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Sample records for vivo mutational events

  1. In vivo somatic mutation systems in the mouse

    International Nuclear Information System (INIS)

    Russell, L.B.

    1979-01-01

    In an effort to meet the need for a fast and cheap in vivo prescreen for inherited mammalian point mutations, a somatic forward-mutation method, originally developed in an x-ray experiment, has more recently been tested in work with chemical mutagens. The method makes use of coat-color mutations because the gene product is usually locally expressed, mosaics can be detected with minimal effort, and opportunities for making comparison with induction of germinal point mutations are greatest.--Following treatment of embryos that are heterozygous at specific coat-color loci, various induced genetic changes can result in expression of the recessive (RS) in clones derived from mutant melanocyte precursor cells. However, other events, such as decrease in the number of precursor cells, or disturbed differentiation, can also result in spots, which with careful classification can usually be distinguished from RS's on the basis of their location and color. When this is done, the relative RS frequencies for a series of compounds at least roughly parallel the relative spermatogonial mutation rates. The fact that easily measurable (though low) RS rates are obtained with compounds that have yielded negative results in spermatogonial tests is not surprising in view of the fact that RS's can be caused by several mechanisms besides point mutation.--In spite of the parallelism observed in one laboratory, the usefulness of the in vivo somatic mutation method as a prescreen could come to be doubted because of major discrepancies between results of similar experiments at different laboratories. However, It appears probable that at least some of these discrepancies are due to failure to discriminate between spots that probably resulted from melanocyte insufficiency and spots that resulted from expression of the recessive.--Reverse somatic mutation systems can potentially avoid some of the pitfalls of forward mutation systems. Such system are still in developmental stages

  2. Impact of Fluoroquinolone Resistance Mutations on Gonococcal Fitness and In Vivo Selection for Compensatory Mutations

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    Kunz, Anjali N.; Begum, Afrin A.; Wu, Hong; D'Ambrozio, Jonathan A.; Robinson, James M.; Shafer, William M.; Bash, Margaret C.; Jerse, Ann E.

    2012-01-01

    Background. Quinolone-resistant Neisseria gonorrhoeae (QRNG) arise from mutations in gyrA (intermediate resistance) or gyrA and parC (resistance). Here we tested the consequence of commonly isolated gyrA91/95 and parC86 mutations on gonococcal fitness. Methods. Mutant gyrA91/95 and parC86 alleles were introduced into wild-type gonococci or an isogenic mutant that is resistant to macrolides due to an mtrR−79 mutation. Wild-type and mutant bacteria were compared for growth in vitro and in competitive murine infection. Results. In vitro growth was reduced with increasing numbers of mutations. Interestingly, the gyrA91/95 mutation conferred an in vivo fitness benefit to wild-type and mtrR−79 mutant gonococci. The gyrA91/95, parC86 mutant, in contrast, showed a slight fitness defect in vivo, and the gyrA91/95, parC86, mtrR−79 mutant was markedly less fit relative to the parent strains. A ciprofloxacin-resistant (CipR) mutant was selected during infection with the gyrA91/95, parC86, mtrR−79 mutant in which the mtrR−79 mutation was repaired and the gyrA91 mutation was altered. This in vivo–selected mutant grew as well as the wild-type strain in vitro. Conclusions. gyrA91/95 mutations may contribute to the spread of QRNG. Further acquisition of a parC86 mutation abrogates this fitness advantage; however, compensatory mutations can occur that restore in vivo fitness and maintain CipR. PMID:22492860

  3. Development of potent in vivo mutagenesis plasmids with broad mutational spectra.

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    Badran, Ahmed H; Liu, David R

    2015-10-07

    Methods to enhance random mutagenesis in cells offer advantages over in vitro mutagenesis, but current in vivo methods suffer from a lack of control, genomic instability, low efficiency and narrow mutational spectra. Using a mechanism-driven approach, we created a potent, inducible, broad-spectrum and vector-based mutagenesis system in E. coli that enhances mutation 322,000-fold over basal levels, surpassing the mutational efficiency and spectra of widely used in vivo and in vitro methods. We demonstrate that this system can be used to evolve antibiotic resistance in wild-type E. coli in mutagenesis of chromosomes, episomes and viruses in vivo, and are applicable to both bacterial and bacteriophage-mediated laboratory evolution platforms.

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

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    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.

  5. Deficiency of the DNA repair protein nibrin increases the basal but not the radiation induced mutation frequency in vivo

    International Nuclear Information System (INIS)

    Wessendorf, Petra; Vijg, Jan; Nussenzweig, André; Digweed, Martin

    2014-01-01

    Highlights: • lacZ mutant frequencies measured in vivo in mouse models of radiosensitive Nijmegen Breakage Syndrome. • Spontaneous mutation frequencies are increased in lymphatic tissue due to Nbn mutation. • Single base transitions, not deletions, dominate the mutation spectrum. • Radiation induced mutation frequencies are not increased due to Nbn mutation. - Abstract: Nibrin (NBN) is a member of a DNA repair complex together with MRE11 and RAD50. The complex is associated particularly with the repair of DNA double strand breaks and with the regulation of cell cycle check points. Hypomorphic mutation of components of the complex leads to human disorders characterised by radiosensitivity and increased tumour occurrence, particularly of the lymphatic system. We have examined here the relationship between DNA damage, mutation frequency and mutation spectrum in vitro and in vivo in mouse models carrying NBN mutations and a lacZ reporter plasmid. We find that NBN mutation leads to increased spontaneous DNA damage in fibroblasts in vitro and high basal mutation rates in lymphatic tissue of mice in vivo. The characteristic mutation spectrum is dominated by single base transitions rather than the deletions and complex rearrangements expected after abortive repair of DNA double strand breaks. We conclude that in the absence of wild type nibrin, the repair of spontaneous errors, presumably arising during DNA replication, makes a major contribution to the basal mutation rate. This applies also to cells heterozygous for an NBN null mutation. Mutation frequencies after irradiation in vivo were not increased in mice with nibrin mutations as might have been expected considering the radiosensitivity of NBS patient cells in vitro. Evidently apoptosis is efficient, even in the absence of wild type nibrin

  6. Deficiency of the DNA repair protein nibrin increases the basal but not the radiation induced mutation frequency in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Wessendorf, Petra [Institute of Medical and Human Genetics, Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353 Berlin (Germany); Vijg, Jan [Albert Einstein College of Medicine, Michael F. Price Center, 1301 Morris Park Avenue, Bronx, NY 10461 (United States); Nussenzweig, André [Laboratory of Genome Integrity, National Cancer Institute, National Institute of Health, 37 Convent Drive, Room 1106, Bethesda, MD 20892 (United States); Digweed, Martin, E-mail: martin.digweed@charite.de [Institute of Medical and Human Genetics, Charité – Universitätsmedizin Berlin, Augustenburger Platz 1, D-13353 Berlin (Germany)

    2014-11-15

    Highlights: • lacZ mutant frequencies measured in vivo in mouse models of radiosensitive Nijmegen Breakage Syndrome. • Spontaneous mutation frequencies are increased in lymphatic tissue due to Nbn mutation. • Single base transitions, not deletions, dominate the mutation spectrum. • Radiation induced mutation frequencies are not increased due to Nbn mutation. - Abstract: Nibrin (NBN) is a member of a DNA repair complex together with MRE11 and RAD50. The complex is associated particularly with the repair of DNA double strand breaks and with the regulation of cell cycle check points. Hypomorphic mutation of components of the complex leads to human disorders characterised by radiosensitivity and increased tumour occurrence, particularly of the lymphatic system. We have examined here the relationship between DNA damage, mutation frequency and mutation spectrum in vitro and in vivo in mouse models carrying NBN mutations and a lacZ reporter plasmid. We find that NBN mutation leads to increased spontaneous DNA damage in fibroblasts in vitro and high basal mutation rates in lymphatic tissue of mice in vivo. The characteristic mutation spectrum is dominated by single base transitions rather than the deletions and complex rearrangements expected after abortive repair of DNA double strand breaks. We conclude that in the absence of wild type nibrin, the repair of spontaneous errors, presumably arising during DNA replication, makes a major contribution to the basal mutation rate. This applies also to cells heterozygous for an NBN null mutation. Mutation frequencies after irradiation in vivo were not increased in mice with nibrin mutations as might have been expected considering the radiosensitivity of NBS patient cells in vitro. Evidently apoptosis is efficient, even in the absence of wild type nibrin.

  7. Retrotransposition and mutation events yield Rap1 GTPases with differential signalling capacity

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    Penzkofer Tobias

    2010-02-01

    Full Text Available Abstract Background Retrotransposition of mRNA transcripts gives occasionally rise to functional retrogenes. Through acquiring tempero-spatial expression patterns distinct from their parental genes and/or functional mutations in their coding sequences, such retrogenes may in principle reshape signalling networks. Results Here we present evidence for such a scenario, involving retrogenes of Rap1 belonging to the Ras family of small GTPases. We identified two murine and one human-specific retrogene of Rap1A and Rap1B, which encode proteins that differ by only a few amino acids from their parental Rap1 proteins. Markedly, human hRap1B-retro and mouse mRap1A-retro1 acquired mutations in the 12th and 59th amino acids, respectively, corresponding to residues mutated in constitutively active oncogenic Ras proteins. Statistical and structural analyses support a functional evolution scenario, where Rap1 isoforms of retrogenic origin are functionally distinct from their parental proteins. Indeed, all retrogene-encoded GTPases have an increased GTP/GDP binding ratio in vivo, indicating that their conformations resemble that of active GTP-bound Rap1. We furthermore demonstrate that these three Rap1 isoforms exhibit distinct affinities for the Ras-binding domain of RalGDS. Finally, when tested for their capacity to induce key cellular processes like integrin-mediated cell adhesion or cell spreading, marked differences are seen. Conclusions Together, these data lend strong support for an evolution scenario, where retrotransposition and subsequent mutation events generated species-specific Rap1 isoforms with differential signaling potential. Expression of the constitutively active human Rap1B-retro in cells like those derived from Ramos Burkitt's lymphoma and bone marrow from a patient with myelodysplastic syndrome (MDS warrants further investigation into its role in disease development.

  8. Adverse events in families with hypertrophic or dilated cardiomyopathy and mutations in the MYBPC3 gene

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    Lehrke Stephanie

    2008-10-01

    Full Text Available Abstract Background Mutations in MYBPC3 encoding myosin binding protein C belong to the most frequent causes of hypertrophic cardiomyopathy (HCM and may also lead to dilated cardiomyopathy (DCM. MYBPC3 mutations initially were considered to cause a benign form of HCM. The aim of this study was to examine the clinical outcome of patients and their relatives with 18 different MYBPC3 mutations. Methods 87 patients with HCM and 71 patients with DCM were screened for MYBPC3 mutations by denaturing gradient gel electrophoresis and sequencing. Close relatives of mutation carriers were genotyped for the respective mutation. Relatives with mutation were then evaluated by echocardiography and magnetic resonance imaging. A detailed family history regarding adverse clinical events was recorded. Results In 16 HCM (18.4% and two DCM (2.8% index patients a mutation was detected. Seven mutations were novel. Mutation carriers exhibited no additional mutations in genes MYH7, TNNT2, TNNI3, ACTC and TPM1. Including relatives of twelve families, a total number of 42 mutation carriers was identified of which eleven (26.2% had at least one adverse event. Considering the twelve families and six single patients with mutations, 45 individuals with cardiomyopathy and nine with borderline phenotype were identified. Among the 45 patients, 23 (51.1% suffered from an adverse event. In eleven patients of seven families an unexplained sudden death was reported at the age between 13 and 67 years. Stroke or a transient ischemic attack occurred in six patients of five families. At least one adverse event occurred in eleven of twelve families. Conclusion MYBPC3 mutations can be associated with cardiac events such as progressive heart failure, stroke and sudden death even at younger age. Therefore, patients with MYBPC3 mutations require thorough clinical risk assessment.

  9. In vivo and in vitro functional characterization of Andersen's syndrome mutations.

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    Bendahhou, Saïd; Fournier, Emmanuel; Sternberg, Damien; Bassez, Guillaume; Furby, Alain; Sereni, Carole; Donaldson, Matthew R; Larroque, Marie-Madeleine; Fontaine, Bertrand; Barhanin, Jacques

    2005-06-15

    The inward rectifier K(+) channel Kir2.1 carries all Andersen's syndrome mutations identified to date. Patients exhibit symptoms of periodic paralysis, cardiac dysrhythmia and multiple dysmorphic features. Here, we report the clinical manifestations found in three families with Andersen's syndrome. Molecular genetics analysis identified two novel missense mutations in the KCNJ2 gene leading to amino acid changes C154F and T309I of the Kir2.1 open reading frame. Patch clamp experiments showed that the two mutations produced a loss of channel function. When co-expressed with Kir2.1 wild-type (WT) channels, both mutations exerted a dominant-negative effect leading to a loss of the inward rectifying K(+) current. Confocal microscopy imaging in HEK293 cells is consistent with a co-assembly of the EGFP-fused mutant proteins with WT channels and proper traffick to the plasma membrane to produce silent channels alone or as hetero-tetramers with WT. Functional expression in C2C12 muscle cell line of newly as well as previously reported Andersen's syndrome mutations confirmed that these mutations act through a dominant-negative effect by altering channel gating or trafficking. Finally, in vivo electromyographic evaluation showed a decrease in muscle excitability in Andersen's syndrome patients. We hypothesize that Andersen's syndrome-associated mutations and hypokalaemic periodic paralysis-associated calcium channel mutations may lead to muscle membrane hypoexcitability via a common mechanism.

  10. The association of JAK2V617F mutation and leukocytosis with thrombotic events in essential thrombocythemia.

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    Hsiao, Hui-Hua; Yang, Ming-Yu; Liu, Yi-Chang; Lee, Ching-Ping; Yang, Wen-Chi; Liu, Ta-Chih; Chang, Chao-Sung; Lin, Sheng-Fung

    2007-11-01

    The Janus kinase 2 mutation, JAK2 (V617F), and megakaryocytic mutations, MPL (W515L/K), have been identified and correlated with a subtype of essential thrombocythemia (ET) patients. We investigated the frequency of mutations in ET patients and analyzed the relationship with their clinical features. Fifty-three ET patients were enrolled in the study. The amplification refractory mutation system was applied for the mutation survey of the JAK2V617F, while the polymerase chain reaction with sequencing was used for the mutation survey of MPLW515L/K. Thirty-five (66%) patients harboring the JAK2 (V617F) mutation, including 3 homozygous and 32 heterozygous changes, but no MPLW515L/K mutation, were found. During follow-up, 17 (32.1%) patients suffered from documented thrombotic events, with 15 having JAK2V617F mutations. Statistical analysis showed that patients with the JAK2 mutation had significantly higher leukocytes, hemoglobin level, and thrombotic event (p = 0.043, p = 0.001, and p = 0.029, respectively). Thrombotic events were also significantly correlated with leukocytosis and older age. The JAK2V617F mutation was noted in a certain population of ET patients and correlated with leukocytosis, high hemoglobin level, and thrombosis. Therefore, detection of the JAK2V617F mutation can affect not only the diagnosis, but also the management of ET patients.

  11. Excess of mutational jackpot events in expanding populations revealed by spatial Luria–Delbrück experiments

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    Fusco, Diana; Gralka, Matti; Kayser, Jona; Anderson, Alex; Hallatschek, Oskar

    2016-01-01

    The genetic diversity of growing cellular populations, such as biofilms, solid tumours or developing embryos, is thought to be dominated by rare, exceptionally large mutant clones. Yet, the emergence of these mutational jackpot events is only understood in well-mixed populations, where they stem from mutations that arise during the first few cell divisions. To study jackpot events in spatially structured populations, we track mutant clones in microbial populations using fluorescence microscopy and population sequencing. High-frequency mutations are found to be massively enriched in microbial colonies compared with well-shaken liquid cultures, as a result of late-occurring mutations surfing at the edge of range expansions. Thus, jackpot events can be generated not only when mutations arise early but also when they occur at favourable locations, which exacerbates their role in adaptation and disease. In particular, because spatial competition with the wild type keeps most mutant clones in a quiescent state, strong selection pressures that kill the wild type promote drug resistance. PMID:27694797

  12. Excess of mutational jackpot events in expanding populations revealed by spatial Luria-Delbrück experiments.

    Science.gov (United States)

    Fusco, Diana; Gralka, Matti; Kayser, Jona; Anderson, Alex; Hallatschek, Oskar

    2016-10-03

    The genetic diversity of growing cellular populations, such as biofilms, solid tumours or developing embryos, is thought to be dominated by rare, exceptionally large mutant clones. Yet, the emergence of these mutational jackpot events is only understood in well-mixed populations, where they stem from mutations that arise during the first few cell divisions. To study jackpot events in spatially structured populations, we track mutant clones in microbial populations using fluorescence microscopy and population sequencing. High-frequency mutations are found to be massively enriched in microbial colonies compared with well-shaken liquid cultures, as a result of late-occurring mutations surfing at the edge of range expansions. Thus, jackpot events can be generated not only when mutations arise early but also when they occur at favourable locations, which exacerbates their role in adaptation and disease. In particular, because spatial competition with the wild type keeps most mutant clones in a quiescent state, strong selection pressures that kill the wild type promote drug resistance.

  13. Parkinson's disease-related LRRK2 G2019S mutation results from independent mutational events in humans.

    Science.gov (United States)

    Lesage, Suzanne; Patin, Etienne; Condroyer, Christel; Leutenegger, Anne-Louise; Lohmann, Ebba; Giladi, Nir; Bar-Shira, Anat; Belarbi, Soraya; Hecham, Nassima; Pollak, Pierre; Ouvrard-Hernandez, Anne-Marie; Bardien, Soraya; Carr, Jonathan; Benhassine, Traki; Tomiyama, Hiroyuki; Pirkevi, Caroline; Hamadouche, Tarik; Cazeneuve, Cécile; Basak, A Nazli; Hattori, Nobutaka; Dürr, Alexandra; Tazir, Meriem; Orr-Urtreger, Avi; Quintana-Murci, Lluis; Brice, Alexis

    2010-05-15

    Mutations in the leucine-rich-repeat kinase 2 (LRRK2) gene have been identified in families with autosomal dominant Parkinson's disease (PD) and in sporadic cases; the G2019S mutation is the single most frequent. Intriguingly, the frequency of this mutation in PD patients varies greatly among ethnic groups and geographic origins: it is present at <0.1% in East Asia, approximately 2% in European-descent patients and can reach frequencies of up to 15-40% in PD Ashkenazi Jews and North African Arabs. To ascertain the evolutionary dynamics of the G2019S mutation in different populations, we genotyped 74 markers spanning a 16 Mb genomic region around G2019S, in 191 individuals carrying the mutation from 126 families of different origins. Sixty-seven families were of North-African Arab origin, 18 were of North/Western European descent, 37 were of Jewish origin, mostly from Eastern Europe, one was from Japan, one from Turkey and two were of mixed origins. We found the G2019S mutation on three different haplotypes. Network analyses of the three carrier haplotypes showed that G2019S arose independently at least twice in humans. In addition, the population distribution of the intra-allelic diversity of the most widespread carrier haplotype, together with estimations of the age of G2019S determined by two different methods, suggests that one of the founding G2019S mutational events occurred in the Near East at least 4000 years ago.

  14. Vascular events in Korean patients with myeloproliferative neoplasms and their relationship to JAK2 mutation.

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    Bang, Soo-Mee; Lee, Jong-Seok; Ahn, Jeong Yeal; Lee, Jae Hoon; Hyun, Myung Soo; Kim, Bong Seog; Park, Moo Rim; Chi, Hyun-Sook; Kim, Ho Young; Kim, Hyo Jung; Lee, Moon Hee; Kim, Hwak; Won, Jong Ho; Yoon, Hwi Joong; Oh, Do-Yeun; Nam, Eun-Mi; Bae, Sung Hwa; Kim, Byoung-Kook

    2009-03-01

    Evaluation of the Janus kinase 2 (JAK2) V617F mutation has been widely used for the diagnosis of myeloproliferative neoplasms (MPN). However, its prognostic relevance to clinical outcome is not completely understood. We investigated the association of JAK2 V617F with vascular events in Korean patients with myeloproliferative neoplasms (MPN). We studied 283 patients from 15 centers, who were diagnosed with MPN. The JAK2 V617F status was evaluated by allele-specific polymerase chain reaction (PCR) and sequencing. The patients' diagnoses were essential thrombocythemia (ET n = 146), polycythemia vera (PV n = 120), primary myelofibrosis (n = 12), and unclassifiable MPN (MPNu n = 5). JAK2 V617F was detected in 89 (61%) patients with ET, 103 (86%) with PV, four (33%) with myelofibrosis, and four (80%) with MPNu. A higher number of leukocytes, haemoglobin levels and BM cellularity as well as an older age, lower platelet counts, and diagnosis of PV were significantly correlated with JAK2 V617F. Eighty-three and 43 episodes of thrombosis and bleeding occurred in 100 patients each before and after the diagnosis. Vascular events more frequently occurred in 37% of patients with JAK2 V617F than in 29% of those without the mutation (p = 0.045). Among 175 patients whose samples were available for sequencing, 28 patients with homozygous JAK2 V617F had vascular events more frequently (57%) than those who were heterozygotes (39%) or had the wild type (27%) (p = 0.03). The multivariate analysis showed that a JAK2 homozygous mutation, hypercholesterolemia and older age were independent risk factors for a vascular event. The results of this study showed that Korean patients with MPN had a similar JAK2 mutation rate and frequency of vascular events when compared to Western patients. The presence of V617F was significantly related to vascular events. Therefore, initial evaluation for the JAK2 mutation and careful monitoring for vascular events should be performed in MPN patients.

  15. Mutations in the RNA-binding domains of tombusvirus replicase proteins affect RNA recombination in vivo

    International Nuclear Information System (INIS)

    Panaviene, Zivile; Nagy, Peter D.

    2003-01-01

    RNA recombination, which is thought to occur due to replicase errors during viral replication, is one of the major driving forces of virus evolution. In this article, we show evidence that the replicase proteins of Cucumber necrosis virus, a tombusvirus, are directly involved in RNA recombination in vivo. Mutations within the RNA-binding domains of the replicase proteins affected the frequency of recombination observed with a prototypical defective-interfering (DI) RNA, a model template for recombination studies. Five of the 17 replicase mutants tested showed delay in the formation of recombinants when compared to the wild-type helper virus. Interestingly, two replicase mutants accelerated recombinant formation and, in addition, these mutants also increased the level of subgenomic RNA synthesis (Virology 308 (2003), 191-205). A trans-complementation system was used to demonstrate that mutation in the p33 replicase protein resulted in altered recombination rate. Isolated recombinants were mostly imprecise (nonhomologous), with the recombination sites clustered around a replication enhancer region and a putative cis-acting element, respectively. These RNA elements might facilitate the proposed template switching events by the tombusvirus replicase. Together with data in the article cited above, results presented here firmly establish that the conserved RNA-binding motif of the replicase proteins is involved in RNA replication, subgenomic RNA synthesis, and RNA recombination

  16. CP-31398 inhibits the growth of p53-mutated liver cancer cells in vitro and in vivo.

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    He, Xing-Xing; Zhang, Yu-Nan; Yan, Jun-Wei; Yan, Jing-Jun; Wu, Qian; Song, Yu-Hu

    2016-01-01

    The tumor suppressor p53 is one of the most frequently mutated genes in hepatocellular carcinoma (HCC). Previous studies demonstrated that CP-31398 restored the native conformation of mutant p53 and trans-activated p53 downstream genes in tumor cells. However, the research on the application of CP-31398 to liver cancer has not been reported. Here, we investigated the effects of CP-31398 on the phenotype of HCC cells carrying p53 mutation. The effects of CP-31398 on the characteristic of p53-mutated HCC cells were evaluated through analyzing cell cycle, cell apoptosis, cell proliferation, and the expression of p53 downstream genes. In tumor xenografts developed by PLC/PRF/5 cells, the inhibition of tumor growth by CP-31398 was analyzed through gross morphology, growth curve, and the expression of p53-related genes. Firstly, we demonstrated that CP-31398 inhibited the growth of p53-mutated liver cancer cells in a dose-dependent and p53-dependent manner. Then, further study showed that CP-31398 re-activated wild-type p53 function in p53-mutated HCC cells, which resulted in inhibitive response of cell proliferation and an induction of cell-cycle arrest and apoptosis. Finally, in vivo data confirmed that CP-31398 blocked the growth of xenografts tumors through transactivation of p53-responsive downstream molecules. Our results demonstrated that CP-31398 induced desired phenotypic change of p53-mutated HCC cells in vitro and in vivo, which revealed that CP-31398 would be developed as a therapeutic candidate for HCC carrying p53 mutation.

  17. Induction of somatic mutations by low-dose X-rays: the challenge in recognizing radiation-induced events.

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    Nagashima, Haruki; Shiraishi, Kumiko; Ohkawa, Saori; Sakamoto, Yuki; Komatsu, Kenshi; Matsuura, Shinya; Tachibana, Akira; Tauchi, Hiroshi

    2017-10-19

    It is difficult to distinguish radiation-induced events from spontaneous events during induction of stochastic effects, especially in the case of low-dose or low-dose-rate exposures. By using a hypersensitive system for detecting somatic mutations at the HPRT1 locus, we investigated the frequency and spectrum of mutations induced by low-dose X-rays. The mutant frequencies induced by doses of >0.15 Gy were statistically significant when compared with the spontaneous frequency, and a clear dose dependency was also observed for mutant frequencies at doses of >0.15 Gy. In contrast, mutant frequencies at doses of 0.2 Gy. Our observations suggest that there could be a critical dose for mutation induction at between 0.1 Gy and 0.2 Gy, where mutagenic events are induced by multiple DNA double-strand breaks (DSBs). These observations also suggest that low-dose radiation delivered at doses of <0.1 Gy may not result in DSB-induced mutations but may enhance spontaneous mutagenesis events. © The Author 2017. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

  18. Diversity of [beta]-globin mutations in Israeli ethnic groups reflects recent historic events

    Energy Technology Data Exchange (ETDEWEB)

    Filon, D.; Oron, V.; Krichevski, S.; Shaag, A.; Goldfarb, A.; Aker, M.; Rachmilewitz, E.A.; Rund, D.; Oppenheim, A. (Hebrew Univ. Hadassah-Medical School, Jerusalem (Israel)) (and others)

    1994-05-01

    The authors characterized nearly 500 [beta]-thalassemia genes from the Israeli population representing a variety of ethnic subgroups. They found 28 different mutations in the [beta]-globin gene, including three mutations ([beta][sup S], [beta][sup C], and [beta][sup O-Arab]) causing hemoglobinopathies. Marked genetic heterogeneity was observed in both the Arab (20 mutations) and Jewish (17 mutations) populations. On the other hand, two ethnic isolates - Druze and Samaritans - had a single mutation each. Fifteen of the [beta]-thalassemia alleles are Mediterranean in type, 5 originated in Kurdistan, 2 are of Indian origin, and 2 sporadic alleles came from Europe. Only one mutant allele-nonsense codon 37-appears to be indigenous to Israel. While human habitation in Israel dates back to early prehistory, the present-day spectrum of [beta]-globin mutations can be largely explained by migration events that occurred in the past millennium. 26 refs., 2 figs., 3 tabs.

  19. Mutational jackpot events generate effective frequency-dependent selection in adapting populations

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    Hallatschek, Oskar

    The site-frequency spectrum is one the most easily measurable quantities that characterize the genetic diversity of a population. While most neutral models predict that site frequency spectra should decay with increasing frequency, a high-frequency uptick has been reported in many populations. Anomalies in the high-frequency tail are particularly unsettling because the highest frequencies can be measured with greatest accuracy. Here, we show that an uptick in the spectrum of neutral mutations generally arises when mutant frequencies are dominated by rare jackpot events, mutational events with large descendant numbers. This leads to an effective pattern of frequency-dependent selection (or unstable internal equilibrium at one half frequency) that causes an accumulation of high-frequency polymorphic sites. We reproduce the known uptick occurring for recurrent hitchhiking (genetic draft) as well as rapid adaptation, and (in the future) generalize the shape of the high-frequency tail to other scenarios that are dominated by jackpot events, such as frequent range expansions. We also tackle (in the future) the inverse approach to use the high-frequency uptick for learning about the tail of the offspring number distribution. Positively selected alleles need to surpass, typically, an u NSF Career Award (PoLS), NIH NIGMS R01, Simons Foundation.

  20. ''In vivo'' methodology for mutation induction in banana, cultivar ''Maca''

    International Nuclear Information System (INIS)

    Tulmann Neto, A.; Domingues, E.T.; Alvarez, A.L.F.; Mendez, B.M.J.; Ando, A.

    1990-01-01

    Full text: The ''Maca'' cultivar is a banana of high acceptability in the south west of Brazil. However, it is very susceptible to several diseases. Due to the difficulties in the application of the traditional plant breeding methods, the Radiation Genetics Section of CENA is utilising the ''in vivo'', and the ''in vitro'' mutation breeding approach. The ''in vivo'' methodology is based on the work of HAMILTON. This method is being utilised in Brazil for rapid banana propagation. Rhizomes (20 cm diameter) were obtained from young field grown plants before flower differentiation. In these rhizomes, only 5-6 leaf sheaths were retained, the others being removed. The rhizomes were maintained in a greenhouse in boxes with vermiculite, covered with plastic. After one week, all leaf sheaths were removed, until the exposure of the meristematic apex with about 2 mm size. This apex was cut off with a scalpel and a cross shaped cut (2,5 cm) was made. This stimulates the development of lateral buds. After four months, the meristematic apices of these new buds were cut off in the same way and immediately the rhizomes were irradiated with gamma rays. Around the eliminated lateral buds callus developed and new lateral buds were formed. The LD 50 in relation to the number of these new buds produced was around 30 Gy. According to the author of the original method, from the callus one can obtain axillary or adventitious buds. In the early stages it is possible, based on the shape, to distinguish both types. The advantage of utilising adventitious buds originating from only one cell to avoid chimerism is well known in mutation breeding. However, it is not certain whether this is the case in the present method. After detachment from rhizomes and rooting in soil, plants with 15-20 cm height were inoculated with Fusarium oxysporum f.sp. cubense. After 3 weeks the plants showed symptoms of the Panama disease and screening could be done at this stage. The total time between the removal of

  1. ''In vivo'' methodology for mutation induction in banana, cultivar ''Maca''

    Energy Technology Data Exchange (ETDEWEB)

    Tulmann Neto, A; Domingues, E T; Alvarez, A L.F.; Mendez, B M.J.; Ando, A [Centre for Nuclear Energy in Agriculture (CENA), University of Sao Paulo, Piracicaba, SP. (Brazil)

    1990-07-01

    Full text: The ''Maca'' cultivar is a banana of high acceptability in the south west of Brazil. However, it is very susceptible to several diseases. Due to the difficulties in the application of the traditional plant breeding methods, the Radiation Genetics Section of CENA is utilising the ''in vivo'', and the ''in vitro'' mutation breeding approach. The ''in vivo'' methodology is based on the work of HAMILTON. This method is being utilised in Brazil for rapid banana propagation. Rhizomes (20 cm diameter) were obtained from young field grown plants before flower differentiation. In these rhizomes, only 5-6 leaf sheaths were retained, the others being removed. The rhizomes were maintained in a greenhouse in boxes with vermiculite, covered with plastic. After one week, all leaf sheaths were removed, until the exposure of the meristematic apex with about 2 mm size. This apex was cut off with a scalpel and a cross shaped cut (2,5 cm) was made. This stimulates the development of lateral buds. After four months, the meristematic apices of these new buds were cut off in the same way and immediately the rhizomes were irradiated with gamma rays. Around the eliminated lateral buds callus developed and new lateral buds were formed. The LD{sub 50} in relation to the number of these new buds produced was around 30 Gy. According to the author of the original method, from the callus one can obtain axillary or adventitious buds. In the early stages it is possible, based on the shape, to distinguish both types. The advantage of utilising adventitious buds originating from only one cell to avoid chimerism is well known in mutation breeding. However, it is not certain whether this is the case in the present method. After detachment from rhizomes and rooting in soil, plants with 15-20 cm height were inoculated with Fusarium oxysporum f.sp. cubense. After 3 weeks the plants showed symptoms of the Panama disease and screening could be done at this stage. The total time between the

  2. Methylenetetrahydrofolate reductase homozygous mutation in a young boy with cerebellar infarction

    Directory of Open Access Journals (Sweden)

    Alberto Spalice

    2009-11-01

    Full Text Available Posterior circulation vascular occlusive disease in children is a rare and uncommonly reported event. Among the numerous risk factors, the methylenetetrahydrofolate reductase (MTHFR mutation is considered to be a common genetic cause of thrombosis in adults and children. Recently, a link between the MTHFR mutation and cerebrovascular disorders was reported in children. Diffusion tensor imaging (DTI is a great improvement on magnetic resonance imaging (MRI, making the in vivo anatomical and pathological study of the brain and its fibers possible. In our patient cerebellar infarction was associated with MTHFR mutation and, in a standard neurological examination, DTI revealed normal white matter tracts.

  3. Preleukemic and second-hit mutational events in an acute myeloid leukemia patient with a novel germline RUNX1 mutation.

    Science.gov (United States)

    Ng, Isaac Ks; Lee, Joanne; Ng, Christopher; Kosmo, Bustamin; Chiu, Lily; Seah, Elaine; Mok, Michelle Meng Huang; Tan, Karen; Osato, Motomi; Chng, Wee-Joo; Yan, Benedict; Tan, Lip Kun

    2018-01-01

    Germline mutations in the RUNX1 transcription factor give rise to a rare autosomal dominant genetic condition classified under the entity: Familial Platelet Disorders with predisposition to Acute Myeloid Leukaemia (FPD/AML). While several studies have identified a myriad of germline RUNX1 mutations implicated in this disorder, second-hit mutational events are necessary for patients with hereditary thrombocytopenia to develop full-blown AML. The molecular picture behind this process remains unclear. We describe a patient of Malay descent with an unreported 7-bp germline RUNX1 frameshift deletion, who developed second-hit mutations that could have brought about the leukaemic transformation from a pre-leukaemic state. These mutations were charted through the course of the treatment and stem cell transplant, showing a clear correlation between her clinical presentation and the mutations present. The patient was a 27-year-old Malay woman who presented with AML on the background of hereditary thrombocytopenia affecting her father and 3 brothers. Initial molecular testing revealed the same novel RUNX1 mutation in all 5 individuals. The patient received standard induction, consolidation chemotherapy, and a haploidentical stem cell transplant from her mother with normal RUNX1 profile. Comprehensive genomic analyses were performed at diagnosis, post-chemotherapy and post-transplant. A total of 8 mutations ( RUNX1 , GATA2 , DNMT3A , BCORL1 , BCOR , 2 PHF6 and CDKN2A ) were identified in the pre-induction sample, of which 5 remained ( RUNX1 , DNMT3A , BCORL1 , BCOR and 1 out of 2 PHF6 ) in the post-treatment sample and none were present post-transplant. In brief, the 3 mutations which were lost along with the leukemic cells at complete morphological remission were most likely acquired leukemic driver mutations that were responsible for the AML transformation from a pre-leukemic germline RUNX1 -mutated state. On the contrary, the 5 mutations that persisted post

  4. Monitoring mutations in people: an in vivo study of people accidentally or occupationally exposed to ionizing radiation

    International Nuclear Information System (INIS)

    Glickman, B.W.

    1996-01-01

    Recent developments in molecular biology and medicine now permit the monitoring of mutation in humans 'in vivo'. The most commonly used approach, and the main one reported in this paper, is the study of mutations at the hypoxanthine phosphoribosyl transferase (hprt) locus in peripheral T-lymphocytes. This paper deals with evidence from the radiological accident in Goiania, Brazil (where several hundred people were accidentally exposed to cesium-137), from a study of Soviet cosmonauts, and from monozygotic twins. The conclusions from Brazil are: mutation at hprt increases with age and is higher in smokers; in adults a linear dose response was found; no radiation-induced mutational fingerprint was found; children are particularly sensitive; the level of mutation dropped over several years (probably reflecting natural T-cell turnover). The conclusions from cosmonauts are: each cosmonaut had a significantly above-average level of mutation, but this may not be due to radiation at all; no 'fingerprint' was found, and there was no apparent dependence on dose. The study of twins showed a very strong correlation of mutant frequencies between one twin and the other, but this correlation decreased with age, presumably due to environmental effects. 1 tab., 2 figs

  5. Characterisation of Neutropenia-Associated Neutrophil Elastase Mutations in a Murine Differentiation Model In Vitro and In Vivo.

    Directory of Open Access Journals (Sweden)

    Michael Wiesmeier

    Full Text Available Severe congenital neutropenia (SCN is characterised by a differentiation block in the bone marrow and low neutrophil numbers in the peripheral blood, which correlates with increased risk of bacterial infections. Several underlying gene defects have been identified in SCN patients. Mutations in the neutrophil elastase (ELANE gene are frequently found in SCN and cyclic neutropenia. Both mislocalization and misfolding of mutant neutrophil elastase protein resulting in ER stress and subsequent induction of the unfolded protein response (UPR have been proposed to be responsible for neutrophil survival and maturation defects. However, the detailed molecular mechanisms still remain unclear, in part due to the lack of appropriate in vitro and in vivo models. Here we used a system of neutrophil differentiation from immortalised progenitor lines by conditional expression of Hoxb8, permitting the generation of mature near-primary neutrophils in vitro and in vivo. NE-deficient Hoxb8 progenitors were reconstituted with murine and human forms of typical NE mutants representative of SCN and cyclic neutropenia, and differentiation of the cells was analysed in vitro and in vivo. ER stress induction by NE mutations could be recapitulated during neutrophil differentiation in all NE mutant-reconstituted Hoxb8 cells. Despite ER stress induction, no change in survival, maturation or function of differentiating cells expressing either murine or human NE mutants was observed. Further analysis of in vivo differentiation of Hoxb8 cells in a murine model of adoptive transfer did not reveal any defects in survival or differentiation in the mouse. Although the Hoxb8 system has been found to be useful for dissection of defects in neutrophil development, our findings indicate that the use of murine systems for analysis of NE-mutation-associated pathogenesis is complicated by differences between humans and mice in the physiology of granulopoiesis, which may go beyond possible

  6. Evidence that expression of a mutated p53 gene attenuates apoptotic cell death in human gastric intestinal-type carcinomas in vivo.

    Science.gov (United States)

    Ishida, M; Gomyo, Y; Ohfuji, S; Ikeda, M; Kawasaki, H; Ito, H

    1997-05-01

    To examine in vivo the validity of the results of experiments in vitro, we analyzed the relationship between p53 gene status and apoptotic cell death of human gastric intestinal-type adenocarcinomas. Surgical specimens were classified into two categories: 18 gastric cancers with nuclear p53 protein (A), and 17 gastric cancers without nuclear p53 protein (B). Polymerase chain reaction-single strand conformation polymorphism disclosed a shifted band that corresponded to a mutation in the p53 gene in 13 cases (72%) in category A and 3 cases (18%) in category B, the frequency being significantly higher in the former (P terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL). The TUNEL index [TI; (the number of TUNEL-positive apoptotic cells/the total number of tumor cells) x 100] was 3.8 +/- 1.4% in category A and 4.9 +/- 1.2% in category B, the value being significantly lower in the former (P gastric cancer, in accordance with the previous in vitro finding that p53 gene mutation provides a possible selective advantage for tumor cell proliferation, and (2) apoptosis is related not only to expression of p53 and the stage of the cell cycle, but also to p53-independent and cell cycle-independent events.

  7. Mutation breeding in vivo and in vitro in vegetatively propagated crops

    International Nuclear Information System (INIS)

    Tulmann Neto, A.; Latado, R.R.; Tsai, S.M.; Derbyshire, M.T.; Yemma, A.F.; Scarpare Filho, J.A.; Ceravolo, L.; Rossi, A.C.; Namekata, T.; Pompeu, J. Jr.; Figueiredo, J.O.; Pio, R.; Tobias Domingues, E.; Santos, P.C.; Boliani, A.

    2001-01-01

    Mutation breeding in vivo and/or in vitro in vegetatively propagated crops as well as somaclonal variation can be used in Brazil in several crops to increase the genetic variability in characteristics of high importance. This was the objective of this research using ornamentals, citrus and bananas. Somaclonal variants can also be useful in these crops, based on the preliminary results observed in banana (Mycosphaerella musicola); where a short plant variant was selected in Brazil and the mutant resistant to yellow sigatoka, selected in Venezuela, showed resistance also in Brazil. Despite the increase in genetic variability in M 1 V 4 generation obtained after in vitro irradiation of meristems in banana, mutants resistant or tolerant to Fusarium were not selected, perhaps due to the limited number of plants evaluated. In citrus the first results from yield trials showed that following bud irradiation, it was possible to select plants of interest, e.g. mutants with a reduced number of seeds in the fruits. In ornamentals mutants induced by gamma rays in this project were released to the farmers. The results obtained in this research showed that biotechnology is a powerful tool that can be used in several ways in association with mutation breeding. (author)

  8. Splice Site Mutations in the ATP7A Gene

    DEFF Research Database (Denmark)

    Skjørringe, Tina; Tümer, Zeynep; Møller, Lisbeth Birk

    2011-01-01

    Menkes disease (MD) is caused by mutations in the ATP7A gene. We describe 33 novel splice site mutations detected in patients with MD or the milder phenotypic form, Occipital Horn Syndrome. We review these 33 mutations together with 28 previously published splice site mutations. We investigate 12...... mutations for their effect on the mRNA transcript in vivo. Transcriptional data from another 16 mutations were collected from the literature. The theoretical consequences of splice site mutations, predicted with the bioinformatics tool Human Splice Finder, were investigated and evaluated in relation...... to in vivo results. Ninety-six percent of the mutations identified in 45 patients with classical MD were predicted to have a significant effect on splicing, which concurs with the absence of any detectable wild-type transcript in all 19 patients investigated in vivo. Sixty-seven percent of the mutations...

  9. CRISPR/Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting.

    Science.gov (United States)

    Courtney, D G; Moore, J E; Atkinson, S D; Maurizi, E; Allen, E H A; Pedrioli, D M L; McLean, W H I; Nesbit, M A; Moore, C B T

    2016-01-01

    CRISPR/Cas9-based therapeutics hold the possibility for permanent treatment of genetic disease. The potency and specificity of this system has been used to target dominantly inherited conditions caused by heterozygous missense mutations through inclusion of the mutated base in the short-guide RNA (sgRNA) sequence. This research evaluates a novel approach for targeting heterozygous single-nucleotide polymorphisms (SNPs) using CRISPR/Cas9. We determined that a mutation within KRT12, which causes Meesmann's epithelial corneal dystrophy (MECD), leads to the occurrence of a novel protospacer adjacent motif (PAM). We designed an sgRNA complementary to the sequence adjacent to this SNP-derived PAM and evaluated its potency and allele specificity both in vitro and in vivo. This sgRNA was found to be highly effective at reducing the expression of mutant KRT12 mRNA and protein in vitro. To assess its activity in vivo we injected a combined Cas9/sgRNA expression construct into the corneal stroma of a humanized MECD mouse model. Sequence analysis of corneal genomic DNA revealed non-homologous end-joining repair resulting in frame-shifting deletions within the mutant KRT12 allele. This study is the first to demonstrate in vivo gene editing of a heterozygous disease-causing SNP that results in a novel PAM, further highlighting the potential for CRISPR/Cas9-based therapeutics.

  10. Mutations within ICP4 acquired during in vitro attenuation do not alter virulence of recombinant Marek's disease viruses in vivo

    Directory of Open Access Journals (Sweden)

    Evin Hildebrandt

    2015-12-01

    Full Text Available Marek's disease (MD is a T-cell lymphoma of chickens caused by the oncogenic Marek's disease virus (MDV. MD is primarily controlled by live-attenuated vaccines generated by repeated in vitro serial passage. Previous efforts to characterize attenuated MDVs identified numerous mutations, particularly a convergence of high-frequency mutations around amino acids 60–63 within ICP4 (RS1, therefore, ICP4 was considered a candidate gene deserving further characterization. Recombinant MDVs were generated containing a single Q63H mutation or double Q63H + S1630P mutations. Despite the repetitive nature of mutations within ICP4, neither recombinant virus decreased virulence, although one mutant reduced in vivo replication and failed to transmit horizontally. Our results indicate that these mutations are insufficient to reduce disease incidence in infected birds, and suggest that variants in ICP4 do not directly alter virulence, but rather may enhance MDV replication rates in vitro, offering an explanation for the widespread occurrence of ICP4 mutations in a variety of attenuated herpesviruses.

  11. Dynamic of Mutational Events in Variable Number Tandem Repeats of Escherichia coli O157:H7

    Directory of Open Access Journals (Sweden)

    A. V. Bustamante

    2013-01-01

    Full Text Available VNTRs regions have been successfully used for bacterial subtyping; however, the hypervariability in VNTR loci is problematic when trying to predict the relationships among isolates. Since few studies have examined the mutation rate of these markers, our aim was to estimate mutation rates of VNTRs specific for verotoxigenic E. coli O157:H7. The knowledge of VNTR mutational rates and the factors affecting them would make MLVA more effective for epidemiological or microbial forensic investigations. For this purpose, we analyzed nine loci performing parallel, serial passage experiments (PSPEs on 9 O157:H7 strains. The combined 9 PSPE population rates for the 8 mutating loci ranged from 4.4 × 10−05 to 1.8 × 10−03 mutations/generation, and the combined 8-loci mutation rate was of 2.5 × 10−03 mutations/generation. Mutations involved complete repeat units, with only one point mutation detected. A similar proportion between single and multiple repeat changes was detected. Of the 56 repeat mutations, 59% were insertions and 41% were deletions, and 72% of the mutation events corresponded to O157-10 locus. For alleles with up to 13 UR, a constant and low mutation rate was observed; meanwhile longer alleles were associated with higher and variable mutation rates. Our results are useful to interpret data from microevolution and population epidemiology studies and particularly point out that the inclusion or not of O157-10 locus or, alternatively, a differential weighting data according to the mutation rates of loci must be evaluated in relation with the objectives of the proposed study.

  12. Quantification of sequence exchange events between PMS2 and PMS2CL provides a basis for improved mutation scanning of Lynch syndrome patients.

    NARCIS (Netherlands)

    Klift, H.M. van der; Tops, C.M.; Bik, E.C.; Boogaard, M.W.; Borgstein, A.M.; Hansson, K.B.; Ausems, M.G.E.M.; Gomez Garcia, E.; Green, A.; Hes, F.J.; Izatt, L.; Hest, L.P. van; Alonso, A.M.; Vriends, A.H.; Wagner, A.; Zelst-Stams, W.A.G. van; Vasen, H.F.; Morreau, H.; Devilee, P.; Wijnen, J.T.

    2010-01-01

    Heterozygous mutations in PMS2 are involved in Lynch syndrome, whereas biallelic mutations are found in Constitutional mismatch repair-deficiency syndrome patients. Mutation detection is complicated by the occurrence of sequence exchange events between the duplicated regions of PMS2 and PMS2CL. We

  13. In Vivo Modeling of the Pathogenic Effect of Copper Transporter Mutations That Cause Menkes and Wilson Diseases, Motor Neuropathy, and Susceptibility to Alzheimer's Disease.

    Science.gov (United States)

    Mercer, Stephen W; Wang, Jianbin; Burke, Richard

    2017-03-10

    Copper is an essential biometal, and several inherited diseases are directly associated with a disruption to normal copper homeostasis. The best characterized are the copper deficiency and toxicity disorders Menkes and Wilson diseases caused by mutations in the p-type Cu-ATPase genes ATP7A and ATP7B , respectively. Missense mutations in the C-terminal portion of ATP7A have also been shown to cause distal motor neuropathy, whereas polymorphisms in ATP7B are associated with increased risk of Alzheimer's disease. We have generated a single, in vivo model for studying multiple pathogenic mutations in ATP7 proteins using Drosophila melanogaster , which has a single orthologue of ATP7A and ATP7B. Four pathogenic ATP7A mutations and two ATP7B mutations were introduced into a genomic ATP7 rescue construct containing an in-frame C-terminal GFP tag. Analysis of the wild type ATP7-GFP transgene confirmed that ATP7 is expressed at the basolateral membrane of larval midgut copper cells and that the transgene can rescue a normally early lethal ATP7 deletion allele to adulthood. Analysis of the gATP7-GFP transgenes containing pathogenic mutations showed that the function of ATP7 was affected, to varying degrees, by all six of the mutations investigated in this study. Of particular interest, the ATP7B K832R Alzheimer's disease susceptibility allele was found, for the first time, to be a loss of function allele. This in vivo system allows us to assess the severity of individual ATP7A / B mutations in an invariant genetic background and has the potential to be used to screen for therapeutic compounds able to restore function to faulty copper transport proteins. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Hierarchical mutational events compensate for glutamate auxotrophy of a Bacillus subtilis gltC mutant.

    Science.gov (United States)

    Dormeyer, Miriam; Lübke, Anastasia L; Müller, Peter; Lentes, Sabine; Reuß, Daniel R; Thürmer, Andrea; Stülke, Jörg; Daniel, Rolf; Brantl, Sabine; Commichau, Fabian M

    2017-06-01

    Glutamate is the major donor of nitrogen for anabolic reactions. The Gram-positive soil bacterium Bacillus subtilis either utilizes exogenously provided glutamate or synthesizes it using the gltAB-encoded glutamate synthase (GOGAT). In the absence of glutamate, the transcription factor GltC activates expression of the GOGAT genes for glutamate production. Consequently, a gltC mutant strain is auxotrophic for glutamate. Using a genetic selection and screening system, we could isolate and differentiate between gltC suppressor mutants in one step. All mutants had acquired the ability to synthesize glutamate, independent of GltC. We identified (i) gain-of-function mutations in the gltR gene, encoding the transcription factor GltR, (ii) mutations in the promoter of the gltAB operon and (iii) massive amplification of the genomic locus containing the gltAB operon. The mutants belonging to the first two classes constitutively expressed the gltAB genes and produced sufficient glutamate for growth. By contrast, mutants that belong to the third class appeared most frequently and solved glutamate limitation by increasing the copy number of the poorly expressed gltAB genes. Thus, glutamate auxotrophy of a B. subtilis gltC mutant can be relieved in multiple ways. Moreover, recombination-dependent amplification of the gltAB genes is the predominant mutational event indicating a hierarchy of mutations. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  15. Loss of Dnmt3a Immortalizes Hematopoietic Stem Cells In Vivo

    Directory of Open Access Journals (Sweden)

    Mira Jeong

    2018-04-01

    Full Text Available Summary: Somatic mutations in DNMT3A are recurrent events across a range of blood cancers. Dnmt3a loss of function in hematopoietic stem cells (HSCs skews divisions toward self-renewal at the expense of differentiation. Moreover, DNMT3A mutations can be detected in the blood of aging individuals, indicating that mutant cells outcompete normal HSCs over time. It is important to understand how these mutations provide a competitive advantage to HSCs. Here we show that Dnmt3a-null HSCs can regenerate over at least 12 transplant generations in mice, far exceeding the lifespan of normal HSCs. Molecular characterization reveals that this in vivo immortalization is associated with gradual and focal losses of DNA methylation at key regulatory regions associated with self-renewal genes, producing a highly stereotypical HSC phenotype in which epigenetic features are further buttressed. These findings lend insight into the preponderance of DNMT3A mutations in clonal hematopoiesis and the persistence of mutant clones after chemotherapy. : Jeong et al. show that a single genetic manipulation, conditional inactivation of the DNA methyltransferase enzyme Dnmt3a, removes all inherent hematopoietic stem cell (HSC self-renewal limits and replicative lifespan. Deletion of Dnmt3a allows HSCs to be propagated indefinitely in vivo. Keywords: DNMT3A, DNA methylation, HSC, self-renewal, leukemia

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

    International Nuclear Information System (INIS)

    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 Chinese 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 ( 3 H-thymidine, autoradiography) or protein synthesis ( 35 S-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

  17. Quantification of sequence exchange events between PMS2 and PMS2CL provides a basis for improved mutation scanning of Lynch syndrome patients.

    Science.gov (United States)

    van der Klift, Heleen M; Tops, Carli M J; Bik, Elsa C; Boogaard, Merel W; Borgstein, Anne-Marijke; Hansson, Kerstin B M; Ausems, Margreet G E M; Gomez Garcia, Encarna; Green, Andrew; Hes, Frederik J; Izatt, Louise; van Hest, Liselotte P; Alonso, Angel M; Vriends, Annette H J T; Wagner, Anja; van Zelst-Stams, Wendy A G; Vasen, Hans F A; Morreau, Hans; Devilee, Peter; Wijnen, Juul T

    2010-05-01

    Heterozygous mutations in PMS2 are involved in Lynch syndrome, whereas biallelic mutations are found in Constitutional mismatch repair-deficiency syndrome patients. Mutation detection is complicated by the occurrence of sequence exchange events between the duplicated regions of PMS2 and PMS2CL. We investigated the frequency of such events with a nonspecific polymerase chain reaction (PCR) strategy, co-amplifying both PMS2 and PMS2CL sequences. This allowed us to score ratios between gene and pseudogene-specific nucleotides at 29 PSV sites from exon 11 to the end of the gene. We found sequence transfer at all investigated PSVs from intron 12 to the 3' end of the gene in 4 to 52% of DNA samples. Overall, sequence exchange between PMS2 and PMS2CL was observed in 69% (83/120) of individuals. We demonstrate that mutation scanning with PMS2-specific PCR primers and MLPA probes, designed on PSVs, in the 3' duplicated region is unreliable, and present an RNA-based mutation detection strategy to improve reliability. Using this strategy, we found 19 different putative pathogenic PMS2 mutations. Four of these (21%) are lying in the region with frequent sequence transfer and are missed or called incorrectly as homozygous with several PSV-based mutation detection methods. (c) 2010 Wiley-Liss, Inc.

  18. Leu452His mutation in lipoprotein lipase gene transfer associated with hypertriglyceridemia in mice in vivo.

    Directory of Open Access Journals (Sweden)

    Kaiyue Sun

    Full Text Available Mutated mouse lipoprotein lipase (LPL containing a leucine (L to histidine (H substitution at position 452 was transferred into mouse liver by hydrodynamics-based gene delivery (HD. Mutated-LPL (MLPL gene transfer significantly increased the concentrations of plasma MLPL and triglyceride (TG but significantly decreased the activity of plasma LPL. Moreover, the gene transfer caused adiposis hepatica and significantly increased TG content in mouse liver. To understand the effects of MLPL gene transfer on energy metabolism, we investigated the expression of key functional genes related to energy metabolism in the liver, epididymal fat, and leg muscles. The mRNA contents of hormone-sensitive lipase (HSL, adipose triglyceride lipase (ATGL, fatty acid-binding protein (FABP, and uncoupling protein (UCP were found to be significantly reduced. Furthermore, we investigated the mechanism by which MLPL gene transfer affected fat deposition in the liver, fat tissue, and muscle. The gene expression and protein levels of forkhead Box O3 (FOXO3, AMP-activated protein kinase (AMPK, and peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α were found to be remarkably decreased in the liver, fat and muscle. These results suggest that the Leu452His mutation caused LPL dysfunction and gene transfer of MLPL in vivo produced resistance to the AMPK/PGC-1α signaling pathway in mice.

  19. IFITM5 mutations and osteogenesis imperfecta.

    Science.gov (United States)

    Hanagata, Nobutaka

    2016-03-01

    Interferon-induced transmembrane protein 5 (IFITM5) is an osteoblast-specific membrane protein that has been shown to be a positive regulatory factor for mineralization in vitro. However, Ifitm5 knockout mice do not exhibit serious bone abnormalities, and thus the function of IFITM5 in vivo remains unclear. Recently, a single point mutation (c.-14C>T) in the 5' untranslated region of IFITM5 was identified in patients with osteogenesis imperfecta type V (OI-V). Furthermore, a single point mutation (c.119C>T) in the coding region of IFITM5 was identified in OI patients with more severe symptoms than patients with OI-V. Although IFITM5 is not directly involved in the formation of bone in vivo, the reason why IFITM5 mutations cause OI remains a major mystery. In this review, the current state of knowledge of OI pathological mechanisms due to IFITM5 mutations will be reviewed.

  20. TERT promoter mutation as an early genetic event activating telomerase in follicular thyroid adenoma (FTA) and atypical FTA.

    Science.gov (United States)

    Wang, Na; Liu, Tiantian; Sofiadis, Anastasios; Juhlin, C Christofer; Zedenius, Jan; Höög, Anders; Larsson, Catharina; Xu, Dawei

    2014-10-01

    The telomerase reverse transcriptase (TERT) promoter mutations C228T and C250T have been found in many malignancies, including in thyroid carcinomas. However, it is unclear how early these mutations occur in thyroid tumorigenesis. The study included primary tumors from 58 patients initially diagnosed with follicular thyroid adenoma (FTA), a benign entity, 18 with atypical FTA (AFTA) having an uncertain malignant potential, and 52 with follicular thyroid carcinoma (FTC). Sanger sequencing was used to investigate the mutational status of the TERT promoter. Telomere length and TERT messenger RNA (mRNA) expression were determined using quantitative polymerase chain reaction (PCR). Telomerase activity was assessed using a Telomerase PCR enzyme-linked immunosorbent assay kit. The C228T mutation was identified in 1 of 58 FTA (2%) and 3 of 18 AFTA (17%) samples. These 4 tumors all expressed TERT mRNA and telomerase activity, whereas the majority of C228T-negative adenomas lacked TERT expression (C228T versus wild-type, P = .008). The C228T mutation was associated with NRAS gene mutations (P = .016). The patient with C228T-mutated FTA later developed a scar recurrence and died of FTC, whereas none of the remaining 57 patients with FTA had recurrence. No recurrence occurred in 3 patients with AFTA who carried C228T during the follow-up period (36-285 months). Nine of the 52 FTCs (17%) exhibited the TERT mutation (8 of 9 C228T and 1 of 9 C250T), and the presence of the mutation was associated with shorter patient survival. TERT promoter mutations may occur as an early genetic event in thyroid follicular tumors that have not developed malignant features on routine histopathological workup. © 2014 American Cancer Society.

  1. CP-31398 prevents the growth of p53-mutated colorectal cancer cells in vitro and in vivo.

    Science.gov (United States)

    He, Xingxing; Kong, Xinjuan; Yan, Junwei; Yan, Jingjun; Zhang, Yunan; Wu, Qian; Chang, Ying; Shang, Haitao; Dou, Qian; Song, Yuhu; Liu, Fang

    2015-03-01

    Rescuing the function of mutant p53 protein is an attractive cancer therapeutic strategy. Small molecule CP-31398 was shown to restore mutant p53 tumor suppressor functions in cancer cells. Here, we determined the effects of CP-31398 on the growth of p53-mutated colorectal cancer (CRC) cells in vitro and in vivo. CRC cells which carry p53 mutation in codon 273 were treated with CP-31398 and the control, and the effects of CP-31398 on cell cycle, cell apoptosis, and proliferation were determined. The expression of p53-responsive downstream genes was evaluated by quantitative reverse transcriptase PCR (RT-PCR) and Western blot. CP-31398 was administrated into xenograft tumors created by the inoculation of HT-29 cells, and then the effect of CP-31398 on the growth of xenograft tumors was examined. CP-31398 induced p53 downstream target molecules in cultured HT-29 cells, which resulted in the inhibition of CRC cell growth assessed by the determination of cell cycle, apoptosis, and cell proliferation. In xenograft tumors, CP-31398 modulated the expression of Bax, Bcl-2, caspase 3, cyclin D, and Mdm2 and then blocked the growth of xenograft tumors. CP-31398 would be developed as a therapeutic candidate for p53-mutated CRC due to the restoration of mutant p53 tumor suppressor functions.

  2. UVA-induced mutational spectra in the laci gene from transgenic mouse skin

    International Nuclear Information System (INIS)

    Gorelick, N.J.; O'Kelly, J.A.; Biedermann, K.A.

    1995-01-01

    The UVB (295-320 nm) component of sunlight was once thought to be the sole cause of photoaging and skin cancer. However, there is now compelling evidence to suggest that chronic irradiation with UVA (320-400 nm) is a significant component of the etiologies of these diseases. To identify acute markers of UVA damage, we investigated UVA-induced mutagenesis in vivo by using a lacI transgenic mouse mutation assay. The backs of adult female C57BL/6 Big Blue reg-sign mice were shaved and exposed daily to a low or a high dose of UVA for 5 consecutive days. One group remained unexposed. The high dose of UVA significantly increased the mutant frequency in skin determined 12 days after the last exposure. Mutant frequencies were (Avg ± SEM, n=7-8/group): 6.1 ± 0.5 x 10 -5 (high dose). DNA sequence analysis of mutant lacI genes demonstrated that the high dose of UVA produced a different mutational spectrum compared to control. The mutational spectrum from the low dose mutants was not different from the control spectrum in skin generated previously; the predominant classes of recovered mutations were GC→At transitions at CpG sites (11/35) and GC →TA transversions (12/35). In contrast, in the high dose group, GC →AT transitions at non-CpG sites predominated (61/97 mutations); three tandem base substitutions (1 GG →AA; 2 CC→TT) were uniquely recovered; and an increased frequency of recovered GC→CG substitutions was observed (12/97 vs. none in controls). The recovered high dose spectrum is consistent with the types of DNA damage generated by UVA as well as by reactive oxygen species. These studies demonstrate that UVA is mutagenic in vivo and that this assay can be used to study early events in UVA-induced skin damage

  3. Analysis of clustered point mutations in the human ribosomal RNA gene promoter by transient expression in vivo

    International Nuclear Information System (INIS)

    Jones, M.H.; Learned, R.M.; Tjian, R.

    1988-01-01

    The authors have mapped the cis regulatory elements required in vivo for initiation at the human rRNA promoter by RNA polymerase I. Transient expression in COS-7 cells was used to evaluate the transcription phenotype of clustered base substitution mutations in the human rRNA promoter. The promoter consists of two major elements: a large upstream region, composed of several domains, that lies between nucleotides -234 and -107 relative to the transcription initiation site and affects transcription up to 100-fold and a core element that lies between nucleotides -45 and +20 and affects transcription up to 1000-fold. The upstream regions is able to retain partial function when positioned within 100-160 nucleotides of the transcription initiation site, but it cannot stimulate transcription from distances of ≥ 600 nucleotides. In addition, they demonstrate, using mouse-human hybrid rRNA promoters, that the sequences responsible for human species-specific transcription in vivo appear to reside in both the core and upstream elements, and sequences from the mouse rRNA promoter cannot be substituted for them

  4. Cre recombinase activity is inhibited in vivo but not ex vivo by a mutation in the asymmetric spacer region of the distal loxP site.

    Science.gov (United States)

    Arguello, Tania; Moraes, Carlos T

    2015-11-01

    The cre/loxP recombination system is a valuable tool used to generate tissue specific genomic rearrangements in mouse models. The deletion of a region of interest flanked by two loxP sites is accomplished by the recombinase (cre) enzyme, which binds to the inverted repeat segments of two loxP sites and recognition of a conserved TA sequence in the asymmetric central spacer region "ATAACTTCGTATA -NNNTANNN-TATACGAAGTTAT. In vivo, we found that a single T to C mutation at position 4 of the central spacer region in the distal (3') loxP site, completely inhibited the recombination reaction in two conditional mouse models. These mice were generated using a mitochondrial methionyl-tRNA formyltransferase (Mtfmt) gene targeted construct and cre transgene under the control of tissue-specific promoters: calcium/calmodulin-dependent kinase II alpha (Camk2a-cre) and myosin light polypeptide 1 (Myl1-cre). Surprisingly, transient transfection of a plasmid expressing cre in dermal fibroblasts derived from the same mutant floxed Mtfmt((loxP/loxP)) mice line, successfully deleted the region of interest. This study demonstrates the sequence specificity required in vivo, the possibility of bypassing this limitation by expressing high levels of cre recombinase ex vivo and raises concerns related to the quality control of large scale production of gene targeted constructs and mice. genesis 53:695-700, 2015. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  5. Partial rescue of in vivo insulin signalling in skeletal muscle by impaired insulin clearance in heterozygous carriers of a mutation in the insulin receptor gene

    DEFF Research Database (Denmark)

    Højlund, K.; Wojtaszewski, Jørgen; Birk, Jesper Bratz

    2006-01-01

    AIMS/HYPOTHESIS: Recently we reported the coexistence of postprandial hypoglycaemia and moderate insulin resistance in heterozygous carriers of the Arg1174Gln mutation in the insulin receptor gene (INSR). Controlled studies of in vivo insulin signalling in humans with mutant INSR are unavailable,...

  6. Gamma-ray mutagenesis of cultured mammalian cells in vitro and in vivo

    International Nuclear Information System (INIS)

    Suzuki, Norio; Okada, Shigefumi

    1977-01-01

    The in vitro assay system used to study the reversion of L5178Y-Ala32 cells from an alanine requiring state to a non-requiring state, has been modified in order to be of use in selected in vivo systems. Gamma-ray induced mutations were compared between cells cultured in vitro and those grown in vivo in the intraperitoneal cavity of mice. The expression time was chosen to be 2 days for cells grown in vitro and 5 days for those grown in vivo. The dose-response curve can be described as cumulative for cells grown in vitro and linear for those grown in vivo. A doserate effect was observed in both systems. The cells grown in vivo were less sensitive to γ-rays with respect to both mutation rate per rad and cell killing as compared to cells grown in vitro. The delayed expression and reduced sensitivity of cells in vivo with respect to induced mutation may be due to factors such as hypoxia and/or reduced availability of essential nutrients. Sensitization in vitro by BUdR was detectable at a concentration as low as 10 -6 M, using an exposure time of 15 h. Under these conditions, BUdR alone did not induce any observable mutations

  7. A pan-cancer analysis of transcriptome changes associated with somatic mutations in U2AF1 reveals commonly altered splicing events.

    Directory of Open Access Journals (Sweden)

    Angela N Brooks

    Full Text Available Although recurrent somatic mutations in the splicing factor U2AF1 (also known as U2AF35 have been identified in multiple cancer types, the effects of these mutations on the cancer transcriptome have yet to be fully elucidated. Here, we identified splicing alterations associated with U2AF1 mutations across distinct cancers using DNA and RNA sequencing data from The Cancer Genome Atlas (TCGA. Using RNA-Seq data from 182 lung adenocarcinomas and 167 acute myeloid leukemias (AML, in which U2AF1 is somatically mutated in 3-4% of cases, we identified 131 and 369 splicing alterations, respectively, that were significantly associated with U2AF1 mutation. Of these, 30 splicing alterations were statistically significant in both lung adenocarcinoma and AML, including three genes in the Cancer Gene Census, CTNNB1, CHCHD7, and PICALM. Cell line experiments expressing U2AF1 S34F in HeLa cells and in 293T cells provide further support that these altered splicing events are caused by U2AF1 mutation. Consistent with the function of U2AF1 in 3' splice site recognition, we found that S34F/Y mutations cause preferences for CAG over UAG 3' splice site sequences. This report demonstrates consistent effects of U2AF1 mutation on splicing in distinct cancer cell types.

  8. The development of (new) in vivo and in vitro techniques of significance for mutation breeding of vegetatively propagated crops

    International Nuclear Information System (INIS)

    Broertjes, C.

    1975-01-01

    Mutation breeding in vegetatively propagated plants is of great potential value 1) to improve the leading results of cross-breeding by altering one or a few important characters, without the rest of the genotype, 2) to induce variability where none is existing or difficult to be introduced in highly developed species and 3) to induce variability in sterile crops or in apomicts. One of the main stumbling-blocks is the chimera formation following the irradiation of the multicellular apices in buds and the subsequent prolonged time and increased labour needed before a mutation can be detected, recovered and compared with the existing cultivars. This problem can be solved by producing plants, ultimately originating from one mutated cell, resulting in solid mutants. The in vivo adventitious bud technique, using detached leaves, has proven its value for mutation breeding. It has been demonstrated in several species that commercial results can be obtained in a relatively short time. Experiments are underway to study the factors which control the process of adventitious bud formation and to make more crops accessible to this method. So far, however, with little success. Many and increasingly more crops can be propagated clonally by in vitro methods, using plant parts (explants of leaves, flowers, flower stalk), callus or other plant material. In some cases it is expected that adventitious plantlets also will originate from one cell. In other cases it is to be investigated which method is of potential value for being used in a mutation breeding programme. In a cooperative project (C. Broertjes, S. Roest and Miss G.S. Bokelmann) it is under investigation which plant part (young flowerheads, flower stalks and leaves) is to be preferred in Chrysanthemum morifolium. Preliminary results will be presented at the meeting. (author)

  9. Precise estimates of mutation rate and spectrum in yeast

    Science.gov (United States)

    Zhu, Yuan O.; Siegal, Mark L.; Hall, David W.; Petrov, Dmitri A.

    2014-01-01

    Mutation is the ultimate source of genetic variation. The most direct and unbiased method of studying spontaneous mutations is via mutation accumulation (MA) lines. Until recently, MA experiments were limited by the cost of sequencing and thus provided us with small numbers of mutational events and therefore imprecise estimates of rates and patterns of mutation. We used whole-genome sequencing to identify nearly 1,000 spontaneous mutation events accumulated over ∼311,000 generations in 145 diploid MA lines of the budding yeast Saccharomyces cerevisiae. MA experiments are usually assumed to have negligible levels of selection, but even mild selection will remove strongly deleterious events. We take advantage of such patterns of selection and show that mutation classes such as indels and aneuploidies (especially monosomies) are proportionately much more likely to contribute mutations of large effect. We also provide conservative estimates of indel, aneuploidy, environment-dependent dominant lethal, and recessive lethal mutation rates. To our knowledge, for the first time in yeast MA data, we identified a sufficiently large number of single-nucleotide mutations to measure context-dependent mutation rates and were able to (i) confirm strong AT bias of mutation in yeast driven by high rate of mutations from C/G to T/A and (ii) detect a higher rate of mutation at C/G nucleotides in two specific contexts consistent with cytosine methylation in S. cerevisiae. PMID:24847077

  10. Ex-vivo assessment and non-invasive in vivo imaging of internal hemorrhages in Aga2/+ mutant mice

    Energy Technology Data Exchange (ETDEWEB)

    Ermolayev, Vladimir [Institute for Biological and Medical Imaging, Helmholtz Zentrum München, Building 56, Ingolstädter Landstraße 1, D-85764 Neuherberg (Germany); Cohrs, Christian M. [Institute for Experimental Genetics, Helmholtz Zentrum München, Ingolstädter Landstraße 1, D-85764 Neuherberg (Germany); Mohajerani, Pouyan; Ale, Angelique [Institute for Biological and Medical Imaging, Helmholtz Zentrum München, Building 56, Ingolstädter Landstraße 1, D-85764 Neuherberg (Germany); Hrabé de Angelis, Martin [Institute for Experimental Genetics, Helmholtz Zentrum München, Ingolstädter Landstraße 1, D-85764 Neuherberg (Germany); Ntziachristos, Vasilis, E-mail: v.ntziachristos@tum.de [Institute for Biological and Medical Imaging, Helmholtz Zentrum München, Building 56, Ingolstädter Landstraße 1, D-85764 Neuherberg (Germany)

    2013-03-08

    Highlights: ► Aga2/+ mice, model for Osteogenesis imperfecta, have type I collagen mutation. ► Aga2/+ mice display both moderate and severe phenotypes lethal 6–11th postnatal. ► Internal hemorrhages studied in Aga2/+ vs. control mice at 6 and 9 days postnatal. ► Anatomical and functional findings in-vivo contrasted to the ex-vivo appearance. -- Abstract: Mutations in type I collagen genes (COL1A1/2) typically lead to Osteogenesis imperfecta, the most common heritable cause of skeletal fractures and bone deformation in humans. Heterozygous Col1a1{sup Aga2/+}, animals with a dominant mutation in the terminal C-propeptide domain of type I collagen develop typical skeletal hallmarks and internal hemorrhages starting from 6 day after birth. The disease progression for Aga2/+ mice, however, is not uniform differing between severe phenotype lethal at the 6–11th day of life, and moderate-to-severe one with survival to adulthood. Herein we investigated whether a new modality that combines X-ray computer tomography with fluorescence tomography in one hybrid system can be employed to study internal bleedings in relation to bone fractures and obtain insights into disease progression. The disease phenotype was characterized on Aga2/+ vs. wild type mice between 6 and 9 days postnatal. Anatomical and functional findings obtained in-vivo were contrasted to the ex-vivo appearance of the same tissues under cryo-slicing.

  11. Ex-vivo assessment and non-invasive in vivo imaging of internal hemorrhages in Aga2/+ mutant mice

    International Nuclear Information System (INIS)

    Ermolayev, Vladimir; Cohrs, Christian M.; Mohajerani, Pouyan; Ale, Angelique; Hrabé de Angelis, Martin; Ntziachristos, Vasilis

    2013-01-01

    Highlights: ► Aga2/+ mice, model for Osteogenesis imperfecta, have type I collagen mutation. ► Aga2/+ mice display both moderate and severe phenotypes lethal 6–11th postnatal. ► Internal hemorrhages studied in Aga2/+ vs. control mice at 6 and 9 days postnatal. ► Anatomical and functional findings in-vivo contrasted to the ex-vivo appearance. -- Abstract: Mutations in type I collagen genes (COL1A1/2) typically lead to Osteogenesis imperfecta, the most common heritable cause of skeletal fractures and bone deformation in humans. Heterozygous Col1a1 Aga2/+ , animals with a dominant mutation in the terminal C-propeptide domain of type I collagen develop typical skeletal hallmarks and internal hemorrhages starting from 6 day after birth. The disease progression for Aga2/+ mice, however, is not uniform differing between severe phenotype lethal at the 6–11th day of life, and moderate-to-severe one with survival to adulthood. Herein we investigated whether a new modality that combines X-ray computer tomography with fluorescence tomography in one hybrid system can be employed to study internal bleedings in relation to bone fractures and obtain insights into disease progression. The disease phenotype was characterized on Aga2/+ vs. wild type mice between 6 and 9 days postnatal. Anatomical and functional findings obtained in-vivo were contrasted to the ex-vivo appearance of the same tissues under cryo-slicing

  12. Crop improvement by using radiation mutation breeding in Korea

    International Nuclear Information System (INIS)

    Lee, Young IL

    1998-01-01

    For crop improvement by the application of radiation technology, induction of mutants by in vivo and in vitro mutagenesis were developed in various crop plants in Korea. Several mutants have been released as recommended cultivars to farmers in rice, soybean, sesame and barley since 1970. Induced mutations were widely used for the introduction of genetic transformation and extending plant genetic resources. High yield, short plant, earliness, resistance to diseases, high protein and oil contents were obtained in the advanced generation of mutation by radiation application to several crops of in vivo and in vitro cultured materials. For induction and selection of promising mutants by in vivo and in vitro mutagenesis, various crops were successively irradiated with radiation to investigate the radiosensitivities, the mutation spectrum and mutation rate for selection of useful mutants. Plant tissue culture methods were developed for in vitro mutagenesis in the seed and the vegetatively propagating crops. Embryogenic callus was obtained from shoot tip culture of sweet potato, and micro propagation was developed from nodal stem culture of potato. The radiosensitivities were investigated in cell, callus, and in vitro plant lets. About 800 lines of mutants were evaluated for the agro-genetic resources. (author). 19 refs., 5 tabs

  13. In Vivo-Selected Compensatory Mutations Restore the Fitness Cost of Mosaic penA Alleles That Confer Ceftriaxone Resistance in Neisseria gonorrhoeae.

