The estimated mutation rate was 4.76 × 10(-3) per locus per generation, with a 95% confidence interval of 1.2 × 10(-4) to 2.7 × 10(-2). The comparison of cv. Bohatýr accessions retrieved from different collections, showed intra-, inter-accession variation and differences in flanking and repeat sequences. Fragment size and sequence alternations were also found in long term in vitro organogenic culture, established at 1983, indicative of somatic mutation process. The evidence of homoplasy was detected across of unrelated pea genotypes, which adversaly affects the reliability of diversity estimates not only for diverse germplasm but also highly bred material. The findings of this study have important implications for Pisum phylogeny studies, variety identification and registration process in pea breeding where mutation rate influences the genetic diversity and the effective population size estimates.

  19. Identification of Lethal Mutations in Yeast Threonyl-tRNA Synthetase Revealing Critical Residues in Its Human Homolog*

    PubMed Central

    Ruan, Zhi-Rong; Fang, Zhi-Peng; Ye, Qing; Lei, Hui-Yan; Eriani, Gilbert; Zhou, Xiao-Long; Wang, En-Duo

    2015-01-01

    Aminoacyl-tRNA synthetases (aaRSs) are a group of ancient enzymes catalyzing aminoacylation and editing reactions for protein biosynthesis. Increasing evidence suggests that these critical enzymes are often associated with mammalian disorders. Therefore, complete determination of the enzymes functions is essential for informed diagnosis and treatment. Here, we show that a yeast knock-out strain for the threonyl-tRNA synthetase (ThrRS) gene is an excellent platform for such an investigation. Saccharomyces cerevisiae ThrRS has a unique modular structure containing four structural domains and a eukaryote-specific N-terminal extension. Using randomly mutated libraries of the ThrRS gene (thrS) and a genetic screen, a set of loss-of-function mutants were identified. The mutations affected the synthetic and editing activities and influenced the dimer interface. The results also highlighted the role of the N-terminal extension for enzymatic activity and protein stability. To gain insights into the pathological mechanisms induced by mutated aaRSs, we systematically introduced the loss-of-function mutations into the human cytoplasmic ThrRS gene. All mutations induced similar detrimental effects, showing that the yeast model could be used to study pathology-associated point mutations in mammalian aaRSs. PMID:25416776

  20. Human Rating Requirements for NASA's Constellation Program

    NASA Technical Reports Server (NTRS)

    Berdich, Debbie

    2008-01-01

    NASA s Constellation Program (CxP) will conduct a series of human space expeditions of increasing scope, starting with missions supporting the International Space Station and expanding to encompass the Moon and Mars. Although human-rating is an integral part of all CxP activities throughout their life cycle, NASA Procedural Requirements document NPR 8705.2B, Human-Rating Requirements (HRR) for Space Flight Systems, defines the additional processes, procedures, and requirements necessary to produce human-rated space systems that protect the safety of crew members and passengers on these NASA missions. In order to be in compliance with 8705.2B the CxP must show appropriate implementation or progression toward the HRR, or justification for an exception. Compliance includes an explanation of how the CxP intends to meet the HRR, analyses to be performed to determine implementation; and a matrix to trace the HRR to CxP requirements. The HRR requires the CxP to establish a human system integration team (HSIT), consisting of astronauts, mission operations personnel, training personnel, ground processing personnel, human factors personnel, and human engineering experts, with clearly defined authority, responsibility, and accountability to lead the human-system integration. For example, per the HRR the HSIT is involved in the evaluation of crew workload, human-in-the-loop usability evaluations, determining associated criteria, and in assessment of how these activities influenced system design. In essence, the HSIT is invaluable in CxP s ability to meet the three fundamental tenets of human rating: the process of designing, evaluating, and assuring that the total system can safely conduct the required human missions; the incorporation of design features and capabilities that accommodate human interaction with the system to enhance overall safety and mission success; and the incorporation of design features and capabilities to enable safe recovery of the crew from hazardous

  1. 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.

  2. How mutation affects evolutionary games on graphs

    PubMed Central

    Allen, Benjamin; Traulsen, Arne; Tarnita, Corina E.; Nowak, Martin A.

    2011-01-01

    Evolutionary dynamics are affected by population structure, mutation rates and update rules. Spatial or network structure facilitates the clustering of strategies, which represents a mechanism for the evolution of cooperation. Mutation dilutes this effect. Here we analyze how mutation influences evolutionary clustering on graphs. We introduce new mathematical methods to evolutionary game theory, specifically the analysis of coalescing random walks via generating functions. These techniques allow us to derive exact identity-by-descent (IBD) probabilities, which characterize spatial assortment on lattices and Cayley trees. From these IBD probabilities we obtain exact conditions for the evolution of cooperation and other game strategies, showing the dual effects of graph topology and mutation rate. High mutation rates diminish the clustering of cooperators, hindering their evolutionary success. Our model can represent either genetic evolution with mutation, or social imitation processes with random strategy exploration. PMID:21473871

  3. Kaplan-Meier Meets Chemical Kinetics: Intrinsic Rate of SOD1 Amyloidogenesis Decreased by Subset of ALS Mutations and Cannot Fully Explain Age of Disease Onset.

    PubMed

    Abdolvahabi, Alireza; Shi, Yunhua; Rasouli, Sanaz; Croom, Corbin M; Aliyan, Amir; Martí, Angel A; Shaw, Bryan F

    2017-06-21

    Over 150 mutations in SOD1 (superoxide dismutase-1) cause amyotrophic lateral sclerosis (ALS), presumably by accelerating SOD1 amyloidogenesis. Like many nucleation processes, SOD1 fibrillization is stochastic (in vitro), which inhibits the determination of aggregation rates (and obscures whether rates correlate with patient phenotypes). Here, we diverged from classical chemical kinetics and used Kaplan-Meier estimators to quantify the probability of apo-SOD1 fibrillization (in vitro) from ∼10 3 replicate amyloid assays of wild-type (WT) SOD1 and nine ALS variants. The probability of apo-SOD1 fibrillization (expressed as a Hazard ratio) is increased by certain ALS-linked SOD1 mutations but is decreased or remains unchanged by other mutations. Despite this diversity, Hazard ratios of fibrillization correlated linearly with (and for three mutants, approximately equaled) Hazard ratios of patient survival (R 2 = 0.67; Pearson's r = 0.82). No correlation exists between Hazard ratios of fibrillization and age of initial onset of ALS (R 2 = 0.09). Thus, Hazard ratios of fibrillization might explain rates of disease progression but not onset. Classical kinetic metrics of fibrillization, i.e., mean lag time and propagation rate, did not correlate as strongly with phenotype (and ALS mutations did not uniformly accelerate mean rate of nucleation or propagation). A strong correlation was found, however, between mean ThT fluorescence at lag time and patient survival (R 2 = 0.93); oligomers of SOD1 with weaker fluorescence correlated with shorter survival. This study suggests that SOD1 mutations trigger ALS by altering a property of SOD1 or its oligomers other than the intrinsic rate of amyloid nucleation (e.g., oligomer stability; rates of intercellular propagation; affinity for membrane surfaces; and maturation rate).

  4. A lack of Birbeck granules in Langerhans cells is associated with a naturally occurring point mutation in the human Langerin gene.

    PubMed

    Verdijk, Pauline; Dijkman, Remco; Plasmeijer, Elsemieke I; Mulder, Aat A; Zoutman, Willem H; Mieke Mommaas, A; Tensen, Cornelis P

    2005-04-01

    A heterozygous mutation in the Langerin gene corresponding to position 837 in the Langerin mRNA was identified in a person deficient in Birbeck granules (BG). This mutation results in an amino acid replacement of tryptophan by arginine at position 264 in the carbohydrate recognition domain of the Langerine protein. Expression of mutated Langerin in human fibroblasts induces tubular-like structures that are negative for BG-specific antibodies and do not resemble the characteristic structural features of BG.

  5. Cabozantinib Is Active against Human Gastrointestinal Stromal Tumor Xenografts Carrying Different KIT Mutations.

    PubMed

    Gebreyohannes, Yemarshet K; Schöffski, Patrick; Van Looy, Thomas; Wellens, Jasmien; Vreys, Lise; Cornillie, Jasmien; Vanleeuw, Ulla; Aftab, Dana T; Debiec-Rychter, Maria; Sciot, Raf; Wozniak, Agnieszka

    2016-12-01

    In the majority of gastrointestinal stromal tumors (GIST), oncogenic signaling is driven by KIT mutations. Advanced GIST is treated with tyrosine kinase inhibitors (TKI) such as imatinib. Acquired resistance to TKI is mainly caused by secondary KIT mutations, but can also be attributed to a switch of KIT dependency to another receptor tyrosine kinase (RTK). We tested the efficacy of cabozantinib, a novel TKI targeting KIT, MET, AXL, and vascular endothelial growth factor receptors (VEGFR), in patient-derived xenograft (PDX) models of GIST, carrying different KIT mutations. NMRI nu/nu mice (n = 52) were bilaterally transplanted with human GIST: UZLX-GIST4 (KIT exon 11 mutation, imatinib sensitive), UZLX-GIST2 (KIT exon 9, imatinib dose-dependent resistance), or UZLX-GIST9 (KIT exon 11 and 17 mutations, imatinib resistant). Mice were grouped as control (untreated), imatinib (50 mg/kg/bid), and cabozantinib (30 mg/kg/qd) and treated orally for 15 days. Cabozantinib resulted in significant tumor regression in UZLX-GIST4 and -GIST2 and delayed tumor growth in -GIST9. In all three models, cabozantinib inhibited the proliferative activity, which was completely absent in UZLX-GIST4 and significantly reduced in -GIST2 and -GIST9. Increased apoptotic activity was observed only in UZLX-GIST4. Cabozantinib inhibited the KIT signaling pathway in UZLX-GIST4 and -GIST2. In addition, compared with both control and imatinib, cabozantinib significantly reduced microvessel density in all models. In conclusion, cabozantinib showed antitumor activity in GIST PDX models through inhibition of tumor growth, proliferation, and angiogenesis, in both imatinib-sensitive and imatinib-resistant models. Mol Cancer Ther; 15(12); 2845-52. ©2016 AACR. ©2016 American Association for Cancer Research.

  6. Density behavior of spatial birth-and-death stochastic evolution of mutating genotypes under selection rates

    NASA Astrophysics Data System (ADS)

    Finkelshtein, D.; Kondratiev, Yu.; Kutoviy, O.; Molchanov, S.; Zhizhina, E.

    2014-10-01

    We consider birth-and-death stochastic evolution of genotypes with different lengths. The genotypes might mutate, which provides a stochastic changing of lengths by a free diffusion law. The birth and death rates are length dependent, which corresponds to a selection effect. We study an asymptotic behavior of a density for an infinite collection of genotypes. The cases of space homogeneous and space heterogeneous densities are considered.

  7. ESA Human rating Requirements:Status

    NASA Astrophysics Data System (ADS)

    Trujillo, M.; Sgobba, T.

    2012-01-01

    The European Space Agency (ESA) human rating safety requirements are based on heritage requirements of the International Space Station as well as the knowledge and experience derived from European participation on international partnerships. This expertise in conjunction with recommendations derived from past accidents (i.e.: Columbia) and lessons learned have led to the identification of m inimum core safety tech nical requirements for hum an rated space syst ems. These requirements apply to th e crewed space vehicle, integrated space system (i.e.: cre wed vehicle on its launcher) and its interfaces with control centres, la unch pad, etc. In 2009, a first draft was issued. Then, in the summer of 2010, ESA established a working group comprised of more than twenty experts (from disciplines including propulsion, pyrotechnics, structures, avionics, human factors and life support among others) across the Agency to review this draft. This paper provides an overview of ESA "Safety technical re quirements for human rated s pace systems" document, its scope a nd structure, as well as the planned steps for verification of these requirements in term s of achieving the identified safety objectives for crew safety in t erms of a quantitative risk evaluation.

  8. Mutation of SALL2 causes recessive ocular coloboma in humans and mice

    PubMed Central

    Kelberman, Daniel; Islam, Lily; Lakowski, Jörn; Bacchelli, Chiara; Chanudet, Estelle; Lescai, Francesco; Patel, Aara; Stupka, Elia; Buck, Anja; Wolf, Stephan; Beales, Philip L.; Jacques, Thomas S.; Bitner-Glindzicz, Maria; Liasis, Alki; Lehmann, Ordan J.; Kohlhase, Jürgen; Nischal, Ken K.; Sowden, Jane C.

    2014-01-01

    Ocular coloboma is a congenital defect resulting from failure of normal closure of the optic fissure during embryonic eye development. This birth defect causes childhood blindness worldwide, yet the genetic etiology is poorly understood. Here, we identified a novel homozygous mutation in the SALL2 gene in members of a consanguineous family affected with non-syndromic ocular coloboma variably affecting the iris and retina. This mutation, c.85G>T, introduces a premature termination codon (p.Glu29*) predicted to truncate the SALL2 protein so that it lacks three clusters of zinc-finger motifs that are essential for DNA-binding activity. This discovery identifies SALL2 as the third member of the Drosophila homeotic Spalt-like family of developmental transcription factor genes implicated in human disease. SALL2 is expressed in the developing human retina at the time of, and subsequent to, optic fissure closure. Analysis of Sall2-deficient mouse embryos revealed delayed apposition of the optic fissure margins and the persistence of an anterior retinal coloboma phenotype after birth. Sall2-deficient embryos displayed correct posterior closure toward the optic nerve head, and upon contact of the fissure margins, dissolution of the basal lamina occurred and PAX2, known to be critical for this process, was expressed normally. Anterior closure was disrupted with the fissure margins failing to meet, or in some cases misaligning leading to a retinal lesion. These observations demonstrate, for the first time, a role for SALL2 in eye morphogenesis and that loss of function of the gene causes ocular coloboma in humans and mice. PMID:24412933

  9. Clustering of Alpers disease mutations and catalytic defects in biochemical variants reveal new features of molecular mechanism of the human mitochondrial replicase, Pol γ

    PubMed Central

    Euro, Liliya; Farnum, Gregory A.; Palin, Eino; Suomalainen, Anu; Kaguni, Laurie S.

