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Sample records for rna-based mutation screening

  1. SELEX-Based Screening of Exosome-Tropic RNA.

    Science.gov (United States)

    Yamashita, Takuma; Shinotsuka, Haruka; Takahashi, Yuki; Kato, Kana; Nishikawa, Makiya; Takakura, Yoshinobu

    2017-01-01

    Cell-derived nanosized vesicles or exosomes are expected to become delivery carriers for functional RNAs, such as small interfering RNA (siRNA). A method to efficiently load functional RNAs into exosomes is required for the development of exosome-based delivery carriers of functional RNAs. However, there is no method to find exosome-tropic exogenous RNA sequences. In this study, we used a systematic evolution of ligands by exponential enrichment (SELEX) method to screen exosome-tropic RNAs that can be used to load functional RNAs into exosomes by conjugation. Pooled single stranded 80-base RNAs, each of which contains a randomized 40-base sequence, were transfected into B16-BL6 murine melanoma cells and exosomes were collected from the cells. RNAs extracted from the exosomes were subjected to next round of SELEX. Cloning and sequencing of RNAs in SELEX-screened RNA pools showed that 29 of 56 clones had a typical RNA sequence. The sequence found by SELEX was enriched in exosomes after transfection to B16-BL6 cells. The results show that the SELEX-based method can be used for screening of exosome-tropic RNAs.

  2. A whole mitochondrial genome screening in a MELAS patient: A novel mitochondrial tRNA{sup Val} mutation

    Energy Technology Data Exchange (ETDEWEB)

    Mezghani, Najla [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia); Mnif, Mouna [Service d' endocrinologie, C.H.U. Habib Bourguiba de Sfax (Tunisia); Kacem, Maha [Service de Medecine interne, C.H.U. Fattouma Bourguiba de Monastir (Tunisia); Mkaouar-Rebai, Emna, E-mail: emna_mkaouar@mail2world.com [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia); Hadj Salem, Ikhlass [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia); Kallel, Nozha; Charfi, Nadia; Abid, Mohamed [Service d' endocrinologie, C.H.U. Habib Bourguiba de Sfax (Tunisia); Fakhfakh, Faiza [Laboratoire de Genetique Moleculaire Humaine, Faculte de Medecine de Sfax, Universite de Sfax (Tunisia)

    2011-04-22

    Highlights: {yields} We report a young Tunisian patient with clinical features of MELAS syndrome. {yields} Reported mitochondrial mutations were absent after a mutational screening of the whole mtDNA. {yields} We described a novel m.1640A>G mutation in the tRNA{sup Val} gene which was absent in 150 controls. {yields} Mitochondrial deletions and POLG1 gene mutations were absent. {yields} The m.1640A>G mutation could be associated to MELAS syndrome. -- Abstract: Mitochondrial encephalopathy, lactic acidosis and strokelike episodes (MELAS) syndrome is a mitochondrial disorder characterized by a wide variety of clinical presentations and a multisystemic organ involvement. In this study, we report a Tunisian girl with clinical features of MELAS syndrome who was negative for the common m.3243A>G mutation, but also for the reported mitochondrial DNA (mtDNA) mutations and deletions. Screening of the entire mtDNA genome showed several known mitochondrial variants besides to a novel transition m.1640A>G affecting a wobble adenine in the anticodon stem region of the tRNA{sup Val}. This nucleotide was conserved and it was absent in 150 controls suggesting its pathogenicity. In addition, no mutations were found in the nuclear polymerase gamma-1 gene (POLG1). These results suggest further investigation nuclear genes encoding proteins responsible for stability and structural components of the mtDNA or to the oxidative phosphorylation machinery to explain the phenotypic variability in the studied family.

  3. An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes

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    Racher, Hilary; Phelps, Ian G.; Toedt, Grischa; Kennedy, Julie; Wunderlich, Kirsten A.; Sorusch, Nasrin; Abdelhamed, Zakia A.; Natarajan, Subaashini; Herridge, Warren; van Reeuwijk, Jeroen; Horn, Nicola; Boldt, Karsten; Parry, David A.; Letteboer, Stef J.F.; Roosing, Susanne; Adams, Matthew; Bell, Sandra M.; Bond, Jacquelyn; Higgins, Julie; Morrison, Ewan E.; Tomlinson, Darren C.; Slaats, Gisela G.; van Dam, Teunis J. P.; Huang, Lijia; Kessler, Kristin; Giessl, Andreas; Logan, Clare V.; Boyle, Evan A.; Shendure, Jay; Anazi, Shamsa; Aldahmesh, Mohammed; Al Hazzaa, Selwa; Hegele, Robert A.; Ober, Carole; Frosk, Patrick; Mhanni, Aizeddin A.; Chodirker, Bernard N.; Chudley, Albert E.; Lamont, Ryan; Bernier, Francois P.; Beaulieu, Chandree L.; Gordon, Paul; Pon, Richard T.; Donahue, Clem; Barkovich, A. James; Wolf, Louis; Toomes, Carmel; Thiel, Christian T.; Boycott, Kym M.; McKibbin, Martin; Inglehearn, Chris F.; Stewart, Fiona; Omran, Heymut; Huynen, Martijn A.; Sergouniotis, Panagiotis I.; Alkuraya, Fowzan S.; Parboosingh, Jillian S.; Innes, A Micheil; Willoughby, Colin E.; Giles, Rachel H.; Webster, Andrew R.; Ueffing, Marius; Blacque, Oliver; Gleeson, Joseph G.; Wolfrum, Uwe; Beales, Philip L.; Gibson, Toby