    Science.gov (United States)

    Vincent, Leah R; Kerr, Samuel R; Tan, Yang; Tomberg, Joshua; Raterman, Erica L; Dunning Hotopp, Julie C; Unemo, Magnus; Nicholas, Robert A; Jerse, Ann E

    2018-04-03

    Resistance to ceftriaxone in Neisseria gonorrhoeae is mainly conferred by mosaic penA alleles that encode penicillin-binding protein 2 (PBP2) variants with markedly lower rates of acylation by ceftriaxone. To assess the impact of these mosaic penA alleles on gonococcal fitness, we introduced the mosaic penA alleles from two ceftriaxone-resistant (Cro r ) clinical isolates (H041 and F89) into a Cro s strain (FA19) by allelic exchange and showed that the resultant Cro r mutants were significantly outcompeted by the Cro s parent strain in vitro and in a murine infection model. Four Cro r compensatory mutants of FA19 penA41 were isolated independently from mice that outcompeted the parent strain both in vitro and in vivo One of these compensatory mutants (LV41C) displayed a unique growth profile, with rapid log growth followed by a sharp plateau/gradual decline at stationary phase. Genome sequencing of LV41C revealed a mutation (G348D) in the acnB gene encoding the bifunctional aconitate hydratase 2/2 methylisocitrate dehydratase. Introduction of the acnB G348D allele into FA19 penA41 conferred both a growth profile that phenocopied that of LV41C and a fitness advantage, although not as strongly as that exhibited by the original compensatory mutant, suggesting the existence of additional compensatory mutations. The mutant aconitase appears to be a functional knockout with lower activity and expression than wild-type aconitase. Transcriptome sequencing (RNA-seq) analysis of FA19 penA41 acnB G348D revealed a large set of upregulated genes involved in carbon and energy metabolism. We conclude that compensatory mutations can be selected in Cro r gonococcal strains that increase metabolism to ameliorate their fitness deficit. IMPORTANCE The emergence of ceftriaxone-resistant (Cro r ) Neisseria gonorrhoeae has led to the looming threat of untreatable gonorrhea. Whether Cro resistance is likely to spread can be predicted from studies that compare the relative fitnesses of

  14. Domain-restricted mutation analysis to identify novel driver events in human cancer

    Directory of Open Access Journals (Sweden)

    Sanket Desai

    2017-10-01

    Full Text Available Analysis of mutational spectra across various cancer types has given valuable insights into tumorigenesis. Different approaches have been used to identify novel drivers from the set of somatic mutations, including the methods which use sequence conservation, geometric localization and pathway information. Recent computational methods suggest use of protein domain information for analysis and understanding of the functional consequence of non-synonymous mutations. Similarly, evidence suggests recurrence at specific position in proteins is robust indicators of its functional impact. Building on this, we performed a systematic analysis of TCGA exome derived somatic mutations across 6089 PFAM domains and significantly mutated domains were identified using randomization approach. Multiple alignment of individual domain allowed us to prioritize for conserved residues mutated at analogous positions across different proteins in a statistically disciplined manner. In addition to the known frequently mutated genes, this analysis independently identifies low frequency Meprin and TRAF-Homology (MATH domain in Speckle Type BTB/POZ (SPOP protein, in prostate adenocarcinoma. Results from this analysis will help generate hypotheses about the downstream molecular mechanism resulting in cancer phenotypes.

  15. Antigen-specific primed cytotoxic T cells eliminate tumour cells in vivo and prevent tumour development, regardless of the presence of anti-apoptotic mutations conferring drug resistance.

    Science.gov (United States)

    Jaime-Sánchez, Paula; Catalán, Elena; Uranga-Murillo, Iratxe; Aguiló, Nacho; Santiago, Llipsy; M Lanuza, Pilar; de Miguel, Diego; A Arias, Maykel; Pardo, Julián

    2018-05-09

    Cytotoxic CD8 + T (Tc) cells are the main executors of transformed and cancer cells during cancer immunotherapy. The latest clinical results evidence a high efficacy of novel immunotherapy agents that modulate Tc cell activity against bad prognosis cancers. However, it has not been determined yet whether the efficacy of these treatments can be affected by selection of tumoural cells with mutations in the cell death machinery, known to promote drug resistance and cancer recurrence. Here, using a model of prophylactic tumour vaccination based on the LCMV-gp33 antigen and the mouse EL4 T lymphoma, we analysed the molecular mechanism employed by Tc cells to eliminate cancer cells in vivo and the impact of mutations in the apoptotic machinery on tumour development. First of all, we found that Tc cells, and perf and gzmB are required to efficiently eliminate EL4.gp33 cells after LCMV immunisation during short-term assays (1-4 h), and to prevent tumour development in the long term. Furthermore, we show that antigen-pulsed chemoresistant EL4 cells overexpressing Bcl-X L or a dominant negative form of caspase-3 are specifically eliminated from the peritoneum of infected animals, as fast as parental EL4 cells. Notably, antigen-specific Tc cells control the tumour growth of the mutated cells, as efficiently as in the case of parental cells. Altogether, expression of the anti-apoptotic mutations does not confer any advantage for tumour cells neither in the short-term survival nor in long-term tumour formation. Although the mechanism involved in the elimination of the apoptosis-resistant tumour cells is not completely elucidated, neither necroptosis nor pyroptosis seem to be involved. Our results provide the first experimental proof that chemoresistant cancer cells with mutations in the main cell death pathways are efficiently eliminated by Ag-specific Tc cells in vivo during immunotherapy and, thus, provide the molecular basis to treat chemoresistant cancer cells with CD8 Tc

  16. Discrimination of three mutational events that result in a disruption of the R122 primary autolysis site of the human cationic trypsinogen (PRSS1 by denaturing high performance liquid chromatography

    Directory of Open Access Journals (Sweden)

    Férec Claude

    2001-11-01

    Full Text Available Abstract Background R122, the primary autolysis site of the human cationic trypsinogen (PRSS1, constitutes an important "self-destruct" or "fail-safe" defensive mechanism against premature trypsin activation within the pancreas. Disruption of this site by a missense mutation, R122H, was found to cause hereditary pancreatitis. In addition to a c.365G>A (CGC>CAC single nucleotide substitution, a c.365~366GC>AT (CGC>CAT gene conversion event in exon 3 of PRSS1 was also found to result in a R122H mutation. This imposes a serious concern on the genotyping of pancreatitis by a widely used polymerase chain reaction-restriction fragment length polymorphism assay, which could only detect the commonest c.365G>A variant. Materials and methods DNA samples containing either the known c.365G>A or c.365~366GC>AT variant in exon 3 of PRSS1 were used as positive controls to establish a denaturing high performance liquid chromatography (DHPLC assay. Results DHPLC could readily discriminate the two known different mutational events resulting in the R122H mutation. More importantly, under the same experimental conditions, it identified a further mutational event that also occurs in the R122 primary autolysis site but results in a different amino acid substitution: c.364C>T (CGC>TGC; R122C. Conclusions A rapid, simple, and low-cost assay for detecting both the known and new mutations occuring in the R122 primary autolysis site of PRSS1 was established. In addition, the newly found R122C variant represents a likely pancreatitis-predisposing mutation.

  17. Biodosimetry of Chernobyl cleanup workers from Estonia and Latvia using the glycophorin A in vivo somatic cell mutation assay

    International Nuclear Information System (INIS)

    Bigbee, W.L.; Jensen, R.H.; Veidebaum, T.

    1997-01-01

    The reactor accident at Chernobyl in 1986 necessitated a massive environmental cleanup that involved over 600,000 workers from all 15 Republics of the former Soviet Union. To determine whether the whole-body radiation received by workers in the course of these decontamination activities resulted in a detectable biological response, over 1,500 blood samples were obtained from cleanup workers sent from two Baltic countries, Estonia and Latvia. Here we report the results of studies of biodosimetry using the glycophorin A (GPA) locus in vivo somatic cell mutation assay applied to 734 blood samples from these workers, to 51 control samples from unexposed Baltic populations and to 94 samples from historical U.S. controls. The data reveal inconsistent evidence that the protracted radiation exposures received by these workers resulted in a significant dose-associated increase in GPA locus mutations compared with the controls. Taken together, these data suggest that the average radiation exposure to these workers does not greatly exceed 10 cGy, the minimum levels at which radiation effects might be detectable by the assay. Although the protracted nature of the exposure may have reduced the efficiency of induction of GPA locus mutations, it is likely that the estimated physical doses for these cleanup worker populations (median reported dose 9.5 cGy) were too low to result in radiation damage to erythroid stem cells that can be detected reliably by this method. 25 refs., 2 figs., 3 tabs

  18. Temporal mechanically-induced signaling events in bone and dorsal root ganglion neurons after in vivo bone loading.

    Directory of Open Access Journals (Sweden)

    Jason A Bleedorn

    Full Text Available Mechanical signals play an integral role in the regulation of bone mass and functional adaptation to bone loading. The osteocyte has long been considered the principle mechanosensory cell type in bone, although recent evidence suggests the sensory nervous system may play a role in mechanosensing. The specific signaling pathways responsible for functional adaptation of the skeleton through modeling and remodeling are not clearly defined. In vitro studies suggest involvement of intracellular signaling through mitogen-activated protein kinase (MAPK, phosphatidylinositol 3-kinase (PI3K/protein kinase B (Akt, and mammalian target of rapamycin (mTOR. However, anabolic signaling responses to bone loading using a whole animal in vivo model have not been studied in detail. Therefore, we examined mechanically-induced signaling events at five time points from 0 to 24 hours after loading using the rat in vivo ulna end-loading model. Western blot analysis of bone for MAPK's, PI3K/Akt, and mTOR signaling, and quantitative reverse transcription polymerase chain reaction (qRT-PCR to estimate gene expression of calcitonin gene-related protein alpha (CGRP-α, brain-derived neurotrophic factor (BDNF, nerve growth factor (NGF, c-jun, and c-fos in dorsal root ganglion (DRG of the brachial intumescence were performed. There was a significant increase in signaling through MAPK's including extracellular signal-related kinase (ERK and c-Jun N-terminal kinase (JNK in loaded limbs at 15 minutes after mechanical loading. Ulna loading did not significantly influence expression of the genes of interest in DRG neurons. Bone signaling and DRG gene expression from the loaded and contralateral limbs was correlated (SR>0.40, P<0.05. However, bone signaling did not correlate with expression of the genes of interest in DRG neurons. These results suggest that signaling through the MAPK pathway may be involved in load-induced bone formation in vivo. Further characterization of the

  19. Endophilin mutations block clathrin-mediated endocytosis but not neurotransmitter release

    DEFF Research Database (Denmark)

    Verstreken, Patrik; Kjaerulff, Ole; Lloyd, Thomas E

    2002-01-01

    We have identified mutations in Drosophila endophilin to study its function in vivo. Endophilin is required presynaptically at the neuromuscular junction, and absence of Endophilin dramatically impairs endocytosis in vivo. Mutant larvae that lack Endophilin fail to take up FM1-43 dye in synaptic ...

  20. In-Vivo Proton Magnetic Resonance Spectroscopy of 2-Hydroxyglutarate in Isocitrate Dehydrogenase-Mutated Gliomas: A Technical Review for Neuroradiologists

    International Nuclear Information System (INIS)

    Kim, Hyeonjin; Kim, Sungjin; Lee, Hyeong Hun; Heo, Hwon

    2016-01-01

    The diagnostic and prognostic potential of an onco-metabolite, 2-hydroxyglutarate (2HG) as a proton magnetic resonance spectroscopy (1H-MRS) detectable biomarker of the isocitrate dehydrogenase (IDH)-mutated (IDH-MT) gliomas has drawn attention of neuroradiologists recently. However, due to severe spectral overlap with background signals, quantification of 2HG can be very challenging. In this technical review for neuroradiologists, first, the biochemistry of 2HG and its significance in the diagnosis of IDH-MT gliomas are summarized. Secondly, various 1H-MRS methods used in the previous studies are outlined. Finally, wereview previous in vivo studies, and discuss the current status of 1H-MRS in the diagnosis of IDH-MT gliomas

  1. In-Vivo Proton Magnetic Resonance Spectroscopy of 2-Hydroxyglutarate in Isocitrate Dehydrogenase-Mutated Gliomas: A Technical Review for Neuroradiologists

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hyeonjin [Department of Radiology, Seoul National University Hospital, Seoul 03080 (Korea, Republic of); Department of Biomedical Sciences, Seoul National University, Seoul 03087 (Korea, Republic of); Institute of Radiation Medicine, Seoul National University Medical Research Center, Seoul 03080 (Korea, Republic of); Kim, Sungjin [Department of Radiology, Seoul National University Hospital, Seoul 03080 (Korea, Republic of); Lee, Hyeong Hun; Heo, Hwon [Department of Biomedical Sciences, Seoul National University, Seoul 03087 (Korea, Republic of)

    2016-11-01

    The diagnostic and prognostic potential of an onco-metabolite, 2-hydroxyglutarate (2HG) as a proton magnetic resonance spectroscopy (1H-MRS) detectable biomarker of the isocitrate dehydrogenase (IDH)-mutated (IDH-MT) gliomas has drawn attention of neuroradiologists recently. However, due to severe spectral overlap with background signals, quantification of 2HG can be very challenging. In this technical review for neuroradiologists, first, the biochemistry of 2HG and its significance in the diagnosis of IDH-MT gliomas are summarized. Secondly, various 1H-MRS methods used in the previous studies are outlined. Finally, wereview previous in vivo studies, and discuss the current status of 1H-MRS in the diagnosis of IDH-MT gliomas.

  2. A deployable in vivo EPR tooth dosimeter for triage after a radiation event involving large populations

    International Nuclear Information System (INIS)

    Williams, Benjamin B.; Dong, Ruhong; Flood, Ann Barry; Grinberg, Oleg; Kmiec, Maciej; Lesniewski, Piotr N.; Matthews, Thomas P.; Nicolalde, Roberto J.; Raynolds, Tim; Salikhov, Ildar K.; Swartz, Harold M.

    2011-01-01

    In order to meet the potential need for emergency large-scale retrospective radiation biodosimetry following an accident or attack, we have developed instrumentation and methodology for in vivo electron paramagnetic resonance spectroscopy to quantify concentrations of radiation-induced radicals within intact teeth. This technique has several very desirable characteristics for triage, including independence from confounding biologic factors, a non-invasive measurement procedure, the capability to make measurements at any time after the event, suitability for use by non-expert operators at the site of an event, and the ability to provide immediate estimates of individual doses. Throughout development there has been a particular focus on the need for a deployable system, including instrumental requirements for transport and field use, the need for high throughput, and use by minimally trained operators. Numerous measurements have been performed using this system in clinical and other non-laboratory settings, including in vivo measurements with unexposed populations as well as patients undergoing radiation therapies. The collection and analyses of sets of three serially-acquired spectra with independent placements of the resonator, in a data collection process lasting approximately 5 min, provides dose estimates with standard errors of prediction of approximately 1 Gy. As an example, measurements were performed on incisor teeth of subjects who had either received no irradiation or 2 Gy total body irradiation for prior bone marrow transplantation; this exercise provided a direct and challenging test of our capability to identify subjects who would be in need of acute medical care. -- Highlights: → Advances in radiation biodosimetry are needed for large-scale emergency response. → Radiation-induced radicals in tooth enamel can be measured using in vivo EPR. → A novel transportable spectrometer was applied in the laboratory and at remote sites. → The current

  3. A deployable in vivo EPR tooth dosimeter for triage after a radiation event involving large populations

    Energy Technology Data Exchange (ETDEWEB)

    Williams, Benjamin B., E-mail: Benjamin.B.Williams@dartmouth.edu [Dartmouth Physically Based Biodosimetry Center for Medical Countermeasures Against Radiation (Dart-Dose CMCR), Dartmouth Medical School, Hanover, NH 03768 (United States); Section of Radiation Oncology, Department of Medicine, Dartmouth Hitchcock Medical Center, Lebanon, NH (United States); Dong, Ruhong [Dartmouth Physically Based Biodosimetry Center for Medical Countermeasures Against Radiation (Dart-Dose CMCR), Dartmouth Medical School, Hanover, NH 03768 (United States); Flood, Ann Barry [Dartmouth Physically Based Biodosimetry Center for Medical Countermeasures Against Radiation (Dart-Dose CMCR), Dartmouth Medical School, Hanover, NH 03768 (United States); Clin-EPR, LLC, Lyme, NH (United States); Grinberg, Oleg [Clin-EPR, LLC, Lyme, NH (United States); Kmiec, Maciej; Lesniewski, Piotr N.; Matthews, Thomas P.; Nicolalde, Roberto J.; Raynolds, Tim [Dartmouth Physically Based Biodosimetry Center for Medical Countermeasures Against Radiation (Dart-Dose CMCR), Dartmouth Medical School, Hanover, NH 03768 (United States); Salikhov, Ildar K. [Clin-EPR, LLC, Lyme, NH (United States); Swartz, Harold M. [Dartmouth Physically Based Biodosimetry Center for Medical Countermeasures Against Radiation (Dart-Dose CMCR), Dartmouth Medical School, Hanover, NH 03768 (United States); Clin-EPR, LLC, Lyme, NH (United States)

    2011-09-15

    In order to meet the potential need for emergency large-scale retrospective radiation biodosimetry following an accident or attack, we have developed instrumentation and methodology for in vivo electron paramagnetic resonance spectroscopy to quantify concentrations of radiation-induced radicals within intact teeth. This technique has several very desirable characteristics for triage, including independence from confounding biologic factors, a non-invasive measurement procedure, the capability to make measurements at any time after the event, suitability for use by non-expert operators at the site of an event, and the ability to provide immediate estimates of individual doses. Throughout development there has been a particular focus on the need for a deployable system, including instrumental requirements for transport and field use, the need for high throughput, and use by minimally trained operators. Numerous measurements have been performed using this system in clinical and other non-laboratory settings, including in vivo measurements with unexposed populations as well as patients undergoing radiation therapies. The collection and analyses of sets of three serially-acquired spectra with independent placements of the resonator, in a data collection process lasting approximately 5 min, provides dose estimates with standard errors of prediction of approximately 1 Gy. As an example, measurements were performed on incisor teeth of subjects who had either received no irradiation or 2 Gy total body irradiation for prior bone marrow transplantation; this exercise provided a direct and challenging test of our capability to identify subjects who would be in need of acute medical care. -- Highlights: > Advances in radiation biodosimetry are needed for large-scale emergency response. > Radiation-induced radicals in tooth enamel can be measured using in vivo EPR. > A novel transportable spectrometer was applied in the laboratory and at remote sites. > The current instrument

  4. BRAF, KRAS and PIK3CA mutations in colorectal serrated polyps and cancer: Primary or secondary genetic events in colorectal carcinogenesis?

    Directory of Open Access Journals (Sweden)

    Schmitt Fernando

    2008-09-01

    Full Text Available Abstract Background BRAF, KRAS and PIK3CA mutations are frequently found in sporadic colorectal cancer (CRC. In contrast to KRAS and PIK3CA mutations, BRAF mutations are associated with tumours harbouring CpG Island methylation phenotype (CIMP, MLH1 methylation and microsatellite instability (MSI. We aimed at determine the frequency of KRAS, BRAF and PIK3CA mutations in the process of colorectal tumourigenesis using a series of colorectal polyps and carcinomas. In the series of polyps CIMP, MLH1 methylation and MSI were also studied. Methods Mutation analyses were performed by PCR/sequencing. Bisulfite treated DNA was used to study CIMP and MLH1 methylation. MSI was detected by pentaplex PCR and Genescan analysis of quasimonomorphic mononucleotide repeats. Chi Square test and Fisher's Exact test were used to perform association studies. Results KRAS, PIK3CA or BRAF occur in 71% of polyps and were mutually exclusive. KRAS mutations occur in 35% of polyps. PIK3CA was found in one of the polyps. V600E BRAF mutations occur in 29% of cases, all of them classified as serrated adenoma. CIMP phenotype occurred in 25% of the polyps and all were mutated for BRAF. MLH1 methylation was not detected and all the polyps were microsatellite stable. The comparison between the frequency of oncogenic mutations in polyps and CRC (MSI and MSS lead us to demonstrate that KRAS and PIK3CA are likely to precede both types of CRC. BRAF mutations are likely to precede MSI carcinomas since the frequency found in serrated polyps is similar to what is found in MSI CRC (P = 0.9112, but statistically different from what is found in microsatellite stable (MSS tumours (P = 0.0191. Conclusion Our results show that BRAF, KRAS and PIK3CA mutations occur prior to malignant transformation demonstrating that these oncogenic alterations are primary genetic events in colorectal carcinogenesis. Further, we show that BRAF mutations occur in association with CIMP phenotype in colorectal

  5. BRAF, KRAS and PIK3CA mutations in colorectal serrated polyps and cancer: Primary or secondary genetic events in colorectal carcinogenesis?

    International Nuclear Information System (INIS)

    Velho, Sérgia; Moutinho, Cátia; Cirnes, Luís; Albuquerque, Cristina; Hamelin, Richard; Schmitt, Fernando; Carneiro, Fátima; Oliveira, Carla; Seruca, Raquel

    2008-01-01

    BRAF, KRAS and PIK3CA mutations are frequently found in sporadic colorectal cancer (CRC). In contrast to KRAS and PIK3CA mutations, BRAF mutations are associated with tumours harbouring CpG Island methylation phenotype (CIMP), MLH1 methylation and microsatellite instability (MSI). We aimed at determine the frequency of KRAS, BRAF and PIK3CA mutations in the process of colorectal tumourigenesis using a series of colorectal polyps and carcinomas. In the series of polyps CIMP, MLH1 methylation and MSI were also studied. Mutation analyses were performed by PCR/sequencing. Bisulfite treated DNA was used to study CIMP and MLH1 methylation. MSI was detected by pentaplex PCR and Genescan analysis of quasimonomorphic mononucleotide repeats. Chi Square test and Fisher's Exact test were used to perform association studies. KRAS, PIK3CA or BRAF occur in 71% of polyps and were mutually exclusive. KRAS mutations occur in 35% of polyps. PIK3CA was found in one of the polyps. V600E BRAF mutations occur in 29% of cases, all of them classified as serrated adenoma. CIMP phenotype occurred in 25% of the polyps and all were mutated for BRAF. MLH1 methylation was not detected and all the polyps were microsatellite stable. The comparison between the frequency of oncogenic mutations in polyps and CRC (MSI and MSS) lead us to demonstrate that KRAS and PIK3CA are likely to precede both types of CRC. BRAF mutations are likely to precede MSI carcinomas since the frequency found in serrated polyps is similar to what is found in MSI CRC (P = 0.9112), but statistically different from what is found in microsatellite stable (MSS) tumours (P = 0.0191). Our results show that BRAF, KRAS and PIK3CA mutations occur prior to malignant transformation demonstrating that these oncogenic alterations are primary genetic events in colorectal carcinogenesis. Further, we show that BRAF mutations occur in association with CIMP phenotype in colorectal serrated polyps and verified that colorectal serrated

  6. Mutation Scanning in a Single and a Stacked Genetically Modified (GM) Event by Real-Time PCR and High Resolution Melting (HRM) Analysis

    Science.gov (United States)

    Ben Ali, Sina-Elisabeth; Madi, Zita Erika; Hochegger, Rupert; Quist, David; Prewein, Bernhard; Haslberger, Alexander G.; Brandes, Christian

    2014-01-01

    Genetic mutations must be avoided during the production and use of seeds. In the European Union (EU), Directive 2001/18/EC requires any DNA construct introduced via transformation to be stable. Establishing genetic stability is critical for the approval of genetically modified organisms (GMOs). In this study, genetic stability of two GMOs was examined using high resolution melting (HRM) analysis and real-time polymerase chain reaction (PCR) employing Scorpion primers for amplification. The genetic variability of the transgenic insert and that of the flanking regions in a single oilseed rape variety (GT73) and a stacked maize (MON88017 × MON810) was studied. The GT73 and the 5' region of MON810 showed no instabilities in the examined regions. However; two out of 100 analyzed samples carried a heterozygous point mutation in the 3' region of MON810 in the stacked variety. These results were verified by direct sequencing of the amplified PCR products as well as by sequencing of cloned PCR fragments. The occurrence of the mutation suggests that the 5' region is more suitable than the 3' region for the quantification of MON810. The identification of the single nucleotide polymorphism (SNP) in a stacked event is in contrast to the results of earlier studies of the same MON810 region in a single event where no DNA polymorphism was found. PMID:25365178

  7. High-throughput in vivo genotoxicity testing: an automated readout system for the somatic mutation and recombination test (SMART.

    Directory of Open Access Journals (Sweden)

    Benoit Lombardot

    Full Text Available Genotoxicity testing is an important component of toxicity assessment. As illustrated by the European registration, evaluation, authorization, and restriction of chemicals (REACH directive, it concerns all the chemicals used in industry. The commonly used in vivo mammalian tests appear to be ill adapted to tackle the large compound sets involved, due to throughput, cost, and ethical issues. The somatic mutation and recombination test (SMART represents a more scalable alternative, since it uses Drosophila, which develops faster and requires less infrastructure. Despite these advantages, the manual scoring of the hairs on Drosophila wings required for the SMART limits its usage. To overcome this limitation, we have developed an automated SMART readout. It consists of automated imaging, followed by an image analysis pipeline that measures individual wing genotoxicity scores. Finally, we have developed a wing score-based dose-dependency approach that can provide genotoxicity profiles. We have validated our method using 6 compounds, obtaining profiles almost identical to those obtained from manual measures, even for low-genotoxicity compounds such as urethane. The automated SMART, with its faster and more reliable readout, fulfills the need for a high-throughput in vivo test. The flexible imaging strategy we describe and the analysis tools we provide should facilitate the optimization and dissemination of our methods.

  8. Repair-resistant mutation in Neurospora

    International Nuclear Information System (INIS)

    Stadler, D.; Macleod, H.; Loo, M.

    1987-01-01

    Chronic UV treatment produces severalfold fewer mutations in Neurospora conidia than does the same total dose of acute UV. Experiments were designed to determine the conditions required for chronic UV mutagenesis. Measurement of the coincidence frequency for two independent mutations revealed the existence of a subset of cells which are mutable by chronic UV. Analysis of forward mutation at the mtr locus showed that the genetic alterations produced by chronic UV were virtually all point mutants, even though the assay system could detect alterations or deletions extending into neighboring genes. A significant fraction of the mutants produced by acute UV were multigenic deletions. The size of the dose-rate effect (acute UV mutation frequency divided by chronic UV mutation frequency) was compared for several different mutation assay systems. Forward mutations (recessive lethals and mtr) gave values ranging from four to nine. For events which were restricted to specific molecular sites (specific reversions and nonsense suppressor mutations), there was a wider range of dose-rate ratios. This suggests that chronic UV mutation may be restricted to certain molecular sequences or configurations

  9. Functional analysis of HNPCC-related missense mutations in MSH2

    International Nuclear Information System (INIS)

    Luetzen, Anne; Wind, Niels de; Georgijevic, Dubravka; Nielsen, Finn Cilius; Rasmussen, Lene Juel

    2008-01-01

    Hereditary nonpolyposis colorectal cancer (HNPCC) is associated with germline mutations in the human DNA mismatch repair (MMR) genes, most frequently MSH2 and MLH1. The majority of HNPCC mutations cause truncations and thus loss of function of the affected polypeptide. However, a significant proportion of MMR mutations found in HNPCC patients are single amino acid substitutions and the functional consequences of many of these mutations in DNA repair are unclear. We have examined the consequences of seven MSH2 missense mutations found in HNPCC families by testing the MSH2 mutant proteins in functional assays as well as by generating equivalent missense mutations in Escherichia coli MutS and analyzing the phenotypes of these mutants. Here we show that two mutant proteins, MSH2-P622L and MSH2-C697F confer multiple biochemical defects, namely in mismatch binding, in vivo interaction with MSH6 and EXO1, and in nuclear localization in the cell. Mutation G674R, located in the ATP-binding region of MSH2, appears to confer resistance to ATP-dependent mismatch release. Mutations D167H and H639R show reduced mismatch binding. Results of in vivo experiments in E. coli with MutS mutants show that one additional mutant, equivalent of MSH2-A834T that do not show any defects in MSH2 assays, is repair deficient. In conclusion, all mutant proteins (except for MSH2-A305T) have defects; either in mismatch binding, ATP-release, mismatch repair activity, subcellular localization or protein-protein interactions

  10. Functional analysis of HNPCC-related missense mutations in MSH2

    Energy Technology Data Exchange (ETDEWEB)

    Luetzen, Anne [Department of Science, Systems and Models, Roskilde University, DK-4000 Roskilde (Denmark); Wind, Niels de; Georgijevic, Dubravka [Department of Toxicogenetics, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden (Netherlands); Nielsen, Finn Cilius [Department of Clinical Biochemistry, Rigshospitalet, DK-2100 Copenhagen (Denmark); Rasmussen, Lene Juel [Department of Science, Systems and Models, Roskilde University, DK-4000 Roskilde (Denmark)], E-mail: ljr@ruc.dk

    2008-10-14

    Hereditary nonpolyposis colorectal cancer (HNPCC) is associated with germline mutations in the human DNA mismatch repair (MMR) genes, most frequently MSH2 and MLH1. The majority of HNPCC mutations cause truncations and thus loss of function of the affected polypeptide. However, a significant proportion of MMR mutations found in HNPCC patients are single amino acid substitutions and the functional consequences of many of these mutations in DNA repair are unclear. We have examined the consequences of seven MSH2 missense mutations found in HNPCC families by testing the MSH2 mutant proteins in functional assays as well as by generating equivalent missense mutations in Escherichia coli MutS and analyzing the phenotypes of these mutants. Here we show that two mutant proteins, MSH2-P622L and MSH2-C697F confer multiple biochemical defects, namely in mismatch binding, in vivo interaction with MSH6 and EXO1, and in nuclear localization in the cell. Mutation G674R, located in the ATP-binding region of MSH2, appears to confer resistance to ATP-dependent mismatch release. Mutations D167H and H639R show reduced mismatch binding. Results of in vivo experiments in E. coli with MutS mutants show that one additional mutant, equivalent of MSH2-A834T that do not show any defects in MSH2 assays, is repair deficient. In conclusion, all mutant proteins (except for MSH2-A305T) have defects; either in mismatch binding, ATP-release, mismatch repair activity, subcellular localization or protein-protein interactions.

  11. First molecular and biochemical analysis of in vivo affinity maturation in an ectothermic vertebrate.

    Science.gov (United States)

    Dooley, Helen; Stanfield, Robyn L; Brady, Rebecca A; Flajnik, Martin F

    2006-02-07

    The cartilaginous fish are the oldest phylogenetic group in which Igs have been found. Sharks produce a unique Ig isotype, IgNAR, a heavy-chain homodimer that does not associate with light chains. Instead, the variable (V) regions of IgNAR bind antigen as soluble single domains. Our group has shown that IgNAR plays an integral part in the humoral response of nurse sharks (Ginglymostoma cirratum) upon antigen challenge. Here, we generated phage-displayed libraries of IgNAR V regions from an immunized animal and found a family of clones derived from the same rearrangement event but differentially mutated during expansion. Because of the cluster organization of shark Ig genes and the paucicopy nature of IgNAR, we were able to construct the putative ancestor of this family. By studying mutations in the context of clone affinities, we found evidence that affinity maturation occurs for this isotype. Subsequently, we were able to identify mutations important in the affinity improvement of this family. Because the family clones were all obtained after immunization, they provide insight into the in vivo maturation mechanisms, in general, and for single-domain antibody fragments.

  12. VNTR diversity in Yersinia pestis isolates from an animal challenge study reveals the potential for in vitro mutations during laboratory cultivation

    Science.gov (United States)

    Vogler, Amy J.; Nottingham, Roxanne; Busch, Joseph D.; Sahl, Jason W.; Shuey, Megan M.; Foster, Jeffrey T.; Schupp, James M.; Smith, Susan; Rocke, Tonie E.; Klein, Paul; Wagner, David M.

    2016-01-01

    Underlying mutation rates and other evolutionary forces shape the population structure of bacteria in nature. Although easily overlooked, similar forces are at work in the laboratory and may influence observed mutations. Here, we investigated tissue samples and Yersinia pestis isolates from a rodent laboratory challenge with strain CO92 using whole genome sequencing and multi-locus variable-number tandem repeat (VNTR) analysis (MLVA). We identified six VNTR mutations that were found to have occurred in vitro during laboratory cultivation rather than in vivo during the rodent challenge. In contrast, no single nucleotide polymorphism (SNP) mutations were observed, either in vivo or in vitro. These results were consistent with previously published mutation rates and the calculated number of Y. pestis generations that occurred during the in vitro versus the in vivo portions of the experiment. When genotyping disease outbreaks, the potential for in vitro mutations should be considered, particularly when highly variable genetic markers such as VNTRs are used.

  13. Mutation of Haemophilus influenzae transforming DNA in vitro with near-ultraviolet radiation: action spectrum

    Energy Technology Data Exchange (ETDEWEB)

    Cabrera-Juarez, E; Setlow, J K [Escuela Nacional de Ciencias Biologicas, Mexico City. Dept. de Bioquimica; Oak Ridge National Lab., Tenn. (USA). Biology Div.)

    1976-05-01

    Mutations were produced in purified transforming DNA from Haemophilus influenzae by near UV radiation and were assayed as mutants among cells transformed with irradiated DNA. The maximum efficiency of mutation induction was at around 334 nm, and the efficiency dropped off steeply at lower and higher wavelengths. The difference between the action spectrum for mutation and that for the oxygen-independent inactivation of transforming DNA, which had a shoulder at 365 nm, indicates that there are different lesions involved in the inactivating and mutagenic effects of near-UV. The presence of histidine during irradiation enhanced the mutagenic effect at 334 and 365 nm, although it protected against inactivation at 365 nm. The effective near-UV wavelengths for in vitro mutation are to some extent the same as the effective wavelengths for mutation in vivo reported previously. These findings indicate that mutations are produced in vivo by near-UV with DNA as the primary target molecule rather than by a secondary non-photochemical reaction between DNA and some other cell component.

  14. Dietary factors and Truncating APC Mutations in Sporadic Colorectal Adenomas

    NARCIS (Netherlands)

    Diergaarde, B.; Tiemersma, E.W.; Braam, H.; Muijen, van G.N.P.; Nagengast, F.M.; Kok, F.J.; Kampman, E.

    2005-01-01

    Inactivating mutations in APC are thought to be early, initiating events in colorectal carcinogenesis. To gain insight into the relationship between diet and inactivating APC mutations, we evaluated associations between dietary factors and the occurrence of these mutations in a Dutch case-control

  15. Dietary factors and truncating APC mutations in sporadic colorectal adenomas.

    NARCIS (Netherlands)

    Diergaarde, B.; Tiemersma, E.W.; Braam, H.; Muijen, G.N.P. van; Nagengast, F.M.; Kok, F.J.; Kampman, E.

    2005-01-01

    Inactivating mutations in APC are thought to be early, initiating events in colorectal carcinogenesis. To gain insight into the relationship between diet and inactivating APC mutations, we evaluated associations between dietary factors and the occurrence of these mutations in a Dutch case-control

  16. Favipiravir elicits antiviral mutagenesis during virus replication in vivo.

    Science.gov (United States)

    Arias, Armando; Thorne, Lucy; Goodfellow, Ian

    2014-10-21

    Lethal mutagenesis has emerged as a novel potential therapeutic approach to treat viral infections. Several studies have demonstrated that increases in the high mutation rates inherent to RNA viruses lead to viral extinction in cell culture, but evidence during infections in vivo is limited. In this study, we show that the broad-range antiviral nucleoside favipiravir reduces viral load in vivo by exerting antiviral mutagenesis in a mouse model for norovirus infection. Increased mutation frequencies were observed in samples from treated mice and were accompanied with lower or in some cases undetectable levels of infectious virus in faeces and tissues. Viral RNA isolated from treated animals showed reduced infectivity, a feature of populations approaching extinction during antiviral mutagenesis. These results suggest that favipiravir can induce norovirus mutagenesis in vivo, which in some cases leads to virus extinction, providing a proof-of-principle for the use of favipiravir derivatives or mutagenic nucleosides in the clinical treatment of noroviruses.

  17. Assay of new systems in vivo mutagenesis for determining the effects of low doses of ionizing radiation

    International Nuclear Information System (INIS)

    Bauluz, C.; Sierra, I.; Martin, L.; Real, A.; Vidania, R. de

    1997-01-01

    Ionizing radiation reacts directly and indirectly with the genetic material in living cells and produces DNA damage. Processing of this damage by correcting enzymes may result in appearing of mutations which, in turn, may lead to carcinogenesis. We have focused on the determination of in vivo mutagenesis induced after exposure to X-rays, aiming at establishing methods to evaluate the effect of low doses of radiation. In vivo mutagenesis has been addressed in the Muta Mouse model that carries a lacZ marker gene and provides a relatively simple assay of appearance of mutations. Mutation frequencies were determined in the lacZ gene copies recovered from mice irradiated with 1Gy or 4Gy of X-rays, acute or fractionated. Liver, spleen and bone marrow DNA samples were isolated at different times after irradiation, ranging from 1 day to 2 months, and evolution of mutations was studied. Results showed different responses depending on the organ and especially on the time of analysis, suggesting that the mutagenic process in vivo is much more complex than previously deduced from in vitro experiments. Therefore, determination of the relationship between dose and mutagenic effect in vivo will require additional studies. (author)

  18. VNTR diversity in Yersinia pestis isolates from an animal challenge study reveals the potential for in vitro mutations during laboratory cultivation.

    Science.gov (United States)

    Vogler, Amy J; Nottingham, Roxanne; Busch, Joseph D; Sahl, Jason W; Shuey, Megan M; Foster, Jeffrey T; Schupp, James M; Smith, Susan R; Rocke, Tonie E; Keim, Paul; Wagner, David M

    2016-11-01

    Underlying mutation rates and other evolutionary forces shape the population structure of bacteria in nature. Although easily overlooked, similar forces are at work in the laboratory and may influence observed mutations. Here, we investigated tissue samples and Yersinia pestis isolates from a rodent laboratory challenge with strain CO92 using whole genome sequencing and multi-locus variable-number tandem repeat (VNTR) analysis (MLVA). We identified six VNTR mutations that were found to have occurred in vitro during laboratory cultivation rather than in vivo during the rodent challenge. In contrast, no single nucleotide polymorphism (SNP) mutations were observed, either in vivo or in vitro. These results were consistent with previously published mutation rates and the calculated number of Y. pestis generations that occurred during the in vitro versus the in vivo portions of the experiment. When genotyping disease outbreaks, the potential for in vitro mutations should be considered, particularly when highly variable genetic markers such as VNTRs are used. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Mutations altering the gammaretrovirus endoproteolytic motif affect glycosylation of the envelope glycoprotein and early events of the virus life cycle

    Energy Technology Data Exchange (ETDEWEB)

    Argaw, Takele; Wilson, Carolyn A., E-mail: carolyn.wilson@fda.hhs.gov

    2015-01-15

    Previously, we found that mutation of glutamine to proline in the endoproteolytic cleavage signal of the PERV-C envelope (RQKK to RPKK) resulted in non-infectious vectors. Here, we show that RPKK results in a non-infectious vector when placed in not only a PERV envelope, but also the envelope of a related gammaretrovirus, FeLV-B. The amino acid substitutions do not prevent envelope precursor cleavage, viral core and genome assembly, or receptor binding. Rather, the mutations result in the formation of hyperglycosylated glycoprotein and a reduction in the reverse transcribed minus strand synthesis and undetectable 2-LTR circular DNA in cells exposed to vectors with these mutated envelopes. Our findings suggest novel functions associated with the cleavage signal sequence that may affect trafficking through the glycosylation machinery of the cell. Further, the glycosylation status of the envelope appears to impact post-binding events of the viral life cycle, either membrane fusion, internalization, or reverse transcription. - Highlights: • Env cleavage signal impacts infectivity of gammaretroviruses. • Non-infectious mutants have hyper-glycosylated envelope that bind target cells. • Non-infectious mutants have defects in the formation of the double-stranded DNA. • Env cleavage motif has functions beyond cleavage of the env precursor.

  20. Mutations in BALB mitochondrial DNA induce CCL20 up-regulation promoting tumorigenic phenotypes

    Energy Technology Data Exchange (ETDEWEB)

    Sligh, James [Department of Medicine—Dermatology Division, University of Arizona, Tucson, AZ 857 24 (United States); University of Arizona Cancer Center, Tucson, AZ 85724 (United States); Janda, Jaroslav [University of Arizona Cancer Center, Tucson, AZ 85724 (United States); Jandova, Jana, E-mail: jjandova@email.arizona.edu [Department of Medicine—Dermatology Division, University of Arizona, Tucson, AZ 857 24 (United States); University of Arizona Cancer Center, Tucson, AZ 85724 (United States)

    2014-11-15

    Highlights: • Alterations in mitochondrial DNA are commonly found in various human cancers. • Mutations in BALB mitochondrial DNA induce up-regulation of chemokine CCL20. • Increased growth and motility of mtBALB cells is associated with CCL20 levels. • mtDNA changes in BALB induce in vivo tumor growth through CCL20 up-regulation. • Mutations in mitochondrial DNA play important roles in keratinocyte neoplasia. - Abstract: mtDNA mutations are common in human cancers and are thought to contribute to the process of neoplasia. We examined the role of mtDNA mutations in skin cancer by generating fibroblast cybrids harboring a mutation in the gene encoding the mitochondrial tRNA for arginine. This somatic mutation (9821insA) was previously reported in UV-induced hyperkeratotic skin tumors in hairless mice and confers specific tumorigenic phenotypes to mutant cybrids. Microarray analysis revealed and RT-PCR along with Western blot analysis confirmed the up-regulation of CCL20 and its receptor CCR6 in mtBALB haplotype containing the mt-Tr 9821insA allele compared to wild type mtB6 haplotype. Based on reported role of CCL20 in cancer progression we examined whether the hyper-proliferation and enhanced motility of mtBALB haplotype would be associated with CCL20 levels. Treatment of both genotypes with recombinant CCL20 (rmCCL20) resulted in enhanced growth and motility of mtB6 cybrids. Furthermore, the acquired somatic alteration increased the in vivo tumor growth of mtBALB cybrids through the up-regulation of CCL20 since neutralizing antibody significantly decreased in vivo tumor growth of these cells; and tumors from anti-CCL20 treated mice injected with mtBALB cybrids showed significantly decreased CCL20 levels. When rmCCL20 or mtBALB cybrids were used as chemotactic stimuli, mtB6 cybrids showed increased motility while anti-CCL20 antibody decreased the migration and in vivo tumor growth of mtBALB cybrids. Moreover, the inhibitors of MAPK signaling and NF

  1. SETBP1 mutations drive leukemic transformation in ASXL1-mutated MDS.

    Science.gov (United States)

    Inoue, D; Kitaura, J; Matsui, H; Hou, H-A; Chou, W-C; Nagamachi, A; Kawabata, K C; Togami, K; Nagase, R; Horikawa, S; Saika, M; Micol, J-B; Hayashi, Y; Harada, Y; Harada, H; Inaba, T; Tien, H-F; Abdel-Wahab, O; Kitamura, T

    2015-04-01

    Mutations in ASXL1 are frequent in patients with myelodysplastic syndrome (MDS) and are associated with adverse survival, yet the molecular pathogenesis of ASXL1 mutations (ASXL1-MT) is not fully understood. Recently, it has been found that deletion of Asxl1 or expression of C-terminal-truncating ASXL1-MTs inhibit myeloid differentiation and induce MDS-like disease in mice. Here, we find that SET-binding protein 1 (SETBP1) mutations (SETBP1-MT) are enriched among ASXL1-mutated MDS patients and associated with increased incidence of leukemic transformation, as well as shorter survival, suggesting that SETBP1-MT play a critical role in leukemic transformation of MDS. We identify that SETBP1-MT inhibit ubiquitination and subsequent degradation of SETBP1, resulting in increased expression. Expression of SETBP1-MT, in turn, inhibited protein phosphatase 2A activity, leading to Akt activation and enhanced expression of posterior Hoxa genes in ASXL1-mutant cells. Biologically, SETBP1-MT augmented ASXL1-MT-induced differentiation block, inhibited apoptosis and enhanced myeloid colony output. SETBP1-MT collaborated with ASXL1-MT in inducing acute myeloid leukemia in vivo. The combination of ASXL1-MT and SETBP1-MT activated a stem cell signature and repressed the tumor growth factor-β signaling pathway, in contrast to the ASXL1-MT-induced MDS model. These data reveal that SETBP1-MT are critical drivers of ASXL1-mutated MDS and identify several deregulated pathways as potential therapeutic targets in high-risk MDS.

  2. Ascorbate/menadione-induced oxidative stress kills cancer cells that express normal or mutated forms of the oncogenic protein Bcr-Abl. An in vitro and in vivo mechanistic study.

    Science.gov (United States)

    Beck, Raphaël; Pedrosa, Rozangela Curi; Dejeans, Nicolas; Glorieux, Christophe; Levêque, Philippe; Gallez, Bernard; Taper, Henryk; Eeckhoudt, Stéphane; Knoops, Laurent; Calderon, Pedro Buc; Verrax, Julien

    2011-10-01

    Numerous studies suggest that generation of oxidative stress could be useful in cancer treatment. In this study, we evaluated, in vitro and in vivo, the antitumor potential of oxidative stress induced by ascorbate/menadione (asc/men). This combination of a reducing agent (ascorbate) and a redox active quinone (menadione) generates redox cycling leading to formation of reactive oxygen species (ROS). Asc/men was tested in several cell types including K562 cells (a stable human-derived leukemia cell line), freshly isolated leukocytes from patients with chronic myeloid leukemia, BaF3 cells (a murine pro-B cell line) transfected with Bcr-Abl and peripheral blood leukocytes derived from healthy donors. Although these latter cells were resistant to asc/men, survival of all the other cell lines was markedly reduced, including the BaF3 cells expressing either wild-type or mutated Bcr-Abl. In a standard in vivo model of subcutaneous tumor transplantation, asc/men provoked a significant delay in the proliferation of K562 and BaF3 cells expressing the T315I mutated form of Bcr-Abl. No effect of asc/men was observed when these latter cells were injected into blood of mice most probably because of the high antioxidant potential of red blood cells, as shown by in vitro experiments. We postulate that cancer cells are more sensitive to asc/men than healthy cells because of their lack of antioxidant enzymes, mainly catalase. The mechanism underlying this cytotoxicity involves the oxidative cleavage of Hsp90 with a subsequent loss of its chaperone function thus leading to degradation of wild-type and mutated Bcr-Abl protein.

  3. Analysis of early initiating event(s) in radiation-induced thymic lymphomagenesis

    International Nuclear Information System (INIS)

    Muto, Masahiro; Ying Chen; Kubo, Eiko; Mita, Kazuei

    1996-01-01

    Since the T cell receptor rearrangement is a sequential process and unique to the progeny of each clone, we investigated the early initiating events in radiation-induced thymic lymphomagenesis by comparing the oncogenic alterations with the pattern of γ T cell receptor (TCR) rearrangements. We reported previously that after leukemogenic irradiation, preneoplastic cells developed, albeit infrequently, from thymic leukemia antigen-2 + (TL-2 + ) thymocytes. Limited numbers of TL-2 + cells from individual irradiated B10.Thy-1.1 mice were injected into B10.Thy-1.2 mice intrathymically, and the common genetic changes among the donor-type T cell lymphomas were investigated with regard to p53 gene and chromosome aberrations. The results indicated that some mutations in the p53 gene had taken place in these lymphomas, but there was no common mutation among the donor-type lymphomas from individual irradiated mice, suggesting that these mutations were late-occurring events in the process of oncogenesis. On the other hand, there were common chromosome aberrations or translocations such as trisomy 15, t(7F; 10C), t(1A; 13D) or t(6A; XB) among the donor-type lymphomas derived from half of the individual irradiated mice. This indicated that the aberrations/translocations, which occurred in single progenitor cells at the early T cell differentiation either just before or after γ T cell receptor rearrangements, might be important candidates for initiating events. In the donor-type lymphomas from the other half of the individual irradiated mice, microgenetic changes were suggested to be initial events and also might take place in single progenitor cells just before or right after γ TCR rearrangements. (author)

  4. Unique mutational profile associated with a loss of TDG expression in the rectal cancer of a patient with a constitutional PMS2 deficiency.

    Science.gov (United States)

    Vasovcak, P; Krepelova, A; Menigatti, M; Puchmajerova, A; Skapa, P; Augustinakova, A; Amann, G; Wernstedt, A; Jiricny, J; Marra, G; Wimmer, K

    2012-07-01

    Cells with DNA repair defects have increased genomic instability and are more likely to acquire secondary mutations that bring about cellular transformation. We describe the frequency and spectrum of somatic mutations involving several tumor suppressor genes in the rectal carcinoma of a 13-year-old girl harboring biallelic, germline mutations in the DNA mismatch repair gene PMS2. Apart from microsatellite instability, the tumor DNA contained a number of C:G→T:A or G:C→A:T transitions in CpG dinucleotides, which often result through spontaneous deamination of cytosine or 5-methylcytosine. Four DNA glycosylases, UNG2, SMUG1, MBD4 and TDG, are involved in the repair of these deamination events. We identified a heterozygous missense mutation in TDG, which was associated with TDG protein loss in the tumor. The CpGs mutated in this patient's tumor are generally methylated in normal colonic mucosa. Thus, it is highly likely that loss of TDG contributed to the supermutator phenotype and that most of the point mutations were caused by deamination of 5-methylcytosine to thymine, which remained uncorrected owing to the TDG deficiency. This case provides the first in vivo evidence of the key role of TDG in protecting the human genome against the deleterious effects of 5-methylcytosine deamination. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Limited importance of the dominant-negative effect of TP53 missense mutations

    International Nuclear Information System (INIS)

    Stoczynska-Fidelus, Ewelina; Liberski, Pawel P; Rieske, Piotr; Szybka, Malgorzata; Piaskowski, Sylwester; Bienkowski, Michal; Hulas-Bigoszewska, Krystyna; Banaszczyk, Mateusz; Zawlik, Izabela; Jesionek-Kupnicka, Dorota; Kordek, Radzislaw

    2011-01-01

    Heterozygosity of TP53 missense mutations is related to the phenomenon of the dominant-negative effect (DNE). To estimate the importance of the DNE of TP53 mutations, we analysed the percentage of cancer cases showing a single heterozygous mutation of TP53 and searched for a cell line with a single heterozygous mutation of this gene. This approach was based on the knowledge that genes with evident DNE, such as EGFR and IDH1, represent nearly 100% of single heterozygous mutations in tumour specimens and cell lines. Genetic analyses (LOH and sequencing) performed for early and late passages of several cell lines originally described as showing single heterozygous TP53 mutations (H-318, G-16, PF-382, MOLT-13, ST-486 and LS-123). Statistical analysis of IARC TP53 and SANGER databases. Genetic analyses of N-RAS, FBXW7, PTEN and STR markers to test cross-contamination and cell line identity. Cell cloning, fluorescence-activated cell sorting and SSCP performed for the PF-382 cell line. A database study revealed TP53 single heterozygous mutations in 35% of in vivo (surgical and biopsy) samples and only 10% of cultured cells (in vitro), although those numbers appeared to be overestimated. We deem that published in vivo TP53 mutation analyses are not as rigorous as studies in vitro, and we did not find any cell line showing a stable, single heterozygous mutation. G16, PF-382 and MOLT-13 cells harboured single heterozygous mutations temporarily. ST-486, H-318 and LS-123 cell lines were misclassified. Specific mutations, such as R175H, R273H, R273L or R273P, which are reported in the literature to exert a DNE, showed the lowest percentage of single heterozygous mutations in vitro (about 5%). We suggest that the currently reported percentage of TP53 single heterozygous mutations in tumour samples and cancer cell lines is overestimated. Thus, the magnitude of the DNE of TP53 mutations is questionable. This scepticism is supported by database investigations showing that retention

  6. Mutations in Plasmodium falciparum K13 propeller gene from Bangladesh (2009-2013).

    Science.gov (United States)

    Mohon, Abu Naser; Alam, Mohammad Shafiul; Bayih, Abebe Genetu; Folefoc, Asongna; Shahinas, Dea; Haque, Rashidul; Pillai, Dylan R

    2014-11-18

    Bangladesh is a malaria hypo-endemic country sharing borders with India and Myanmar. Artemisinin combination therapy (ACT) remains successful in Bangladesh. An increase of artemisinin-resistant malaria parasites on the Thai-Cambodia and Thai-Myanmar borders is worrisome. K13 propeller gene (PF3D7_1343700 or PF13_0238) mutations have been linked to both in vitro artemisinin resistance and in vivo slow parasite clearance rates. This group undertook to evaluate if mutations seen in Cambodia have emerged in Bangladesh where ACT use is now standard for a decade. Samples were obtained from Plasmodium falciparum-infected malaria patients from Upazila health complexes (UHC) between 2009 and 2013 in seven endemic districts of Bangladesh. These districts included Khagrachari (Matiranga UHC), Rangamati (Rajasthali UHC), Cox's Bazar (Ramu and Ukhia UHC), Bandarban (Lama UHC), Mymensingh (Haluaghat UHC), Netrokona (Durgapur and Kalmakanda UHC), and Moulvibazar (Sreemangal and Kamalganj UHC). Out of 296 microscopically positive P. falciparum samples, 271 (91.6%) were confirmed as mono-infections by both real-time PCR and nested PCR. The K13 propeller gene from 253 (93.4%) samples was sequenced bi-directionally. One non-synonymous mutation (A578S) was found in Bangladeshi clinical isolates. The A578S mutation was confirmed and lies adjacent to the C580Y mutation, the major mutation causing delayed parasite clearance in Cambodia. Based on computational modeling A578S should have a significant effect on tertiary structure of the protein. The data suggest that P. falciparum in Bangladesh remains free of the C580Y mutation linked to delayed parasite clearance. However, the mutation A578S is present and based on structural analysis could affect K13 gene function. Further in vivo clinical studies are required to validate the effect of this mutation.

  7. Are There Mutator Polymerases?

    Directory of Open Access Journals (Sweden)

    Miguel Garcia-Diaz

    2003-01-01

    Full Text Available DNA polymerases are involved in different cellular events, including genome replication and DNA repair. In the last few years, a large number of novel DNA polymerases have been discovered, and the biochemical analysis of their properties has revealed a long list of intriguing features. Some of these polymerases have a very low fidelity and have been suggested to play mutator roles in different processes, like translesion synthesis or somatic hypermutation. The current view of these processes is reviewed, and the current understanding of DNA polymerases and their role as mutator enzymes is discussed.

  8. SF3B1-initiating mutations in MDS-RSs target lymphomyeloid hematopoietic stem cells.

    Science.gov (United States)

    Mortera-Blanco, Teresa; Dimitriou, Marios; Woll, Petter S; Karimi, Mohsen; Elvarsdottir, Edda; Conte, Simona; Tobiasson, Magnus; Jansson, Monika; Douagi, Iyadh; Moarii, Matahi; Saft, Leonie; Papaemmanuil, Elli; Jacobsen, Sten Eirik W; Hellström-Lindberg, Eva

    2017-08-17

    Mutations in the RNA splicing gene SF3B1 are found in >80% of patients with myelodysplastic syndrome with ring sideroblasts (MDS-RS). We investigated the origin of SF3B1 mutations within the bone marrow hematopoietic stem and progenitor cell compartments in patients with MDS-RS. Screening for recurrently mutated genes in the mononuclear cell fraction revealed mutations in SF3B1 in 39 of 40 cases (97.5%), combined with TET2 and DNMT3A in 11 (28%) and 6 (15%) patients, respectively. All recurrent mutations identified in mononuclear cells could be tracked back to the phenotypically defined hematopoietic stem cell (HSC) compartment in all investigated patients and were also present in downstream myeloid and erythroid progenitor cells. While in agreement with previous studies, little or no evidence for clonal ( SF3B1 mutation) involvement could be found in mature B cells, consistent involvement at the pro-B-cell progenitor stage was established, providing definitive evidence for SF3B1 mutations targeting lymphomyeloid HSCs and compatible with mutated SF3B1 negatively affecting lymphoid development. Assessment of stem cell function in vitro as well as in vivo established that only HSCs and not investigated progenitor populations could propagate the SF3B1 mutated clone. Upon transplantation into immune-deficient mice, SF3B1 mutated MDS-RS HSCs differentiated into characteristic ring sideroblasts, the hallmark of MDS-RS. Our findings provide evidence of a multipotent lymphomyeloid HSC origin of SF3B1 mutations in MDS-RS patients and provide a novel in vivo platform for mechanistically and therapeutically exploring SF3B1 mutated MDS-RS. © 2017 by The American Society of Hematology.

  9. Circulating mutational portrait of cancer: manifestation of aggressive clonal events in both early and late stages

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    Meng Yang

    2017-05-01

    Full Text Available Abstract Background Solid tumors residing in tissues and organs leave footprints in circulation through circulating tumor cells (CTCs and circulating tumor DNAs (ctDNA. Characterization of the ctDNA portraits and comparison with tumor DNA mutational portraits may reveal clinically actionable information on solid tumors that is traditionally achieved through more invasive approaches. Methods We isolated ctDNAs from plasma of patients of 103 lung cancer and 74 other solid tumors of different tissue origins. Deep sequencing using the Guardant360 test was performed to identify mutations in 73 clinically actionable genes, and the results were associated with clinical characteristics of the patient. The mutation profiles of 37 lung cancer cases with paired ctDNA and tumor genomic DNA sequencing were used to evaluate clonal representation of tumor in circulation. Five lung cancer cases with longitudinal ctDNA sampling were monitored for cancer progression or response to treatments. Results Mutations in TP53, EGFR, and KRAS genes are most prevalent in our cohort. Mutation rates of ctDNA are similar in early (I and II and late stage (III and IV cancers. Mutation in DNA repair genes BRCA1, BRCA2, and ATM are found in 18.1% (32/177 of cases. Patients with higher mutation rates had significantly higher mortality rates. Lung cancer of never smokers exhibited significantly higher ctDNA mutation rates as well as higher EGFR and ERBB2 mutations than ever smokers. Comparative analysis of ctDNA and tumor DNA mutation data from the same patients showed that key driver mutations could be detected in plasma even when they were present at a minor clonal population in the tumor. Mutations of key genes found in the tumor tissue could remain in circulation even after frontline radiotherapy and chemotherapy suggesting these mutations represented resistance mechanisms. Longitudinal sampling of five lung cancer cases showed distinct changes in ctDNA mutation portraits that

  10. Revertant mutation releases confined lethal mutation, opening Pandora's box: a novel genetic pathogenesis.

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    Yasushi Ogawa

    2014-05-01

    Full Text Available When two mutations, one dominant pathogenic and the other "confining" nonsense, coexist in the same allele, theoretically, reversion of the latter may elicit a disease, like the opening of Pandora's box. However, cases of this hypothetical pathogenic mechanism have never been reported. We describe a lethal form of keratitis-ichthyosis-deafness (KID syndrome caused by the reversion of the GJB2 nonsense mutation p.Tyr136X that would otherwise have confined the effect of another dominant lethal mutation, p.Gly45Glu, in the same allele. The patient's mother had the identical misssense mutation which was confined by the nonsense mutation. The biological relationship between the parents and the child was confirmed by genotyping of 15 short tandem repeat loci. Haplotype analysis using 40 SNPs spanning the >39 kbp region surrounding the GJB2 gene and an extended SNP microarray analysis spanning 83,483 SNPs throughout chromosome 13 in the family showed that an allelic recombination event involving the maternal allele carrying the mutations generated the pathogenic allele unique to the patient, although the possibility of coincidental accumulation of spontaneous point mutations cannot be completely excluded. Previous reports and our mutation screening support that p.Gly45Glu is in complete linkage disequilibrium with p.Tyr136X in the Japanese population. Estimated from statisitics in the literature, there may be approximately 11,000 p.Gly45Glu carriers in the Japanese population who have this second-site confining mutation, which acts as natural genetic protection from the lethal disease. The reversion-triggered onset of the disesase shown in this study is a previously unreported genetic pathogenesis based on Mendelian inheritance.

  11. Volatility of Mutator Phenotypes at Single Cell Resolution.

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    Scott R Kennedy

    2015-04-01

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

  12. Mutation breeding techniques and behaviour of irradiated shoot apices of potato

    International Nuclear Information System (INIS)

    Harten, A.M. van.

    1978-01-01

    The author describes part of the investigations being carried out at the Institute of Plant Breeding, Wageningen into mutation breeding in potato; in particular, efforts to produce a di(ha)ploid tester clone for reliable mutation frequency data are described, the formation of adventitious roots and shoots from potato leaves, leaflets and stem parts in vivo is studied, and damage and recovery of irradiated potato tuber eyes is investigated. (G.T.H.)

  13. In Vivo-Selected Compensatory Mutations Restore the Fitness Cost of Mosaic penA Alleles That Confer Ceftriaxone Resistance in Neisseria gonorrhoeae

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    Leah R. Vincent

    2018-04-01

    Full Text Available Resistance to ceftriaxone in Neisseria gonorrhoeae is mainly conferred by mosaic penA alleles that encode penicillin-binding protein 2 (PBP2 variants with markedly lower rates of acylation by ceftriaxone. To assess the impact of these mosaic penA alleles on gonococcal fitness, we introduced the mosaic penA alleles from two ceftriaxone-resistant (Cror clinical isolates (H041 and F89 into a Cros strain (FA19 by allelic exchange and showed that the resultant Cror mutants were significantly outcompeted by the Cros parent strain in vitro and in a murine infection model. Four Cror compensatory mutants of FA19 penA41 were isolated independently from mice that outcompeted the parent strain both in vitro and in vivo. One of these compensatory mutants (LV41C displayed a unique growth profile, with rapid log growth followed by a sharp plateau/gradual decline at stationary phase. Genome sequencing of LV41C revealed a mutation (G348D in the acnB gene encoding the bifunctional aconitate hydratase 2/2 methylisocitrate dehydratase. Introduction of the acnBG348D allele into FA19 penA41 conferred both a growth profile that phenocopied that of LV41C and a fitness advantage, although not as strongly as that exhibited by the original compensatory mutant, suggesting the existence of additional compensatory mutations. The mutant aconitase appears to be a functional knockout with lower activity and expression than wild-type aconitase. Transcriptome sequencing (RNA-seq analysis of FA19 penA41 acnBG348D revealed a large set of upregulated genes involved in carbon and energy metabolism. We conclude that compensatory mutations can be selected in Cror gonococcal strains that increase metabolism to ameliorate their fitness deficit.

  14. Penetrance of Hypertrophic Cardiomyopathy in Children Who Are Mutation Positive

    NARCIS (Netherlands)

    Vermeer, Alexa M. C.; Clur, Sally-Ann B.; Blom, Nico A.; Wilde, Arthur A. M.; Christiaans, Imke

    2017-01-01

    Objectives To investigate the presence of hypertrophic cardiomyopathy (HCM) at first cardiac evaluation and during follow-up and cardiac events in predictively tested children who are mutation positive. Study design The study included 119 predictively tested children who were mutation positive, with

  15. Isolation and propagation of mutations by in vitro culture. Part of a coordinated programme on improvement of vegetatively propagated crops and tree crops through radiation induced mutations

    International Nuclear Information System (INIS)

    Asahira, T.

    1974-01-01

    Tuberous roots of two Dahlia cultivars were irradiated with 1000 - 2000 R of x-rays. Chlorophyll and flower colour mutations were scored on M 1 plants and the subsequent vegetatively propagated generations. The project aimed at the development of experimental methods suitable for easy isolation of mutated tissue chimeras deriving from mutagen treatment. In comparing in vivo methods such as leaf bud cutting and root propagation with in vitro methods using explants from leaves and florets, the problems encountered by in vitro culture were so manifold, that this method is not considered to be at present of much promise. These experimental results should not discourage from developing and using in vitro culture methods for mutant isolation in other plant species, particularly those were in vivo adventitious bud techniques are not available. Besides the main result obtained, the following valuable observations were made: a) The genotype used for mutation induction is not only relevant for the number and the spectrum of mutations, but also size of chimerical sectors. b) Flower colour changes, which were investigated in some detail, revealed that pigments were lost as groups e.g. all buteins in one case and all cyanins plus pelargonins in another case. c) When using the leaf bud cutting method the greatest frequency of mutations was recovered from the axillary bud of the leaf, that is at the youngest primordial stage at the time of mutagen treatment

  16. Axonal transport of TDP-43 mRNA granules in neurons is impaired by ALS-causing mutations

    Science.gov (United States)

    Carrasco, Monica A.; Williams, Luis A.; Winborn, Christina S.; Han, Steve S. W.; Kiskinis, Evangelos; Winborn, Brett; Freibaum, Brian D.; Kanagaraj, Anderson; Clare, Alison J.; Badders, Nisha M.; Bilican, Bilada; Chaum, Edward; Chandran, Siddharthan; Shaw, Christopher E.; Eggan, Kevin C.; Maniatis, Tom; Taylor, J. Paul

    2014-01-01

    Summary The RNA binding protein TDP-43 regulates RNA metabolism at multiple levels, including transcription, RNA splicing, and mRNA stability. TDP-43 is a major component of the cytoplasmic inclusions characteristic of amyotrophic lateral sclerosis and some types of frontotemporal lobar degeneration. The importance of TDP-43 in disease is underscored by the fact that dominant missense mutations are sufficient to cause disease, although the role of TDP-43 in pathogenesis is unknown. Here we show that TDP-43 forms cytoplasmic mRNP granules that undergo bidirectional, microtubule-dependent transport in neurons in vitro and in vivo and facilitate delivery of target mRNA to distal neuronal compartments. TDP-43 mutations impair this mRNA transport function in vivo and in vitro, including in stem cell-derived motor neurons from ALS patients bearing any one of three different TDP-43 ALS-causing mutations. Thus, TDP43 mutations that cause ALS lead to partial loss of a novel cytoplasmic function of TDP-43. PMID:24507191

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

    Science.gov (United States)

    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.

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

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    Benjamin Werner

    2018-06-01

    Full Text Available 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.

  19. The effects of extremely low frequency magnetic fields on mutation induction in mice

    Energy Technology Data Exchange (ETDEWEB)

    Wilson, James W. [Department of Genetics, University of Leicester, Leicester LE1 7RH (United Kingdom); Haines, Jackie; Sienkiewicz, Zenon [Centre for Radiation, Chemical and Environmental Hazards, Public Health England, Chilton, Didcot, Oxfordshire OX11 0RQ (United Kingdom); Dubrova, Yuri E., E-mail: yed2@le.ac.uk [Department of Genetics, University of Leicester, Leicester LE1 7RH (United Kingdom)

    2015-03-15

    Highlights: • The effects of 50 Hz magnetic fields on mutation induction in mice were analyzed. • The frequency of ESTR mutation was established in sperm and blood. • Exposure to 10–300 μT for 2 and 15 h did not result in mutation induction. • Mutagenic effects of 50 Hz magnetic fields are likely to be negligible. - Abstract: The growing human exposure to extremely low frequency (ELF) magnetic fields has raised a considerable concern regarding their genotoxic effects. The aim of this study was to evaluate the in vivo effects of ELF magnetic fields irradiation on mutation induction in the germline and somatic tissues of male mice. Seven week old BALB/c × CBA/Ca F{sub 1} hybrid males were exposed to 10, 100 or 300 μT of 50 Hz magnetic fields for 2 or 15 h. Using single-molecule PCR, the frequency of mutation at the mouse Expanded Simple Tandem Repeat (ESTR) locus Ms6-hm was established in sperm and blood samples of exposed and matched sham-treated males. ESTR mutation frequency was also established in sperm and blood samples taken from male mice exposed to 1 Gy of acute X-rays. The frequency of ESTR mutation in DNA samples extracted from blood of mice exposed to magnetic fields did not significantly differ from that in sham-treated controls. However, there was a marginally significant increase in mutation frequency in sperm but this was not dose-dependent. In contrast, acute exposure X-rays led to significant increases in mutation frequency in sperm and blood of exposed males. The results of our study suggest that, within the range of doses analyzed here, the in vivo mutagenic effects of ELF magnetic fields are likely to be minor if not negligible.

  20. The effects of extremely low frequency magnetic fields on mutation induction in mice

    International Nuclear Information System (INIS)

    Wilson, James W.; Haines, Jackie; Sienkiewicz, Zenon; Dubrova, Yuri E.

    2015-01-01

    Highlights: • The effects of 50 Hz magnetic fields on mutation induction in mice were analyzed. • The frequency of ESTR mutation was established in sperm and blood. • Exposure to 10–300 μT for 2 and 15 h did not result in mutation induction. • Mutagenic effects of 50 Hz magnetic fields are likely to be negligible. - Abstract: The growing human exposure to extremely low frequency (ELF) magnetic fields has raised a considerable concern regarding their genotoxic effects. The aim of this study was to evaluate the in vivo effects of ELF magnetic fields irradiation on mutation induction in the germline and somatic tissues of male mice. Seven week old BALB/c × CBA/Ca F 1 hybrid males were exposed to 10, 100 or 300 μT of 50 Hz magnetic fields for 2 or 15 h. Using single-molecule PCR, the frequency of mutation at the mouse Expanded Simple Tandem Repeat (ESTR) locus Ms6-hm was established in sperm and blood samples of exposed and matched sham-treated males. ESTR mutation frequency was also established in sperm and blood samples taken from male mice exposed to 1 Gy of acute X-rays. The frequency of ESTR mutation in DNA samples extracted from blood of mice exposed to magnetic fields did not significantly differ from that in sham-treated controls. However, there was a marginally significant increase in mutation frequency in sperm but this was not dose-dependent. In contrast, acute exposure X-rays led to significant increases in mutation frequency in sperm and blood of exposed males. The results of our study suggest that, within the range of doses analyzed here, the in vivo mutagenic effects of ELF magnetic fields are likely to be minor if not negligible

  1. Flow-cytometric measurements of somatic cell mutations in Thorotrast patients

    International Nuclear Information System (INIS)

    Umeki, Shigeko; Kyoizumi, Seishi; Kusunoki, Yoichiro; Nakamura, Nori; Sasaki, Masao; Mori, Takesaburo; Ishikawa, Yuichi; Cologne, J.B.; Akiyama, Mitoshi.

    1992-10-01

    Exposure to ionizing radiation is a well-recognized risk factor for cancer development. Because ionizing radiation can induce mutations, an accurate way of measuring somatic mutation frequencies could be a useful tool for evaluating cancer risk. In the present study, we have examined in vivo somatic mutation frequencies at the erythrocyte glycophorin A and T-cell receptor loci in 18 Thorotrast patients. These persons have been continuously irradiated with alpha particles emitted from the internal deposition of thorium dioxide and thus have increased risks of certain malignant tumors. When compared with controls, the Thorotrast patients showed a significantly higher frequency of mutants at the lymphocyte T-cell receptor loci but not at the erythrocyte glycophorin A loci. (author)

  2. Same β-globin gene mutation is present on nine different β-thalassemia chromosomes in a Sardinian population

    International Nuclear Information System (INIS)

    Pirastu, M.; Galanello, R.; Doherty, M.A.; Tuveri, T.; Cao, A.; Kan, Y.W.

    1987-01-01

    The predominant β-thalassemia in Sardinia is the β 0 type in which no β-globin chains are synthesized in the homozygous state. The authors determined the β-thalassemia mutations in this population by the oligonucleotide-probe method and defined the chromosome haplotypes on which the mutation resides. The same β/sup 39(CAG→TAG)/ nonsense mutation was found on nine different chromosome haplotypes. Although this mutation may have arisen more than once, the multiple haplotypes could also be generated by crossing over and gene conversion events. These findings underscore the frequency of mutational events in the β-globin gene region

  3. E-cadherin destabilization accounts for the pathogenicity of missense mutations in hereditary diffuse gastric cancer.

    Directory of Open Access Journals (Sweden)

    Joana Simões-Correia

    Full Text Available E-cadherin is critical for the maintenance of tissue architecture due to its role in cell-cell adhesion. E-cadherin mutations are the genetic cause of Hereditary Diffuse Gastric Cancer (HDGC and missense mutations represent a clinical burden, due to the uncertainty of their pathogenic role. In vitro and in vivo, most mutations lead to loss-of-function, although the causal factor is unknown for the majority. We hypothesized that destabilization could account for the pathogenicity of E-cadherin missense mutations in HDGC, and tested our hypothesis using in silico and in vitro tools. FoldX algorithm was used to calculate the impact of each mutation in E-cadherin native-state stability, and the analysis was complemented with evolutionary conservation, by SIFT. Interestingly, HDGC patients harbouring germline E-cadherin destabilizing mutants present a younger age at diagnosis or death, suggesting that the loss of native-state stability of E-cadherin accounts for the disease phenotype. To elucidate the biological relevance of E-cadherin destabilization in HDGC, we investigated a group of newly identified HDGC-associated mutations (E185V, S232C and L583R, of which L583R is predicted to be destabilizing. We show that this mutation is not functional in vitro, exhibits shorter half-life and is unable to mature, due to premature proteasome-dependent degradation, a phenotype reverted by stabilization with the artificial mutation L583I (structurally tolerated. Herein we report E-cadherin structural models suitable to predict the impact of the majority of cancer-associated missense mutations and we show that E-cadherin destabilization leads to loss-of-function in vitro and increased pathogenicity in vivo.

  4. Artemisinin resistance without pfkelch13 mutations in Plasmodium falciparum isolates from Cambodia.

    Science.gov (United States)

    Mukherjee, Angana; Bopp, Selina; Magistrado, Pamela; Wong, Wesley; Daniels, Rachel; Demas, Allison; Schaffner, Stephen; Amaratunga, Chanaki; Lim, Pharath; Dhorda, Mehul; Miotto, Olivo; Woodrow, Charles; Ashley, Elizabeth A; Dondorp, Arjen M; White, Nicholas J; Wirth, Dyann; Fairhurst, Rick; Volkman, Sarah K

    2017-05-12

    Artemisinin resistance is associated with delayed parasite clearance half-life in vivo and correlates with ring-stage survival under dihydroartemisinin in vitro. Both phenotypes are associated with mutations in the PF3D7_1343700 pfkelch13 gene. Recent spread of artemisinin resistance and emerging piperaquine resistance in Southeast Asia show that artemisinin combination therapy, such as dihydroartemisinin-piperaquine, are losing clinical effectiveness, prompting investigation of drug resistance mechanisms and development of strategies to surmount emerging anti-malarial resistance. Sixty-eight parasites isolates with in vivo clearance data were obtained from two Tracking Resistance to Artemisinin Collaboration study sites in Cambodia, culture-adapted, and genotyped for pfkelch13 and other mutations including pfmdr1 copy number; and the RSA 0-3h survival rates and response to antimalarial drugs in vitro were measured for 36 of these isolates. Among these 36 parasites one isolate demonstrated increased ring-stage survival for a PfKelch13 mutation (D584V, RSA 0-3h  = 8%), previously associated with slow clearance but not yet tested in vitro. Several parasites exhibited increased ring-stage survival, yet lack pfkelch13 mutations, and one isolate showed evidence for piperaquine resistance. This study of 68 culture-adapted Plasmodium falciparum clinical isolates from Cambodia with known clearance values, associated the D584V PfKelch13 mutation with increased ring-stage survival and identified parasites that lack pfkelch13 mutations yet exhibit increased ring-stage survival. These data suggest mutations other than those found in pfkelch13 may be involved in conferring artemisinin resistance in P. falciparum. Piperaquine resistance was also detected among the same Cambodian samples, consistent with reports of emerging piperaquine resistance in the field. These culture-adapted parasites permit further investigation of mechanisms of both artemisinin and piperaquine

  5. Somatic point mutation calling in low cellularity tumors.

    Directory of Open Access Journals (Sweden)

    Karin S Kassahn

    Full Text Available Somatic mutation calling from next-generation sequencing data remains a challenge due to the difficulties of distinguishing true somatic events from artifacts arising from PCR, sequencing errors or mis-mapping. Tumor cellularity or purity, sub-clonality and copy number changes also confound the identification of true somatic events against a background of germline variants. We have developed a heuristic strategy and software (http://www.qcmg.org/bioinformatics/qsnp/ for somatic mutation calling in samples with low tumor content and we show the superior sensitivity and precision of our approach using a previously sequenced cell line, a series of tumor/normal admixtures, and 3,253 putative somatic SNVs verified on an orthogonal platform.