    2011-01-01

    Mutations in Pol γ represent a major cause of human mitochondrial diseases, especially those affecting the nervous system in adults and in children. Recessive mutations in Pol γ represent nearly half of those reported to date, and they are nearly uniformly distributed along the length of the POLG1 gene (Human DNA Polymerase gamma Mutation Database); the majority of them are linked to the most severe form of POLG syndrome, Alpers–Huttenlocher syndrome. In this report, we assess the structure–function relationships for recessive disease mutations by reviewing existing biochemical data on site-directed mutagenesis of the human, Drosophila and yeast Pol γs, and their homologs from the family A DNA polymerase group. We do so in the context of a molecular model of Pol γ in complex with primer–template DNA, which we have developed based upon the recently solved crystal structure of the apoenzyme form. We present evidence that recessive mutations cluster within five distinct functional modules in the catalytic core of Pol γ. Our results suggest that cluster prediction can be used as a diagnosis-supporting tool to evaluate the pathogenic role of new Pol γ variants. PMID:21824913

  10. The human Cx26-D50A and Cx26-A88V mutations causing keratitis-ichthyosis-deafness syndrome display increased hemichannel activity

    PubMed Central

    Mhaske, Pallavi V.; Levit, Noah A.; Li, Leping; Wang, Hong-Zhan; Lee, Jack R.; Shuja, Zunaira; Brink, Peter R.

    2013-01-01

    Mutations in the human gene encoding connexin 26 (Cx26 or GJB2) cause either nonsyndromic deafness or syndromic deafness associated with skin diseases. That distinct clinical disorders can be caused by different mutations within the same gene suggests that different channel activities influence the ear and skin. Here we use three different expression systems to examine the functional characteristics of two Cx26 mutations causing either mild (Cx26-D50A) or lethal (Cx26-A88V) keratitis-ichthyosis-deafness (KID) syndrome. In either cRNA-injected Xenopus oocytes, transfected HeLa cells, or transfected primary human keratinocytes, we show that both Cx26-D50A and Cx26-A88V form active hemichannels that significantly increase membrane current flow compared with wild-type Cx26. This increased membrane current accelerated cell death in low extracellular calcium solutions and was not due to increased mutant protein expression. Elevated mutant hemichannel currents could be blocked by increased extracellular calcium concentration. These results show that these two mutations exhibit a shared gain of functional activity and support the hypothesis that increased hemichannel activity is a common feature of human Cx26 mutations responsible for KID syndrome. PMID:23447037

  11. A Foxp2 Mutation Implicated in Human Speech Deficits Alters Sequencing of Ultrasonic Vocalizations in Adult Male Mice

    PubMed Central

    Chabout, Jonathan; Sarkar, Abhra; Patel, Sheel R.; Radden, Taylor; Dunson, David B.; Fisher, Simon E.; Jarvis, Erich D.

    2016-01-01

    Development of proficient spoken language skills is disrupted by mutations of the FOXP2 transcription factor. A heterozygous missense mutation in the KE family causes speech apraxia, involving difficulty producing words with complex learned sequences of syllables. Manipulations in songbirds have helped to elucidate the role of this gene in vocal learning, but findings in non-human mammals have been limited or inconclusive. Here, we performed a systematic study of ultrasonic vocalizations (USVs) of adult male mice carrying the KE family mutation. Using novel statistical tools, we found that Foxp2 heterozygous mice did not have detectable changes in USV syllable acoustic structure, but produced shorter sequences and did not shift to more complex syntax in social contexts where wildtype animals did. Heterozygous mice also displayed a shift in the position of their rudimentary laryngeal motor cortex (LMC) layer-5 neurons. Our findings indicate that although mouse USVs are mostly innate, the underlying contributions of FoxP2 to sequencing of vocalizations are conserved with humans. PMID:27812326

  12. Probing the changes in gene expression due to α-crystallin mutations in mouse models of hereditary human cataract.

    PubMed

    Andley, Usha P; Tycksen, Eric; McGlasson-Naumann, Brittney N; Hamilton, Paul D

    2018-01-01

    The mammalian eye lens expresses a high concentration of crystallins (α, β and γ-crystallins) to maintain the refractive index essential for lens transparency. Crystallins are long-lived proteins that do not turnover throughout life. The structural destabilization of crystallins by UV exposure, glycation, oxidative stress and mutations in crystallin genes leads to protein aggregation and development of cataracts. Several destabilizing mutations in crystallin genes are linked with human autosomal dominant hereditary cataracts. To investigate the mechanism by which the α-crystallin mutations Cryaa-R49C and Cryab-R120G lead to cataract formation, we determined whether these mutations cause an altered expression of specific transcripts in the lens at an early postnatal age by RNA-seq analysis. Using knock-in mouse models previously generated in our laboratory, in the present work, we identified genes that exhibited altered abundance in the mutant lenses, including decreased transcripts for Clic5, an intracellular water channel in Cryaa-R49C heterozygous mutant lenses, and increased transcripts for Eno1b in Cryab-R120G heterozygous mutant lenses. In addition, RNA-seq analysis revealed increased histones H2B, H2A, and H4 gene expression in Cryaa-R49C mutant lenses, suggesting that the αA-crystallin mutation regulates histone expression via a transcriptional mechanism. Additionally, these studies confirmed the increased expression of histones H2B, H2A, and H4 by proteomic analysis of Cryaa-R49C knock-in and Cryaa;Cryab gene knockout lenses reported previously. Taken together, these findings offer additional insight into the early transcriptional changes caused by Cryaa and Cryab mutations associated with autosomal dominant human cataracts, and indicate that the transcript levels of certain genes are affected by the expression of mutant α-crystallin in vivo.

  13. 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.

  14. Mutations in the PP2A regulatory subunit B family genes PPP2R5B, PPP2R5C and PPP2R5D cause human overgrowth.

    PubMed

    Loveday, Chey; Tatton-Brown, Katrina; Clarke, Matthew; Westwood, Isaac; Renwick, Anthony; Ramsay, Emma; Nemeth, Andrea; Campbell, Jennifer; Joss, Shelagh; Gardner, McKinlay; Zachariou, Anna; Elliott, Anna; Ruark, Elise; van Montfort, Rob; Rahman, Nazneen

    2015-09-01

    Overgrowth syndromes comprise a group of heterogeneous disorders characterised by excessive growth parameters, often in association with intellectual disability. To identify new causes of human overgrowth, we have been undertaking trio-based exome sequencing studies in overgrowth patients and their unaffected parents. Prioritisation of functionally relevant genes with multiple unique de novo mutations revealed four mutations in protein phosphatase 2A (PP2A) regulatory subunit B family genes protein phosphatase 2, regulatory Subunit B', beta (PPP2R5B); protein phosphatase 2, regulatory Subunit B', gamma (PPP2R5C); and protein phosphatase 2, regulatory Subunit B', delta (PPP2R5D). This observation in 3 related genes in 111 individuals with a similar phenotype is greatly in excess of the expected number, as determined from gene-specific de novo mutation rates (P = 1.43 × 10(-10)). Analysis of exome-sequencing data from a follow-up series of overgrowth probands identified a further pathogenic mutation, bringing the total number of affected individuals to 5. Heterozygotes shared similar phenotypic features including increased height, increased head circumference and intellectual disability. The mutations clustered within a region of nine amino acid residues in the aligned protein sequences (P = 1.6 × 10(-5)). We mapped the mutations onto the crystal structure of the PP2A holoenzyme complex to predict their molecular and functional consequences. These studies suggest that the mutations may affect substrate binding, thus perturbing the ability of PP2A to dephosphorylate particular protein substrates. PP2A is a major negative regulator of v-akt murine thymoma viral oncogene homolog 1 (AKT). Thus, our data further expand the list of genes encoding components of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT signalling cascade that are disrupted in human overgrowth conditions. © The Author 2015. Published by Oxford University Press.

  15. Mutation in TDRD9 causes non-obstructive azoospermia in infertile men.

    PubMed

    Arafat, Maram; Har-Vardi, Iris; Harlev, Avi; Levitas, Eliahu; Zeadna, Atif; Abofoul-Azab, Maram; Dyomin, Victor; Sheffield, Val C; Lunenfeld, Eitan; Huleihel, Mahmoud; Parvari, Ruti

    2017-09-01

    Azoospermia is diagnosed when sperm cells are completely absent in the ejaculate even after centrifugation. It is identified in approximately 1% of all men and in 10%-20% of infertile males. Non-obstructive azoospermia (NOA) is characterised by the absence of sperm due to either a Sertoli cell-only pattern, maturation arrest, hypospermatogenesis or mixed patterns. NOA is a severe form of male infertility, with limited treatment options and low fertility success rates. In the majority of patients, the cause for NOA is not known and mutations in only a few genes were shown to be causative. We investigated the cause of maturation arrest in five azoospermic infertile men of a large consanguineous Bedouin family. Using whole genome genotyping and exome sequencing we identified a 4 bp deletion frameshift mutation in TDRD9 as the causative mutation with a Lod Score of 3.42. We demonstrate that the mutation results in a frameshift as well as exon skipping. Immunofluorescent staining with anti-TDRD9 antibody directed towards the N terminus demonstrated the presence of the protein in testicular biopsies of patients with an intracellular distribution comparable to a control biopsy. The mutation does not cause female infertility. This is the first report of a recessive deleterious mutation in TDRD9 in humans. The clinical phenotype recapitulates that observed in the Tdrd9 knockout mice where this gene was demonstrated to participate in long interspersed element-1 retrotransposon silencing. If this function is preserved in human, our data underscore the importance of maintaining DNA stability in the human male germ line. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  16. Methylation, expression, and mutation analysis of the cell cycle control genes in human brain tumors.

    PubMed

    Yin, Dong; Xie, Dong; Hofmann, Wolf-Karsten; Miller, Carl W; Black, Keith L; Koeffler, H Phillip

    2002-11-28

    Methylation status of the p15(INK4B), p16(INK4A), p14(ARF) and retinoblastoma (RB) genes was studied using methylation specific polymerase chain reaction (MSP) in 85 human brain tumors of various subtypes and four normal brain samples. These genes play an important role in the control of the cell cycle. Twenty-four out of 85 cases (28%) had at least one of these genes methylated. The frequency of p14(ARF) methylation was 15 out of 85 (18%) cases, and the expression of p14(ARF) in methylated gliomas was significantly lower than in unmethylated gliomas. The incidence of methylation of p15(INK4B), p16(INK4A) and RB gene was 4%, 7%, and 4%, respectively. Samples with p14(ARF) methylation did not have p16(INK4A) methylation even though both genes physically overlap. None of the target genes was methylated in the normal brain samples. In addition, the p53 gene was mutated in 19 out of 85 (22%) samples as determined by single strand conformation polymorphism (SSCP) analysis and DNA sequencing. Thirty out of 85 (35%) brain tumors had either a p53 mutation or methylation of p14(ARF). Also, the p14(ARF) expression in p53 wild-type gliomas was lower than levels in p53 mutated gliomas. This finding is consistent with wild-type p53 being able to autoregulate its levels by down-regulating expression of p14(ARF). In summary, inactivation of the apoptosis pathway that included the p14(ARF) and p53 genes by hypermethylation and mutation, respectively, occurred frequently in human brain tumors. Down-regulation of p14(ARF) in gliomas was associated with hypermethylation of its promoter and the presence of a wild-type p53 in these samples.

  17. 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.

  18. Age-Dependent Recombination Rates in Human Pedigrees

    PubMed Central

    Hussin, Julie; Roy-Gagnon, Marie-Hélène; Gendron, Roxanne; Andelfinger, Gregor; Awadalla, Philip

    2011-01-01

    In humans, chromosome-number abnormalities have been associated with altered recombination and increased maternal age. Therefore, age-related effects on recombination are of major importance, especially in relation to the mechanisms involved in human trisomies. Here, we examine the relationship between maternal age and recombination rate in humans. We localized crossovers at high resolution by using over 600,000 markers genotyped in a panel of 69 French-Canadian pedigrees, revealing recombination events in 195 maternal meioses. Overall, we observed the general patterns of variation in fine-scale recombination rates previously reported in humans. However, we make the first observation of a significant decrease in recombination rates with advancing maternal age in humans, likely driven by chromosome-specific effects. The effect appears to be localized in the middle section of chromosomal arms and near subtelomeric regions. We postulate that, for some chromosomes, protection against non-disjunction provided by recombination becomes less efficient with advancing maternal age, which can be partly responsible for the higher rates of aneuploidy in older women. We propose a model that reconciles our findings with reported associations between maternal age and recombination in cases of trisomies. PMID:21912527

  19. Mutations of human NARS2, encoding the mitochondrial asparaginyl-tRNA synthetase, cause nonsyndromic deafness and Leigh syndrome.

    PubMed

    Simon, Mariella; Richard, Elodie M; Wang, Xinjian; Shahzad, Mohsin; Huang, Vincent H; Qaiser, Tanveer A; Potluri, Prasanth; Mahl, Sarah E; Davila, Antonio; Nazli, Sabiha; Hancock, Saege; Yu, Margret; Gargus, Jay; Chang, Richard; Al-Sheqaih, Nada; Newman, William G; Abdenur, Jose; Starr, Arnold; Hegde, Rashmi; Dorn, Thomas; Busch, Anke; Park, Eddie; Wu, Jie; Schwenzer, Hagen; Flierl, Adrian; Florentz, Catherine; Sissler, Marie; Khan, Shaheen N; Li, Ronghua; Guan, Min-Xin; Friedman, Thomas B; Wu, Doris K; Procaccio, Vincent; Riazuddin, Sheikh; Wallace, Douglas C; Ahmed, Zubair M; Huang, Taosheng; Riazuddin, Saima

    2015-03-01

    Here we demonstrate association of variants in the mitochondrial asparaginyl-tRNA synthetase NARS2 with human hearing loss and Leigh syndrome. A homozygous missense mutation ([c.637G>T; p.Val213Phe]) is the underlying cause of nonsyndromic hearing loss (DFNB94) and compound heterozygous mutations ([c.969T>A; p.Tyr323*] + [c.1142A>G; p.Asn381Ser]) result in mitochondrial respiratory chain deficiency and Leigh syndrome, which is a neurodegenerative disease characterized by symmetric, bilateral lesions in the basal ganglia, thalamus, and brain stem. The severity of the genetic lesions and their effects on NARS2 protein structure cosegregate with the phenotype. A hypothetical truncated NARS2 protein, secondary to the Leigh syndrome mutation p.Tyr323* is not detectable and p.Asn381Ser further decreases NARS2 protein levels in patient fibroblasts. p.Asn381Ser also disrupts dimerization of NARS2, while the hearing loss p.Val213Phe variant has no effect on NARS2 oligomerization. Additionally we demonstrate decreased steady-state levels of mt-tRNAAsn in fibroblasts from the Leigh syndrome patients. In these cells we show that a decrease in oxygen consumption rates (OCR) and electron transport chain (ETC) activity can be rescued by overexpression of wild type NARS2. However, overexpression of the hearing loss associated p.Val213Phe mutant protein in these fibroblasts cannot complement the OCR and ETC defects. Our findings establish lesions in NARS2 as a new cause for nonsyndromic hearing loss and Leigh syndrome.