    2015-01-01

    Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin-proteasome system, 12 G-protein-coupled receptors, and three pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localise to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1/CEP90 and C21orf2/LRRC76 as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2-variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease. PMID:26167768

  4. MicroRNA genes and their target 3'-untranslated regions are infrequently somatically mutated in ovarian cancers.

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    Georgina L Ryland

    Full Text Available MicroRNAs are key regulators of gene expression and have been shown to have altered expression in a variety of cancer types, including epithelial ovarian cancer. MiRNA function is most often achieved through binding to the 3'-untranslated region of the target protein coding gene. Mutation screening using massively-parallel sequencing of 712 miRNA genes in 86 ovarian cancer cases identified only 5 mutated miRNA genes, each in a different case. One mutation was located in the mature miRNA, and three mutations were predicted to alter the secondary structure of the miRNA transcript. Screening of the 3'-untranslated region of 18 candidate cancer genes identified one mutation in each of AKT2, EGFR, ERRB2 and CTNNB1. The functional effect of these mutations is unclear, as expression data available for AKT2 and EGFR showed no increase in gene transcript. Mutations in miRNA genes and 3'-untranslated regions are thus uncommon in ovarian cancer.

  5. A Systematic Genetic Screen to Dissect the MicroRNA Pathway in Drosophila.

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    Pressman, Sigal; Reinke, Catherine A; Wang, Xiaohong; Carthew, Richard W

    2012-04-01

    A central goal of microRNA biology is to elucidate the genetic program of miRNA function and regulation. However, relatively few of the effectors that execute miRNA repression have been identified. Because such genes may function in many developmental processes, mutations in them are expected to be pleiotropic and thus are discarded in most standard genetic screens. Here, we describe a systematic screen designed to identify all Drosophila genes in ∼40% of the genome that function in the miRNA pathway. To identify potentially pleiotropic genes, the screen analyzed clones of homozygous mutant cells in heterozygous animals. We identified 45 mutations representing 24 genes, and we molecularly characterized 9 genes. These include 4 previously known genes that encode core components of the miRNA pathway, including Drosha, Pasha, Dicer-1, and Ago1. The rest are new genes that function through chromatin remodeling, signaling, and mRNA decapping. The results suggest genetic screens that use clonal analysis can elucidate the miRNA program and that ∼100 genes are required to execute the miRNA program.

  6. RNA2DMut: a web tool for the design and analysis of RNA structure mutations.

    Science.gov (United States)

    Moss, Walter N

    2018-03-01

    With the widespread application of high-throughput sequencing, novel RNA sequences are being discovered at an astonishing rate. The analysis of function, however, lags behind. In both the cis - and trans -regulatory functions of RNA, secondary structure (2D base-pairing) plays essential regulatory roles. In order to test RNA function, it is essential to be able to design and analyze mutations that can affect structure. This was the motivation for the creation of the RNA2DMut web tool. With RNA2DMut, users can enter in RNA sequences to analyze, constrain mutations to specific residues, or limit changes to purines/pyrimidines. The sequence is analyzed at each base to determine the effect of every possible point mutation on 2D structure. The metrics used in RNA2DMut rely on the calculation of the Boltzmann structure ensemble and do not require a robust 2D model of RNA structure for designing mutations. This tool can facilitate a wide array of uses involving RNA: for example, in designing and evaluating mutants for biological assays, interrogating RNA-protein interactions, identifying key regions to alter in SELEX experiments, and improving RNA folding and crystallization properties for structural biology. Additional tools are available to help users introduce other mutations (e.g., indels and substitutions) and evaluate their effects on RNA structure. Example calculations are shown for five RNAs that require 2D structure for their function: the MALAT1 mascRNA, an influenza virus splicing regulatory motif, the EBER2 viral noncoding RNA, the Xist lncRNA repA region, and human Y RNA 5. RNA2DMut can be accessed at https://rna2dmut.bb.iastate.edu/. © 2018 Moss; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  7. Efficient algorithms for probing the RNA mutation landscape.