  6. The relationship of the factor V Leiden mutation or the deletion-deletion polymorphism of the angiotensin converting enzyme to postoperative thromboembolic events following total joint arthroplasty

    Directory of Open Access Journals (Sweden)

    Fang Carrie

    2001-04-01

    Full Text Available Abstract Background Although all patients undergoing total joint arthroplasty are subjected to similar risk factors that predispose to thromboembolism, only a subset of patients develop this complication. The objective of this study was to determine whether a specific genetic profile is associated with a higher risk of developing a postoperative thromboembolic complication. Specifically, we examined if the Factor V Leiden (FVL mutation or the deletion polymorphism of the angiotensin-converting enzyme (ACE gene increased a patient's risk for postoperative thromboembolic events. The FVL mutation has been associated with an increased risk of idiopathic thromboembolism and the deletion polymorphism of the ACE gene has been associated with increased vascular tone, attenuated fibrinolysis and increased platelet aggregation. Methods The presence of these genetic profiles was determined for 38 patients who had a postoperative symptomatic pulmonary embolus or proximal deep venous thrombosis and 241 control patients without thrombosis using molecular biological techniques. Results The Factor V Leiden mutation was present in none of the 38 experimental patients and in 3% or 8 of the 241 controls (p = 0.26. Similarly there was no difference detected in the distribution of polymorphisms for the ACE gene with the deletion-deletion genotype present in 36% or 13 of the 38 experimental patients and in 31% or 74 of the 241 controls (p = 0.32. Conclusions Our results suggest that neither of these potentially hypercoaguable states are associated with an increased risk of symptomatic thromboembolic events following total hip or knee arthroplasty in patients receiving pharmacological thromboprophylaxis.

  7. Mutations in the DNA-binding domain of NR2E3 affect in vivo dimerization and interaction with CRX.

    Directory of Open Access Journals (Sweden)

    Raphael Roduit

    Full Text Available BACKGROUND: NR2E3 (PNR is an orphan nuclear receptor essential for proper photoreceptor determination and differentiation. In humans, mutations in NR2E3 have been associated with the recessively inherited enhanced short wavelength sensitive (S- cone syndrome (ESCS and, more recently, with autosomal dominant retinitis pigmentosa (adRP. NR2E3 acts as a suppressor of the cone generation program in late mitotic retinal progenitor cells. In adult rod photoreceptors, NR2E3 represses cone-specific gene expression and acts in concert with the transcription factors CRX and NRL to activate rod-specific genes. NR2E3 and CRX have been shown to physically interact in vitro through their respective DNA-binding domains (DBD. The DBD also contributes to homo- and heterodimerization of nuclear receptors. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed NR2E3 homodimerization and NR2E3/CRX complex formation in an in vivo situation by Bioluminescence Resonance Energy Transfer (BRET(2. NR2E3 wild-type protein formed homodimers in transiently transfected HEK293T cells. NR2E3 homodimerization was impaired in presence of disease-causing mutations in the DBD, except for the p.R76Q and p.R104W mutant proteins. Strikingly, the adRP-linked p.G56R mutant protein interacted with CRX with a similar efficiency to that of NR2E3 wild-type and p.R311Q proteins. In contrast, all other NR2E3 DBD-mutant proteins did not interact with CRX. The p.G56R mutant protein was also more effective in abolishing the potentiation of rhodospin gene transactivation by the NR2E3 wild-type protein. In addition, the p.G56R mutant enhanced the transrepression of the M- and S-opsin promoter, while all other NR2E3 DBD-mutants did not. CONCLUSIONS/SIGNIFICANCE: These results suggest different disease mechanisms in adRP- and ESCS-patients carrying NR2E3 mutations. Titration of CRX by the p.G56R mutant protein acting as a repressor in trans may account for the severe clinical phenotype in adRP patients.

  8. In vivo therapeutic responses contingent on Fanconi anemia/BRCA2 status of the tumor.

    Science.gov (United States)

    van der Heijden, Michiel S; Brody, Jonathan R; Dezentje, David A; Gallmeier, Eike; Cunningham, Steven C; Swartz, Michael J; DeMarzo, Angelo M; Offerhaus, G Johan A; Isacoff, William H; Hruban, Ralph H; Kern, Scott E

    2005-10-15

    BRCA2, FANCC, and FANCG gene mutations are present in a subset of pancreatic cancer. Defects in these genes could lead to hypersensitivity to interstrand cross-linkers in vivo and a more optimal treatment of pancreatic cancer patients based on the genetic profile of the tumor. Two retrovirally complemented pancreatic cancer cell lines having defects in the Fanconi anemia pathway, PL11 (FANCC-mutated) and Hs766T (FANCG-mutated), as well as several parental pancreatic cancer cell lines with or without mutations in the Fanconi anemia/BRCA2 pathway, were assayed for in vitro and in vivo sensitivities to various chemotherapeutic agents. A distinct dichotomy of drug responses was observed. Fanconi anemia-defective cancer cells were hypersensitive to the cross-linking agents mitomycin C (MMC), cisplatin, chlorambucil, and melphalan but not to 5-fluorouracil, gemcitabine, doxorubicin, etoposide, vinblastine, or paclitaxel. Hypersensitivity to cross-linking agents was confirmed in vivo; FANCC-deficient xenografts of PL11 and BRCA2-deficient xenografts of CAPAN1 regressed on treatment with two different regimens of MMC whereas Fanconi anemia-proficient xenografts did not. The MMC response comprised cell cycle arrest, apoptosis, and necrosis. Xenografts of PL11 also regressed after a single dose of cyclophosphamide whereas xenografts of genetically complemented PL11(FANCC) did not. MMC or other cross-linking agents as a clinical therapy for pancreatic cancer patients with tumors harboring defects in the Fanconi anemia/BRCA2 pathway should be specifically investigated.

  9. A novel deleterious PTEN mutation in a patient with early-onset bilateral breast cancer

    International Nuclear Information System (INIS)

    Pradella, Laura Maria; Gasparre, Giuseppe; Turchetti, Daniela; Evangelisti, Cecilia; Ligorio, Claudia; Ceccarelli, Claudio; Neri, Iria; Zuntini, Roberta; Amato, Laura Benedetta; Ferrari, Simona; Martelli, Alberto Maria

    2014-01-01

    An early age at Breast Cancer (BC) onset may be a hallmark of inherited predisposition, but BRCA1/2 mutations are only found in a minority of younger BC patients. Among the others, a fraction may carry mutations in rarer BC genes, such as TP53, STK11, CDH1 and PTEN. As the identification of women harboring such mutations allows for targeted risk-management, the knowledge of associated manifestations and an accurate clinical and family history evaluation are warranted. We describe the case of a woman who developed an infiltrating ductal carcinoma of the right breast at the age of 32, a contralateral BC at age 36 and another BC of the right breast at 40. When she was 39 years-old, during a dermatological examination, mucocutaneous features suggestive of Cowden Syndrome, a disorder associated to germ-line PTEN mutations, were noticed. PTEN genetic testing revealed the novel c.71A > T (p.Asp24Val) mutation, whose deleterious effect, suggested by conservation data and in silico tools, was definitely demonstrated by the incapacity of mutant PTEN to inhibit Akt phosphorylation when used to complement PTEN-null cells. In BC tissue, despite the absence of LOH or somatic mutations of PTEN, Akt phosphorylation was markedly increased in comparison to normal tissue, thus implying additional somatic events into the deregulation of the PI3K/Akt/mTOR pathway and, presumably, into carcinogenesis. Hence, known oncogenic mutations in PIK3CA (exons 10 and 21) and AKT1 (exon 2) were screened in tumor DNA with negative results, which suggests that the responsible somatic event(s) is a different, uncommon one. This case stresses the importance of clinical/genetic assessment of early-onset BC patients in order to identify mutation carriers, who are at high risk of new events, so requiring tailored management. Moreover, it revealed a novel PTEN mutation with pathogenic effect, pointing out, however, the need for further efforts to elucidate the molecular steps of PTEN

  10. Coexistence of K-ras mutations and HPV infection in colon cancer

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    Tezol Ayda

    2006-05-01

    Full Text Available Abstract Background Activation of the ras genes or association with human papillomavirus infection have been extensively studied in colorectal cancer. However, the correlation between K-ras mutations and HPV in colorectal cancer has not been investigated yet. In this study we aimed to investigate the presence of K-ras mutations and their correlation with HPV infection in colon cancer. Methods K-ras mutations were analyzed by a mutagenic PCR assay and digestion with specific restriction enzymes to distinguish the wild-type and mutant codons. HPV infection was analyzed by PCR amplification and hybridization with specific probes by Southern blotting. Stattistical analyses were performed by the chi-square and Fisher's exact tests Results HPV gene fragments were detected in 43 tumors and 17 normal tissue samples. HPV 18 was the prevalent type in the tumor tissue. A mutation at codon 12 of the K-ras gene was present in 31 patients. 56% of the HPV-positive tumors also harbored a K-ras mutation. Codon 13 mutations were not observed. These data indicate that infection with high risk HPV types and mutational activation of the K-ras gene are frequent events in colorectal carcinogenesis. Conclusion Our findings suggest that mutational activation of the K-ras gene is a common event in colon carcinogenesis and that HPV infection may represent an important factor in the development of the premalignant lesions leading to the neoplastic phenotype.

  11. Screening of Duchenne muscular dystrophy (DMD mutations and investigating its mutational mechanism in Chinese patients.

    Directory of Open Access Journals (Sweden)

    Chen Chen

    Full Text Available Duchenne muscular dystrophy (DMD is a common X-linked recessive disease of muscle degeneration and death. In order to provide accurate and reliable genetic counseling and prenatal diagnosis, we screened DMD mutations in a cohort of 119 Chinese patients using multiplex ligation-dependent probe amplification (MLPA and denaturing high performance liquid chromatography (DHPLC followed by Sanger sequencing. In these unrelated DMD patients, we identified 11 patients with DMD small mutations (9.2% and 81 patients with DMD deletions/duplications (del/dup (68.1%, of which 64 (79.0% were deletions, 16 (19.8% were duplications, and one (1.2% was both deletion and duplication. Furthermore, we analyzed the frequency of DMD breakpoint in the 64 deletion cases by calculating exon-deletion events of certain exon interval that revealed a novel mutation hotspot boundary. To explore why DMD rearrangement breakpoints were predisposed to specific regions (hotspot, we precisely characterized junction sequences of breakpoints at the nucleotide level in 21 patients with exon deleted/duplicated in DMD with a high-resolution SNP microarray assay. There were no exactly recurrent breakpoints and there was also no significant difference between single-exon del/dup and multiple-exon del/dup cases. The data from the current study provided a comprehensive strategy to detect DMD mutations for clinical practice, and identified two deletion hotspots at exon 43-55 and exon 10-23 by calculating exon-deletion events of certain exon interval. Furthermore, this is the first study to characterize DMD breakpoint at the nucleotide level in a Chinese population. Our observations provide better understanding of the mechanism for DMD gene rearrangements.

  12. Sequential mutations in Notch1, Fbxw7, and Tp53 in radiation-induced mouse thymic lymphomas.

    Science.gov (United States)

    Jen, Kuang-Yu; Song, Ihn Young; Banta, Karl Luke; Wu, Di; Mao, Jian-Hua; Balmain, Allan

    2012-01-19

    T-cell acute lymphoblastic lymphomas commonly demonstrate activating Notch1 mutations as well as mutations or deletions in Fbxw7. However, because Fbxw7 targets Notch1 for degradation, genetic alterations in these genes are expected to be mutually exclusive events in lymphomagenesis. Previously, by using a radiation-induced Tp53-deficient mouse model for T-cell acute lymphoblastic lymphoma, we reported that loss of heterozygosity at the Fbxw7 locus occurs frequently in a Tp53-dependent manner. In the current study, we show that these thymic lymphomas also commonly exhibit activating Notch1 mutations in the proline-glutamic acid-serine-threonine (PEST) domain. Moreover, concurrent activating Notch1 PEST domain mutations and single-copy deletions at the Fbxw7 locus occur with high frequency in the same individual tumors, indicating that these changes are not mutually exclusive events. We further demonstrate that although Notch1 PEST domain mutations are independent of Tp53 status, they are completely abolished in mice with germline Fbxw7 haploinsufficiency. Therefore, Notch1 PEST domain mutations only occur when Fbxw7 expression levels are intact. These data suggest a temporal sequence of mutational events involving these important cancer-related genes, with Notch1 PEST domain mutations occurring first, followed by Fbxw7 deletion, and eventually by complete loss of Tp53.

  13. Studies of human mutation rates, December 1, 1985--November 30, 1986

    International Nuclear Information System (INIS)

    Neel, J.V.

    1985-01-01

    This program seeks to quantify native human mutation rates and to determine how man's activities may affect these rates. The program is divided into six tasks, i.e. The American Indian mutation rate, monitoring populations for frequency of mutation by electrophoresis of blood proteins, application of molecular biological approaches to the detection and study of mutational events in human populations, development of two-dimensional electrophoresis for identification of mutant proteins, co-operative program with the Radiation Effects Research Foundation in Hiroshima and Nagasaki, Japan, and statistical problems associated with the estimation of mutation rates. Progress of each of the above tasks is related in detail. (DT)

  14. Mature Microsatellites: Mechanisms Underlying Dinucleotide Microsatellite Mutational Biases in Human Cells

    OpenAIRE

    Baptiste, Beverly A.; Ananda, Guruprasad; Strubczewski, Noelle; Lutzkanin, Andrew; Khoo, Su Jen; Srikanth, Abhinaya; Kim, Nari; Makova, Kateryna D.; Krasilnikova, Maria M.; Eckert, Kristin A.

    2013-01-01

    Dinucleotide microsatellites are dynamic DNA sequences that affect genome stability. Here, we focused on mature microsatellites, defined as pure repeats of lengths above the threshold and unlikely to mutate below it in a single mutational event. We investigated the prevalence and mutational behavior of these sequences by using human genome sequence data, human cells in culture, and purified DNA polymerases. Mature dinucleotides (?10 units) are present within exonic sequences of >350 genes, re...

  15. In vitro and in vivo growth alter the population dynamic and properties of a Jeryl Lynn mumps vaccine.

    Science.gov (United States)

    Connaughton, Sarah M; Wheeler, Jun X; Vitková, Eva; Minor, Philip; Schepelmann, Silke

    2015-08-26

    Mumps vaccines are live attenuated viruses. They are known to vary in effectiveness, degree of attenuation and adverse event profile. However, the underlying reasons are poorly understood. We studied two closely related mumps vaccines which originate from the same attenuated Jeryl Lynn-5 strain but have different efficacies. Jeryl Lynn-Canine Kidney (JL-CK), produced on primary canine kidney cells, is less effective than RIT4385, which is produced on chicken embryo fibroblasts. JL-CK and RIT4385 could be distinguished by a number of in vitro and in vivo properties. JL-CK produced heterogeneous, generally smaller plaques than RIT4385, but gave 100-fold higher titres when grown in cells and showed a higher degree of hydrocephalus formation in neonatal rat brains. Sanger sequencing of JL-CK identified 14 regions of heterogeneity throughout the genome. Plaque purification of JL-CK demonstrated the presence of five different Jeryl Lynn-5 variants encompassing the 14 mutations. One JL-CK mutation was associated with a small plaque phenotype, the effects of the others in vitro or in vivo were less clear. Only 4% of the JL-CK population corresponded to the parental Jeryl Lynn-5 strain. Next generation sequencing of JL-CK and virus before and after growth in cell lines or neonatal rat brains showed that propagation in vitro or in vivo altered the population dramatically. Our findings indicate that growth of JL-CK in primary canine kidney cells resulted in the selection of a mixture of mumps virus variants that have different biological properties compared to the parent Jeryl Lynn-5 virus. We also report three previously unknown heterogenic regions within the N gene of the RIT4385 vaccine. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  16. Confirmation of emergence of mutations associated with atovaquone-proguanil resistance in unexposed Plasmodium falciparum isolates from Africa

    OpenAIRE

    Happi, Christian T; Gbotosho, Grace O; Folarin, Onikepe A; Milner, Danny; Sarr, Ousmane; Sowunmi, Akintunde; Kyle, Dennis E; Milhous, Wilbur K; Wirth, Dyann F; Oduola, Ayoade MJ

    2006-01-01

    Abstract Background In vitro and in vivo resistance of Plasmodium falciparum to atovaquone or atovaquone-proguanil hydrochloride combination has been associated to two point mutations in the parasite cytochrome b (cytb) gene (Tyr268Ser and Tyr268Asn). However, little is known about the prevalence of codon-268 mutations in natural populations of P. falciparum without previous exposure to the drug in Africa. Methods The prevalence of codon-268 mutations in the cytb gene of African P. falciparum...

  17. Herpes simplex virus mutant generation and dual-detection methods for gaining insight into latent/lytic cycles in vivo.

    Science.gov (United States)

    Sawtell, Nancy M; Thompson, Richard L

    2014-01-01

    Two important components to a useful strategy to examine viral gene regulation in vivo are (1) a highly efficient protocol to generate viral mutants that limits undesired mutation and retains full replication competency in vivo and (2) an efficient system to detect and quantify viral promoter activity in rare cells in vivo. Our strategy and protocols for generating, characterizing, and employing HSV viral promoter/reporter mutants in vivo are provided in this two-part chapter.

  18. Functional significance of SRJ domain mutations in CITED2.

    Directory of Open Access Journals (Sweden)

    Chiann-mun Chen

    Full Text Available CITED2 is a transcriptional co-activator with 3 conserved domains shared with other CITED family members and a unique Serine-Glycine Rich Junction (SRJ that is highly conserved in placental mammals. Loss of Cited2 in mice results in cardiac and aortic arch malformations, adrenal agenesis, neural tube and placental defects, and partially penetrant defects in left-right patterning. By screening 1126 sporadic congenital heart disease (CHD cases and 1227 controls, we identified 19 variants, including 5 unique non-synonymous sequence variations (N62S, R92G, T166N, G180-A187del and A187T in patients. Many of the CHD-specific variants identified in this and previous studies cluster in the SRJ domain. Transient transfection experiments show that T166N mutation impairs TFAP2 co-activation function and ES cell proliferation. We find that CITED2 is phosphorylated by MAPK1 in vitro at T166, and that MAPK1 activation enhances the coactivation function of CITED2 but not of CITED2-T166N. In order to investigate the functional significance in vivo, we generated a T166N mutation of mouse Cited2. We also used PhiC31 integrase-mediated cassette exchange to generate a Cited2 knock-in allele replacing the mouse Cited2 coding sequence with human CITED2 and with a mutant form deleting the entire SRJ domain. Mouse embryos expressing only CITED2-T166N or CITED2-SRJ-deleted alleles surprisingly show no morphological abnormalities, and mice are viable and fertile. These results indicate that the SRJ domain is dispensable for these functions of CITED2 in mice and that mutations clustering in the SRJ region are unlikely to be the sole cause of the malformations observed in patients with sporadic CHD. Our results also suggest that coding sequence mutations observed in case-control studies need validation using in vivo models and that predictions based on structural conservation and in vitro functional assays, or even in vivo global loss of function models, may be

  19. Mutations in Plasmodium falciparum K13 propeller gene from Bangladesh (2009–2013)

    OpenAIRE

    Mohon, Abu Naser; Alam, Mohammad Shafiul; Bayih, Abebe Genetu; Folefoc, Asongna; Shahinas, Dea; Haque, Rashidul; Pillai, Dylan R

    2014-01-01

    Background Bangladesh is a malaria hypo-endemic country sharing borders with India and Myanmar. Artemisinin combination therapy (ACT) remains successful in Bangladesh. An increase of artemisinin-resistant malaria parasites on the Thai-Cambodia and Thai-Myanmar borders is worrisome. K13 propeller gene (PF3D7_1343700 or PF13_0238) mutations have been linked to both in vitro artemisinin resistance and in vivo slow parasite clearance rates. This group undertook to evaluate if mutations seen in Ca...

  20. Advances in in vivo EPR Tooth Biodosimetry: Meeting the targets for initial triage following a large-scale radiation event

    International Nuclear Information System (INIS)

    Flood, Ann Barry; Schreiber, Wilson; Du, Gaixin; Wood, Victoria A.; Kmiec, Maciej M.; Petryakov, Sergey V.; Williams, Benjamin B.; Swartz, Harold M.; Demidenko, Eugene; Boyle, Holly K.; Dong, Ruhong; Geimer, Shireen; Jarvis, Lesley A.; Kobayashi, Kyo; Nicolalde; Roberto, J.; Crist, Jason; Gupta, Ankit; Raynolds, Timothy; Brugger, Spencer; Budzioh, Pawel; Carr, Brandon; Feldman, Matthew; Gimi, Barjor; Grinberg, Oleg; Krymov, Vladimir; Lesniewski, Piotr; Mariani, Michael; Meaney, Paul M.; Rychert, Kevin M.; Salikhov, Ildar; Tipikin, Dmitriy S.; Tseytlin, Mark; Edwards, Brian R.; Herring, Christopher D.; Lindsay, Catherine; Rosenbaum, Traci; Ali, Arif; Carlson, David; Froncisz, Wojciech; Hirata, Hiroshi; Sidabras, Jason; Swarts, Steven G.

    2016-01-01

    Several important recent advances in the development and evolution of in vivo Tooth Biodosimetry using Electron Paramagnetic Resonance (EPR) allow its performance to meet or exceed the U.S. targeted requirements for accuracy and ease of operation and throughput in a large-scale radiation event. Ergonomically based changes to the magnet, coupled with the development of rotation of the magnet and advanced software to automate collection of data, have made it easier and faster to make a measurement. From start to finish, measurements require a total elapsed time of 5 min, with data acquisition taking place in less than 3 min. At the same time, the accuracy of the data for triage of large populations has improved, as indicated using the metrics of sensitivity, specificity and area under the ROC curve. Applying these standards to the intended population, EPR in vivo Tooth Biodosimetry has approximately the same diagnostic accuracy as the purported 'gold standard' (dicentric chromosome assay). Other improvements include miniaturisation of the spectrometer, leading to the creation of a significantly lighter and more compact prototype that is suitable for transporting for Point of Care (POC) operation and that can be operated off a single standard power outlet. Additional advancements in the resonator, including use of a disposable sensing loop attached to the incisor tooth, have resulted in a biodosimetry method where measurements can be made quickly with a simple 5-step workflow and by people needing only a few minutes of training (which can be built into the instrument as a training video). In sum, recent advancements allow this prototype to meet or exceed the US Federal Government's recommended targets for POC biodosimetry in large-scale events. (authors)

  1. Stable cytotoxic T cell escape mutation in hepatitis C virus is linked to maintenance of viral fitness.

    Directory of Open Access Journals (Sweden)

    Luke Uebelhoer

    2008-09-01

    Full Text Available Mechanisms by which hepatitis C virus (HCV evades cellular immunity to establish persistence in chronically infected individuals are not clear. Mutations in human leukocyte antigen (HLA class I-restricted epitopes targeted by CD8(+ T cells are associated with persistence, but the extent to which these mutations affect viral fitness is not fully understood. Previous work showed that the HCV quasispecies in a persistently infected chimpanzee accumulated multiple mutations in numerous class I epitopes over a period of 7 years. During the acute phase of infection, one representative epitope in the C-terminal region of the NS3/4A helicase, NS3(1629-1637, displayed multiple serial amino acid substitutions in major histocompatibility complex (MHC anchor and T cell receptor (TCR contact residues. Only one of these amino acid substitutions at position 9 (P9 of the epitope was stable in the quasispecies. We therefore assessed the effect of each mutation observed during in vivo infection on viral fitness and T cell responses using an HCV subgenomic replicon system and a recently developed in vitro infectious virus cell culture model. Mutation of a position 7 (P7 TCR-contact residue, I1635T, expectedly ablated the T cell response without affecting viral RNA replication or virion production. In contrast, two mutations at the P9 MHC-anchor residue abrogated antigen-specific T cell responses, but additionally decreased viral RNA replication and virion production. The first escape mutation, L1637P, detected in vivo only transiently at 3 mo after infection, decreased viral production, and reverted to the parental sequence in vitro. The second P9 variant, L1637S, which was stable in vivo through 7 years of follow-up, evaded the antigen-specific T cell response and did not revert in vitro despite being less optimal in virion production compared to the parental virus. These studies suggest that HCV escape mutants emerging early in infection are not necessarily

  2. Prognostic implications of mutation-specific QTc standard deviation in congenital long QT syndrome.

    Science.gov (United States)

    Mathias, Andrew; Moss, Arthur J; Lopes, Coeli M; Barsheshet, Alon; McNitt, Scott; Zareba, Wojciech; Robinson, Jennifer L; Locati, Emanuela H; Ackerman, Michael J; Benhorin, Jesaia; Kaufman, Elizabeth S; Platonov, Pyotr G; Qi, Ming; Shimizu, Wataru; Towbin, Jeffrey A; Michael Vincent, G; Wilde, Arthur A M; Zhang, Li; Goldenberg, Ilan

    2013-05-01

    Individual corrected QT interval (QTc) may vary widely among carriers of the same long QT syndrome (LQTS) mutation. Currently, neither the mechanism nor the implications of this variable penetrance are well understood. To hypothesize that the assessment of QTc variance in patients with congenital LQTS who carry the same mutation provides incremental prognostic information on the patient-specific QTc. The study population comprised 1206 patients with LQTS with 95 different mutations and ≥ 5 individuals who carry the same mutation. Multivariate Cox proportional hazards regression analysis was used to assess the effect of mutation-specific standard deviation of QTc (QTcSD) on the risk of cardiac events (comprising syncope, aborted cardiac arrest, and sudden cardiac death) from birth through age 40 years in the total population and by genotype. Assessment of mutation-specific QTcSD showed large differences among carriers of the same mutations (median QTcSD 45 ms). Multivariate analysis showed that each 20 ms increment in QTcSD was associated with a significant 33% (P = .002) increase in the risk of cardiac events after adjustment for the patient-specific QTc duration and the family effect on QTc. The risk associated with QTcSD was pronounced among patients with long QT syndrome type 1 (hazard ratio 1.55 per 20 ms increment; P<.001), whereas among patients with long QT syndrome type 2, the risk associated with QTcSD was not statistically significant (hazard ratio 0.99; P = .95; P value for QTcSD-by-genotype interaction = .002). Our findings suggest that mutations with a wider variation in QTc duration are associated with increased risk of cardiac events. These findings appear to be genotype-specific, with a pronounced effect among patients with the long QT syndrome type 1 genotype. Copyright © 2013. Published by Elsevier Inc.

  3. Mutagenic Organized Recombination Process by Homologous IN vivo Grouping (MORPHING) for directed enzyme evolution.

    Science.gov (United States)

    Gonzalez-Perez, David; Molina-Espeja, Patricia; Garcia-Ruiz, Eva; Alcalde, Miguel

    2014-01-01

    Approaches that depend on directed evolution require reliable methods to generate DNA diversity so that mutant libraries can focus on specific target regions. We took advantage of the high frequency of homologous DNA recombination in Saccharomyces cerevisiae to develop a strategy for domain mutagenesis aimed at introducing and in vivo recombining random mutations in defined segments of DNA. Mutagenic Organized Recombination Process by Homologous IN vivo Grouping (MORPHING) is a one-pot random mutagenic method for short protein regions that harnesses the in vivo recombination apparatus of yeast. Using this approach, libraries can be prepared with different mutational loads in DNA segments of less than 30 amino acids so that they can be assembled into the remaining unaltered DNA regions in vivo with high fidelity. As a proof of concept, we present two eukaryotic-ligninolytic enzyme case studies: i) the enhancement of the oxidative stability of a H2O2-sensitive versatile peroxidase by independent evolution of three distinct protein segments (Leu28-Gly57, Leu149-Ala174 and Ile199-Leu268); and ii) the heterologous functional expression of an unspecific peroxygenase by exclusive evolution of its native 43-residue signal sequence.

  4. Exact, time-independent estimation of clone size distributions in normal and mutated cells.

    Science.gov (United States)

    Roshan, A; Jones, P H; Greenman, C D

    2014-10-06

    Biological tools such as genetic lineage tracing, three-dimensional confocal microscopy and next-generation DNA sequencing are providing new ways to quantify the distribution of clones of normal and mutated cells. Understanding population-wide clone size distributions in vivo is complicated by multiple cell types within observed tissues, and overlapping birth and death processes. This has led to the increased need for mathematically informed models to understand their biological significance. Standard approaches usually require knowledge of clonal age. We show that modelling on clone size independent of time is an alternative method that offers certain analytical advantages; it can help parametrize these models, and obtain distributions for counts of mutated or proliferating cells, for example. When applied to a general birth-death process common in epithelial progenitors, this takes the form of a gambler's ruin problem, the solution of which relates to counting Motzkin lattice paths. Applying this approach to mutational processes, alternative, exact, formulations of classic Luria-Delbrück-type problems emerge. This approach can be extended beyond neutral models of mutant clonal evolution. Applications of these approaches are twofold. First, we resolve the probability of progenitor cells generating proliferating or differentiating progeny in clonal lineage tracing experiments in vivo or cell culture assays where clone age is not known. Second, we model mutation frequency distributions that deep sequencing of subclonal samples produce.

  5. The rad2 mutation affects the molecular nature of UV and acridine-mustard-induced mutations in the ADE2 gene of Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Ivanov, E.L.; Kovaltzova, S.V.; Kassinova, G.V.; Gracheva, L.M.; Korolev, V.G.; Zakharov, I.A.

    1986-01-01

    The authors have studied the molecular nature of ade2 mutations induced by UV light and bifunctional acridine-mustard (BAM) in wild-type (RAD) and in excision-deficient (rad2) strains of the yeast, Saccharomyces cerevisiae. In the RAD strain, UV causes 45% GC → AT transitions among all mutations; in the rad2 strain this value is 77%. BAM was shown to be highly specific for frameshift mutagenesis: 60% frameshifts in the RAD strain, and as many as 84% frameshifts in the rad2 strain were induced. Therefore, the rad2 mutation affects the specificity of UV- and BAM-induced mutagenesis in yeast. Experimental data agree with the view that the majority of mutations in the RAD strain are induced by a prereplicative mechanism, whereas mutations in the rad2 strain are predominantly postreplicative events. (Auth.)

  6. Age-related cancer mutations associated with clonal hematopoietic expansion

    Science.gov (United States)

    Xie, Mingchao; Lu, Charles; Wang, Jiayin; McLellan, Michael D.; Johnson, Kimberly J.; Wendl, Michael C.; McMichael, Joshua F.; Schmidt, Heather K.; Yellapantula, Venkata; Miller, Christopher A.; Ozenberger, Bradley A.; Welch, John S.; Link, Daniel C.; Walter, Matthew J.; Mardis, Elaine R.; Dipersio, John F.; Chen, Feng; Wilson, Richard K.; Ley, Timothy J.; Ding, Li

    2015-01-01

    Several genetic alterations characteristic of leukemia and lymphoma have been detected in the blood of individuals without apparent hematological malignancies. We analyzed blood-derived sequence data from 2,728 individuals within The Cancer Genome Atlas, and discovered 77 blood-specific mutations in cancer-associated genes, the majority being associated with advanced age. Remarkably, 83% of these mutations were from 19 leukemia/lymphoma-associated genes, and nine were recurrently mutated (DNMT3A, TET2, JAK2, ASXL1, TP53, GNAS, PPM1D, BCORL1 and SF3B1). We identified 14 additional mutations in a very small fraction of blood cells, possibly representing the earliest stages of clonal expansion in hematopoietic stem cells. Comparison of these findings to mutations in hematological malignancies identified several recurrently mutated genes that may be disease initiators. Our analyses show that the blood cells of more than 2% of individuals (5–6% of people older than 70 years) contain mutations that may represent premalignant, initiating events that cause clonal hematopoietic expansion. PMID:25326804

  7. Identification of seven novel mutations including the first two genomic rearrangements in SLC26A3 mutated in congenital chloride diarrhea.

    Science.gov (United States)

    Höglund, P; Sormaala, M; Haila, S; Socha, J; Rajaram, U; Scheurlen, W; Sinaasappel, M; de Jonge, H; Holmberg, C; Yoshikawa, H; Kere, J

    2001-09-01

    Congenital chloride diarrhea (CLD) is an autosomal recessive disorder characterized by defective intestinal electrolyte absorption, resulting in voluminous osmotic diarrhea with high chloride content. A variety of mutations in the solute carrier family 26, member 3 gene (SLC26A3, previously known as CLD or DRA) are responsible for the disease. Since the identification of the SLC26A3 gene and the determination of its genomic structure, altogether three founder and 17 private mutations have been characterized within miscellaneous ethnic groups. We screened for mutations in seven unrelated families with CLD. The diagnoses were confirmed by fecal chloride measurements. The combined PCR-SSCP and sequencing analyses revealed altogether seven novel mutations including two missense mutations (S206P, D468V), two splicing defects (IVS12-1G>C, IVS13-2delA), one nonsense mutation (Q436X), one insertion/deletion mutation (2104-2105delGGins29-bp), and an intragenic deletion of SLC26A3 exons 7 and 8. Two previously identified mutations were also found. This is the first report of rearrangement mutations in SLC26A3. Molecular features predisposing SLC26A3 for the two rearrangements may include repetitive elements and palindromic-like sequences. The increasingly wide diversity of SLC26A3 mutations suggests that mutations in the SLC26A3 gene may not be rare events. Copyright 2001 Wiley-Liss, Inc.

  8. The Impact of ESR1 Mutations on the Treatment of Metastatic Breast Cancer.

    Science.gov (United States)

    Pejerrey, Sasha M; Dustin, Derek; Kim, Jin-Ah; Gu, Guowei; Rechoum, Yassine; Fuqua, Suzanne A W

    2018-05-07

    After nearly 20 years of research, it is now established that mutations within the estrogen receptor (ER) gene, ESR1, frequently occur in metastatic breast cancer and influence response to hormone therapy. Though early studies presented differing results, sensitive sequencing techniques now show that ESR1 mutations occur at a frequency between 20 and 40% depending on the assay method. Recent studies have focused on several "hot spot mutations," a cluster of mutations found in the hormone-binding domain of the ESR1 gene. Throughout the course of treatment, tumor evolution can occur, and ESR1 mutations emerge and become enriched in the metastatic setting. Sensitive techniques to continually monitor mutant burden in vivo are needed to effectively treat patients with mutant ESR1. The full impact of these mutations on tumor response to different therapies remains to be determined. However, recent studies indicate that mutant-bearing tumors may be less responsive to specific hormonal therapies, and suggest that aromatase inhibitor (AI) therapy may select for the emergence of ESR1 mutations. Additionally, different mutations may respond discretely to targeted therapies. The need for more preclinical mechanistic studies on ESR1 mutations and the development of better agents to target these mutations are urgently needed. In the future, sequential monitoring of ESR1 mutational status will likely direct personalized therapeutic regimens appropriate to each tumor's unique mutational landscape.

  9. Presenilin 1 mutation decreases both calcium and contractile responses in cerebral arteries.

    Science.gov (United States)

    Toussay, Xavier; Morel, Jean-Luc; Biendon, Nathalie; Rotureau, Lolita; Legeron, François-Pierre; Boutonnet, Marie-Charlotte; Cho, Yoon H; Macrez, Nathalie

    2017-10-01

    Mutations or upregulation in presenilin 1 (PS1) gene are found in familial early-onset Alzheimer's disease or sporadic late-onset Alzheimer's disease, respectively. PS1 has been essentially studied in neurons and its mutation was shown to alter intracellular calcium (Ca 2+ ) signals. Here, we showed that PS1 is expressed in smooth muscle cells (SMCs) of mouse cerebral arteries, and we assessed the effects of the deletion of exon 9 of PS1 (PS1dE9) on Ca 2+ signals and contractile responses of vascular SMC. Agonist-induced contraction of cerebral vessels was significantly decreased in PS1dE9 both in vivo and ex vivo. Spontaneous activity of Ca 2+ sparks through ryanodine-sensitive channels (RyR) was unchanged, whereas the RyR-mediated Ca 2+ -release activated by caffeine was shorter in PS1dE9 SMC when compared with control. Moreover, PS1dE9 mutation decreased the caffeine-activated capacitive Ca 2+ entry, and inhibitors of SERCA pumps reversed the effects of PS1dE9 on Ca 2+ signals. PS1dE9 mutation also leads to the increased expression of SERCA3, phospholamban, and RyR3. These results show that PS1 plays a crucial role in the cerebrovascular system and the vascular reactivity is decreased through altered Ca 2+ signals in PS1dE9 mutant mice. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. A meta-analysis of the relationship between FGFR3 and TP53 mutations in bladder cancer.

    Science.gov (United States)

    Neuzillet, Yann; Paoletti, Xavier; Ouerhani, Slah; Mongiat-Artus, Pierre; Soliman, Hany; de The, Hugues; Sibony, Mathilde; Denoux, Yves; Molinie, Vincent; Herault, Aurélie; Lepage, May-Linda; Maille, Pascale; Renou, Audrey; Vordos, Dimitri; Abbou, Claude-Clément; Bakkar, Ashraf; Asselain, Bernard; Kourda, Nadia; El Gaaied, Amel; Leroy, Karen; Laplanche, Agnès; Benhamou, Simone; Lebret, Thierry; Allory, Yves; Radvanyi, François

    2012-01-01

    TP53 and FGFR3 mutations are the most common mutations in bladder cancers. FGFR3 mutations are most frequent in low-grade low-stage tumours, whereas TP53 mutations are most frequent in high-grade high-stage tumours. Several studies have reported FGFR3 and TP53 mutations to be mutually exclusive events, whereas others have reported them to be independent. We carried out a meta-analysis of published findings for FGFR3 and TP53 mutations in bladder cancer (535 tumours, 6 publications) and additional unpublished data for 382 tumours. TP53 and FGFR3 mutations were not independent events for all tumours considered together (OR = 0.25 [0.18-0.37], p = 0.0001) or for pT1 tumours alone (OR = 0.47 [0.28-0.79], p = 0.0009). However, if the analysis was restricted to pTa tumours or to muscle-invasive tumours alone, FGFR3 and TP53 mutations were independent events (OR = 0.56 [0.23-1.36] (p = 0.12) and OR = 0.99 [0.37-2.7] (p = 0.35), respectively). After stratification of the tumours by stage and grade, no dependence was detected in the five tumour groups considered (pTaG1 and pTaG2 together, pTaG3, pT1G2, pT1G3, pT2-4). These differences in findings can be attributed to the putative existence of two different pathways of tumour progression in bladder cancer: the CIS pathway, in which FGFR3 mutations are rare, and the Ta pathway, in which FGFR3 mutations are frequent. TP53 mutations occur at the earliest stage of the CIS pathway, whereas they occur would much later in the Ta pathway, at the T1G3 or muscle-invasive stage.

  11. The Rapid Emergence of Tigecycline Resistance in blaKPC–2 Harboring Klebsiella pneumoniae, as Mediated in Vivo by Mutation in tetA During Tigecycline Treatment

    Directory of Open Access Journals (Sweden)

    Xiaoxing Du

    2018-04-01

    Full Text Available Tigecycline is one of the last resort treatments for carbapenem-resistant Klebsiella pneumoniae (CRKP infections. Tigecycline resistance often occurs during the clinical treatment of CRKP, yet its mechanism has still not been clearly elucidated. This study presents an analysis of a tigecycline resistance mechanism that developed in clinical isolates from a 56-year-old female patient infected with CRKP during tigecycline treatment. Consecutive clonal consistent K. pneumoniae isolates were obtained during tigecycline treatment. Whole genome sequencing of the isolates was performed, and putative single nucleotide polymorphisms and insertion and deletion mutations were analyzed in susceptible and resistant isolates. The identified gene of interest was examined through experiments involving transformations and conjugations. Four isolates, two of which were susceptible and two resistant, were collected from the patient. All of the isolates belonged to Sequence Type 11 (ST11 and were classified as extensively drug resistant (XDR. One amino acid substitution S251A in TetA was identified in the tigecycline-resistant isolates. Subsequent transformation experiments confirmed the contribution of the TetA variant (S251A to tigecycline resistance. The transfer capacity of tigecycline resistance via this mutation was confirmed by conjugation experiments. Using southern blot hybridization and PCR assays, we further proved that the tetA gene was located on a transferable plasmid of ca. 65 kb in an Escherichia coli EC600 transconjugant. Our results provide direct in vivo evidence that evolution in the tetA gene can lead to tigecycline treatment failure in CRKP clinical strains that carry tetA. Moreover, the transfer capacity of tigecycline resistance mediated by mutated tetA is a threat.

  12. Delineation of Ehlers-Danlos syndrome phenotype due to the c.934C>T, p.(Arg312Cys) mutation in COL1A1: Report on a three-generation family without cardiovascular events, and literature review.

    Science.gov (United States)

    Colombi, Marina; Dordoni, Chiara; Venturini, Marina; Zanca, Arianna; Calzavara-Pinton, Piergiacomo; Ritelli, Marco

    2017-02-01

    Classical Ehlers-Danlos syndrome (cEDS) is a rare connective tissue disorder primarily characterized by hyperextensible skin, defective wound healing, abnormal scars, easy bruising, and generalized joint hypermobility; arterial dissections are rarely observed. Mutations in COL5A1 and COL5A2 encoding type V collagen account for more than 90% of the patients so far characterized. In addition, cEDS phenotype was reported in a small number of patients carrying the c.934C>T mutation in COL1A1 that results in an uncommon substitution of a non-glycine residue in one Gly-Xaa-Yaa repeat of the pro-α1(I)-chain p.(Arg312Cys), which leads to disturbed collagen fibrillogenesis due to delayed removal of the type I procollagen N-propeptide. This specific mutation has been associated with propensity to arterial rupture in early adulthood; indeed, in literature the individuals harboring this mutation are also referred to as "(classic) vascular-like" EDS patients. Herein, we describe a three-generation cEDS family with six adults carrying the p.(Arg312Cys) substitution, which show a variable and prevalent cutaneous involvement without any major vascular event. These data, together with those available in literature, suggest that vascular events are not a diagnostic handle to differentiate patients with the p.(Arg312Cys) COL1A1 mutation from those with COL5A1 and COL5A2 defects, and highlight that during the diagnostic process the presence of at least the p.(Arg312Cys) substitution in COL1A1 should be investigated in cEDS patients without type V collagen mutations. Nevertheless, for these patients, as well as for those affected with cEDS, a periodical vascular surveillance should be carried out together with cardiovascular risk factors monitoring. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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

    Science.gov (United States)

    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.

  14. Mutations of the KRAS oncogene in endometrial hyperplasia and carcinoma.

    Directory of Open Access Journals (Sweden)

    Wiesława Niklińska

    2009-05-01

    Full Text Available The aim of this study was to examine the prevalence and clinicopathological significance of KRAS point mutation in endometrial hyperplasia and carcinoma. We analysed KRAS in 11 cases of complex atypical hyperplasia and in 49 endometrial carcinomas using polymerase chain reaction associated with restriction fragment length polymorphism (PCR-RFPL. Point mutations at codon 12 of KRAS oncogene were identified in 7 of 49 (14,3% tumor specimens and in 2 of 11 (18,2% hyperplasias. No correlation was found between KRAS gene mutation and age at onset, histology, grade of differentiation and clinical stage. We conclude that KRAS mutation is a relatively common event in endometrial carcinogenesis, but with no prognostic value.

  15. TBX3 regulates splicing in vivo: a novel molecular mechanism for Ulnar-mammary syndrome.

    Directory of Open Access Journals (Sweden)

    Pavan Kumar P

    2014-03-01

    Full Text Available TBX3 is a member of the T-box family of transcription factors with critical roles in development, oncogenesis, cell fate, and tissue homeostasis. TBX3 mutations in humans cause complex congenital malformations and Ulnar-mammary syndrome. Previous investigations into TBX3 function focused on its activity as a transcriptional repressor. We used an unbiased proteomic approach to identify TBX3 interacting proteins in vivo and discovered that TBX3 interacts with multiple mRNA splicing factors and RNA metabolic proteins. We discovered that TBX3 regulates alternative splicing in vivo and can promote or inhibit splicing depending on context and transcript. TBX3 associates with alternatively spliced mRNAs and binds RNA directly. TBX3 binds RNAs containing TBX binding motifs, and these motifs are required for regulation of splicing. Our study reveals that TBX3 mutations seen in humans with UMS disrupt its splicing regulatory function. The pleiotropic effects of TBX3 mutations in humans and mice likely result from disrupting at least two molecular functions of this protein: transcriptional regulation and pre-mRNA splicing.

  16. TBX3 regulates splicing in vivo: a novel molecular mechanism for Ulnar-mammary syndrome.

    Science.gov (United States)

    Kumar P, Pavan; Franklin, Sarah; Emechebe, Uchenna; Hu, Hao; Moore, Barry; Lehman, Chris; Yandell, Mark; Moon, Anne M

    2014-03-01

    TBX3 is a member of the T-box family of transcription factors with critical roles in development, oncogenesis, cell fate, and tissue homeostasis. TBX3 mutations in humans cause complex congenital malformations and Ulnar-mammary syndrome. Previous investigations into TBX3 function focused on its activity as a transcriptional repressor. We used an unbiased proteomic approach to identify TBX3 interacting proteins in vivo and discovered that TBX3 interacts with multiple mRNA splicing factors and RNA metabolic proteins. We discovered that TBX3 regulates alternative splicing in vivo and can promote or inhibit splicing depending on context and transcript. TBX3 associates with alternatively spliced mRNAs and binds RNA directly. TBX3 binds RNAs containing TBX binding motifs, and these motifs are required for regulation of splicing. Our study reveals that TBX3 mutations seen in humans with UMS disrupt its splicing regulatory function. The pleiotropic effects of TBX3 mutations in humans and mice likely result from disrupting at least two molecular functions of this protein: transcriptional regulation and pre-mRNA splicing.

  17. Diploid yeast cells yield homozygous spontaneous mutations

    Science.gov (United States)

    Esposito, M. S.; Bruschi, C. V.; Brushi, C. V. (Principal Investigator)

    1993-01-01

    A leucine-requiring hybrid of Saccharomyces cerevisiae, homoallelic at the LEU1 locus (leu1-12/leu1-12) and heterozygous for three chromosome-VII genetic markers distal to the LEU1 locus, was employed to inquire: (1) whether spontaneous gene mutation and mitotic segregation of heterozygous markers occur in positive nonrandom association and (2) whether homozygous LEU1/LEU1 mutant diploids are generated. The results demonstrate that gene mutation of leu1-12 to LEU1 and mitotic segregation of heterozygous chromosome-VII markers occur in strong positive nonrandom association, suggesting that the stimulatory DNA lesion is both mutagenic and recombinogenic. In addition, genetic analysis of diploid Leu+ revertants revealed that approximately 3% of mutations of leu1-12 to LEU1 result in LEU1/LEU1 homozygotes. Red-white sectored Leu+ colonies exhibit genotypes that implicate post-replicational chromatid breakage and exchange near the site of leu1-12 reversion, chromosome loss, and subsequent restitution of diploidy, in the sequence of events leading to mutational homozygosis. By analogy, diploid cell populations can yield variants homozygous for novel recessive gene mutations at biologically significant rates. Mutational homozygosis may be relevant to both carcinogenesis and the evolution of asexual diploid organisms.

  18. Formalin fixation increases deamination mutation signature but should not lead to false positive mutations in clinical practice.

    Directory of Open Access Journals (Sweden)

    Leah M Prentice

    Full Text Available Genomic analysis of cancer tissues is an essential aspect of personalized oncology treatment. Though it has been suggested that formalin fixation of patient tissues may be suboptimal for molecular studies, this tissue processing approach remains the industry standard. Therefore clinical molecular laboratories must be able to work with formalin fixed, paraffin embedded (FFPE material. This study examines the effects of pre-analytic variables introduced by routine pathology processing on specimens used for clinical reports produced by next-generation sequencing technology. Tissue resected from three colorectal cancer patients was subjected to 2, 15, 24, and 48 hour fixation times in neutral buffered formalin. DNA was extracted from all tissues twice, once with uracil-N-glycosylase (UNG treatment to counter deamination effects, and once without. Of note, deamination events at methylated cytosine, as found at CpG sites, remains unaffected by UNG. After extraction a two-step PCR targeted sequencing method was performed using the Illumina MiSeq and the data was analyzed via a custom-built bioinformatics pipeline, including filtration of reads with mapping quality T/A mutations that is not represented in DNA treated with UNG. This suggests these errors may be due to deamination events triggered by a longer fixation time. However the allelic frequency of these events remained below the limit of detection for reportable mutations in this assay (<2%. We do however recommend that suspected intratumoral heterogeneity events be verified by re-sequencing the same FFPE block.

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

    Science.gov (United States)

    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.

  20. Adenovirus sequences required for replication in vivo.

    OpenAIRE

    Wang, K; Pearson, G D

    1985-01-01

    We have studied the in vivo replication properties of plasmids carrying deletion mutations within cloned adenovirus terminal sequences. Deletion mapping located the adenovirus DNA replication origin entirely within the first 67 bp of the adenovirus inverted terminal repeat. This region could be further subdivided into two functional domains: a minimal replication origin and an adjacent auxillary region which boosted the efficiency of replication by more than 100-fold. The minimal origin occup...

  1. Haplotype analysis suggest that the MLH1 c.2059C > T mutation is a Swedish founder mutation.

    Science.gov (United States)

    von Salomé, Jenny; Liu, Tao; Keihäs, Markku; Morak, Moni; Holinski-Feder, Elke; Berry, Ian R; Moilanen, Jukka S; Baert-Desurmont, Stéphanie; Lindblom, Annika; Lagerstedt-Robinson, Kristina

    2017-12-29

    Lynch syndrome (LS) predisposes to a spectrum of cancers and increases the lifetime risk of developing colorectal- or endometrial cancer to over 50%. Lynch syndrome is dominantly inherited and is caused by defects in DNA mismatch-repair genes MLH1, MSH2, MSH6 or PMS2, with the vast majority detected in MLH1 and MSH2. Recurrent LS-associated variants observed in apparently unrelated individuals, have either arisen de novo in different families due to mutation hotspots, or are inherited from a founder (a common ancestor) that lived several generations back. There are variants that recur in some populations while also acting as founders in other ethnic groups. Testing for founder mutations can facilitate molecular diagnosis of Lynch Syndrome more efficiently and more cost effective than screening for all possible mutations. Here we report a study of the missense mutation MLH1 c.2059C > T (p.Arg687Trp), a potential founder mutation identified in eight Swedish families and one Finnish family with Swedish ancestors. Haplotype analysis confirmed that the Finnish and Swedish families shared a haplotype of between 0.9 and 2.8 Mb. While MLH1 c.2059C > T exists worldwide, the Swedish haplotype was not found among mutation carriers from Germany or France, which indicates a common founder in the Swedish population. The geographic distribution of MLH1 c.2059C > T in Sweden suggests a single, ancient mutational event in the northern part of Sweden.

  2. RAC1 Missense Mutations in Developmental Disorders with Diverse Phenotypes.

    Science.gov (United States)

    Reijnders, Margot R F; Ansor, Nurhuda M; Kousi, Maria; Yue, Wyatt W; Tan, Perciliz L; Clarkson, Katie; Clayton-Smith, Jill; Corning, Ken; Jones, Julie R; Lam, Wayne W K; Mancini, Grazia M S; Marcelis, Carlo; Mohammed, Shehla; Pfundt, Rolph; Roifman, Maian; Cohn, Ronald; Chitayat, David; Millard, Tom H; Katsanis, Nicholas; Brunner, Han G; Banka, Siddharth

    2017-09-07

    RAC1 is a widely studied Rho GTPase, a class of molecules that modulate numerous cellular functions essential for normal development. RAC1 is highly conserved across species and is under strict mutational constraint. We report seven individuals with distinct de novo missense RAC1 mutations and varying degrees of developmental delay, brain malformations, and additional phenotypes. Four individuals, each harboring one of c.53G>A (p.Cys18Tyr), c.116A>G (p.Asn39Ser), c.218C>T (p.Pro73Leu), and c.470G>A (p.Cys157Tyr) variants, were microcephalic, with head circumferences between -2.5 to -5 SD. In contrast, two individuals with c.151G>A (p.Val51Met) and c.151G>C (p.Val51Leu) alleles were macrocephalic with head circumferences of +4.16 and +4.5 SD. One individual harboring a c.190T>G (p.Tyr64Asp) allele had head circumference in the normal range. Collectively, we observed an extraordinary spread of ∼10 SD of head circumferences orchestrated by distinct mutations in the same gene. In silico modeling, mouse fibroblasts spreading assays, and in vivo overexpression assays using zebrafish as a surrogate model demonstrated that the p.Cys18Tyr and p.Asn39Ser RAC1 variants function as dominant-negative alleles and result in microcephaly, reduced neuronal proliferation, and cerebellar abnormalities in vivo. Conversely, the p.Tyr64Asp substitution is constitutively active. The remaining mutations are probably weakly dominant negative or their effects are context dependent. These findings highlight the importance of RAC1 in neuronal development. Along with TRIO and HACE1, a sub-category of rare developmental disorders is emerging with RAC1 as the central player. We show that ultra-rare disorders caused by private, non-recurrent missense mutations that result in varying phenotypes are challenging to dissect, but can be delineated through focused international collaboration. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  3. Neighborhood properties are important determinants of temperature sensitive mutations.

    Directory of Open Access Journals (Sweden)

    Svetlana Lockwood

    Full Text Available Temperature-sensitive (TS mutants are powerful tools to study gene function in vivo. These mutants exhibit wild-type activity at permissive temperatures and reduced activity at restrictive temperatures. Although random mutagenesis can be used to generate TS mutants, the procedure is laborious and unfeasible in multicellular organisms. Further, the underlying molecular mechanisms of the TS phenotype are poorly understood. To elucidate TS mechanisms, we used a machine learning method-logistic regression-to investigate a large number of sequence and structure features. We developed and tested 133 features, describing properties of either the mutation site or the mutation site neighborhood. We defined three types of neighborhood using sequence distance, Euclidean distance, and topological distance. We discovered that neighborhood features outperformed mutation site features in predicting TS mutations. The most predictive features suggest that TS mutations tend to occur at buried and rigid residues, and are located at conserved protein domains. The environment of a buried residue often determines the overall structural stability of a protein, thus may lead to reversible activity change upon temperature switch. We developed TS prediction models based on logistic regression and the Lasso regularized procedure. Through a ten-fold cross-validation, we obtained the area under the curve of 0.91 for the model using both sequence and structure features. Testing on independent datasets suggested that the model predicted TS mutations with a 50% precision. In summary, our study elucidated the molecular basis of TS mutants and suggested the importance of neighborhood properties in determining TS mutations. We further developed models to predict TS mutations derived from single amino acid substitutions. In this way, TS mutants can be efficiently obtained through experimentally introducing the predicted mutations.

  4. Low-dose radiation attenuates chemical mutagenesis in vivo. Cross adaptation

    International Nuclear Information System (INIS)

    Kakinuma, Shizuko; Yamauchi, Kazumi; Amasaki, Yoshiko; Nishimura, Mayumi; Shimada, Yoshiya

    2009-01-01

    The biological effects of low-dose radiation are not only of social concern but also of scientific interest. The radioadaptive response, which is defined as an increased radioresistance by prior exposure to low-dose radiation, has been extensively studied both in vitro and in vivo. Here we briefly review the radioadaptive response with respect to mutagenesis, survival rate, and carcinogenesis in vivo, and introduce our recent findings of cross adaptation in mouse thymic cells, that is, the suppressive effect of repeated low-dose radiation on mutation induction by the alkylating agent N-ethyl-N-nitrosourea. (author)

  5. [Comparative mutagenesis of human cells in vivo and in vitro]. Progress report, January 1-December 30, 1985

    International Nuclear Information System (INIS)

    1985-01-01

    Annual progress report is made on project focusing on the comparative mutagenesis of human cells in vivo and in vitro. The study employs the HGPRT gene to explore the changes in nucleotide sequence which has occurred in spontaneous mutations or mutations induced by MNNG or ICR191. Reports on the individual projects have been abstracted and indexed for the Energy Data Base. (DT)

  6. The Versatile Mutational Resistome of Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Carla López-Causapé

    2018-04-01

    Full Text Available One of the most striking features of Pseudomonas aeruginosa is its outstanding capacity for developing antimicrobial resistance to nearly all available antipseudomonal agents through the selection of chromosomal mutations, leading to the failure of the treatment of severe hospital-acquired or chronic infections. Recent whole-genome sequencing (WGS data obtained from in vitro assays on the evolution of antibiotic resistance, in vivo monitoring of antimicrobial resistance development, analysis of sequential cystic fibrosis isolates, and characterization of widespread epidemic high-risk clones have provided new insights into the evolutionary dynamics and mechanisms of P. aeruginosa antibiotic resistance, thus motivating this review. Indeed, the analysis of the WGS mutational resistome has proven to be useful for understanding the evolutionary dynamics of classical resistance pathways and to describe new mechanisms for the majority of antipseudomonal classes, including β-lactams, aminoglycosides, fluoroquinolones, or polymixins. Beyond addressing a relevant scientific question, the analysis of the P. aeruginosa mutational resistome is expected to be useful, together with the analysis of the horizontally-acquired resistance determinants, for establishing the antibiotic resistance genotype, which should correlate with the antibiotic resistance phenotype and as such, it should be useful for the design of therapeutic strategies and for monitoring the efficacy of administered antibiotic treatments. However, further experimental research and new bioinformatics tools are still needed to overcome the interpretation limitations imposed by the complex interactions (including those leading to collateral resistance or susceptibility between the 100s of genes involved in the mutational resistome, as well as the frequent difficulties for differentiating relevant mutations from simple natural polymorphisms.

  7. The Versatile Mutational Resistome of Pseudomonas aeruginosa.

    Science.gov (United States)

    López-Causapé, Carla; Cabot, Gabriel; Del Barrio-Tofiño, Ester; Oliver, Antonio

    2018-01-01

    One of the most striking features of Pseudomonas aeruginosa is its outstanding capacity for developing antimicrobial resistance to nearly all available antipseudomonal agents through the selection of chromosomal mutations, leading to the failure of the treatment of severe hospital-acquired or chronic infections. Recent whole-genome sequencing (WGS) data obtained from in vitro assays on the evolution of antibiotic resistance, in vivo monitoring of antimicrobial resistance development, analysis of sequential cystic fibrosis isolates, and characterization of widespread epidemic high-risk clones have provided new insights into the evolutionary dynamics and mechanisms of P. aeruginosa antibiotic resistance, thus motivating this review. Indeed, the analysis of the WGS mutational resistome has proven to be useful for understanding the evolutionary dynamics of classical resistance pathways and to describe new mechanisms for the majority of antipseudomonal classes, including β-lactams, aminoglycosides, fluoroquinolones, or polymixins. Beyond addressing a relevant scientific question, the analysis of the P. aeruginosa mutational resistome is expected to be useful, together with the analysis of the horizontally-acquired resistance determinants, for establishing the antibiotic resistance genotype, which should correlate with the antibiotic resistance phenotype and as such, it should be useful for the design of therapeutic strategies and for monitoring the efficacy of administered antibiotic treatments. However, further experimental research and new bioinformatics tools are still needed to overcome the interpretation limitations imposed by the complex interactions (including those leading to collateral resistance or susceptibility) between the 100s of genes involved in the mutational resistome, as well as the frequent difficulties for differentiating relevant mutations from simple natural polymorphisms.

  8. Allogeneic Transplant in ELANE and MEFV Mutation Positive Severe Cyclic Neutropenia: Review of Prognostic Factors for Secondary Severe Events

    Directory of Open Access Journals (Sweden)

    Onyemaechi N. Okolo

    2017-01-01

    Full Text Available Objective and Importance. Cyclic neutropenia (CyN is a rare autosomal dominant inherited disorder due to the mutation ELANE primarily affecting bone marrow stem cells and is characterized by recurrent neutropenia every 2 to 4 weeks. Symptoms vary from benign to severe, including death. Postulations on the cause of wide spectrum in symptom presentation include the possibility of other genetic mutations, such as MEFV. Recommended treatment for CyN is G-CSF to keep ANC higher to minimize risk of infection. Case. A 25-year-old male diagnosed with CyN, on G-CSF but worsening quality of life. Pretransplant investigations revealed ELANE mutation positive severe CyN along with familial Mediterranean fever (MEFV mutation. Intervention. Bone marrow transplantation as treatment for dual mutation (ELANE and MEFV mutation positive severe CyN. Conclusion. BMT may be considered as an alternative treatment for severe CyN in patients who are refractory to G-CSF. It is postulated that in our patient the combined mutations (CyN and MEFV may have contributed to the severity of this individual’s symptoms. We suggest CyN patients who present with severe symptoms have evaluation with ELANE mutation testing, Periodic Fever Syndromes Panel, and routine marrow assessment with FISH, conventional cytogenetics, and morphological evaluation for MDS/AML.

  9. JAK2 mutation in a patient with CLL with coexistent myeloproliferative neoplasm (MPN).

    Science.gov (United States)

    Kodali, Srinivas; Chen, Chi; Rathnasabapathy, Chenthilmurugan; Wang, Jen Chin

    2009-12-01

    JAK2 mutation has not been described in patients with chronic lymphocytic leukemia (CLL). We found JAK2 mutation in a patient with CLL and coexisting myeloproliferative neoplasm (MPN). In this patient, we demonstrated the presence of the JAK2 mutation in CD34(+) progenitor cells, myeloid lineage cells, megakaryocytes, B lymphocytes but not in T lymphocytes. This case represents the first case report of JAK2 mutation in CLL and may also suggest that, JAK2 mutation most likely represents a secondary event from primary gene mutations involving the primitive stem cells which give rise to MPN and CLL. Furthermore, in this case, we believe that we are the first to demonstrate that JAK2 mutation in myeloid and B lymphoid cells but not T lymphocytes in a case of coexisting CLL and MPN.

  10. Sequence analysis of LACI mutations obtained from lung cells of control and radon-exposed Big Blue trademark transgenic mice

    International Nuclear Information System (INIS)

    Jostes, R.F.; Cross, F.T.; Stillwell, L.

    1995-01-01

    We have exposed Stratagene Big Blue trademark transgenic mice by inhalation to 310, 640 and 960 Working Level Months (WLM) of radon progency. Twelve LacI mutations have been isolated from the lung tissue of a mouse from the 960-WLM group and the LacI gene sequenced. Mutations are scored only if they occur unambiguously in both strands of the mutant gene; the entire gene is evaluated. In addition, sixteen LacI mutations were isolated from the lung tissue of a mouse from the 640-WLM group; seven have been completely sequenced. Nine LacI mutations from the lung tissue of unirradiated control mice have been sequenced. Sequence data from the unirradiated mice are similar to that found in lung tissue at Stratagene; predominately G:C to A:T transitions in the protein associated region. The mutation spectrum from radon-irradiated mice is markedly different from that obtained with the control, unirradiated mice. Small deletions and insertions compromise 53% of the mutations in the irradiated mice. No multiple events have been noted in the spontaneous mutations; six of the mutations obtained from radon-irradiated mice (26%) have multiple events within the gene. In some, deletions, insertions are base changes occur together. The mutational events in the irradiated mice are approximately equally distributed throughout the gene. The breakpoint rejoining regions of large deletions obtained from the radon-irradiated mice are being studied at the University of California, San Francisco

  11. Age-related mutations associated with clonal hematopoietic expansion and malignancies.

    Science.gov (United States)

    Xie, Mingchao; Lu, Charles; Wang, Jiayin; McLellan, Michael D; Johnson, Kimberly J; Wendl, Michael C; McMichael, Joshua F; Schmidt, Heather K; Yellapantula, Venkata; Miller, Christopher A; Ozenberger, Bradley A; Welch, John S; Link, Daniel C; Walter, Matthew J; Mardis, Elaine R; Dipersio, John F; Chen, Feng; Wilson, Richard K; Ley, Timothy J; Ding, Li

    2014-12-01

    Several genetic alterations characteristic of leukemia and lymphoma have been detected in the blood of individuals without apparent hematological malignancies. The Cancer Genome Atlas (TCGA) provides a unique resource for comprehensive discovery of mutations and genes in blood that may contribute to the clonal expansion of hematopoietic stem/progenitor cells. Here, we analyzed blood-derived sequence data from 2,728 individuals from TCGA and discovered 77 blood-specific mutations in cancer-associated genes, the majority being associated with advanced age. Remarkably, 83% of these mutations were from 19 leukemia and/or lymphoma-associated genes, and nine were recurrently mutated (DNMT3A, TET2, JAK2, ASXL1, TP53, GNAS, PPM1D, BCORL1 and SF3B1). We identified 14 additional mutations in a very small fraction of blood cells, possibly representing the earliest stages of clonal expansion in hematopoietic stem cells. Comparison of these findings to mutations in hematological malignancies identified several recurrently mutated genes that may be disease initiators. Our analyses show that the blood cells of more than 2% of individuals (5-6% of people older than 70 years) contain mutations that may represent premalignant events that cause clonal hematopoietic expansion.

  12. The FTD-like syndrome causing TREM2 T66M mutation impairs microglia function, brain perfusion, and glucose metabolism.

    Science.gov (United States)

    Kleinberger, Gernot; Brendel, Matthias; Mracsko, Eva; Wefers, Benedikt; Groeneweg, Linda; Xiang, Xianyuan; Focke, Carola; Deußing, Maximilian; Suárez-Calvet, Marc; Mazaheri, Fargol; Parhizkar, Samira; Pettkus, Nadine; Wurst, Wolfgang; Feederle, Regina; Bartenstein, Peter; Mueggler, Thomas; Arzberger, Thomas; Knuesel, Irene; Rominger, Axel; Haass, Christian

    2017-07-03

    Genetic variants in the triggering receptor expressed on myeloid cells 2 (TREM2) increase the risk for several neurodegenerative diseases including Alzheimer's disease and frontotemporal dementia (FTD). Homozygous TREM2 missense mutations, such as p.T66M, lead to the FTD-like syndrome, but how they cause pathology is unknown. Using CRISPR/Cas9 genome editing, we generated a knock-in mouse model for the disease-associated Trem2 p.T66M mutation. Consistent with a loss-of-function mutation, we observe an intracellular accumulation of immature mutant Trem2 and reduced generation of soluble Trem2 similar to patients with the homozygous p.T66M mutation. Trem2 p.T66M knock-in mice show delayed resolution of inflammation upon in vivo lipopolysaccharide stimulation and cultured macrophages display significantly reduced phagocytic activity. Immunohistochemistry together with in vivo TSPO small animal positron emission tomography (μPET) demonstrates an age-dependent reduction in microglial activity. Surprisingly, perfusion magnetic resonance imaging and FDG-μPET imaging reveal a significant reduction in cerebral blood flow and brain glucose metabolism. Thus, we demonstrate that a TREM2 loss-of-function mutation causes brain-wide metabolic alterations pointing toward a possible function of microglia in regulating brain glucose metabolism. © 2017 The Authors.

  13. Mutation at the Human D1S80 Minisatellite Locus

    Directory of Open Access Journals (Sweden)

    Kuppareddi Balamurugan

    2012-01-01

    Full Text Available Little is known about the general biology of minisatellites. The purpose of this study is to examine repeat mutations from the D1S80 minisatellite locus by sequence analysis to elucidate the mutational process at this locus. This is a highly polymorphic minisatellite locus, located in the subtelomeric region of chromosome 1. We have analyzed 90,000 human germline transmission events and found seven (7 mutations at this locus. The D1S80 alleles of the parentage trio, the child, mother, and the alleged father were sequenced and the origin of the mutation was determined. Using American Association of Blood Banks (AABB guidelines, we found a male mutation rate of 1.04×10-4 and a female mutation rate of 5.18×10-5 with an overall mutation rate of approximately 7.77×10-5. Also, in this study, we found that the identified mutations are in close proximity to the center of the repeat array rather than at the ends of the repeat array. Several studies have examined the mutational mechanisms of the minisatellites according to infinite allele model (IAM and the one-step stepwise mutation model (SMM. In this study, we found that this locus fits into the one-step mutation model (SMM mechanism in six out of seven instances similar to STR loci.

  14. The use of radiations to induce useful mutations in fruit trees

    International Nuclear Information System (INIS)

    Donini, B.

    1976-01-01

    The researches carried out at Casaccia in this field had covered the problems of technique of mutagenic treatment, mechanism of mutation induction and methodology of somatic mutation isolation. To enhance the efficiency of somatic mutation induction several conditions during and after the treatment have been studied. More experience has been gained with regard to the induction of somatic mutation which raises from genetical event or by tissue arrangement of a chimaeric shoot apex. To increase the size of mutated sector treatment have been carried out on the primordia in a very early stages and to improve the methodology of somatic mutation special techniques have been adapted of handling the material in the propagation. Possibility for early detection of mutants has been explored in cherry by establishing a correlation between mutants and hormonal content. By using the above mentioned techniques, useful mutants have been isolated in cherries, grapes, olives and peaches. (author)

  15. A meta-analysis of the relationship between FGFR3 and TP53 mutations in bladder cancer.

    Directory of Open Access Journals (Sweden)

    Yann Neuzillet

    Full Text Available TP53 and FGFR3 mutations are the most common mutations in bladder cancers. FGFR3 mutations are most frequent in low-grade low-stage tumours, whereas TP53 mutations are most frequent in high-grade high-stage tumours. Several studies have reported FGFR3 and TP53 mutations to be mutually exclusive events, whereas others have reported them to be independent. We carried out a meta-analysis of published findings for FGFR3 and TP53 mutations in bladder cancer (535 tumours, 6 publications and additional unpublished data for 382 tumours. TP53 and FGFR3 mutations were not independent events for all tumours considered together (OR = 0.25 [0.18-0.37], p = 0.0001 or for pT1 tumours alone (OR = 0.47 [0.28-0.79], p = 0.0009. However, if the analysis was restricted to pTa tumours or to muscle-invasive tumours alone, FGFR3 and TP53 mutations were independent events (OR = 0.56 [0.23-1.36] (p = 0.12 and OR = 0.99 [0.37-2.7] (p = 0.35, respectively. After stratification of the tumours by stage and grade, no dependence was detected in the five tumour groups considered (pTaG1 and pTaG2 together, pTaG3, pT1G2, pT1G3, pT2-4. These differences in findings can be attributed to the putative existence of two different pathways of tumour progression in bladder cancer: the CIS pathway, in which FGFR3 mutations are rare, and the Ta pathway, in which FGFR3 mutations are frequent. TP53 mutations occur at the earliest stage of the CIS pathway, whereas they occur would much later in the Ta pathway, at the T1G3 or muscle-invasive stage.

  16. NetNorM: Capturing cancer-relevant information in somatic exome mutation data with gene networks for cancer stratification and prognosis.

    Science.gov (United States)

    Le Morvan, Marine; Zinovyev, Andrei; Vert, Jean-Philippe

    2017-06-01

    Genome-wide somatic mutation profiles of tumours can now be assessed efficiently and promise to move precision medicine forward. Statistical analysis of mutation profiles is however challenging due to the low frequency of most mutations, the varying mutation rates across tumours, and the presence of a majority of passenger events that hide the contribution of driver events. Here we propose a method, NetNorM, to represent whole-exome somatic mutation data in a form that enhances cancer-relevant information using a gene network as background knowledge. We evaluate its relevance for two tasks: survival prediction and unsupervised patient stratification. Using data from 8 cancer types from The Cancer Genome Atlas (TCGA), we show that it improves over the raw binary mutation data and network diffusion for these two tasks. In doing so, we also provide a thorough assessment of somatic mutations prognostic power which has been overlooked by previous studies because of the sparse and binary nature of mutations.

  17. Genome Analysis of a Transmissible Lineage of Pseudomonas aeruginosa Reveals Pathoadaptive Mutations and Distinct Evolutionary Paths of Hypermutators

    DEFF Research Database (Denmark)

    Marvig, Rasmus Lykke; Johansen, Helle Krogh; Molin, Søren

    2013-01-01

    Genome sequencing of bacterial pathogens has advanced our understanding of their evolution, epidemiology, and response to antibiotic therapy. However, we still have only a limited knowledge of the molecular changes in in vivo evolving bacterial populations in relation to long-term, chronic...... targeted by mutations to optimize pathogen fitness (pathoadaptive mutations). These genes were related to antibiotic resistance, the cell envelope, or regulatory functions, and we find that the prevalence of pathoadaptive mutations correlates with evolutionary success of co-evolving sub-lineages. The long...... likelihood to acquire mutations and identify two homopolymer-containing genes preferentially mutated in hypermutators. This homopolymer facilitated differential mutagenesis provides a novel genome-wide perspective on the different evolutionary trajectories of hypermutators, which may help explain...

  18. Gene-mutation assays in lambda-lacZ transgenic mice : comparison of lacZ with endogenous genes in splenocytes and small intestinal epithelium

    NARCIS (Netherlands)

    Delft, J.H.M. van; Bergmans, A.; Dam, F.J. van; Tates, A.D.; Howard, L.; Winton, D.J.; Baan, R.A.