  20. Human Rating the Orion Parachute System

    NASA Technical Reports Server (NTRS)

    Machin, Ricardo A.; Fisher, Timothy E.; Evans, Carol T.; Stewart, Christine E.

    2011-01-01

    Human rating begins with design. Converging on the requirements and identifying the risks as early as possible in the design process is essential. Understanding of the interaction between the recovery system and the spacecraft will in large part dictate the achievable reliability of the final design. Component and complete system full-scale flight testing is critical to assure a realistic evaluation of the performance and reliability of the parachute system. However, because testing is so often difficult and expensive, comprehensive analysis of test results and correlation to accurate modeling completes the human rating process. The National Aeronautics and Space Administration (NASA) Orion program uses parachutes to stabilize and decelerate the Crew Exploration Vehicle (CEV) spacecraft during subsonic flight in order to deliver a safe water landing. This paper describes the approach that CEV Parachute Assembly System (CPAS) will take to human rate the parachute recovery system for the CEV.

  1. Identification of the structural mutation responsible for the dibucaine-resistant (atypical) variant form of human serum cholinesterase

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

    McGuire, M.C.; Nogueira, C.P.; Bartels, C.F.

    1989-02-01

    A point mutation in the gene for human serum cholinesterase was identified that changes Asp-70 to Gly in the atypical form of serum cholinesterase. The mutation in nucleotide 209, which changes codon 70 from GAT to GGT, was found by sequencing a genomic clone and sequencing selected regions of DNA amplified by the polymerase chain reaction. The entire coding sequences for usual and atypical cholinesterases were compared, and no other consistent base differences were found. The nucleotide-209 mutation was detected in all five atypical cholinesterase families examined. There was complete concordance between this mutation and serum cholinesterase phenotypes for allmore » 14 heterozygous and 6 homozygous atypical subjects tested. The mutation causes the loss of a Sau3A1 restriction site; the resulting DNA fragment length polymorphism was verified by electrophoresis of {sup 32}P-labeled DNA restriction fragments from usual and atypical subjects. Dot-blot hybridization analysis with a 19-mer allele-specific probe to the DNA amplified by the polymerase chain reaction distinguished between the usual and atypical genotypes. The authors conclude that the Asp-70 {yields} Gly mutation accounts for reduced affinity of atypical cholinesterase for choline esters and that Asp-70 must be an important component of the anionic site. Heterogeneity in atypical alleles may exist, but the Asp-70 point mutation may represent an appreciable portion of the atypical gene pool.« less

  2. Pathogenic mutations of the human mitochondrial citrate carrier SLC25A1 lead to impaired citrate export required for lipid, dolichol, ubiquinone and sterol synthesis.

    PubMed

    Majd, Homa; King, Martin S; Smith, Anthony C; Kunji, Edmund R S

    2018-01-01

    Missense mutations of the human mitochondrial citrate carrier, encoded by the SLC25A1 gene, lead to an autosomal recessive neurometabolic disorder characterised by neonatal-onset encephalopathy with severe muscular weakness, intractable seizures, respiratory distress, and lack of psychomotor development, often resulting in early death. Here, we have measured the effect of all twelve known pathogenic mutations on the transport activity. The results show that nine mutations abolish transport of citrate completely, whereas the other three reduce the transport rate by >70%, indicating that impaired citrate transport is the most likely primary cause of the disease. Some mutations may be detrimental to the structure of the carrier, whereas others may impair key functional elements, such as the substrate binding site and the salt bridge network on the matrix side of the carrier. To understand the consequences of impaired citrate transport on metabolism, the substrate specificity was also determined, showing that the human citrate carrier predominantly transports citrate, isocitrate, cis-aconitate, phosphoenolpyruvate and malate. Although D-2- and L-2 hydroxyglutaric aciduria is a metabolic hallmark of the disease, it is unlikely that the citrate carrier plays a significant role in the removal of hydroxyglutarate from the cytosol for oxidation to oxoglutarate in the mitochondrial matrix. In contrast, computer simulations of central metabolism predict that the export of citrate from the mitochondrion cannot be fully compensated by other pathways, restricting the cytosolic production of acetyl-CoA that is required for the synthesis of lipids, sterols, dolichols and ubiquinone, which in turn explains the severe disease phenotypes. Copyright © 2017. Published by Elsevier B.V.

  3. Characterization of Somatic Mutations in Air Pollution-Related Lung Cancer.

    PubMed

    Yu, Xian-Jun; Yang, Min-Jun; Zhou, Bo; Wang, Gui-Zhen; Huang, Yun-Chao; Wu, Li-Chuan; Cheng, Xin; Wen, Zhe-Sheng; Huang, Jin-Yan; Zhang, Yun-Dong; Gao, Xiao-Hong; Li, Gao-Feng; He, Shui-Wang; Gu, Zhao-Hui; Ma, Liang; Pan, Chun-Ming; Wang, Ping; Chen, Hao-Bin; Hong, Zhi-Peng; Wang, Xiao-Lu; Mao, Wen-Jing; Jin, Xiao-Long; Kang, Hui; Chen, Shu-Ting; Zhu, Yong-Qiang; Gu, Wen-Yi; Liu, Zi; Dong, Hui; Tian, Lin-Wei; Chen, Sai-Juan; Cao, Yi; Wang, Sheng-Yue; Zhou, Guang-Biao

    2015-06-01

    Air pollution has been classified as Group 1 carcinogenic to humans, but the underlying tumorigenesis remains unclear. In Xuanwei City of Yunnan Province, the lung cancer incidence is among the highest in China attributed to severe air pollution generated by combustion of smoky coal, providing a unique opportunity to dissect lung carcinogenesis of air pollution. Here we analyzed the somatic mutations of 164 non-small cell lung cancers (NSCLCs) from Xuanwei and control regions (CR) where smoky coal was not used. Whole genome sequencing revealed a mean of 289 somatic exonic mutations per tumor and the frequent C:G → A:T nucleotide substitutions in Xuanwei NSCLCs. Exome sequencing of 2010 genes showed that Xuanwei and CR NSCLCs had a mean of 68 and 22 mutated genes per tumor, respectively (p < 0.0001). We found 167 genes (including TP53, RYR2, KRAS, CACNA1E) which had significantly higher mutation frequencies in Xuanwei than CR patients, and mutations in most genes in Xuanwei NSCLCs differed from those in CR cases. The mutation rates of 70 genes (e.g., RYR2, MYH3, GPR144, CACNA1E) were associated with patients' lifetime benzo(a)pyrene exposure. This study uncovers the mutation spectrum of air pollution-related lung cancers, and provides evidence for pollution exposure-genomic mutation relationship at a large scale.

  4. 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.

  5. Frequency and spectrum of c-Ki-ras mutations in human sporadic colon carcinoma, carcinomas arising in ulcerative colitis, and pancreatic adenocarcinoma

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

    Burmer, G.C.; Rabinovitch, P.S.; Loeb, L.A.

    1991-06-01

    Sporadic colon carcinomas, carcinomas arising in chronic ulcerative colitis, and pancreatic adenocarcinomas have been analyzed for the presence of c-Ki-ras mutations by a combination of histological enrichment, cell sorting, polymerase chain reaction, and direct sequencing. Although 60% (37/61) of sporadic colon carcinomas contained mutations in codon 12, only 1 of 17 specimens of dysplasia or carcinoma from ulcerative colitis patients contained c-Ki-ras mutations, despite a high frequency of aneuploid tumors. In contrast, a higher percentage (16/20 = 80%) of pancreatic adenocarcinomas contained mutations in c-Ki-ras 2, despite a lower frequency of DNA aneuploidy in these neoplasms. Moreover, the spectrum ofmore » mutations differed between sporadic colon carcinoma, where the predominant mutation was a G to A transition, and pancreatic carcinomas, which predominantly contained G to C or T transversions. These results suggest that the etiology of ras mutations is different in these three human neoplasms.« less

  6. 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.

  7. Role of the uridine/cytidine kinase 2 mutation in cellular sensitiveness toward 3'-ethynylcytidine treatment of human cancer cells.

    PubMed

    Sato, Akira; Takano, Takeshi; Hiramoto, Akiko; Naito, Tomoharu; Matsuda, Akira; Fukushima, Masakazu; Wataya, Yusuke; Kim, Hye-Sook

    2017-08-01

    A nucleosidic medicine, 1-(3-C-ethynyl-β-D-ribo-pentofuranosyl)cytosine [3'-ethynylcytidine (ECyd)], is a potent inhibitor of RNA polymerase I and shows anticancer activity to various human solid tumors in vitro and in vivo. ECyd is phosphorylated to 3'-ethyntlcytidine 5'-monophosphate by uridine/cytidine kinase 2 (UCK2) and subsequently further to diphosphate and triphosphate (3'-ethyntlcytidine 5'-diphosphate, 3'-ethyntlcytidine 5'-triphosphate). 3'-Ethyntlcytidine 5'-triphosphate is an active metabolite that can inhibit RNA polymerase I competitively, causing cancer cell death. Here, to identify the UCK2 mutation for detecting responder or nonresponder to ECyd, we investigated the relationship between point mutation of the UCK2 gene and response to ECyd in various human solid tumors. We identified several functional point mutations including the splice-site mutation of the UCK2 gene IVS5+5 G>A. In addition, we found that the IVS5+5 G>A variant generates an aberrant mRNA transcript, namely, truncated mRNA was produced and normal mRNA levels were markedly decreased in the ECyd-resistant cancer cell line HT1080. We concluded that these findings strongly suggest that the IVS5+5 G>A variant would affect the expression level of the UCK2 transcript, resulting in decreased sensitivity to ECyd.

  8. [Breeding of transgenic mice expressing human tau isoform with P301L mutation and identification of homozygous transgenic mice].

    PubMed

    Wang, Yan-yan; Chen, Ru-zhui; Zhu, Xiao-nani; Liu, Jing; Li, Zhi-hui; Liu, Xiu-juan; Li, Zhi-hui; Na, Xin; Liang, Shan-shan; Qiu, Guo-guang; Zhang, Wei; Wang, Hai; Wang, Xue-lan

    2012-05-01

    To establish homozygous transgenic mouse strain expressing human tau isoform with P301L mutation. Five transgenic mice expressing human tau isoform with P301L mutation were obtained by microinjection into male nuclei. Homozygote and hemizygote were identified by PCR and real-time fluorescent quantitative PCR. Ninety five homozygous transgenic mice were selected, and the results indicated that homozygous transgenic mice were superior to hemizygote in simulating the changes of biological characteristics. Exogenous gene tau is able to stably transmit to next generation and the combination of SYBR Green real-time fluorescent quantitative PCR with the traditional mating is a fast, reliable and economical way to screen homozygous and hemizygous transgenic mice.

  9. 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.

  10. A consistent two-mutation model of bone cancer for two data sets of radium-injected beagles.

    PubMed

    Bijwaard, H; Brugmans, M J P; Leenhouts, H P

    2002-09-01

    A two-mutation carcinogenesis model has been applied to model osteosarcoma incidence in two data sets of beagles injected with 226Ra. Taking age-specific retention into account, the following results have been obtained: (1) a consistent and well-fitting solution for all age and dose groups, (2) mutation rates that are linearly dependent on dose rate, with an exponential decrease for the second mutation at high dose rates, (3) a linear-quadratic dose-effect relationship, which indicates that care should be taken when extrapolating linearly, (4) highest cumulative incidences for injection at young adult age, and highest risks for injection doses of a few kBq kg(-1) at these ages, and (5) when scaled appropriately, the beagle model compares fairly well with a description for radium dial painters, suggesting that a consistent model description of bone cancer induction in beagles and humans may be possible.

  11. Repair of naturally occurring mismatches can induce mutations in flanking DNA

    PubMed Central

    Chen, Jia; Miller, Brendan F; Furano, Anthony V

    2014-01-01

    ‘Normal’ genomic DNA contains hundreds of mismatches that are generated daily by the spontaneous deamination of C (U/G) and methyl-C (T/G). Thus, a mutagenic effect of their repair could constitute a serious genetic burden. We show here that while mismatches introduced into human cells on an SV40-based episome were invariably repaired, this process induced mutations in flanking DNA at a significantly higher rate than no mismatch controls. Most mutations involved the C of TpC, the substrate of some single strand-specific APOBEC cytidine deaminases, similar to the mutations that can typify the ‘mutator phenotype’ of numerous tumors. siRNA knockdowns and chromatin immunoprecipitation showed that TpC preferring APOBECs mediate the mutagenesis, and siRNA knockdowns showed that both the base excision and mismatch repair pathways are involved. That naturally occurring mispairs can be converted to mutators, represents an heretofore unsuspected source of genetic changes that could underlie disease, aging, and evolutionary change. DOI: http://dx.doi.org/10.7554/eLife.02001.001 PMID:24843013

  12. Monitoring humans for somatic mutation in the endogenous PIG-a gene using red blood cells.

    PubMed

    Dobrovolsky, Vasily N; Elespuru, Rosalie K; Bigger, C Anita H; Robison, Timothy W; Heflich, Robert H

    2011-12-01

    The endogenous X-linked PIG-A gene is involved in the synthesis of glycosyl phosphatidyl inositol (GPI) anchors that tether specific protein markers to the exterior of mammalian cell cytoplasmic membranes. Earlier studies in rodent models indicate that Pig-a mutant red blood cells (RBCs) can be induced in animals treated with genotoxic agents, and that flow cytometry can be used to identify rare RBCs deficient in the GPI-anchored protein, CD59, as a marker of Pig-a gene mutation. We investigated if a similar approach could be used for detecting gene mutation in humans. We first determined the frequency of spontaneous CD59-deficient RBCs (presumed PIG-A mutants) in 97 self-identified healthy volunteers. For most subjects, the frequency of CD59-deficient RBCs was low (average of 5.1 ± 4.9 × 10(-6) ; median of 3.8 × 10(-6) and mutant frequency less than 8 × 10(-6) for 75% of subjects), with a statistically significant difference in median mutant frequencies between males and females. PIG-A RBC mutant frequency displayed poor correlation with the age and no correlation with the smoking status of the subjects. Also, two individuals had markedly increased CD59-deficient RBC frequencies of ∼300 × 10(-6) and ∼100 × 10(-6) . We then monitored PIG-A mutation in 10 newly diagnosed cancer patients undergoing chemotherapy with known genotoxic drugs. The frequency of CD59-deficient RBCs in the blood of the patients was measured before the start of chemotherapy and three times over a period of ∼6 months while on/after chemotherapy. Responses were generally weak, most observations being less than the median mutant frequency for both males and females; the greatest response was an approximate three-fold increase in the frequency of CD59-deficient RBCs in one patient treated with a combination of cisplatin and etoposide. These results suggest that the RBC PIG-A assay can be adopted to measuring somatic cell mutation in humans. Further research is necessary to determine

  13. Development of a human live attenuated West Nile infectious DNA vaccine: Identification of a minimal mutation set conferring the attenuation level acceptable for a human vaccine

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

    Yamshchikov, Vladimir, E-mail: yaximik@gmail.com

    ABSTRACT: For the development of a human West Nile (WN) infectious DNA (iDNA) vaccine, we created highly attenuated chimeric virus W1806 with the serological identity of highly virulent WN-NY99. Earlier, we attempted to utilize mutations found in the E protein of the SA14-14-2 vaccine to bring safety of W1806 to the level acceptable for human use (). Here, we analyzed effects of the SA14-14-2 changes on growth properties and neurovirulence of W1806. A set including the E138K, K279M, K439R and G447D changes was identified as the perspective subset for satisfying the target safety profile without compromising immunogenicity of the vaccinemore » candidate. The genetic stability of the attenuated phenotype was found to be unsatisfactory being dependent on a subset of attenuating changes incorporated in W1806. Elucidation of underlying mechanisms influencing selection of pathways for restoration of the envelope protein functionality will facilitate resolution of the emerged genetic stability issue. - Highlights: •Effect of mutations in E on properties of WN1806 is determined. •A subset of attenuating mutations suitable for a human vaccine is defined. •Mechanism of attenuation is proposed and illustrated. •Underlying mechanisms of neurovirulence reversion are suggested.« less

  14. Disease-associated mitochondrial mutations and the evolution of primate mitogenomes

    PubMed Central

    Tavares, William Corrêa

    2017-01-01

    Several human diseases have been associated with mutations in mitochondrial genes comprising a set of confirmed and reported mutations according to the MITOMAP database. An analysis of complete mitogenomes across 139 primate species showed that most confirmed disease-associated mutations occurred in aligned codon positions and gene regions under strong purifying selection resulting in a strong evolutionary conservation. Only two confirmed variants (7.1%), coding for the same amino acids accounting for severe human diseases, were identified without apparent pathogenicity in non-human primates, like the closely related Bornean orangutan. Conversely, reported disease-associated mutations were not especially concentrated in conserved codon positions, and a large fraction of them occurred in highly variable ones. Additionally, 88 (45.8%) of reported mutations showed similar variants in several non-human primates and some of them have been present in extinct species of the genus Homo. Considering that recurrent mutations leading to persistent variants throughout the evolutionary diversification of primates are less likely to be severely damaging to fitness, we suggest that these 88 mutations are less likely to be pathogenic. Conversely, 69 (35.9%) of reported disease-associated mutations occurred in extremely conserved aligned codon positions which makes them more likely to damage the primate mitochondrial physiology. PMID:28510580

  15. Biallelic CHP1 mutation causes human autosomal recessive ataxia by impairing NHE1 function

    PubMed Central

    Mendoza-Ferreira, Natalia; Coutelier, Marie; Janzen, Eva; Hosseinibarkooie, Seyyedmohsen; Löhr, Heiko; Schneider, Svenja; Milbradt, Janine; Karakaya, Mert; Riessland, Markus; Pichlo, Christian; Torres-Benito, Laura; Singleton, Andrew; Zuchner, Stephan; Brice, Alexis; Durr, Alexandra; Hammerschmidt, Matthias; Stevanin, Giovanni

    2018-01-01

    Objective: To ascertain the genetic and functional basis of complex autosomal recessive cerebellar ataxia (ARCA) presented by 2 siblings of a consanguineous family characterized by motor neuropathy, cerebellar atrophy, spastic paraparesis, intellectual disability, and slow ocular saccades. Methods: Combined whole-genome linkage analysis, whole-exome sequencing, and focused screening for identification of potential causative genes were performed. Assessment of the functional consequences of the mutation on protein function via subcellular fractionation, size-exclusion chromatography, and fluorescence microscopy were done. A zebrafish model, using Morpholinos, was generated to study the pathogenic effect of the mutation in vivo. Results: We identified a biallelic 3-bp deletion (p.K19del) in CHP1 that cosegregates with the disease. Neither focused screening for CHP1 variants in 2 cohorts (ARCA: N = 319 and NeurOmics: N = 657) nor interrogating GeneMatcher yielded additional variants, thus revealing the scarcity of CHP1 mutations. We show that mutant CHP1 fails to integrate into functional protein complexes and is prone to aggregation, thereby leading to diminished levels of soluble CHP1 and reduced membrane targeting of NHE1, a major Na+/H+ exchanger implicated in syndromic ataxia-deafness. Chp1 deficiency in zebrafish, resembling the affected individuals, led to movement defects, cerebellar hypoplasia, and motor axon abnormalities, which were ameliorated by coinjection with wild-type, but not mutant, human CHP1 messenger RNA. Conclusions: Collectively, our results identified CHP1 as a novel ataxia-causative gene in humans, further expanding the spectrum of ARCA-associated loci, and corroborated the crucial role of NHE1 within the pathogenesis of these disorders. PMID:29379881

  16. 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

  17. 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.

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

    DTIC Science & Technology

    2016-12-24

    Te, and Michael Lynch Department of Biology , Indiana University, Bloomington, IN *Corresponding author: E-mail: longhongan@gmail.com. Accepted...GBE The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access...fungal mutation spectrum. Supplementary Material Supplementary data are available at Genome Biology and Evolution online. Acknowledgments This research

  19. Explosive mutation accumulation triggered by heterozygous human Pol ε proofreading-deficiency is driven by suppression of mismatch repair

    PubMed Central

    Campbell, Brittany B; Ungerleider, Nathan; Light, Nicholas; Wu, Tong; LeCompte, Kimberly G; Goksenin, A Yasemin; Bunnell, Bruce A; Tabori, Uri; Shlien, Adam

    2018-01-01

    Tumors defective for DNA polymerase (Pol) ε proofreading have the highest tumor mutation burden identified. A major unanswered question is whether loss of Pol ε proofreading by itself is sufficient to drive this mutagenesis, or whether additional factors are necessary. To address this, we used a combination of next generation sequencing and in vitro biochemistry on human cell lines engineered to have defects in Pol ε proofreading and mismatch repair. Absent mismatch repair, monoallelic Pol ε proofreading deficiency caused a rapid increase in a unique mutation signature, similar to that observed in tumors from patients with biallelic mismatch repair deficiency and heterozygous Pol ε mutations. Restoring mismatch repair was sufficient to suppress the explosive mutation accumulation. These results strongly suggest that concomitant suppression of mismatch repair, a hallmark of colorectal and other aggressive cancers, is a critical force for driving the explosive mutagenesis seen in tumors expressing exonuclease-deficient Pol ε. PMID:29488881

  20. Explosive mutation accumulation triggered by heterozygous human Pol ε proofreading-deficiency is driven by suppression of mismatch repair.

    PubMed

    Hodel, Karl P; de Borja, Richard; Henninger, Erin E; Campbell, Brittany B; Ungerleider, Nathan; Light, Nicholas; Wu, Tong; LeCompte, Kimberly G; Goksenin, A Yasemin; Bunnell, Bruce A; Tabori, Uri; Shlien, Adam; Pursell, Zachary F

    2018-02-28

    Tumors defective for DNA polymerase (Pol) ε proofreading have the highest tumor mutation burden identified. A major unanswered question is whether loss of Pol ε proofreading by itself is sufficient to drive this mutagenesis, or whether additional factors are necessary. To address this, we used a combination of next generation sequencing and in vitro biochemistry on human cell lines engineered to have defects in Pol ε proofreading and mismatch repair. Absent mismatch repair, monoallelic Pol ε proofreading deficiency caused a rapid increase in a unique mutation signature, similar to that observed in tumors from patients with biallelic mismatch repair deficiency and heterozygous Pol ε mutations. Restoring mismatch repair was sufficient to suppress the explosive mutation accumulation. These results strongly suggest that concomitant suppression of mismatch repair, a hallmark of colorectal and other aggressive cancers, is a critical force for driving the explosive mutagenesis seen in tumors expressing exonuclease-deficient Pol ε. © 2018, Hodel et al.

  1. Identification of the structural mutation responsible for the dibucaine-resistant (atypical) variant form of human serum cholinesterase.

    PubMed Central

    McGuire, M C; Nogueira, C P; Bartels, C F; Lightstone, H; Hajra, A; Van der Spek, A F; Lockridge, O; La Du, B N

    1989-01-01

    A point mutation in the gene for human serum cholinesterase was identified that changes Asp-70 to Gly in the atypical form of serum cholinesterase. The mutation in nucleotide 209, which changes codon 70 from GAT to GGT, was found by sequencing a genomic clone and sequencing selected regions of DNA amplified by the polymerase chain reaction. The entire coding sequences for usual and atypical cholinesterases were compared, and no other consistent base differences were found. A polymorphic site near the C terminus of the coded region was detected, but neither allele at this locus segregated consistently with the atypical trait. The nucleotide-209 mutation was detected in all five atypical cholinesterase families examined. There was complete concordance between this mutation and serum cholinesterase phenotypes for all 14 heterozygous and 6 homozygous atypical subjects tested. The mutation causes the loss of a Sau3A1 restriction site; the resulting DNA fragment length polymorphism was verified by electrophoresis of 32P-labeled DNA restriction fragments from usual and atypical subjects. Dot-blot hybridization analysis with a 19-mer allele-specific probe to the DNA amplified by the polymerase chain reaction distinguished between the usual and atypical genotypes. We conclude that the Asp-70----Gly mutation (acidic to neutral amino acid substitution) accounts for reduced affinity of atypical cholinesterase for choline esters and that Asp-70 must be an important component of the anionic site. Heterogeneity in atypical alleles may exist, but the Asp-70 point mutation may represent an appreciable portion of the atypical gene pool. Images PMID:2915989

  2. Exon skipping and gene transfer restore dystrophin expression in human induced pluripotent stem cells-cardiomyocytes harboring DMD mutations.

    PubMed

    Dick, Emily; Kalra, Spandan; Anderson, David; George, Vinoj; Ritso, Morten; Laval, Steven H; Barresi, Rita; Aartsma-Rus, Annemieke; Lochmüller, Hanns; Denning, Chris

    2013-10-15

    With an incidence of ∼1:3,500 to 5,000 in male children, Duchenne muscular dystrophy (DMD) is an X-linked disorder in which progressive muscle degeneration occurs and affected boys usually die in their twenties or thirties. Cardiac involvement occurs in 90% of patients and heart failure accounts for up to 40% of deaths. To enable new therapeutics such as gene therapy and exon skipping to be tested in human cardiomyocytes, we produced human induced pluripotent stem cells (hiPSC) from seven patients harboring mutations across the DMD gene. Mutations were retained during differentiation and analysis indicated the cardiomyocytes showed a dystrophic gene expression profile. Antisense oligonucleotide-mediated skipping of exon 51 restored dystrophin expression to ∼30% of normal levels in hiPSC-cardiomyocytes carrying exon 47-50 or 48-50 deletions. Alternatively, delivery of a dystrophin minigene to cardiomyocytes with a deletion in exon 35 or a point mutation in exon 70 allowed expression levels similar to those seen in healthy cells. This demonstrates that DMD hiPSC-cardiomyocytes provide a novel tool to evaluate whether new therapeutics can restore dystrophin expression in the heart.

  3. Exon Skipping and Gene Transfer Restore Dystrophin Expression in Human Induced Pluripotent Stem Cells-Cardiomyocytes Harboring DMD Mutations

    PubMed Central

    Dick, Emily; Kalra, Spandan; Anderson, David; George, Vinoj; Ritso, Morten; Laval, Steven H.; Barresi, Rita; Aartsma-Rus, Annemieke; Lochmüller, Hanns

    2013-01-01

    With an incidence of ∼1:3,500 to 5,000 in male children, Duchenne muscular dystrophy (DMD) is an X-linked disorder in which progressive muscle degeneration occurs and affected boys usually die in their twenties or thirties. Cardiac involvement occurs in 90% of patients and heart failure accounts for up to 40% of deaths. To enable new therapeutics such as gene therapy and exon skipping to be tested in human cardiomyocytes, we produced human induced pluripotent stem cells (hiPSC) from seven patients harboring mutations across the DMD gene. Mutations were retained during differentiation and analysis indicated the cardiomyocytes showed a dystrophic gene expression profile. Antisense oligonucleotide-mediated skipping of exon 51 restored dystrophin expression to ∼30% of normal levels in hiPSC-cardiomyocytes carrying exon 47–50 or 48–50 deletions. Alternatively, delivery of a dystrophin minigene to cardiomyocytes with a deletion in exon 35 or a point mutation in exon 70 allowed expression levels similar to those seen in healthy cells. This demonstrates that DMD hiPSC-cardiomyocytes provide a novel tool to evaluate whether new therapeutics can restore dystrophin expression in the heart. PMID:23829870

  4. Heterogeneous distribution of P53 immunoreactivity in human lung adenocarcinoma correlates with MDM2 protein expression, rather than with P53 gene mutation.