    1998-01-01

    Comparison of results derived from transgenic animal gene-mutation assays with those from mutation analyses in endogenous genes is an important step in the validation of the former. We have used λlacZ transgenic mice to study alkylation-induced mutagenesis in vivo in (a) lacZ and hprt in spleen

  19. Diverse growth hormone receptor gene mutations in Laron syndrome.

    Science.gov (United States)

    Berg, M A; Argente, J; Chernausek, S; Gracia, R; Guevara-Aguirre, J; Hopp, M; Pérez-Jurado, L; Rosenbloom, A; Toledo, S P; Francke, U

    1993-01-01

    To better understand the molecular genetic basis and genetic epidemiology of Laron syndrome (growth-hormone insensitivity syndrome), we analyzed the growth-hormone receptor (GHR) genes of seven unrelated affected individuals from the United States, South America, Europe, and Africa. We amplified all nine GHR gene exons and splice junctions from these individuals by PCR and screened the products for mutations by using denaturing gradient gel electrophoresis (DGGE). We identified a single GHR gene fragment with abnormal DGGE results for each affected individual, sequenced this fragment, and, in each case, identified a mutation likely to cause Laron syndrome, including two nonsense mutations (R43X and R217X), two splice-junction mutations, (189-1 G to T and 71 + 1 G to A), and two frameshift mutations (46 del TT and 230 del TA or AT). Only one of these mutations, R43X, has been previously reported. Using haplotype analysis, we determined that this mutation, which involves a CpG dinucleotide hot spot, likely arose as a separate event in this case, relative to the two prior reports of R43X. Aside from R43X, the mutations we identified are unique to patients from particular geographic regions. Ten GHR gene mutations have now been described in this disorder. We conclude that Laron syndrome is caused by diverse GHR gene mutations, including deletions, RNA processing defects, translational stop codons, and missense codons. All the identified mutations involve the extracellular domain of the receptor, and most are unique to particular families or geographic areas. Images Figure 1 Figure 2 PMID:8488849

  20. Herpesvirus telomerase RNA (vTR with a mutated template sequence abrogates herpesvirus-induced lymphomagenesis.

    Directory of Open Access Journals (Sweden)

    Benedikt B Kaufer

    2011-10-01

    Full Text Available Telomerase reverse transcriptase (TERT and telomerase RNA (TR represent the enzymatically active components of telomerase. In the complex, TR provides the template for the addition of telomeric repeats to telomeres, a protective structure at the end of linear chromosomes. Human TR with a mutation in the template region has been previously shown to inhibit proliferation of cancer cells in vitro. In this report, we examined the effects of a mutation in the template of a virus encoded TR (vTR on herpesvirus-induced tumorigenesis in vivo. For this purpose, we used the oncogenic avian herpesvirus Marek's disease virus (MDV as a natural virus-host model for lymphomagenesis. We generated recombinant MDV in which the vTR template sequence was mutated from AATCCCAATC to ATATATATAT (vAU5 by two-step Red-mediated mutagenesis. Recombinant viruses harboring the template mutation replicated with kinetics comparable to parental and revertant viruses in vitro. However, mutation of the vTR template sequence completely abrogated virus-induced tumor formation in vivo, although the virus was able to undergo low-level lytic replication. To confirm that the absence of tumors was dependent on the presence of mutant vTR in the telomerase complex, a second mutation was introduced in vAU5 that targeted the P6.1 stem loop, a conserved region essential for vTR-TERT interaction. Absence of vTR-AU5 from the telomerase complex restored virus-induced lymphoma formation. To test if the attenuated vAU5 could be used as an effective vaccine against MDV, we performed vaccination-challenge studies and determined that vaccination with vAU5 completely protected chickens from lethal challenge with highly virulent MDV. Taken together, our results demonstrate 1 that mutation of the vTR template sequence can completely abrogate virus-induced tumorigenesis, likely by the inhibition of cancer cell proliferation, and 2 that this strategy could be used to generate novel vaccine candidates

  1. Distinct mutational signatures characterize concurrent loss of polymerase proofreading and mismatch repair.

    Science.gov (United States)

    Haradhvala, N J; Kim, J; Maruvka, Y E; Polak, P; Rosebrock, D; Livitz, D; Hess, J M; Leshchiner, I; Kamburov, A; Mouw, K W; Lawrence, M S; Getz, G

    2018-05-01

    Fidelity of DNA replication is maintained using polymerase proofreading and the mismatch repair pathway. Tumors with loss of function of either mechanism have elevated mutation rates with characteristic mutational signatures. Here we report that tumors with concurrent loss of both polymerase proofreading and mismatch repair function have mutational patterns that are not a simple sum of the signatures of the individual alterations, but correspond to distinct, previously unexplained signatures: COSMIC database signatures 14 and 20. We then demonstrate that in all five cases in which the chronological order of events could be determined, polymerase epsilon proofreading alterations precede the defect in mismatch repair. Overall, we illustrate that multiple distinct mutational signatures can result from different combinations of a smaller number of mutational processes (of either damage or repair), which can influence the interpretation and discovery of mutational signatures.

  2. KIAA1549-BRAF fusions and IDH mutations can coexist in diffuse gliomas of adults.

    Science.gov (United States)

    Badiali, Manuela; Gleize, Vincent; Paris, Sophie; Moi, Loredana; Elhouadani, Selma; Arcella, Antonietta; Morace, Roberta; Antonelli, Manila; Buttarelli, Francesca Romana; Figarella-Branger, Dominique; Kim, Young-Ho; Ohgaki, Hiroko; Mokhtari, Karima; Sanson, Marc; Giangaspero, Felice

    2012-11-01

    KIAA1549-BRAF fusion gene and isocitrate dehydrogenase (IDH) mutations are considered two mutually exclusive genetic events in pilocytic astrocytomas and diffuse gliomas, respectively. We investigated the presence of the KIAA1549-BRAF fusion gene in conjunction with IDH mutations and 1p/19q loss in 185 adult diffuse gliomas. Moreover BRAF(v600E) mutation was also screened. The KIAA1549-BRAF fusion gene was evaluated by reverse-transcription polymerase chain reaction (RT-PCR) and sequencing. We found IDH mutations in 125 out 175 cases (71.4%). There were KIAA1549-BRAF fusion gene in 17 out of 180 (9.4%) cases and BRAF(v600E) in 2 out of 133 (1.5%) cases. In 11 of these 17 cases, both IDH mutations and the KIAA1549-BRAF fusion were present, as independent molecular events. Moreover, 6 of 17 cases showed co-presence of 1p/19q loss, IDH mutations and KIAA1549-BRAF fusion. Among the 17 cases with KIAA1549-BRAF fusion gene 15 (88.2%) were oligodendroglial neoplasms. Similarly, the two cases with BRAF(v600E) mutation were both oligodendroglioma and one had IDH mutations and 1p/19q co-deletion. Our results suggest that in a small fraction of diffuse gliomas, KIAA1549-BRAF fusion gene and BRAF(v600E) mutation may be responsible for deregulation of the Ras-RAF-ERK signaling pathway. Such alterations are more frequent in oligodendroglial neoplasm and may be co-present with IDH mutations and 1p/19q loss. © 2012 The Authors; Brain Pathology © 2012 International Society of Neuropathology.

  3. KRAS exon 2 mutations influence activity of regorafenib in an SW48-based disease model of colorectal cancer.

    Science.gov (United States)

    Camaj, Peter; Primo, Stefano; Wang, Yan; Heinemann, Volker; Zhao, Yue; Laubender, Ruediger Paul; Stintzing, Sebastian; Giessen-Jung, Clemens; Jung, Andreas; Gamba, Sebastian; Bruns, Christiane Josephine; Modest, Dominik Paul

    2015-01-01

    To investigate the impact of KRAS mutation variants on the activity of regorafenib in SW48 colorectal cancer cells. Activity of regorafenib was evaluated in isogenic SW48 KRAS wild-type (WT) and mutant cells. Subcutaneous xenografts (KRAS WT and G12C mutant variants) in NOD/SCID mice were analyzed to elucidate the effect of regorafenib treatment in vivo. Compared with KRAS WT cells, all mutant variants seemed associated with some degree of resistance to regorafenib-treatment in vitro. In vivo, activation of apoptosis (TUNEL) and reduction of proliferation (Ki67) after treatment with regorafenib were more pronounced in KRAS WT tumors as compared with G12C variants. In SW48 cells, exon 2 mutations of the KRAS gene may influence antitumor effects of regorafenib.

  4. Evolution of the Pseudomonas aeruginosa Aminoglycoside Mutational Resistome In Vitro and in the Cystic Fibrosis Setting.

    Science.gov (United States)

    López-Causapé, Carla; Rubio, Rosa; Cabot, Gabriel; Oliver, Antonio

    2018-04-01

    Inhaled administration of high doses of aminoglycosides is a key maintenance treatment of Pseudomonas aeruginosa chronic respiratory infections in cystic fibrosis (CF). We analyzed the dynamics and mechanisms of stepwise high-level tobramycin resistance development in vitro and compared the results with those of isogenic pairs of susceptible and resistant clinical isolates. Resistance development correlated with fusA1 mutations in vitro and in vivo. pmrB mutations, conferring polymyxin resistance, were also frequently selected in vitro In contrast, mutational overexpression of MexXY, a hallmark of aminoglycoside resistance in CF, was not observed in in vitro evolution experiments. Copyright © 2018 American Society for Microbiology.

  5. Epidural Analgesia with Ropivacaine during Labour in a Patient with a SCN5A Gene Mutation

    Directory of Open Access Journals (Sweden)

    A. L. M. J. van der Knijff-van Dortmont

    2016-01-01

    Full Text Available SCN5A gene mutations can lead to ion channel defects which can cause cardiac conduction disturbances. In the presence of specific ECG characteristics, this mutation is called Brugada syndrome. Many drugs are associated with adverse events, making anesthesia in patients with SCN5A gene mutations or Brugada syndrome challenging. In this case report, we describe a pregnant patient with this mutation who received epidural analgesia using low dose ropivacaine and sufentanil during labour.

  6. Kin-Driver: a database of driver mutations in protein kinases.

    Science.gov (United States)

    Simonetti, Franco L; Tornador, Cristian; Nabau-Moretó, Nuria; Molina-Vila, Miguel A; Marino-Buslje, Cristina

    2014-01-01

    Somatic mutations in protein kinases (PKs) are frequent driver events in many human tumors, while germ-line mutations are associated with hereditary diseases. Here we present Kin-driver, the first database that compiles driver mutations in PKs with experimental evidence demonstrating their functional role. Kin-driver is a manual expert-curated database that pays special attention to activating mutations (AMs) and can serve as a validation set to develop new generation tools focused on the prediction of gain-of-function driver mutations. It also offers an easy and intuitive environment to facilitate the visualization and analysis of mutations in PKs. Because all mutations are mapped onto a multiple sequence alignment, analogue positions between kinases can be identified and tentative new mutations can be proposed for studying by transferring annotation. Finally, our database can also be of use to clinical and translational laboratories, helping them to identify uncommon AMs that can correlate with response to new antitumor drugs. The website was developed using PHP and JavaScript, which are supported by all major browsers; the database was built using MySQL server. Kin-driver is available at: http://kin-driver.leloir.org.ar/ © The Author(s) 2014. Published by Oxford University Press.

  7. Introducing Pitt-Hopkins syndrome-associated mutations of TCF4 to Drosophila daughterless

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    Laura Tamberg

    2015-12-01

    Full Text Available Pitt-Hopkins syndrome (PTHS is caused by haploinsufficiency of Transcription factor 4 (TCF4, one of the three human class I basic helix-loop-helix transcription factors called E-proteins. Drosophila has a single E-protein, Daughterless (Da, homologous to all three mammalian counterparts. Here we show that human TCF4 can rescue Da deficiency during fruit fly nervous system development. Overexpression of Da or TCF4 specifically in adult flies significantly decreases their survival rates, indicating that these factors are crucial even after development has been completed. We generated da transgenic fruit fly strains with corresponding missense mutations R578H, R580W, R582P and A614V found in TCF4 of PTHS patients and studied the impact of these mutations in vivo. Overexpression of wild type Da as well as human TCF4 in progenitor tissues induced ectopic sensory bristles and the rough eye phenotype. By contrast, overexpression of DaR580W and DaR582P that disrupt DNA binding reduced the number of bristles and induced the rough eye phenotype with partial lack of pigmentation, indicating that these act dominant negatively. Compared to the wild type, DaR578H and DaA614V were less potent in induction of ectopic bristles and the rough eye phenotype, respectively, suggesting that these are hypomorphic. All studied PTHS-associated mutations that we introduced into Da led to similar effects in vivo as the same mutations in TCF4 in vitro. Consequently, our Drosophila models of PTHS are applicable for further studies aiming to unravel the molecular mechanisms of this disorder.

  8. In vivo evaluation of the effect of stimulus distribution on FIR statistical efficiency in event-related fMRI.

    Science.gov (United States)

    Jansma, J Martijn; de Zwart, Jacco A; van Gelderen, Peter; Duyn, Jeff H; Drevets, Wayne C; Furey, Maura L

    2013-05-15

    Technical developments in MRI have improved signal to noise, allowing use of analysis methods such as Finite impulse response (FIR) of rapid event related functional MRI (er-fMRI). FIR is one of the most informative analysis methods as it determines onset and full shape of the hemodynamic response function (HRF) without any a priori assumptions. FIR is however vulnerable to multicollinearity, which is directly related to the distribution of stimuli over time. Efficiency can be optimized by simplifying a design, and restricting stimuli distribution to specific sequences, while more design flexibility necessarily reduces efficiency. However, the actual effect of efficiency on fMRI results has never been tested in vivo. Thus, it is currently difficult to make an informed choice between protocol flexibility and statistical efficiency. The main goal of this study was to assign concrete fMRI signal to noise values to the abstract scale of FIR statistical efficiency. Ten subjects repeated a perception task with five random and m-sequence based protocol, with varying but, according to literature, acceptable levels of multicollinearity. Results indicated substantial differences in signal standard deviation, while the level was a function of multicollinearity. Experiment protocols varied up to 55.4% in standard deviation. Results confirm that quality of fMRI in an FIR analysis can significantly and substantially vary with statistical efficiency. Our in vivo measurements can be used to aid in making an informed decision between freedom in protocol design and statistical efficiency. Published by Elsevier B.V.

  9. Evidence for in vivo phosphorylation of the Grb2 SH2-domain binding site on focal adhesion kinase by Src-family protein-tyrosine kinases.

    Science.gov (United States)

    Schlaepfer, D D; Hunter, T

    1996-10-01

    Focal adhesion kinase (FAK) is a nonreceptor protein-tyrosine kinase (PTK) that associates with integrin receptors and participates in extracellular matrix-mediated signal transduction events. We showed previously that the c-Src nonreceptor PTK and the Grb2 SH2/SH3 adaptor protein bound directly to FAK after fibronectin stimulation (D. D. Schlaepfer, S.K. Hanks, T. Hunter, and P. van der Geer, Nature [London] 372:786-791, 1994). Here, we present evidence that c-Src association with FAK is required for Grb2 binding to FAK. Using a tryptic phosphopeptide mapping approach, the in vivo phosphorylation of the Grb2 binding site on FAK (Tyr-925) was detected after fibronectin stimulation of NIH 3T3 cells and was constitutively phosphorylated in v-Src-transformed NIH 3T3 cells. In vitro, c-Src phosphorylated FAK Tyr-925 in a glutathione S-transferase-FAK C-terminal domain fusion protein, whereas FAK did not. Using epitope-tagged FAK constructs, transiently expressed in human 293 cells, we determined the effect of site-directed mutations on c-Src and Grb2 binding to FAK. Mutation of FAK Tyr-925 disrupted Grb2 binding, whereas mutation of the c-Src binding site on FAK (Tyr-397) disrupted both c-Src and Grb2 binding to FAK in vivo. These results support a model whereby Src-family PTKs are recruited to FAK and focal adhesions following integrin-induced autophosphorylation and exposure of FAK Tyr-397. Src-family binding and phosphorylation of FAK at Tyr-925 creates a Grb2 SH2-domain binding site and provides a link to the activation of the Ras signal transduction pathway. In Src-transformed cells, this pathway may be constitutively activated as a result of FAK Tyr-925 phosphorylation in the absence of integrin stimulation.

  10. Analysis of Escherichia coli nicotinate mononucleotide adenylyltransferase mutants in vivo and in vitro

    Directory of Open Access Journals (Sweden)

    Rydén-Aulin Monica

    2005-09-01

    Full Text Available Abstract Background Adenylation of nicotinate mononucleotide to nicotinate adenine dinucleotide is the penultimate step in NAD+ synthesis. In Escherichia coli, the enzyme nicotinate mononucleotide adenylyltransferase is encoded by the nadD gene. We have earlier made an initial characterization in vivo of two mutant enzymes, NadD72 and NadD74. Strains with either mutation have decreased intracellular levels of NAD+, especially for one of the alleles, nadD72. Results In this study these two mutant proteins have been further characterized together with ten new mutant variants. Of the, in total, twelve mutations four are in a conserved motif in the C-terminus and eight are in the active site. We have tested the activity of the enzymes in vitro and their effect on the growth phenotype in vivo. There is a very good correlation between the two data sets. Conclusion The mutations in the C-terminus did not reveal any function for the conserved motif. On the other hand, our data has lead us to assign amino acid residues His-19, Arg-46 and Asp-109 to the active site. We have also shown that the nadD gene is essential for growth in E. coli.

  11. Mutations in the Gene PRRT2 Cause Paroxysmal Kinesigenic Dyskinesia with Infantile Convulsions

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    Hsien-Yang Lee

    2012-01-01

    Full Text Available Paroxysmal kinesigenic dyskinesia with infantile convulsions (PKD/IC is an episodic movement disorder with autosomal-dominant inheritance and high penetrance, but the causative genetic mutation is unknown. We have now identified four truncating mutations involving the gene PRRT2 in the vast majority (24/25 of well-characterized families with PKD/IC. PRRT2 truncating mutations were also detected in 28 of 78 additional families. PRRT2 encodes a proline-rich transmembrane protein of unknown function that has been reported to interact with the t-SNARE, SNAP25. PRRT2 localizes to axons but not to dendritic processes in primary neuronal culture, and mutants associated with PKD/IC lead to dramatically reduced PRRT2 levels, leading ultimately to neuronal hyperexcitability that manifests in vivo as PKD/IC.

  12. Mice with an Oncogenic HRAS Mutation are Resistant to High-Fat Diet-Induced Obesity and Exhibit Impaired Hepatic Energy Homeostasis

    Directory of Open Access Journals (Sweden)

    Daiju Oba

    2018-01-01

    Full Text Available Costello syndrome is a “RASopathy” that is characterized by growth retardation, dysmorphic facial appearance, hypertrophic cardiomyopathy and tumor predisposition. >80% of patients with Costello syndrome harbor a heterozygous germline G12S mutation in HRAS. Altered metabolic regulation has been suspected because patients with Costello syndrome exhibit hypoketotic hypoglycemia and increased resting energy expenditure, and their growth is severely retarded. To examine the mechanisms of energy reprogramming by HRAS activation in vivo, we generated knock-in mice expressing a heterozygous Hras G12S mutation (HrasG12S/+ mice as a mouse model of Costello syndrome. On a high-fat diet, HrasG12S/+ mice developed a lean phenotype with microvesicular hepatic steatosis, resulting in early death compared with wild-type mice. Under starvation conditions, hypoketosis and elevated blood levels of long-chain fatty acylcarnitines were observed, suggesting impaired mitochondrial fatty acid oxidation. Our findings suggest that the oncogenic Hras mutation modulates energy homeostasis in vivo.

  13. A duck hepatitis B virus strain with a knockout mutation in the putative X ORF shows similar infectivity and in vivo growth characteristics to wild-type virus

    International Nuclear Information System (INIS)

    Meier, P.; Scougall, C.A.; Will, H.; Burrell, C.J.; Jilbert, A.R.

    2003-01-01

    Hepadnaviruses including human hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) express X proteins, HBx and DHBx, respectively. Both HBx and DHBx are transcriptional activators and modulate cellular signaling in in vitro assays. To test whether the DHBx protein plays a role in virus infection, we compared the in vivo infectivity and growth characteristics of a DHBV3 strain with a stop codon in the X-like ORF (DHBV3-X-K.O.) to those of the wild-type DHBV3 strain. Here we report that the two strains showed no significant difference in (i) their ability to induce infection that resulted in stable viraemia measured by serum surface antigen (DHBsAg) and DHBV DNA, and detection of viral proteins and replicative DNA intermediates in the liver; (ii) the rate of spread of infection in liver and extrahepatic sites after low-dose virus inoculation; and (iii) the ability to produce transient or persistent infection under balanced age/dose conditions designed to detect small differences between the strains. Thus, none of the infection parameters assayed were detectably affected by the X-ORF knockout mutation, raising the question whether DHBx expression plays a physiological role during in vivo infection with wild-type DHBV

  14. Complementation pattern of lexB and recA mutations in Escherichia coli K12; mapping of tif-1, lexB and recA mutations

    International Nuclear Information System (INIS)

    Morand, P.; Goze, A.; Devoret, R.

    1977-01-01

    Three lexB mutations, whose phenotypes have been previously characterized, are studied here in relation to a few recA mutations as to their complementation pattern and relative location. The restoration of resistance to UV-light and to X-rays in the hetero-allelic diploid bacteria was used as a test for dominance and complementation. The wild type allele was always dominant over the mutant allele. Only partial complementation was found between lexB and two rexA alleles. There was no complementation between the recA alleles. All the data taken together strongly suggest that the complementations found are intragenic: lexB and recA mutations are in one gene. Mapping of lexB, recA and tif-1 mutations in relation to srl-1 and cysC by phage P1 transduction shows that lexB and the tif-1 mutations form a cluster proximal to srl-1 whereas recA mutations are located at the other extremity of the gene. Variability with temperature of cotransduction frequencies as well as their extended range of values prevent a meaningful calculation of the length of the recA gene. Our hypothesis is that the recA protein has two functional regions called A and B respectively defined at the genetical level by recA and lexB mutations and that it is, in vivo, an oligomeric protein forming a complex with the lexA protein. This complex is postulated to be multifunctional: recombination and control of exonuclease V are effected by the A region while the B region and lexA protein effect induced DNA repair and lysogenic induction. (orig.) [de

  15. Identifying uniformly mutated segments within repeats.

    Science.gov (United States)

    Sahinalp, S Cenk; Eichler, Evan; Goldberg, Paul; Berenbrink, Petra; Friedetzky, Tom; Ergun, Funda

    2004-12-01

    Given a long string of characters from a constant size alphabet we present an algorithm to determine whether its characters have been generated by a single i.i.d. random source. More specifically, consider all possible n-coin models for generating a binary string S, where each bit of S is generated via an independent toss of one of the n coins in the model. The choice of which coin to toss is decided by a random walk on the set of coins where the probability of a coin change is much lower than the probability of using the same coin repeatedly. We present a procedure to evaluate the likelihood of a n-coin model for given S, subject a uniform prior distribution over the parameters of the model (that represent mutation rates and probabilities of copying events). In the absence of detailed prior knowledge of these parameters, the algorithm can be used to determine whether the a posteriori probability for n=1 is higher than for any other n>1. Our algorithm runs in time O(l4logl), where l is the length of S, through a dynamic programming approach which exploits the assumed convexity of the a posteriori probability for n. Our test can be used in the analysis of long alignments between pairs of genomic sequences in a number of ways. For example, functional regions in genome sequences exhibit much lower mutation rates than non-functional regions. Because our test provides means for determining variations in the mutation rate, it may be used to distinguish functional regions from non-functional ones. Another application is in determining whether two highly similar, thus evolutionarily related, genome segments are the result of a single copy event or of a complex series of copy events. This is particularly an issue in evolutionary studies of genome regions rich with repeat segments (especially tandemly repeated segments).

  16. Kinetics of mutation induction by ultraviolet light in excision-deficient yeast.

    Science.gov (United States)

    Eckardt, F; Haynes, R H

    1977-02-01

    We have measured the frequency of UV-induced reversions (locus plus suppressor) for the ochre alleles ade2-1 and lys2-1 and forward mutations (ade2 adex double auxotrophs) in an excision-deficient strain of Saccharomyces cerevisiae (rad2-20). For very low UV doses, both mutational systems exhibit linear induction kinetics. However, as the dose increases, a strikingly different response is observed: in the selective reversion system a transition to higher order induction kinetics occurs near 9 ergs/mm2 (25% survival), whereas in the nonselective forward system the mutation frequency passes through a maximum near 14 ergs/mm2 (4.4% survival) and then declines. This contrast in kinetics cannot be explained in any straightforward way by current models of induced mutagenesis, which have been developed primarily on the basis of bacterial data. The bacterial models are designed to accommodate the quadratic induction kinetics that are frequently observed in these systems. We have derived a mathematical expression for mutation frequency that enables us to fit both the forward and reversion data on the assumptions that mutagenesis is basically a "single event" Poisson process, and that mutation and killing are not necessarily independent of one another. In particular, the dose-response relations are consistent with the idea that the sensitivity of the revertants is about 25% less than that of the original cell population, whereas the sensitivity of the forward mutants is about 29% greater than the population average. We argue that this relatively small differential sensitivity of mutant and nonmutant cells is associated with events that take place during mutation expression and clonal growth.

  17. Kinetics of mutation induction by ultraviolet light in excision-deficient yeast

    International Nuclear Information System (INIS)

    Eckardt, F.; Haynes, R.H.

    1977-01-01

    We have measured the frequency of uv-induced reversions (locus plus suppressor) for the ochre alleles ade 2-1 and lys 2-1 and forward mutations (ade2 adex double auxotrophs) in an excision-deficient strain of Saccharomyces cerevisiae (rad 2-20). For very low uv doses, both mutational systems exhibit linear induction kinetics. However, as the dose increases, a strikingly different response is observed: in the selective reversion system a transition to higher order induction kinetics occurs near 9 ergs/mm 2 (25 percent survival), whereas in the nonselective forward system the mutation frequency passes through a maximum near 14 ergs/mm 2 (4.4 percent survival) and then declines. This contrast in kinetics cannot be explained in any straightforward way by current models of induced mutagenesis, which have been developed primarily on the basis of bacterial data. The bacterial models are designed to accommodate the quadratic induction kinetics that are frequently observed in these systems. We have derived a mathematical expression for mutation frequency that enables us to fit both the forward and reversion data on the assumptions that mutagenesis is basically a ''single event'' Poisson process, and that mutation and killing are not necessarily independent of one another. In particular, the dose-response relations are consistent with the idea that the sensitivity of the revertants is about 25 percent less than that of the original cell population, whereas the sensitivity of the forward mutants is about 29 percent greater than the population average. We argue that this relatively small differential sensitivity of mutant and nonmutant cells is associated with events that take place during mutation expression and clonal growth

  18. Human immunodeficiency virus type 1 evolution in vivo tracked by DNA heteroduplex mobility assays

    NARCIS (Netherlands)

    Delwart, E. L.; Sheppard, H. W.; Walker, B. D.; Goudsmit, J.; Mullins, J. I.

    1994-01-01

    High mutation rates and strong selective pressures imposed on human immunodeficiency viruses in vivo result in the formation of pools of genetic variants known as quasispecies. DNA heteroduplex mobility and tracking analyses were used to monitor the generation of HIV sequence diversity, to estimate

  19. Induction of in vivo mutation in chrysanthemum (Dendranthema grandiflora Tzvelev) cv. Pink Repin breeding

    International Nuclear Information System (INIS)

    Neto, Augusto Tulmann; Latado, Rodrigo Rocha

    1997-01-01

    Mutation induction was used with the objective of obtaining mutants for flower colour of chrysanthemum, cv. Repin (pink colour). Rooted cuttings were irradiated with 20 Gy of gamma rays and before the selection the cutting back method was used to advance the generations. The frequency of colour mutants observed was 5.8%. Among the mutants obtainedthe white and dark-pink-coloured ones were evaluated in yield trial and post-harvest. The results indicated that these mutants mantained the same agronomical characteristics showed by the control, with the exception of plant height in the white mutant that was shorter. Due to commercial interest of the producers, these mutants were multiplied and released as new cultivars. The white flower colour mutant was named Cristiane and the dark-pink, Ingrid. This was the first example of cultivars from an ornamental plant released by mutation breeding in Brazil. (author)

  20. Brief Report: Human Acute Myeloid Leukemia Reprogramming to Pluripotency Is a Rare Event and Selects for Patient Hematopoietic Cells Devoid of Leukemic Mutations.

    Science.gov (United States)

    Lee, Jong-Hee; Salci, Kyle R; Reid, Jennifer C; Orlando, Luca; Tanasijevic, Borko; Shapovalova, Zoya; Bhatia, Mickie

    2017-09-01

    Induced pluripotent stem cell reprogramming has provided critical insights into disease processes by modeling the genetics and related clinical pathophysiology. Human cancer represents highly diverse genetics, as well as inter- and intra-patient heterogeneity, where cellular model systems capable of capturing this disease complexity would be invaluable. Acute myeloid leukemia (AML) represents one of most heterogeneous cancers and has been divided into genetic subtypes correlated with unique risk stratification over the decades. Here, we report our efforts to induce pluripotency from the heterogeneous population of human patients that represents this disease in the clinic. Using robust optimized reprogramming methods, we demonstrate that reprogramming of AML cells harboring leukemic genomic aberrations is a rare event with the exception of those with de novo mixed-lineage leukemia (MLL) mutations that can be reprogrammed and model drug responses in vitro. Our findings indicate that unlike hematopoietic cells devoid of genomic aberrations, AML cells harboring driver mutations are refractory to reprogramming. Expression of MLL fusion proteins in AML cells did not contribute to induced reprogramming success, which continued to select for patient derived cells devoid of AML patient-specific aberrations. Our study reveals that unanticipated blockades to achieving pluripotency reside within the majority of transformed AML patient cells. Stem Cells 2017;35:2095-2102. © 2017 AlphaMed Press.

  1. Deep sequencing of natural and experimental populations of Drosophila melanogaster reveals biases in the spectrum of new mutations.

    Science.gov (United States)

    Assaf, Zoe June; Tilk, Susanne; Park, Jane; Siegal, Mark L; Petrov, Dmitri A

    2017-12-01

    Mutations provide the raw material of evolution, and thus our ability to study evolution depends fundamentally on having precise measurements of mutational rates and patterns. We generate a data set for this purpose using (1) de novo mutations from mutation accumulation experiments and (2) extremely rare polymorphisms from natural populations. The first, mutation accumulation (MA) lines are the product of maintaining flies in tiny populations for many generations, therefore rendering natural selection ineffective and allowing new mutations to accrue in the genome. The second, rare genetic variation from natural populations allows the study of mutation because extremely rare polymorphisms are relatively unaffected by the filter of natural selection. We use both methods in Drosophila melanogaster , first generating our own novel data set of sequenced MA lines and performing a meta-analysis of all published MA mutations (∼2000 events) and then identifying a high quality set of ∼70,000 extremely rare (≤0.1%) polymorphisms that are fully validated with resequencing. We use these data sets to precisely measure mutational rates and patterns. Highlights of our results include: a high rate of multinucleotide mutation events at both short (∼5 bp) and long (∼1 kb) genomic distances, showing that mutation drives GC content lower in already GC-poor regions, and using our precise context-dependent mutation rates to predict long-term evolutionary patterns at synonymous sites. We also show that de novo mutations from independent MA experiments display similar patterns of single nucleotide mutation and well match the patterns of mutation found in natural populations. © 2017 Assaf et al.; Published by Cold Spring Harbor Laboratory Press.

  2. Analysis of mutations in the human HPRT gene induced by accelerated heavy-ion irradiation

    International Nuclear Information System (INIS)

    Kagawa, Yasuhiro; Yatagai, Fumio; Hanaoka, Fumio; Suzuki, Masao; Kase, Youko; Kobayashi, Akiko; Hirano, Masahiko; Kato, Takesi; Watanabe, Masami.

    1995-01-01

    Multiplex PCR analysis of HPRT(-) mutations in human embryo (HE) cells induced by 230 keV/μm carbon-ion irradiation showed no large deletion around the exon regions of the locus gene in contrast to the irradiations at different LETs. To identify these mutations, the sequence alterations in a cDNA of hprt gene were determined for 18 mutant clones in this study. Missing of exon 6 was the most frequent mutational event (10 clones), and missing of both exons 6 and 8 was next most frequent event (6 clones), then base substitutions (2 clones). These characteristics were not seen in a similar analysis of spontaneous mutations, which showed base substitution (5 clones), frameshift (2 clones), missing of both exons 2 and 3 (2 clones), and a single unidentified clone. Direct sequencing and restriction enzyme digestion of the genomic DNA of the mutants which showed missing of exons 6 and 8 in the cDNA, supports the possibility that they were induced by aberrant mRNA splicing. (author)

  3. Arsenic trioxide promotes mitochondrial DNA mutation and cell apoptosis in primary APL cells and NB4 cell line.

    Science.gov (United States)

    Meng, Ran; Zhou, Jin; Sui, Meng; Li, ZhiYong; Feng, GuoSheng; Yang, BaoFeng

    2010-01-01

    This study aimed to investigate the effects of arsenic trioxide (As(2)O(3)) on the mitochondrial DNA (mtDNA) of acute promyelocytic leukemia (APL) cells. The NB4 cell line was treated with 2.0 micromol/L As(2)O(3) in vitro, and the primary APL cells were treated with 2.0 micromol/L As(2)O(3) in vitro and 0.16 mg kg(-1) d(-1) As(2)O(3) in vivo. The mitochondrial DNA of all the cells above was amplified by PCR, directly sequenced and analyzed by Sequence Navigatore and Factura software. The apoptosis rates were assayed by flow cytometry. Mitochondrial DNA mutation in the D-loop region was found in NB4 and APL cells before As(2)O(3) use, but the mutation spots were remarkably increased after As(2)O(3) treatment, which was positively correlated to the rates of cellular apoptosis, the correlation coefficient: r (NB4-As2O3)=0.973818, and r (APL-As2O3)=0.934703. The mutation types include transition, transversion, codon insertion or deletion, and the mutation spots in all samples were not constant and regular. It is revealed that As(2)O(3) aggravates mtDNA mutation in the D-loop region of acute promyelocytic leukemia cells both in vitro and in vivo. Mitochondrial DNA might be one of the targets of As(2)O(3) in APL treatment.

  4. Frequent beneficial mutations during single-colony serial transfer of Streptococcus pneumoniae.

    Directory of Open Access Journals (Sweden)

    Kathleen E Stevens

    2011-08-01

    Full Text Available The appearance of new mutations within a population provides the raw material for evolution. The consistent decline in fitness observed in classical mutation accumulation studies has provided support for the long-held view that deleterious mutations are more common than beneficial mutations. Here we present results of a study using a mutation accumulation design with the bacterium Streptococcus pneumoniae in which the fitness of the derived populations increased. This rise in fitness was associated specifically with adaptation to survival during brief stationary phase periods between single-colony population bottlenecks. To understand better the population dynamics behind this unanticipated adaptation, we developed a maximum likelihood model describing the processes of mutation and stationary-phase selection in the context of frequent population bottlenecks. Using this model, we estimate that the rate of beneficial mutations may be as high as 4.8×10(-4 events per genome for each time interval corresponding to the pneumococcal generation time. This rate is several orders of magnitude higher than earlier estimates of beneficial mutation rates in bacteria but supports recent results obtained through the propagation of small populations of Escherichia coli. Our findings indicate that beneficial mutations may be relatively frequent in bacteria and suggest that in S. pneumoniae, which develops natural competence for transformation, a steady supply of such mutations may be available for sampling by recombination.

  5. Mutations in FUS cause FALS and SALS in French and French Canadian populations.

    Science.gov (United States)

    Belzil, V V; Valdmanis, P N; Dion, P A; Daoud, H; Kabashi, E; Noreau, A; Gauthier, J; Hince, P; Desjarlais, A; Bouchard, J-P; Lacomblez, L; Salachas, F; Pradat, P-F; Camu, W; Meininger, V; Dupré, N; Rouleau, G A

    2009-10-13

    The identification of mutations in the TARDBP and more recently the identification of mutations in the FUS gene as the cause of amyotrophic lateral sclerosis (ALS) is providing the field with new insight about the mechanisms involved in this severe neurodegenerative disease. To extend these recent genetic reports, we screened the entire gene in a cohort of 200 patients with ALS. An additional 285 patients with sporadic ALS were screened for variants in exon 15 for which mutations were previously reported. In total, 3 different mutations were identified in 4 different patients, including 1 3-bp deletion in exon 3 of a patient with sporadic ALS and 2 missense mutations in exon 15 of 1 patient with familial ALS and 2 patients with sporadic ALS. Our study identified sporadic patients with mutations in the FUS gene. The accumulation and description of different genes and mutations helps to develop a more comprehensive picture of the genetic events underlying amyotrophic lateral sclerosis.

  6. Experiments on the role of deleterious mutations as stepping stones in adaptive evolution

    Science.gov (United States)

    Covert, Arthur W.; Lenski, Richard E.; Wilke, Claus O.; Ofria, Charles

    2013-01-01

    Many evolutionary studies assume that deleterious mutations necessarily impede adaptive evolution. However, a later mutation that is conditionally beneficial may interact with a deleterious predecessor before it is eliminated, thereby providing access to adaptations that might otherwise be inaccessible. It is unknown whether such sign-epistatic recoveries are inconsequential events or an important factor in evolution, owing to the difficulty of monitoring the effects and fates of all mutations during experiments with biological organisms. Here, we used digital organisms to compare the extent of adaptive evolution in populations when deleterious mutations were disallowed with control populations in which such mutations were allowed. Significantly higher fitness levels were achieved over the long term in the control populations because some of the deleterious mutations served as stepping stones across otherwise impassable fitness valleys. As a consequence, initially deleterious mutations facilitated the evolution of complex, beneficial functions. We also examined the effects of disallowing neutral mutations, of varying the mutation rate, and of sexual recombination. Populations evolving without neutral mutations were able to leverage deleterious and compensatory mutation pairs to overcome, at least partially, the absence of neutral mutations. Substantially raising or lowering the mutation rate reduced or eliminated the long-term benefit of deleterious mutations, but introducing recombination did not. Our work demonstrates that deleterious mutations can play an important role in adaptive evolution under at least some conditions. PMID:23918358

  7. Generation and Characterization of a Transgenic Mouse Carrying a Functional Human β-Globin Gene with the IVSI-6 Thalassemia Mutation

    Directory of Open Access Journals (Sweden)

    Giulia Breveglieri

    2015-01-01

    Full Text Available 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 αmu-globin2/βhu-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.

  8. Generation and Characterization of a Transgenic Mouse Carrying a Functional Human β-Globin Gene with the IVSI-6 Thalassemia Mutation

    Science.gov (United States)

    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

  9. Effect of low dose radiation on somatic intrachromosomal recombination in vivo and in vitro

    International Nuclear Information System (INIS)

    Hooker, A.M.; Cormack, J.; Morley, A.A.; Sykes, P.J.; Bhat, M.

    2003-01-01

    Full text: High doses of ionising radiation are mutagenic in a wide range of mutation assays. The majority of radiation exposure studies in in vivo mouse mutation assays have been performed at high doses, eg greater than 1 Gy. However, these doses are not relevant to the low doses of ionising radiation that the majority of the population might likely come into contact with. Radiation protection levels tend to be based on a simple linear no-threshold model which suggests that any radiation above zero is potentially harmful. The pKZ1 recombination mutagenesis mouse model has proven to be a sensitive assay for the detection of mutations caused by low doses of chemical agents. In pKZ1 mice, somatic intrachromosomal recombination (SICR) inversion events can be detected in cells using histochemistry for the E. coli LacZ transgene. We exposed pKZ1 mice to a single radiation dose ranging from 0.001 to 2 Gy. A significant increase in SICR was observed in spleen at the two highest doses of 0.1 and 2 Gy and a significant reduction in SICR below the endogenous frequency was observed at the two lowest doses of 0.01 and 0.001 Gy. After exposing a pKZ1 cell line to the same dose range, a similar J curve response was observed with significant increases in SICR observed at the 3 highest doses and a significant decrease below the endogenous frequency at the lowest dose (0.001 Gy). The next experiments will be to determine the dose where the SICR frequency returns to the endogenous level. The important question posed by these results is 'Is a reduction below the endogenous SICR level caused by low doses of ionising radiation anti-mutagenic?' Studies now need to be performed to investigate the effect of low doses of radiation on other mutation end-points, and the mechanism for the reduction in SICR

  10. Strikingly different penetrance of LHON in two Chinese families with primary mutation G11778A is independent of mtDNA haplogroup background and secondary mutation G13708A

    International Nuclear Information System (INIS)

    Wang Huawei; Jia Xiaoyun; Ji Yanli; Kong Qingpeng; Zhang Qingjiong; Yao Yonggang; Zhang Yaping

    2008-01-01

    The penetrance of Leber's hereditary optic neuropathy (LHON) in families with primary mitochondrial DNA (mtDNA) mutations is very complex. Matrilineal and nuclear genetic background, as well as environmental factors, have been reported to be involved in different affected pedigrees. Here we describe two large Chinese families that show a striking difference in the penetrance of LHON, in which 53.3% and 15.0% of members were affected (P < 0.02), respectively. Analysis of the complete mtDNA genome of the two families revealed the presence of the primary mutation G11778A and several other variants suggesting the same haplogroup status G2a. The family with higher penetrance contained a previously described secondary mutation G13708A, which presents a polymorphism in normal Chinese samples and does not affect in vivo mitochondrial oxidative metabolism as described in a previous study. Evolutionary analysis failed to indicate any putatively pathogenic mutation that cosegregated with G11778A in these two pedigrees. Our results suggest that the variable penetrance of LHON in the two Chinese families is independent of both their mtDNA haplotype background and a secondary mutation G13708A. As a result, it is likely that unknown nuclear gene involvement and/or other factors contribute to the strikingly different penetrance of LHON

  11. Col4a1 mutations cause progressive retinal neovascular defects and retinopathy.

    Science.gov (United States)

    Alavi, Marcel V; Mao, Mao; Pawlikowski, Bradley T; Kvezereli, Manana; Duncan, Jacque L; Libby, Richard T; John, Simon W M; Gould, Douglas B

    2016-01-27

    Mutations in collagen, type IV, alpha 1 (COL4A1), a major component of basement membranes, cause multisystem disorders in humans and mice. In the eye, these include anterior segment dysgenesis, optic nerve hypoplasia and retinal vascular tortuosity. Here we investigate the retinal pathology in mice carrying dominant-negative Col4a1 mutations. To this end, we examined retinas longitudinally in vivo using fluorescein angiography, funduscopy and optical coherence tomography. We assessed retinal function by electroretinography and studied the retinal ultrastructural pathology. Retinal examinations revealed serous chorioretinopathy, retinal hemorrhages, fibrosis or signs of pathogenic angiogenesis with chorioretinal anastomosis in up to approximately 90% of Col4a1 mutant eyes depending on age and the specific mutation. To identify the cell-type responsible for pathogenesis we generated a conditional Col4a1 mutation and determined that primary vascular defects underlie Col4a1-associated retinopathy. We also found focal activation of Müller cells and increased expression of pro-angiogenic factors in retinas from Col4a1(+/Δex41)mice. Together, our findings suggest that patients with COL4A1 and COL4A2 mutations may be at elevated risk of retinal hemorrhages and that retinal examinations may be useful for identifying patients with COL4A1 and COL4A2 mutations who are also at elevated risk of hemorrhagic strokes.

  12. Metabolic Alterations Caused by KRAS Mutations in Colorectal Cancer Contribute to Cell Adaptation to Glutamine Depletion by Upregulation of Asparagine Synthetase

    Directory of Open Access Journals (Sweden)

    Kosuke Toda

    2016-11-01

    Full Text Available A number of clinical trials have shown that KRAS mutations of colorectal cancer (CRC can predict a lack of responses to anti-epidermal growth factor receptor–based therapy. Recently, there have been several studies to elucidate metabolism reprogramming in cancer. However, it remains to be investigated how mutated KRAS can coordinate the metabolic shift to sustain CRC tumor growth. In this study, we found that KRAS mutation in CRC caused alteration in amino acid metabolism. KRAS mutation causes a marked decrease in aspartate level and an increase in asparagine level in CRC. Using several human CRC cell lines and clinical specimens of primary CRC, we demonstrated that the expression of asparagine synthetase (ASNS, an enzyme that synthesizes asparagine from aspartate, was upregulated by mutated KRAS and that ASNS expression was induced by KRAS-activated signaling pathway, in particular PI3K-AKT-mTOR pathway. Importantly, we demonstrated that KRAS-mutant CRC cells could become adaptive to glutamine depletion through asparagine biosynthesis by ASNS and that asparagine addition could rescue the inhibited growth and viability of cells grown under the glutamine-free condition in vitro. Notably, a pronounced growth suppression of KRAS-mutant CRC was observed upon ASNS knockdown in vivo. Furthermore, combination of L-asparaginase plus rapamycin markedly suppressed the growth of KRAS-mutant CRC xenografts in vivo, whereas either L-asparaginase or rapamycin alone was not effective. These results indicate ASNS might be a novel therapeutic target against CRCs with mutated KRAS.

  13. Multiple origins of knockdown resistance mutations in the Afrotropical mosquito vector Anopheles gambiae.

    Directory of Open Access Journals (Sweden)

    João Pinto

    2007-11-01

    Full Text Available How often insecticide resistance mutations arise in natural insect populations is a fundamental question for understanding the evolution of resistance and also for modeling its spread. Moreover, the development of resistance is regarded as a favored model to study the molecular evolution of adaptive traits. In the malaria vector Anopheles gambiae two point mutations (L1014F and L1014S in the voltage-gated sodium channel gene, that confer knockdown resistance (kdr to DDT and pyrethroid insecticides, have been described. In order to determine whether resistance alleles result from single or multiple mutation events, genotyping of the kdr locus and partial sequencing of the upstream intron-1 was performed on a total of 288 A. gambiae S-form collected from 28 localities in 15 countries. Knockdown resistance alleles were found to be widespread in West Africa with co-occurrence of both 1014S and 1014F in West-Central localities. Differences in intron-1 haplotype composition suggest that kdr alleles may have arisen from at least four independent mutation events. Neutrality tests provided evidence for a selective sweep acting on this genomic region, particularly in West Africa. The frequency and distribution of these kdr haplotypes varied geographically, being influenced by an interplay between different mutational occurrences, gene flow and local selection. This has important practical implications for the management and sustainability of malaria vector control programs.

  14. Oncogenetic tree model of somatic mutations and DNA methylation in colon tumors.

    Science.gov (United States)

    Sweeney, Carol; Boucher, Kenneth M; Samowitz, Wade S; Wolff, Roger K; Albertsen, Hans; Curtin, Karen; Caan, Bette J; Slattery, Martha L

    2009-01-01

    Our understanding of somatic alterations in colon cancer has evolved from a concept of a series of events taking place in a single sequence to a recognition of multiple pathways. An oncogenetic tree is a model intended to describe the pathways and sequence of somatic alterations in carcinogenesis without assuming that tumors will fall in mutually exclusive categories. We applied this model to data on colon tumor somatic alterations. An oncogenetic tree model was built using data on mutations of TP53, KRAS2, APC, and BRAF genes, methylation at CpG sites of MLH1 and TP16 genes, methylation in tumor (MINT) markers, and microsatellite instability (MSI) for 971 colon tumors from a population-based series. Oncogenetic tree analysis resulted in a reproducible tree with three branches. The model represents methylation of MINT markers as initiating a branch and predisposing to MSI, methylation of MHL1 and TP16, and BRAF mutation. APC mutation is the first alteration in an independent branch and is followed by TP53 mutation. KRAS2 mutation was placed a third independent branch, implying that it neither depends on, nor predisposes to, the other alterations. Individual tumors were observed to have alteration patterns representing every combination of one, two, or all three branches. The oncogenetic tree model assumptions are appropriate for the observed heterogeneity of colon tumors, and the model produces a useful visual schematic of the sequence of events in pathways of colon carcinogenesis.

  15. In vivo therapeutic responses contingent on Fanconi anemia/BRCA2 status of the tumor

    NARCIS (Netherlands)

    van der Heijden, Michiel S.; Brody, Jonathan R.; Dezentje, David A.; Gallmeier, Eike; Cunningham, Steven C.; Swartz, Michael J.; DeMarzo, Angelo M.; Offerhaus, G. Johan A.; Isacoff, William H.; Hruban, Ralph H.; Kern, Scott E.

    2005-01-01

    Purpose: BRCA2, FANCC, and FANCG gene mutations are present in a subset of pancreatic cancer. Defects in these genes could lead to hypersensitivity to interstrand cross-linkers in vivo and a more optimal treatment of pancreatic cancer patients based on the genetic profile of the tumor. Experimental

  16. Kinome-wide Decoding of Network-Attacking Mutations Rewiring Cancer Signaling

    DEFF Research Database (Denmark)

    Creixell, Pau; Schoof, Erwin M; Simpson, Craig D.

    2015-01-01

    Cancer cells acquire pathological phenotypes through accumulation of mutations that perturb signaling networks. However, global analysis of these events is currently limited. Here, we identify six types of network-attacking mutations (NAMs), including changes in kinase and SH2 modulation, network...... and experimentally validated several NAMs, including PKCγ M501I and PKD1 D665N, which encode specificity switches analogous to the appearance of kinases de novo within the kinome. We discover mutant molecular logic gates, a drift toward phospho-threonine signaling, weakening of phosphorylation motifs, and kinase...

  17. Deleterious ABCA7 mutations and transcript rescue mechanisms in early onset Alzheimer's disease.

    Science.gov (United States)

    De Roeck, Arne; Van den Bossche, Tobi; van der Zee, Julie; Verheijen, Jan; De Coster, Wouter; Van Dongen, Jasper; Dillen, Lubina; Baradaran-Heravi, Yalda; Heeman, Bavo; Sanchez-Valle, Raquel; Lladó, Albert; Nacmias, Benedetta; Sorbi, Sandro; Gelpi, Ellen; Grau-Rivera, Oriol; Gómez-Tortosa, Estrella; Pastor, Pau; Ortega-Cubero, Sara; Pastor, Maria A; Graff, Caroline; Thonberg, Håkan; Benussi, Luisa; Ghidoni, Roberta; Binetti, Giuliano; de Mendonça, Alexandre; Martins, Madalena; Borroni, Barbara; Padovani, Alessandro; Almeida, Maria Rosário; Santana, Isabel; Diehl-Schmid, Janine; Alexopoulos, Panagiotis; Clarimon, Jordi; Lleó, Alberto; Fortea, Juan; Tsolaki, Magda; Koutroumani, Maria; Matěj, Radoslav; Rohan, Zdenek; De Deyn, Peter; Engelborghs, Sebastiaan; Cras, Patrick; Van Broeckhoven, Christine; Sleegers, Kristel

    2017-09-01

    Premature termination codon (PTC) mutations in the ATP-Binding Cassette, Sub-Family A, Member 7 gene (ABCA7) have recently been identified as intermediate-to-high penetrant risk factor for late-onset Alzheimer's disease (LOAD). High variability, however, is observed in downstream ABCA7 mRNA and protein expression, disease penetrance, and onset age, indicative of unknown modifying factors. Here, we investigated the prevalence and disease penetrance of ABCA7 PTC mutations in a large early onset AD (EOAD)-control cohort, and examined the effect on transcript level with comprehensive third-generation long-read sequencing. We characterized the ABCA7 coding sequence with next-generation sequencing in 928 EOAD patients and 980 matched control individuals. With MetaSKAT rare variant association analysis, we observed a fivefold enrichment (p = 0.0004) of PTC mutations in EOAD patients (3%) versus controls (0.6%). Ten novel PTC mutations were only observed in patients, and PTC mutation carriers in general had an increased familial AD load. In addition, we observed nominal risk reducing trends for three common coding variants. Seven PTC mutations were further analyzed using targeted long-read cDNA sequencing on an Oxford Nanopore MinION platform. PTC-containing transcripts for each investigated PTC mutation were observed at varying proportion (5-41% of the total read count), implying incomplete nonsense-mediated mRNA decay (NMD). Furthermore, we distinguished and phased several previously unknown alternative splicing events (up to 30% of transcripts). In conjunction with PTC mutations, several of these novel ABCA7 isoforms have the potential to rescue deleterious PTC effects. In conclusion, ABCA7 PTC mutations play a substantial role in EOAD, warranting genetic screening of ABCA7 in genetically unexplained patients. Long-read cDNA sequencing revealed both varying degrees of NMD and transcript-modifying events, which may influence ABCA7 dosage, disease severity, and may

  18. Loss-of-function mutations in SLC30A8 protect against type 2 diabetes

    DEFF Research Database (Denmark)

    Flannick, Jason; Thorleifsson, Gudmar; Beer, Nicola L

    2014-01-01

    Loss-of-function mutations protective against human disease provide in vivo validation of therapeutic targets, but none have yet been described for type 2 diabetes (T2D). Through sequencing or genotyping of ~150,000 individuals across 5 ancestry groups, we identified 12 rare protein-truncating va...

  19. The p16INK4alpha/p19ARF gene mutations are infrequent and are mutually exclusive to p53 mutations in Indian oral squamous cell carcinomas.

    Science.gov (United States)

    Kannan, K; Munirajan, A K; Krishnamurthy, J; Bhuvarahamurthy, V; Mohanprasad, B K; Panishankar, K H; Tsuchida, N; Shanmugam, G

    2000-03-01

    Eighty-seven untreated primary oral squamous cell carcinomas (SCCs) associated with betel quid and tobacco chewing from Indian patients were analysed for the presence of mutations in the commonly shared exon 2 of p16INK4alpha/p19ARF genes. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and sequencing analysis were used to detect mutations. SSCP analysis indicated that only 9% (8/87) of the tumours had mutation in p16INK4alpha/p19ARF genes. Seventy-two tumours studied here were previously analysed for p53 mutations and 21% (15/72) of them were found to have mutations in p53 gene. Only one tumour was found to have mutation at both p53 and p16INK4alpha/p19ARF genes. Thus, the mutation rates observed were 21% for p53, 9% for p16INK4alpha/p19ARF, and 1% for both. Sequencing analysis revealed two types of mutations; i) G to C (GCAG to CCAG) transversion type mutation at intron 1-exon 2 splice junction and ii) another C to T transition type mutation resulting in CGA to TGA changing arginine to a termination codon at p16INK4alpha gene codon 80 and the same mutation will alter codon 94 of p19ARF gene from CCG to CTG (proline to leucine). These results suggest that p16INK4alpha/p19ARF mutations are less frequent than p53 mutations in Indian oral SCCs. The p53 and p16INK4alpha/p19ARF mutational events are independent and are mutually exclusive suggesting that mutational inactivation of either p53 or p16INK4alpha/p19ARF may alleviate the need for the inactivation of the other gene.

  20. Apoptotic Activity of MeCP2 Is Enhanced by C-Terminal Truncating Mutations.

    Directory of Open Access Journals (Sweden)

    Alison A Williams

    Full Text Available Methyl-CpG binding protein 2 (MeCP2 is a widely abundant, multifunctional protein most highly expressed in post-mitotic neurons. Mutations causing Rett syndrome and related neurodevelopmental disorders have been identified along the entire MECP2 locus, but symptoms vary depending on mutation type and location. C-terminal mutations are prevalent, but little is known about the function of the MeCP2 C-terminus. We employ the genetic efficiency of Drosophila to provide evidence that expression of p.Arg294* (more commonly identified as R294X, a human MECP2 E2 mutant allele causing truncation of the C-terminal domains, promotes apoptosis of identified neurons in vivo. We confirm this novel finding in HEK293T cells and then use Drosophila to map the region critical for neuronal apoptosis to a small sequence at the end of the C-terminal domain. In vitro studies in mammalian systems previously indicated a role of the MeCP2 E2 isoform in apoptosis, which is facilitated by phosphorylation at serine 80 (S80 and decreased by interactions with the forkhead protein FoxG1. We confirm the roles of S80 phosphorylation and forkhead domain transcription factors in affecting MeCP2-induced apoptosis in Drosophila in vivo, thus indicating mechanistic conservation between flies and mammalian cells. Our findings are consistent with a model in which C- and N-terminal interactions are required for healthy function of MeCP2.

  1. Loss of Optineurin In Vivo Results in Elevated Cell Death and Alters Axonal Trafficking Dynamics

    Science.gov (United States)

    Paulus, Jeremiah D.; Link, Brian A.

    2014-01-01

    Mutations in Optineurin have been associated with ALS, glaucoma, and Paget’s disease of bone in humans, but little is known about how these mutations contribute to disease. Most of the cellular consequences of Optineurin loss have come from in vitro studies, and it remains unclear whether these same defects would be seen in vivo. To answer this question, we assessed the cellular consequences of Optineurin loss in zebrafish embryos to determine if they showed the same defects as have been described in the in vitro studies. We found that loss of Optineurin resulted in increased cell death, as well as subtle cell morphology, cell migration and vesicle trafficking defects. However, unlike experiments on cells in culture, we found no indication that the Golgi apparatus was disrupted or that NF-κB target genes were upregulated. Therefore, we conclude that in vivo loss of Optineurin shows some, but not all, of the defects seen in in vitro work. PMID:25329564

  2. Metabolic Profiling of IDH Mutation and Malignant Progression in Infiltrating Glioma

    Science.gov (United States)

    Jalbert, Llewellyn E.; Elkhaled, Adam; Phillips, Joanna J.; Neill, Evan; Williams, Aurelia; Crane, Jason C.; Olson, Marram P.; Molinaro, Annette M.; Berger, Mitchel S.; Kurhanewicz, John; Ronen, Sabrina M.; Chang, Susan M.; Nelson, Sarah J.

    2017-03-01

    Infiltrating low grade gliomas (LGGs) are heterogeneous in their behavior and the strategies used for clinical management are highly variable. A key factor in clinical decision-making is that patients with mutations in the isocitrate dehydrogenase 1 and 2 (IDH1/2) oncogenes are more likely to have a favorable outcome and be sensitive to treatment. Because of their relatively long overall median survival, more aggressive treatments are typically reserved for patients that have undergone malignant progression (MP) to an anaplastic glioma or secondary glioblastoma (GBM). In the current study, ex vivo metabolic profiles of image-guided tissue samples obtained from patients with newly diagnosed and recurrent LGG were investigated using proton high-resolution magic angle spinning spectroscopy (1H HR-MAS). Distinct spectral profiles were observed for lesions with IDH-mutated genotypes, between astrocytoma and oligodendroglioma histologies, as well as for tumors that had undergone MP. Levels of 2-hydroxyglutarate (2HG) were correlated with increased mitotic activity, axonal disruption, vascular neoplasia, and with several brain metabolites including the choline species, glutamate, glutathione, and GABA. The information obtained in this study may be used to develop strategies for in vivo characterization of infiltrative glioma, in order to improve disease stratification and to assist in monitoring response to therapy.

  3. Famine versus feast: understanding the metabolism of tumors in vivo.

    Science.gov (United States)

    Mayers, Jared R; Vander Heiden, Matthew G

    2015-03-01

    To fuel unregulated proliferation, cancer cells alter metabolism to support macromolecule biosynthesis. Cell culture studies have revealed how different oncogenic mutations and nutrients impact metabolism. Glucose and glutamine are the primary fuels used in vitro; however, recent studies have suggested that utilization of other amino acids as well as lipids and protein can also be important to cancer cells. Early investigations of tumor metabolism are translating these findings to the biology of whole tumors and suggest that additional complexity exists beyond nutrient availability alone in vivo. Whole-body metabolism and tumor heterogeneity also influence the metabolism of tumor cells, and successful targeting of metabolism for cancer therapy will require an understanding of tumor metabolism in vivo. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    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.

  5. Requirement of the propeptide for in vivo formation of active yeast carboxypeptidase Y

    DEFF Research Database (Denmark)

    Ramos, C; Winther, Jakob R.; Kielland-Brandt, Morten

    1994-01-01

    Deletions have been constructed in the region encoding the 91-amino acid propeptide of the vacuolar enzyme carboxypeptidase Y of Saccharomyces cerevisiae, and in vivo effects of these mutations on the intracellular transport of the mutant proenzymes have been examined. Deletions did not include...... the vacuolar targeting signal, and none of the mutated forms of procarboxypeptidase Y was found to be secreted. All deletions, however, resulted in a decreased rate of transport of the truncated proenzymes from the endoplasmic reticulum to the Golgi apparatus. Up to 29 residues close to the N terminus can...... be removed without completely eliminating transport of the mutated proenzymes to the vacuole. However, the C-terminal part of the propeptide contains elements which are essential, since two small deletions, of 9 and 15 residues, respectively, within this area resulted in loss of carboxypeptidase Y activity...

  6. Rare mutations predisposing to familial adenomatous polyposis in Greek FAP patients

    International Nuclear Information System (INIS)

    Mihalatos, Markos; Fountzilas, George; Agnantis, Niki J; Nasioulas, Georgios; Apessos, Angela; Dauwerse, Hans; Velissariou, Voula; Psychias, Aristidis; Koliopanos, Alexander; Petropoulos, Konstantinos; Triantafillidis, John K; Danielidis, Ioannis

    2005-01-01

    Familial Adenomatous Polyposis (FAP) is caused by germline mutations in the APC (Adenomatous Polyposis Coli) gene. The vast majority of APC mutations are point mutations or small insertions / deletions which lead to truncated protein products. Splicing mutations or gross genomic rearrangements are less common inactivating events of the APC gene. In the current study genomic DNA or RNA from ten unrelated FAP suspected patients was examined for germline mutations in the APC gene. Family history and phenotype were used in order to select the patients. Methods used for testing were dHPLC (denaturing High Performance Liquid Chromatography), sequencing, MLPA (Multiplex Ligation – dependent Probe Amplification), Karyotyping, FISH (Fluorescence In Situ Hybridization) and RT-PCR (Reverse Transcription – Polymerase Chain Reaction). A 250 Kbp deletion in the APC gene starting from intron 5 and extending beyond exon 15 was identified in one patient. A substitution of the +5 conserved nucleotide at the splice donor site of intron 9 in the APC gene was shown to produce frameshift and inefficient exon skipping in a second patient. Four frameshift mutations (1577insT, 1973delAG, 3180delAAAA, 3212delA) and a nonsense mutation (C1690T) were identified in the rest of the patients. Screening for APC mutations in FAP patients should include testing for splicing defects and gross genomic alterations

  7. DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo.

    Science.gov (United States)

    Zubradt, Meghan; Gupta, Paromita; Persad, Sitara; Lambowitz, Alan M; Weissman, Jonathan S; Rouskin, Silvi

    2017-01-01

    Coupling of structure-specific in vivo chemical modification to next-generation sequencing is transforming RNA secondary structure studies in living cells. The dominant strategy for detecting in vivo chemical modifications uses reverse transcriptase truncation products, which introduce biases and necessitate population-average assessments of RNA structure. Here we present dimethyl sulfate (DMS) mutational profiling with sequencing (DMS-MaPseq), which encodes DMS modifications as mismatches using a thermostable group II intron reverse transcriptase. DMS-MaPseq yields a high signal-to-noise ratio, can report multiple structural features per molecule, and allows both genome-wide studies and focused in vivo investigations of even low-abundance RNAs. We apply DMS-MaPseq for the first analysis of RNA structure within an animal tissue and to identify a functional structure involved in noncanonical translation initiation. Additionally, we use DMS-MaPseq to compare the in vivo structure of pre-mRNAs with their mature isoforms. These applications illustrate DMS-MaPseq's capacity to dramatically expand in vivo analysis of RNA structure.

  8. ISVASE: identification of sequence variant associated with splicing event using RNA-seq data.

    Science.gov (United States)

    Aljohi, Hasan Awad; Liu, Wanfei; Lin, Qiang; Yu, Jun; Hu, Songnian

    2017-06-28

    Exon recognition and splicing precisely and efficiently by spliceosome is the key to generate mature mRNAs. About one third or a half of disease-related mutations affect RNA splicing. Software PVAAS has been developed to identify variants associated with aberrant splicing by directly using RNA-seq data. However, it bases on the assumption that annotated splicing site is normal splicing, which is not true in fact. We develop the ISVASE, a tool for specifically identifying sequence variants associated with splicing events (SVASE) by using RNA-seq data. Comparing with PVAAS, our tool has several advantages, such as multi-pass stringent rule-dependent filters and statistical filters, only using split-reads, independent sequence variant identification in each part of splicing (junction), sequence variant detection for both of known and novel splicing event, additional exon-exon junction shift event detection if known splicing events provided, splicing signal evaluation, known DNA mutation and/or RNA editing data supported, higher precision and consistency, and short running time. Using a realistic RNA-seq dataset, we performed a case study to illustrate the functionality and effectiveness of our method. Moreover, the output of SVASEs can be used for downstream analysis such as splicing regulatory element study and sequence variant functional analysis. ISVASE is useful for researchers interested in sequence variants (DNA mutation and/or RNA editing) associated with splicing events. The package is freely available at https://sourceforge.net/projects/isvase/ .

  9. Prevalence and clinical correlates of JAK2 mutations in Down syndrome acute lymphoblastic leukemia

    Science.gov (United States)

    Gaikwad, Amos; Rye, Cassia L.; Devidas, Meenakshi; Heerema, Nyla A.; Carroll, Andrew J.; Izraeli, Shai; Plon, Sharon E.; Basso, Giuseppe; Pession, Andrea; Rabin, Karen R.

    2009-01-01

    Summary Recurrent, prognostically significant chromosomal abnormalities occur in approximately 75% of pediatric acute lymphoblastic leukemia (ALL), but only infrequently in children with Down syndrome (DS) and ALL. Recently, novel somatic activating mutations in Janus kinase 2 (JAK2) were reported in 18% of DS ALL. Here we report identification and clinical correlates of JAK2 mutations in an independent cohort. JAK2 activating mutations occurred in 10 of 53 DS ALL cases (18.9%). Mutations were overrepresented in males (p<0.03), occurred once in association with high hyperdiploidy, and were not significantly correlated with age, initial white blood count, or event-free survival. Our results confirm significance of JAK-STAT pathway activation in DS ALL. PMID:19120350

  10. Consortium for Osteogenesis Imperfecta Mutations in the Helical Domain of Type I Collagen: Regions Rich in Lethal Mutations Align With Collagen Binding Sites for Integrins and Proteoglycans

    Science.gov (United States)

    Marini, Joan C.; Forlino, Antonella; Cabral, Wayne A.; Barnes, Aileen M.; San Antonio, James D.; Milgrom, Sarah; Hyland, James C.; Körkkö, Jarmo; Prockop, Darwin J.; De Paepe, Anne; Coucke, Paul; Symoens, Sofie; Glorieux, Francis H.; Roughley, Peter J.; Lund, Alan M.; Kuurila-Svahn, Kaija; Hartikka, Heini; Cohn, Daniel H.; Krakow, Deborah; Mottes, Monica; Schwarze, Ulrike; Chen, Diana; Yang, Kathleen; Kuslich, Christine; Troendle, James; Dalgleish, Raymond; Byers, Peter H.

    2014-01-01

    Osteogenesis imperfecta (OI) is a generalized disorder of connective tissue characterized by fragile bones and easy susceptibility to fracture. Most cases of OI are caused by mutations in type I collagen. We have identified and assembled structural mutations in type I collagen genes (COL1A1 and COL1A2, encoding the proα1(I) and proα2(I) chains, respectively) that result in OI. Quantitative defects causing type I OI were not included. Of these 832 independent mutations, 682 result in substitution for glycine residues in the triple helical domain of the encoded protein and 150 alter splice sites. Distinct genotype–phenotype relationships emerge for each chain. One-third of the mutations that result in glycine substitutions in α1(I) are lethal, especially when the substituting residues are charged or have a branched side chain. Substitutions in the first 200 residues are nonlethal and have variable outcome thereafter, unrelated to folding or helix stability domains. Two exclusively lethal regions (helix positions 691–823 and 910–964) align with major ligand binding regions (MLBRs), suggesting crucial interactions of collagen monomers or fibrils with integrins, matrix metalloproteinases (MMPs), fibronectin, and cartilage oligomeric matrix protein (COMP). Mutations in COL1A2 are predominantly nonlethal (80%). Lethal substitutions are located in eight regularly spaced clusters along the chain, supporting a regional model. The lethal regions align with proteoglycan binding sites along the fibril, suggesting a role in fibril–matrix interactions. Recurrences at the same site in α2(I) are generally concordant for outcome, unlike α1(I). Splice site mutations comprise 20% of helical mutations identified in OI patients, and may lead to exon skipping, intron inclusion, or the activation of cryptic splice sites. Splice site mutations in COL1A1 are rarely lethal; they often lead to frameshifts and the mild type I phenotype. In α2(I), lethal exon skipping events are

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

    Science.gov (United States)

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

  12. TARDBP and FUS mutations associated with amyotrophic lateral sclerosis: summary and update.

    Science.gov (United States)

    Lattante, Serena; Rouleau, Guy A; Kabashi, Edor

    2013-06-01

    Mutations in the TAR DNA Binding Protein gene (TARDBP), encoding the protein TDP-43, were identified in amyotrophic lateral sclerosis (ALS) patients. Interestingly, TDP-43 positive inclusion bodies were first discovered in ubiquitin-positive, tau-negative ALS and frontotemporal dementia (FTD) inclusion bodies, and subsequently observed in the majority of neurodegenerative disorders. To date, 47 missense and one truncating mutations have been described in a large number of familial (FALS) and sporadic (SALS) patients. Fused in sarcoma (FUS) was found to be responsible for a previously identified ALS6 locus, being mutated in both FALS and SALS patients. TARDBP and FUS have a structural and functional similarity and most of mutations in both genes are also clustered in the C-terminus of the proteins. The molecular mechanisms through which mutant TDP-43 and FUS may cause motor neuron degeneration are not well understood. Both proteins play an important role in mRNA transport, axonal maintenance, and motor neuron development. Functional characterization of these mutations in in vitro and in vivo systems is helping to better understand how motor neuron degeneration occurs. This report summarizes the biological and clinical relevance of TARDBP and FUS mutations in ALS. All the data reviewed here have been submitted to a database based on the Leiden Open (source) Variation Database (LOVD) and is accessible online at www.lovd.nl/TARDBP, www.lovd.nl/FUS. © 2013 Wiley Periodicals, Inc.

  13. Ghrelin receptor mutations--too little height and too much hunger

    DEFF Research Database (Denmark)

    Holst, Birgitte; Schwartz, Thue W

    2006-01-01

    The ghrelin receptor is known from in vitro studies to signal in the absence of the hormone ghrelin at almost 50% of its maximal capacity. But, as for many other 7-transmembrane receptors, the in vivo importance of this ligand-independent signaling has remained unclear. In this issue of the JCI......, Pantel et al. find that a natural mutation in the ghrelin receptor, Ala204Glu, which is associated with a selective loss of constitutive activity without affecting ghrelin affinity, potency, or efficacy, segregates in 2 families with the development of short stature (see the related article beginning...... on page 760). By combination of the observations from this study with those related to the phenotype of subjects carrying another natural ghrelin receptor mutation, Phe279Leu, having identical molecular-pharmacological properties, it is proposed that selective lack of ghrelin receptor constitutive...

  14. Strikingly different penetrance of LHON in two Chinese families with primary mutation G11778A is independent of mtDNA haplogroup background and secondary mutation G13708A

    Energy Technology Data Exchange (ETDEWEB)

    Wang Huawei [Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223 (China)]|[Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming 650091 (China); Jia Xiaoyun; Ji Yanli [State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060 (China); Kong Qingpeng [State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223 (China); Zhang Qingjiong [State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060 (China)], E-mail: qingjiongzhang@yahoo.com; Yao Yonggang [Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223 (China)]|[State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223 (China)], E-mail: ygyaozh@yahoo.com; Zhang Yaping [Laboratory for Conservation and Utilization of Bio-resource, Yunnan University, Kunming 650091 (China)]|[State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223 (China)

    2008-08-25

    The penetrance of Leber's hereditary optic neuropathy (LHON) in families with primary mitochondrial DNA (mtDNA) mutations is very complex. Matrilineal and nuclear genetic background, as well as environmental factors, have been reported to be involved in different affected pedigrees. Here we describe two large Chinese families that show a striking difference in the penetrance of LHON, in which 53.3% and 15.0% of members were affected (P < 0.02), respectively. Analysis of the complete mtDNA genome of the two families revealed the presence of the primary mutation G11778A and several other variants suggesting the same haplogroup status G2a. The family with higher penetrance contained a previously described secondary mutation G13708A, which presents a polymorphism in normal Chinese samples and does not affect in vivo mitochondrial oxidative metabolism as described in a previous study. Evolutionary analysis failed to indicate any putatively pathogenic mutation that cosegregated with G11778A in these two pedigrees. Our results suggest that the variable penetrance of LHON in the two Chinese families is independent of both their mtDNA haplotype background and a secondary mutation G13708A. As a result, it is likely that unknown nuclear gene involvement and/or other factors contribute to the strikingly different penetrance of LHON.

  15. Uncommon EGFR mutations in cytological specimens of 1,874 newly diagnosed Indonesian lung cancer patients

    Science.gov (United States)

    Syahruddin, Elisna; Wulandari, Laksmi; Sri Muktiati, Nunuk; Rima, Ana; Soeroso, Noni; Ermayanti, Sabrina; Levi, Michael; Hidajat, Heriawaty; Widjajahakim, Grace; Utomo, Ahmad Rusdan Handoyo

    2018-01-01

    Purpose We aimed to evaluate the distribution of individual epidermal growth factor receptor (EGFR) mutation subtypes found in routine cytological specimens. Patients and methods A retrospective audit was performed on EGFR testing results of 1,874 consecutive cytological samples of newly diagnosed or treatment-naïve Indonesian lung cancer patients (years 2015–2016). Testing was performed by ISO15189 accredited central laboratory. Results Overall test failure rate was 5.1%, with the highest failure (7.1%) observed in pleural effusion and lowest (1.6%) in needle aspiration samples. EGFR mutation frequency was 44.4%. Tyrosine kinase inhibitor (TKI)-sensitive common EGFR mutations (ins/dels exon 19, L858R) and uncommon mutations (G719X, T790M, L861Q) contributed 57.1% and 29%, respectively. Approximately 13.9% of mutation-positive patients carried a mixture of common and uncommon mutations. Women had higher EGFR mutation rate (52.9%) vs men (39.1%; pcytological techniques yielded similar success rate to detect EGFR mutations. Uncommon EGFR mutations were frequent events in Indonesian lung cancer patients. PMID:29615847

  16. Multiple Hotspot Mutations Scanning by Single Droplet Digital PCR.

    Science.gov (United States)

    Decraene, Charles; Silveira, Amanda B; Bidard, François-Clément; Vallée, Audrey; Michel, Marc; Melaabi, Samia; Vincent-Salomon, Anne; Saliou, Adrien; Houy, Alexandre; Milder, Maud; Lantz, Olivier; Ychou, Marc; Denis, Marc G; Pierga, Jean-Yves; Stern, Marc-Henri; Proudhon, Charlotte

    2018-02-01

    Progress in the liquid biopsy field, combined with the development of droplet digital PCR (ddPCR), has enabled noninvasive monitoring of mutations with high detection accuracy. However, current assays detect a restricted number of mutations per reaction. ddPCR is a recognized method for detecting alterations previously characterized in tumor tissues, but its use as a discovery tool when the mutation is unknown a priori remains limited. We established 2 ddPCR assays detecting all genomic alterations within KRAS exon 2 and EGFR exon 19 mutation hotspots, which are of clinical importance in colorectal and lung cancer, with use of a unique pair of TaqMan ® oligoprobes. The KRAS assay scanned for the 7 most common mutations in codons 12/13 but also all other mutations found in that region. The EGFR assay screened for all in-frame deletions of exon 19, which are frequent EGFR-activating events. The KRAS and EGFR assays were highly specific and both reached a limit of detection of <0.1% in mutant allele frequency. We further validated their performance on multiple plasma and formalin-fixed and paraffin-embedded tumor samples harboring a panel of different KRAS or EGFR mutations. This method presents the advantage of detecting a higher number of mutations with single-reaction ddPCRs while consuming a minimum of patient sample. This is particularly useful in the context of liquid biopsy because the amount of circulating tumor DNA is often low. This method should be useful as a discovery tool when the tumor tissue is unavailable or to monitor disease during therapy. © 2017 American Association for Clinical Chemistry.

  17. Isolation of temperature-sensitive mutations in murC of Staphylococcus aureus.

    Science.gov (United States)

    Ishibashi, Mihoko; Kurokawa, Kenji; Nishida, Satoshi; Ueno, Kohji; Matsuo, Miki; Sekimizu, Kazuhisa

    2007-09-01

    Enzymes in the bacterial peptidoglycan biosynthesis pathway are important targets for novel antibiotics. Of 750 temperature-sensitive (TS) mutants of Gram-positive Staphylococcus aureus, six were complemented by the murC gene, which encodes the UDP-N-acetylmuramic acid:l-alanine ligase. Each mutation resulted in a single amino acid substitution and, in all cases, the TS phenotype was suppressed by high osmotic stress. In mutant strains with the G222E substitution, a decrease in the viable cell number immediately after shift to the restrictive temperature was observed. These results suggest that S. aureus MurC protein is essential for cell growth. The MurC H343Y mutation is located in the putative alanine recognition pocket. Consistent with this, allele-specific suppression was observed of the H343Y mutation by multiple copies of the aapA gene, which encodes an alanine transporter. The results suggest an in vivo role for the H343 residue of S. aureus MurC protein in high-affinity binding to L-alanine.