    PubMed

    Koga, T; Hashimoto, S; Sugio, K; Yoshino, I; Nakagawa, K; Yonemitsu, Y; Sugimachi, K; Sueishi, K

    2001-07-20

    Although the tumor suppressor p53 protein (P53) immunoreactivity and its gene (p53) mutation were reported to be significant prognostic indicators for human lung adenocarcinomas, little is known regarding the relationship between the heterogeneous distribution of P53 and its genetic status in each tumor focus and the clinicopathological significance. To determine how P53 is heterogeneously stabilized in patients, we compared P53 expression to both the p53 allelic mutation in exon 2 approximately 9 by polymerase chain reaction-single strand conformation polymorphism using microdissected DNA fractions, and the immunohistochemical MDM2 expression. Of the 48 positive to P53 in 118 lung adenocarcinomas examined, 10 with heterogeneous P53 expression were closely examined. The higher P53 expression foci in 7 of 10 cases were less differentiated, histologically in respective cases, and were frequently associated with fibrous stroma. Two had genetic mutations in exon 7 of the p53 gene in both the high and low P53 expression foci of cancer tissue indicating no apparent correlation between heterogeneous P53 expression and the occurrence of gene mutation. Immunohistochemical expression of MDM2 was significantly lower in high P53 expression areas (p < 0.05, the mean labeling indices of high and low P53 expression areas being 4.2 +/- 5.4% and 13.6 +/- 12.2%, respectively). In addition, among all the 118 cases examined, MDM2 expression was significantly suppressed in cases of p53 gene mutation, simultaneously with P53 overexpression, as compared with cases without both the p53 mutation and expression (p < 0.001). These findings suggest that the heterogeneous stabilization of P53 in human lung adenocarcinomas could be partly due to suppressed MDM2 expression. The overexpression of non-mutated P53 may afford a protective mechanism in human lung adenocarcinomas. Copyright 2001 Wiley-Liss, Inc.

  5. Ataxia telangiectasia mutated (ATM) modulates long interspersed element-1 (L1) retrotransposition in human neural stem cells

    PubMed Central

    Coufal, Nicole G.; Garcia-Perez, Josè Luis; Peng, Grace E.; Marchetto, Maria C. N.; Muotri, Alysson R.; Mu, Yangling; Carson, Christian T.; Macia, Angela; Moran, John V.; Gage, Fred H.

    2011-01-01

    Long interspersed element-1 (L1) retrotransposons compose ∼20% of the mammalian genome, and ongoing L1 retrotransposition events can impact genetic diversity by various mechanisms. Previous studies have demonstrated that endogenous L1 retrotransposition can occur in the germ line and during early embryonic development. In addition, recent data indicate that engineered human L1s can undergo somatic retrotransposition in human neural progenitor cells and that an increase in human-specific L1 DNA content can be detected in the brains of normal controls, as well as in Rett syndrome patients. Here, we demonstrate an increase in the retrotransposition efficiency of engineered human L1s in cells that lack or contain severely reduced levels of ataxia telangiectasia mutated, a serine/threonine kinase involved in DNA damage signaling and neurodegenerative disease. We demonstrate that the increase in L1 retrotransposition in ataxia telangiectasia mutated-deficient cells most likely occurs by conventional target-site primed reverse transcription and generate either longer, or perhaps more, L1 retrotransposition events per cell. Finally, we provide evidence suggesting an increase in human-specific L1 DNA copy number in postmortem brain tissue derived from ataxia telangiectasia patients compared with healthy controls. Together, these data suggest that cellular proteins involved in the DNA damage response may modulate L1 retrotransposition. PMID:22159035

  6. Long-Range Structural Effects of a Charcot-Marie-Tooth Disease-Causing Mutation in Human Glycyl-TRNA Synthetase

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

    Xie, W.; Nangle, L.A.; Zhang, W.

    2009-06-04

    Functional expansion of specific tRNA synthetases in higher organisms is well documented. These additional functions may explain why dominant mutations in glycyl-tRNA synthetase (GlyRS) and tyrosyl-tRNA synthetase cause Charcot-Marie-Tooth (CMT) disease, the most common heritable disease of the peripheral nervous system. At least 10 disease-causing mutant alleles of GlyRS have been annotated. These mutations scatter broadly across the primary sequence and have no apparent unifying connection. Here we report the structure of wild type and a CMT-causing mutant (G526R) of homodimeric human GlyRS. The mutation is at the site for synthesis of glycyl-adenylate, but the rest of the two structuresmore » are closely similar. Significantly, the mutant form diffracts to a higher resolution and has a greater dimer interface. The extra dimer interactions are located {approx}30 {angstrom} away from the G526R mutation. Direct experiments confirm the tighter dimer interaction of the G526R protein. The results suggest the possible importance of subtle, long-range structural effects of CMT-causing mutations at the dimer interface. From analysis of a third crystal, an appended motif, found in higher eukaryote GlyRSs, seems not to have a role in these long-range effects.« less

  7. A nonsense loss-of-function mutation in PCSK1 contributes to dominantly inherited human obesity.

    PubMed

    Philippe, J; Stijnen, P; Meyre, D; De Graeve, F; Thuillier, D; Delplanque, J; Gyapay, G; Sand, O; Creemers, J W; Froguel, P; Bonnefond, A

    2015-02-01

    A significant proportion of severe familial forms of obesity remain genetically elusive. Taking advantage of our unique cohort of multigenerational obese families, we aimed to assess the contribution of rare mutations in 29 common obesity-associated genes to familial obesity, and to evaluate in these families the putative presence of nine known monogenic forms of obesity. Through next-generation sequencing, we sequenced the coding regions of 34 genes involved in polygenic and/or monogenic forms of obesity in 201 participants (75 normal weight individuals, 54 overweight individuals and 72 individuals with obesity class I, II or III) from 13 French families. In vitro functional analyses were performed to investigate the mutation PCSK1-p.Arg80* which was identified in a family. A novel heterozygous nonsense variant in PCSK1 (p.Arg80*), encoding a propeptide truncated to less than two exons (out of 14), was found to co-segregate with obesity in a three-generation family. We demonstrated that this mutation inhibits PCSK1 enzyme activity and that this inhibition most likely does not involve a strong physical interaction. Furthermore, both mutations PCSK1-p.Asn180Ser and POMC-p.Phe144Leu, which had previously been reported to be associated with severe obesity, were also identified in this study, but did not co-segregate with obesity. Finally, we did not identify any rare mutations co-segregating with obesity in common obesity susceptibility genes, except for CADM2 and QPCTL, where we found two novel variants (p.Arg81His and p.Leu98Pro, respectively) in three obese individuals. We showed for the first time that a nonsense mutation in PCSK1 was likely to cause dominantly inherited human obesity, due to the inhibiting properties of the propeptide fragment encoded by the null allele. Furthermore, the present family sequencing design challenged the contribution of previously reported mutations to monogenic or at least severe obesity.

  8. Mutation detection using ENDO1: application to disease diagnostics in humans and TILLING and Eco-TILLING in plants.

    PubMed

    Triques, Karine; Piednoir, Elodie; Dalmais, Marion; Schmidt, Julien; Le Signor, Christine; Sharkey, Mark; Caboche, Michel; Sturbois, Bénédicte; Bendahmane, Abdelhafid

    2008-04-23

    Most enzymatic mutation detection methods are based on the cleavage of heteroduplex DNA by a mismatch-specific endonuclease at mismatch sites and the analysis of the digestion product on a DNA sequencer. Important limitations of these methods are the availability of a mismatch-specific endonuclease, their sensitivity in detecting one allele in pool of DNA, the cost of the analysis and the ease by which the technique could be implemented in a standard molecular biology laboratory. The co-agroinfiltration of ENDO1 and p19 constructs into N. benthamiana leaves allowed high level of transient expression of a mismatch-specific and sensitive endonuclease, ENDO1 from Arabidopsis thaliana. We demonstrate the broad range of uses of the produced enzyme in detection of mutations. In human, we report the diagnosis of the G1691A mutation in Leiden factor-V gene associated with venous thrombosis and the fingerprinting of HIV-1 quasispecies in patients subjected to antiretroviral treatments. In plants, we report the use of ENDO1 system for detection of mutant alleles of Retinoblastoma-related gene by TILLING in Pisum sativum and discovery of natural sequence variations by Eco-TILLING in Arabidopsis thaliana. We introduce a cost-effective tool based on a simplified purification protocol of a mismatch-specific and sensitive endonuclease, ENDO1. Especially, we report the successful applications of ENDO1 in mutation diagnostics in humans, fingerprinting of complex population of viruses, and in TILLING and Eco-TILLING in plants.

  9. TP53 mutation and human papilloma virus status of oral squamous cell carcinomas in young adult patients.

    PubMed

    Braakhuis, B J M; Rietbergen, M M; Buijze, M; Snijders, P J F; Bloemena, E; Brakenhoff, R H; Leemans, C R

    2014-09-01

    Little is known about the molecular carcinogenesis of oral squamous cell carcinoma (OSCC) in young adult patients. The aim of this study was to investigate the detailed TP53 mutation and human papilloma virus (HPV) status of OSCC in patients, younger than 45 years. TP53 mutations were determined with direct sequencing on paraffin-embedded carcinoma tissue from 31 young patients and compared with two older age OSCC reference groups: one from the same institute (N = 87) and an independent one (N = 675). Biologically active tumour HPV was detected by p16-immunohistochemistry followed by a HPV-DNA GP5 + /6 + -PCR. HPV16 was present in one OSCC (3%). TP53 mutations were found in 14 (45%) OSCC: five were missense and nine resulted in a truncated protein. Six of these latter were insertions or deletions of one or more nucleotides leading to frameshift, one was at a splice site and two resulted in a stop codon. The percentage of truncating mutations (64% of all mutations) was higher than that observed in the institute's reference group (44%, P = 0.23) and in the independent reference group (24%, P = 0.002). This study shows that TP53 mutations are common in OSCC of young adult patients; infection with biologically active HPV is rare. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. Divergence times in Caenorhabditis and Drosophila inferred from direct estimates of the neutral mutation rate.

    PubMed

    Cutter, Asher D

    2008-04-01

    Accurate inference of the dates of common ancestry among species forms a central problem in understanding the evolutionary history of organisms. Molecular estimates of divergence time rely on the molecular evolutionary prediction that neutral mutations and substitutions occur at the same constant rate in genomes of related species. This underlies the notion of a molecular clock. Most implementations of this idea depend on paleontological calibration to infer dates of common ancestry, but taxa with poor fossil records must rely on external, potentially inappropriate, calibration with distantly related species. The classic biological models Caenorhabditis and Drosophila are examples of such problem taxa. Here, I illustrate internal calibration in these groups with direct estimates of the mutation rate from contemporary populations that are corrected for interfering effects of selection on the assumption of neutrality of substitutions. Divergence times are inferred among 6 species each of Caenorhabditis and Drosophila, based on thousands of orthologous groups of genes. I propose that the 2 closest known species of Caenorhabditis shared a common ancestor <24 MYA (Caenorhabditis briggsae and Caenorhabditis sp. 5) and that Caenorhabditis elegans diverged from its closest known relatives <30 MYA, assuming that these species pass through at least 6 generations per year; these estimates are much more recent than reported previously with molecular clock calibrations from non-nematode phyla. Dates inferred for the common ancestor of Drosophila melanogaster and Drosophila simulans are roughly concordant with previous studies. These revised dates have important implications for rates of genome evolution and the origin of self-fertilization in Caenorhabditis.

  11. Mutation-selection equilibrium in games with multiple strategies.

    PubMed

    Antal, Tibor; Traulsen, Arne; Ohtsuki, Hisashi; Tarnita, Corina E; Nowak, Martin A

    2009-06-21

    In evolutionary games the fitness of individuals is not constant but depends on the relative abundance of the various strategies in the population. Here we study general games among n strategies in populations of large but finite size. We explore stochastic evolutionary dynamics under weak selection, but for any mutation rate. We analyze the frequency dependent Moran process in well-mixed populations, but almost identical results are found for the Wright-Fisher and Pairwise Comparison processes. Surprisingly simple conditions specify whether a strategy is more abundant on average than 1/n, or than another strategy, in the mutation-selection equilibrium. We find one condition that holds for low mutation rate and another condition that holds for high mutation rate. A linear combination of these two conditions holds for any mutation rate. Our results allow a complete characterization of nxn games in the limit of weak selection.

  12. SLITRK6 mutations cause myopia and deafness in humans and mice

    PubMed Central

    Tekin, Mustafa; Chioza, Barry A.; Matsumoto, Yoshifumi; Diaz-Horta, Oscar; Cross, Harold E.; Duman, Duygu; Kokotas, Haris; Moore-Barton, Heather L.; Sakoori, Kazuto; Ota, Maya; Odaka, Yuri S.; Foster, Joseph; Cengiz, F. Basak; Tokgoz-Yilmaz, Suna; Tekeli, Oya; Grigoriadou, Maria; Petersen, Michael B.; Sreekantan-Nair, Ajith; Gurtz, Kay; Xia, Xia-Juan; Pandya, Arti; Patton, Michael A.; Young, Juan I.; Aruga, Jun; Crosby, Andrew H.