  18. TAL effector nucleases induce mutations at a pre-selected location in the genome of primary barley transformants

    DEFF Research Database (Denmark)

    Wendt, Toni; Holm, Preben Bach; Starker, Colby G

    2013-01-01

    , and their broad targeting range. Here we report the assembly of several TALENs for a specific genomic locus in barley. The cleavage activity of individual TALENs was first tested in vivo using a yeast-based, single-strand annealing assay. The most efficient TALEN was then selected for barley transformation....... Analysis of the resulting transformants showed that TALEN-induced double strand breaks led to the introduction of short deletions at the target site. Additional analysis revealed that each barley transformant contained a range of different mutations, indicating that mutations occurred independently...

  19. Enhanced susceptibility of a transposable-element-bearing strain of Drosophila melanogaster to somatic eye-color mutations by ethyl nitrosourea, methyl nitrosourea, and X-rays

    International Nuclear Information System (INIS)

    Ryo, H.; Kondo, S.; Rasmuson, B.

    1983-01-01

    A strain of Drosophila with the genes z and w + plus a transposable element (TE) is about 3 times more sensitive than a strain without TE toward somatic eye-color mutations after larval exposure to ethyl nitrosourea, methyl nitrosourea and X-rays. The assay system with TE is simple, reliable, and sensitive for detecting somatic mutations induced in vivo by mutagens. (orig.)

  20. A mutational analysis of Caenorhabditis elegans in space

    Energy Technology Data Exchange (ETDEWEB)

    Zhao Yang [Department of Medical Genetics, University of British Columbia, Life Sciences Centre, Room 1364-2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3 (Canada); Lai, Kenneth [Department of Medical Genetics, University of British Columbia, Life Sciences Centre, Room 1364-2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3 (Canada); Cheung, Iris [Department of Medical Genetics, University of British Columbia, Life Sciences Centre, Room 1364-2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3 (Canada); Youds, Jillian [Department of Medical Genetics, University of British Columbia, Life Sciences Centre, Room 1364-2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3 (Canada); Tarailo, Maja [Department of Medical Genetics, University of British Columbia, Life Sciences Centre, Room 1364-2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3 (Canada); Tarailo, Sanja [Department of Medical Genetics, University of British Columbia, Life Sciences Centre, Room 1364-2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3 (Canada); Rose, Ann [Department of Medical Genetics, University of British Columbia, Life Sciences Centre, Room 1364-2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3 (Canada)]. E-mail: arose@gene.nce.ubc.ca

    2006-10-10

    The International Caenorhabditis elegans Experiment First Flight (ICE-First) was a project using C. elegans as a model organism to study the biological effects of short duration spaceflight (11 days in the International Space Station). As a member of the ICE-First research team, our group focused on the mutational effects of spaceflight. Several approaches were taken to measure mutational changes that occurred during the spaceflight including measurement of the integrity of poly-G/poly-C tracts, determination of the mutation frequency in the unc-22 gene, analysis of lethal mutations captured by the genetic balancer eT1(III;V), and identification of alterations in telomere length. By comparing the efficiency, sensitivity, and convenience of these methods, we deduced that the eT1 balancer system is well-suited for capturing, maintaining and recovering mutational events that occur over several generations during spaceflight. In the course of this experiment, we have extended the usefulness of the eT1 balancer system by identifying the physical breakpoints of the eT1 translocation and have developed a PCR assay to follow the eT1 chromosomes. C. elegans animals were grown in a defined liquid media during the spaceflight. This is the first analysis of genetic changes in C. elegans grown in the defined media. Although no significant difference in mutation rate was detected between spaceflight and control samples, which is not surprising given the short duration of the spaceflight, we demonstrate here the utility of worms as an integrating biological dosimeter for spaceflight.

  1. A mutational analysis of Caenorhabditis elegans in space

    International Nuclear Information System (INIS)

    Zhao Yang; Lai, Kenneth; Cheung, Iris; Youds, Jillian; Tarailo, Maja; Tarailo, Sanja; Rose, Ann

    2006-01-01

    The International Caenorhabditis elegans Experiment First Flight (ICE-First) was a project using C. elegans as a model organism to study the biological effects of short duration spaceflight (11 days in the International Space Station). As a member of the ICE-First research team, our group focused on the mutational effects of spaceflight. Several approaches were taken to measure mutational changes that occurred during the spaceflight including measurement of the integrity of poly-G/poly-C tracts, determination of the mutation frequency in the unc-22 gene, analysis of lethal mutations captured by the genetic balancer eT1(III;V), and identification of alterations in telomere length. By comparing the efficiency, sensitivity, and convenience of these methods, we deduced that the eT1 balancer system is well-suited for capturing, maintaining and recovering mutational events that occur over several generations during spaceflight. In the course of this experiment, we have extended the usefulness of the eT1 balancer system by identifying the physical breakpoints of the eT1 translocation and have developed a PCR assay to follow the eT1 chromosomes. C. elegans animals were grown in a defined liquid media during the spaceflight. This is the first analysis of genetic changes in C. elegans grown in the defined media. Although no significant difference in mutation rate was detected between spaceflight and control samples, which is not surprising given the short duration of the spaceflight, we demonstrate here the utility of worms as an integrating biological dosimeter for spaceflight

  2. The influence of DNA repair inhibitors on the mutation rate

    International Nuclear Information System (INIS)

    Auzinger, Th.; Hruby, R.

    1980-12-01

    The simultaneous influence of gamma-radiation and DNA-repair inhibiting substances on the mutation frequency of mice was investigated in vivo with the micronucleus test. The detergens Tween 80, vitamin A, and the antiphlogisticum phenylbutazone were used as DNA-repair inhibiting substances. Using the same irradiation doses, a statistic significant increase of mutagenicity respectively micronucleus frequency was found in high concentrations of Tween 80 and in all used dosages of vitamin A, but not in phenylbutazone and in low concentrations of tween. (auth.)

  3. Sodium channel SCN8A (Nav1.6: properties and de novo mutations in epileptic encephalopathy and intellectual disability

    Directory of Open Access Journals (Sweden)

    Janelle Elizabeth O'Brien

    2013-10-01

    Full Text Available The sodium channel Nav1.6, encoded by the gene SCN8A, is one of the major voltage-gated channels in human brain. The sequences of sodium channels have been highly conserved during evolution, and minor changes in biophysical properties can have a major impact in vivo. Insight into the role of Nav1.6 has come from analysis of spontaneous and induced mutations of mouse Scn8a during the past 18 years. Only within the past year has the role of SCN8A in human disease become apparent from whole exome and genome sequences of patients with sporadic disease. Unique features of Nav1.6 include its contribution to persistent current, resurgent current, repetitive neuronal firing, and subcellular localization at the axon initial segment and nodes of Ranvier. Loss of Nav1.6 activity results in reduced neuronal excitability, while gain-of-function mutations can increase neuronal excitability. Mouse Scn8a (med mutants exhibit movement disorders including ataxia, tremor and dystonia. Thus far, more than ten human de novo mutations have been identified in patients with two types of disorders, epileptic encephalopathy and intellectual disability. We review these human mutations as well as the unique features of Nav1.6 that contribute to its role in determining neuronal excitability in vivo. A supplemental figure illustrating the positions of amino acid residues within the 4 domains and 24 transmembrane segments of Nav1.6 is provided to facilitate the location of novel mutations within the channel protein.

  4. Experimental evolution, genetic analysis and genome re-sequencing reveal the mutation conferring artemisinin resistance in an isogenic lineage of malaria parasites

    KAUST Repository

    Hunt, Paul

    2010-09-16

    Background: Classical and quantitative linkage analyses of genetic crosses have traditionally been used to map genes of interest, such as those conferring chloroquine or quinine resistance in malaria parasites. Next-generation sequencing technologies now present the possibility of determining genome-wide genetic variation at single base-pair resolution. Here, we combine in vivo experimental evolution, a rapid genetic strategy and whole genome re-sequencing to identify the precise genetic basis of artemisinin resistance in a lineage of the rodent malaria parasite, Plasmodium chabaudi. Such genetic markers will further the investigation of resistance and its control in natural infections of the human malaria, P. falciparum.Results: A lineage of isogenic in vivo drug-selected mutant P. chabaudi parasites was investigated. By measuring the artemisinin responses of these clones, the appearance of an in vivo artemisinin resistance phenotype within the lineage was defined. The underlying genetic locus was mapped to a region of chromosome 2 by Linkage Group Selection in two different genetic crosses. Whole-genome deep coverage short-read re-sequencing (IlluminaSolexa) defined the point mutations, insertions, deletions and copy-number variations arising in the lineage. Eight point mutations arise within the mutant lineage, only one of which appears on chromosome 2. This missense mutation arises contemporaneously with artemisinin resistance and maps to a gene encoding a de-ubiquitinating enzyme.Conclusions: This integrated approach facilitates the rapid identification of mutations conferring selectable phenotypes, without prior knowledge of biological and molecular mechanisms. For malaria, this model can identify candidate genes before resistant parasites are commonly observed in natural human malaria populations. 2010 Hunt et al; licensee BioMed Central Ltd.

  5. Gene Mutation Profiles in Primary Diffuse Large B Cell Lymphoma of Central Nervous System: Next Generation Sequencing Analyses

    Science.gov (United States)

    Todorovic Balint, Milena; Jelicic, Jelena; Mihaljevic, Biljana; Kostic, Jelena; Stanic, Bojana; Balint, Bela; Pejanovic, Nadja; Lucic, Bojana; Tosic, Natasa; Marjanovic, Irena; Stojiljkovic, Maja; Karan-Djurasevic, Teodora; Perisic, Ognjen; Rakocevic, Goran; Popovic, Milos; Raicevic, Sava; Bila, Jelena; Antic, Darko; Andjelic, Bosko; Pavlovic, Sonja

    2016-01-01

    The existence of a potential primary central nervous system lymphoma-specific genomic signature that differs from the systemic form of diffuse large B cell lymphoma (DLBCL) has been suggested, but is still controversial. We investigated 19 patients with primary DLBCL of central nervous system (DLBCL CNS) using the TruSeq Amplicon Cancer Panel (TSACP) for 48 cancer-related genes. Next generation sequencing (NGS) analyses have revealed that over 80% of potentially protein-changing mutations were located in eight genes (CTNNB1, PIK3CA, PTEN, ATM, KRAS, PTPN11, TP53 and JAK3), pointing to the potential role of these genes in lymphomagenesis. TP53 was the only gene harboring mutations in all 19 patients. In addition, the presence of mutated TP53 and ATM genes correlated with a higher total number of mutations in other analyzed genes. Furthermore, the presence of mutated ATM correlated with poorer event-free survival (EFS) (p = 0.036). The presence of the mutated SMO gene correlated with earlier disease relapse (p = 0.023), inferior event-free survival (p = 0.011) and overall survival (OS) (p = 0.017), while mutations in the PTEN gene were associated with inferior OS (p = 0.048). Our findings suggest that the TP53 and ATM genes could be involved in the molecular pathophysiology of primary DLBCL CNS, whereas mutations in the PTEN and SMO genes could affect survival regardless of the initial treatment approach. PMID:27164089

  6. Somatic mutations associated with MRI-derived volumetric features in glioblastoma

    Energy Technology Data Exchange (ETDEWEB)

    Gutman, David A.; Dunn, William D. [Emory University School of Medicine, Departments of Neurology, Atlanta, GA (United States); Emory University School of Medicine, Biomedical Informatics, Atlanta, GA (United States); Grossmann, Patrick; Alexander, Brian M. [Harvard Medical School, Department of Radiation Oncology, Dana-Farber Cancer Institute, Brigham and Women' s Hospital, Boston, MA (United States); Cooper, Lee A.D. [Emory University School of Medicine, Biomedical Informatics, Atlanta, GA (United States); Georgia Institute of Technology, Department of Biomedical Engineering, Atlanta, GA (United States); Holder, Chad A. [Emory University School of Medicine, Radiology and Imaging Sciences, Atlanta, GA (United States); Ligon, Keith L. [Brigham and Women' s Hospital, Harvard Medical School, Pathology, Dana-Farber Cancer Institute, Boston, MA (United States); Aerts, Hugo J.W.L. [Harvard Medical School, Department of Radiation Oncology, Dana-Farber Cancer Institute, Brigham and Women' s Hospital, Boston, MA (United States); Brigham and Women' s Hospital, Harvard Medical School, Radiology, Dana-Farber Cancer Institute, Boston, MA (United States)

    2015-12-15

    MR imaging can noninvasively visualize tumor phenotype characteristics at the macroscopic level. Here, we investigated whether somatic mutations are associated with and can be predicted by MRI-derived tumor imaging features of glioblastoma (GBM). Seventy-six GBM patients were identified from The Cancer Imaging Archive for whom preoperative T1-contrast (T1C) and T2-FLAIR MR images were available. For each tumor, a set of volumetric imaging features and their ratios were measured, including necrosis, contrast enhancing, and edema volumes. Imaging genomics analysis assessed the association of these features with mutation status of nine genes frequently altered in adult GBM. Finally, area under the curve (AUC) analysis was conducted to evaluate the predictive performance of imaging features for mutational status. Our results demonstrate that MR imaging features are strongly associated with mutation status. For example, TP53-mutated tumors had significantly smaller contrast enhancing and necrosis volumes (p = 0.012 and 0.017, respectively) and RB1-mutated tumors had significantly smaller edema volumes (p = 0.015) compared to wild-type tumors. MRI volumetric features were also found to significantly predict mutational status. For example, AUC analysis results indicated that TP53, RB1, NF1, EGFR, and PDGFRA mutations could each be significantly predicted by at least one imaging feature. MRI-derived volumetric features are significantly associated with and predictive of several cancer-relevant, drug-targetable DNA mutations in glioblastoma. These results may shed insight into unique growth characteristics of individual tumors at the macroscopic level resulting from molecular events as well as increase the use of noninvasive imaging in personalized medicine. (orig.)

  7. Somatic mutations associated with MRI-derived volumetric features in glioblastoma

    International Nuclear Information System (INIS)

    Gutman, David A.; Dunn, William D.; Grossmann, Patrick; Alexander, Brian M.; Cooper, Lee A.D.; Holder, Chad A.; Ligon, Keith L.; Aerts, Hugo J.W.L.

    2015-01-01

    MR imaging can noninvasively visualize tumor phenotype characteristics at the macroscopic level. Here, we investigated whether somatic mutations are associated with and can be predicted by MRI-derived tumor imaging features of glioblastoma (GBM). Seventy-six GBM patients were identified from The Cancer Imaging Archive for whom preoperative T1-contrast (T1C) and T2-FLAIR MR images were available. For each tumor, a set of volumetric imaging features and their ratios were measured, including necrosis, contrast enhancing, and edema volumes. Imaging genomics analysis assessed the association of these features with mutation status of nine genes frequently altered in adult GBM. Finally, area under the curve (AUC) analysis was conducted to evaluate the predictive performance of imaging features for mutational status. Our results demonstrate that MR imaging features are strongly associated with mutation status. For example, TP53-mutated tumors had significantly smaller contrast enhancing and necrosis volumes (p = 0.012 and 0.017, respectively) and RB1-mutated tumors had significantly smaller edema volumes (p = 0.015) compared to wild-type tumors. MRI volumetric features were also found to significantly predict mutational status. For example, AUC analysis results indicated that TP53, RB1, NF1, EGFR, and PDGFRA mutations could each be significantly predicted by at least one imaging feature. MRI-derived volumetric features are significantly associated with and predictive of several cancer-relevant, drug-targetable DNA mutations in glioblastoma. These results may shed insight into unique growth characteristics of individual tumors at the macroscopic level resulting from molecular events as well as increase the use of noninvasive imaging in personalized medicine. (orig.)

  8. Identification of Constrained Cancer Driver Genes Based on Mutation Timing

    Science.gov (United States)

    Sakoparnig, Thomas; Fried, Patrick; Beerenwinkel, Niko

    2015-01-01

    Cancer drivers are genomic alterations that provide cells containing them with a selective advantage over their local competitors, whereas neutral passengers do not change the somatic fitness of cells. Cancer-driving mutations are usually discriminated from passenger mutations by their higher degree of recurrence in tumor samples. However, there is increasing evidence that many additional driver mutations may exist that occur at very low frequencies among tumors. This observation has prompted alternative methods for driver detection, including finding groups of mutually exclusive mutations and incorporating prior biological knowledge about gene function or network structure. Dependencies among drivers due to epistatic interactions can also result in low mutation frequencies, but this effect has been ignored in driver detection so far. Here, we present a new computational approach for identifying genomic alterations that occur at low frequencies because they depend on other events. Unlike passengers, these constrained mutations display punctuated patterns of occurrence in time. We test this driver–passenger discrimination approach based on mutation timing in extensive simulation studies, and we apply it to cross-sectional copy number alteration (CNA) data from ovarian cancer, CNA and single-nucleotide variant (SNV) data from breast tumors and SNV data from colorectal cancer. Among the top ranked predicted drivers, we find low-frequency genes that have already been shown to be involved in carcinogenesis, as well as many new candidate drivers. The mutation timing approach is orthogonal and complementary to existing driver prediction methods. It will help identifying from cancer genome data the alterations that drive tumor progression. PMID:25569148

  9. Reduced rates of gene loss, gene silencing, and gene mutation in Dnmt1-deficient embryonic stem cells

    NARCIS (Netherlands)

    Chan, M.F.; van Amerongen, R.; Nijjar, T.; Cuppen, E.; Jones, P.A.; Laird, P.W.

    2001-01-01

    Tumor suppressor gene inactivation is a crucial event in oncogenesis. Gene inactivation mechanisms include events resulting in loss of heterozygosity (LOH), gene mutation, and transcriptional silencing. The contribution of each of these different pathways varies among tumor suppressor genes and by

  10. Comprehensive mutational screening in a cohort of Danish families with hereditary congenital cataract

    DEFF Research Database (Denmark)

    Hansen, Lars; Mikkelsen, Annemette; Nürnberg, Peter

    2009-01-01

    , and a gene conversion is the most likely mutational event causing this variant. Ten families had microcornea cataract, and a mutation was identified in eight of those. Most families displayed mixed phenotypes with nuclear, lamellar, and polar opacities and no apparent genotype-phenotype correlation emerged......PURPOSE: Identification of the causal mutations in 28 unrelated families and individuals with hereditary congenital cataract identified from a national Danish register of hereditary eye diseases. Seven families have been published previously, and the data of the remaining 21 families are presented...... together with an overview of the results in all families. METHODS: A combined screening approach of linkage analysis and sequencing of 17 cataract genes were applied to mutation analyses of total 28 families. RESULTS: The study revealed a disease locus in seven of eight families that were amenable...

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

    Directory of Open Access Journals (Sweden)

    Mariana Maschietto

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

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

    Science.gov (United States)

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

    2014-01-01

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

  13. Fitness loss and germline mutations in barn swallows breeding in Chernobyl

    Energy Technology Data Exchange (ETDEWEB)

    Ellegren, Hans; Lindgren, Gabriella; Primmer, C.R. [Swedish Univ. of Agricultural Sciences, Animal Breeding and Genetics Dept., Uppsala (Sweden); Moeller, A.P. [Universite Pierre et Marie Curie. Lab. d`Ecologie, Paris, 75 (France)

    1997-10-09

    The severe nuclear accident at Chernobyl in 1986 resulted in the worst reported accidental exposure of radioactive material to free-living organisms. Short-term effects on human populations inhabiting polluted areas include increased incidence of thyroid cancer, infant leukaemia, and congenital malformations in newborns. Two recent studies have reported, although with some controversy, that germline mutation rates were increased in humans and voles living close to Chernobyl, but little is known about the viability of the organisms affected. Here we report an increased frequency of partial albinism, a morphological aberration associated with a loss of fitness, among barn swallows, Hirundo rustica, breeding close to Chernobyl. Heretability estimates indicate that mutations causing albinism were at least partly of germline origin. Furthermore, evidence for an increased germline mutation rate was obtained from segregation analysis at two hypervariable microsatellite loci, indicating that mutation events in barn swallows from Chernobyl were two- to tenfold higher than in birds from control areas in Ukraine and Italy. (author).

  14. Fitness loss and germline mutations in barn swallows breeding in Chernobyl

    International Nuclear Information System (INIS)

    Ellegren, Hans; Lindgren, Gabriella; Primmer, C.R.; Moeller, A.P.

    1997-01-01

    The severe nuclear accident at Chernobyl in 1986 resulted in the worst reported accidental exposure of radioactive material to free-living organisms. Short-term effects on human populations inhabiting polluted areas include increased incidence of thyroid cancer, infant leukaemia, and congenital malformations in newborns. Two recent studies have reported, although with some controversy, that germline mutation rates were increased in humans and voles living close to Chernobyl, but little is known about the viability of the organisms affected. Here we report an increased frequency of partial albinism, a morphological aberration associated with a loss of fitness, among barn swallows, Hirundo rustica, breeding close to Chernobyl. Heretability estimates indicate that mutations causing albinism were at least partly of germline origin. Furthermore, evidence for an increased germline mutation rate was obtained from segregation analysis at two hypervariable microsatellite loci, indicating that mutation events in barn swallows from Chernobyl were two- to tenfold higher than in birds from control areas in Ukraine and Italy. (author)

  15. The prognostic value of p53 mutation in pediatric marrow hypoplasia

    Directory of Open Access Journals (Sweden)

    Sharaf Alzahraa EA

    2011-06-01

    Full Text Available Abstract Background The tumor suppressor gene p53 is involved in the control of cell proliferation, particularly in stressed cells. p 53 gene mutations are the most frequent genetic event found in human cancers. Fanconi Anemia (FA is the most common representative of inherited bone marrow failure syndromes (IBMFS with a leukemic propensity. P 53 DNA alteration has not been studied before in Egyptian children with FA. Patients and methods we investigated p53 mutation in the bone marrow and peripheral blood of forty children, FA (n = 10, acquired aplastic anemia (AAA (n = 10, and immune thrombocytopenia (ITP as a control (n = 20, using real-time PCR by TaqMan probe assay Results Mutation of p53 gene was demonstrated in the BM of 90% (9/10 of children with FA, compared to 10% (1/10 in AAA (p Conclusion mutation of p53 gene in hypoplastic marrow especially FA may represent an early indicator of significant DNA genetic alteration with cancer propensity.

  16. Reversal or protection by light of the ethidium bromide induced petite mutation in yeast

    International Nuclear Information System (INIS)

    Hixon, S.C.; Burnham, A.D.; Irons, R.L.

    1979-01-01

    An intermediate in the ethidium bromide (EB) induced petite mutation pathway may be destabilized by daylight light to cause a reversion to the normal grande phenotype. Starved cells preincubated in the dark for up to 6 h with 100μg/ml EB could be reverted to grandes after one hour of light exposure, whereas similarly treated cells maintained in the dark expresse the petite mutation in more than 80 percent of the population. In addition, the production of petite mutants by EB in buffer could be prevented if cell suspensions were exposed to light immediately upon the addition of EB. Photoreversal of the EB-derived petite mutation in growing cells as less efficient presumably because the availability of an energy source caused a continuation of mutation events beyond the light revertible step to a non-reversible fixation of the mutation. Cells treated with EB in growth and reversal of the mutation. This may be due to the cold inhibition of an enzyme which comes into play beyond the light sensitive step in the mutation pathway. (orig.) [de

  17. 'In vivo' and high resolution spectroscopy in solids by NMR: an instrument for transgenic plants study

    International Nuclear Information System (INIS)

    Colnago, L.A.; Herrmann, P.S.P.; Bernardes Filho, R.

    1995-01-01

    This work has developed a study on transgenic plants using two different techniques of nuclear magnetic resonance, in vivo NMR and high resolution NMR. In order to understand the gene mutations and characterize the plants constituents, NMR spectral data were analysed and discussed, then the results were presented

  18. Herpes Simplex Virus 1 Mutant with Point Mutations in UL39 Is Impaired for Acute Viral Replication in Mice, Establishment of Latency, and Explant-Induced Reactivation.

    Science.gov (United States)

    Mostafa, Heba H; Thompson, Thornton W; Konen, Adam J; Haenchen, Steve D; Hilliard, Joshua G; Macdonald, Stuart J; Morrison, Lynda A; Davido, David J

    2018-04-01

    greater detail the events that modulate HSV-1 replication and pathogenesis. In the current study, we identified a neuroattenuated HSV-1 mutant (i.e., KOS-NA) that contains novel mutations in the UL39 gene, which codes for the large subunit of ribonucleotide reductase (also known as ICP6). This mutant form of ICP6 was responsible for the attenuation of KOS-NA in vivo and resulted in diminished ICP6 protein levels and antiapoptotic effect. Thus, we have determined that subtle alteration of the UL39 gene regulates expression and functions of ICP6 and severely impacts HSV-1 pathogenesis, potentially making KOS-NA a promising vaccine candidate against HSV-1. Copyright © 2018 American Society for Microbiology.

  19. Mutations in PIK3CA are infrequent in neuroblastoma

    International Nuclear Information System (INIS)

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

    2006-01-01

    mutations in the Ras/Raf-MAPK/PI3K signaling cascades occur infrequently in neuroblastoma. Further, despite compelling evidence for MYC and RAS cooperation in vitro and in vivo to promote tumourigenesis, activation of RAS signal transduction does not constitute a preferred secondary pathway in neuroblastomas with MYCN deregulation in either human tumors or murine models

  20. Assessment of the in vitro and in vivo genotoxicity of extracts and indole monoterpene alkaloid from the roots of Galianthe thalictroides (Rubiaceae).

    Science.gov (United States)

    Fernandes, L M; Garcez, W S; Mantovani, M S; Figueiredo, P O; Fernandes, C A; Garcez, F R; Guterres, Z R

    2013-09-01

    Roots of Galianthe thalictroides K. Schum. (Rubiaceae) are used in folk medicine in the State of Mato Grosso do Sul, Brazil, for treating and preventing cancer. To gain information about the genotoxicity of extracts (aqueous and EtOH), the CHCl₃ phase resulting from partition of the EtOH extract and the indole monoterpene alkaloid 1 obtained from this plant. The genotoxicity of 1 and extracts was evaluated in vivo through the Drosophila melanogaster wing Somatic Mutation and Recombination Test - SMART, while in vitro cytotoxic (MTT) and Comet assays were performed only with alkaloid 1. The results obtained with the SMART test indicated that the aqueous extract had no genotoxic activity. The EtOH extract was not genotoxic to ST descendants but genotoxic to HB ones. The CHCl₃ phase was genotoxic and cytotoxic. Alkaloid 1 showed significant mutational events with SMART, in the cytotoxicity assay (MTT), it showed a high cytotoxicity for human hepatoma cells (HepG2), whereas for the Comet assay, not showing genotoxic activity. The ethanol extract was shown to be genotoxic to HB descendants in the SMART assay, while the results obtained in this test for the monoterpene indole alkaloid 1 isolated from this extract. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Activating mutations in FGFR3 and HRAS reveal a shared genetic origin for congenital disorders and testicular tumors

    DEFF Research Database (Denmark)

    Goriely, Anne; Hansen, Ruth M S; Taylor, Indira B

    2009-01-01

    Genes mutated in congenital malformation syndromes are frequently implicated in oncogenesis, but the causative germline and somatic mutations occur in separate cells at different times of an organism's life. Here we unify these processes to a single cellular event for mutations arising in male germ...... cells that show a paternal age effect. Screening of 30 spermatocytic seminomas for oncogenic mutations in 17 genes identified 2 mutations in FGFR3 (both 1948A>G, encoding K650E, which causes thanatophoric dysplasia in the germline) and 5 mutations in HRAS. Massively parallel sequencing of sperm DNA...... a common 'selfish' pathway supporting proliferation in the testis, leading to diverse phenotypes in the next generation including fetal lethality, congenital syndromes and cancer predisposition....

  2. Thyroid hyperfunctioning adenomas with and without Gsp/TSH receptor mutations show similar clinical features.

    Science.gov (United States)

    Arturi, F; Capula, C; Chiefari, E; Filetti, S; Russo, D

    1998-01-01

    Activating mutations of Gs alpha protein (gsp) and TSH receptor (TSH-R) identified in autonomously hyperfunctioning thyroid adenomas have been proposed as the primary event responsible for this disease. Since mutations have not been detected in 100% (ranging from less than 10% to 90%) of the patients, we evaluated whether the presence of gsp and TSH-R mutations cause differences in the clinical and biochemical parameters of the affected patients. Fifteen consecutive patients (11 women and 4 men) with autonomously hyperfunctioning thyroid adenomas who underwent thyroidectomy, previously examined for the presence of gsp or TSH-R mutations, were investigated. In all of the patients we examined plasma free T3, free T4, TSH levels and ultrasound volume of the nodules. The patients with mutations in gsp or TSH-R were similar to the patients without mutations for clinical presentation, sex distribution and mean age. Furthermore, basal serum FT3, TSH and tumor volume in the patients with mutations were not significantly different from the group without mutations. Our preliminary data demonstrate that no significant differences are present in the two groups of patients examined, suggesting that factors other than gsp or TSH-R mutations play a role in the clinical presentation of the disease.

  3. Acquired copy-neutral loss of heterozygosity of chromosome 1p as a molecular event associated with marrow fibrosis in MPL-mutated myeloproliferative neoplasms.

    Science.gov (United States)

    Rumi, Elisa; Pietra, Daniela; Guglielmelli, Paola; Bordoni, Roberta; Casetti, Ilaria; Milanesi, Chiara; Sant'Antonio, Emanuela; Ferretti, Virginia; Pancrazzi, Alessandro; Rotunno, Giada; Severgnini, Marco; Pietrelli, Alessandro; Astori, Cesare; Fugazza, Elena; Pascutto, Cristiana; Boveri, Emanuela; Passamonti, Francesco; De Bellis, Gianluca; Vannucchi, Alessandro; Cazzola, Mario

    2013-05-23

    We studied mutations of MPL exon 10 in patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF), first investigating a cohort of 892 consecutive patients. MPL mutation scanning was performed on granulocyte genomic DNA by using a high-resolution melt assay, and the mutant allele burden was evaluated by using deep sequencing. Somatic mutations of MPL, all but one involving codon W515, were detected in 26/661 (4%) patients with ET, 10/187 (5%) with PMF, and 7/44 (16%) patients with post-ET myelofibrosis. Comparison of JAK2 (V617F)-mutated and MPL-mutated patients showed only minor phenotypic differences. In an extended group of 62 MPL-mutated patients, the granulocyte mutant allele burden ranged from 1% to 95% and was significantly higher in patients with PMF or post-ET myelofibrosis compared with those with ET. Patients with higher mutation burdens had evidence of acquired copy-neutral loss of heterozygosity (CN-LOH) of chromosome 1p in granulocytes, consistent with a transition from heterozygosity to homozygosity for the MPL mutation in clonal cells. A significant association was found between MPL-mutant allele burden greater than 50% and marrow fibrosis. These observations suggest that acquired CN-LOH of chromosome 1p involving the MPL location may represent a molecular mechanism of fibrotic transformation in MPL-mutated myeloproliferative neoplasms.

  4. Live cell imaging unveils multiple domain requirements for in vivo dimerization of the glucocorticoid receptor.

    Directory of Open Access Journals (Sweden)

    Diego M Presman

    2014-03-01

    Full Text Available Glucocorticoids are essential for life, but are also implicated in disease pathogenesis and may produce unwanted effects when given in high doses. Glucocorticoid receptor (GR transcriptional activity and clinical outcome have been linked to its oligomerization state. Although a point mutation within the GR DNA-binding domain (GRdim mutant has been reported as crucial for receptor dimerization and DNA binding, this assumption has recently been challenged. Here we have analyzed the GR oligomerization state in vivo using the number and brightness assay. Our results suggest a complete, reversible, and DNA-independent ligand-induced model for GR dimerization. We demonstrate that the GRdim forms dimers in vivo whereas adding another mutation in the ligand-binding domain (I634A severely compromises homodimer formation. Contrary to dogma, no correlation between the GR monomeric/dimeric state and transcriptional activity was observed. Finally, the state of dimerization affected DNA binding only to a subset of GR binding sites. These results have major implications on future searches for therapeutic glucocorticoids with reduced side effects.

  5. BRF1 mutations alter RNA polymerase III–dependent transcription and cause neurodevelopmental anomalies

    Science.gov (United States)

    Hög, Friederike; Dentici, Maria Lisa; Tan, Perciliz L.; Sowada, Nadine; Medeira, Ana; Gueneau, Lucie; Thiele, Holger; Kousi, Maria; Lepri, Francesca; Wenzeck, Larissa; Blumenthal, Ian; Radicioni, Antonio; Schwarzenberg, Tito Livio; Mandriani, Barbara; Fischetto, Rita; Morris-Rosendahl, Deborah J.; Altmüller, Janine; Reymond, Alexandre; Nürnberg, Peter; Merla, Giuseppe; Dallapiccola, Bruno; Katsanis, Nicholas; Cramer, Patrick; Kubisch, Christian

    2015-01-01

    RNA polymerase III (Pol III) synthesizes tRNAs and other small noncoding RNAs to regulate protein synthesis. Dysregulation of Pol III transcription has been linked to cancer, and germline mutations in genes encoding Pol III subunits or tRNA processing factors cause neurogenetic disorders in humans, such as hypomyelinating leukodystrophies and pontocerebellar hypoplasia. Here we describe an autosomal recessive disorder characterized by cerebellar hypoplasia and intellectual disability, as well as facial dysmorphic features, short stature, microcephaly, and dental anomalies. Whole-exome sequencing revealed biallelic missense alterations of BRF1 in three families. In support of the pathogenic potential of the discovered alleles, suppression or CRISPR-mediated deletion of brf1 in zebrafish embryos recapitulated key neurodevelopmental phenotypes; in vivo complementation showed all four candidate mutations to be pathogenic in an apparent isoform-specific context. BRF1 associates with BDP1 and TBP to form the transcription factor IIIB (TFIIIB), which recruits Pol III to target genes. We show that disease-causing mutations reduce Brf1 occupancy at tRNA target genes in Saccharomyces cerevisiae and impair cell growth. Moreover, BRF1 mutations reduce Pol III–related transcription activity in vitro. Taken together, our data show that BRF1 mutations that reduce protein activity cause neurodevelopmental anomalies, suggesting that BRF1-mediated Pol III transcription is required for normal cerebellar and cognitive development. PMID:25561519

  6. Somatic Mutational Landscape of Splicing Factor Genes and Their Functional Consequences across 33 Cancer Types

    Directory of Open Access Journals (Sweden)

    Michael Seiler

    2018-04-01

    Full Text Available Summary: Hotspot mutations in splicing factor genes have been recently reported at high frequency in hematological malignancies, suggesting the importance of RNA splicing in cancer. We analyzed whole-exome sequencing data across 33 tumor types in The Cancer Genome Atlas (TCGA, and we identified 119 splicing factor genes with significant non-silent mutation patterns, including mutation over-representation, recurrent loss of function (tumor suppressor-like, or hotspot mutation profile (oncogene-like. Furthermore, RNA sequencing analysis revealed altered splicing events associated with selected splicing factor mutations. In addition, we were able to identify common gene pathway profiles associated with the presence of these mutations. Our analysis suggests that somatic alteration of genes involved in the RNA-splicing process is common in cancer and may represent an underappreciated hallmark of tumorigenesis. : Seiler et al. report that 119 splicing factor genes carry putative driver mutations over 33 tumor types in TCGA. The most common mutations appear to be mutually exclusive and are associated with lineage-independent altered splicing. Samples with these mutations show deregulation of cell-autonomous pathways and immune infiltration. Keywords: splicing, SF3B1, U2AF1, SRSF2, RBM10, FUBP1, cancer, mutation

  7. Association of KIT exon 9 mutations with nongastric primary site and aggressive behavior: KIT mutation analysis and clinical correlates of 120 gastrointestinal stromal tumors.

    Science.gov (United States)

    Antonescu, Cristina R; Sommer, Gunhild; Sarran, Lisa; Tschernyavsky, Sylvia J; Riedel, Elyn; Woodruff, James M; Robson, Mark; Maki, Robert; Brennan, Murray F; Ladanyi, Marc; DeMatteo, Ronald P; Besmer, Peter

    2003-08-15

    Activating mutations of the KIT juxtamembrane region are the most common genetic events in gastrointestinal stromal tumors (GISTs) and have been noted as independent prognostic factors. The impact of KIT mutation in other regions, such as the extracellular or kinase domains, is not well-defined and fewer than 30 cases have been published to date. One hundred twenty GISTs, confirmed by KIT immunoreactivity, were evaluated for the presence of KIT exon 9, 11, 13, and 17 mutations. The relation between the presence/type of KIT mutation and clinicopathological factors was analyzed using Fisher's exact test and log-rank test. Forty-four % of the tumors were located in the stomach, 47% in the small bowel, 6% in the rectum, and 3% in the retroperitoneum. Overall, KIT mutations were detected in 78% of patients as follows: 67% in exon 11, 11% in exon 9, and none in exon 13 or 17. The types of KIT exon 11 mutations were heterogeneous and clustered in the classic "hot spot" at the 5' end of exon 11. Seven % of cases showed internal tandem duplications (ITD) at the 3' end of exon 11, in a region that we designate as a second hot spot for KIT mutations. Interestingly, these cases were associated with: female predominance, stomach location, occurrence in older patients, and favorable outcome. There were significant associations between exon 9 mutations and large tumor size (P < 0.001) and extragastric location (P = 0.02). Ten of these 13 patients with more than 1-year follow-up have developed recurrent disease. Most KIT-expressing GISTs show KIT mutations that are preferentially located within the classic hot spot of exon 11. In addition, we found an association between a second hot spot at the 3'end of exon 11, characterized by ITDs, and a subgroup of clinically indolent gastric GISTs in older females. KIT exon 9 mutations seem to define a distinct subset of GISTs, located predominantly in the small bowel and associated with an unfavorable clinical course.

  8. Rates of Mutation and Host Transmission for an Escherichia coli Clone over 3 Years

    Science.gov (United States)

    Reeves, Peter R.; Liu, Bin; Zhou, Zhemin; Li, Dan; Guo, Dan; Ren, Yan; Clabots, Connie; Lan, Ruiting; Johnson, James R.; Wang, Lei

    2011-01-01

    Although over 50 complete Escherichia coli/Shigella genome sequences are available, it is only for closely related strains, for example the O55:H7 and O157:H7 clones of E. coli, that we can assign differences to individual evolutionary events along specific lineages. Here we sequence the genomes of 14 isolates of a uropathogenic E. coli clone that persisted for 3 years within a household, including a dog, causing a urinary tract infection (UTI) in the dog after 2 years. The 20 mutations observed fit a single tree that allows us to estimate the mutation rate to be about 1.1 per genome per year, with minimal evidence for adaptive change, including in relation to the UTI episode. The host data also imply at least 6 host transfer events over the 3 years, with 2 lineages present over much of that period. To our knowledge, these are the first direct measurements for a clone in a well-defined host community that includes rates of mutation and host transmission. There is a concentration of non-synonymous mutations associated with 2 transfers to the dog, suggesting some selection pressure from the change of host. However, there are no changes to which we can attribute the UTI event in the dog, which suggests that this occurrence after 2 years of the clone being in the household may have been due to chance, or some unknown change in the host or environment. The ability of a UTI strain to persist for 2 years and also to transfer readily within a household has implications for epidemiology, diagnosis, and clinical intervention. PMID:22046404

  9. Factors affecting mutational specificity in mammalian cells: Informal [technical] progress report, February 1, 1987-January 31, 1988

    International Nuclear Information System (INIS)

    Strauss, B.S.

    1986-01-01

    We analyzed the different c-H-ras mutations produced in cells after treatment with chemical carcinogens. The overall goal of this work is an understanding of the changes produced by environmental mutagens and carcinogens to learn how closely the results obtained with bacterial and cellular assay systems apply to the in vivo situation

  10. To a cultural perspective of mixed reality events: a case study of event overflow in operas and concerts in mixed reality

    Science.gov (United States)

    Lucas, Jean-François; Cornish, Tracy; Margolis, Todd

    2012-12-01

    Mixed reality defines the sharing of a space-time between the real and the virtual world. The definition of this concept is further extended when virtual worlds such as Second Life® (SL) are included. Through cultural events such as concerts and operas, we will see that the main goal of these kinds of projects is not simply to offer a video and audio broadcast of these events in the digital dimension. The current challenge is to create interactions between the individuals who are in different shared spaces. By studying the unfolding of these events in its various phases-before, during, and after-we examine the culture of the event. We question how the culture of the event can be transposed in a mixed reality display, and how this kind of event can affect people on both sides of the "membrane" made by the technical configuration. Beyond the alignments and adjustments that we can see between the different individuals involved in these events, we examine more broadly the changes and mutations of the culture of the event in this specific configuration.

  11. Hotspot mutations in KIT receptor differentially modulate its allosterically coupled conformational dynamics: impact on activation and drug sensitivity.

    Directory of Open Access Journals (Sweden)

    Isaure Chauvot de Beauchêne

    2014-07-01

    Full Text Available Receptor tyrosine kinase KIT controls many signal transduction pathways and represents a typical allosterically regulated protein. The mutation-induced deregulation of KIT activity impairs cellular physiological functions and causes serious human diseases. The impact of hotspots mutations (D816H/Y/N/V and V560G/D localized in crucial regulatory segments, the juxtamembrane region (JMR and the activation (A- loop, on KIT internal dynamics was systematically studied by molecular dynamics simulations. The mutational outcomes predicted in silico were correlated with in vitro and in vivo activation rates and drug sensitivities of KIT mutants. The allosteric regulation of KIT in the native and mutated forms is described in terms of communication between the two remote segments, JMR and A-loop. A strong correlation between the communication profile and the structural and dynamical features of KIT in the native and mutated forms was established. Our results provide new insight on the determinants of receptor KIT constitutive activation by mutations and resistance of KIT mutants to inhibitors. Depiction of an intra-molecular component of the communication network constitutes a first step towards an integrated description of vast communication pathways established by KIT in physiopathological contexts.

  12. The impact of KRAS mutations on VEGF-A production and tumour vascular network

    International Nuclear Information System (INIS)

    Figueras, Agnès; Arbos, Maria Antonia; Quiles, Maria Teresa; Viñals, Francesc; Germà, Josep Ramón; Capellà, Gabriel

    2013-01-01

    The malignant potential of tumour cells may be influenced by the molecular nature of KRAS mutations being codon 13 mutations less aggressive than codon 12 ones. Their metabolic profile is also different, with an increased anaerobic glycolytic metabolism in cells harbouring codon 12 KRAS mutations compared with cells containing codon 13 mutations. We hypothesized that this distinct metabolic behaviour could be associated with different HIF-1α expression and a distinct angiogenic profile. Codon13 KRAS mutation (ASP13) or codon12 KRAS mutation (CYS12) NIH3T3 transfectants were analyzed in vitro and in vivo. Expression of HIF-1α, and VEGF-A was studied at RNA and protein levels. Regulation of VEGF-A promoter activity was assessed by means of luciferase assays using different plasmid constructs. Vascular network was assessed in tumors growing after subcutaneous inoculation. Non parametric statistics were used for analysis of results. Our results show that in normoxic conditions ASP13 transfectants exhibited less HIF-1α protein levels and activity than CYS12. In contrast, codon 13 transfectants exhibited higher VEGF-A mRNA and protein levels and enhanced VEGF-A promoter activity. These differences were due to a differential activation of Sp1/AP2 transcription elements of the VEGF-A promoter associated with increased ERKs signalling in ASP13 transfectants. Subcutaneous CYS12 tumours expressed less VEGF-A and showed a higher microvessel density (MVD) than ASP13 tumours. In contrast, prominent vessels were only observed in the latter. Subtle changes in the molecular nature of KRAS oncogene activating mutations occurring in tumour cells have a major impact on the vascular strategy devised providing with new insights on the role of KRAS mutations on angiogenesis

  13. mtDNA mutation C1494T, haplogroup A, and hearing loss in Chinese

    International Nuclear Information System (INIS)

    Wang Chengye; Kong Qingpeng; Yao Yonggang; Zhang Yaping

    2006-01-01

    Mutation C1494T in mitochondrial 12S rRNA gene was recently reported in two large Chinese families with aminoglycoside-induced and nonsyndromic hearing loss (AINHL) and was claimed to be pathogenic. This mutation, however, was first reported in a sample from central China in our previous study that was aimed to reconstruct East Asian mtDNA phylogeny. All these three mtDNAs formed a subclade defined by mutation C1494T in mtDNA haplogroup A. It thus seems that mutation C1494T is a haplogroup A-associated mutation and this matrilineal background may contribute a high risk for the penetrance of mutation C1494T in Chinese with AINHL. To test this hypothesis, we first genotyped mutation C1494T in 553 unrelated individuals from three regional Chinese populations and performed an extensive search for published complete or near-complete mtDNA data sets (>3000 mtDNAs), we then screened the C1494T mutation in 111 mtDNAs with haplogroup A status that were identified from 1823 subjects across China. The search for published mtDNA data sets revealed no other mtDNA besides the above-mentioned three carrying mutation C1494T. None of the 553 randomly selected individuals and the 111 haplogroup A mtDNAs was found to bear this mutation. Therefore, our results suggest that C1494T is a very rare event. The mtDNA haplogroup A background in general is unlikely to play an active role in the penetrance of mutation C1494T in AINHL

  14. Functional analysis of propeptide as an intramolecular chaperone for in vivo folding of subtilisin nattokinase.

    Science.gov (United States)

    Jia, Yan; Liu, Hui; Bao, Wei; Weng, Meizhi; Chen, Wei; Cai, Yongjun; Zheng, Zhongliang; Zou, Guolin

    2010-12-01

    Here, we show that during in vivo folding of the precursor, the propeptide of subtilisin nattokinase functions as an intramolecular chaperone (IMC) that organises the in vivo folding of the subtilisin domain. Two residues belonging to β-strands formed by conserved regions of the IMC are crucial for the folding of the subtilisin domain through direct interactions. An identical protease can fold into different conformations in vivo due to the action of a mutated IMC, resulting in different kinetic parameters. Some interfacial changes involving conserved regions, even those induced by the subtilisin domain, blocked subtilisin folding and altered its conformation. Insight into the interaction between the subtilisin and IMC domains is provided by a three-dimensional structural model. Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  15. Mutation Spectrum and Phenotypic Features in Noonan Syndrome with PTPN11 Mutations: Definition of Two Novel Mutations.

    Science.gov (United States)

    Atik, Tahir; Aykut, Ayca; Hazan, Filiz; Onay, Huseyin; Goksen, Damla; Darcan, Sukran; Tukun, Ajlan; Ozkinay, Ferda

    2016-06-01

    To evaluate the spectrum of PTPN11 gene mutations in Noonan syndrome patients and to study the genotype-phenotype associations. In this study, twenty Noonan syndrome patients with PTPN11 mutations were included. The patients underwent a detailed clinical and physical evaluation. To identify inherited cases, parents of all mutation positive patients were analyzed. Thirteen different PTPN11 mutations, two of them being novel, were detected in the study group. These mutations included eleven missense mutations: p.G60A, p.D61N, p.Y62D, p.Y63C, p.E69Q, p.Q79R, p.Y279C,p.N308D, p.N308S, p.M504V, p.Q510R and two novel missense mutations: p.I56V and p.I282M. The frequency of cardiac abnormalities and short stature were found to be 80 % and 80 %, respectively. Mental retardation was not observed in patients having exon 8 mutations. No significant correlations were detected between other phenotypic features and genotypes. By identifying genotype-phenotype correlations, this study provides information on phenotypes observed in NS patients with different PTPN11 mutations.

  16. Mutation at codon 442 in the rpoB gene of Mycobacterium leprae does not confer resistance to rifampicin.

    Science.gov (United States)

    Lavania, Mallika; Hena, Abu; Reja, Hasanoor; Nigam, Astha; Biswas, Nibir Kumar; Singh, Itu; Turankar, Ravindra P; Gupta, Ud; Kumar, Senthil; Rewaria, Latika; Patra, Pradip K R; Sengupta, Utpal; Bhattacharya, Basudeb

    2016-03-01

    Rifampicin is the major drug in the treatment of leprosy. The rifampicin resistance of Mycobacterium leprae results from a mutation in the rpoB gene, encoding the β subunit of RNA polymerase. As M. leprae is a non-cultivable organism observation of its growth using mouse food-pad (MFP) is the only Gold Standard assay used for confirmation of "in-vivo" drug resistance. Any mutation at molecular level has to be verified by MFP assay for final confirmation of drug resistance in leprosy. In the present study, M. leprae strains showing a mutation only at codon 442 Gln-His and along with mutation either at codon 424 Val-Gly or at 438 Gln-Val within the Rifampicin Resistance Determining Region (RRDR) confirmed by DNA sequencing and by high resolution melting (HRM) analysis were subjected for its growth in MFP. The M. leprae strain having the new mutation at codon 442 Gln-His was found to be sensitive to all the three drugs and strains having additional mutations at 424 Val-Gly and 438 Gln-Val were conferring resistance with Multi drug therapy (MDT) in MFP. These results indicate that MFP is the gold standard method for confirming the mutations detected by molecular techniques.

  17. Long-term follow-up of patients with a clinically benign extrahepatic biliary stenosis and K-ras mutation in endobiliary brush cytology

    NARCIS (Netherlands)

    van Heek, N. Tjarda; Rauws, Erik A. J.; Caspers, Eric; Drillenburg, Paul; Gouma, Dirk J.; Offerhaus, G. Johan A.

    2002-01-01

    Background. K-ras mutations in endobiliary brush cytology are an early event in carcinogenesis and justify a suspicion of malignancy in patients with extrahepatic biliary stenosis. However, K-ras mutations have been detected in specimens obtained by brushing of clinically benign extrahepatic biliary

  18. Cystic fibrosis transmembrane conductance regulator intracellular processing, trafficking, and opportunities for mutation-specific treatment.

    LENUS (Irish Health Repository)

    Rogan, Mark P

    2012-02-01

    Recent advances in basic science have greatly expanded our understanding of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the chloride and bicarbonate channel that is encoded by the gene, which is mutated in patients with CF. We review the structure, function, biosynthetic processing, and intracellular trafficking of CFTR and discuss the five classes of mutations and their impact on the CF phenotype. The therapeutic discussion is focused on the significant progress toward CFTR mutation-specific therapies. We review the results of encouraging clinical trials examining orally administered therapeutics, including agents that promote read-through of class I mutations (premature termination codons); correctors, which overcome the CFTR misfolding that characterizes the common class II mutation F508del; and potentiators, which enhance the function of class III or IV mutated CFTR at the plasma membrane. Long-term outcomes from successful mutation-specific treatments could finally answer the question that has been lingering since and even before the CFTR gene discovery: Will therapies that specifically restore CFTR-mediated chloride secretion slow or arrest the deleterious cascade of events leading to chronic infection, bronchiectasis, and end-stage lung disease?

  19. In vitro and in vivo assessment of direct effects of simulated solar and galactic cosmic radiation on human hematopoietic stem/progenitor cells.

    Science.gov (United States)

    Rodman, C; Almeida-Porada, G; George, S K; Moon, J; Soker, S; Pardee, T; Beaty, M; Guida, P; Sajuthi, S P; Langefeld, C D; Walker, S J; Wilson, P F; Porada, C D

    2017-06-01

    Future deep space missions to Mars and near-Earth asteroids will expose astronauts to chronic solar energetic particles (SEP) and galactic cosmic ray (GCR) radiation, and likely one or more solar particle events (SPEs). Given the inherent radiosensitivity of hematopoietic cells and short latency period of leukemias, space radiation-induced hematopoietic damage poses a particular threat to astronauts on extended missions. We show that exposing human hematopoietic stem/progenitor cells (HSC) to extended mission-relevant doses of accelerated high-energy protons and iron ions leads to the following: (1) introduces mutations that are frequently located within genes involved in hematopoiesis and are distinct from those induced by γ-radiation; (2) markedly reduces in vitro colony formation; (3) markedly alters engraftment and lineage commitment in vivo; and (4) leads to the development, in vivo, of what appears to be T-ALL. Sequential exposure to protons and iron ions (as typically occurs in deep space) proved far more deleterious to HSC genome integrity and function than either particle species alone. Our results represent a critical step for more accurately estimating risks to the human hematopoietic system from space radiation, identifying and better defining molecular mechanisms by which space radiation impairs hematopoiesis and induces leukemogenesis, as well as for developing appropriately targeted countermeasures.

  20. Partial uniparental isodisomy of chromosome 16 unmasks a deleterious biallelic mutation in IFT140 that causes Mainzer-Saldino syndrome.

    Science.gov (United States)

    Helm, Benjamin M; Willer, Jason R; Sadeghpour, Azita; Golzio, Christelle; Crouch, Eric; Vergano, Samantha Schrier; Katsanis, Nicholas; Davis, Erica E

    2017-07-19

    The ciliopathies represent an umbrella group of >50 clinical entities that share both clinical features and molecular etiology underscored by structural and functional defects of the primary cilium. Despite the advances in gene discovery, this group of entities continues to pose a diagnostic challenge, in part due to significant genetic and phenotypic heterogeneity and variability. We consulted a pediatric case from asymptomatic, non-consanguineous parents who presented as a suspected ciliopathy due to a constellation of retinal, renal, and skeletal findings. Although clinical panel sequencing of genes implicated in nephrotic syndromes yielded no likely causal mutation, an oligo-SNP microarray identified a ~20-Mb region of homozygosity, with no altered gene dosage, on chromosome 16p13. Intersection of the proband's phenotypes with known disease genes within the homozygous region yielded a single candidate, IFT140, encoding a retrograde intraflagellar transport protein implicated previously in several ciliopathies, including the phenotypically overlapping Mainzer-Saldino syndrome (MZSDS). Sanger sequencing yielded a maternally inherited homozygous c.634G>A; p.Gly212Arg mutation altering the exon 6 splice donor site. Functional studies in cells from the proband showed that the locus produced two transcripts: a majority message containing a mis-splicing event that caused a premature termination codon and a minority message homozygous for the p.Gly212Arg allele. Zebrafish in vivo complementation studies of the latter transcript demonstrated a loss of function effect. Finally, we conducted post-hoc trio-based whole exome sequencing studies to (a) test the possibility of other causal loci in the proband and (b) explain the Mendelian error of segregation for the IFT140 mutation. We show that the proband harbors a chromosome 16 maternal heterodisomy, with segmental isodisomy at 16p13, likely due to a meiosis I error in the maternal gamete. Using clinical phenotyping

  1. The industrial melanism mutation in British peppered moths is a transposable element.

    Science.gov (United States)

    Van't Hof, Arjen E; Campagne, Pascal; Rigden, Daniel J; Yung, Carl J; Lingley, Jessica; Quail, Michael A; Hall, Neil; Darby, Alistair C; Saccheri, Ilik J

    2016-06-02

    Discovering the mutational events that fuel adaptation to environmental change remains an important challenge for evolutionary biology. The classroom example of a visible evolutionary response is industrial melanism in the peppered moth (Biston betularia): the replacement, during the Industrial Revolution, of the common pale typica form by a previously unknown black (carbonaria) form, driven by the interaction between bird predation and coal pollution. The carbonaria locus has been coarsely localized to a 200-kilobase region, but the specific identity and nature of the sequence difference controlling the carbonaria-typica polymorphism, and the gene it influences, are unknown. Here we show that the mutation event giving rise to industrial melanism in Britain was the insertion of a large, tandemly repeated, transposable element into the first intron of the gene cortex. Statistical inference based on the distribution of recombined carbonaria haplotypes indicates that this transposition event occurred around 1819, consistent with the historical record. We have begun to dissect the mode of action of the carbonaria transposable element by showing that it increases the abundance of a cortex transcript, the protein product of which plays an important role in cell-cycle regulation, during early wing disc development. Our findings fill a substantial knowledge gap in the iconic example of microevolutionary change, adding a further layer of insight into the mechanism of adaptation in response to natural selection. The discovery that the mutation itself is a transposable element will stimulate further debate about the importance of 'jumping genes' as a source of major phenotypic novelty.

  2. Cellular and molecular events leading to the development of skin cancer

    International Nuclear Information System (INIS)

    Melnikova, Vladislava O.; Ananthaswamy, Honnavara N.

    2005-01-01

    The transition from a normal cell to a neoplastic cell is a complex process and involves both genetic and epigenetic changes. The process of carcinogenesis begins when the DNA is damaged, which then leads to a cascade of events leading to the development of a tumor. Ultraviolet (UV) radiation causes DNA damage, inflammation, erythema, sunburn, immunosuppression, photoaging, gene mutations, and skin cancer. Upon DNA damage, the p53 tumor suppressor protein undergoes phosphorylation and translocation to the nucleus and aids in DNA repair or causes apoptosis. Excessive UV exposure overwhelms DNA repair mechanisms leading to induction of p53 mutations and loss of Fas-FasL interaction. Keratinocytes carrying p53 mutations acquire a growth advantage by virtue of their increased resistance to apoptosis. Thus, resistance to cell death is a key event in photocarcinogenesis and conversely, elimination of cells containing excessive UV-induced DNA damage is a key step in protecting against skin cancer development. Apoptosis-resistant keratinocytes undergo clonal expansion that eventually leads to formation of actinic keratoses and squamous cell carcinomas. In this article, we will review some of the cellular and molecular mechanisms involved in initiation and progression of UV-induced skin cancer

  3. Cellular and molecular events leading to the development of skin cancer

    Energy Technology Data Exchange (ETDEWEB)

    Melnikova, Vladislava O. [Department of Immunology, University of Texas M.D. Anderson Cancer Center, P.O. Box 301402, Unit 902, Houston, TX 77030 (United States); Ananthaswamy, Honnavara N. [Department of Immunology, University of Texas M.D. Anderson Cancer Center, P.O. Box 301402, Unit 902, Houston, TX 77030 (United States)]. E-mail: hanantha@mdanderson.org

    2005-04-01

    The transition from a normal cell to a neoplastic cell is a complex process and involves both genetic and epigenetic changes. The process of carcinogenesis begins when the DNA is damaged, which then leads to a cascade of events leading to the development of a tumor. Ultraviolet (UV) radiation causes DNA damage, inflammation, erythema, sunburn, immunosuppression, photoaging, gene mutations, and skin cancer. Upon DNA damage, the p53 tumor suppressor protein undergoes phosphorylation and translocation to the nucleus and aids in DNA repair or causes apoptosis. Excessive UV exposure overwhelms DNA repair mechanisms leading to induction of p53 mutations and loss of Fas-FasL interaction. Keratinocytes carrying p53 mutations acquire a growth advantage by virtue of their increased resistance to apoptosis. Thus, resistance to cell death is a key event in photocarcinogenesis and conversely, elimination of cells containing excessive UV-induced DNA damage is a key step in protecting against skin cancer development. Apoptosis-resistant keratinocytes undergo clonal expansion that eventually leads to formation of actinic keratoses and squamous cell carcinomas. In this article, we will review some of the cellular and molecular mechanisms involved in initiation and progression of UV-induced skin cancer.

  4. Toxicological Evaluation of Low Molecular Weight Fucoidan in Vitro and in Vivo

    Directory of Open Access Journals (Sweden)

    Pai-An Hwang

    2016-06-01

    Full Text Available For a long time, fucoidan has been well known for its pharmacological activities, and recently low molecular weight fucoidan (LMF has been used in food supplements and pharmaceutical products. In the present study, LMF was extracted from Laminaria japonica by enzyme hydrolysis. The toxicity of LMF in mouse and rat models was determined by many methods, such as total arsenic content, bacterial reverse mutation assay, chromosome aberration assay, and in vivo micronucleus assay. The present findings showed that LMF at 5000 μg/mL exhibited no mutagenicity. It also produced no formatting disruption of red blood cells in vivo. At 2000 mg/kg BW/day there were no toxicological indications. LMF is expected to be used as a safe food supplement.

  5. Disease-associated mutations identify a novel region in human STING necessary for the control of type I interferon signaling.

    Science.gov (United States)

    Melki, Isabelle; Rose, Yoann; Uggenti, Carolina; Van Eyck, Lien; Frémond, Marie-Louise; Kitabayashi, Naoki; Rice, Gillian I; Jenkinson, Emma M; Boulai, Anaïs; Jeremiah, Nadia; Gattorno, Marco; Volpi, Sefano; Sacco, Olivero; Terheggen-Lagro, Suzanne W J; Tiddens, Harm A W M; Meyts, Isabelle; Morren, Marie-Anne; De Haes, Petra; Wouters, Carine; Legius, Eric; Corveleyn, Anniek; Rieux-Laucat, Frederic; Bodemer, Christine; Callebaut, Isabelle; Rodero, Mathieu P; Crow, Yanick J

    2017-08-01

    Gain-of-function mutations in transmembrane protein 173 (TMEM173) encoding stimulator of interferon genes (STING) underlie a recently described type I interferonopathy called STING-associated vasculopathy with onset in infancy (SAVI). We sought to define the molecular and cellular pathology relating to 3 individuals variably exhibiting the core features of the SAVI phenotype including systemic inflammation, destructive skin lesions, and interstitial lung disease. Genetic analysis, conformational studies, in vitro assays and ex vivo flow-cytometry were performed. Molecular and in vitro data demonstrate that the pathology in these patients is due to amino acid substitutions at positions 206, 281, and 284 of the human STING protein. These mutations confer cGAMP-independent constitutive activation of type I interferon signaling through TBK1 (TANK-binding kinase), independent from the alternative STING pathway triggered by membrane fusion of enveloped RNA viruses. This constitutive activation was abrogated by ex vivo treatment with the janus kinase 1/2 inhibitor ruxolitinib. Structural analysis indicates that the 3 disease-associated mutations at positions 206, 281, and 284 of the STING protein define a novel cluster of amino acids with functional importance in the regulation of type I interferon signaling. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  6. Sun exposure causes somatic second-hit mutations and angiofibroma development in tuberous sclerosis complex

    Science.gov (United States)

    Tyburczy, Magdalena E.; Wang, Ji-an; Li, Shaowei; Thangapazham, Rajesh; Chekaluk, Yvonne; Moss, Joel; Kwiatkowski, David J.; Darling, Thomas N.

    2014-01-01

    Tuberous sclerosis complex (TSC) is characterized by the formation of tumors in multiple organs and is caused by germline mutation in one of two tumor suppressor genes, TSC1 and TSC2. As for other tumor suppressor gene syndromes, the mechanism of somatic second-hit events in TSC tumors is unknown. We grew fibroblast-like cells from 29 TSC skin tumors from 22 TSC subjects and identified germline and second-hit mutations in TSC1/TSC2 using next-generation sequencing. Eighteen of 22 (82%) subjects had a mutation identified, and 8 of the 18 (44%) subjects were mosaic with mutant allele frequencies of 0 to 19% in normal tissue DNA. Multiple tumors were available from four patients, and in each case, second-hit mutations in TSC2 were distinct indicating they arose independently. Most remarkably, 7 (50%) of the 14 somatic point mutations were CC>TT ultraviolet ‘signature’ mutations, never seen as a TSC germline mutation. These occurred exclusively in facial angiofibroma tumors from sun-exposed sites. These results implicate UV-induced DNA damage as a cause of second-hit mutations and development of TSC facial angiofibromas and suggest that measures to limit UV exposure in TSC children and adults should reduce the frequency and severity of these lesions. PMID:24271014

  7. The myopathy-causing mutation DNM2-S619L leads to defective tubulation in vitro and in developing zebrafish

    Directory of Open Access Journals (Sweden)

    Elizabeth M. Gibbs

    2014-01-01

    Full Text Available DNM2 is a ubiquitously expressed GTPase that regulates multiple subcellular processes. Mutations in DNM2 are a common cause of centronuclear myopathy, a severe disorder characterized by altered skeletal muscle structure and function. The precise mechanisms underlying disease-associated DNM2 mutations are unresolved. We examined the common DNM2-S619L mutation using both in vitro and in vivo approaches. Expression of DNM2-S619L in zebrafish led to the accumulation of aberrant vesicular structures and to defective excitation-contraction coupling. Expression of DNM2-S619L in COS7 cells resulted in defective BIN1-dependent tubule formation. These data suggest that DNM2-S619L causes disease, in part, by interfering with membrane tubulation.

  8. MPL mutation profile in JAK2 mutation-negative patients with myeloproliferative disorders.

    Science.gov (United States)

    Ma, Wanlong; Zhang, Xi; Wang, Xiuqiang; Zhang, Zhong; Yeh, Chen-Hsiung; Uyeji, Jennifer; Albitar, Maher

    2011-03-01

    Mutations in the thrombopoietin receptor gene (myeloproliferative leukemia, MPL) have been reported in patients with JAK2 V617F-negative chronic myeloproliferative disorders (MPDs). We evaluated the prevalence of MPL mutations relative to JAK2 mutations in patients with suspected MPDs. A total of 2790 patient samples submitted for JAK2 mutation analysis were tested using real-time polymerase chain reaction and bidirectional sequencing of plasma RNA. JAK2 V617F-negative samples were tested for JAK2 exons 12 to 14 mutations, and those with negative results were then tested for mutations in MPL exons 10 and 11. Of the 2790 patients, 529 (18.96%) had V617F, 12 (0.43%) had small insertions or deletions in exon 12, and 7 (0.25%) had other JAK2 mutations in exons 12 to 14. Of the 2242 JAK2 mutation-negative patients, 68 (3.03%) had MPL mutations. W515L was the predominant MPL mutation (n=46; 68%), and 10 (15%) patients had other W515 variants. The remaining MPL mutations (n=12, 17%) were detected at other locations in exons 10 and 11 and included 3 insertion/deletion mutations. The S505N mutation, associated with familial MPD, was detected in 3 patients. Overall, for every 100 V617F mutations in patients with suspected MPDs, there were 12.9 MPL mutations, 2.3 JAK2 exon 12 mutations, and 1.3 JAK2 exons 13 to 14 mutations. These findings suggest that MPL mutation screening should be performed before JAK2 exons 12 to 14 testing in JAK2 V617F-negative patients with suspected MPDs.

  9. Mutations in LRRC50 predispose zebrafish and humans to seminomas.

    Directory of Open Access Journals (Sweden)

    Sander G Basten

    2013-04-01

    Full Text Available Seminoma is a subclass of human testicular germ cell tumors (TGCT, the most frequently observed cancer in young men with a rising incidence. Here we describe the identification of a novel gene predisposing specifically to seminoma formation in a vertebrate model organism. Zebrafish carrying a heterozygous nonsense mutation in Leucine-Rich Repeat Containing protein 50 (lrrc50 also called dnaaf1, associated previously with ciliary function, are found to be highly susceptible to the formation of seminomas. Genotyping of these zebrafish tumors shows loss of heterozygosity (LOH of the wild-type lrrc50 allele in 44.4% of tumor samples, correlating with tumor progression. In humans we identified heterozygous germline LRRC50 mutations in two different pedigrees with a family history of seminomas, resulting in a nonsense Arg488* change and a missense Thr590Met change, which show reduced expression of the wild-type allele in seminomas. Zebrafish in vivo complementation studies indicate the Thr590Met to be a loss-of-function mutation. Moreover, we show that a pathogenic Gln307Glu change is significantly enriched in individuals with seminoma tumors (13% of our cohort. Together, our study introduces an animal model for seminoma and suggests LRRC50 to be a novel tumor suppressor implicated in human seminoma pathogenesis.

  10. Frequent occurrence of mitochondrial DNA mutations in Barrett's metaplasia without the presence of dysplasia.

    Directory of Open Access Journals (Sweden)

    Soong Lee

    Full Text Available BACKGROUND: Barrett's esophagus (BE is one of the most common premalignant lesions and can progress to esophageal adenocarcinoma (EA. The numerous molecular events may play a role in the neoplastic transformation of Barrett's mucosa such as the change of DNA ploidy, p53 mutation and alteration of adhesion molecules. However, the molecular mechanism of the progression of BE to EA remains unclear and most studies of mitochondrial DNA (mtDNA mutations in BE have performed on BE with the presence of dysplasia. METHODS/FINDINGS: Thus, the current study is to investigate new molecular events (Barrett's esophageal tissue-specific-mtDNA alterations/instabilities in mitochondrial genome and causative factors for their alterations using the corresponding adjacent normal mucosal tissue (NT and tissue (BT from 34 patients having Barrett's metaplasia without the presence of dysplasia. Eighteen patients (53% exhibited mtDNA mutations which were not found in adjacent NT. mtDNA copy number was about 3 times higher in BT than in adjacent NT. The activity of the mitochondrial respiratory chain enzyme complexes in tissues from Barrett's metaplasia without the presence of dysplasia was impaired. Reactive oxygen species (ROS level in BT was significantly higher than those in corresponding samples. CONCLUSION/SIGNIFICANCE: High ROS level in BT may contribute to the development of mtDNA mutations, which may play a crucial role in disease progression and tumorigenesis in BE.

  11. Comprehensive mutational profiling of core binding factor acute myeloid leukemia.

    Science.gov (United States)

    Duployez, Nicolas; Marceau-Renaut, Alice; Boissel, Nicolas; Petit, Arnaud; Bucci, Maxime; Geffroy, Sandrine; Lapillonne, Hélène; Renneville, Aline; Ragu, Christine; Figeac, Martin; Celli-Lebras, Karine; Lacombe, Catherine; Micol, Jean-Baptiste; Abdel-Wahab, Omar; Cornillet, Pascale; Ifrah, Norbert; Dombret, Hervé; Leverger, Guy; Jourdan, Eric; Preudhomme, Claude

    2016-05-19

    Acute myeloid leukemia (AML) with t(8;21) or inv(16) have been recognized as unique entities within AML and are usually reported together as core binding factor AML (CBF-AML). However, there is considerable clinical and biological heterogeneity within this group of diseases, and relapse incidence reaches up to 40%. Moreover, translocations involving CBFs are not sufficient to induce AML on its own and the full spectrum of mutations coexisting with CBF translocations has not been elucidated. To address these issues, we performed extensive mutational analysis by high-throughput sequencing in 215 patients with CBF-AML enrolled in the Phase 3 Trial of Systematic Versus Response-adapted Timed-Sequential Induction in Patients With Core Binding Factor Acute Myeloid Leukemia and Treating Patients with Childhood Acute Myeloid Leukemia with Interleukin-2 trials (age, 1-60 years). Mutations in genes activating tyrosine kinase signaling (including KIT, N/KRAS, and FLT3) were frequent in both subtypes of CBF-AML. In contrast, mutations in genes that regulate chromatin conformation or encode members of the cohesin complex were observed with high frequencies in t(8;21) AML (42% and 18%, respectively), whereas they were nearly absent in inv(16) AML. High KIT mutant allele ratios defined a group of t(8;21) AML patients with poor prognosis, whereas high N/KRAS mutant allele ratios were associated with the lack of KIT or FLT3 mutations and a favorable outcome. In addition, mutations in epigenetic modifying or cohesin genes were associated with a poor prognosis in patients with tyrosine kinase pathway mutations, suggesting synergic cooperation between these events. These data suggest that diverse cooperating mutations may influence CBF-AML pathophysiology as well as clinical behavior and point to potential unique pathogenesis of t(8;21) vs inv(16) AML. © 2016 by The American Society of Hematology.

  12. Mediator independently orchestrates multiple steps of preinitiation complex assembly in vivo.

    Science.gov (United States)

    Eyboulet, Fanny; Wydau-Dematteis, Sandra; Eychenne, Thomas; Alibert, Olivier; Neil, Helen; Boschiero, Claire; Nevers, Marie-Claire; Volland, Hervé; Cornu, David; Redeker, Virginie; Werner, Michel; Soutourina, Julie

    2015-10-30

    Mediator is a large multiprotein complex conserved in all eukaryotes, which has a crucial coregulator function in transcription by RNA polymerase II (Pol II). However, the molecular mechanisms of its action in vivo remain to be understood. Med17 is an essential and central component of the Mediator head module. In this work, we utilised our large collection of conditional temperature-sensitive med17 mutants to investigate Mediator's role in coordinating preinitiation complex (PIC) formation in vivo at the genome level after a transfer to a non-permissive temperature for 45 minutes. The effect of a yeast mutation proposed to be equivalent to the human Med17-L371P responsible for infantile cerebral atrophy was also analyzed. The ChIP-seq results demonstrate that med17 mutations differentially affected the global presence of several PIC components including Mediator, TBP, TFIIH modules and Pol II. Our data show that Mediator stabilizes TFIIK kinase and TFIIH core modules independently, suggesting that the recruitment or the stability of TFIIH modules is regulated independently on yeast genome. We demonstrate that Mediator selectively contributes to TBP recruitment or stabilization to chromatin. This study provides an extensive genome-wide view of Mediator's role in PIC formation, suggesting that Mediator coordinates multiple steps of a PIC assembly pathway. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. A single mutation in the envelope protein modulates flavivirus antigenicity, stability, and pathogenesis.

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    Leslie Goo

    2017-02-01

    Full Text Available The structural flexibility or 'breathing' of the envelope (E protein of flaviviruses allows virions to sample an ensemble of conformations at equilibrium. The molecular basis and functional consequences of virus conformational dynamics are poorly understood. Here, we identified a single mutation at residue 198 (T198F of the West Nile virus (WNV E protein domain I-II hinge that regulates virus breathing. The T198F mutation resulted in a ~70-fold increase in sensitivity to neutralization by a monoclonal antibody targeting a cryptic epitope in the fusion loop. Increased exposure of this otherwise poorly accessible fusion loop epitope was accompanied by reduced virus stability in solution at physiological temperatures. Introduction of a mutation at the analogous residue of dengue virus (DENV, but not Zika virus (ZIKV, E protein also increased accessibility of the cryptic fusion loop epitope and decreased virus stability in solution, suggesting that this residue modulates the structural ensembles sampled by distinct flaviviruses at equilibrium in a context dependent manner. Although the T198F mutation did not substantially impair WNV growth kinetics in vitro, studies in mice revealed attenuation of WNV T198F infection. Overall, our study provides insight into the molecular basis and the in vitro and in vivo consequences of flavivirus breathing.

  14. Biophysical analysis of a lethal laminin alpha-1 mutation reveals altered self-interaction

    KAUST Repository

    Patel, Trushar R.; Nikodemus, Denise; Besong, Tabot M.D.; Reuten, Raphael; Meier, Markus; Harding, Stephen E.; Winzor, Donald J.; Koch, Manuel; Stetefeld, Jö rg

    2015-01-01

    Laminins are key basement membrane molecules that influence several biological activities and are linked to a number of diseases. They are secreted as heterotrimeric proteins consisting of one α, one β, and one γ chain, followed by their assembly into a polymer-like sheet at the basement membrane. Using sedimentation velocity, dynamic light scattering, and surface plasmon resonance experiments, we studied self-association of three laminin (LM) N-terminal fragments α-1 (hLM α-1 N), α-5 (hLM α-5 N) and β-3 (hLM β-3 N) originating from the short arms of the human laminin αβγ heterotrimer. Corresponding studies of the hLM α-1 N C49S mutant, equivalent to the larval lethal C56S mutant in zebrafish, have shown that this mutation causes enhanced self-association behavior, an observation that provides a plausible explanation for the inability of laminin bearing this mutation to fulfill functional roles in vivo, and hence for the deleterious pathological consequences of the mutation on lens function.