    2013-01-01

    Myopia is by far the most common human eye disorder that is known to have a clear, albeit poorly defined, heritable component. In this study, we describe an autosomal-recessive syndrome characterized by high myopia and sensorineural deafness. Our molecular investigation in 3 families led to the identification of 3 homozygous nonsense mutations (p.R181X, p.S297X, and p.Q414X) in SLIT and NTRK-like family, member 6 (SLITRK6), a leucine-rich repeat domain transmembrane protein. All 3 mutant SLITRK6 proteins displayed defective cell surface localization. High-resolution MRI of WT and Slitrk6-deficient mouse eyes revealed axial length increase in the mutant (the endophenotype of myopia). Additionally, mutant mice exhibited auditory function deficits that mirrored the human phenotype. Histological investigation of WT and Slitrk6-deficient mouse retinas in postnatal development indicated a delay in synaptogenesis in Slitrk6-deficient animals. Taken together, our results showed that SLITRK6 plays a crucial role in the development of normal hearing as well as vision in humans and in mice and that its disruption leads to a syndrome characterized by severe myopia and deafness. PMID:23543054

  13. Mutations that Cause Human Disease: A Computational/Experimental Approach

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

    Beernink, P; Barsky, D; Pesavento, B

    International genome sequencing projects have produced billions of nucleotides (letters) of DNA sequence data, including the complete genome sequences of 74 organisms. These genome sequences have created many new scientific opportunities, including the ability to identify sequence variations among individuals within a species. These genetic differences, which are known as single nucleotide polymorphisms (SNPs), are particularly important in understanding the genetic basis for disease susceptibility. Since the report of the complete human genome sequence, over two million human SNPs have been identified, including a large-scale comparison of an entire chromosome from twenty individuals. Of the protein coding SNPs (cSNPs), approximatelymore » half leads to a single amino acid change in the encoded protein (non-synonymous coding SNPs). Most of these changes are functionally silent, while the remainder negatively impact the protein and sometimes cause human disease. To date, over 550 SNPs have been found to cause single locus (monogenic) diseases and many others have been associated with polygenic diseases. SNPs have been linked to specific human diseases, including late-onset Parkinson disease, autism, rheumatoid arthritis and cancer. The ability to predict accurately the effects of these SNPs on protein function would represent a major advance toward understanding these diseases. To date several attempts have been made toward predicting the effects of such mutations. The most successful of these is a computational approach called ''Sorting Intolerant From Tolerant'' (SIFT). This method uses sequence conservation among many similar proteins to predict which residues in a protein are functionally important. However, this method suffers from several limitations. First, a query sequence must have a sufficient number of relatives to infer sequence conservation. Second, this method does not make use of or provide any information on protein structure, which can be used to

  14. The morbid anatomy of the human genome: chromosomal location of mutations causing disease.

    PubMed Central

    McKusick, V A; Amberger, J S

    1993-01-01

    Information is given in tabular form derived from a synopsis of the human gene map which has been updated continuously since 1973 as part of Mendelian Inheritance in Man (Johns Hopkins University Press, 10th ed, 1992) and of OMIM (Online Mendelian Inheritance in Man, available generally since 1987). The part of the synopsis reproduced here consists of chromosome by chromosome gene lists of loci for which there are associated disorders (table 1), a pictorial representation of this information (fig 1a-d), and an index of disorders for which the causative mutations have been mapped (table 2). In table 1, information on genes that have been located to specific chromosomal positions and are also the site of disease producing mutations is arranged by chromosome, starting with chromosome 1 and with the end of the short arm of the chromosome in each case. In table 2 an alphabetized list of these disorders and the chromosomal location of the mutation in each case are provided. Both in the 'Disorder' field of table 1 and in table 2, the numbers 1, 2, or 3 in parentheses after the name of the disorder indicate that its chromosomal location was determined by mapping of the wildtype gene (1), by mapping of the clinical phenotype (2), or by both strategies (3). PMID:8423603

  15. Human Empathy, Personality and Experience Affect the Emotion Ratings of Dog and Human Facial Expressions.

    PubMed

    Kujala, Miiamaaria V; Somppi, Sanni; Jokela, Markus; Vainio, Outi; Parkkonen, Lauri

    2017-01-01

    Facial expressions are important for humans in communicating emotions to the conspecifics and enhancing interpersonal understanding. Many muscles producing facial expressions in humans are also found in domestic dogs, but little is known about how humans perceive dog facial expressions, and which psychological factors influence people's perceptions. Here, we asked 34 observers to rate the valence, arousal, and the six basic emotions (happiness, sadness, surprise, disgust, fear, and anger/aggressiveness) from images of human and dog faces with Pleasant, Neutral and Threatening expressions. We investigated how the subjects' personality (the Big Five Inventory), empathy (Interpersonal Reactivity Index) and experience of dog behavior affect the ratings of dog and human faces. Ratings of both species followed similar general patterns: human subjects classified dog facial expressions from pleasant to threatening very similarly to human facial expressions. Subjects with higher emotional empathy evaluated Threatening faces of both species as more negative in valence and higher in anger/aggressiveness. More empathetic subjects also rated the happiness of Pleasant humans but not dogs higher, and they were quicker in their valence judgments of Pleasant human, Threatening human and Threatening dog faces. Experience with dogs correlated positively with ratings of Pleasant and Neutral dog faces. Personality also had a minor effect on the ratings of Pleasant and Neutral faces in both species. The results imply that humans perceive human and dog facial expression in a similar manner, and the perception of both species is influenced by psychological factors of the evaluators. Especially empathy affects both the speed and intensity of rating dogs' emotional facial expressions.

  16. Human Empathy, Personality and Experience Affect the Emotion Ratings of Dog and Human Facial Expressions

    PubMed Central

    Kujala, Miiamaaria V.; Somppi, Sanni; Jokela, Markus; Vainio, Outi; Parkkonen, Lauri

    2017-01-01

    Facial expressions are important for humans in communicating emotions to the conspecifics and enhancing interpersonal understanding. Many muscles producing facial expressions in humans are also found in domestic dogs, but little is known about how humans perceive dog facial expressions, and which psychological factors influence people’s perceptions. Here, we asked 34 observers to rate the valence, arousal, and the six basic emotions (happiness, sadness, surprise, disgust, fear, and anger/aggressiveness) from images of human and dog faces with Pleasant, Neutral and Threatening expressions. We investigated how the subjects’ personality (the Big Five Inventory), empathy (Interpersonal Reactivity Index) and experience of dog behavior affect the ratings of dog and human faces. Ratings of both species followed similar general patterns: human subjects classified dog facial expressions from pleasant to threatening very similarly to human facial expressions. Subjects with higher emotional empathy evaluated Threatening faces of both species as more negative in valence and higher in anger/aggressiveness. More empathetic subjects also rated the happiness of Pleasant humans but not dogs higher, and they were quicker in their valence judgments of Pleasant human, Threatening human and Threatening dog faces. Experience with dogs correlated positively with ratings of Pleasant and Neutral dog faces. Personality also had a minor effect on the ratings of Pleasant and Neutral faces in both species. The results imply that humans perceive human and dog facial expression in a similar manner, and the perception of both species is influenced by psychological factors of the evaluators. Especially empathy affects both the speed and intensity of rating dogs’ emotional facial expressions. PMID:28114335

  17. Molecular dynamics simulations and principal component analysis on human laforin mutation W32G and W32G/K87A.

    PubMed

    Srikumar, P S; Rohini, K; Rajesh, Perumbilavil Kaithamanakallam

    2014-06-01

    Mutations in human laforin lead to an autosomal neurodegenerative disorder Lafora disease. In N-terminal carbohydrate binding domain of laforin, two mutations W32G and K87A are reported as highly disease causing laforin mutants. Experimental studies reported that mutations are responsible for the abolishment of glycogen binding which is a critical function of laforin. Our current computational study focused on the role of conformational changes in human laforin structure due to existing single mutation W32G and prepared double mutation W32G/K87A related to loss of glycogen binding. We performed 10 ns molecular dynamics (MD) simulation studies in the Gromacs package for both mutations and analyzed the trajectories. From the results, the global properties like root mean square deviation, root mean square fluctuation, radius of gyration, solvent accessible surface area and hydrogen bonds showed structural changes in atomic level observed in W32G and W32G/K87A laforin mutants. The conformational change induced by mutants influenced the loss of the overall stability of the native laforin. Moreover, the change in overall motion of protein was analyzed by principal component analysis and results showed protein clusters expanded more than native and also change in direction in case of double mutant in conformational space. Overall, our report provides theoretical information on loss of structure-function relationship due to flexible nature of laforin mutants. In conclusion, comparative MD simulation studies support the experimental data on W32G and W32G/K87A related to the lafora disease mechanism on glycogen binding.

  18. Evolutionary pattern of mutation in the factor IX genes of great apes: How does it compare to the pattern of recent germline mutation in patients with hemophilia B?

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

    Grouse, L.H.; Ketterling, R.P.; Sommer, S.S.

    Most mutations causing hemophilia B have arisen within the past 150 years. By correcting for multiple biases, the underlying rates of spontaneous germline mutation have been estimated in the factor IX gene. From these rates, an underlying pattern of mutation has emerged. To determine if this pattern compares to a underlying pattern found in the great apes, sequence changes were determined in intronic regions of the factor IX gene. The following species were studied: Gorilla gorilla, Pan troglodytes (chimpanzee), Pongo pygmacus (orangutan) and Homo sapiens. Intronic sequences at least 200 bp from a splice junction were randomly chosen, amplified bymore » cross-species PCR, and sequenced. These regions are expected to be subject to little if any selective pressure. Early diverged species of Old World monkeys were also studied to help determine the direction of mutational changes. A total of 62 sequence changes were observed. Initial data suggest that the average pattern since evolution of the great apes has a paucity of transitions at CpG dinucleotides and an excess of microinsertions to microdeletions when compared to the pattern observed in humans during the past 150 years (p<.05). A larger study is in progress to confirm these results.« less

  19. Seamless correction of the sickle cell disease mutation of the HBB gene in human induced pluripotent stem cells using TALENs.

    PubMed

    Sun, Ning; Zhao, Huimin

    2014-05-01

    Sickle cell disease (SCD) is the most common human genetic disease which is caused by a single mutation of human β-globin (HBB) gene. The lack of long-term treatment makes the development of reliable cell and gene therapies highly desirable. Disease-specific patient-derived human induced pluripotent stem cells (hiPSCs) have great potential for developing novel cell and gene therapies. With the disease-causing mutations corrected in situ, patient-derived hiPSCs can restore normal cell functions and serve as a renewable autologous cell source for the treatment of genetic disorders. Here we successfully utilized transcription activator-like effector nucleases (TALENs), a recently emerged novel genome editing tool, to correct the SCD mutation in patient-derived hiPSCs. The TALENs we have engineered are highly specific and generate minimal off-target effects. In combination with piggyBac transposon, TALEN-mediated gene targeting leaves no residual ectopic sequences at the site of correction and the corrected hiPSCs retain full pluripotency and a normal karyotype. Our study demonstrates an important first step of using TALENs for the treatment of genetic diseases such as SCD, which represents a significant advance toward hiPSC-based cell and gene therapies. © 2013 Wiley Periodicals, Inc.

  20. Mutation screening of X-chromosomal neuroligin genes: no mutations in 196 autism probands.

    PubMed

    Vincent, John B; Kolozsvari, Debbie; Roberts, Wendy S; Bolton, Patrick F; Gurling, Hugh M D; Scherer, Stephen W

    2004-08-15

    Autism, a childhood neuropsychiatric disorder with a strong genetic component, is currently the focus of considerable attention within the field of human genetics as well many other medical-related disciplines. A recent study has implicated two X-chromosomal neuroligin genes, NLGN3 and NLGN4, as having an etiological role in autism, having identified a frameshift mutation in one gene and a substitution mutation in the other, segregating in multiplex autism spectrum families (Jamain et al. [2003: Nat Genet 34:27-29]). The function of neuroligin as a trigger for synapse formation would suggest that such mutations would likely result in some form of pathological manifestation. Our own study, screening a larger sample of 196 autism probands, failed to identify any mutations that would affect the coding regions of these genes. Our findings suggest that mutations in these two genes are infrequent in autism. Copyright 2004 Wiley-Liss, Inc.

  1. The mutation-drift balance in spatially structured populations.

    PubMed

    Schneider, David M; Martins, Ayana B; de Aguiar, Marcus A M

    2016-08-07

    In finite populations the action of neutral mutations is balanced by genetic drift, leading to a stationary distribution of alleles that displays a transition between two different behaviors. For small mutation rates most individuals will carry the same allele at equilibrium, whereas for high mutation rates of the alleles will be randomly distributed with frequencies close to one half for a biallelic gene. For well-mixed haploid populations the mutation threshold is μc=1/2N, where N is the population size. In this paper we study how spatial structure affects this mutation threshold. Specifically, we study the stationary allele distribution for populations placed on regular networks where connected nodes represent potential mating partners. We show that the mutation threshold is sensitive to spatial structure only if the number of potential mates is very small. In this limit, the mutation threshold decreases substantially, increasing the diversity of the population at considerably low mutation rates. Defining kc as the degree of the network for which the mutation threshold drops to half of its value in well-mixed populations we show that kc grows slowly as a function of the population size, following a power law. Our calculations and simulations are based on the Moran model and on a mapping between the Moran model with mutations and the voter model with opinion makers. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Mutations in the evolutionarily highly conserved KEOPS complex genes cause nephrotic syndrome with microcephaly

    PubMed Central

    Braun, Daniela A.; Rao, Jia; Mollet, Geraldine; Schapiro, David; Daugeron, Marie-Claire; Tan, Weizhen; Gribouval, Olivier; Boyer, Olivia; Revy, Patrick; Jobst-Schwan, Tilman; Schmidt, Johanna Magdalena; Lawson, Jennifer A.; Schanze, Denny; Ashraf, Shazia; Boddaert, Nathalie; Collinet, Bruno; Martin, Gaëlle; Liger, Dominique; Lovric, Svjetlana; Furlano, Monica; Guerrera, I. Chiara; Sanchez-Ferras, Oraly; Menten, Björn; Vergult, Sarah; De Rocker, Nina; Airik, Merlin; Hermle, Tobias; Shril, Shirlee; Widmeier, Eugen; Gee, Heon Yung; Choi, Won-Il; Sadowski, Carolin E.; Pabst, Werner L.; Warejko, Jillian; Daga, Ankana; LeBerre, Tamara Basta; Matejas, Verena; Behnam, Babak; Beeson, Brendan; Begtrup, Amber; Bruce, Malcolm; Ch'ng, Gaik-Siew; Lin, Shuan-Pei; Chang, Jui-Hsing; Chen, Chao-Huei; Cho, Megan T.; Gipson, Patrick E.; Hsu, Chyong-Hsin; Kari, Jameela A.; Ke, Yu-Yuan; Kiraly-Borri, Cathy; Lai, Wai-ming; Lemyre, Emmanuelle; Littlejohn, Rebecca Okasha; Masri, Amira; Moghtaderi, Mastaneh; Nakamura, Kazuyuki; Praet, Marleen; Prasad, Chitra; Prytula, Agnieszka; Roeder, Elizabeth; Rump, Patrick; Schnur, Rhonda E.; Shiihara, Takashi; Sinha, Manish; Soliman, Neveen A; Soulami, Kenza; Sweetser, David A.; Tsai, Wen-Hui; Tsai, Jeng-Daw; Vester, Udo; Viskochil, David H.; Vatanavicharn, Nithiwat; Waxler, Jessica L.; Wolf, Matthias T.F.; Wong, Sik-Nin; Poduri, Annapurna; Truglio, Gessica; Mane, Shrikant; Lifton, Richard P.; Bouchard, Maxime; Kannu, Peter; Chitayat, David; Magen, Daniella; Calleweart, Bert; van Tilbeurgh, Herman; Zenker, Martin; Antignac, Corinne; Hildebrandt, Friedhelm