  15. Biophysical analysis of a lethal laminin alpha-1 mutation reveals altered self-interaction

    KAUST Repository

    Patel, Trushar R.

    2015-07-26

    Laminins are key basement membrane molecules that influence several biological activities and are linked to a number of diseases. They are secreted as heterotrimeric proteins consisting of one α, one β, and one γ chain, followed by their assembly into a polymer-like sheet at the basement membrane. Using sedimentation velocity, dynamic light scattering, and surface plasmon resonance experiments, we studied self-association of three laminin (LM) N-terminal fragments α-1 (hLM α-1 N), α-5 (hLM α-5 N) and β-3 (hLM β-3 N) originating from the short arms of the human laminin αβγ heterotrimer. Corresponding studies of the hLM α-1 N C49S mutant, equivalent to the larval lethal C56S mutant in zebrafish, have shown that this mutation causes enhanced self-association behavior, an observation that provides a plausible explanation for the inability of laminin bearing this mutation to fulfill functional roles in vivo, and hence for the deleterious pathological consequences of the mutation on lens function.

  16. Analyses of Dynein Heavy Chain Mutations Reveal Complex Interactions Between Dynein Motor Domains and Cellular Dynein Functions

    Science.gov (United States)

    Sivagurunathan, Senthilkumar; Schnittker, Robert R.; Razafsky, David S.; Nandini, Swaran; Plamann, Michael D.; King, Stephen J.

    2012-01-01

    Cytoplasmic dynein transports cargoes for a variety of crucial cellular functions. However, since dynein is essential in most eukaryotic organisms, the in-depth study of the cellular function of dynein via genetic analysis of dynein mutations has not been practical. Here, we identify and characterize 34 different dynein heavy chain mutations using a genetic screen of the ascomycete fungus Neurospora crassa, in which dynein is nonessential. Interestingly, our studies show that these mutations segregate into five different classes based on the in vivo localization of the mutated dynein motors. Furthermore, we have determined that the different classes of dynein mutations alter vesicle trafficking, microtubule organization, and nuclear distribution in distinct ways and require dynactin to different extents. In addition, biochemical analyses of dynein from one mutant strain show a strong correlation between its in vitro biochemical properties and the aberrant intracellular function of that altered dynein. When the mutations were mapped to the published dynein crystal structure, we found that the three-dimensional structural locations of the heavy chain mutations were linked to particular classes of altered dynein functions observed in cells. Together, our data indicate that the five classes of dynein mutations represent the entrapment of dynein at five separate points in the dynein mechanochemical and transport cycles. We have developed N. crassa as a model system where we can dissect the complexities of dynein structure, function, and interaction with other proteins with genetic, biochemical, and cell biological studies. PMID:22649085

  17. Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke.

    Science.gov (United States)

    Talley Watts, Lora; Zheng, Wei; Garling, R Justin; Frohlich, Victoria C; Lechleiter, James Donald

    2015-06-23

    In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

  18. Genetic analysis of suppressors of the PF10 mutation in Chlamydomonas reinhardtii

    International Nuclear Information System (INIS)

    Dutcher, S.K.; Gibbons, W.; Inwood, W.B.

    1988-01-01

    A mutation at the PF10 locus of the unicellular green alga Chlamydomonas reinhardtii leads to abnormal cell motility. The asymmetric form of the ciliary beat stroke characteristic of wild-type flagella is modified by this mutation to a nearly symmetric beat. We report here that this abnormal motility is a conditional phenotype that depends on light intensity. In the absence of light or under low light intensities, the motility is more severely impaired than at higher light intensities. By UV mutagenesis we obtained 11 intragenic and 70 extragenic strains that show reversion of the pf10 motility phenotype observed in low light. The intragenic events reverted the motility phenotype of the pf10 mutation completely. The extragenic events define at least seven suppressor loci; these map to linkage groups IV, VII, IX, XI, XII and XVII. Suppressor mutations at two of the seven loci (LIS1 and LIS2) require light for their suppressor activity. Forty-eight of the 70 extragenic suppressors were examined in heterozygous diploid cells; 47 of these mutants were recessive to the wild-type allele and one mutant (bop5-1) was dominant to the wild-type allele. Complementation analysis of the 47 recessive mutants showed unusual patterns. Most mutants within a recombinationally defined group failed to complement one another, although there were pairs that showed intra-allelic complementation. Additionally, some of the mutants at each recombinationally defined locus failed to complement mutants at other loci. They define dominant enhancers of one another

  19. Whole-exome sequencing of muscle-invasive bladder cancer identifies recurrent mutations of UNC5C and prognostic importance of DNA repair gene mutations on survival.

    Science.gov (United States)

    Yap, Kai Lee; Kiyotani, Kazuma; Tamura, Kenji; Antic, Tatjana; Jang, Miran; Montoya, Magdeline; Campanile, Alexa; Yew, Poh Yin; Ganshert, Cory; Fujioka, Tomoaki; Steinberg, Gary D; O'Donnell, Peter H; Nakamura, Yusuke

    2014-12-15

    Because of suboptimal outcomes in muscle-invasive bladder cancer even with multimodality therapy, determination of potential genetic drivers offers the possibility of improving therapeutic approaches and discovering novel prognostic indicators. Using pTN staging, we case-matched 81 patients with resected ≥pT2 bladder cancers for whom perioperative chemotherapy use and disease recurrence status were known. Whole-exome sequencing was conducted in 43 cases to identify recurrent somatic mutations and targeted sequencing of 10 genes selected from the initial screening in an additional 38 cases was completed. Mutational profiles along with clinicopathologic information were correlated with recurrence-free survival (RFS) in the patients. We identified recurrent novel somatic mutations in the gene UNC5C (9.9%), in addition to TP53 (40.7%), KDM6A (21.0%), and TSC1 (12.3%). Patients who were carriers of somatic mutations in DNA repair genes (one or more of ATM, ERCC2, FANCD2, PALB2, BRCA1, or BRCA2) had a higher overall number of somatic mutations (P = 0.011). Importantly, after a median follow-up of 40.4 months, carriers of somatic mutations (n = 25) in any of these six DNA repair genes had significantly enhanced RFS compared with noncarriers [median, 32.4 vs. 14.8 months; hazard ratio of 0.46, 95% confidence interval (CI), 0.22-0.98; P = 0.0435], after adjustment for pathologic pTN staging and independent of adjuvant chemotherapy usage. Better prognostic outcomes of individuals carrying somatic mutations in DNA repair genes suggest these mutations as favorable prognostic events in muscle-invasive bladder cancer. Additional mechanistic investigation into the previously undiscovered role of UNC5C in bladder cancer is warranted. ©2014 American Association for Cancer Research.

  20. Characterization of phospholipase C gamma enzymes with gain-of-function mutations.

    Science.gov (United States)

    Everett, Katy L; Bunney, Tom D; Yoon, Youngdae; Rodrigues-Lima, Fernando; Harris, Richard; Driscoll, Paul C; Abe, Koichiro; Fuchs, Helmut; de Angelis, Martin Hrabé; Yu, Philipp; Cho, Wohnwa; Katan, Matilda

    2009-08-21

    Phospholipase C gamma isozymes (PLC gamma 1 and PLC gamma 2) have a crucial role in the regulation of a variety of cellular functions. Both enzymes have also been implicated in signaling events underlying aberrant cellular responses. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we have recently identified single point mutations in murine PLC gamma 2 that lead to spontaneous inflammation and autoimmunity. Here we describe further, mechanistic characterization of two gain-of-function mutations, D993G and Y495C, designated as ALI5 and ALI14. The residue Asp-993, mutated in ALI5, is a conserved residue in the catalytic domain of PLC enzymes. Analysis of PLC gamma 1 and PLC gamma 2 with point mutations of this residue showed that removal of the negative charge enhanced PLC activity in response to EGF stimulation or activation by Rac. Measurements of PLC activity in vitro and analysis of membrane binding have suggested that ALI5-type mutations facilitate membrane interactions without compromising substrate binding and hydrolysis. The residue mutated in ALI14 (Tyr-495) is within the spPH domain. Replacement of this residue had no effect on folding of the domain and enhanced Rac activation of PLC gamma 2 without increasing Rac binding. Importantly, the activation of the ALI14-PLC gamma 2 and corresponding PLC gamma 1 variants was enhanced in response to EGF stimulation and bypassed the requirement for phosphorylation of critical tyrosine residues. ALI5- and ALI14-type mutations affected basal activity only slightly; however, their combination resulted in a constitutively active PLC. Based on these data, we suggest that each mutation could compromise auto-inhibition in the inactive PLC, facilitating the activation process; in addition, ALI5-type mutations could enhance membrane interaction in the activated state.

  1. Mutation in LIM2 Is Responsible for Autosomal Recessive Congenital Cataracts.

    Directory of Open Access Journals (Sweden)

    Bushra Irum

    Full Text Available To identify the molecular basis of non-syndromic autosomal recessive congenital cataracts (arCC in a consanguineous family.All family members participating in the study received a comprehensive ophthalmic examination to determine their ocular phenotype and contributed a blood sample, from which genomic DNA was extracted. Available medical records and interviews with the family were used to compile the medical history of the family. The symptomatic history of the individuals exhibiting cataracts was confirmed by slit-lamp biomicroscopy. A genome-wide linkage analysis was performed to localize the disease interval. The candidate gene, LIM2 (lens intrinsic membrane protein 2, was sequenced bi-directionally to identify the disease-causing mutation. The physical changes caused by the mutation were analyzed in silico through homology modeling, mutation and bioinformatic algorithms, and evolutionary conservation databases. The physiological importance of LIM2 to ocular development was assessed in vivo by real-time expression analysis of Lim2 in a mouse model.Ophthalmic examination confirmed the diagnosis of nuclear cataracts in the affected members of the family; the inheritance pattern and cataract development in early infancy indicated arCC. Genome-wide linkage analysis localized the critical interval to chromosome 19q with a two-point logarithm of odds (LOD score of 3.25. Bidirectional sequencing identified a novel missense mutation, c.233G>A (p.G78D in LIM2. This mutation segregated with the disease phenotype and was absent in 192 ethnically matched control chromosomes. In silico analysis predicted lower hydropathicity and hydrophobicity but higher polarity of the mutant LIM2-encoded protein (MP19 compared to the wild-type. Moreover, these analyses predicted that the mutation would disrupt the secondary structure of a transmembrane domain of MP19. The expression of Lim2, which was detected in the mouse lens as early as embryonic day 15 (E15

  2. Oncogenic Activation of Fibroblast Growth Factor Receptor-3 and RAS Genes as Non-Overlapping Mutual Exclusive Events in Urinary Bladder Cancer.

    Science.gov (United States)

    Pandith, Arshad A; Hussain, Aashaq; Khan, Mosin S; Shah, Zafar A; Wani, M Saleem; Siddiqi, Mushtaq A

    2016-01-01

    Urinary bladder cancer is a common malignancy in the West and ranks as the 7th most common cancer in our region of Kashmir, India. FGFR3 mutations are frequent in superficial urothelial carcinoma (UC) differing from the RAS gene mutational pattern. The aim of this study was to analyze the frequency and association of FGFR3 and RAS gene mutations in UC cases. Paired tumor and adjacent normal tissue specimens of 65 consecutive UC patients were examined. DNA preparations were evaluated for the occurrence of FGFR3 and RAS gene mutations by PCR-SCCP and DNA sequencing. Somatic point mutations of FGFR3 were identified in 32.3% (21 of 65). The pattern and distribution were significantly associated with low grade/stage (<0.05). The overall mutations in exon 1 and 2 in all the forms of RAS genes aggregated to 21.5% and showed no association with any clinic-pathological parameters. In total, 53.8% (35 of 65) of the tumors studied had mutations in either a RAS or FGFR3 gene, but these were totally mutually exclusive in and none of the samples showed both the mutational events in mutually exclusive RAS and FGFR3. We conclude that RAS and FGFR3 mutations in UC are mutually exclusive and non-overlapping events which reflect activation of oncogenic pathways through different elements.

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

    Science.gov (United States)

    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.

  4. A targeted mutation within the feline leukemia virus (FeLV) envelope protein immunosuppressive domain to improve a canarypox virus-vectored FeLV vaccine.

    Science.gov (United States)

    Schlecht-Louf, Géraldine; Mangeney, Marianne; El-Garch, Hanane; Lacombe, Valérie; Poulet, Hervé; Heidmann, Thierry

    2014-01-01

    We previously delineated a highly conserved immunosuppressive (IS) domain within murine and primate retroviral envelope proteins that is critical for virus propagation in vivo. The envelope-mediated immunosuppression was assessed by the ability of the proteins, when expressed by allogeneic tumor cells normally rejected by engrafted mice, to allow these cells to escape, at least transiently, immune rejection. Using this approach, we identified key residues whose mutation (i) specifically abolishes immunosuppressive activity without affecting the "mechanical" function of the envelope protein and (ii) significantly enhances humoral and cellular immune responses elicited against the virus. The objective of this work was to study the immunosuppressive activity of the envelope protein (p15E) of feline leukemia virus (FeLV) and evaluate the effect of its abolition on the efficacy of a vaccine against FeLV. Here we demonstrate that the FeLV envelope protein is immunosuppressive in vivo and that this immunosuppressive activity can be "switched off" by targeted mutation of a specific amino acid. As a result of the introduction of the mutated envelope sequence into a previously well characterized canarypox virus-vectored vaccine (ALVAC-FeLV), the frequency of vaccine-induced FeLV-specific gamma interferon (IFN-γ)-producing cells was increased, whereas conversely, the frequency of vaccine-induced FeLV-specific interleukin-10 (IL-10)-producing cells was reduced. This shift in the IFN-γ/IL-10 response was associated with a higher efficacy of ALVAC-FeLV against FeLV infection. This study demonstrates that FeLV p15E is immunosuppressive in vivo, that the immunosuppressive domain of p15E can modulate the FeLV-specific immune response, and that the efficacy of FeLV vaccines can be enhanced by inhibiting the immunosuppressive activity of the IS domain through an appropriate mutation.

  5. Evaluating the Genetic, Hormonal, and Exogenous Factors Affecting Somatic Copy Number Variation in Breast Cancer

    Science.gov (United States)

    2016-10-01

    assess genomic instability in different mammary epithelial populations in vivo and in vitro, 2) determine how mutations in heritable breast cancer genes...respectively, located on chromosome 6. When loci harboring the shRNAs are deleted by a spontaneous mutation event, affected cells become GFP and/or RFP...assay adapted from the yeast genetics literature, we will determine whether baseline deletion rates in normal human mammary epithelial cells (HMECs

  6. Amnionless function is required for cubilin brush-border expression and intrinsic factor-cobalamin (vitamin B12) absorption in vivo

    Science.gov (United States)

    He, Qianchuan; Madsen, Mette; Kilkenney, Adam; Gregory, Brittany; Christensen, Erik I.; Vorum, Henrik; Højrup, Peter; Schäffer, Alejandro A.; Kirkness, Ewen F.; Tanner, Stephan M.; de la Chapelle, Albert; Giger, Urs; Moestrup, Søren K.; Fyfe, John C.

    2005-01-01

    Amnionless (AMN) and cubilin gene products appear to be essential functional subunits of an endocytic receptor called cubam. Mutation of either gene causes autosomal recessive Imerslund-Gräsbeck syndrome (I-GS, OMIM no. 261100) in humans, a disorder characterized by selective intestinal malabsorption of cobalamin (vitamin B12) and urinary loss of several specific low-molecular-weight proteins. Vital insight into the molecular pathology of I-GS has been obtained from studies of dogs with a similar syndrome. In this work, we show that I-GS segregates in a large canine kindred due to an in-frame deletion of 33 nucleotides in exon 10 of AMN. In a second, unrelated I-GS kindred, affected dogs exhibit a homozygous substitution in the AMN translation initiation codon. Studies in vivo demonstrated that both mutations abrogate AMN expression and block cubilin processing and targeting to the apical membrane. The essential features of AMN dysfunction observed in vivo are recapitulated in a heterologous cell-transfection system, thus validating the system for analysis of AMN-cubilin interactions. Characterization of canine AMN mutations that cause I-GS establishes the canine model as an ortholog of the human disorder well suited to studies of AMN function and coevolution with cubilin. (Blood. 2005;106:1447-1453) PMID:15845892

  7. Inhibition of Axl improves the targeted therapy against ALK-mutated neuroblastoma

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Fei [Department of Neurology, Sichuan Medical Science Institute and Sichuan Provincial Hospital, Chengdu 610072 (China); Li, Hongling [Department of Radiotherapy, Shanghai First People’s Hospital, Shanghai Jiao Tong University, Shanghai 201620 (China); Sun, Yong, E-mail: sunfanqi2010@163.com [Department of Burn and Plastic Surgery, Huai’an First People’s Hospital, Nanjing Medical University, Huai’an 223300 (China)

    2014-11-28

    Highlights: • First reported Axl is co-expressed with ALK in neuroblastoma tissues and cell lines. • Axl activation promotes cell growth and impairs the efficiency of ALK inhibitor. • Further found silence of Axl leads to increased sensitivity to ALK inhibitors. • Axl inhibitor promotes the efficiency of targeted therapy in vitro and in vivo. • Axl activation should be considered in the clinical application of ALK inhibitors. - Abstract: Neuroblastoma (NB) patients harboring mutated ALK can be expected to potentially benefit from targeted therapy based on ALK tyrosine kinase inhibitor (TKI), such as crizotinib and ceritinib. However, the effect of the treatment varies with different individuals, although with the same genic changes. Axl receptor tyrosine kinase is expressed in a variety of human cancers, but little data are reported in NB, particularly in which carrying mutated ALK. In this study, we focus on the roles of Axl in ALK-mutated NB for investigating rational therapeutic strategy. We found that Axl is expressed in ALK-positive NB tissues and cell lines, and could be effectively activated by its ligand GAS6. Ligand-dependent Axl activation obviously rescued crizotinib-mediated suppression of cell proliferation in ALK-mutated NB cells. Genetic inhibition of Axl with specific small interfering RNA markedly increased the sensitivity of cells to ALK-TKIs. Furthermore, a small-molecule inhibitor of Axl significantly enhanced ALK-targeted therapy, as an increased frequency of apoptosis was observed in NB cells co-expressing ALK and Axl. Taken together, our results demonstrated that activation of Axl could lead to insensitivity to ALK inhibitors, and dual inhibition of ALK and Axl might be a potential therapeutic strategy against ALK-mutated NB.

  8. Rasfonin, a novel 2-pyrone derivative, induces ras-mutated Panc-1 pancreatic tumor cell death in nude mice.

    Science.gov (United States)

    Xiao, Z; Li, L; Li, Y; Zhou, W; Cheng, J; Liu, F; Zheng, P; Zhang, Y; Che, Y

    2014-05-22

    Rasfonin is a novel 2-pyrone derivative reported to induce apoptosis in ras-dependent cells. In this study, its effects on ras-mutated pancreatic cancer cells were investigated in vitro and in vivo. Two human pancreatic cancer cell lines Panc-1 (mutated K-ras) and BxPC-3 (wild-type K-ras) were selected to test the effects of rasfonin on cell proliferation, clone formation, migration and invasion in vitro. Immunoblotting was used to detect the expressions of EGFR-Ras-Raf-MEK-ERK signaling pathway proteins. Ras activity was measured using a pull-down ELISA kit and guanine exchange factor (GEF)/GTPase-activating proteins (GAP) activity was measured by [(3)H]-GDP radiometric ligand binding. For an in vivo study, CD1 nude mice bearing Panc-1 cells were treated with rasfonin or Salirasib (FTS). We found that rasfonin suppressed proliferation more strongly in Panc-1 cells (IC50=5.5 μM) than BxPC-3 cells (IC50=10 μM) in vitro. Clone formation, migration and invasion by Panc-1 cells were also reduced by rasfonin. Rasfonin had little effect on the farnesylation of Ras, but it strongly downregulated Ras activity and consequently phosphorylation of c-Raf/MEK/ERK. Further experiments indicated that rasfonin reduced Son of sevenless (Sos1) expression but did not alter GEF and GAP activities. The in vivo experiments also revealed that rasfonin (30 mg/kg) delayed the growth of xenograft tumors originating from Panc-1 cells. Tumor weight was ultimately decreased after 20 days of treatment of rasfonin. Rasfonin is a robust inhibitor of pancreatic cancers with the K-ras mutation. The reduction of Sos1 expression and the consequently depressed Ras-MAPK activity could be important in its anticancer activity.

  9. Drosophila studies support a role for a presynaptic synaptotagmin mutation in a human congenital myasthenic syndrome.

    Directory of Open Access Journals (Sweden)

    Mallory C Shields

    Full Text Available During chemical transmission, the function of synaptic proteins must be coordinated to efficiently release neurotransmitter. Synaptotagmin 2, the Ca2+ sensor for fast, synchronized neurotransmitter release at the human neuromuscular junction, has recently been implicated in a dominantly inherited congenital myasthenic syndrome associated with a non-progressive motor neuropathy. In one family, a proline residue within the C2B Ca2+-binding pocket of synaptotagmin is replaced by a leucine. The functional significance of this residue has not been investigated previously. Here we show that in silico modeling predicts disruption of the C2B Ca2+-binding pocket, and we examine the in vivo effects of the homologous mutation in Drosophila. When expressed in the absence of native synaptotagmin, this mutation is lethal, demonstrating for the first time that this residue plays a critical role in synaptotagmin function. To achieve expression similar to human patients, the mutation is expressed in flies carrying one copy of the wild type synaptotagmin gene. We now show that Drosophila carrying this mutation developed neurological and behavioral manifestations similar to those of human patients and provide insight into the mechanisms underlying these deficits. Our Drosophila studies support a role for this synaptotagmin point mutation in disease etiology.

  10. Active Center Control of Termination by RNA Polymerase III and tRNA Gene Transcription Levels In Vivo.

    Directory of Open Access Journals (Sweden)

    Keshab Rijal

    2016-08-01

    Full Text Available The ability of RNA polymerase (RNAP III to efficiently recycle from termination to reinitiation is critical for abundant tRNA production during cellular proliferation, development and cancer. Yet understanding of the unique termination mechanisms used by RNAP III is incomplete, as is its link to high transcription output. We used two tRNA-mediated suppression systems to screen for Rpc1 mutants with gain- and loss- of termination phenotypes in S. pombe. 122 point mutation mutants were mapped to a recently solved 3.9 Å structure of yeast RNAP III elongation complex (EC; they cluster in the active center bridge helix and trigger loop, as well as the pore and funnel, the latter of which indicate involvement of the RNA cleavage domain of the C11 subunit in termination. Purified RNAP III from a readthrough (RT mutant exhibits increased elongation rate. The data strongly support a kinetic coupling model in which elongation rate is inversely related to termination efficiency. The mutants exhibit good correlations of terminator RT in vitro and in vivo, and surprisingly, amounts of transcription in vivo. Because assessing in vivo transcription can be confounded by various parameters, we used a tRNA reporter with a processing defect and a strong terminator. By ruling out differences in RNA decay rates, the data indicate that mutants with the RT phenotype synthesize more RNA than wild type cells, and than can be accounted for by their increased elongation rate. Finally, increased activity by the mutants appears unrelated to the RNAP III repressor, Maf1. The results show that the mobile elements of the RNAP III active center, including C11, are key determinants of termination, and that some of the mutations activate RNAP III for overall transcription. Similar mutations in spontaneous cancer suggest this as an unforeseen mechanism of RNAP III activation in disease.

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

    Science.gov (United States)

    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. Nationwide experience of catecholaminergic polymorphic ventricular tachycardia caused by RyR2 mutations

    DEFF Research Database (Denmark)

    Broendberg, Anders Krogh; Nielsen, Jens Cosedis; Bjerre, Jesper

    2017-01-01

    probands, 18 symptomatic and 10 asymptomatic relatives with a RyR2 mutation. Twenty (87%) probands and 10 (36%) relatives had severe presenting symptoms (sudden cardiac death (SCD), aborted SCD (ASCD) or syncope).As compared with symptomatic relatives, probands had lower age at onset of symptoms (16 years...... (IQR, 10-33) vs 43 years (IQR, 25-54), pnear-fatal events (ASCD, SCD) (16vs5, p... events in the majority of probands and also occurred in 36% of relatives identified through family screening. Probands were younger at disease onset and more prone to fatal or near-fatal events than relatives....

  13. Active site mutations in yeast protein disulfide isomerase cause dithiothreitol sensitivity and a reduced rate of protein folding in the endoplasmic reticulum

    DEFF Research Database (Denmark)

    Holst, B; Tachibana, C; Winther, Jakob R.

    1997-01-01

    Aspects of protein disulfide isomerase (PDI) function have been studied in yeast in vivo. PDI contains two thioredoxin-like domains, a and a', each of which contains an active-site CXXC motif. The relative importance of the two domains was analyzed by rendering each one inactive by mutation to SGAS....... Such mutations had no significant effect on growth. The domains however, were not equivalent since the rate of folding of carboxypeptidase Y (CPY) in vivo was reduced by inactivation of the a domain but not the a' domain. To investigate the relevance of PDI redox potential, the G and H positions of each CGHC......-deleted strains overexpressing the yeast PDI homologue EUG1 are viable. Exchanging the wild-type Eug1p C(L/I)HS active site sequences for C(L/I)HC increased the growth rate significantly, however, further highlighting the importance of the oxidizing function for optimal growth....

  14. Allogeneic Transplant in ELANE and MEFV Mutation Positive Severe Cyclic Neutropenia: Review of Prognostic Factors for Secondary Severe Events

    OpenAIRE

    Okolo, Onyemaechi N.; Katsanis, Emmanuel; Yun, Seongseok; Reveles, Candace Y.; Anwer, Faiz

    2017-01-01

    Objective and Importance. Cyclic neutropenia (CyN) is a rare autosomal dominant inherited disorder due to the mutation ELANE primarily affecting bone marrow stem cells and is characterized by recurrent neutropenia every 2 to 4 weeks. Symptoms vary from benign to severe, including death. Postulations on the cause of wide spectrum in symptom presentation include the possibility of other genetic mutations, such as MEFV. Recommended treatment for CyN is G-CSF to keep ANC higher to minimize risk o...

  15. PIK3CA Mutation in Colorectal Cancer: Relationship with Genetic and Epigenetic Alterations

    Directory of Open Access Journals (Sweden)

    Katsuhiko Nosho

    2008-06-01

    Full Text Available Somatic PIK3CA mutations are often present in colorectal cancer. Mutant PIK3CA activates AKT signaling, which up-regulates fatty acid synthase (FASN. Microsatellite instability (MSI and CpG island methylator phenotype (CIMP are important molecular classifiers in colorectal cancer. However, the relationship between PIK3CA mutation, MSI and CIMP remains uncertain. Using Pyrosequencing technology, we detected PIK3CA mutations in 91 (15% of 590 population-based colorectal cancers. To determine CIMP status, we quantified DNA methylation in eight CIMP-specific promoters [CACNA1G, CDKN2A (p16, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1] by real-time polymerase chain reaction (MethyLight. PIK3CA mutation was significantly associated with mucinous tumors [P = .0002; odds ratio (OR = 2.44], KRAS mutation (P < .0001; OR = 2.68, CIMP-high (P = .03; OR = 2.08, phospho–ribosomal protein S6 expression (P = .002; OR = 2.19, and FASN expression (P = .02; OR = 1.85 and inversely with p53 expression (P = .01; OR = 0.54 and β-catenin (CTNNB1 alteration (P = .004; OR = 0.43. In addition, PIK3CA G-to-A mutations were associated with MGMT loss (P = .001; OR = 3.24 but not with MGMT promoter methylation. In conclusion, PIK3CA mutation is significantly associated with other key molecular events in colorectal cancer, and MGMT loss likely contributes to the development of PIK3CA G>A mutation. In addition, Pyrosequencing is useful in detecting PIK3CA mutation in archival paraffin tumor tissue. PIK3CA mutational data further emphasize heterogeneity of colorectal cancer at the molecular level.

  16. SMARCB1/INI1 germline mutations contribute to 10% of sporadic schwannomatosis

    Directory of Open Access Journals (Sweden)

    Bourdon Violaine

    2011-01-01

    Full Text Available Abstract Background Schwannomatosis is a disease characterized by multiple non-vestibular schwannomas. Although biallelic NF2 mutations are found in schwannomas, no germ line event is detected in schwannomatosis patients. In contrast, germline mutations of the SMARCB1 (INI1 tumor suppressor gene were described in familial and sporadic schwannomatosis patients. Methods To delineate the SMARCB1 gene contribution, the nine coding exons were sequenced in a series of 56 patients affected with a variable number of non-vestibular schwannomas. Results Nine variants scattered along the sequence of SMARCB1 were identified. Five of them were classified as deleterious. All five patients carrying a SMARCB1 mutation had more multiple schwannomas, corresponding to 10.2% of patients with schwannomatosis. They were also diagnosed before 35 years of age. Conclusions These results suggest that patients with schwannomas have a significant probability of carrying a SMARCB1 mutation. Combined with data available from other studies, they confirm the clinical indications for genetic screening of the SMARCB1 gene.

  17. CHCHD10 mutations p.R15L and p.G66V cause motoneuron disease by haploinsufficiency.

    Science.gov (United States)

    Brockmann, Sarah J; Freischmidt, Axel; Oeckl, Patrick; Müller, Kathrin; Ponna, Srinivas K; Helferich, Anika M; Paone, Christoph; Reinders, Jörg; Kojer, Kerstin; Orth, Michael; Jokela, Manu; Auranen, Mari; Udd, Bjarne; Hermann, Andreas; Danzer, Karin M; Lichtner, Peter; Walther, Paul; Ludolph, Albert C; Andersen, Peter M; Otto, Markus; Kursula, Petri; Just, Steffen; Weishaupt, Jochen H

    2018-02-15

    Mutations in the mitochondrially located protein CHCHD10 cause motoneuron disease by an unknown mechanism. In this study, we investigate the mutations p.R15L and p.G66V in comparison to wild-type CHCHD10 and the non-pathogenic variant p.P34S in vitro, in patient cells as well as in the vertebrate in vivo model zebrafish. We demonstrate a reduction of CHCHD10 protein levels in p.R15L and p.G66V mutant patient cells to approximately 50%. Quantitative real-time PCR revealed that expression of CHCHD10 p.R15L, but not of CHCHD10 p.G66V, is already abrogated at the mRNA level. Altered secondary structure and rapid protein degradation are observed with regard to the CHCHD10 p.G66V mutant. In contrast, no significant differences in expression, degradation rate or secondary structure of non-pathogenic CHCHD10 p.P34S are detected when compared with wild-type protein. Knockdown of CHCHD10 expression in zebrafish to about 50% causes motoneuron pathology, abnormal myofibrillar structure and motility deficits in vivo. Thus, our data show that the CHCHD10 mutations p.R15L and p.G66V cause motoneuron disease primarily based on haploinsufficiency of CHCHD10. © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  18. Genetic mutation analysis of HBV covalently closed circular DNA in peripheral blood mononuclear cells from chronic hepatitis B patients with nucleos(tide analog-resistant mutations in serum virions

    Directory of Open Access Journals (Sweden)

    Zhong-bin LI

    2012-06-01

    Full Text Available Objective  To analyze the characteristics of genetic mutations in reverse-transcriptase (RT domain of HBV covalently closed circular DNA (cccDNA in peripheral blood mononuclear cells (PBMCs obtained from chronic hepatitis B (CHB patients with drug-resistant mutations in serum virions during nucleoside/nucleotide analog (NA therapy. Methods  A total of 30 CHB patients admitted to 302 Hospital of PLA from July 2010 to August 2011 were included in this study. All the patients were confirmed to harbor the drug-resistant mutations in serum virions during an NA therapy longer than 6 months. Total DNA was extracted from PBMCs isolated from 30 whole blood samples at the same time point as that of serum analysis. Plasmid-safe ATP-dependent DNase (PSAD digestion in combination with rolling circle amplification and gap-spanning semi-nested PCR were used to amplify the RT region of HBV cccDNA. NA-resistant-associated mutations were analyzed at nine sites. Results  HBV cccDNA was efficiently amplified in 16 out of 30 (53.3% PBMC samples, and the detection rate was not correlated with HBeAg-positive rate, serum ALT level or HBV DNA load. Five of 16 (31.3% patients were sustained to have genotype B HBV infection, and 11 of 16 (68.8% were of genotype C HBV infection, and the result was consistent with the genotyping results using serum HBV. Different from drug-resistant mutations detected in the serum virions, the viruses detected in HBV cccDNA of 16 PBMC samples were all wild-type viruses without NA-resistant-associated mutations in RT region. Conclusions  During NA antiviral treatment, if drug-resistant mutations occur in serum HBV DNA of CHB patients, the dominant species of HBV cccDNA in PBMCs from the same patient is still the original wild-type strains. It is speculated that PBMCs might be the potential "repository" of HBV wild-type strain in vivo.

  19. Darwinism for the Genomic Age: Connecting Mutation to Diversification.

    Science.gov (United States)

    Hua, Xia; Bromham, Lindell

    2017-01-01

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

  20. Calmodulin Mutations Associated with Recurrent Cardiac Arrest in Infants

    Science.gov (United States)

    Crotti, Lia; Johnson, Christopher N.; Graf, Elisabeth; De Ferrari, Gaetano M.; Cuneo, Bettina F.; Ovadia, Marc; Papagiannis, John; Feldkamp, Michael D.; Rathi, Subodh G.; Kunic, Jennifer D.; Pedrazzini, Matteo; Wieland, Thomas; Lichtner, Peter; Beckmann, Britt-Maria; Clark, Travis; Shaffer, Christian; Benson, D. Woodrow; Kääb, Stefan; Meitinger, Thomas; Strom, Tim M.; Chazin, Walter J.; Schwartz, Peter J.; George, Alfred L.

    2013-01-01

    Background Life-threatening disorders of heart rhythm may arise during infancy and can result in the sudden and tragic death of a child. We performed exome sequencing on two unrelated infants presenting with recurrent cardiac arrest to discover a genetic cause. Methods and Results We ascertained two unrelated infants (probands) with recurrent cardiac arrest and dramatically prolonged QTc interval who were both born to healthy parents. The two parent-child trios were investigated using exome sequencing to search for de novo genetic variants. We then performed follow-up candidate gene screening on an independent cohort of 82 subjects with congenital long-QT syndrome without an identified genetic cause. Biochemical studies were performed to determine the functional consequences of mutations discovered in two genes encoding calmodulin. We discovered three heterozygous de novo mutations in either CALM1 or CALM2, two of the three human genes encoding calmodulin, in the two probands and in two additional subjects with recurrent cardiac arrest. All mutation carriers were infants who exhibited life-threatening ventricular arrhythmias combined variably with epilepsy and delayed neurodevelopment. Mutations altered residues in or adjacent to critical calcium binding loops in the calmodulin carboxyl-terminal domain. Recombinant mutant calmodulins exhibited several fold reductions in calcium binding affinity. Conclusions Human calmodulin mutations disrupt calcium ion binding to the protein and are associated with a life-threatening condition in early infancy. Defects in calmodulin function will disrupt important calcium signaling events in heart affecting membrane ion channels, a plausible molecular mechanism for potentially deadly disturbances in heart rhythm during infancy. PMID:23388215

  1. Mutations in conserved amino acids in the KCNQ1 channel and risk of cardiac events in type-1 long-QT syndrome

    DEFF Research Database (Denmark)

    Jons, Christian; Moss, Arthur J; Lopes, Coeli M

    2009-01-01

    BACKGROUND: Type-1 long-QT syndrome (LQT1) is caused by mutations in the KCNQ1 gene. The purpose of this study was to investigate whether KCNQ1 mutations in highly conserved amino acid residues within the voltage-gated potassium channel family are associated with an increased risk of cardiac even...

  2. Detection of Hepatitis B Virus M204I Mutation by Quantum Dot-Labeled DNA Probe

    Directory of Open Access Journals (Sweden)

    Cheng Zhang

    2017-04-01

    Full Text Available Quantum dots (QDs are semiconductor nanoparticles with a diameter of less than 10 nm, which have been widely used as fluorescent probes in biochemical analysis and vivo imaging because of their excellent optical properties. Sensitive and convenient detection of hepatitis B virus (HBV gene mutations is important in clinical diagnosis. Therefore, we developed a sensitive, low-cost and convenient QDs-mediated fluorescent method for the detection of HBV gene mutations in real serum samples from chronic hepatitis B (CHB patients who had received lamivudine or telbivudine antiviral therapy. We also evaluated the efficiency of this method for the detection of drug-resistant mutations compared with direct sequencing. In CHB, HBV DNA from the serum samples of patients with poor response or virological breakthrough can be hybridized to probes containing the M204I mutation to visualize fluorescence under fluorescence microscopy, where fluorescence intensity is related to the virus load, in our method. At present, the limits of the method used to detect HBV genetic variations by fluorescence quantum dots is 103 IU/mL. These results show that QDs can be used as fluorescent probes to detect viral HBV DNA polymerase gene variation, and is a simple readout system without complex and expensive instruments, which provides an attractive platform for the detection of HBV M204I mutation.

  3. Detection of induced male germline mutation: Correlations and comparisons between traditional germline mutation assays, transgenic rodent assays and expanded simple tandem repeat instability assays

    Energy Technology Data Exchange (ETDEWEB)

    Singer, Timothy M. [Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Programme, 0803A, Health Canada, Ottawa, Ont., K1A 0K9 (Canada); Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ont., K1S 5B6 (Canada); Lambert, Iain B. [Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ont., K1S 5B6 (Canada); Williams, Andrew [Biostatistics and Epidemiology Division, Safe Environments Programme, 6604B, Health Canada, Ottawa, Ont., K1A 0K9 (Canada); Douglas, George R. [Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Programme, 0803A, Health Canada, Ottawa, Ont., K1A 0K9 (Canada); Yauk, Carole L. [Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Programme, 0803A, Health Canada, Ottawa, Ont., K1A 0K9 (Canada)]. E-mail: carole_yauk@hc-sc.gc.ca

    2006-06-25

    Several rodent assays are capable of monitoring germline mutation. These include traditional assays, such as the dominant lethal (DL) assay, the morphological specific locus (SL) test and the heritable translocation (HT) assay, and two assays that have been developed more recently-the expanded simple tandem repeat (ESTR) and transgenic rodent (TGR) mutation assays. In this paper, we have compiled the limited amount of experimental data that are currently available to make conclusions regarding the comparative ability of the more recently developed assays to detect germline mutations induced by chemical and radiological agents. The data suggest that ESTR and TGR assays are generally comparable with SL in detecting germline mutagenicity induced by alkylating agents and radiation, though TGR offered less sensitivity than ESTR in some cases. The DL and HT assays detect clastogenic events and are most susceptible to mutations arising in post-spermatogonial cells, and they may not provide the best comparisons with TGR and ESTR instability. The measurement of induced ESTR instability represents a relatively sensitive method of identifying agents causing germline mutation in rodents, and may also be useful for bio-monitoring exposed individuals in the human population. Any future use of the TGR and ESTR germline mutation assays in a regulatory testing context will entail more robust and extensive characterization of assay performance. This will require substantially more data, including experiments measuring multiple endpoints, a greatly expanded database of chemical agents and a focus on characterizing stage-specific activity of mutagens in these assays, preferably by sampling epididymal sperm exposed at defined pre-meiotic, meiotic and post-meiotic stages of development.

  4. Detection of induced male germline mutation: Correlations and comparisons between traditional germline mutation assays, transgenic rodent assays and expanded simple tandem repeat instability assays

    International Nuclear Information System (INIS)

    Singer, Timothy M.; Lambert, Iain B.; Williams, Andrew; Douglas, George R.; Yauk, Carole L.

    2006-01-01

    Several rodent assays are capable of monitoring germline mutation. These include traditional assays, such as the dominant lethal (DL) assay, the morphological specific locus (SL) test and the heritable translocation (HT) assay, and two assays that have been developed more recently-the expanded simple tandem repeat (ESTR) and transgenic rodent (TGR) mutation assays. In this paper, we have compiled the limited amount of experimental data that are currently available to make conclusions regarding the comparative ability of the more recently developed assays to detect germline mutations induced by chemical and radiological agents. The data suggest that ESTR and TGR assays are generally comparable with SL in detecting germline mutagenicity induced by alkylating agents and radiation, though TGR offered less sensitivity than ESTR in some cases. The DL and HT assays detect clastogenic events and are most susceptible to mutations arising in post-spermatogonial cells, and they may not provide the best comparisons with TGR and ESTR instability. The measurement of induced ESTR instability represents a relatively sensitive method of identifying agents causing germline mutation in rodents, and may also be useful for bio-monitoring exposed individuals in the human population. Any future use of the TGR and ESTR germline mutation assays in a regulatory testing context will entail more robust and extensive characterization of assay performance. This will require substantially more data, including experiments measuring multiple endpoints, a greatly expanded database of chemical agents and a focus on characterizing stage-specific activity of mutagens in these assays, preferably by sampling epididymal sperm exposed at defined pre-meiotic, meiotic and post-meiotic stages of development

  5. Mutations in plant breeding: a glance back and a look forward

    International Nuclear Information System (INIS)

    Gustafsson, A.

    1975-01-01

    This brief retrospect of previous shortcomings and procedures really indicates the need for a better co-ordination and co-operation in plant breeding. We now know which mutagens are essential in practical mutation work, and we include radiations as well as chemicals. We now know that there is no principal difference between natural and induced variation. We can easily induce mutational events covering the gap between gross chromosomal rearrangements and DNA-base substitutions. We also know that induced variation may in the future fill and replace the loss of natural variability, or may even extend the limits of variation. We know that mutation and recombination--as evidenced, for instance, by the successful work in barley--will be united into routine procedures, also leading to new breakthroughs in plant improvement. Moreover, ingenious techniques of mass testing are under way, combining traditional and prospective ideas. The mutation method has come to stay, not alone but with gene recombination, with heterosis and with polyploidy. We say that the present is the past's future. To glance back is then to look forward; failures give rise to progress. A synthesis of breeding methods is around the corner

  6. Production of infectious genotype 1b virus particles in cell culture and impairment by replication enhancing mutations.

    Directory of Open Access Journals (Sweden)

    Thomas Pietschmann

    2009-06-01

    Full Text Available With the advent of subgenomic hepatitis C virus (HCV replicons, studies of the intracellular steps of the viral replication cycle became possible. These RNAs are capable of self-amplification in cultured human hepatoma cells, but save for the genotype 2a isolate JFH-1, efficient replication of these HCV RNAs requires replication enhancing mutations (REMs, previously also called cell culture adaptive mutations. These mutations cluster primarily in the central region of non-structural protein 5A (NS5A, but may also reside in the NS3 helicase domain or at a distinct position in NS4B. Most efficient replication has been achieved by combining REMs residing in NS3 with distinct REMs located in NS4B or NS5A. However, in spite of efficient replication of HCV genomes containing such mutations, they do not support production of infectious virus particles. By using the genotype 1b isolate Con1, in this study we show that REMs interfere with HCV assembly. Strongest impairment of virus formation was found with REMs located in the NS3 helicase (E1202G and T1280I as well as NS5A (S2204R, whereas a highly adaptive REM in NS4B still allowed virus production although relative levels of core release were also reduced. We also show that cells transfected with the Con1 wild type genome or the genome containing the REM in NS4B release HCV particles that are infectious both in cell culture and in vivo. Our data provide an explanation for the in vitro and in vivo attenuation of cell culture adapted HCV genomes and may open new avenues for the development of fully competent culture systems covering the therapeutically most relevant HCV genotypes.

  7. Case Report: Exome sequencing reveals recurrent RETSAT mutations and a loss-of-function POLDIP2 mutation in a rare undifferentiated tongue sarcoma [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Jason Y. K. Chan

    2018-04-01

    Full Text Available Soft tissue sarcoma of the tongue represents a very rare head and neck cancer with connective tissue features, and the genetics underlying this rare cancer are largely unknown. There are less than 20 cases reported in the literature thus far. Here, we reported the first whole-exome characterization (>×200 depth of an undifferentiated sarcoma of the tongue in a 31-year-old male. Even with a very good sequencing depth, only 19 nonsynonymous mutations were found, indicating a relatively low mutation rate of this rare cancer (lower than that of human papillomavirus (HPV-positive head and neck cancer. Yet, among the few genes that are somatically mutated in this HPV-negative undifferentiated tongue sarcoma, a noticeable deleterious frameshift mutation (with a very high allele frequency of >93% of a gene for DNA replication and repair, namely POLDIP2 (DNA polymerase delta interacting protein 2, and two recurrent mutations of the adipogenesis and adipocyte differentiation gene RETSAT (retinol saturase, were identified. Thus, somatic events likely affecting adipogenesis and differentiation, as well as potential stem mutations to POLDIP2, may be implicated in the formation of this rare cancer. This identified somatic whole-exome sequencing profile appears to be distinct from that of other reported adult sarcomas from The Cancer Genome Atlas, suggesting a potential unique genetic profile for this rare sarcoma of the tongue. Interestingly, this low somatic mutation rate is unexpectedly found to be accompanied by multiple tumor protein p53 and NOTCH1 germline mutations of the patient’s blood DNA. This may explain the very early age of onset of head and neck cancer, with likely hereditary predisposition. Our findings are, to our knowledge, the first to reveal a unique genetic profile of this very rare undifferentiated sarcoma of the tongue.

  8. A Probability-based Evolutionary Algorithm with Mutations to Learn Bayesian Networks

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    Sho Fukuda

    2014-12-01

    Full Text Available Bayesian networks are regarded as one of the essential tools to analyze causal relationship between events from data. To learn the structure of highly-reliable Bayesian networks from data as quickly as possible is one of the important problems that several studies have been tried to achieve. In recent years, probability-based evolutionary algorithms have been proposed as a new efficient approach to learn Bayesian networks. In this paper, we target on one of the probability-based evolutionary algorithms called PBIL (Probability-Based Incremental Learning, and propose a new mutation operator. Through performance evaluation, we found that the proposed mutation operator has a good performance in learning Bayesian networks

  9. Ex vivo generation of a functional and regenerative wound epithelium from axolotl (Ambystoma mexicanum) skin.

    Science.gov (United States)

    Ferris, Donald R; Satoh, Akira; Mandefro, Berhan; Cummings, Gillian M; Gardiner, David M; Rugg, Elizabeth L

    2010-10-01

    Urodele amphibians (salamanders) are unique among adult vertebrates in their ability to regenerate structurally complete and fully functional limbs. Regeneration is a stepwise process that requires interactions between keratinocytes, nerves and fibroblasts. The formation of a wound epithelium covering the amputation site is an early and necessary event in the process but the molecular mechanisms that underlie the role of the wound epithelium in regeneration remain unclear. We have developed an ex vivo model that recapitulates many features of in vivo wound healing. The model comprises a circular explant of axolotl (Ambystoma mexicanum) limb skin with a central circular, full thickness wound. Re-epithelialization of the wound area is rapid (typically <11 h) and is dependent on metalloproteinase activity. The ex vivo wound epithelium is viable, responds to neuronal signals and is able to participate in ectopic blastema formation and limb regeneration. This ex vivo model provides a reproducible and tractable system in which to study the cellular and molecular events that underlie wound healing and regeneration. © 2010 The Authors. Journal compilation © 2010 Japanese Society of Developmental Biologists.

  10. Evaluation of in vitro and in vivo genotoxicity of single-walled carbon nanotubes.

    Science.gov (United States)

    Kim, Jin Sik; Song, Kyung Seuk; Yu, Il Je

    2015-08-01

    Single-walled carbon nanotubes (SWCNTs) have extensive potential industrial applications due to their unique physical and chemical properties; yet this also increases the chance of human and environment exposure to SWCNTs. Due to the current lack of hazardous effect information on SWNCTs, a standardized genotoxicity battery test was conducted to clarify the genetic toxicity potential of SWCNTs (diameter: 1-1.2 nm, length: ∼20 μm) according to Organization for Economic Cooperation and Development test guidelines 471 (bacterial reverse mutation test), 473 (in vitro chromosome aberration test), and 474 (in vivo micronuclei test) with a good laboratory practice system. The test results showed that the SWCNTs did not induce significant bacterial reverse mutations at 31.3-500 μg/plate in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 or in Escherichia coli strain WP2uvrA, with and without a metabolic activation system. Furthermore, the in vitro chromosome aberration test showed no significant increase in structural or numerical chromosome aberration frequencies at SWCNT dose levels of 12.5-50 μg/ml in the presence and absence of metabolic activation. However, dose-dependent cell growth inhibition was found at all the SWCNT dose levels and statistically significant cytotoxic effects observed at certain concentrations in the presence and absence of metabolic activation. Finally, the SWCNTs did not evoke significant in vivo micronuclei frequencies in the polychromatic erythrocytes of an imprinting control region mice at 25-100 mg/kg. Thus, according to the results of the present study, the SWCNTs were not found to have a genotoxic effect on the in vitro and in vivo test systems. © The Author(s) 2013.

  11. Hereditary sensory and autonomic neuropathy type IID caused by an SCN9A mutation.

    Science.gov (United States)

    Yuan, Junhui; Matsuura, Eiji; Higuchi, Yujiro; Hashiguchi, Akihiro; Nakamura, Tomonori; Nozuma, Satoshi; Sakiyama, Yusuke; Yoshimura, Akiko; Izumo, Shuji; Takashima, Hiroshi

    2013-04-30

    To identify the clinical features of Japanese patients with suspected hereditary sensory and autonomic neuropathy (HSAN) on the basis of genetic diagnoses. On the basis of clinical, in vivo electrophysiologic, and pathologic findings, 9 Japanese patients with sensory and autonomic nervous dysfunctions were selected. Eleven known HSAN disease-causing genes and 5 related genes were screened using a next-generation sequencer. A homozygous mutation, c.3993delGinsTT, was identified in exon 22 of SCN9A from 2 patients/families. The clinical phenotype was characterized by adolescent or congenital onset with loss of pain and temperature sensation, autonomic nervous dysfunctions, hearing loss, and hyposmia. Subsequently, this mutation was discovered in one of patient 1's sisters, who also exhibited sensory and autonomic nervous system dysfunctions, with recurrent fractures being the most predominant feature. Nerve conduction studies revealed definite asymmetric sensory nerve involvement in patient 1. In addition, sural nerve pathologic findings showed loss of large myelinated fibers in patient 1, whereas the younger patient showed normal sural nerve pathology. We identified a novel homozygous mutation in SCN9A from 2 Japanese families with autosomal recessive HSAN. This loss-of-function SCN9A mutation results in disturbances in the sensory, olfactory, and autonomic nervous systems. We propose that SCN9A mutation results in the new entity of HSAN type IID, with additional symptoms including hyposmia, hearing loss, bone dysplasia, and hypogeusia.

  12. TWIST1 a new determinant of epithelial to mesenchymal transition in EGFR mutated lung adenocarcinoma.

    Directory of Open Access Journals (Sweden)

    Karine Pallier

    Full Text Available Metastasis is a multistep process and the main cause of mortality in lung cancer patients. We previously showed that EGFR mutations were associated with a copy number gain at a locus encompassing the TWIST1 gene on chromosome 7. TWIST1 is a highly conserved developmental gene involved in embryogenesis that may be reactivated in cancers promoting both malignant conversion and cancer progression through an epithelial to mesenchymal transition (EMT. The aim of this study was to investigate the possible implication of TWIST1 reactivation on the acquisition of a mesenchymal phenotype in EGFR mutated lung cancer. We studied a series of consecutive lung adenocarcinoma from Caucasian non-smokers for which surgical frozen samples were available (n = 33 and showed that TWIST1 expression was linked to EGFR mutations (P<0.001, to low CDH1 expression (P<0.05 and low disease free survival (P = 0.044. To validate that TWIST1 is a driver of EMT in EGFR mutated lung cancer, we used five human lung cancer cell lines and demonstrated that EMT and the associated cell mobility were dependent upon TWIST1 expression in cells with EGFR mutation. Moreover a decrease of EGFR pathway stimulation through EGF retrieval or an inhibition of TWIST1 expression by small RNA technology reversed the phenomenon. Collectively, our in vivo and in vitro findings support that TWIST1 collaborates with the EGF pathway in promoting EMT in EGFR mutated lung adenocarcinoma and that large series of EGFR mutated lung cancer patients are needed to further define the prognostic role of TWIST1 reactivation in this subgroup.

  13. Discordance between bovine leukemia virus tax immortalization in vitro and oncogenicity in vivo.

    Science.gov (United States)

    Twizere, J C; Kerkhofs, P; Burny, A; Portetelle, D; Kettmann, R; Willems, L

    2000-11-01

    Bovine leukemia virus (BLV) Tax protein, a transcriptional activator of viral expression, is essential for viral replication in vivo. Tax is believed to be involved in leukemogenesis because of its second function, immortalization of primary cells in vitro. These activities of Tax can be dissociated on the basis of point mutations within specific regions of the protein. For example, mutation of the phosphorylation sites at serines 106 and 293 abrogates immortalization potential in vitro but maintains transcriptional activity. This type of mutant is thus particularly useful for unraveling the role of Tax immortalization activity during leukemogenesis independently of viral replication. In this report, we describe the biological properties of BLV recombinant proviruses mutated in the Tax phosphorylation sites (BLVTax106+293). Titration of the proviral loads by semiquantitative PCR revealed that the BLV mutants propagated at wild-type levels in vivo. Furthermore, two animals (sheep 480 and 296) infected with BLVTax106+293 developed leukemia or lymphosarcoma after 16 and 36 months, respectively. These periods of time are within the normal range of latencies preceding the onset of pathogenesis induced by wild-type viruses. The phenotype of the mutant-infected cells was characteristic of a B lymphocyte (immunoglobulin M positive) expressing CD11b and CD5 (except at the final stage for the latter marker), a pattern that is typical of wild-type virus-infected target cells. Interestingly, the transformed B lymphocytes from sheep 480 also coexpressed the CD8 marker, a phenotype rarely observed in tumor biopsies from chronic lymphocytic leukemia patients. Finally, direct sequencing of the tax gene demonstrated that the leukemic cells did not harbor revertant proviruses. We conclude that viruses expressing a Tax mutant unable to transform primary cells in culture are still pathogenic in the sheep animal model. Our data thus provide a clear example of the discordant conclusions

  14. Mutation in the factor VII hepatocyte nuclear factor 4α-binding site contributes to factor VII deficiency.

    Science.gov (United States)

    Zheng, Xing-Wu; Kudaravalli, Rama; Russell, Theresa T; DiMichele, Donna M; Gibb, Constance; Russell, J Eric; Margaritis, Paris; Pollak, Eleanor S

    2011-10-01

    Severe coagulant factor VII (FVII) deficiency in postpubertal dizygotic twin males results from two point mutations in the FVII gene, a promoter region T→C transition at -60 and a His-to-Arg substitution at amino acid 348; both mutations prevent persistence of plasma functional FVII. This report documents longitudinal laboratory measurements from infancy to adulthood of FVII coagulant activity (FVII:C) in the twin FVII-deficient patients; it also details specific biochemical analyses of the -60 T→C mutation. The results revealed FVII:C levels of less than 1% in infancy that remain severely decreased through puberty and into adulthood. In-vitro analyses utilizing hepatocyte nuclear factor 4α (HNF4α) co-transfection and a chromatin immunoprecipitation assay indicate that the -60 T→C mutation severely diminishes functional interaction between the FVII promoter and transcription factor HNF4α. The importance of interaction between the FVII gene and HNF4α in normal FVII expression provides an in-vivo illustration of the regulated expression of an autosomal gene encoding a coagulation protein. The constancy of FVII:C and peripubertal patient symptomatology reported here illustrates androgen-independent expression in contrast to expression with an analogous mutation in the promoter region of the gene encoding coagulation FIX.

  15. KRAS Mutation and Epithelial-Macrophage Interplay in Pancreatic Neoplastic Transformation.

    Science.gov (United States)

    Bishehsari, Faraz; Zhang, Lijuan; Barlass, Usman; Preite, Nailliw; Turturro, Sanja; Najor, Matthew S; Shetuni, Brandon B; Zayas, Janet P; Mahdavinia, Mahboobeh; Abukhdeir, Abde M; Keshavarzian, Ali

    2018-05-14

    Pancreatic ductal adenocarcinoma (PDA) is characterized by epithelial mutations in KRAS and prominent tumor-associated inflammation, including macrophage infiltration. But knowledge of early interactions between neoplastic epithelium and macrophages in PDA carcinogenesis is limited. Using a pancreatic organoid model, we found that the expression of mutant KRAS in organoids increased i) ductal to acinar gene expression ratios, ii) epithelial cells proliferation, and iii) colony formation capacity in vitro, and endowed pancreatic cells with the ability to generate neoplastic tumors in vivo. KRAS mutations induced a pro-tumorigenic phenotype in macrophages. Altered macrophages decreased epithelial Pigment Epithelial Derived Factor (PEDF) expression and induced a cancerous phenotype. We validated our findings using annotated patient samples from The Cancer Genome Atlas (TCGA) as well as in our human PDA specimens. Epithelium-macrophage cross talk occurs early in pancreatic carcinogenesis where KRAS directly induces cancer-related phenotypes in epithelium, and also promotes a pro-tumorigenic phenotype in macrophages, in turn augmenting neoplastic growth. This article is protected by copyright. All rights reserved. © 2018 UICC.

  16. Sentinel and other mutational effects in offspring of cancer survivors

    International Nuclear Information System (INIS)

    Mulvihill, J.J.

    1990-01-01

    To date, no agent has been documented to cause germ cell mutation in human beings, with the possible exception of radiation causing abnormal meiotic chromosomes in testes. For studies in humans, mutation epidemiologists prefer the cohort approach, starting with an exposed population and looking for mutations that may be expressed in offspring as variants in health, chromosomes, proteins, or nucleic acids. Currently patients with cancer are the cohort exposed to the largest doses of potential mutagens, i.e., radiotherapy and drugs. In 12 large studies with over 825 patients and 1573 pregnancies, 46 (4%) of 1240 liveborns had a major birth defect, a rate comparable to that in the general population. One of these was a classic sentinel phenotype, i.e., a new sporadic case of a dominant mendelian syndrome. In collaboration with 5 U.S. cancer registries, we interviewed a retrospective cohort of 2383 patients diagnosed with cancer under age 20 years, from 1945 through 1975. Records were sought to verify major genetic disease, defined as a cytogenetic or single gene disorder or 1 of 15 isolated birth defects. In 2308 offspring of survivors, 5 had a chromosomal syndrome, 11 had a single gene disorder, and 62 had at least one major malformation. Among 4722 offspring of sibling controls, the respective numbers were 7, 12, and 127, nonsignificant differences. 7% of the parents of the offspring with possibly new mutations received potentially mutagenic therapy, compared with 12% of parents of normal children. Since pregnancy in or by cancer survivors is still a rare event, future efforts to document germ cell mutation may be best studied through international cooperation coupled with diverse laboratory measures of mutation

  17. The effect of SOD1 mutation on cellular bioenergetic profile and viability in response to oxidative stress and influence of mutation-type.

    Directory of Open Access Journals (Sweden)

    Katie Richardson

    Full Text Available Amyotrophic Lateral Sclerosis (ALS is a fatal neurodegenerative disorder characterized by the progressive degeneration of motor neurons. Substantial evidence implicates oxidative stress and mitochondrial dysfunction as early events in disease progression. Our aim was to ascertain whether mutation of the SOD1 protein increases metabolic functional susceptibility to oxidative stress. Here we used a motor neuron-like cell line (NSC34 stably transfected with various human mutant SOD1 transgenes (G93A, G37R, H48Q to investigate the impact of oxidative stress on cell viability and metabolic function within intact cells. NSC34 cells expressing mutant SOD1 showed a dose dependent reduction in cell viability when exposed to oxidative stress induced by hydrogen peroxide, with variation between mutations. The G93A transfectants showed greater cell death and LDH release compared to cells transfected with the other SOD1 mutations, and H48Q showed an accelerated decline at later time points. Differences in mitochondrial bioenergetics, including mitochondrial respiration, coupling efficiency and proton leak, were identified between the mutations, consistent with the differences observed in viability. NSC34 cells expressing G93A SOD1 displayed reduced coupled respiration and mitochondrial membrane potential compared to controls. Furthermore, the G93A mutation had significantly increased metabolic susceptibility to oxidative stress, with hydrogen peroxide increasing ROS production, reducing both cellular oxygen consumption and glycolytic flux in the cell. This study highlights bioenergetic defects within a cellular model of ALS and suggests that oxidative stress is not only detrimental to oxygen consumption but also glycolytic flux, which could lead to an energy deficit in the cell.

  18. Spectrum of mutations in homozygous familial hypercholesterolemia in India, with four novel mutations.

    Science.gov (United States)

    Setia, Nitika; Saxena, Renu; Arora, Anjali; Verma, Ishwar C

    2016-12-01

    Homozygous familial hypercholesterolemia (FH) is a rare but serious, inherited disorder of lipid metabolism characterized by very high total and LDL cholesterol levels from birth. It presents as cutaneous and tendon xanthomas since childhood, with or without cardiac involvement. FH is commonly caused by mutations in three genes, i.e. LDL receptor (LDLR), apolipoprotein B (ApoB) and PCSK9. We aimed to determine the spectrum of mutations in cases of homozygous FH in Asian Indians and evaluate if there was any similarity to the mutations observed in Caucasians. Sixteen homozygous FH subjects from eleven families were analyzed for mutations by Sanger sequencing. Large rearrangements in LDLR gene were evaluated by multiplex ligation probe dependent amplification (MLPA) technique. Ten mutations were observed in LDLR gene, of which four mutations were novel. No mutation was detected in ApoB gene and common PCSK9 mutation (p.D374Y). Fourteen cases had homozygous mutations; one had compound heterozygous mutation, while no mutation was detected in one clinically homozygous case. We report an interesting "Triple hit" case with features of homozygous FH. The spectrum of mutations in the Asian Indian population is quite heterogeneous. Of the mutations identified, 40% were novel. No mutation was observed in exons 3, 9 and 14 of LDLR gene, which are considered to be hot spots in studies done on Asian Indians in South Africa. Early detection followed by aggressive therapy, and cascade screening of extended families has been initiated to reduce the morbidity and mortality in these patients. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  19. Exploring sex differences in the adult zebra finch brain: In vivo diffusion tensor imaging and ex vivo super-resolution track density imaging.

    Science.gov (United States)

    Hamaide, Julie; De Groof, Geert; Van Steenkiste, Gwendolyn; Jeurissen, Ben; Van Audekerke, Johan; Naeyaert, Maarten; Van Ruijssevelt, Lisbeth; Cornil, Charlotte; Sijbers, Jan; Verhoye, Marleen; Van der Linden, Annemie

    2017-02-01

    Zebra finches are an excellent model to study the process of vocal learning, a complex socially-learned tool of communication that forms the basis of spoken human language. So far, structural investigation of the zebra finch brain has been performed ex vivo using invasive methods such as histology. These methods are highly specific, however, they strongly interfere with performing whole-brain analyses and exclude longitudinal studies aimed at establishing causal correlations between neuroplastic events and specific behavioral performances. Therefore, the aim of the current study was to implement an in vivo Diffusion Tensor Imaging (DTI) protocol sensitive enough to detect structural sex differences in the adult zebra finch brain. Voxel-wise comparison of male and female DTI parameter maps shows clear differences in several components of the song control system (i.e. Area X surroundings, the high vocal center (HVC) and the lateral magnocellular nucleus of the anterior nidopallium (LMAN)), which corroborate previous findings and are in line with the clear behavioral difference as only males sing. Furthermore, to obtain additional insights into the 3-dimensional organization of the zebra finch brain and clarify findings obtained by the in vivo study, ex vivo DTI data of the male and female brain were acquired as well, using a recently established super-resolution reconstruction (SRR) imaging strategy. Interestingly, the SRR-DTI approach led to a marked reduction in acquisition time without interfering with the (spatial and angular) resolution and SNR which enabled to acquire a data set characterized by a 78μm isotropic resolution including 90 diffusion gradient directions within 44h of scanning time. Based on the reconstructed SRR-DTI maps, whole brain probabilistic Track Density Imaging (TDI) was performed for the purpose of super resolved track density imaging, further pushing the resolution up to 40μm isotropic. The DTI and TDI maps realized atlas

  20. 13-week dietary study and in vitro and in vivo genotoxicity studies of a structuring fat produced through a microalgal fermentation process

    Directory of Open Access Journals (Sweden)

    R.A. Matulka

    Full Text Available Microalgae are increasingly being utilized as food ingredients for a variety of applications, including as sources of protein, egg and dairy substitutes, and cooking oils. The dietary safety of a new structuring fat produced using a heterotrophic fermentation process by a strain of Prototheca moriformis was evaluated in a 13-week dietary toxicity study and compared with kokum fat, a structuring fat of similar composition used in the food industry and derived from Garcinia indica seeds. The algal structuring fat was evaluated for its genotoxic potential using both in vitro and in vivo assays. No treatment-related adverse events occurred in rats consuming algal structuring fat or kokum fat in the 13-week study; no treatment-related effects were reported for body weight, food consumption, urinalysis, hematology, clinical chemistry, gross pathology, organ weights, or histopathology. While statistically significant effects occurred in some parameters, none were dose-related or considered adverse. Overall, the NOAELs for the algal structuring fat and the kokum fat were 100 000 ppm, the highest concentrations tested. The algal structuring fat was not mutagenic in the bacterial reverse mutation assay in the Salmonella typhimurium or Escherichia coli strains tested and was not clastogenic in the in vivo mouse bone marrow chromosome aberration assay. Keywords: Microalgae, Stearic acid, Subchronic toxicity, Kokum, Prototheca moriformis, Structuring fat

  1. AIP mutations in young patients with acromegaly and the Tampico Giant: the Mexican experience.

    Science.gov (United States)

    Ramírez-Rentería, Claudia; Hernández-Ramírez, Laura C; Portocarrero-Ortiz, Lesly; Vargas, Guadalupe; Melgar, Virgilio; Espinosa, Etual; Espinosa-de-Los-Monteros, Ana Laura; Sosa, Ernesto; González, Baldomero; Zúñiga, Sergio; Unterländer, Martina; Burger, Joachim; Stals, Karen; Bussell, Anne-Marie; Ellard, Sian; Dang, Mary; Iacovazzo, Donato; Kapur, Sonal; Gabrovska, Plamena; Radian, Serban; Roncaroli, Federico; Korbonits, Márta; Mercado, Moisés

    2016-08-01

    Although aryl hydrocarbon receptor-interacting protein (AIP) mutations are rare in sporadic acromegaly, their prevalence among young patients is nonnegligible. The objectives of this study were to evaluate the frequency of AIP mutations in a cohort of Mexican patients with acromegaly with disease onset before the age of 30 and to search for molecular abnormalities in the AIP gene in teeth obtained from the "Tampico Giant". Peripheral blood DNA from 71 patients with acromegaly (51 females) with disease onset T (p.Arg304Ter), well-known truncating mutation was identified; in one of these two cases and her identical twin sister, the mutation proved to be a de novo event, since neither of their parents were found to be carriers. In the remaining three patients, new mutations were identified: a frameshift mutation (c.976_977insC, p.Gly326AfsTer), an in-frame deletion (c.872_877del, p.Val291_Leu292del) and a nonsense mutation (c.868A > T, p.Lys290Ter), which are predicted to be pathogenic based on in silico analysis. Patients with AIP mutations tended to have an earlier onset of acromegaly and harboured larger and more invasive tumours. A previously described genetic variant of unknown significance (c.869C > T, p.Ala299Val) was identified in DNA from the Tampico Giant. The prevalence of AIP mutations in young Mexican patients with acromegaly is similar to that of European cohorts. Our results support the need for genetic evaluation of patients with early onset acromegaly.

  2. Contracting in vivo research: what are the issues?

    Science.gov (United States)

    Underwood, Wendy J

    2007-07-01

    As a result of increasing internal and external pressures, research institutions are using contract research organizations for the conduct of in vivo research. Many issues arise when contracting animal research, including concern regarding animal health and welfare. Each sponsor institution should develop a program for outsourced in vivo research that evaluates and ensures appropriate care and use of research animals. Each sponsoring institution should consider establishing a policy and procedure for how outsourced in vivo studies will be approved, conducted, and monitored. An approved list of contract facilities can be established on the basis of accepted standards for animal care and use. Written contracts should include confidentiality agreements, the delineation of animal ownership, and the expectation to comply with all applicable regulations and guidelines for research animal care and use. Finally, a process for communication of adverse study or animal welfare events should be established. Thorough evaluation of contract organizations will help ensure appropriate research animal care and use.

  3. JAK2 V617F, MPL, and CALR mutations in essential thrombocythaemia and major thrombotic complications: a single-institute retrospective analysis.

    Science.gov (United States)

    Pósfai, Éva; Marton, Imelda; Király, Péter Attila; Kotosz, Balázs; Kiss-László, Zsuzsanna; Széll, Márta; Borbényi, Zita

    2015-07-01

    Thrombo-haemorrhagic events are the main cause of morbidity and mortality in essential thrombocythemia. The aim of this study was to estimate the incidence of thrombotic events and the impact of the JAK2V617F, MPL (W515L, W515K, W515R, W515A and S505N) and CALR (type-1, type-2) mutations on 101 essential thrombocythaemia patients (72 females and 29 males with a mean age of 61 years) diagnosed in a Southern Hungarian regional academic centre. The incidence of major thrombosis was 13.86 %. Sixty percent of the patients carried the JAK2V617F mutation. The MPL mutations were analysed by sequencing and the W515L was the only one we could identify with an incidence of 3.96 %. Type-2 CALR mutation could be identified in 3 cases among the patients who had JAK2/MPL-unmutated ET. Statistical analyses revealed that the JAK2V617F mutation was associated with significantly increased levels of platelet (p = 0.042), haemoglobin (p = 0.000), red blood cell (p = 0.000) and haematocrit (p = 0.000) and hepatomegaly (p = 0.045) at diagnosis compared to JAK2V617F negative counterparts, however there was no significant association between the JAK2V617F mutation status (relative risk: 1.297, 95 % CI 0.395-4.258; p = 0.668) and subsequent thrombotic complications. The impact of JAK2V617F, MPL W515L and CALR mutations on the clinical findings at the diagnosis of ET was obvious, but their statistically significant role in the prediction of thrombotic events could not be proven in this study. Our results indirectly support the concept that, besides the quantitative and qualitative changes in the platelets, the mechanisms leading to thrombosis are more complex and multifactorial.

  4. Mutant IDH1 Promotes Glioma Formation In Vivo

    Directory of Open Access Journals (Sweden)

    Beatrice Philip

    2018-05-01

    Full Text Available Summary: Isocitrate dehydrogenase 1 (IDH1 is the most commonly mutated gene in grade II–III glioma and secondary glioblastoma (GBM. A causal role for IDH1R132H in gliomagenesis has been proposed, but functional validation in vivo has not been demonstrated. In this study, we assessed the role of IDH1R132H in glioma development in the context of clinically relevant cooperating genetic alterations in vitro and in vivo. Immortal astrocytes expressing IDH1R132H exhibited elevated (R-2-hydroxyglutarate levels, reduced NADPH, increased proliferation, and anchorage-independent growth. Although not sufficient on its own, IDH1R132H cooperated with PDGFA and loss of Cdkn2a, Atrx, and Pten to promote glioma development in vivo. These tumors resembled proneural human mutant IDH1 GBM genetically, histologically, and functionally. Our findings support the hypothesis that IDH1R132H promotes glioma development. This model enhances our understanding of the biology of IDH1R132H-driven gliomas and facilitates testing of therapeutic strategies designed to combat this deadly disease. : Philip et al. show that mutant IDH1 cooperates with PDGFA and loss of Cdkn2a, Atrx, and Pten to promote gliomagenesis in vivo in a mouse model of glioma. These tumors resemble proneural human mutant IDH1 glioblastoma and exhibit enhanced sensitivity to PARP inhibition in combination with chemotherapy. Keywords: IDH1, Cdkn2a, Atrx, Pten, glioma, mouse model, RCAS/TVA

  5. Epidermal growth factor receptor (EGFR) mutations in lung cancer: preclinical and clinical data

    Energy Technology Data Exchange (ETDEWEB)

    Jorge, S.E.D.C.; Kobayashi, S.S.; Costa, D.B. [Harvard Medical School, Beth Israel Deaconess Medical Center, Department of Medicine, Division of Hematology/Oncology, Boston, MA (United States)

    2014-09-05

    Lung cancer leads cancer-related mortality worldwide. Non-small-cell lung cancer (NSCLC), the most prevalent subtype of this recalcitrant cancer, is usually diagnosed at advanced stages, and available systemic therapies are mostly palliative. The probing of the NSCLC kinome has identified numerous nonoverlapping driver genomic events, including epidermal growth factor receptor (EGFR) gene mutations. This review provides a synopsis of preclinical and clinical data on EGFR mutated NSCLC and EGFR tyrosine kinase inhibitors (TKIs). Classic somatic EGFR kinase domain mutations (such as L858R and exon 19 deletions) make tumors addicted to their signaling cascades and generate a therapeutic window for the use of ATP-mimetic EGFR TKIs. The latter inhibit these kinases and their downstream effectors, and induce apoptosis in preclinical models. The aforementioned EGFR mutations are stout predictors of response and augmentation of progression-free survival when gefitinib, erlotinib, and afatinib are used for patients with advanced NSCLC. The benefits associated with these EGFR TKIs are limited by the mechanisms of tumor resistance, such as the gatekeeper EGFR-T790M mutation, and bypass activation of signaling cascades. Ongoing preclinical efforts for treating resistance have started to translate into patient care (including clinical trials of the covalent EGFR-T790M TKIs AZD9291 and CO-1686) and hold promise to further boost the median survival of patients with EGFR mutated NSCLC.

  6. Epidermal growth factor receptor (EGFR) mutations in lung cancer: preclinical and clinical data

    International Nuclear Information System (INIS)

    Jorge, S.E.D.C.; Kobayashi, S.S.; Costa, D.B.

    2014-01-01

    Lung cancer leads cancer-related mortality worldwide. Non-small-cell lung cancer (NSCLC), the most prevalent subtype of this recalcitrant cancer, is usually diagnosed at advanced stages, and available systemic therapies are mostly palliative. The probing of the NSCLC kinome has identified numerous nonoverlapping driver genomic events, including epidermal growth factor receptor (EGFR) gene mutations. This review provides a synopsis of preclinical and clinical data on EGFR mutated NSCLC and EGFR tyrosine kinase inhibitors (TKIs). Classic somatic EGFR kinase domain mutations (such as L858R and exon 19 deletions) make tumors addicted to their signaling cascades and generate a therapeutic window for the use of ATP-mimetic EGFR TKIs. The latter inhibit these kinases and their downstream effectors, and induce apoptosis in preclinical models. The aforementioned EGFR mutations are stout predictors of response and augmentation of progression-free survival when gefitinib, erlotinib, and afatinib are used for patients with advanced NSCLC. The benefits associated with these EGFR TKIs are limited by the mechanisms of tumor resistance, such as the gatekeeper EGFR-T790M mutation, and bypass activation of signaling cascades. Ongoing preclinical efforts for treating resistance have started to translate into patient care (including clinical trials of the covalent EGFR-T790M TKIs AZD9291 and CO-1686) and hold promise to further boost the median survival of patients with EGFR mutated NSCLC

  7. The Role of gsp Mutations on the Development of Adrenal Cortical Tumors and Adrenal Hyperplasias

    Directory of Open Access Journals (Sweden)

    Maria Candida Barisson Villares Fragoso

    2016-07-01

    Full Text Available Somatic GNAS point mutations, commonly known as gsp mutations, are involved in the pathogenesis of McCune Albright syndrome and have also been described in autonomous hormone-producing tumors, such as somatotropinoma, corticotrophoma, thyroid cancer, ovarian and testicular Leydig cell tumors and primary macronodular adrenocortical hyperplasia (PMAH. [1-3]The involvement of gsp mutations in adrenal tumors was first described by Lyons et al. in 1990. Since then, several studies have detected the presence of gsp mutations in adrenal tumors, but none of them could explain its presence along or the mechanism that leads to tumor formation and hormone hypersecretion. As a result, the molecular pathogenesis of the majority of sporadic adrenocortical tumors remains unclear. [3] PMAH has also been reported with gsp somatic mutations in a few cases. Fragoso et al. in 2003 identified two distinct gsp somatic mutations affecting arginine residues on codon 201 of GNAS in a few patients with PMAH who lacked any features or manifestations of McCune Albright syndrome. Followed by this discovery, other studies have continued looking for gsp mutations based on strong prior evidence demonstrating that increased cAMP signaling is sufficient for cell proliferation and cortisol production. [2, 4] With consideration for the previously reported findings, we conjecture that although somatic activating mutations in GNAS are a rare molecular event, these mutations could probably be sufficient to induce the development of macronodule hyperplasia and variable cortisol secretion.In this manuscript, we revised the presence of gsp mutations associated with adrenal cortical tumors and hyperplasia.

  8. Mutation Supply and Relative Fitness Shape the Genotypes of Ciprofloxacin-Resistant Escherichia coli.

    Science.gov (United States)

    Huseby, Douglas L; Pietsch, Franziska; Brandis, Gerrit; Garoff, Linnéa; Tegehall, Angelica; Hughes, Diarmaid

    2017-05-01

    Ciprofloxacin is an important antibacterial drug targeting Type II topoisomerases, highly active against Gram-negatives including Escherichia coli. The evolution of resistance to ciprofloxacin in E. coli always requires multiple genetic changes, usually including mutations affecting two different drug target genes, gyrA and parC. Resistant mutants selected in vitro or in vivo can have many different mutations in target genes and efflux regulator genes that contribute to resistance. Among resistant clinical isolates the genotype, gyrA S83L D87N, parC S80I is significantly overrepresented suggesting that it has a selective advantage. However, the evolutionary or functional significance of this high frequency resistance genotype is not fully understood. By combining experimental data and mathematical modeling, we addressed the reasons for the predominance of this specific genotype. The experimental data were used to model trajectories of mutational resistance evolution under different conditions of drug exposure and population bottlenecks. We identified the order in which specific mutations are selected in the clinical genotype, showed that the high frequency genotype could be selected over a range of drug selective pressures, and was strongly influenced by the relative fitness of alternative mutations and factors affecting mutation supply. Our data map for the first time the fitness landscape that constrains the evolutionary trajectories taken during the development of clinical resistance to ciprofloxacin and explain the predominance of the most frequently selected genotype. This study provides strong support for the use of in vitro competition assays as a tool to trace evolutionary trajectories, not only in the antibiotic resistance field. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  9. MSH6 mutations arise in glioblastomas during temozolomide therapy and mediate temozolomide resistance

    Science.gov (United States)

    Yip, Stephen; Miao, Jiangyong; Cahill, Daniel P.; Iafrate, A. John; Aldape, Ken; Nutt, Catherine L.; Louis, David N.

    2009-01-01

    Purpose Over the past few years, the alkylating agent temozolomide (TMZ) has become the standard-of-care therapy for patients with glioblastoma, the most common brain tumor. Recently, large-scale cancer genome sequencing efforts have identified a hypermutation phenotype and inactivating MSH6 mismatch repair gene mutations in recurrent, post-TMZ glioblastomas, particularly those growing more rapidly during TMZ treatment. This study aimed to clarify the timing and role of MSH6 mutations in mediating glioblastoma TMZ resistance. Experimental Design MSH6 sequence and microsatellite instability (MSI) status were determined in matched pre- and post-chemotherapy glioblastomas identified by The Cancer Genome Atlas (TCGA) as having post-treatment MSH6 mutations. TMZ-resistant lines were derived in vitro via selective growth under TMZ and the MSH6 gene was sequenced in resistant clones. The role of MSH6 inactivation in mediating resistance was explored using lentiviral shRNA knockdown and MSH6 reconstitution. Results MSH6 mutations were confirmed in post-treatment TCGA glioblastomas but absent in matched pre-treatment tumors. The post-treatment hypermutation phenotype displayed a signature bias toward CpC transitions and was not associated with MSI. In vitro modeling via exposure of an MSH6-wildtype glioblastoma line to TMZ resulted in resistant clones; one clone showed an MSH6 mutation, Thr1219Ile, that had been independently noted in two treated TCGA glioblastomas. Knockdown of MSH6 in the glioblastoma line U251 increased resistance to TMZ cytotoxicity and reconstitution restored cytotoxicity in MSH6-null glioma cells. Conclusions MSH6 mutations are selected for in glioblastomas during TMZ therapy both in vitro and in vivo, and are causally associated with TMZ resistance. PMID:19584161

  10. Identification of syncytial mutations in a clinical isolate of herpes simplex virus 2

    International Nuclear Information System (INIS)

    Muggeridge, Martin I.; Grantham, Michael L.; Johnson, F. Brent

    2004-01-01

    Small polykaryocytes resulting from cell fusion are found in herpes simplex virus (HSV) lesions in patients, but their significance for viral spread and pathogenesis is unclear. Although syncytial variants causing extensive fusion in tissue culture can be readily isolated from laboratory strains, they are rarely found in clinical isolates, suggesting that extensive cell fusion may be deleterious in vivo. Syncytial mutations have previously been identified for several laboratory strains, but not for clinical isolates of HSV type 2. To address this deficiency, we studied a recent syncytial clinical isolate, finding it to be a mixture of two syncytial and one nonsyncytial strain. The two syncytial strains have novel mutations in glycoprotein B, and in vitro cell fusion assays confirmed that they are responsible for syncytium formation. This panel of clinical strains may be ideal for examining the effect of increased cell fusion on pathogenesis

  11. TP53 Mutations in Nonsmall Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Akira Mogi

    2011-01-01

    Full Text Available The tumor suppressor gene TP53 is frequently mutated in human cancers. Abnormality of the TP53 gene is one of the most significant events in lung cancers and plays an important role in the tumorigenesis of lung epithelial cells. Human lung cancers are classified into two major types, small cell lung cancer (SCLC and nonsmall cell lung cancer (NSCLC. The latter accounts for approximately 80% of all primary lung cancers, and the incidence of NSCLC is increasing yearly. Most clinical studies suggest that NSCLC with TP53 alterations carries a worse prognosis and may be relatively more resistant to chemotherapy and radiation. A deep understanding of the role of TP53 in lung carcinogenesis may lead to a more reasonably targeted clinical approach, which should be exploited to enhance the survival rates of patients with lung cancer. This paper will focus on the role of TP53 in the molecular pathogenesis, epidemiology, and therapeutic strategies of TP53 mutation in NSCLC.

  12. Gain and loss of function of ALS-related mutations of TARDBP (TDP-43) cause motor deficits in vivo.

    Science.gov (United States)

    Kabashi, Edor; Lin, Li; Tradewell, Miranda L; Dion, Patrick A; Bercier, Valérie; Bourgouin, Patrick; Rochefort, Daniel; Bel Hadj, Samar; Durham, Heather D; Vande Velde, Christine; Rouleau, Guy A; Drapeau, Pierre

    2010-02-15

    TDP-43 has been found in inclusion bodies of multiple neurological disorders, including amyotrophic lateral sclerosis, frontotemporal dementia, Parkinson's disease and Alzheimer's disease. Mutations in the TDP-43 encoding gene, TARDBP, have been subsequently reported in sporadic and familial ALS patients. In order to investigate the pathogenic nature of these mutants, the effects of three consistently reported TARDBP mutations (A315T, G348C and A382T) were tested in cell lines, primary cultured motor neurons and living zebrafish embryos. Each of the three mutants and wild-type (WT) human TDP-43 localized to nuclei when expressed in COS1 and Neuro2A cells by transient transfection. However, when expressed in motor neurons from dissociated spinal cord cultures these mutant TARDBP alleles, but less so for WT TARDBP, were neurotoxic, concomitant with perinuclear localization and aggregation of TDP-43. Finally, overexpression of mutant, but less so of WT, human TARDBP caused a motor phenotype in zebrafish (Danio rerio) embryos consisting of shorter motor neuronal axons, premature and excessive branching as well as swimming deficits. Interestingly, knock-down of zebrafisfh tardbp led to a similar phenotype, which was rescued by co-expressing WT but not mutant human TARDBP. Together these approaches showed that TARDBP mutations cause motor neuron defects and toxicity, suggesting that both a toxic gain of function as well as a novel loss of function may be involved in the molecular mechanism by which mutant TDP-43 contributes to disease pathogenesis.

  13. CRISPRscan: designing highly efficient sgRNAs for CRISPR/Cas9 targeting in vivo

    Science.gov (United States)

    Moreno-Mateos, Miguel A.; Vejnar, Charles E.; Beaudoin, Jean-Denis; Fernandez, Juan P.; Mis, Emily K.; Khokha, Mustafa K.; Giraldez, Antonio J.

    2015-01-01

    CRISPR/Cas9 technology provides a powerful system for genome engineering. However, variable activity across different single guide RNAs (sgRNAs) remains a significant limitation. We have analyzed the molecular features that influence sgRNA stability, activity and loading into Cas9 in vivo. We observe that guanine enrichment and adenine depletion increase sgRNA stability and activity, while loading, nucleosome positioning and Cas9 off-target binding are not major determinants. We additionally identified truncated and 5′ mismatch-containing sgRNAs as efficient alternatives to canonical sgRNAs. Based on these results, we created a predictive sgRNA-scoring algorithm (CRISPRscan.org) that effectively captures the sequence features affecting Cas9/sgRNA activity in vivo. Finally, we show that targeting Cas9 to the germ line using a Cas9-nanos-3′-UTR fusion can generate maternal-zygotic mutants, increase viability and reduce somatic mutations. Together, these results provide novel insights into the determinants that influence Cas9 activity and a framework to identify highly efficient sgRNAs for genome targeting in vivo. PMID:26322839

  14. High-risk Long QT Syndrome Mutations in the Kv7.1 (KCNQ1) Pore Disrupt the Molecular Basis for Rapid K+ Permeation

    Science.gov (United States)

    Burgess, Don E.; Bartos, Daniel C.; Reloj, Allison R.; Campbell, Kenneth S.; Johnson, Jonathan N.; Tester, David J.; Ackerman, Michael J.; Fressart, Véronique; Denjoy, Isabelle; Guicheney, Pascale; Moss, Arthur J.; Ohno, Seiko; Horie, Minoru; Delisle, Brian P.

    2012-01-01

    Type 1 long QT syndrome (LQT1) syndrome is caused by loss-of-function mutations in the KCNQ1, which encodes the K+ channel (Kv7.1) that underlies the slowly activating delayed rectifier K+ current in the heart. Intragenic risk stratification suggests LQT1 mutations that disrupt conserved amino acid residues in the pore are an independent risk factor for LQT1-related cardiac events. The purpose of this study is to determine possible molecular mechanisms that underlie the loss-of-function for these high-risk mutations. Extensive genotype-phenotype analyses of LQT1 patients showed that T322M-, T322A-, or G325R-Kv7.1 confer a high risk for LQT1-related cardiac events. Heterologous expression of these mutations with KCNE1 revealed they generated non-functional channels and caused dominant negative suppression of WT-Kv7.1 current. Molecular dynamic simulations (MDS) of analogous mutations in KcsA (T85M-, T85A-, and G88R-KcsA) demonstrated that they disrupted the symmetrical distribution of the carbonyl oxygen atoms in the selectivity filter, which upset the balance between the strong attractive and K+-K+ repulsive forces required for rapid K+ permeation. We conclude high-risk LQT1 mutations in the pore likely disrupt the architectural and physical properties of the K+ channel selectivity filter. PMID:23092362

  15. High-risk long QT syndrome mutations in the Kv7.1 (KCNQ1) pore disrupt the molecular basis for rapid K(+) permeation.

    Science.gov (United States)

    Burgess, Don E; Bartos, Daniel C; Reloj, Allison R; Campbell, Kenneth S; Johnson, Jonathan N; Tester, David J; Ackerman, Michael J; Fressart, Véronique; Denjoy, Isabelle; Guicheney, Pascale; Moss, Arthur J; Ohno, Seiko; Horie, Minoru; Delisle, Brian P

    2012-11-13

    Type 1 long QT syndrome (LQT1) is caused by loss-of-function mutations in the KCNQ1 gene, which encodes the K(+) channel (Kv7.1) that underlies the slowly activating delayed rectifier K(+) current in the heart. Intragenic risk stratification suggests LQT1 mutations that disrupt conserved amino acid residues in the pore are an independent risk factor for LQT1-related cardiac events. The purpose of this study is to determine possible molecular mechanisms that underlie the loss of function for these high-risk mutations. Extensive genotype-phenotype analyses of LQT1 patients showed that T322M-, T322A-, or G325R-Kv7.1 confers a high risk for LQT1-related cardiac events. Heterologous expression of these mutations with KCNE1 revealed they generated nonfunctional channels and caused dominant negative suppression of WT-Kv7.1 current. Molecular dynamics simulations of analogous mutations in KcsA (T85M-, T85A-, and G88R-KcsA) demonstrated that they disrupted the symmetrical distribution of the carbonyl oxygen atoms in the selectivity filter, which upset the balance between the strong attractive and K(+)-K(+) repulsive forces required for rapid K(+) permeation. We conclude high-risk LQT1 mutations in the pore likely disrupt the architectural and physical properties of the K(+) channel selectivity filter.

  16. Characterization of a mutation commonly associated with persistent stuttering: evidence for a founder mutation

    Science.gov (United States)

    Fedyna, Alison; Drayna, Dennis; Kang, Changsoo

    2010-01-01

    Stuttering is a disorder which affects the fluency of speech. It has been shown to have high heritability, and has recently been linked to mutations in the GNPTAB gene. One such mutation, Glu1200Lys, has been repeatedly observed in unrelated families and individual cases. Eight unrelated individuals carrying this mutation were analyzed in an effort to distinguish whether these arise from repeated mutation at the same site, or whether they represent a founder mutation with a single origin. Results show that all 12 chromosomes carrying this mutation share a common haplotype in this region, indicating it is a founder mutation. Further analysis estimated the age of this allele to be ~572 generations. Construction of a cladogram tracing the mutation through our study sample also supports the founder mutation hypothesis. PMID:20944643

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

    Directory of Open Access Journals (Sweden)

    Valentina Guerrini

    Full Text Available 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.

  18. MET amplification, expression, and exon 14 mutations in colorectal adenocarcinoma.

    Science.gov (United States)

    Zhang, Meng; Li, Guichao; Sun, Xiangjie; Ni, Shujuan; Tan, Cong; Xu, Midie; Huang, Dan; Ren, Fei; Li, Dawei; Wei, Ping; Du, Xiang

    2018-04-08

    MET amplification, expression, and splice mutations at exon 14 result in dysregulation of the MET signaling pathway. The aim of this study was to identify the relationship between MET amplification, protein or mRNA expression, and mutations in colorectal cancer (CRC). MET immunohistochemistry (IHC) was used for MET protein expression analysis and fluorescence in situ hybridization (FISH) was used for MET amplification detection. Both analyses were performed in tissue microarrays (TMA) containing 294 of colorectal adenocarcinoma tissue samples and 131 samples of adjacent normal epithelial tissue. MET mRNA expression was examined by real-time quantitative polymerase chain reaction (qRT-PCR) in 72 fresh colorectal adenocarcinoma tissue samples and adjacent normal colon tissue. PCR sequencing was performed to screen for MET exon 14 splice mutations in 59 fresh CRC tissue samples. Our results showed that MET protein expression was higher in colorectal tumor tissue than in adjacent normal intestinal epithelium. Positive MET protein expression was associated with significantly poorer overall survival (OS) and disease-free survival (DFS). Multivariate analysis revealed that positive MET protein expression was an independent risk factor for DFS, but not for OS. MET mRNA expression was upregulated in tumor tissues compared with the adjacent normal tissues. The incidence of MET amplification was 4.4%. None of the patients was positive for MET mutation. Collectively, MET was overexpressed in colorectal adenocarcinoma, and its positive protein expression predicted a poorer outcome in CRC patients. Furthermore, according to our results, MET amplification and 14 exon mutation are extremely rare events in colorectal adenocarcinoma. Copyright © 2018. Published by Elsevier Inc.

  19. Risk-reducing mastectomy and salpingo-oophorectomy in unaffected BRCA mutation carriers: uptake and timing

    DEFF Research Database (Denmark)

    Skytte, A-B; Gerdes, Anne-Marie Axø; Andersen, M K

    2010-01-01

    from 306 healthy BRCA carriers with no personal history of ovarian or breast cancer. We found a 10-year uptake of 75% for risk-reducing salpingo-oophorectomy and 50% for risk-reducing mastectomy by time to event analysis. Age and childbirth influenced this decision. The uptake rate has not changed......Once female carriers of a BRCA mutation are identified they have to make decisions on risk management. The aim of this study is to outline the uptake of risk-reducing surgery in the Danish population of BRCA mutation positive women and to search for factors affecting this decision. We analysed data...

  20. Interleukin-7 receptor-α gene mutations are not detected in adult T-cell acute lymphoblastic leukemia

    International Nuclear Information System (INIS)

    Rozovski, Uri; Li, Ping; Harris, David; Ohanian, Maro; Kantarjian, Hagop; Estrov, Zeev

    2014-01-01

    Somatic mutations in cancer cell genes are classified according to their functional significance. Those that provide the malignant cells with significant advantage are collectively referred to as driver mutations and those that do not, are the passenger mutations. Accordingly, analytical criteria to distinguish driver mutations from passenger mutations have been recently suggested. Recent studies revealed mutations in interleukin-7 receptor-α (IL7R) gene in 10% of pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients and in only a few cases of pediatric B-ALL. IL7R mutations are also frequently found in patients with lung cancer, but whereas in pediatric T-ALL IL7R mutations are “drivers” (consisting of gain-of-function mutations within a narrow 50-base pair interval at exon 6 that confer cytokine-independent cell growth and promote tumor transformation), in lung cancer, mutations are substitution mutations randomly distributed across the gene and are probably only “passenger” events. Because the treatment response of adult T-ALL is significantly poorer than that of childhood T-ALL and because exon 6 IL7R mutations play a role in the pathogenesis of childhood T-ALL, we sought to determine how the pattern of IL7R mutations varies between adult and childhood T-ALL. To that end, we sequenced the 50-base pair interval in exon 6 of the IL7R of DNA obtained from bone marrow samples of 35 randomly selected adult patients with T-ALL. Our analysis revealed that none of these 35 samples carried an IL7R mutation in exon 6. Whether differences in the genetic makeup of adult and childhood T-ALL explain the differential response to therapy remains to be determined

  1. Measuring in-vivo and in-situ ex-vivo the 3D deformation of the lamina cribrosa microstructure under elevated intraocular pressure

    Science.gov (United States)

    Wei, Junchao; Yang, Bin; Voorhees, Andrew P.; Tran, Huong; Brazile, Bryn; Wang, Bo; Schuman, Joel; Smith, Matthew A.; Wollstein, Gadi; Sigal, Ian A.

    2018-02-01

    Elevated intraocular pressure (IOP) deforms the lamina cribrosa (LC), a structure within the optic nerve head (ONH) in the back of the eye. Evidence suggests that these deformations trigger events that eventually cause irreversible blindness, and have therefore been studied in-vivo using optical coherence tomography (OCT), and ex-vivo using OCT and a diversity of techniques. To the best of our knowledge, there have been no in-situ ex-vivo studies of LC mechanics. Our goal was two-fold: to introduce a technique for measuring 3D LC deformations from OCT, and to determine whether deformations of the LC induced by elevated IOP differ between in-vivo and in-situ ex-vivo conditions. A healthy adult rhesus macaque monkey was anesthetized and IOP was controlled by inserting a 27- gauge needle into the anterior chamber of the eye. Spectral domain OCT was used to obtain volumetric scans of the ONH at normal and elevated IOPs. To improve the visibility of the LC microstructure the scans were first processed using a novel denoising technique. Zero-normalized cross-correlation was used to find paired corresponding locations between images. For each location pair, the components of the 3D strain tensor were determined using non-rigid image registration. A mild IOP elevation from 10 to 15mmHg caused LC effective strains as large as 3%, and about 50% larger in-vivo than in-situ ex-vivo. The deformations were highly heterogeneous, with substantial 3D components, suggesting that accurate measurement of LC microstructure deformation requires high-resolution volumes. This technique will help improve understanding of LC biomechanics and how IOP contributes to glaucoma.

  2. Delayed manifestation and transmission bias of de novo chromosome mutations. Their relevance for radiation health effect

    International Nuclear Information System (INIS)

    Sasaki, Masao S.

    2006-01-01

    The origin and transmission of de novo chromosome mutations were reviewed on the basis of our chromosome studies in retinoblastoma patients and male infertility. In a series of 264 sporadic retinoblastoma families, gross chromosome rearrangements involving the RB1 locus were identified in 23 cases (8.7%), of which 16 were non-mosaic and 7 were mosaic mutations. The newly formed chromosome mutations, whether they were non-mosaic or mosaic, had a strong bias towards paternally derived chromosome, indicating that they shared a common mechanism where a pre-mutational event or instability is carried over to zygote by sperm and manifested as gross chromosome mutation at the early stages of development. The de novo chromosome mutations are preferentially transmitted through female carriers. This transmission bias is consistent with the finding of higher frequencies of translocation carriers in infertile men (7.69% versus 0.27% in general populations) in whom meiotic progression is severely suppressed, possibly through activation of meiotic checkpoints. Such a meiotic surveillance mechanism may minimize the spreading of newly-arisen chromosome mutations in populations. A quantitative model of meiotic surveillance mechanism is proposed and successfully applied to the published data on ''humped'' dose-response curves for radiation-induced spermatogonial reciprocal translocations in several mammalian species. (author)

  3. Introduction of a point mutation into an HLA class I single-chain trimer induces enhancement of CTL priming and antitumor immunity

    Directory of Open Access Journals (Sweden)

    Masanori Matsui

    2014-01-01

    Full Text Available We previously discovered one particular HLA-A*02:01 mutant that enhanced peptide-specific cytotoxic T lymphocyte (CTL recognition in vitro compared to wild-type HLA-A*02:01. This mutant contains a single amino acid substitution from histidine to leucine at position 74 (H74L that is located in the peptide-binding groove. To investigate the effect of the H74L mutation on the in vivo CTL priming, we took advantage of the technology of the HLA class I single-chain trimer (SCT in which three components involving a peptide, β2 microglobulin and the HLA class I heavy chain are joined together via flexible linkers. We generated recombinant adenovirus expressing SCT comprised influenza A matrix protein (FMP-derived peptide, β2 microglobulin and the H74L heavy chain. HLA-A*02:01 transgenic mice were immunized with the adenovirus, and the induction of peptide-specific CTLs and antitumor immunity was investigated. It was clearly shown that the H74L mutation enabled the HLA-A*02:01 SCT molecule to dramatically enhance both in vivo priming of FMP-specific CTLs and protection against a lethal challenge of tumor cells expressing FMP. These data present the first evidence that a simple point mutation in the HLA class I heavy chain of SCT is beneficial for improving CTL-based immunotherapy and prophylaxis to control tumors.

  4. Maintenance of EGFR and EGFRvIII expressions in an in vivo and in vitro model of human glioblastoma multiforme

    DEFF Research Database (Denmark)

    Stockhausen, Marie-Thérése; Broholm, Helle; Villingshøj, Mette

    2011-01-01

    Glioblastoma multiforme (GBM) is the most common, and most aggressive primary brain tumor among adults. A vast majority of the tumors express high levels of the epidermal growth factor receptor (EGFR) as a consequence of gene amplification. Furthermore, gene amplification is often associated...... with mutation of EGFR, and the constitutive activated deletion variant EGFRvIII is the most common EGFR mutation found in GBM. Activated EGFR signaling, through overexpression and/or mutation, is involved in increased tumorigenic potential. As such, EGFR is an attractive target for GBM therapy. However......, clinical studies with EGFR inhibitors have shown inconsistent results, and as such, further knowledge regarding the role of EGFR and EGFRvIII in GBM is needed. For this, an appropriate in vivo/in vitro tumor model is required. Here, we report the establishment of an experimental GBM model in which...

  5. A Semantic Web-based System for Mining Genetic Mutations in Cancer Clinical Trials.

    Science.gov (United States)

    Priya, Sambhawa; Jiang, Guoqian; Dasari, Surendra; Zimmermann, Michael T; Wang, Chen; Heflin, Jeff; Chute, Christopher G

    2015-01-01

    Textual eligibility criteria in clinical trial protocols contain important information about potential clinically relevant pharmacogenomic events. Manual curation for harvesting this evidence is intractable as it is error prone and time consuming. In this paper, we develop and evaluate a Semantic Web-based system that captures and manages mutation evidences and related contextual information from cancer clinical trials. The system has 2 main components: an NLP-based annotator and a Semantic Web ontology-based annotation manager. We evaluated the performance of the annotator in terms of precision and recall. We demonstrated the usefulness of the system by conducting case studies in retrieving relevant clinical trials using a collection of mutations identified from TCGA Leukemia patients and Atlas of Genetics and Cytogenetics in Oncology and Haematology. In conclusion, our system using Semantic Web technologies provides an effective framework for extraction, annotation, standardization and management of genetic mutations in cancer clinical trials.

  6. Detection of K76T Mutation in pfcrt Gene as an Applicable Ge-netic Marker for Prediction of Chloroquine Resistant falciparum Malaria in Isolates from an Endemic District of Iran

    Directory of Open Access Journals (Sweden)

    A Raeisi

    2008-04-01

    Full Text Available Background: This study investigated the association between pfcrt, T76 allele and chloroquine resistance in patients with falciparum malaria. Molecular assays for point mutations on drugs resistance-related genes are applied tools for monitoring emerging resistance and surveillance malaria control strategies in endemic areas. The mutant genotype at codon 76 of Plasmodium falciparum chloroquine resistance transporter gene (pfcrt has been proposed as a molecular marker for the faster detection of chloroquine resistance in field. Methods: In 64 samples from patients with uncomplicated falciparum malaria from Sarbaz district in southeast of Iran,  the clinical response to chloroquine and the prevalence of K76T  mutations in pfcrt gene were investigated by in vivo and nested-PCR  followed restriction enzyme digestion methods. Results:  The occurrence of the K76T mutation was very high (60 of 64, i.e. 93.75% among these filed isolates. Only 4 of 64 isolates harbored wild type K76 codon and no case was a mixed of K76 and 76T codons. All of the 22 (100% chloroquine-resistant and 16.7% of sensitive isolates were found to harbor the 76T mutation and none was found to contain the wild type (K76 allele. Conclusions: The frequency of chloroquine resistance associated point mutation K76T, in pfcrt gene in this region suggest that detection of this mutation can be applied for predicting chloroquine resistance in epidemiologic settings with sufficiently high sensitivity to make it an attractive alternative to time and labor-consuming in vivo trials.

  7. Do BRCA1/2 mutation carriers have an earlier onset of natural menopause?

    Science.gov (United States)

    van Tilborg, Theodora C; Broekmans, Frank J; Pijpe, Anouk; Schrijver, Lieske H; Mooij, Thea M; Oosterwijk, Jan C; Verhoef, Senno; Gómez Garcia, Encarna B; van Zelst-Stams, Wendy A; Adank, Muriel A; van Asperen, Christi J; van Doorn, Helena C; van Os, Theo A; Bos, Anna M; Rookus, Matti A; Ausems, Margreet G

    2016-08-01

    It has been hypothesized that BRCA1/2 mutation carriers have an earlier age at natural menopause (ANM), although to date findings are inconclusive. This study assessed the influence of BRCA mutation status on ANM, and aimed to explore the reasons of inconsistency in the literature. Cross-sectional assessment from an ongoing nationwide cohort study among members of BRCA1/2 mutated families. Information was obtained by a standardized questionnaire. Kaplan-Meier curves were constructed, and Cox regression was used to assess the association between BRCA1/2 mutation status and ANM. Adjustments were made for birth cohort, family, smoking, use of hormonal contraceptives, and parity. A total of 1,208 BRCA1/2 mutation carriers and 2,211 proven noncarriers were included. Overall, no association was found between BRCA1/2 mutation status and ANM (adjusted hazard ratio [HR] = 1.06 [95% CI, 0.87-1.30]). We examined if the null finding was due to informative censoring by uptake of risk-reducing salpingo-oophorectomy. Indeed, within the oldest birth cohort, in which the percentage of surgical menopause events was lowest and comparable between carriers and noncarriers, the HR for earlier natural menopause in carriers was 1.45 (95% CI, 1.09-1.94). The second oldest birth cohort, however, demonstrated a decreased HR (0.67 [95% CI, 0.46-0.98]), and thus no trend over birth cohorts was found. Various types of selection bias hamper the comparison of ANM between BRCA1/2 mutation carriers and noncarriers, genetically tested in the clinic.

  8. Prevalence and clinical significance of mediator complex subunit 12 mutations in 362 Han Chinese samples with uterine leiomyoma.

    Science.gov (United States)

    Wu, Juan; Zou, Yang; Luo, Yong; Guo, Jiu-Bai; Liu, Fa-Ying; Zhou, Jiang-Yan; Zhang, Zi-Yu; Wan, Lei; Huang, Ou-Ping

    2017-07-01

    Uterine leiomyomas (ULs) are the most common gynecological benign tumors originating from the myometrium. Prevalent mutations in the mediator complex subunit 12 (MED12) gene have been identified in ULs, and functional evidence has revealed that these mutations may promote the development of ULs. However, whether MED12 mutations are associated with certain clinical characteristics in ULs remains largely unknown. In the present study, the potential mutations of MED12 and its paralogous gene, mediator complex subunit 12-like (MED12L), were screened in 362 UL tumors from Han Chinese patients. A total of 158 out of 362 UL tumors (43.6%) were identified as harboring MED12 somatic mutations, and the majority of these mutations were restricted to the 44th residue. MED12 mutations were also observed in 2 out of 145 (1.4%) adjacent control myometrium. Furthermore, the mutation spectrum of MED12 in the concurrent leiomyomas was noticeably different. Correlation analysis of MED12 mutations with the available clinical features indicated that patients with mutated MED12 tended to have smaller cervical diameters. By contrast, no MED12L mutation was identified in the present samples. In summary, the present study demonstrated the presence of prevalent MED12 somatic mutations in UL samples, and the MED12 mutation was associated with smaller cervical diameters. The low mutation frequency of MED12 in adjacent control myometrium indicated that MED12 mutation may be an early event in the pathogenesis of ULs. Furthermore, MED12 mutation status in concurrent tumors from multiple leiomyomas supported several prior observations that the majority of these tumors arose independently.

  9. Activation of the MAPK pathway is a common event in uveal melanomas although it rarely occurs through mutation of BRAF or RAS.

    Science.gov (United States)

    Zuidervaart, W; van Nieuwpoort, F; Stark, M; Dijkman, R; Packer, L; Borgstein, A-M; Pavey, S; van der Velden, P; Out, C; Jager, M J; Hayward, N K; Gruis, N A

    2005-06-06

    In contrast to cutaneous melanoma, there is no evidence that BRAF mutations are involved in the activation of the mitogen-activated protein kinase (MAPK) pathway in uveal melanoma, although there is increasing evidence that this pathway is activated frequently in the latter tumours. In this study, we performed mutation analysis of the RAS and BRAF genes in a panel of 11 uveal melanoma cell lines and 19 primary uveal melanoma tumours. In addition, Western blot and immunohistochemical analyses were performed on downstream members of the MAPK pathway in order to assess the contribution of each of these components. No mutations were found in any of the three RAS gene family members and only one cell line carried a BRAF mutation (V599E). Despite this, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), ERK and ELK were constitutively activated in all samples. These data suggest that activation of the MAPK pathway is commonly involved in the development of uveal melanoma, but occurs through a mechanism different to that of cutaneous melanoma.

  10. A Human Neural Crest Stem Cell-Derived Dopaminergic Neuronal Model Recapitulates Biochemical Abnormalities in GBA1 Mutation Carriers

    Directory of Open Access Journals (Sweden)

    Shi-Yu Yang

    2017-03-01

    Full Text Available Numerically the most important risk factor for the development of Parkinson's disease (PD is the presence of mutations in the glucocerebrosidase GBA1 gene. In vitro and in vivo studies show that GBA1 mutations reduce glucocerebrosidase (GCase activity and are associated with increased α-synuclein levels, reflecting similar changes seen in idiopathic PD brain. We have developed a neural crest stem cell-derived dopaminergic neuronal model that recapitulates biochemical abnormalities in GBA1 mutation-associated PD. Cells showed reduced GCase protein and activity, impaired macroautophagy, and increased α-synuclein levels. Advantages of this approach include easy access to stem cells, no requirement to reprogram, and retention of the intact host genome. Treatment with a GCase chaperone increased GCase protein levels and activity, rescued the autophagic defects, and decreased α-synuclein levels. These results provide the basis for further investigation of GCase chaperones or similar drugs to slow the progression of PD.

  11. Base substitutions, frameshifts, and small deletions constitute ionizing radiation-induced point mutations in mammalian cells

    International Nuclear Information System (INIS)

    Grosovsky, A.J.; de Boer, J.G.; de Jong, P.J.; Drobetsky, E.A.; Glickman, B.W.

    1988-01-01

    The relative role of point mutations and large genomic rearrangements in ionizing radiation-induced mutagenesis has been an issue of long-standing interest. Recent studies using Southern blotting analysis permit the partitioning of ionizing radiation-induced mutagenesis in mammalian cells into detectable deletions and major genomic rearrangements and into point mutations. The molecular nature of these point mutations has been left unresolved; they may include base substitutions as well as small deletions, insertions, and frame-shifts below the level of resolution of Southern blotting analysis. In this investigation, we have characterized a collection of ionizing radiation-induced point mutations at the endogenous adenine phosphoribosyltransferase (aprt) locus of Chinese hamster ovary cells at the DNA sequence level. Base substitutions represented approximately equal to 2/3 of the point mutations analyzed. Although the collection of mutants is relatively small, every possible type of base substitution event has been recovered. These mutations are well distributed throughout the coding sequence with only one multiple occurrence. Small deletions represented the remainder of characterized mutants; no insertions have been observed. Sequence-directed mechanisms mediated by direct repeats could account for some of the observed deletions, while others appear to be directly attributable to radiation-induced strand breakage

  12. Limited ability of DNA polymerase kappa to suppress benzo[a]pyrene-induced genotoxicity in vivo.

    Science.gov (United States)

    Masumura, Kenichi; Toyoda-Hokaiwado, Naomi; Niimi, Naoko; Grúz, Petr; Wada, Naoko A; Takeiri, Akira; Jishage, Kou-Ichi; Mishima, Masayuki; Nohmi, Takehiko

    2017-12-01

    DNA polymerase kappa (Polk) is a specialized DNA polymerase involved in translesion DNA synthesis. To understand the protective roles against genotoxins in vivo, we established inactivated Polk knock-in gpt delta (inactivated Polk KI) mice that possessed reporter genes for mutations and expressed inactive Polk. In this study, we examined genotoxicity of benzo[a]pyrene (BP) to determine whether Polk actually suppressed BP-induced genotoxicity as predicted by biochemistry and in vitro cell culture studies. Seven-week-old inactivated Polk KI and wild-type (WT) mice were treated with BP at doses of 5, 15, or 50 mg/(kg·day) for three consecutive days by intragastric gavage, and mutations in the colon and micronucleus formation in the peripheral blood were examined. Surprisingly, no differences were observed in the frequencies of mutations and micronucleus formation at 5 or 50 mg/kg doses. Inactivated Polk KI mice exhibited approximately two times higher gpt mutant frequency than did WT mice only at the 15 mg/kg dose. The frequency of micronucleus formation was slightly higher in inactivated Polk KI than in WT mice at the same dose, but it was statistically insignificant. The results suggest that Polk has a limited ability to suppress BP-induced genotoxicity in the colon and bone marrow and also that the roles of specialized DNA polymerases in mutagenesis and carcinogenesis should be examined not only by in vitro assays but also by in vivo mouse studies. We also report the spontaneous mutagenesis in inactivated Polk KI mice at young and old ages. Environ. Mol. Mutagen. 58:644-653, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  13. p110α Hot Spot Mutations E545K and H1047R Exert Metabolic Reprogramming Independently of p110α Kinase Activity.

    Science.gov (United States)

    Chaudhari, Aditi; Krumlinde, Daniel; Lundqvist, Annika; Akyürek, Levent M; Bandaru, Sashidhar; Skålén, Kristina; Ståhlman, Marcus; Borén, Jan; Wettergren, Yvonne; Ejeskär, Katarina; Rotter Sopasakis, Victoria

    2015-10-01

    The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) catalytic subunit p110α is the most frequently mutated kinase in human cancer, and the hot spot mutations E542K, E545K, and H1047R are the most common mutations in p110α. Very little is known about the metabolic consequences of the hot spot mutations of p110α in vivo. In this study, we used adenoviral gene transfer in mice to investigate the effects of the E545K and H1047R mutations on hepatic and whole-body glucose metabolism. We show that hepatic expression of these hot spot mutations results in rapid hepatic steatosis, paradoxically accompanied by increased glucose tolerance, and marked glycogen accumulation. In contrast, wild-type p110α expression does not lead to hepatic accumulation of lipids or glycogen despite similar degrees of upregulated glycolysis and expression of lipogenic genes. The reprogrammed metabolism of the E545K and H1047R p110α mutants was surprisingly not dependent on altered p110α lipid kinase activity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  14. Adaptive error detection for HDR/PDR brachytherapy: Guidance for decision making during real-time in vivo point dosimetry

    DEFF Research Database (Denmark)

    Kertzscher Schwencke, Gustavo Adolfo Vladimir; Andersen, Claus E.; Tanderup, Kari

    2014-01-01

    Purpose:This study presents an adaptive error detection algorithm (AEDA) for real-timein vivo point dosimetry during high dose rate (HDR) or pulsed dose rate (PDR) brachytherapy (BT) where the error identification, in contrast to existing approaches, does not depend on an a priori reconstruction ......, and the AEDA’s capacity to distinguish between true and false error scenarios. The study further shows that the AEDA can offer guidance in decision making in the event of potential errors detected with real-time in vivo point dosimetry....... of the dosimeter position reconstruction. Given its nearly exclusive dependence on stable dosimeter positioning, the AEDA allows for a substantially simplified and time efficient real-time in vivo BT dosimetry implementation. Methods:In the event of a measured potential treatment error, the AEDA proposes the most...

  15. The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation.

    Science.gov (United States)

    Silva, John-Paul; Vetterlein, Olivia; Jose, Joby; Peters, Shirley; Kirby, Hishani

    2015-02-27

    Human immunoglobulin G isotype 4 (IgG4) antibodies (Abs) are potential candidates for immunotherapy when reduced effector functions are desirable. IgG4 Abs are dynamic molecules able to undergo a process known as Fab arm exchange (FAE). This results in functionally monovalent, bispecific antibodies (bsAbs) with unknown specificity and hence, potentially, reduced therapeutic efficacy. IgG4 FAE is suggested to be an important biological mechanism that provides the basis for the anti-inflammatory activity attributed to IgG4 Abs. To date, the mechanism of FAE is not entirely understood and studies measuring FAE in ex vivo matrices have been hampered by the presence and abundance of endogenous IgG4 wild-type (WT) Abs. Using representative humanized WT IgG4 monoclonal Abs, namely, anti-IL-6 and anti-TNF, and a core-hinge stabilized serine 228 to proline (S228P) anti-IL-6 IgG4 mutant, it is demonstrated for the first time how anti-IgG4 affinity chromatography can be used to prepare physiologically relevant matrices for assessing and quantifying FAE. A novel method for quantifying FAE using a single MSD immunoassay is also reported and confirms previous findings that, dependent on the redox conditions, the S228P mutation can prevent IgG4 FAE to undetectable levels both in vitro and in vivo. Together, the findings and novel methodologies will allow researchers to monitor and quantify FAE of their own IgG4 molecules in physiologically relevant matrices. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Dual HER2\\PIK3CA targeting overcomes single-agent acquired resistance in HER2 amplified uterine serous carcinoma cell lines in vitro and in vivo

    Science.gov (United States)

    Lopez, Salvatore; Cocco, Emiliano; Black, Jonathan; Bellone, Stefania; Bonazzoli, Elena; Predolini, Federica; Ferrari, Francesca; Schwab, Carlton L.; English, Diana P.; Ratner, Elena; Silasi, Dan-Arin; Azodi, Masoud; Schwartz, Peter E.; Terranova, Corrado; Angioli, Roberto; Santin, Alessandro D.

    2015-01-01

    HER2/neu gene amplification and PIK3CA driver mutations are common in uterine serous carcinoma (USC), and may represent ideal therapeutic targets against this aggressive variant of endometrial cancer. We examined the sensitivity to neratinib, taselisib and the combination of the two compounds in in vitro and in vivo experiments using PIK3CA mutated and PIK3CA-wild type HER2/neu amplified USC cell lines. Cell viability and cell cycle distribution were assessed using flow-cytometry assays. Downstream signaling was assessed by immunoblotting. Preclinical efficacy of single versus dual inhibition was evaluated in vivo using two USC-xenografts. We found both single agent neratinib and taselisib to be active but only transiently effective in controlling the in vivo growth of USC xenografts harboring HER2/neu gene amplification with or without oncogenic PIK3CA mutations. In contrast, the combination of the two inhibitors caused a stronger and long lasting growth inhibition in both USC xenografts when compared to single agent therapy. Combined targeting of HER2 and PIK3CA was associated with a significant and dose-dependent increase in the percentage of cells in the G0/G1 phase of the cell cycle and a dose-dependent decline in the phosphorylation of S6. Importantly, dual inhibition therapy initiated after tumor progression in single agent-treated mice was still remarkably effective at inducing tumor regression in both large PIK3CA or pan-ErbB inhibitor-resistant USC xenografts. Dual HER2/PIK3CA blockade may represent a novel therapeutic option for USC patients harboring tumors with HER2/neu gene amplification and mutated or wild type PIK3CA resistant to chemotherapy. PMID:26333383

  17. Dual HER2/PIK3CA Targeting Overcomes Single-Agent Acquired Resistance in HER2-Amplified Uterine Serous Carcinoma Cell Lines In Vitro and In Vivo.

    Science.gov (United States)

    Lopez, Salvatore; Cocco, Emiliano; Black, Jonathan; Bellone, Stefania; Bonazzoli, Elena; Predolini, Federica; Ferrari, Francesca; Schwab, Carlton L; English, Diana P; Ratner, Elena; Silasi, Dan-Arin; Azodi, Masoud; Schwartz, Peter E; Terranova, Corrado; Angioli, Roberto; Santin, Alessandro D

    2015-11-01

    HER2/neu gene amplification and PIK3CA driver mutations are common in uterine serous carcinoma (USC) and may represent ideal therapeutic targets against this aggressive variant of endometrial cancer. We examined the sensitivity to neratinib, taselisib, and the combination of the two compounds in in vitro and in vivo experiments using PIK3CA-mutated and PIK3CA wild-type HER2/neu-amplified USC cell lines. Cell viability and cell-cycle distribution were assessed using flow-cytometry assays. Downstream signaling was assessed by immunoblotting. Preclinical efficacy of single versus dual inhibition was evaluated in vivo using two USC xenografts. We found both single-agent neratinib and taselisib to be active but only transiently effective in controlling the in vivo growth of USC xenografts harboring HER2/neu gene amplification with or without oncogenic PIK3CA mutations. In contrast, the combination of the two inhibitors caused a stronger and long-lasting growth inhibition in both USC xenografts when compared with single-agent therapy. Combined targeting of HER2 and PIK3CA was associated with a significant and dose-dependent increase in the percentage of cells in the G0-G1 phase of the cell cycle and a dose-dependent decline in the phosphorylation of S6. Importantly, dual inhibition therapy initiated after tumor progression in single-agent-treated mice was still remarkably effective at inducing tumor regression in both large PIK3CA and pan-ErbB inhibitor-resistant USC xenografts. Dual HER2/PIK3CA blockade may represent a novel therapeutic option for USC patients harboring tumors with HER2/neu gene amplification and mutated or wild-type PIK3CA resistant to chemotherapy. ©2015 American Association for Cancer Research.

  18. Evidence that adaptation in Drosophila is not limited by mutation at single sites.

    Directory of Open Access Journals (Sweden)

    Talia Karasov

    2010-06-01

    Full Text Available Adaptation in eukaryotes is generally assumed to be mutation-limited because of small effective population sizes. This view is difficult to reconcile, however, with the observation that adaptation to anthropogenic changes, such as the introduction of pesticides, can occur very rapidly. Here we investigate adaptation at a key insecticide resistance locus (Ace in Drosophila melanogaster and show that multiple simple and complex resistance alleles evolved quickly and repeatedly within individual populations. Our results imply that the current effective population size of modern D. melanogaster populations is likely to be substantially larger (> or = 100-fold than commonly believed. This discrepancy arises because estimates of the effective population size are generally derived from levels of standing variation and thus reveal long-term population dynamics dominated by sharp--even if infrequent--bottlenecks. The short-term effective population sizes relevant for strong adaptation, on the other hand, might be much closer to census population sizes. Adaptation in Drosophila may therefore not be limited by waiting for mutations at single sites, and complex adaptive alleles can be generated quickly without fixation of intermediate states. Adaptive events should also commonly involve the simultaneous rise in frequency of independently generated adaptive mutations. These so-called soft sweeps have very distinct effects on the linked neutral polymorphisms compared to the standard hard sweeps in mutation-limited scenarios. Methods for the mapping of adaptive mutations or association mapping of evolutionarily relevant mutations may thus need to be reconsidered.

  19. A Novel MAPT Mutation, G55R, in a Frontotemporal Dementia Patient Leads to Altered Tau Function

    Science.gov (United States)

    Guzman, Elmer; Barczak, Anna; Chodakowska-Żebrowska, Małgorzata; Barcikowska, Maria; Feinstein, Stuart

    2013-01-01

    Over two dozen mutations in the gene encoding the microtubule associated protein tau cause a variety of neurodegenerative dementias known as tauopathies, including frontotemporal dementia (FTD), PSP, CBD and Pick's disease. The vast majority of these mutations map to the C-terminal region of tau possessing microtubule assembly and microtubule dynamics regulatory activities as well as the ability to promote pathological tau aggregation. Here, we describe a novel and non-conservative tau mutation (G55R) mapping to an alternatively spliced exon encoding part of the N-terminal region of the protein in a patient with the behavioral variant of FTD. Although less well understood than the C-terminal region of tau, the N-terminal region can influence both MT mediated effects as well as tau aggregation. The mutation changes an uncharged glycine to a basic arginine in the midst of a highly conserved and very acidic region. In vitro, 4-repeat G55R tau nucleates microtubule assembly more effectively than wild-type 4-repeat tau; surprisingly, this effect is tau isoform specific and is not observed in a 3-repeat G55R tau versus 3-repeat wild-type tau comparison. In contrast, the G55R mutation has no effect upon the abilities of tau to regulate MT growing and shortening dynamics or to aggregate. Additionally, the mutation has no effect upon kinesin translocation in a microtubule gliding assay. Together, (i) we have identified a novel tau mutation mapping to a mutation deficient region of the protein in a bvFTD patient, and (ii) the G55R mutation affects the ability of tau to nucleate microtubule assembly in vitro in a 4-repeat tau isoform specific manner. This altered capability could markedly affect in vivo microtubule function and neuronal cell biology. We consider G55R to be a candidate mutation for bvFTD since additional criteria required to establish causality are not yet available for assessment. PMID:24086739

  20. Mutation of the regulatory phosphorylation site of tobacco nitrate reductase results in constitutive activation of the enzyme in vivo and nitrite accumulation.

    Science.gov (United States)

    Lillo, Cathrine; Lea, Unni S; Leydecker, Marie-Thérèse; Meyer, Christian

    2003-09-01

    In wild-type Nicotiana plumbaginifolia and other higher plants, nitrate reductase (NR) is rapidly inactivated/activated in response to dark/light transitions. Inactivation of NR is believed to be caused by phosphorylation at a special conserved regulatory Ser residue, Ser 521, and interactions with divalent cations and inhibitory 14-3-3 proteins. A transgenic N. plumbaginifolia line (S(521)) was constructed where the Ser 521 had been changed by site-directed mutagenesis into Asp. This mutation resulted in complete abolishment of inactivation in response to light/dark transitions or other treatments known to inactivate NR. During prolonged darkness, NR in wild-type plants is in the inactivated form, whereas NR in the S(521) line is always in the active form. Differences in degradation rate between NR from S(521) and lines with non-mutated NR were not found. Kinetic constants like Km values for NADH and NO3(-) were not changed, but a slightly different pH profile was observed for mutated NR as opposed to non-mutated NR. Under optimal growth conditions, the phenotype of the S(521) plants was not different from the wild type (WT). However, when plants were irrigated with high nitrate concentration, 150 mM, the transgenic plants accumulated nitrite in darkness, and young leaves showed chlorosis.

  1. Deletion mutations of bacteriophage

    International Nuclear Information System (INIS)

    Ryo, Yeikou

    1975-01-01

    Resolution of mutation mechanism with structural changes of DNA was discussed through the studies using bacteriophage lambda. One of deletion mutations inductions of phage lambda is the irradiation of ultraviolet ray. It is not clear if the inductions are caused by errors in reparation of ultraviolet-induced damage or by the activation of int gene. Because the effective site of int gene lies within the regions unnecessary for existing, it is considered that int gene is connected to deletion mutations induction. A certain system using prophage complementarity enables to detect deletion mutations at essential hereditary sites and to solve the relations of deletion mutations with other recombination system, DNA reproduction and repairment system. Duplication and multiplication of hereditary elements were discussed. If lambda deletion mutations of the system, which can control recombination, reproduction and repairment of added DNA, are constructed, mutations mechanism with great changes of DNA structure can be solved by phage lambda. (Ichikawa, K.)

  2. The F309S mutation increases factor VIII secretion in human cell line

    Directory of Open Access Journals (Sweden)

    Daianne Maciely Carvalho Fantacini

    2016-06-01

    Full Text Available ABSTRACT OBJECTIVES: The capacity of a human cell line to secrete recombinant factor VIII with a F309S point mutation was investigated, as was the effect of the addition of chemical chaperones (betaine and sodium-4-phenylbutyrate on the secretion of factor VIII. METHODS: This work used a vector with a F309S mutation in the A1 domain to investigate FVIII production in the HEK 293 human cell line. Factor VIII activity was measured by chromogenic assay. Furthermore, the effects of chemical drugs on the culture were evaluated. RESULTS: The addition of the F309S mutation to a previously described FVIII variant increased FVIII secretion by 4.5 fold. Moreover, the addition of betaine or sodium-4-phenylbutyrate increased the secretion rate of FVIIIΔB proteins in HEK 293 cells, but the same effect was not seen for FVIIIΔB-F309S indicating that all the recombinant protein produced had been efficiently secreted. CONCLUSION: Bioengineering factor VIII expressed in human cells may lead to an efficient production of recombinant factor VIII and contribute toward low-cost coagulation factor replacement therapy for hemophilia A. FVIII-F309S produced in human cells can be effective in vivo.

  3. Calreticulin Mutations in Myeloproliferative Neoplasms

    Directory of Open Access Journals (Sweden)

    Noa Lavi

    2014-10-01

    Full Text Available With the discovery of the JAK2V617F mutation in patients with Philadelphia chromosome-negative (Ph− myeloproliferative neoplasms (MPNs in 2005, major advances have been made in the diagnosis of MPNs, in understanding of their pathogenesis involving the JAK/STAT pathway, and finally in the development of novel therapies targeting this pathway. Nevertheless, it remains unknown which mutations exist in approximately one-third of patients with non-mutated JAK2 or MPL essential thrombocythemia (ET and primary myelofibrosis (PMF. At the end of 2013, two studies identified recurrent mutations in the gene encoding calreticulin (CALR using whole-exome sequencing. These mutations were revealed in the majority of ET and PMF patients with non-mutated JAK2 or MPL but not in polycythemia vera patients. Somatic 52-bp deletions (type 1 mutations and recurrent 5-bp insertions (type 2 mutations in exon 9 of the CALR gene (the last exon encoding the C-terminal amino acids of the protein calreticulin were detected and found always to generate frameshift mutations. All detected mutant calreticulin proteins shared a novel amino acid sequence at the C-terminal. Mutations in CALR are acquired early in the clonal history of the disease, and they cause activation of JAK/STAT signaling. The CALR mutations are the second most frequent mutations in Ph− MPN patients after the JAK2V617F mutation, and their detection has significantly improved the diagnostic approach for ET and PMF. The characteristics of the CALR mutations as well as their diagnostic, clinical, and pathogenesis implications are discussed in this review.

  4. Characterization of in vivo DNA-binding events of plant transcription factors by ChIP-seq

    NARCIS (Netherlands)

    Mourik, Van Hilda; Muiño, J.M.; Pajoro, Alice; Angenent, G.C.; Kaufmann, Kerstin

    2015-01-01

    Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) is a powerful technique for genome-wide identification of in vivo binding sites of DNA-binding proteins. The technique had been used to study many DNA-binding proteins in a broad variety of species. The basis of the

  5. Tissue-specific in vivo genetic toxicity of nine polycyclic aromatic hydrocarbons assessed using the Muta™Mouse transgenic rodent assay

    Energy Technology Data Exchange (ETDEWEB)

    Long, Alexandra S., E-mail: alexandra.long@hc-sc.gc.ca [Faculty of Graduate and Postdoctoral Studies, Department of Biology, University of Ottawa, Ottawa, ON (Canada); Mechanistic Studies Division, Environmental Health Science and Research Bureau, Health Canada, Ottawa, ON (Canada); Lemieux, Christine L. [Air Health Science Division, Water and Air Quality Bureau, Health Canada, Ottawa, ON (Canada); Arlt, Volker M. [Analytical and Environmental Sciences Division, MRC-PHE Centre for Environment and Health, King' s College London, London (United Kingdom); White, Paul A. [Faculty of Graduate and Postdoctoral Studies, Department of Biology, University of Ottawa, Ottawa, ON (Canada); Mechanistic Studies Division, Environmental Health Science and Research Bureau, Health Canada, Ottawa, ON (Canada)

    2016-01-01

    Test batteries to screen chemicals for mutagenic hazard include several endpoints regarded as effective for detecting genotoxic carcinogens. Traditional in vivo methods primarily examine clastogenic endpoints in haematopoietic tissues. Although this approach is effective for identifying systemically distributed clastogens, some mutagens may not induce clastogenic effects; moreover, genotoxic effects may be restricted to the site of contact and/or related tissues. An OECD test guideline for transgenic rodent (TGR) gene mutation assays was released in 2011, and the TGR assays permit assessment of mutagenicity in any tissue. This study assessed the responses of two genotoxicity endpoints following sub-chronic oral exposures of male Muta™Mouse to 9 carcinogenic polycyclic aromatic hydrocarbons (PAHs). Clastogenicity was assessed via induction of micronuclei in peripheral blood, and mutagenicity via induction of lacZ transgene mutations in bone marrow, glandular stomach, small intestine, liver, and lung. Additionally, the presence of bulky PAH-DNA adducts was examined. Five of the 9 PAHs elicited positive results across all endpoints in at least one tissue, and no PAHs were negative or equivocal across all endpoints. All PAHs were positive for lacZ mutations in at least one tissue (sensitivity = 100%), and for 8 PAHs, one or more initial sites of chemical contact (i.e., glandular stomach, liver, small intestine) yielded a greater response than bone marrow. Five PAHs were positive in the micronucleus assay (sensitivity = 56%). Furthermore, all PAHs produced DNA adducts in at least one tissue. The results demonstrate the utility of the TGR assay for mutagenicity assessment, especially for compounds that may not be systemically distributed. - Highlights: • The Muta™Mouse is a reliable tool for in vivo mutagenicity assessment of PAHs. • All 9 PAHs induced lacZ transgene mutations in small intestine. • Only 5 of 9 PAHs induced lacZ mutations and micronuclei in

  6. Most ultraviolet irradiation induced mutations in the nematode Caenorhabditis elegans are chromosomal rearrangements

    International Nuclear Information System (INIS)

    Stewart, H.I.; Rosenbluth, R.E.; Baillie, D.L.

    1991-01-01

    In this study the utility of 254-nm ultraviolet light (UV) as a magnetic tool in C.elegans is determined. It is demonstrated that irradiation of adult hermaphrodites provides a simple method for the induction of heritable chromosomal rearrangements. A screening protocol was employed that identifies either recessive lethal mutations in the 40 map unit region balanced by the translocation eT1(III;V), or unc-36(III) duplications. Mutations were recovered in 3% of the chromosomes screened after a dose of 120 J/m 2 . This rate resembles that for 1500 R γ-ray-induced mutations selected in a similar manner. The mutations were classified either as lethals [mapping to Linkage Group (LG)III or LGV] or as putative unc-36 duplications. In contrast to the majority of UV-induced mutations analysed in micro-organisms, a large fraction of the C.elegans UV-induced mutations were found to be not simple intragenic lesions, but deficiencies for more than one adjacent gene or more complex events. Preliminary evidence for this conclusion came from the high frequency of mutations that had a dominant effect causing reduced numbers of adult progeny. Subsequently 6 out of 9 analysed LGV mutations were found to be deficiencies. Other specific rearrangements also identified were: one translocation, sT5(II;III), and two unc-36 duplications, sDp8 and sDp9. It was concluded that UV irradiation can easily be used as an additional tool for the analysis of C.elegans chromosomes, and that C.elegans should prove to be a useful organism in which to study the mechanisms whereby UV acts as a mutagen in cells of complex eukaryotes. (author). 46 refs.; 5 figs.; 4 tabs

  7. Visualization of multidrug resistance in vivo

    International Nuclear Information System (INIS)

    Hendrikse, N.H.; Franssen, E.J.F.; Graaf, W.T.A. van der; Vries, E.G.E. de; Vaalburg, W.

    1999-01-01

    function non-invasively. Results obtained in MRP 2 mutated GY/TR rats have demonstrated visualization of MRP-mediated transport. This tracer permits the study of MRP transport function abnormalities in vivo, e.g. in Dubin-Johnson patients, who are MRP 2 gene deficient. Results obtained show the feasibility of using SPET and PET to study the functionality of MDR transporters in vivo. (orig.)

  8. Identification of somatic mutations in postmortem human brains by whole genome sequencing and their implications for psychiatric disorders.

    Science.gov (United States)

    Nishioka, Masaki; Bundo, Miki; Ueda, Junko; Katsuoka, Fumiki; Sato, Yukuto; Kuroki, Yoko; Ishii, Takao; Ukai, Wataru; Murayama, Shigeo; Hashimoto, Eri; Nagasaki, Masao; Yasuda, Jun; Kasai, Kiyoto; Kato, Tadafumi; Iwamoto, Kazuya

    2018-04-01

    Somatic mutations in the human brain are hypothesized to contribute to the functional diversity of brain cells as well as the pathophysiology of neuropsychiatric diseases. However, there are still few reports on somatic mutations in non-neoplastic human brain tissues. This study attempted to unveil the landscape of somatic mutations in the human brain. We explored the landscape of somatic mutations in human brain tissues derived from three individuals with no neuropsychiatric diseases by whole-genome deep sequencing at a depth of around 100. The candidate mutations underwent multi-layered filtering, and were validated by ultra-deep target amplicon sequencing at a depth of around 200 000. Thirty-one somatic mutations were identified in the human brain, demonstrating the utility of whole-genome sequencing of bulk brain tissue. The mutations were enriched in neuron-expressed genes, and two-thirds of the identified somatic single nucleotide variants in the brain tissues were cytosine-to-thymine transitions, half of which were in CpG dinucleotides. Our developed filtering and validation approaches will be useful to identify somatic mutations in the human brain. The vulnerability of neuron-expressed genes to mutational events suggests their potential relevance to neuropsychiatric diseases. © 2017 The Authors. Psychiatry and Clinical Neurosciences published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Psychiatry and Neurology.

  9. Functional features of gene expression profiles differentiating gastrointestinal stromal tumours according to KIT mutations and expression

    International Nuclear Information System (INIS)

    Ostrowski, Jerzy; Dobosz, Anna Jerzak Vel; Jarosz, Dorota; Ruka, Wlodzimierz; Wyrwicz, Lucjan S; Polkowski, Marcin; Paziewska, Agnieszka; Skrzypczak, Magdalena; Goryca, Krzysztof; Rubel, Tymon; Kokoszyñska, Katarzyna; Rutkowski, Piotr; Nowecki, Zbigniew I

    2009-01-01

    Gastrointestinal stromal tumours (GISTs) represent a heterogeneous group of tumours of mesenchymal origin characterized by gain-of-function mutations in KIT or PDGFRA of the type III receptor tyrosine kinase family. Although mutations in either receptor are thought to drive an early oncogenic event through similar pathways, two previous studies reported the mutation-specific gene expression profiles. However, their further conclusions were rather discordant. To clarify the molecular characteristics of differentially expressed genes according to GIST receptor mutations, we combined microarray-based analysis with detailed functional annotations. Total RNA was isolated from 29 frozen gastric GISTs and processed for hybridization on GENECHIP ® HG-U133 Plus 2.0 microarrays (Affymetrix). KIT and PDGFRA were analyzed by sequencing, while related mRNA levels were analyzed by quantitative RT-PCR. Fifteen and eleven tumours possessed mutations in KIT and PDGFRA, respectively; no mutation was found in three tumours. Gene expression analysis identified no discriminative profiles associated with clinical or pathological parameters, even though expression of hundreds of genes differentiated tumour receptor mutation and expression status. Functional features of genes differentially expressed between the two groups of GISTs suggested alterations in angiogenesis and G-protein-related and calcium signalling. Our study has identified novel molecular elements likely to be involved in receptor-dependent GIST development and allowed confirmation of previously published results. These elements may be potential therapeutic targets and novel markers of KIT mutation status

  10. Environmental modulation of somatic mutations: nature of interactions. Final report, 1 June 1974--31 May 1977

    International Nuclear Information System (INIS)

    Mericle, L.W.

    1977-05-01

    Research on this project has had as a major goal a combined ecologic-genetic investigation of somatic mutations in order to evaluate the impacts of certain changing environmental parameters. The ultimate aim, to better understand how such environmental-mutation interactions operate and to assure the information obtained be extrapolatable to conditions and events in nature. Higher plants delineate reproductive tissues late in development from meristematic, somatic tissues. Moreover, the prevailing method of reproduction may be without sexual fusion of gametes and/or wholly asexual (vegetative). Therefore, somatic mutations can have as far-reaching genetic significance for a plant population as when germ cells, themselves, are directly affected. Our data show diurnal temperature differences (DTD) of greater than or equal to 22.2 C-degrees to be very effective mutagenic agents in the Tradescantia somatic mutation system. Further, these ranges of DTD were found to occur often in important seed production areas. A DTD of 22.2 in magnitude can increase mutations 10-fold. And, durations short as 1-day can induce significant increases in mutation rate. Whether interaction of 22.2 DTD with low-level radiation (800 mR/day) is synergistic or attenuative is still debatable. We believe, however, that spontaneous, and 22.2 DTD induced, mutations occur mainly via the genetic mechanism of somatic crossing-over; mutations from acute ionizing radiation (e.g., 30-60 R γ) via chromosome breakage, producing micronuclei. Requirements for maximizing the Discriminatory Response Capability (DRC) in the Tradescantia somatic mutation system are set forth

  11. Utility of BRAF V600E mutation detection in cytologically indeterminate thyroid nodules

    Directory of Open Access Journals (Sweden)

    Rowe Leslie R

    2006-04-01

    Full Text Available Abstract Background Fine needle aspiration (FNA is widely utilized for evaluation of patients with thyroid nodules. However, approximately 30% are indeterminate for malignancy. Recently, a mutation in the BRAF gene has been reported to be the most common genetic event in papillary thyroid carcinoma (PTC. In this retrospective study, we assessed the utility of BRAF V600E mutation detection for refining indeterminate preoperative cytologic diagnoses in patients with PTC. Methods Archival indeterminate thyroid FNAs and corresponding formalin-fixed, paraffin-embedded (FFPE surgical samples with PTC were identified in our patient files. DNA extracted from slide scape lysates and 5 μm FFPE sections were evaluated for the BRAF V600E mutation using LightCycler PCR and fluorescent melting curve analysis (LCPCR. Amplification products that showed deviation from the wild-type genomic DNA melting peak, discordant FNA and FFPE matched pairs, and all benign control samples, underwent direct DNA sequencing. Results A total of 19 indeterminate thyroid FNAs demonstrating PTC on FFPE surgical samples were included in the study. Using BRAF mutation analysis, the preoperative diagnosis of PTC was confirmed in 3/19 (15.8% FNA samples that could not be conclusively diagnosed on cytology alone. However, 9/19 (47.4% FFPE tissue samples were positive for the V600E mutation. Of the discordant pairs, 5/6 FNAs contained less than 50% tumor cells. Conclusion When used with indeterminate FNA samples, BRAF mutation analysis may be a useful adjunct technique for confirming the diagnosis of malignancy in an otherwise equivocal case. However, overall tumor cell content of some archival FNA smear slides is a limiting factor for mutation detection.

  12. In vivo studies of genomic packaging in the dsRNA bacteriophage Φ8

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    Mindich Leonard

    2005-03-01

    Full Text Available Abstract Background Φ8 is a bacteriophage containing a genome of three segments of double-stranded RNA inside a polyhedral capsid enveloped in a lipid-containing membrane. Plus strand RNA binds and is packaged by empty procapsids. Whereas Φ6, another member of the Cystoviridae, shows high stringency, serial dependence and precision in its genomic packaging in vitro and in vivo, Φ8 packaging is more flexible. Unique sequences (pac near the 5' ends of plus strands are necessary and sufficient for Φ6 genomic packaging and the RNA binding sites are located on P1, the major structural protein of the procapsid. Results In this paper the boundaries of the Φ8 pac sequences have been explored by testing the in vivo packaging efficacy of transcripts containing deletions or changes in the RNA sequences. The pac sequences have been localized to the 5' untranslated regions of the viral transcripts. Major changes in the pac sequences are either tolerated or ameliorated by suppressor mutations in the RNA sequence. Changes in the genomic packaging program can be established as a result of mutations in P1, the major structural protein of the procapsid and the determinant of RNA binding specificity. Conclusion Although Φ8 is distantly related to bacteriophage Φ6, and does not show sequence similarity, it has a similar genomic packaging program. This program, however, is less stringent than that of Φ6.

  13. Critical mutation rate has an exponential dependence on population size in haploid and diploid populations.

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    Elizabeth Aston

    Full Text Available 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

  14. Inducible transgenics. New lessons on events governing the induction and commitment in mammary tumorigenesis

    International Nuclear Information System (INIS)

    Hulit, James; Di Vizio, Dolores; Pestell, Richard G

    2001-01-01

    Breast cancer arises from multiple genetic events that together contribute to the established, irreversible malignant phenotype. The development of inducible tissue-specific transgenics has allowed a careful dissection of the events required for induction and subsequent maintenance of tumorigenesis. Mammary gland targeted expression of oncogenic Ras or c-Myc is sufficient for the induction of mammary gland tumorigenesis in the rodent, and when overexpressed together the rate of tumor onset is substantially enhanced. In an exciting recent finding, D'Cruz et al discovered tetracycline-regulated c-Myc overexpression in the mammary gland induced invasive mammary tumors that regressed upon withdrawal of c-Myc expression. Almost one-half of the c-Myc-induced tumors harbored K-ras or N-ras gene point mutations, correlating with tumor persistence on withdrawal of c-Myc transgene expression. These findings suggest maintenance of tumorigenesis may involve a second mutation within the Ras pathway

  15. Tempo and mode of genomic mutations unveil human evolutionary history.

    Science.gov (United States)

    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.

  16. Calcium dynamics of cortical astrocytic networks in vivo.

    Directory of Open Access Journals (Sweden)

    Hajime Hirase

    2004-04-01

    Full Text Available Large and long-lasting cytosolic calcium surges in astrocytes have been described in cultured cells and acute slice preparations. The mechanisms that give rise to these calcium events have been extensively studied in vitro. However, their existence and functions in the intact brain are unknown. We have topically applied Fluo-4 AM on the cerebral cortex of anesthetized rats, and imaged cytosolic calcium fluctuation in astrocyte populations of superficial cortical layers in vivo, using two-photon laser scanning microscopy. Spontaneous [Ca(2+](i events in individual astrocytes were similar to those observed in vitro. Coordination of [Ca(2+](i events among astrocytes was indicated by the broad cross-correlograms. Increased neuronal discharge was associated with increased astrocytic [Ca(2+](i activity in individual cells and a robust coordination of [Ca(2+](i signals in neighboring astrocytes. These findings indicate potential neuron-glia communication in the intact brain.

  17. Molecular nature of X-ray-induced mutations compared with that of spontaneous ones in human c-hprt gene integrated into mammalian chromosomal DNA

    International Nuclear Information System (INIS)

    Kimura, Hiroshi; Kato, Takesi.

    1992-01-01

    X-ray-induced mutations were analysed at molecular levels in comparison with spontaneous mutations. Altered sequences were determined tentatively of 30 independent X-ray-induced mutations in a cDNA of the human hprt gene which was integrated into mammalian chromosome as a part of a shuttle vector. Mutations consisted of base substitutions (37 %), frameshifts (27 %), deletions (27 %) and others (10 %). All these mutational events were distributed randomly over the gene without there being hot spots. The spectrum and distribution of X-ray-induced mutations resembled those of spontaneous mutations. Among base substitutions, transversions were predominant and base substitution mutations occurred more at A:T sites than at G:C sites, which is also the case in spontaneous mutations. Most of the frameshift and deletion mutations induced by X-rays, as well as those spontaneously arising, were characterized by the existence of short direct repeats of several identical bases in a row at the sites of the mutations. A slippage misalignment mechanism in replication well accounts for the generation of these classes of mutations. Judging from the data accumulated so far, it can be concluded that X-ray-induced mutations at molecular levels are similar to those spontaneously occurring. (author)

  18. ERK mediated upregulation of death receptor 5 overcomes the lack of p53 functionality in the diaminothiazole DAT1 induced apoptosis in colon cancer models: efficiency of DAT1 in Ras-Raf mutated cells.

    Science.gov (United States)

    Thamkachy, Reshma; Kumar, Rohith; Rajasekharan, K N; Sengupta, Suparna

    2016-03-08

    p53 is a tumour suppressor protein that plays a key role in many steps of apoptosis, and malfunctioning of this transcription factor leads to tumorigenesis. Prognosis of many tumours also depends upon the p53 status. Most of the clinically used anticancer compounds activate p53 dependent pathway of apoptosis and hence require p53 for their mechanism of action. Further, Ras/Raf/MEK/ERK axis is an important signaling pathway activated in many cancers. Dependence of diaminothiazoles, compounds that have gained importance recently due to their anticancer and anti angiogenic activities, were tested in cancer models with varying p53 or Ras/Raf mutational status. In this study we have used p53 mutated and knock out colon cancer cells and xenograft tumours to study the role of p53 in apoptosis mediated by diaminothiazoles. Colon cancer cell lines with varying mutational status for Ras or Raf were also used. We have also examined the toxicity and in vivo efficacy of a lead diaminothiazole 4-Amino-5-benzoyl-2-(4-methoxy phenylamino)thiazole (DAT1) in colon cancer xenografts. We have found that DAT1 is active in both in vitro and in vivo models with nonfunctional p53. Earlier studies have shown that extrinsic pathway plays major role in DAT1 mediated apoptosis. In this study, we have found that DAT1 is causing p53 independent upregulation of the death receptor 5 by activating the Ras/Raf/MEK/ERK signaling pathway both in wild type and p53 suppressed colon cancer cells. These findings are also confirmed by the in vivo results. Further, DAT1 is more efficient to induce apoptosis in colon cancer cells with mutated Ras or Raf. Minimal toxicity in both acute and subacute studies along with the in vitro and in vivo efficacy of DAT1 in cancers with both wild type and nonfunctional p53 place it as a highly beneficial candidate for cancer chemotherapy. Besides, efficiency in cancer cells with mutations in the Ras oncoprotein or its downstream kinase Raf raise interest in

  19. BCR-ABL1 mutation development during first-line treatment with dasatinib or imatinib for chronic myeloid leukemia in chronic phase.

    Science.gov (United States)

    Hughes, T P; Saglio, G; Quintás-Cardama, A; Mauro, M J; Kim, D-W; Lipton, J H; Bradley-Garelik, M B; Ukropec, J; Hochhaus, A

    2015-09-01

    BCR-ABL1 mutations are a common, well-characterized mechanism of resistance to imatinib as first-line treatment of chronic myeloid leukemia in chronic phase (CML-CP). Less is known about mutation development during first-line treatment with dasatinib and nilotinib, despite increased use because of higher response rates compared with imatinib. Retrospective analyses were conducted to characterize mutation development in patients with newly diagnosed CML-CP treated with dasatinib (n=259) or imatinib (n=260) in DASISION (Dasatinib versus Imatinib Study in Treatment-Naive CML-CP), with 3-year minimum follow-up. Mutation screening, including patients who discontinued treatment and patients who had a clinically relevant on-treatment event (no confirmed complete cytogenetic response (cCCyR) and no major molecular response (MMR) within 12 months; fivefold increase in BCR-ABL1 with loss of MMR; loss of CCyR), yielded a small number of patients with mutations (dasatinib, n=17; imatinib, n=18). Dasatinib patients had a narrower spectrum of mutations (4 vs 12 sites for dasatinib vs imatinib), fewer phosphate-binding loop mutations (1 vs 9 mutations), fewer multiple mutations (1 vs 6 patients) and greater occurrence of T315I (11 vs 0 patients). This trial was registered at www.clinicaltrials.gov as NCT00481247.

  20. Longevity in vivo of primary cell wall cellulose synthases.

    Science.gov (United States)

    Hill, Joseph Lee; Josephs, Cooper; Barnes, William J; Anderson, Charles T; Tien, Ming

    2018-02-01

    Our work focuses on understanding the lifetime and thus stability of the three main cellulose synthase (CESA) proteins involved in primary cell wall synthesis of Arabidopsis. It had long been thought that a major means of CESA regulation was via their rapid degradation. However, our studies here have uncovered that AtCESA proteins are not rapidly degraded. Rather, they persist for an extended time in the plant cell. Plant cellulose is synthesized by membrane-embedded cellulose synthase complexes (CSCs). The CSC is composed of cellulose synthases (CESAs), of which three distinct isozymes form the primary cell wall CSC and another set of three isozymes form the secondary cell wall CSC. We determined the stability over time of primary cell wall (PCW) CESAs in Arabidopsis thaliana seedlings, using immunoblotting after inhibiting protein synthesis with cycloheximide treatment. Our work reveals very slow turnover for the Arabidopsis PCW CESAs in vivo. Additionally, we show that the stability of all three CESAs within the PCW CSC is altered by mutations in individual CESAs, elevated temperature, and light conditions. Together, these results suggest that CESA proteins are very stable in vivo, but that their lifetimes can be modulated by intrinsic and environmental cues.