    2018-01-01

    Galloway-Mowat syndrome (GAMOS) is a severe autosomal-recessive disease characterized by the combination of early-onset steroid-resistant nephrotic syndrome (SRNS) and microcephaly with brain anomalies. To date, mutations of WDR73 are the only known monogenic cause of GAMOS and in most affected individuals the molecular diagnosis remains elusive. We here identify recessive mutations of OSGEP, TP53RK, TPRKB, or LAGE3, encoding the 4 subunits of the KEOPS complex in 33 individuals of 30 families with GAMOS. CRISPR/Cas9 knockout in zebrafish and mice recapitulates the human phenotype of microcephaly and results in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibits cell proliferation, which human mutations fail to rescue, and knockdown of either gene activates DNA damage response signaling and induces apoptosis. OSGEP and TP53RK molecularly interact and co-localize with the actin-regulating ARP2/3 complex. Furthermore, knockdown of OSGEP and TP53RK induces defects of the actin cytoskeleton and reduces migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identify 4 novel monogenic causes of GAMOS, describe the first link between KEOPS function and human disease, and delineate potential pathogenic mechanisms. PMID:28805828

  3. Inactivating KISS1 mutation and hypogonadotropic hypogonadism.

    PubMed

    Topaloglu, A Kemal; Tello, Javier A; Kotan, L Damla; Ozbek, Mehmet N; Yilmaz, M Bertan; Erdogan, Seref; Gurbuz, Fatih; Temiz, Fatih; Millar, Robert P; Yuksel, Bilgin

    2012-02-16

    Gonadotropin-releasing hormone (GnRH) is the central regulator of gonadotropins, which stimulate gonadal function. Hypothalamic neurons that produce kisspeptin and neurokinin B stimulate GnRH release. Inactivating mutations in the genes encoding the human kisspeptin receptor (KISS1R, formerly called GPR54), neurokinin B (TAC3), and the neurokinin B receptor (TACR3) result in pubertal failure. However, human kisspeptin loss-of-function mutations have not been described, and contradictory findings have been reported in Kiss1-knockout mice. We describe an inactivating mutation in KISS1 in a large consanguineous family that results in failure of pubertal progression, indicating that functional kisspeptin is important for puberty and reproduction in humans. (Funded by the Scientific and Technological Research Council of Turkey [TÜBİTAK] and others.).

  4. Molecular modeling of retinoschisin with functional analysis of pathogenic mutations from human X-linked retinoschisis

    PubMed Central

    Sergeev, Y.V.; Caruso, R.C.; Meltzer, M.R.; Smaoui, N.; MacDonald, I.M.; Sieving, P.A.

    2010-01-01

    Gene mutations that encode retinoschisin (RS1) cause X-linked retinoschisis (XLRS), a form of juvenile macular and retinal degeneration that affects males. RS1 is an adhesive protein which is proposed to preserve the structural and functional integrity of the retina, but there is very little evidence of the mechanism by which protein changes are related to XLRS disease. Here, we report molecular modeling of the RS1 protein and consider perturbations caused by mutations found in human XLRS subjects. In 60 XLRS patients who share 27 missense mutations, we then evaluated possible correlations of the molecular modeling with retinal function as determined by the electroretinogram (ERG) a- and b-waves. The b/a-wave ratio reflects visual-signal transfer in retina. We sorted the ERG b/a-ratios by patient age and by the mutation impact on protein structure. The majority of RS1 mutations caused minimal structure perturbation and targeted the protein surface. These patients' b/a-ratios were similar across younger and older subjects. Maximum structural perturbations from either the removal or insertion of cysteine residues or changes in the hydrophobic core were associated with greater difference in the b/a-ratio with age, with a significantly smaller ratio at younger ages, analogous to the ERG changes with age observed in mice with no RS1-protein expression due to a recombinant RS1-knockout gene. The molecular modeling suggests an association between the predicted structural alteration and/or damage to retinoschisin and the severity of XLRS as measured by the ERG analogous to the RS1-knockout mouse. PMID:20061330

  5. Aflatoxin B1-induced DNA adduct formation and p53 mutations in CYP450-expressing human liver cell lines.

    PubMed

    Macé, K; Aguilar, F; Wang, J S; Vautravers, P; Gómez-Lechón, M; Gonzalez, F J; Groopman, J; Harris, C C; Pfeifer, A M

    1997-07-01

    Epidemiological evidence has been supporting a relationship between dietary aflatoxin B1 (AFB1) exposure, development of human primary hepatocellular carcinoma (HCC) and mutations in the p53 tumor suppressor gene. However, the correlation between the observed p53 mutations, the AFB1 DNA adducts and their activation pathways has not been elucidated. Development of relevant cellular in vitro models, taking into account species and tissue specificity, could significantly contribute to the knowledge of cytotoxicity and genotoxicity mechanisms of chemical procarcinogens, such as AFB1, in humans. For this purpose a non-tumorigenic SV40-immortalized human liver epithelial cell line (THLE cells) which retained most of the phase II enzymes, but had markedly reduced phase I activities was used for stable expression of the human CYP1A2, CYP2A6, CYP2B6 and CYP3A4 cDNA. The four genetically engineered cell lines (T5-1A2, T5-2A6, T5-2B6 and T5-3A4) produced high levels of the specific CYP450 proteins and showed comparable or higher catalytic activities related to the CYP450 expression when compared to human hepatocytes. The T5-1A2, T5-2A6, T5-2B6 and T5-3A4 cell lines exhibited a very high sensitivity to the cytotoxic effects of AFB1 and were approximately 125-, 2-, 2- and 15-fold, respectively, more sensitive than the control T5-neo cells, transfected with an expressing vector which does not contain CYP450 cDNA. In the CYP450-expressing cells, nanomolar doses of AFB1-induced DNA adduct formation including AFB1-N7-guanine, -pyrimidyl and -diol adducts. In addition, the T5-1A2 cells showed AFM1-DNA adducts. At similar levels of total DNA adducts, both the T5-1A2 and T5-3A4 cells showed, at codon 249 of the p53 gene, AGG to AGT transversions at a relative frequency of 15x10(-6). In contrast, only the T5-3A4 cells showed CCC to ACC transversion at codon 250 at a high frequency, whereas the second most frequent mutations found in the T5-1A2 cells were C to T transitions at the first

  6. Identification of cis-suppression of human disease mutations by comparative genomics

    PubMed Central

    Jordan, Daniel M.; Frangakis, Stephan G.; Golzio, Christelle; Cassa, Christopher A.; Kurtzberg, Joanne; Davis, Erica E.; Sunyaev, Shamil R.; Katsanis, Nicholas

    2015-01-01

    Patterns of amino acid conservation have served as a tool for understanding protein evolution1. The same principles have also found broad application in human genomics, driven by the need to interpret the pathogenic potential of variants in patients2. Here we performed a systematic comparative genomics analysis of human disease-causing missense variants. We found that an appreciable fraction of disease-causing alleles are fixed in the genomes of other species, suggesting a role for genomic context. We developed a model of genetic interactions that predicts most of these to be simple pairwise compensations. Functional testing of this model on two known human disease genes3,4 revealed discrete cis amino acid residues that, although benign on their own, could rescue the human mutations in vivo. This approach was also applied to ab initio gene discovery to support the identification of a de novo disease driver in BTG2 that is subject to protective cis-modification in more than 50 species. Finally, on the basis of our data and models, we developed a computational tool to predict candidate residues subject to compensation. Taken together, our data highlight the importance of cis-genomic context as a contributor to protein evolution; they provide an insight into the complexity of allele effect on phenotype; and they are likely to assist methods for predicting allele pathogenicity5,6. PMID:26123021

  7. Identification of cis-suppression of human disease mutations by comparative genomics.

    PubMed

    Jordan, Daniel M; Frangakis, Stephan G; Golzio, Christelle; Cassa, Christopher A; Kurtzberg, Joanne; Davis, Erica E; Sunyaev, Shamil R; Katsanis, Nicholas

    2015-08-13

    Patterns of amino acid conservation have served as a tool for understanding protein evolution. The same principles have also found broad application in human genomics, driven by the need to interpret the pathogenic potential of variants in patients. Here we performed a systematic comparative genomics analysis of human disease-causing missense variants. We found that an appreciable fraction of disease-causing alleles are fixed in the genomes of other species, suggesting a role for genomic context. We developed a model of genetic interactions that predicts most of these to be simple pairwise compensations. Functional testing of this model on two known human disease genes revealed discrete cis amino acid residues that, although benign on their own, could rescue the human mutations in vivo. This approach was also applied to ab initio gene discovery to support the identification of a de novo disease driver in BTG2 that is subject to protective cis-modification in more than 50 species. Finally, on the basis of our data and models, we developed a computational tool to predict candidate residues subject to compensation. Taken together, our data highlight the importance of cis-genomic context as a contributor to protein evolution; they provide an insight into the complexity of allele effect on phenotype; and they are likely to assist methods for predicting allele pathogenicity.

  8. Rate-distortion theory and human perception.

    PubMed

    Sims, Chris R

    2016-07-01

    The fundamental goal of perception is to aid in the achievement of behavioral objectives. This requires extracting and communicating useful information from noisy and uncertain sensory signals. At the same time, given the complexity of sensory information and the limitations of biological information processing, it is necessary that some information must be lost or discarded in the act of perception. Under these circumstances, what constitutes an 'optimal' perceptual system? This paper describes the mathematical framework of rate-distortion theory as the optimal solution to the problem of minimizing the costs of perceptual error subject to strong constraints on the ability to communicate or transmit information. Rate-distortion theory offers a general and principled theoretical framework for developing computational-level models of human perception (Marr, 1982). Models developed in this framework are capable of producing quantitatively precise explanations for human perceptual performance, while yielding new insights regarding the nature and goals of perception. This paper demonstrates the application of rate-distortion theory to two benchmark domains where capacity limits are especially salient in human perception: discrete categorization of stimuli (also known as absolute identification) and visual working memory. A software package written for the R statistical programming language is described that aids in the development of models based on rate-distortion theory. Copyright © 2016 The Author. Published by Elsevier B.V. All rights reserved.

  9. SMARCB1 mutations in schwannomatosis and genotype correlations with rhabdoid tumors.

    PubMed

    Smith, Miriam J; Wallace, Andrew J; Bowers, Naomi L; Eaton, Helen; Evans, D Gareth R

    2014-09-01

    Mutations in the SMARCB1 gene are involved in several human tumor-predisposing syndromes. They were established as an underlying cause of the tumor suppressor syndrome schwannomatosis in 2008. There is a much higher rate of mutation detection in familial disease than in sporadic disease. We have performed extensive genetic testing on a cohort of familial and sporadic patients who fulfilled clinical diagnostic criteria for schwannomatosis. In our updated cohort, we identified novel mutations within the SMARCB1 gene as well as several recurrent mutations. Of the schwannomatosis screens reported to date, including those in our updated cohort, SMARCB1 mutations have been found in 45% of familial probands and 9% of sporadic patients. The exon 1 mutation, c.41C>A p.Pro14His (10% in our series), and the 3' untranslated region mutation, c.*82C>T (27%), are the most common changes reported in patients with schwannomatosis to date, indicating the presence of mutation hot spots at both 5' and 3' portions of the gene. Comparison with germline SMARCB1 mutations in patients with rhabdoid tumors showed that the schwannomatosis mutations were significantly more likely to occur at either end of the gene and be nontruncating mutations (P < 0.0001). SMARCB1 mutations are found in a significant proportion of schwannomatosis patients, and an even higher proportion of rhabdoid patients. Whereas SMARCB1 alone seems to account for rhabdoid disease, there is likely to be substantial heterogeneity in schwannomatosis even for familial disease. There is a clear genotype-phenotype correlation, with germline rhabdoid mutations being significantly more likely to be centrally placed, involve multiple exon deletions, and be truncating mutations. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Pituitary resistance to thyroid hormone associated with a base mutation in the hormone-binding domain of the human 3,5,3'-triiodothyronine receptor-beta.

    PubMed

    Sasaki, S; Nakamura, H; Tagami, T; Miyoshi, Y; Nogimori, T; Mitsuma, T; Imura, H

    1993-05-01

    Point mutations in the human T3 receptor-beta (TR beta) gene causing single amino acid substitutions have been identified in several different kindreds with generalized resistance to thyroid hormone. Until now, no study has been reported on the TR gene in cases of pituitary resistance (PRTH). In the present study, we analyzed the TR beta gene in a 30-yr-old Japanese female with PRTH. She exhibited clinical features of hyperthyroidism, elevated serum thyroid hormone levels accompanied by inappropriately increased secretion of TSH, mildly elevated basal metabolic rate, and increased urinary excretion of hydroxyproline. No pituitary tumor was detected. DNA fragments of exons 3-8 of the genomic TR beta gene were generated by the polymerase chain reaction and analyzed by a single stranded conformation polymorphism method. Exon 7 of the patient's TR beta gene showed an abnormal band, suggesting the existence of mutation(s). By subcloning and sequencing the DNA, a point mutation was identified in one allele at nucleotide 1297 (C to T), which altered the 333rd amino acid, arginine, to tryptophan. Neither of her apparently normal parents had any mutations of the TR beta gene. In vitro translation products of the mutant TR beta gene showed remarkably decreased T3-binding activity (Ka, 2.1 x 10(8) M-1; normal TR beta Ka, 1.1 x 10(10) M-1). Since the molecular defect detected in a patient with PRTH is similar to that seen in subjects with generalized resistance to thyroid hormone, both types of the syndrome may represent a continuous spectrum of the same etiological defect with variable tissue resistance to thyroid hormone.

  11. Mutation of the human mitochondrial phenylalanine-tRNA synthetase causes infantile-onset epilepsy and cytochrome c oxidase deficiency.