  1. Mutation Analysis in Classical Phenylketonuria Patients Followed by Detecting Haplotypes Linked to Some PAH Mutations.

    Science.gov (United States)

    Dehghanian, Fatemeh; Silawi, Mohammad; Tabei, Seyed M B

    2017-02-01

    Deficiency of phenylalanine hydroxylase (PAH) enzyme and elevation of phenylalanine in body fluids cause phenylketonuria (PKU). The gold standard for confirming PKU and PAH deficiency is detecting causal mutations by direct sequencing of the coding exons and splicing involved sequences of the PAH gene. Furthermore, haplotype analysis could be considered as an auxiliary approach for detecting PKU causative mutations before direct sequencing of the PAH gene by making comparisons between prior detected mutation linked-haplotypes and new PKU case haplotypes with undetermined mutations. In this study, 13 unrelated classical PKU patients took part in the study detecting causative mutations. Mutations were identified by polymerase chain reaction (PCR) and direct sequencing in all patients. After that, haplotype analysis was performed by studying VNTR and PAHSTR markers (linked genetic markers of the PAH gene) through application of PCR and capillary electrophoresis (CE). Mutation analysis was performed successfully and the detected mutations were as follows: c.782G>A, c.754C>T, c.842C>G, c.113-115delTCT, c.688G>A, and c.696A>G. Additionally, PAHSTR/VNTR haplotypes were detected to discover haplotypes linked to each mutation. Mutation detection is the best approach for confirming PAH enzyme deficiency in PKU patients. Due to the relatively large size of the PAH gene and high cost of the direct sequencing in developing countries, haplotype analysis could be used before DNA sequencing and mutation detection for a faster and cheaper way via identifying probable mutated exons.

  2. Autosomal mutations affecting Y chromosome loops in Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Petrucci Romano

    2008-04-01

    Full Text Available Abstract Background The Y chromosome of Drosophila melanogaster harbors several genes required for male fertility. The genes for these fertility factors are very large in size and contain conspicuous amounts of repetitive DNA and transposons. Three of these loci (ks-1, kl-3 and kl-5 have the ability to develop giant lampbrush-like loops in primary spermatocytes, a cytological manifestation of their active state in these cells. Y-loops bind a number of non-Y encoded proteins, but the mechanisms regulating their development and their specific functions are still to be elucidated. Results Here we report the results of a screen of 726 male sterile lines to identify novel autosomal genes controlling Y-loop function. We analyzed mutant testis preparations both in vivo and by immunofluorescence using antibodies directed against Y-loop-associated proteins. This screen enabled us to isolate 17 mutations at 15 loci whose wild-type function is required for proper Y-loop morphogenesis. Six of these loci are likely to specifically control loop development, while the others display pleiotropic effects on both loops and meiotic processes such as spermiogenesis, sperm development and maturation. We also determined the map position of the mutations affecting exclusively Y-loop morphology. Conclusion Our cytological screening permitted us to identify novel genetic functions required for male spermatogenesis, some of which show pleiotropic effects. Analysis of these mutations also shows that loop development can be uncoupled from meiosis progression. These data represent a useful framework for the characterization of Y-loop development at a molecular level and for the study of the genetic control of heterochromatin.

  3. Retrospective study of adjuvant icotinib in postoperative lung cancer patients harboring epidermal growth factor receptor mutations.

    Science.gov (United States)

    Yao, Shuyang; Zhi, Xiuyi; Wang, Ruotian; Qian, Kun; Hu, Mu; Zhang, Yi

    2016-09-01

    Epidermal growth factor receptor (EGFR) mutations occur in about 50% of Asian patients with non-small cell lung cancer (NSCLC). Patients with advanced NSCLC and EGFR mutations derive clinical benefit from treatment with EGFR-tyrosine kinase inhibitors (TKIs). This study assessed the efficacy and safety of adjuvant icotinib without chemotherapy in EGFR-mutated NSCLC patients undergoing resection of stage IB-IIIA. Our retrospective study enrolled 20 patients treated with icotinib as adjuvant therapy. Survival factors were evaluated by univariate and Cox regression analysis. The median follow-up time was 30 months (range 24-41). At the data cut-off, five patients (25%) had recurrence or metastasis and one patient had died of the disease. The two-year disease-free survival (DFS) rate was 85%. No recurrence occurred in the high-risk stage IB subgroup during the follow-up period. In univariate analysis, the micropapillary pattern had a statistically significant effect on DFS ( P = 0.040). Multivariate logistic regression analysis showed that there was no independent predictor. Drug related adverse events (AEs) occurred in nine patients (45.0%). The most common AEs were skin-related events and diarrhea, but were relatively mild. No grade 3 AEs or occurrences of intolerable toxicity were observed. Icotinib as adjuvant therapy is effective in patients harboring EGFR mutations after complete resection, with an acceptable AE profile. Further trials with larger sample sizes might confirm the efficiency of adjuvant TKI in selected patients. © 2016 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

  4. In vitro and in vivo genotoxic effects of somatic cell nuclear transfer cloned cattle meat.

    Science.gov (United States)

    Lee, Nam-Jin; Yang, Byoung-Chul; Jung, Yu-Ri; Lee, Jung-Won; Im, Gi-Sun; Seong, Hwan-Hoo; Park, Jin-Ki; Kang, Jong-Koo; Hwang, Seongsoo

    2011-09-01

    Although the nutritional composition and health status after consumption of the meat and milk derived from both conventionally bred (normal) and somatic cell nuclear transferred (cloned) animals and their progeny are not different, little is known about their food safeties like genetic toxicity. This study is performed to examine both in vitro (bacterial mutation and chromosome aberration) and in vivo (micronucleus) genotoxicity studies of cloned cattle meat. The concentrations of both normal and cloned cattle meat extracts (0-10×) were tested to five strains of bacteria (Salmonella typhimurium: TA98, TA100, TA1535, and TA1537; Escherichia coli: WP2uvrA) for bacterial mutation and to Chinese hamster lung (CHL/IU) cells for chromosome aberration, respectively. For micronucleus test, ICR mice were divided into five dietary groups: commercial pellets (control), pellets containing 5% (N-5) and 10% (N-10) normal cattle meat, and pellets containing 5% (C-5) and 10% (C-10) cloned cattle meat. No test substance-related genotoxicity was noted in the five bacterial strains, CHL/IU cells, or mouse bone marrow cells, suggesting that the cloned cattle meat potentially may be safe in terms of mutagenic hazards. Thus, it can be postulated that the cloned cattle meat do not induce any harmful genotoxic effects in vitro and in vivo. Copyright © 2011 Elsevier Ltd. All rights reserved.

  5. Detection of somatic mutations by high-resolution DNA melting (HRM) analysis in multiple cancers.

    Science.gov (United States)

    Gonzalez-Bosquet, Jesus; Calcei, Jacob; Wei, Jun S; Garcia-Closas, Montserrat; Sherman, Mark E; Hewitt, Stephen; Vockley, Joseph; Lissowska, Jolanta; Yang, Hannah P; Khan, Javed; Chanock, Stephen

    2011-01-17

    Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM) curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each). HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples.

  6. Detection of somatic mutations by high-resolution DNA melting (HRM analysis in multiple cancers.

    Directory of Open Access Journals (Sweden)

    Jesus Gonzalez-Bosquet

    Full Text Available Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each. HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples.

  7. Simulating the Emergence and Survival of Mutations Using a Self Regulating Multitype Branching Processes

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    Charles J. Mode

    2011-01-01

    Full Text Available It is difficult for an experimenter to study the emergence and survival of mutations, because mutations are rare events so that large experimental population must be maintained to ensure a reasonable chance that a mutation will be observed. In his famous book, The Genetical Theory of Natural Selection, Sir R. A. Fisher introduced branching processes into evolutionary genetics as a framework for studying the emergence and survival of mutations in an evolving population. During the lifespan of Fisher, computer technology had not advanced to a point at which it became an effective tool for simulating the phenomenon of the emergence and survival of mutations, but given the wide availability of personal desktop and laptop computers, it is now possible and financially feasible for investigators to perform Monte Carlo Simulation experiments. In this paper all computer simulation experiments were carried out within a framework of self regulating multitype branching processes, which are part of a stochastic working paradigm. Emergence and survival of mutations could also be studied within a deterministic paradigm, which raises the issue as to what sense are predictions based on the stochastic and deterministic models are consistent. To come to grips with this issue, a technique was used such that a deterministic model could be embedded in a branching process so that the predictions of both the stochastic and deterministic compared based on the same assigned values of parameters.

  8. Empirical evaluation of cell critical volume dose vs. cell response function for pink mutations in tradescantia

    International Nuclear Information System (INIS)

    Varma, M.N.; Bond, V.P.

    1982-01-01

    Microdosimetric spectra for 0.43, 1.8, and 14.7 MeV neutrons, and for 215 kVp x rays and 1250 keV gammas were used in conjunction with relative biological effectiveness (RBE) values for pink mutations in Tradescantia to obtain an effectiveness function (i.e., a cell critical volume dose vs. cell response function). This effectiveness function (or hit size weighting function) provides the probability of inducing a biological effect of interest (in the present study, pink mutations in Tradescantia) as a function of lineal energy density y. In a preliminary analysis the critical value of y above which pink mutations are seen was 4.5 keV/μm, and the value of y at which the probability reaches unity was 115 keV/μm. Idealized but approximate event size distributions for mono-LET particles ranging from 10 to 5000 keV/μm were generated, and these distributions were weighted by the effectiveness function to determine the pink mutation frequencies. Results are compared with measured pink mutation frequencies for 11 keV/μm ( 12 C) and 31 keV/μm ( 20 Ne) ions

  9. The relationship between religion and thought-action fusion: use of an in vivo paradigm.

    Science.gov (United States)

    Berman, Noah C; Abramowitz, Jonathan S; Pardue, Caleb M; Wheaton, Michael G

    2010-07-01

    Research has demonstrated that higher levels of religiosity are positively correlated with thought-action fusion (TAF), a set of cognitive biases found to be associated with obsessive-compulsive symptoms. However, previous studies have exclusively relied on a nomothetic approach to measuring TAF using a single self-report instrument, the thought-action fusion scale. The current study examined the relationship between religiosity and TAF using an in vivo behaviorally-based assessment in which participants thought about and wrote down thoughts of negative events involving loved ones. Forty-three highly religious Protestant Christians were compared to 30 Atheists/Agnostics on their in vivo ratings of anxiety, estimates of likelihood, and moral wrongness related to the negative thoughts. Results indicated that compared to the non-religious participants, those who were highly religious believed that writing and thinking about the negative events was more morally wrong and increased the likelihood of the event. Results are discussed in terms of the potential relationship between certain religious teachings and TAF-related beliefs about the importance, significance, and influence of thoughts. Copyright 2010 Elsevier Ltd. All rights reserved.

  10. The Oenothera plastome mutator: effect of UV irradiation and nitroso-methyl urea on mutation frequencies

    International Nuclear Information System (INIS)

    Sears, B.B.; Sokalski, M.B.

    1991-01-01

    Oenothera plants homozygous for a recessive plastome mutator allele (pm) showed spontaneous mutation frequencies for plastome genes that are 200-fold higher than spontaneous levels. Mutations occurred at high frequencies in plants grown in the field, in a glasshouse, or as leaf tip cultures under fluorescent light, indicating that the plastome mutator activity is UV-independent. However, the chlorotic sectors became visible at an earlier stage of development when seedlings were irradiated, compared to seedlings that were not exposed to UV. These results imply that the rate of sorting-out was increased by the irradiation treatment, possibly due to a decrease in the effective number of multiplication-competent plastids, or a reduction in the extent of cytoplasmic mixing. Nitroso-methyl urea treatment of seeds had a dramatic effect on mutation frequency in both wild-type and plastome mutator samples. When the background mutation rates were low, the combination of the plastome mutator nucleus and the chemical mutagenesis treatment resulted in a synergistic effect, suggesting that the plastome mutator may involve a cpDNA repair pathway. (author)

  11. Confirmation of emergence of mutations associated with atovaquone-proguanil resistance in unexposed Plasmodium falciparum isolates from Africa

    Directory of Open Access Journals (Sweden)

    Kyle Dennis E

    2006-10-01

    Full Text Available Abstract Background In vitro and in vivo resistance of Plasmodium falciparum to atovaquone or atovaquone-proguanil hydrochloride combination has been associated to two point mutations in the parasite cytochrome b (cytb gene (Tyr268Ser and Tyr268Asn. However, little is known about the prevalence of codon-268 mutations in natural populations of P. falciparum without previous exposure to the drug in Africa. Methods The prevalence of codon-268 mutations in the cytb gene of African P. falciparum isolates from Nigeria, Malawi and Senegal, where atovaquone-proguanil has not been introduced for treatment of malaria was assessed. Genotyping of the cytb gene in isolates of P. falciparum was performed by PCR-restriction fragment length polymorphism and confirmed by sequencing. Results 295 samples from Nigeria (111, Malawi (91 and Senegal (93 were successfully analyzed for detection of either mutant Tyr268Ser or Tyr268Asn. No case of Ser268 or Asn268 was detected in cytb gene of parasites from Malawi or Senegal. However, Asn268 was detected in five out of 111 (4.5% unexposed P. falciparum isolates from Nigeria. In addition, one out of these five mutant Asn268 isolates showed an additional cytb mutation leading to a Pro266Thr substitution inside the ubiquinone reduction site. Conclusion No Tyr268Ser mutation is found in cytb of P. falciparum isolates from Nigeria, Malawi or Senegal. This study reports for the first time cytb Tyr268Asn mutation in unexposed P. falciparum isolates from Nigeria. The emergence in Africa of P. falciparum isolates with cytb Tyr268Asn mutation is a matter of serious concern. Continuous monitoring of atovaquone-proguanil resistant P. falciparum in Africa is warranted for the rational use of this new antimalarial drug, especially in non-immune travelers.

  12. Confirmation of emergence of mutations associated with atovaquone-proguanil resistance in unexposed Plasmodium falciparum isolates from Africa.

    Science.gov (United States)

    Happi, Christian T; Gbotosho, Grace O; Folarin, Onikepe A; Milner, Danny; Sarr, Ousmane; Sowunmi, Akintunde; Kyle, Dennis E; Milhous, Wilbur K; Wirth, Dyann F; Oduola, Ayoade M J

    2006-10-04

    In vitro and in vivo resistance of Plasmodium falciparum to atovaquone or atovaquone-proguanil hydrochloride combination has been associated to two point mutations in the parasite cytochrome b (cytb) gene (Tyr268Ser and Tyr268Asn). However, little is known about the prevalence of codon-268 mutations in natural populations of P. falciparum without previous exposure to the drug in Africa. The prevalence of codon-268 mutations in the cytb gene of African P. falciparum isolates from Nigeria, Malawi and Senegal, where atovaquone-proguanil has not been introduced for treatment of malaria was assessed. Genotyping of the cytb gene in isolates of P. falciparum was performed by PCR-restriction fragment length polymorphism and confirmed by sequencing. 295 samples from Nigeria (111), Malawi (91) and Senegal (93) were successfully analyzed for detection of either mutant Tyr268Ser or Tyr268Asn. No case of Ser268 or Asn268 was detected in cytb gene of parasites from Malawi or Senegal. However, Asn268 was detected in five out of 111 (4.5%) unexposed P. falciparum isolates from Nigeria. In addition, one out of these five mutant Asn268 isolates showed an additional cytb mutation leading to a Pro266Thr substitution inside the ubiquinone reduction site. No Tyr268Ser mutation is found in cytb of P. falciparum isolates from Nigeria, Malawi or Senegal. This study reports for the first time cytb Tyr268Asn mutation in unexposed P. falciparum isolates from Nigeria. The emergence in Africa of P. falciparum isolates with cytb Tyr268Asn mutation is a matter of serious concern. Continuous monitoring of atovaquone-proguanil resistant P. falciparum in Africa is warranted for the rational use of this new antimalarial drug, especially in non-immune travelers.

  13. Mutations causative of familial hypercholesterolaemia

    DEFF Research Database (Denmark)

    Benn, Marianne; Watts, Gerald F; Tybjærg-Hansen, Anne

    2016-01-01

    causing mutations in 98 098 participants from the general population, the Copenhagen General Population Study. METHODS AND RESULTS: We genotyped for LDLR[W23X;W66G;W556S] and APOB[R3500Q] accounting for 38.7% of pathogenic FH mutations in Copenhagen. Clinical FH assessment excluded mutation information......-cholesterol concentration to discriminate between mutation carriers and non-carriers was 4.4 mmol/L. CONCLUSION: Familial hypercholesterolaemia-causing mutations are estimated to occur in 1:217 in the general population and are best identified by a definite or probable phenotypic diagnosis of FH based on the DLCN criteria....... The prevalence of the four FH mutations was 0.18% (1:565), suggesting a total prevalence of FH mutations of 0.46% (1:217). Using the Dutch Lipid Clinic Network (DLCN) criteria, odds ratios for an FH mutation were 439 (95% CI: 170-1 138) for definite FH, 90 (53-152) for probable FH, and 18 (13-25) for possible FH...

  14. Mutations within the nuclear localization signal of the porcine reproductive and respiratory syndrome virus nucleocapsid protein attenuate virus replication

    International Nuclear Information System (INIS)

    Lee, Changhee; Hodgins, Douglas; Calvert, Jay G.; Welch, Siao-Kun W.; Jolie, Rika; Yoo, Dongwan

    2006-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is an RNA virus replicating in the cytoplasm, but the nucleocapsid (N) protein is specifically localized to the nucleus and nucleolus in virus-infected cells. A 'pat7' motif of 41-PGKK(N/S)KK has previously been identified in the N protein as the functional nuclear localization signal (NLS); however, the biological consequences of N protein nuclear localization are unknown. In the present study, the role of N protein nuclear localization during infection was investigated in pigs using an NLS-null mutant virus. When two lysines at 43 and 44 at the NLS locus were substituted to glycines, the modified NLS with 41-PGGGNKK restricted the N protein to the cytoplasm. This NLS-null mutation was introduced into a full-length infectious cDNA clone of PRRSV. Upon transfection of cells, the NLS-null full-length clone induced cytopathic effects and produced infectious progeny. The NLS-null virus grew to a titer 100-fold lower than that of wild-type virus. To examine the response to NLS-null PRRSV in the natural host, three groups of pigs, consisting of seven animals per group, were intranasally inoculated with wild-type, placebo, or NLS-null virus, and the animals were maintained for 4 weeks. The NLS-null-infected pigs had a significantly shorter mean duration of viremia than wild-type-infected pigs but developed significantly higher titers of neutralizing antibodies. Mutations occurred at the NLS locus in one pig during viremia, and four types of mutations were identified: 41-PGRGNKK, 41-PGGRNKK, and 41-PGRRNKK, and 41-PGKKSKK. Both wild-type and NLS-null viruses persisted in the tonsils for at least 4 weeks, and the NLS-null virus persisting in the tonsils was found to be mutated to either 41-PGRGNKK or 41-PGGRNKK in all pigs. No other mutation was found in the N gene. All types of reversions which occurred during viremia and persistence were able to translocate the mutated N proteins to the nucleus, indicating a

  15. Position of nonmuscle myosin heavy chain IIA (NMMHC-IIA) mutations predicts the natural history of MYH9-related disease

    DEFF Research Database (Denmark)

    Pecci, A.; Panza, E.; Pujol-Moix, N.

    2008-01-01

    MYH9-related disease (MYH9-RD) is a rare autosomal-dominant disorder caused by mutations in MYH9, the gene for the heavy chain of nonmuscle myosin IIA (NMMHC-IIA). All patients present from birth with macrothrombocytopenia, but in infancy or adult life, some of them develop sensorineural deafness...... to 50 unrelated pedigrees. The risk of noncongenital manifestations associated with different genotypes was estimated over time by event-free survival analysis. We demonstrated that all subjects with mutations in the motor domain of NMMHC-IIA present with severe thrombocytopenia and develop nephritis...... and deafness before the age of 40 years, while those with mutations in the tail domain have a much lower risk of noncongenital complications and significantly higher platelet counts. We also evaluated the clinical course of patients with mutations in the four most frequently affected residues of NMMHC...

  16. Analysis of PIK3CA Mutations and Activation Pathways in Triple Negative Breast Cancer.

    Directory of Open Access Journals (Sweden)

    Paolo Cossu-Rocca

    Full Text Available Triple Negative Breast Cancer (TNBC accounts for 12-24% of all breast carcinomas, and shows worse prognosis compared to other breast cancer subtypes. Molecular studies demonstrated that TNBCs are a heterogeneous group of tumors with different clinical and pathologic features, prognosis, genetic-molecular alterations and treatment responsivity. The PI3K/AKT is a major pathway involved in the regulation of cell survival and proliferation, and is the most frequently altered pathway in breast cancer, apparently with different biologic impact on specific cancer subtypes. The most common genetic abnormality is represented by PIK3CA gene activating mutations, with an overall frequency of 20-40%. The aims of our study were to investigate PIK3CA gene mutations on a large series of TNBC, to perform a wider analysis on genetic alterations involving PI3K/AKT and BRAF/RAS/MAPK pathways and to correlate the results with clinical-pathologic data.PIK3CA mutation analysis was performed by using cobas® PIK3CA Mutation Test. EGFR, AKT1, BRAF, and KRAS genes were analyzed by sequencing. Immunohistochemistry was carried out to identify PTEN loss and to investigate for PI3K/AKT pathways components.PIK3CA mutations were detected in 23.7% of TNBC, whereas no mutations were identified in EGFR, AKT1, BRAF, and KRAS genes. Moreover, we observed PTEN loss in 11.3% of tumors. Deregulation of PI3K/AKT pathways was revealed by consistent activation of pAKT and p-p44/42 MAPK in all PIK3CA mutated TNBC.Our data shows that PIK3CA mutations and PI3K/AKT pathway activation are common events in TNBC. A deeper investigation on specific TNBC genomic abnormalities might be helpful in order to select patients who would benefit from current targeted therapy strategies.

  17. Analysis of PIK3CA Mutations and Activation Pathways in Triple Negative Breast Cancer.

    Science.gov (United States)

    Cossu-Rocca, Paolo; Orrù, Sandra; Muroni, Maria Rosaria; Sanges, Francesca; Sotgiu, Giovanni; Ena, Sara; Pira, Giovanna; Murgia, Luciano; Manca, Alessandra; Uras, Maria Gabriela; Sarobba, Maria Giuseppina; Urru, Silvana; De Miglio, Maria Rosaria

    2015-01-01

    Triple Negative Breast Cancer (TNBC) accounts for 12-24% of all breast carcinomas, and shows worse prognosis compared to other breast cancer subtypes. Molecular studies demonstrated that TNBCs are a heterogeneous group of tumors with different clinical and pathologic features, prognosis, genetic-molecular alterations and treatment responsivity. The PI3K/AKT is a major pathway involved in the regulation of cell survival and proliferation, and is the most frequently altered pathway in breast cancer, apparently with different biologic impact on specific cancer subtypes. The most common genetic abnormality is represented by PIK3CA gene activating mutations, with an overall frequency of 20-40%. The aims of our study were to investigate PIK3CA gene mutations on a large series of TNBC, to perform a wider analysis on genetic alterations involving PI3K/AKT and BRAF/RAS/MAPK pathways and to correlate the results with clinical-pathologic data. PIK3CA mutation analysis was performed by using cobas® PIK3CA Mutation Test. EGFR, AKT1, BRAF, and KRAS genes were analyzed by sequencing. Immunohistochemistry was carried out to identify PTEN loss and to investigate for PI3K/AKT pathways components. PIK3CA mutations were detected in 23.7% of TNBC, whereas no mutations were identified in EGFR, AKT1, BRAF, and KRAS genes. Moreover, we observed PTEN loss in 11.3% of tumors. Deregulation of PI3K/AKT pathways was revealed by consistent activation of pAKT and p-p44/42 MAPK in all PIK3CA mutated TNBC. Our data shows that PIK3CA mutations and PI3K/AKT pathway activation are common events in TNBC. A deeper investigation on specific TNBC genomic abnormalities might be helpful in order to select patients who would benefit from current targeted therapy strategies.

  18. Systematic reconstruction of autism biology from massive genetic mutation profiles.

    Science.gov (United States)

    Luo, Weijun; Zhang, Chaolin; Jiang, Yong-Hui; Brouwer, Cory R

    2018-04-01

    Autism spectrum disorder (ASD) affects 1% of world population and has become a pressing medical and social problem worldwide. As a paradigmatic complex genetic disease, ASD has been intensively studied and thousands of gene mutations have been reported. Because these mutations rarely recur, it is difficult to (i) pinpoint the fewer disease-causing versus majority random events and (ii) replicate or verify independent studies. A coherent and systematic understanding of autism biology has not been achieved. We analyzed 3392 and 4792 autism-related mutations from two large-scale whole-exome studies across multiple resolution levels, that is, variants (single-nucleotide), genes (protein-coding unit), and pathways (molecular module). These mutations do not recur or replicate at the variant level, but significantly and increasingly do so at gene and pathway levels. Genetic association reveals a novel gene + pathway dual-hit model, where the mutation burden becomes less relevant. In multiple independent analyses, hundreds of variants or genes repeatedly converge to several canonical pathways, either novel or literature-supported. These pathways define recurrent and systematic ASD biology, distinct from previously reported gene groups or networks. They also present a catalog of novel ASD risk factors including 118 variants and 72 genes. At a subpathway level, most variants disrupt the pathway-related gene functions, and in the same gene, they tend to hit residues extremely close to each other and in the same domain. Multiple interacting variants spotlight key modules, including the cAMP (adenosine 3',5'-monophosphate) second-messenger system and mGluR (metabotropic glutamate receptor) signaling regulation by GRKs (G protein-coupled receptor kinases). At a superpathway level, distinct pathways further interconnect and converge to three biology themes: synaptic function, morphology, and plasticity.

  19. A case of early onset rectal cancer of Lynch syndrome with a novel deleterious PMS2 mutation.

    Science.gov (United States)

    Nomura, Sachio; Fujimoto, Yoshiya; Yamamoto, Noriko; Sato, Yuri; Ashihara, Yuumi; Kita, Mizuho; Yamaguchi, Junya; Ishikawa, Yuichi; Ueno, Masashi; Arai, Masami

    2015-10-01

    Heterozygous deleterious mutation of the PMS2 gene is a cause of Lynch syndrome, an autosomal dominant cancer disease. However, the frequency of PMS2 mutation is rare compared with that of the other causative genes; MSH2, MLH1 and MSH6. PMS2 mutation has so far only been reported once from a Japanese facility. Detection of PMS2 mutation is relatively complicated due to the existence of 15 highly homologous pseudogenes, and its gene conversion event with the pseudogene PMS2CL. Therefore, for PMS2 mutation analysis, it is crucial to clearly distinguish PMS2 from its pseudogenes. We report here a novel deleterious 11 bp deletion mutation of exon 11 of PMS2 distinguished from PMS2CL in a 34-year-old Japanese female with rectal cancer. PMS2 mutated at c.1492del11 results in a truncated 500 amino acid protein rather than the wild-type protein of 862 amino acids. This is supported by the fact that, although there is usually concordance between MLH1 and PMS2 expression, cells were immunohistochemically positive for MLH1, whereas PMS2 could not be immunohistochemically stained using an anti-C-terminal PMS2 antibody, or effective PMS2 mRNA degradation with NMD caused by the frameshift mutation. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  20. A VESICLE TRAFFICKING PROTEIN αSNAP REGULATES PANETH CELL DIFFERENTIATION IN VIVO

    Science.gov (United States)

    Lechuga, Susana; Naydenov, Nayden G.; Feygin, Alex; Jimenez, Antonio J.; Ivanov, Andrei I.

    2017-01-01

    A soluble N-ethylmaleimide-sensitive factor-attachment protein alpha (αSNAP) is a multifunctional scaffolding protein that regulates intracellular vesicle trafficking and signaling. In cultured intestinal epithelial cells, αSNAP has been shown to be essential for cell survival, motility, and adhesion; however, its physiologic functions in the intestinal mucosa remain unknown. In the present study, we used a mouse with a spontaneous hydrocephalus with hop gait (hyh) mutation of αSNAP to examine the roles of this trafficking protein in regulating intestinal epithelial homeostasis in vivo. Homozygous hyh mice demonstrated decreased expression of αSNAP protein in the intestinal epithelium, but did not display gross abnormalities of epithelial architecture in the colon and ileum. Such αSNAP depletion attenuated differentiation of small intestinal epithelial enteroids ex vivo. Furthermore, αSNAP-deficient mutant animals displayed reduced formation of lysozyme granules in small intestinal crypts and decreased expression of lysozyme and defensins in the intestinal mucosa, which is indicative of defects in Paneth cell differentiation. By contrast, development of Goblet cells, enteroendocrine cells, and assembly of enterocyte apical junctions was not altered in hyh mutant mice. Our data revealed a novel role of αSNAP in the intestinal Paneth cell differentiation in vivo. PMID:28359759

  1. Mutator activity in Schizophyllum commune

    Energy Technology Data Exchange (ETDEWEB)

    Shneyour, Y.; Koltin, Y. (Tel Aviv Univ. (Israel). Dept. of Microbiology)

    1983-01-01

    A strain with an elevated level of spontaneous mutations and an especially high rate of reversion at a specific locus (pab/sup -/) was identified. The mutator trait is recessive. UV sensitivity and the absence of a UV-specific endonucleolytic activity were associated with the enhancement of the mutation rate in mutator strains. The endonuclease associated with the regulation of the mutation rate also acted on single-stranded DNA. The molecular weight of this enzyme is about 38,000 daltons.

  2. Mutation and premating isolation

    Science.gov (United States)

    Woodruff, R. C.; Thompson, J. N. Jr

    2002-01-01

    While premating isolation might be traceable to different genetic mechanisms in different species, evidence supports the idea that as few as one or two genes may often be sufficient to initiate isolation. Thus, new mutation can theoretically play a key role in the process. But it has long been thought that a new isolation mutation would fail, because there would be no other individuals for the isolation-mutation-carrier to mate with. We now realize that premeiotic mutations are very common and will yield a cluster of progeny carrying the same new mutant allele. In this paper, we discuss the evidence for genetically simple premating isolation barriers and the role that clusters of an isolation mutation may play in initiating allopatric, and even sympatric, species divisions.

  3. Constitutional abnormalities of IDH1 combined with secondary mutations predispose a patient with Maffucci syndrome to acute lymphoblastic leukemia.

    Science.gov (United States)

    Hirabayashi, Shinsuke; Seki, Masafumi; Hasegawa, Daisuke; Kato, Motohiro; Hyakuna, Nobuyuki; Shuo, Takuya; Kimura, Shunsuke; Yoshida, Kenichi; Kataoka, Keisuke; Fujii, Yoichi; Shiraishi, Yuichi; Chiba, Kenichi; Tanaka, Hiroko; Kiyokawa, Nobutaka; Miyano, Satoru; Ogawa, Seishi; Takita, Junko; Manabe, Atsushi

    2017-12-01

    Maffucci syndrome is a nonhereditary disorder caused by somatic mosaic isocitrate dehydrogenase 1 or 2 (IDH1 or IDH2) mutations and is characterized by multiple enchondromas along with hemangiomas. Malignant transformation of enchondromas to chondrosarcomas and secondary neoplasms, such as brain tumors or acute myeloid leukemia, are serious complications. A 15-year-old female with Maffucci syndrome developed B-cell precursor acute lymphoblastic leukemia (BCP-ALL). A somatic mutation in IDH1 was detected in hemangioma and leukemic cells. KRAS mutation and deletion of IKZF1 were detected in leukemic cells. Patients with Maffucci syndrome may, therefore, be at risk of BCP-ALL associated with secondary genetic events that affect lymphocyte differentiation. © 2017 Wiley Periodicals, Inc.

  4. Mutation profiles of phenylketonuria in Quebec populations: Evidence of stratification and novel mutations

    Energy Technology Data Exchange (ETDEWEB)

    Rozen, R.; Mascisch, A.; Scriver, C.R. (McGill Univ., Montreal (Canada)); Lambert, M. (Hopital Ste-Justine, Montreal (Canada)); Laframboise, R. (Centre Hospitalier Universite Laval, Quebec (Canada))

    1994-08-01

    Independent phenylketonuria (PKU) chromosomes (n=109) representing 80% of a proband cohort in Quebec province carry 18 different identified mutations in 20 different mutation/haplotype combinations. The study reported here, the third in a series on Quebec populations, was done in the Montreal region and predominantly on French Canadians. It has identified three novel mutations (A309D, D338Y, and 1054/1055delG [352fs]) and one unusual mutation/RFLP haplotype combination (E280K on Hp 2). The relative frequencies and distribution of PKU mutations were then compared in three regions and population subsets (eastern Quebec, French Canadian; western Quebec, French Canadian; and Montreal, non-French Canadian). The distributions of the prevalent and rare mutations are nonrandom and provide evidence for genetic stratification. The latter and the presence of eight unusual mutation/haplotype combinations in Quebec families with European ancestries (the aforementioned four and M1V, 165T, S349P, and R408W on Hp 1) corroborate demographic and anthropologic evidence, from elsewhere, for different origins of French Canadians in eastern and western Quebec. 29 refs., 1 fig., 1 tab.

  5. Hyperfunctioning thyroid nodules in toxic multinodular goiter share activating thyrotropin receptor mutations with solitary toxic adenoma.

    Science.gov (United States)

    Tonacchera, M; Chiovato, L; Pinchera, A; Agretti, P; Fiore, E; Cetani, F; Rocchi, R; Viacava, P; Miccoli, P; Vitti, P

    1998-02-01

    Toxic multinodular goiter is a cause of nonautoimmune hyperthyroidism and is believed to differ in its nature and pathogenesis from toxic adenoma. Gain-of-function mutations of the TSH receptor gene have been identified as a cause of toxic adenoma. The pathogenesis at the molecular level of hyperfunctioning nodules in toxic multinodular goiter has yet not been reported. Six patients with a single hot nodule within a multinodular goiter and 11 patients with toxic thyroid adenoma were enrolled in our study. At histology five hyperfunctioning nodules in multinodular goiters showed the features of adenomas, and one was identified as a hyperplastic nodule. The entire exon 10 of the TSH receptor gene was directly sequenced after PCR amplification from genomic DNA obtained from surgical specimens. Functional studies of mutated receptors were performed in COS-7 cells. Five out of 6 (83%) hyperfunctioning nodules within toxic multinodular goiters harbored a TSH receptor mutation. A TSH receptor mutation was also evident in the hyperfunctioning nodule that at histology had the features of noncapsulated hyperplastic nodule. Among toxic adenomas, 8 out of 11 (72%) nodules harbored a TSH receptor mutation. All the mutations were heterozygotic and somatic. Nonfunctioning nodules, whether adenomas or hyperplastic nodules present in association with hyperfunctioning nodules in the same multinodular goiters, had no TSH receptor mutation. All the mutations identified had constitutive activity as assessed by cAMP production after expression in COS-7 cells. Hyperfunctioning thyroid nodules in multinodular goiters recognize the same pathogenetic event (TSH receptor mutation) as toxic adenoma. Other mechanisms are implicated in the growth of nonfunctioning thyroid nodules coexistent in the same gland.

  6. Impact of JAK2V617F Mutational Status on Phenotypic Features in Essential Thrombocythemia and Primary Myelofibrosis

    Directory of Open Access Journals (Sweden)

    İpek Yönal

    2016-05-01

    Full Text Available Objective: The JAK2V617F mutation is present in the majority of patients with essential thrombocythemia (ET and primary myelofibrosis (PMF. The impact of this mutation on disease phenotype in ET and PMF is still a matter of discussion. This study aims to determine whether there are differences in clinical presentation and disease outcome between ET and PMF patients with and without the JAK2V617F mutation. Materials and Methods: In this single-center study, a total of 184 consecutive Philadelphia-negative chronic myeloproliferative neoplasms, 107 cases of ET and 77 cases of PMF, were genotyped for JAK2V617F mutation using the JAK2 Ipsogen MutaScreen assay, which involves allele-specific polymerase chain reaction. Results: ET patients positive for JAK2V617F mutation had higher hemoglobin (Hb and hematocrit (Hct levels, lower platelet counts, and more prevalent splenomegaly at diagnosis compared to patients negative for the JAK2V617F mutation, but rates of major thrombotic events, arterial thrombosis, and venous thrombosis were comparable between the groups. At presentation, PMF patients with JAK2V617F mutation had significantly higher Hb and Hct levels and leukocyte counts than patients without the mutation. Similar to the findings of ET patients, thromboembolic rates were similar in PMF patients with and without theJAK2V617F mutation. For ET and PMF patients, no difference was observed in rates of death with respect to JAK2V617F mutational status. Moreover, leukemic transformation rate was not different in our PMF patients with and without JAK2V617F mutation. Conclusion: We conclude that JAK2V617F-mutated ET patients express a polycythemia vera-like phenotype and JAK2V617F mutation in PMF patients is associated with a more pronounced myeloproliferative phenotype.

  7. In-Vivo Synthetic Aperture and Plane Wave High Frame Rate Cardiac Imaging

    DEFF Research Database (Denmark)

    Stuart, Matthias Bo; Jensen, Jonas; Brandt, Andreas Hjelm

    2014-01-01

    A comparison of synthetic aperture imaging using spherical and plane waves with low number of emission events is presented. For both wave types, a 90 degree sector is insonified using 15 emission events giving a frame rate of 200 frames per second. Field II simulations of point targets show simil.......43 for spherical and 0.70 for plane waves. All measures are well within FDA limits for cardiac imaging. In-vivo images of the heart of a healthy 28-year old volunteer are shown....

  8. Better plants through mutations

    International Nuclear Information System (INIS)

    1988-01-01

    This is a public relations film describing problems associated with the genetic improvement of crop plants through induced mutations. Mutations are the ultimate source of genetic variation in plants. Mutation induction is now established as a practical tool in plant breeding. The Joint FAO/IAEA Division and the IAEA's laboratory at Seibersdorf have supported research and practical implementation of mutation breeding of both seed propagated and vegetatively propagated plants. Plant biotechnology based on in vitro culture and recombinant DNA technology will make a further significant contribution to plant breeding

  9. CRISPR/Cas9 – Mediated Precise Targeted Integration In Vivo Using a Double Cut Donor with Short Homology Arms

    Directory of Open Access Journals (Sweden)

    Xuan Yao

    2017-06-01

    Full Text Available Precisely targeted genome editing is highly desired for clinical applications. However, the widely used homology-directed repair (HDR-based genome editing strategies remain inefficient for certain in vivo applications. We here demonstrate a microhomology-mediated end-joining (MMEJ-based strategy for precisely targeted gene integration in transfected neurons and hepatocytes in vivo with efficiencies up to 20%, much higher (up to 10 fold than HDR-based strategy in adult mouse tissues. As a proof of concept of its therapeutic potential, we demonstrate the efficacy of MMEJ-based strategy in correction of Fah mutation and rescue of Fah−/− liver failure mice, offering an efficient approach for precisely targeted gene therapies.

  10. Predictable Phenotypes of Antibiotic Resistance Mutations.

    Science.gov (United States)

    Knopp, M; Andersson, D I

    2018-05-15

    Antibiotic-resistant bacteria represent a major threat to our ability to treat bacterial infections. Two factors that determine the evolutionary success of antibiotic resistance mutations are their impact on resistance level and the fitness cost. Recent studies suggest that resistance mutations commonly show epistatic interactions, which would complicate predictions of their stability in bacterial populations. We analyzed 13 different chromosomal resistance mutations and 10 host strains of Salmonella enterica and Escherichia coli to address two main questions. (i) Are there epistatic interactions between different chromosomal resistance mutations? (ii) How does the strain background and genetic distance influence the effect of chromosomal resistance mutations on resistance and fitness? Our results show that the effects of combined resistance mutations on resistance and fitness are largely predictable and that epistasis remains rare even when up to four mutations were combined. Furthermore, a majority of the mutations, especially target alteration mutations, demonstrate strain-independent phenotypes across different species. This study extends our understanding of epistasis among resistance mutations and shows that interactions between different resistance mutations are often predictable from the characteristics of the individual mutations. IMPORTANCE The spread of antibiotic-resistant bacteria imposes an urgent threat to public health. The ability to forecast the evolutionary success of resistant mutants would help to combat dissemination of antibiotic resistance. Previous studies have shown that the phenotypic effects (fitness and resistance level) of resistance mutations can vary substantially depending on the genetic context in which they occur. We conducted a broad screen using many different resistance mutations and host strains to identify potential epistatic interactions between various types of resistance mutations and to determine the effect of strain

  11. FBXW7 and NOTCH1 mutations in childhood T cell acute lymphoblastic leukaemia and T cell non-Hodgkin lymphoma.

    Science.gov (United States)

    Park, Myoung-Ja; Taki, Tomohiko; Oda, Megumi; Watanabe, Tomoyuki; Yumura-Yagi, Keiko; Kobayashi, Ryoji; Suzuki, Nobuhiro; Hara, Junichi; Horibe, Keizo; Hayashi, Yasuhide

    2009-04-01

    Mutation analysis of FBXW7 and NOTCH1 genes was performed in 55 T cell acute lymphoblastic leukaemia (T-ALL) and 14 T cell non-Hodgkin lymphoma (T-NHL) patients who were treated on the Japan Association of Childhood Leukaemia Study (JACLS) protocols ALL-97 and NHL-98. FBXW7 and/or NOTCH1 mutations were found in 22 (40.0%) of 55 T-ALL and 7 (50.0%) of 14 T-NHL patients. FBXW7 mutations were found in 8 (14.6%) of 55 T-ALL and 3 (21.4%) of 14 T-NHL patients, and NOTCH1 mutations in 17 (30.9%) of 55 T-ALL and 6 (42.9%) of 14 T-NHL patients. Three (5.4%) T-ALL and two (1.4%) T-NHL patients had mutations in both FBXW7 and NOTCH1. FBXW7 mutations included one insertion, one deletion, one deletion/insertion and nine missense mutations. NOTCH1 mutations were detected in the heterodimerization domain (HD) in 15 cases, in the PEST domain in seven cases, and in both the HD and PEST domains in one case. Five-year event-free survival and overall survival for patients with FBXW7 and/or NOTCH1 mutations were 95.5% (95% CI, 71.9-99.4%) and 100% respectively, suggesting that T-ALL patients with FBXW7 and/or NOTCH1 mutation represent a good prognosis compared to those without FBXW7 and/or NOTCH1 mutations (63.6%, P = 0.007 and 78.8%, P = 0.023, respectively).

  12. A novel mutation in TFL1 homolog affecting determinacy in cowpea (Vigna unguiculata).

    Science.gov (United States)

    Dhanasekar, P; Reddy, K S

    2015-02-01

    Mutations in the widely conserved Arabidopsis Terminal Flower 1 (TFL1) gene and its homologs have been demonstrated to result in determinacy across genera, the knowledge of which is lacking in cowpea. Understanding the molecular events leading to determinacy of apical meristems could hasten development of cowpea varieties with suitable ideotypes. Isolation and characterization of a novel mutation in cowpea TFL1 homolog (VuTFL1) affecting determinacy is reported here for the first time. Cowpea TFL1 homolog was amplified using primers designed based on conserved sequences in related genera and sequence variation was analysed in three gamma ray-induced determinate mutants, their indeterminate parent "EC394763" and two indeterminate varieties. The analyses of sequence variation exposed a novel SNP distinguishing the determinate mutants from the indeterminate types. The non-synonymous point mutation in exon 4 at position 1,176 resulted from transversion of cytosine (C) to adenine (A) leading to an amino acid change (Pro-136 to His) in determinate mutants. The effect of the mutation on protein function and stability was predicted to be detrimental using different bioinformatics/computational tools. The functionally significant novel substitution mutation is hypothesized to affect determinacy in the cowpea mutants. Development of suitable regeneration protocols in this hitherto recalcitrant crop and subsequent complementation assay in mutants or over-expressing assay in parents could decisively conclude the role of the SNP in regulating determinacy in these cowpea mutants.

  13. Structural defect linked to nonrandom mutations in the matrix gene of Biden strain subacute sclerosing panencephalitis virus defined by cDNA cloning and expression of chimeric genes

    International Nuclear Information System (INIS)

    Ayata, M.; Hirano, A.; Wong, T.C.

    1989-01-01

    Biken strain, a nonproductive measles viruslike agent isolated from a subacute sclerosing panencephalitis (SSPE) patient, contains a posttranscriptional defect affecting matrix (M) protein. A putative M protein was translated in vitro with RNA from Biken strain-infected cells. A similar protein was detected in vivo by an antiserum against a peptide synthesized from the cloned M gene of Edmonston strain measles virus. By using a novel method, full-length cDNAs of the Biken M gene were selectively cloned. The cloned Biken M gene contained an open reading frame which encoded 8 extra carboxy-terminal amino acid residues and 20 amino acid substitutions predicted to affect both the hydrophobicity and secondary structure of the gene product. The cloned gene was expressed in vitro and in vivo into a 37,500 M r protein electrophoretically and antigenically distinct from the M protein of Edmonston strain but identical to the M protein in Biken strain-infected cells. Chimeric M proteins synthesized in vitro and in vivo showed that the mutations in the carboxy-proximal region altered the local antigenicity and those in the amino region affected the overall protein conformation. The protein expressed from the Biken M gene was unstable in vivo. Instability was attributed to multiple mutations. These results offer insights into the basis of the defect in Biken strain and pose intriguing questions about the evolutionary origins of SSPE viruses in general

  14. Hypoxia induces mitochondrial mutagenesis and dysfunction in inflammatory arthritis.

    LENUS (Irish Health Repository)

    Biniecka, Monika

    2012-02-01

    OBJECTIVE: To assess the levels and spectrum of mitochondrial DNA (mtDNA) point mutations in synovial tissue from patients with inflammatory arthritis in relation to in vivo hypoxia and oxidative stress levels. METHODS: Random Mutation Capture assay was used to quantitatively evaluate alterations of the synovial mitochondrial genome. In vivo tissue oxygen levels (tPO(2)) were measured at arthroscopy using a Licox probe. Synovial expression of lipid peroxidation (4-hydroxynonenal [4-HNE]) and mitochondrial cytochrome c oxidase subunit II (CytcO II) deficiency were assessed by immunohistochemistry. In vitro levels of mtDNA point mutations, reactive oxygen species (ROS), mitochondrial membrane potential, and markers of oxidative DNA damage (8-oxo-7,8-dihydro-2\\'-deoxyguanine [8-oxodG]) and lipid peroxidation (4-HNE) were determined in human synoviocytes under normoxia and hypoxia (1%) in the presence or absence of superoxide dismutase (SOD) or N-acetylcysteine (NAC) or a hydroxylase inhibitor (dimethyloxalylglycine [DMOG]). Patients were categorized according to their in vivo tPO(2) level (<20 mm Hg or >20 mm Hg), and mtDNA point mutations, immunochemistry features, and stress markers were compared between groups. RESULTS: The median tPO(2) level in synovial tissue indicated significant hypoxia (25.47 mm Hg). Higher frequency of mtDNA mutations was associated with reduced in vivo oxygen tension (P = 0.05) and with higher synovial 4-HNE cytoplasmic expression (P = 0.04). Synovial expression of CytcO II correlated with in vivo tPO(2) levels (P = 0.03), and levels were lower in patients with tPO(2) <20 mm Hg (P < 0.05). In vitro levels of mtDNA mutations, ROS, mitochondrial membrane potential, 8-oxo-dG, and 4-HNE were higher in synoviocytes exposed to 1% hypoxia (P < 0.05); all of these increased levels were rescued by SOD and DMOG and, with the exception of ROS, by NAC. CONCLUSION: These findings demonstrate that hypoxia-induced mitochondrial dysfunction drives

  15. Fragile X founder chromosomes in Italy: A few initial events and possible explanation for their heterogeneity

    Energy Technology Data Exchange (ETDEWEB)

    Chiurazzi, P.; Genuardi, M.; Kozak, L.; Neri, G. [Universita Cattolica and Centro Ricerche per la Disabilita Mentale e Motoria, Roma (Italy)] [and others

    1996-07-12

    A total of 137 fragile X and 235 control chromosomes from various regions of Italy were haplotyped by analyzing two neighbouring marker microsatellites, FRAXAC1 and DXS548. The number of CGG repeats at the 5{prime} end of the FMR1 gene was also assessed in 141 control chromosomes and correlated with their haplotypes. Significant linkage disequilibrium between some {open_quotes}major{close_quotes} haplotypes and fragile X was observed, while other {open_quotes}minor{close_quotes} haplotypes may have originated by subsequent mutation at the marker microsatellite loci and/or recombination between them. Recent evidence suggests that the initial mechanism leading to CGG instability might consist of rare (10{sup -6/-7}) CGG repeat slippage events and/or loss of a stabilizing AGG via A-to-C transversion. Also, the apparently high variety of fragile X chromosomes may be partly due to the relatively high mutation rate (10{sup -4/-5}) of the microsatellite markers used in haplotyping. Our fragile X sample also showed a higher than expected heterozygosity when compared to the control sample and we suggest that this might be explained by the chance occurrence of the few founding events on different chromosomes, irrespective of their actual frequency in the population. Alternatively, a local mechanism could enhance the microsatellite mutation rate only on fragile X chromosomes, or fragile X mutations might occur more frequently on certain background haplotypes. 59 refs., 4 figs.

  16. Targeted next-generation sequencing extends the phenotypic and mutational spectrums for EYS mutations.

    Science.gov (United States)

    Gu, Shun; Tian, Yuanyuan; Chen, Xue; Zhao, Chen

    2016-01-01

    We aim to determine genetic lesions with a phenotypic correlation in four Chinese families with autosomal recessive retinitis pigmentosa (RP). Medical histories were carefully reviewed. All patients received comprehensive ophthalmic evaluations. The next-generation sequencing (NGS) approach targeting a panel of 205 retinal disease-relevant genes and 15 candidate genes was selectively performed on probands from the four recruited families for mutation detection. Online predictive software and crystal structure modeling were also applied to test the potential pathogenic effects of identified mutations. Of the four families, two were diagnosed with RP sino pigmento (RPSP). Patients with RPSP claimed to have earlier RP age of onset but slower disease progression. Five mutations in the eyes shut homolog (EYS) gene, involving two novel (c.7228+1G>A and c.9248G>A) and three recurrent mutations (c.4957dupA, c.6416G>A and c.6557G>A), were found as RP causative in the four families. The missense variant c.5093T>C was determined to be a variant of unknown significance (VUS) due to the variant's colocalization in the same allele with the reported pathogenic mutation c.6416G>A. The two novel variants were further confirmed absent in 100 unrelated healthy controls. Online predictive software indicated potential pathogenicity of the three missense mutations. Further, crystal structural modeling suggested generation of two abnormal hydrogen bonds by the missense mutation p.G2186E (c.6557G>A) and elongation of its neighboring β-sheet induced by p.G3083D (c.9248G>A), which could alter the tertiary structure of the eys protein and thus interrupt its physicochemical properties. Taken together, with the targeted NGS approach, we reveal novel EYS mutations and prove the efficiency of targeted NGS in the genetic diagnoses of RP. We also first report the correlation between EYS mutations and RPSP. The genotypic-phenotypic relationship in all Chinese patients carrying mutations in the EYS

  17. Human lymphoma mutations reveal CARD11 as the switch between self-antigen–induced B cell death or proliferation and autoantibody production

    Science.gov (United States)

    Jeelall, Yogesh S.; Wang, James Q.; Law, Hsei-Di; Domaschenz, Heather; Fung, Herman K.H.; Kallies, Axel; Nutt, Stephen L.

    2012-01-01

    Self-tolerance and immunity are actively acquired in parallel through a poorly understood ability of antigen receptors to switch between signaling death or proliferation of antigen-binding lymphocytes in different contexts. It is not known whether this tolerance-immunity switch requires global rewiring of the signaling apparatus or if it can arise from a single molecular change. By introducing individual CARD11 mutations found in human lymphomas into antigen-activated mature B lymphocytes in mice, we find here that lymphoma-derived CARD11 mutations switch the effect of self-antigen from inducing B cell death into T cell–independent proliferation, Blimp1-mediated plasmablast differentiation, and autoantibody secretion. Our findings demonstrate that regulation of CARD11 signaling is a critical switch governing the decision between death and proliferation in antigen-stimulated mature B cells and that mutations in this switch represent a powerful initiator for aberrant B cell responses in vivo. PMID:23027925

  18. Mutations in TGFbeta-RII and BAX mediate tumor progression in the later stages of colorectal cancer with microsatellite instability

    International Nuclear Information System (INIS)

    Yashiro, Masakazu; Hirakawa, Kosei; Boland, C Richard

    2010-01-01

    Microsatellite instability (MSI) occurs in 15% of colorectal cancers (CRC). The genetic targets for mutation in the MSI phenotype include somatic mutations in the transforming growth factor beta receptor typeII (TGFbetaRII), BAX, hMSH3 and hMSH6. It is not clear how mutations of these genes mediate tumor progression in the MSI pathway, and the temporal sequence of these mutations remains uncertain. In this study, early stage CRCs were examined for frameshift mutations in these target genes, and compared with late stage tumors and CRC cell lines. We investigated 6 CRC cell lines and 71 sporadic CRCs, including 61 early stage cancers and 10 late stage cancers. Mutations of repetitive mononucleotide tracts in the coding regions of TGFbetaRII, BAX, hMSH3, hMSH6, IGFIIR and Fas antigen were identified by direct sequencing. Thirteen (18.3%) of 71 CRC, including 9/61 (14.7%) early stage cancers and 4/10 (40%) late stage cancers, were identified as MSI and analyzed for frameshift mutations. No mutation in the target genes was observed in any of the 9 early stage MSI CRCs. In contrast, frameshift mutations of TGFbetaRII, BAX, hMSH3 and hMSH6 were present in 3/4 late stage MSI tumors. There is a statistical association (p = 0.014) between mutation in any one gene and tumor stage. TGFbetaRII, BAX, hMSH3 and hMSH6 mutations are relatively late events in the genesis of MSI CRCs. The frameshift mutations in these target genes might mediate progression from early to late stage cancer, rather than mediating the adenoma to carcinoma transition

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

    Science.gov (United States)

    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

  20. Psychological distress in women at risk of hereditary breast/ovarian or HNPCC cancers in the absence of demonstrated mutations.

    Science.gov (United States)

    Geirdal, Amy Østertun; Reichelt, Jon G; Dahl, Alv A; Heimdal, Ketil; Maehle, Lovise; Stormorken, Astrid; Møller, Pål

    2005-01-01

    To examine psychological distress in women at risk of familial breast-ovarian cancer (FBOC) or hereditary non-polyposis colorectal cancer (HNPCC) with absence of demonstrated mutations in the family (unknown mutation). Two-hundred and fifty three consecutive women at risk of FBOC and 77 at risk of HNPCC and with no present or past history of cancer. They were aware of their risk and had received genetic counseling. Comparisons were made between these two groups, normal controls, and women who were identified to be BRCA1 mutation carriers. The questionnaires Beck Hopelessness Scale (BHS), General Health Questionnaire (GHQ-28), Hospital Anxiety and Depression Scale (HADS) and Impact of Event Scale (IES) were employed to assess psychological distress. No significant differences concerning psychological distress were observed between women with FBOC and women with HNPCC. Compared to mutation carriers for BRCA1, the level of anxiety and depression was significantly higher in the FBOC group with absence of demonstrated mutation. Compared to normal controls, the level of anxiety was higher, while the level of depression was lower in the groups with unknown mutation. Women in the absence of demonstrated mutations have higher anxiety and depression levels than women with known mutation-carrier status. Access to genetic testing may be of psychologically benefit to women at risk for FBOC or HNPCC.

  1. Comprehensive Mutation Analysis of PMS2 in a Large Cohort of Probands Suspected of Lynch Syndrome or Constitutional Mismatch Repair Deficiency Syndrome.

    Science.gov (United States)

    van der Klift, Heleen M; Mensenkamp, Arjen R; Drost, Mark; Bik, Elsa C; Vos, Yvonne J; Gille, Hans J J P; Redeker, Bert E J W; Tiersma, Yvonne; Zonneveld, José B M; García, Encarna Gómez; Letteboer, Tom G W; Olderode-Berends, Maran J W; van Hest, Liselotte P; van Os, Theo A; Verhoef, Senno; Wagner, Anja; van Asperen, Christi J; Ten Broeke, Sanne W; Hes, Frederik J; de Wind, Niels; Nielsen, Maartje; Devilee, Peter; Ligtenberg, Marjolijn J L; Wijnen, Juul T; Tops, Carli M J

    2016-11-01

    Monoallelic PMS2 germline mutations cause 5%-15% of Lynch syndrome, a midlife cancer predisposition, whereas biallelic PMS2 mutations cause approximately 60% of constitutional mismatch repair deficiency (CMMRD), a rare childhood cancer syndrome. Recently improved DNA- and RNA-based strategies are applied to overcome problematic PMS2 mutation analysis due to the presence of pseudogenes and frequent gene conversion events. Here, we determined PMS2 mutation detection yield and mutation spectrum in a nationwide cohort of 396 probands. Furthermore, we studied concordance between tumor IHC/MSI (immunohistochemistry/microsatellite instability) profile and mutation carrier state. Overall, we found 52 different pathogenic PMS2 variants explaining 121 Lynch syndrome and nine CMMRD patients. In vitro mismatch repair assays suggested pathogenicity for three missense variants. Ninety-one PMS2 mutation carriers (70%) showed isolated loss of PMS2 in their tumors, for 31 (24%) no or inconclusive IHC was available, and eight carriers (6%) showed discordant IHC (presence of PMS2 or loss of both MLH1 and PMS2). Ten cases with isolated PMS2 loss (10%; 10/97) harbored MLH1 mutations. We confirmed that recently improved mutation analysis provides a high yield of PMS2 mutations in patients with isolated loss of PMS2 expression. Application of universal tumor prescreening methods will however miss some PMS2 germline mutation carriers. © 2016 WILEY PERIODICALS, INC.

  2. Generation and analysis of knock-in mice carrying pseudohypoaldosteronism type II-causing mutations in the cullin 3 gene.

    Science.gov (United States)

    Araki, Yuya; Rai, Tatemitsu; Sohara, Eisei; Mori, Takayasu; Inoue, Yuichi; Isobe, Kiyoshi; Kikuchi, Eriko; Ohta, Akihito; Sasaki, Sei; Uchida, Shinichi

    2015-10-21

    Pseudohypoaldosteronism type II (PHAII) is a hereditary hypertensive disease caused by mutations in four different genes: with-no-lysine kinases (WNK) 1 and 4, Kelch-like family member 3 (KLHL3), and cullin 3 (Cul3). Cul3 and KLHL3 form an E3 ligase complex that ubiquitinates and reduces the expression level of WNK4. PHAII-causing mutations in WNK4 and KLHL3 impair WNK4 ubiquitination. However, the molecular pathogenesis of PHAII caused by Cul3 mutations is unclear. In cultured cells and human leukocytes, PHAII-causing Cul3 mutations result in the skipping of exon 9, producing mutant Cul3 protein lacking 57 amino acids. However, whether this phenomenon occurs in the kidneys and is responsible for the pathogenesis of PHAII in vivo is unknown. We generated knock-in mice carrying a mutation in the C-terminus of intron 8 of Cul3, c.1207-1G>A, which corresponds to a PHAII-causing mutation in the human Cul3 gene. Heterozygous Cul3(G(-1)A/+) knock-in mice did not exhibit PHAII phenotypes, and the skipping of exon 9 was not evident in their kidneys. However, the level of Cul3 mRNA expression in the kidneys of heterozygous knock-in mice was approximately half that of wild-type mice. Furthermore, homozygous knock-in mice were nonviable. It suggested that the mutant allele behaved like a knockout allele and did not produce Cul3 mRNA lacking exon 9. A reduction in Cul3 expression alone was not sufficient to develop PHAII in the knock-in mice. Our findings highlighted the pathogenic role of mutant Cul3 protein and provided insight to explain why PHAII-causing mutations in Cul3 cause kidney-predominant PHAII phenotypes. © 2015. Published by The Company of Biologists Ltd.

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

    International Nuclear Information System (INIS)

    Fu, Liang-Yu; Wang, Guang-Zhong; Ma, Bin-Guang; Zhang, Hong-Yu

    2011-01-01

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

  4. Targeted sequencing identifies associations between IL7R-JAK mutations and epigenetic modulators in T-cell acute lymphoblastic leukemia

    Science.gov (United States)

    Vicente, Carmen; Schwab, Claire; Broux, Michaël; Geerdens, Ellen; Degryse, Sandrine; Demeyer, Sofie; Lahortiga, Idoya; Elliott, Alannah; Chilton, Lucy; La Starza, Roberta; Mecucci, Cristina; Vandenberghe, Peter; Goulden, Nicholas; Vora, Ajay; Moorman, Anthony V.; Soulier, Jean; Harrison, Christine J.; Clappier, Emmanuelle; Cools, Jan

    2015-01-01

    T-cell acute lymphoblastic leukemia is caused by the accumulation of multiple oncogenic lesions, including chromosomal rearrangements and mutations. To determine the frequency and co-occurrence of mutations in T-cell acute lymphoblastic leukemia, we performed targeted re-sequencing of 115 genes across 155 diagnostic samples (44 adult and 111 childhood cases). NOTCH1 and CDKN2A/B were mutated/deleted in more than half of the cases, while an additional 37 genes were mutated/deleted in 4% to 20% of cases. We found that IL7R-JAK pathway genes were mutated in 27.7% of cases, with JAK3 mutations being the most frequent event in this group. Copy number variations were also detected, including deletions of CREBBP or CTCF and duplication of MYB. FLT3 mutations were rare, but a novel extracellular mutation in FLT3 was detected and confirmed to be transforming. Furthermore, we identified complex patterns of pairwise associations, including a significant association between mutations in IL7R-JAK genes and epigenetic regulators (WT1, PRC2, PHF6). Our analyses showed that IL7R-JAK genetic lesions did not confer adverse prognosis in T-cell acute lymphoblastic leukemia cases enrolled in the UK ALL2003 trial. Overall, these results identify interconnections between the T-cell acute lymphoblastic leukemia genome and disease biology, and suggest a potential clinical application for JAK inhibitors in a significant proportion of patients with T-cell acute lymphoblastic leukemia. PMID:26206799

  5. Mitochondrial DNA mutations in mutator mice confer respiration defects and B-cell lymphoma development.

    Directory of Open Access Journals (Sweden)

    Takayuki Mito

    Full Text Available Mitochondrial DNA (mtDNA mutator mice are proposed to express premature aging phenotypes including kyphosis and hair loss (alopecia due to their carrying a nuclear-encoded mtDNA polymerase with a defective proofreading function, which causes accelerated accumulation of random mutations in mtDNA, resulting in expression of respiration defects. On the contrary, transmitochondrial mito-miceΔ carrying mtDNA with a large-scale deletion mutation (ΔmtDNA also express respiration defects, but not express premature aging phenotypes. Here, we resolved this discrepancy by generating mtDNA mutator mice sharing the same C57BL/6J (B6J nuclear background with that of mito-miceΔ. Expression patterns of premature aging phenotypes are very close, when we compared between homozygous mtDNA mutator mice carrying a B6J nuclear background and selected mito-miceΔ only carrying predominant amounts of ΔmtDNA, in their expression of significant respiration defects, kyphosis, and a short lifespan, but not the alopecia. Therefore, the apparent discrepancy in the presence and absence of premature aging phenotypes in mtDNA mutator mice and mito-miceΔ, respectively, is partly the result of differences in the nuclear background of mtDNA mutator mice and of the broad range of ΔmtDNA proportions of mito-miceΔ used in previous studies. We also provided direct evidence that mtDNA abnormalities in homozygous mtDNA mutator mice are responsible for respiration defects by demonstrating the co-transfer of mtDNA and respiration defects from mtDNA mutator mice into mtDNA-less (ρ(0 mouse cells. Moreover, heterozygous mtDNA mutator mice had a normal lifespan, but frequently developed B-cell lymphoma, suggesting that the mtDNA abnormalities in heterozygous mutator mice are not sufficient to induce a short lifespan and aging phenotypes, but are able to contribute to the B-cell lymphoma development during their prolonged lifespan.

  6. The rem mutations in the ATP-binding groove of the Rad3/XPD helicase lead to Xeroderma pigmentosum-Cockayne syndrome-like phenotypes.

    Science.gov (United States)

    Herrera-Moyano, Emilia; Moriel-Carretero, María; Montelone, Beth A; Aguilera, Andrés

    2014-12-01

    The eukaryotic TFIIH complex is involved in Nucleotide Excision Repair and transcription initiation. We analyzed three yeast mutations of the Rad3/XPD helicase of TFIIH known as rem (recombination and mutation phenotypes). We found that, in these mutants, incomplete NER reactions lead to replication fork breaking and the subsequent engagement of the homologous recombination machinery to restore them. Nevertheless, the penetrance varies among mutants, giving rise to a phenotype gradient. Interestingly, the mutations analyzed reside at the ATP-binding groove of Rad3 and in vivo experiments reveal a gain of DNA affinity upon damage of the mutant Rad3 proteins. Since mutations at the ATP-binding groove of XPD in humans are present in the Xeroderma pigmentosum-Cockayne Syndrome (XP-CS), we recreated rem mutations in human cells, and found that these are XP-CS-like. We propose that the balance between the loss of helicase activity and the gain of DNA affinity controls the capacity of TFIIH to open DNA during NER, and its persistence at both DNA lesions and promoters. This conditions NER efficiency and transcription resumption after damage, which in human cells would explain the XP-CS phenotype, opening new perspectives to understand the molecular basis of the role of XPD in human disease.

  7. Stereochemical criteria for prediction of the effects of proline mutations on protein stability.

    Directory of Open Access Journals (Sweden)

    Kanika Bajaj

    2007-12-01

    Full Text Available When incorporated into a polypeptide chain, proline (Pro differs from all other naturally occurring amino acid residues in two important respects. The phi dihedral angle of Pro is constrained to values close to -65 degrees and Pro lacks an amide hydrogen. Consequently, mutations which result in introduction of Pro can significantly affect protein stability. In the present work, we describe a procedure to accurately predict the effect of Pro introduction on protein thermodynamic stability. Seventy-seven of the 97 non-Pro amino acid residues in the model protein, CcdB, were individually mutated to Pro, and the in vivo activity of each mutant was characterized. A decision tree to classify the mutation as perturbing or nonperturbing was created by correlating stereochemical properties of mutants to activity data. The stereochemical properties including main chain dihedral angle phi and main chain amide H-bonds (hydrogen bonds were determined from 3D models of the mutant proteins built using MODELLER. We assessed the performance of the decision tree on a large dataset of 163 single-site Pro mutations of T4 lysozyme, 74 nsSNPs, and 52 other Pro substitutions from the literature. The overall accuracy of this algorithm was found to be 81% in the case of CcdB, 77% in the case of lysozyme, 76% in the case of nsSNPs, and 71% in the case of other Pro substitution data. The accuracy of Pro scanning mutagenesis for secondary structure assignment was also assessed and found to be at best 69%. Our prediction procedure will be useful in annotating uncharacterized nsSNPs of disease-associated proteins and for protein engineering and design.

  8. Stereochemical criteria for prediction of the effects of proline mutations on protein stability.

    Science.gov (United States)

    Bajaj, Kanika; Madhusudhan, M S; Adkar, Bharat V; Chakrabarti, Purbani; Ramakrishnan, C; Sali, Andrej; Varadarajan, Raghavan

    2007-12-01

    When incorporated into a polypeptide chain, proline (Pro) differs from all other naturally occurring amino acid residues in two important respects. The phi dihedral angle of Pro is constrained to values close to -65 degrees and Pro lacks an amide hydrogen. Consequently, mutations which result in introduction of Pro can significantly affect protein stability. In the present work, we describe a procedure to accurately predict the effect of Pro introduction on protein thermodynamic stability. Seventy-seven of the 97 non-Pro amino acid residues in the model protein, CcdB, were individually mutated to Pro, and the in vivo activity of each mutant was characterized. A decision tree to classify the mutation as perturbing or nonperturbing was created by correlating stereochemical properties of mutants to activity data. The stereochemical properties including main chain dihedral angle phi and main chain amide H-bonds (hydrogen bonds) were determined from 3D models of the mutant proteins built using MODELLER. We assessed the performance of the decision tree on a large dataset of 163 single-site Pro mutations of T4 lysozyme, 74 nsSNPs, and 52 other Pro substitutions from the literature. The overall accuracy of this algorithm was found to be 81% in the case of CcdB, 77% in the case of lysozyme, 76% in the case of nsSNPs, and 71% in the case of other Pro substitution data. The accuracy of Pro scanning mutagenesis for secondary structure assignment was also assessed and found to be at best 69%. Our prediction procedure will be useful in annotating uncharacterized nsSNPs of disease-associated proteins and for protein engineering and design.

  9. Evidence in Latin America of recurrence of V388M, a phenylketonuria mutation with high in vitro residual activity

    Energy Technology Data Exchange (ETDEWEB)

    Desviat, L.R.; Perez, B.; De Lucca, M. [Universidad Autonoma de Madrid, (Spain)] [and others

    1995-08-01

    Phenylketonuria mutation V388M is frequent in the Iberian Peninsula. In vitro, the V388M mutant enzyme has similar immunoreactive protein and phenylalanine hydroxylase mRNA and had 43% residual activity, which correlates well with the mild phenotype exhibited by the homozygous patients. In Spain it has been detected in 5.7% of the mutant alleles and is always associated with haplotype 1.7. This mutation is also present in high frequency in some Latin American countries (Brazil, 9% Chile, 13%). It is interesting that in Chile most of the alleles bearing this mutation carry haplotype 4.3, although in Brazil it is found only on the background of haplotype 1.7. The origin of V388M in Spain on haplotype 1.7 and in Chile on haplotype 4.3 is clearly different. Recurrence is the most plausible explanation, because the mutation involves a CpG dinucleotide, and a recombination event transferring the mutation from haplotype 1 to 4 is unlikely. 29 refs., 2 figs., 3 tabs.

  10. Mutations induced by ultraviolet light

    International Nuclear Information System (INIS)

    Pfeifer, Gerd P.; You, Young-Hyun; Besaratinia, Ahmad

    2005-01-01

    The different ultraviolet (UV) wavelength components, UVA (320-400 nm), UVB (280-320 nm), and UVC (200-280 nm), have distinct mutagenic properties. A hallmark of UVC and UVB mutagenesis is the high frequency of transition mutations at dipyrimidine sequences containing cytosine. In human skin cancers, about 35% of all mutations in the p53 gene are transitions at dipyrimidines within the sequence 5'-TCG and 5'-CCG, and these are localized at several mutational hotspots. Since 5'-CG sequences are methylated along the p53 coding sequence in human cells, these mutations may be derived from sunlight-induced pyrimidine dimers forming at sequences that contain 5-methylcytosine. Cyclobutane pyrimidine dimers (CPDs) form preferentially at dipyrimidines containing 5-methylcytosine when cells are irradiated with UVB or sunlight. In order to define the contribution of 5-methylcytosine to sunlight-induced mutations, the lacI and cII transgenes in mouse fibroblasts were used as mutational targets. After 254 nm UVC irradiation, only 6-9% of the base substitutions were at dipyrimidines containing 5-methylcytosine. However, 24-32% of the solar light-induced mutations were at dipyrimidines that contain 5-methylcytosine and most of these mutations were transitions. Thus, CPDs forming preferentially at dipyrimidines with 5-methylcytosine are responsible for a considerable fraction of the mutations induced by sunlight in mammalian cells. Using mouse cell lines harboring photoproduct-specific photolyases and mutational reporter genes, we showed that CPDs (rather than 6-4 photoproducts or other lesions) are responsible for the great majority of UVB-induced mutations. An important component of UVB mutagenesis is the deamination of cytosine and 5-methylcytosine within CPDs. The mutational specificity of long-wave UVA (340-400 nm) is distinct from that of the shorter wavelength UV and is characterized mainly by G to T transversions presumably arising through mechanisms involving oxidized DNA

  11. Mutation of Gly717Phe in human topoisomerase 1B has an effect on enzymatic function, reactivity to the camptothecin anticancer drug and on the linker domain orientation

    DEFF Research Database (Denmark)

    Wang, Zhenxing; D'Annessa, Ilda; Tesauro, Cinzia

    2015-01-01

    –DNA covalent adduct. In this work the role of the Gly717 residue, located in a α-helix structure bridging the active site and the linker domain, has been investigated mutating it in Phe. The mutation gives rise to drug resistance in vivo as observed through a viability assay of yeast cells. In vitro activity...... assays show that the mutant is characterized by a fast religation rate, only partially reduced by the presence of the drug. Comparative molecular dynamics simulations of the native and mutant proteins indicate that the mutation of Gly717 affects the motion orientation of the linker domain, changing its...... interaction with the DNA substrate, likely affecting the strand rotation and religation rate. The mutation also causes a slight rearrangement of the active site and of the drug binding site, providing an additional explanation for the lowered effect of camptothecin toward the mutant....

  12. Inflammation, gene mutation and photoimmunosuppression in response to UVR-induced oxidative damage contributes to photocarcinogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Halliday, Gary M. [Dermatology Research Laboratories, Division of Medicine, Melanoma and Skin Cancer Research Institute, Royal Prince Alfred Hospital at the University of Sydney, Sydney, NSW (Australia)]. E-mail: garyh@med.usyd.edu.au

    2005-04-01

    Ultraviolet (UV) radiation causes inflammation, gene mutation and immunosuppression in the skin. These biological changes are responsible for photocarcinogenesis. UV radiation in sunlight is divided into two wavebands, UVB and UVA, both of which contribute to these biological changes, and therefore probably to skin cancer in humans and animal models. Oxidative damage caused by UV contributes to inflammation, gene mutation and immunosuppression. This article reviews evidence for the hypothesis that UV oxidative damage to these processes contributes to photocarcinogenesis. UVA makes a larger impact on oxidative stress in the skin than UVB by inducing reactive oxygen and nitrogen species which damage DNA, protein and lipids and which also lead to NAD+ depletion, and therefore energy loss from the cell. Lipid peroxidation induces prostaglandin production that in association with UV-induced nitric oxide production causes inflammation. Inflammation drives benign human solar keratosis (SK) to undergo malignant conversion into squamous cell carcinoma (SCC) probably because the inflammatory cells produce reactive oxygen species, thus increasing oxidative damage to DNA and the immune system. Reactive oxygen or nitrogen appears to cause the increase in mutational burden as SK progress into SCC in humans. UVA is particularly important in causing immunosuppression in both humans and mice, and UV lipid peroxidation induced prostaglandin production and UV activation of nitric oxide synthase is important mediators of this event. Other immunosuppressive events are likely to be initiated by UV oxidative stress. Antioxidants have also been shown to reduce photocarcinogenesis. While most of this evidence comes from studies in mice, there is supporting evidence in humans that UV-induced oxidative damage contributes to inflammation, gene mutation and immunosuppression. Available evidence implicates oxidative damage as an important contributor to sunlight-induced carcinogenesis in humans.

  13. Inflammation, gene mutation and photoimmunosuppression in response to UVR-induced oxidative damage contributes to photocarcinogenesis

    International Nuclear Information System (INIS)

    Halliday, Gary M.

    2005-01-01

    Ultraviolet (UV) radiation causes inflammation, gene mutation and immunosuppression in the skin. These biological changes are responsib