    PubMed

    Almalki, Abdulraheem; Alston, Charlotte L; Parker, Alasdair; Simonic, Ingrid; Mehta, Sarju G; He, Langping; Reza, Mojgan; Oliveira, Jorge M A; Lightowlers, Robert N; McFarland, Robert; Taylor, Robert W; Chrzanowska-Lightowlers, Zofia M A

    2014-01-01

    Mitochondrial aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in protein synthesis since they charge tRNAs with their cognate amino acids. Mutations in the genes encoding mitochondrial aaRSs have been associated with a wide spectrum of human mitochondrial diseases. Here we report the identification of pathogenic mutations (a partial genomic deletion and a highly conserved p. Asp325Tyr missense variant) in FARS2, the gene encoding mitochondrial phenylalanyl-tRNA synthetase, in a patient with early-onset epilepsy and isolated complex IV deficiency in muscle. The biochemical defect was expressed in myoblasts but not in fibroblasts and associated with decreased steady state levels of COXI and COXII protein and reduced steady state levels of the mt-tRNA(Phe) transcript. Functional analysis of the recombinant mutant p. Asp325Tyr FARS2 protein showed an inability to bind ATP and consequently undetectable aminoacylation activity using either bacterial tRNA or human mt-tRNA(Phe) as substrates. Lentiviral transduction of cells with wildtype FARS2 restored complex IV protein levels, confirming that the p.Asp325Tyr mutation is pathogenic, causing respiratory chain deficiency and neurological deficits on account of defective aminoacylation of mt-tRNA(Phe). © 2013. Published by Elsevier B.V. All rights reserved.

  12. Actionable mutations in canine hemangiosarcoma.

    PubMed

    Wang, Guannan; Wu, Ming; Maloneyhuss, Martha A; Wojcik, John; Durham, Amy C; Mason, Nicola J; Roth, David B

    2017-01-01

    Angiosarcomas (AS) are rare in humans, but they are a deadly subtype of soft tissue sarcoma. Discovery sequencing in AS, especially the visceral form, is hampered by the rarity of cases. Most diagnostic material exists as archival formalin fixed, paraffin embedded tissue which serves as a poor source of high quality DNA for genome-wide sequencing. We approached this problem through comparative genomics. We hypothesized that exome sequencing a histologically similar tumor, hemangiosarcoma (HSA), that occurs in approximately 50,000 dogs per year, may lead to the identification of potential oncogenic drivers and druggable targets that could also occur in angiosarcoma. Splenic hemangiosarcomas are common in dogs, which allowed us to collect a cohort of archived matched tumor and normal tissue samples suitable for whole exome sequencing. Mapping of the reads to the latest canine reference genome (Canfam3) demonstrated that >99% of the targeted exomal regions were covered, with >80% at 20X coverage and >90% at 10X coverage. Sequence analysis of 20 samples identified somatic mutations in PIK3CA, TP53, PTEN, and PLCG1, all of which correspond to well-known tumor drivers in human cancer, in more than half of the cases. In one case, we identified a mutation in PLCG1 identical to a mutation observed previously in this gene in human visceral AS. Activating PIK3CA mutations present novel therapeutic targets, and clinical trials of targeted inhibitors are underway in human cancers. Our results lay a foundation for similar clinical trials in canine HSA, enabling a precision medicine approach to this disease.

  13. Mutations in the growth hormone releasing hormone receptor: a new form of dwarfism in humans.

    PubMed

    Baumann, G

    1999-06-01

    We describe a recently identified new form of dwarfism due to isolated growth hormone (GH) deficiency, secondary to inactivating mutations in the GH-releasing hormone receptor (GHRHR) gene. The identical nonsense mutations in the extracellular domain of the GHRHR (E72X or E50X, depending on whether the signal peptide is included in the numbering) has been independently described in three families residing on or originating from the Indian subcontinent (Pakistan, the Bombay region, and Delft near Sri Lanka). Another inactivating mutation, involving the donor splice site of intron 1, has been identified in a population in north-eastern Brazil. Genetic transmission is autosomal recessive; the gene is located on the short arm of chromosome 7. Affected subjects have severe isolated GH deficiency and postnatal growth failure, with a mean adult height of 130 cm for men and 114 cm for women (7-8 standard deviations below the norm). Dwarfism is proportional; a characteristic feature is relative microcephaly, which results in a 'miniaturized adult', eumorphic aspect. Bone age and puberty are delayed, but fertility appears normal. This new syndrome corresponds to the human homologue of the previously identified 'little mouse'.

  14. Heterozygous Mutations Causing Partial Prohormone Convertase 1 Deficiency Contribute to Human Obesity

    PubMed Central

    Creemers, John W.M.; Choquet, Hélène; Stijnen, Pieter; Vatin, Vincent; Pigeyre, Marie; Beckers, Sigri; Meulemans, Sandra; Than, Manuel E.; Yengo, Loïc; Tauber, Maithé; Balkau, Beverley; Elliott, Paul; Jarvelin, Marjo-Riitta; Van Hul, Wim; Van Gaal, Luc; Horber, Fritz; Pattou, François; Froguel, Philippe; Meyre, David

    2012-01-01

    Null mutations in the PCSK1 gene, encoding the proprotein convertase 1/3 (PC1/3), cause recessive monogenic early onset obesity. Frequent coding variants that modestly impair PC1/3 function mildly increase the risk for common obesity. The aim of this study was to determine the contribution of rare functional PCSK1 mutations to obesity. PCSK1 exons were sequenced in 845 nonconsanguineous extremely obese Europeans. Eight novel nonsynonymous PCSK1 mutations were identified, all heterozygous. Seven mutations had a deleterious effect on either the maturation or the enzymatic activity of PC1/3 in cell lines. Of interest, five of these novel mutations, one of the previously described frequent variants (N221D), and the mutation found in an obese mouse model (N222D), affect residues at or near the structural calcium binding site Ca-1. The prevalence of the newly identified mutations was assessed in 6,233 obese and 6,274 lean European adults and children, which showed that carriers of any of these mutations causing partial PCSK1 deficiency had an 8.7-fold higher risk to be obese than wild-type carriers. These results provide the first evidence of an increased risk of obesity in heterozygous carriers of mutations in the PCSK1 gene. Furthermore, mutations causing partial PCSK1 deficiency are present in 0.83% of extreme obesity phenotypes. PMID:22210313

  15. Heterozygous mutations causing partial prohormone convertase 1 deficiency contribute to human obesity.

    PubMed

    Creemers, John W M; Choquet, Hélène; Stijnen, Pieter; Vatin, Vincent; Pigeyre, Marie; Beckers, Sigri; Meulemans, Sandra; Than, Manuel E; Yengo, Loïc; Tauber, Maithé; Balkau, Beverley; Elliott, Paul; Jarvelin, Marjo-Riitta; Van Hul, Wim; Van Gaal, Luc; Horber, Fritz; Pattou, François; Froguel, Philippe; Meyre, David

    2012-02-01

    Null mutations in the PCSK1 gene, encoding the proprotein convertase 1/3 (PC1/3), cause recessive monogenic early onset obesity. Frequent coding variants that modestly impair PC1/3 function mildly increase the risk for common obesity. The aim of this study was to determine the contribution of rare functional PCSK1 mutations to obesity. PCSK1 exons were sequenced in 845 nonconsanguineous extremely obese Europeans. Eight novel nonsynonymous PCSK1 mutations were identified, all heterozygous. Seven mutations had a deleterious effect on either the maturation or the enzymatic activity of PC1/3 in cell lines. Of interest, five of these novel mutations, one of the previously described frequent variants (N221D), and the mutation found in an obese mouse model (N222D), affect residues at or near the structural calcium binding site Ca-1. The prevalence of the newly identified mutations was assessed in 6,233 obese and 6,274 lean European adults and children, which showed that carriers of any of these mutations causing partial PCSK1 deficiency had an 8.7-fold higher risk to be obese than wild-type carriers. These results provide the first evidence of an increased risk of obesity in heterozygous carriers of mutations in the PCSK1 gene. Furthermore, mutations causing partial PCSK1 deficiency are present in 0.83% of extreme obesity phenotypes.

  16. Comparative analysis of KRAS codon 12, 13, 18, 61, and 117 mutations using human MCF10A isogenic cell lines

    PubMed Central

    Stolze, Britta; Reinhart, Stefanie; Bulllinger, Lars; Fröhling, Stefan; Scholl, Claudia

    2015-01-01

    KRAS mutations occur in one third of human cancers and cluster in several hotspots, with codons 12 and 13 being most commonly affected. It has been suggested that the position and type of amino acid exchange influence the transforming capacity of mutant KRAS proteins. We used MCF10A human mammary epithelial cells to establish isogenic cell lines that express different cancer-associated KRAS mutations (G12C, G12D, G12V, G13C, G13D, A18D, Q61H, K117N) at physiological or elevated levels, and investigated the biochemical and functional consequences of the different variants. The overall effects of low-expressing mutants were moderate compared to overexpressed variants, but allowed delineation of biological functions that were related to specific alleles rather than KRAS expression level. None of the mutations induced morphological changes, migratory abilities, or increased phosphorylation of ERK, PDK1, and AKT. KRAS-G12D, G12V, G13D, and K117N mediated EGF-independent proliferation, whereas anchorage-independent growth was primarily induced by K117N and Q61H. Both codon 13 mutations were associated with increased EGFR expression. Finally, global gene expression analysis of MCF10A-G13D versus MCF10A-G12D revealed distinct transcriptional changes. Together, we describe a useful resource for investigating the function of multiple KRAS mutations and provide insights into the differential effects of these variants in MCF10A cells. PMID:25705018

  17. Missense mutation of the COQ2 gene causes defects of bioenergetics and de novo pyrimidine synthesis.

    PubMed

    López-Martín, José M; Salviati, Leonardo; Trevisson, Eva; Montini, Giovanni; DiMauro, Salvatore; Quinzii, Catarina; Hirano, Michio; Rodriguez-Hernandez, Angeles; Cordero, Mario D; Sánchez-Alcázar, José A; Santos-Ocaña, Carlos; Navas, Plácido

    2007-05-01

    Coenzyme Q(10) (CoQ(10)) deficiency has been associated with an increasing number of clinical phenotypes that respond to CoQ(10) supplementation. In two siblings with encephalomyopathy, nephropathy and severe CoQ(10) deficiency, a homozygous mutation was identified in the CoQ(10) biosynthesis gene COQ2, encoding polyprenyl-pHB transferase. To confirm the pathogenicity of this mutation, we have demonstrated that human wild-type, but not mutant COQ2, functionally complements COQ2 defective yeast. In addition, an equivalent mutation introduced in the yeast COQ2 gene also decreases both CoQ(6) concentration and growth in respiratory-chain dependent medium. Polyprenyl-pHB transferase activity was 33-45% of controls in COQ2 mutant fibroblasts. CoQ-dependent mitochondrial complexes activities were restored in deficient fibroblasts by CoQ(10) supplementation, and growth rate was restored in these cells by either CoQ(10) or uridine supplementation. This work is the first direct demonstration of the pathogenicity of a COQ2 mutation involved in human disease, and establishes yeast as a useful model to study human CoQ(10) deficiency. Moreover, we demonstrate that CoQ(10) deficiency in addition to the bioenergetics defect also impairs de novo pyrimidine synthesis, which may contribute to the pathogenesis of the disease.

  18. Induction of mutations by bismuth-212 alpha particles at two genetic loci in human B-lymphoblasts.

    PubMed

    Metting, N F; Palayoor, S T; Macklis, R M; Atcher, R W; Liber, H L; Little, J B

    1992-12-01

    The human lymphoblast cell line TK6 was exposed to the alpha-particle-emitting radon daughter 212Bi by adding DTPA-chelated 212Bi directly to the cell suspension. Cytotoxicity and mutagenicity at two genetic loci were measured, and the molecular nature of mutant clones was studied by Southern blot analysis. Induced mutant fractions were 2.5 x 10(-5)/Gy at the hprt locus and 3.75 x 10(-5)/Gy at the tk locus. Molecular analysis of HPRT- mutant DNAs showed a high frequency (69%) of clones with partial or full deletions of the hprt gene among radiation-induced mutants compared with spontaneous mutants (31%). Chi-squared analyses of mutational spectra show a significant difference (P < or = 0.005) between spontaneous mutants and alpha-particle-induced mutants. Comparison with published studies of accelerator-produced heavy-ion exposures of TK6 cells indicates that the induction of mutations at the hprt locus, and perhaps a subset of mutations at the tk locus, is a simple linear function of particle fluence regardless of the ion species or its LET.

  19. Reshaping the folding energy landscape of human carbonic anhydrase II by a single point genetic mutation Pro237His.

    PubMed

    Jiang, Yan; Su, Jing-Tan; Zhang, Jun; Wei, Xiang; Yan, Yong-Bin; Zhou, Hai-Meng

    2008-01-01

    Human carbonic anhydrase (HCA) II participates in a variety of important biological processes, and it has long been known that genetic mutations of HCA II are closely correlated to human disease. In this research, we investigated the effects of a genetic single point mutation P237, which is located on the surface of the molecule and does not participate in the HCA II catalysis, on HCA II activity, stability and folding. Spectroscopic studies revealed that the mutation caused more buried Trp residues to become accessible by solvent and caused the NMR signals to become less dispersed, but did not affect the secondary structure or the hydrophobic exposure of the protein. The mutant was less stable than the wild type enzyme against heat- and GdnHCl-induced inactivation, but its pH adaptation was similar to the wild type. The mutation slightly decreased the stability of the molten globular intermediate, but gradually affected the stability of the native state by a 10-fold reduction of the Gibbs free energy for the transition from the native state to the intermediate. This might have led to an accumulation of the aggregation-prone molten globular intermediate, which further trapped the proteins into the off-pathway aggregates during refolding and reduced the levels of active enzyme in vivo. The results herein suggested that the correct positioning of the long loop around P237 might be crucial to the folding of HCA II, particularly the formation of the active site.

  20. [Observation and analysis on mutation of routine STR locus].

    PubMed

    Li, Qiu-yang; Feng, Wei-jun; Yang, Qin-gen

    2005-05-01

    To observe and analyze the characteristic of mutation at STR locus. 27 mutant genes observed in 1211 paternity testing cases were checked by PAGE-silver stained and PowerPlex 16 System Kit and validated by sequencing. Mutant genes locate on 15 loci. The pattern of mutation was accord with stepwise mutation model. The mutation ratio of male-to-female was 8:1 and correlated to the age of father. Mutation rate is correlated to the geometric mean of the number of homogeneous repeats of locus. The higher the mean, the higher the mutation rate. These loci are not so appropriate for use in paternity testing.