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    Jérôme Waldispühl

    Full Text Available The diversity and importance of the role played by RNAs in the regulation and development of the cell are now well-known and well-documented. This broad range of functions is achieved through specific structures that have been (presumably optimized through evolution. State-of-the-art methods, such as McCaskill's algorithm, use a statistical mechanics framework based on the computation of the partition function over the canonical ensemble of all possible secondary structures on a given sequence. Although secondary structure predictions from thermodynamics-based algorithms are not as accurate as methods employing comparative genomics, the former methods are the only available tools to investigate novel RNAs, such as the many RNAs of unknown function recently reported by the ENCODE consortium. In this paper, we generalize the McCaskill partition function algorithm to sum over the grand canonical ensemble of all secondary structures of all mutants of the given sequence. Specifically, our new program, RNAmutants, simultaneously computes for each integer k the minimum free energy structure MFE(k and the partition function Z(k over all secondary structures of all k-point mutants, even allowing the user to specify certain positions required not to mutate and certain positions required to base-pair or remain unpaired. This technically important extension allows us to study the resilience of an RNA molecule to pointwise mutations. By computing the mutation profile of a sequence, a novel graphical representation of the mutational tendency of nucleotide positions, we analyze the deleterious nature of mutating specific nucleotide positions or groups of positions. We have successfully applied RNAmutants to investigate deleterious mutations (mutations that radically modify the secondary structure in the Hepatitis C virus cis-acting replication element and to evaluate the evolutionary pressure applied on different regions of the HIV trans-activation response

  8. Mutational analysis of the mitochondrial 12S rRNA and tRNASer(UCN) genes in Tunisian patients with nonsyndromic hearing loss

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    Mkaouar-Rebai, Emna; Tlili, Abdelaziz; Masmoudi, Saber; Louhichi, Nacim; Charfeddine, Ilhem; Amor, Mohamed Ben; Lahmar, Imed; Driss, Nabil; Drira, Mohamed; Ayadi, Hammadi; Fakhfakh, Faiza

    2006-01-01

    We explored the mitochondrial 12S rRNA and the tRNA Ser(UCN) genes in 100 Tunisian families affected with NSHL and in 100 control individuals. We identified the mitochondrial A1555G mutation in one out of these 100 families and not in the 100 control individuals. Members of this family harbouring the A1555G mutation showed phenotypic heterogeneity which could be explained by an eventual nuclear-mitochondrial interaction. So, we have screened three nuclear genes: GJB2, GJB3, and GJB6 but we have not found correlation between the phenotypic heterogeneity and variants detected in these genes. We explored also the entire mitochondrial 12S rRNA and the tRNA Ser(UCN) genes. We detected five novel polymorphisms: T742C, T794A, A813G, C868T, and C954T, and 12 known polymorphisms in the mitochondrial 12S rRNA gene. None of the 100 families or the 100 controls were found to carry mutations in the tRNA Ser(UCN) gene. We report here First mutational screening of the mitochondrial 12S rRNA and the tRNA Ser(UCN) genes in the Tunisian population which describes the second family harbouring the A1555G mutation in Africa and reveals novel polymorphisms in the mitochondrial 12S rRNA gene

  9. Advanced cell-based modeling of the royal disease: characterization of the mutated F9 mRNA.

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    Martorell, L; Luce, E; Vazquez, J L; Richaud-Patin, Y; Jimenez-Delgado, S; Corrales, I; Borras, N; Casacuberta-Serra, S; Weber, A; Parra, R; Altisent, C; Follenzi, A; Dubart-Kupperschmitt, A; Raya, A; Vidal, F; Barquinero, J

    2017-11-01

    Essentials The Royal disease (RD) is a form of hemophilia B predicted to be caused by a splicing mutation. We generated an iPSC-based model of the disease allowing mechanistic studies at the RNA level. F9 mRNA analysis in iPSC-derived hepatocyte-like cells showed the predicted abnormal splicing. Mutated F9 mRNA level was very low but we also found traces of wild type transcripts. Background The royal disease is a form of hemophilia B (HB) that affected many descendants of Queen Victoria in the 19th and 20th centuries. It was found to be caused by the mutation F9 c.278-3A>G. Objective To generate a physiological cell model of the disease and to study F9 expression at the RNA level. Methods Using fibroblasts from skin biopsies of a previously identified hemophilic patient bearing the F9 c.278-3A>G mutation and his mother, we generated induced pluripotent stem cells (iPSCs). Both the patient's and mother's iPSCs were differentiated into hepatocyte-like cells (HLCs) and their F9 mRNA was analyzed using next-generation sequencing (NGS). Results and Conclusion We demonstrated the previously predicted aberrant splicing of the F9 transcript as a result of an intronic nucleotide substitution leading to a frameshift and the generation of a premature termination codon (PTC). The F9 mRNA level in the patient's HLCs was significantly reduced compared with that of his mother, suggesting that mutated transcripts undergo nonsense-mediated decay (NMD), a cellular mechanism that degrades PTC-containing mRNAs. We also detected small proportions of correctly spliced transcripts in the patient's HLCs, which, combined with genetic variability in splicing and NMD machineries, could partially explain some clinical variability among affected members of the European royal families who had lifespans above the average. This work allowed the demonstration of the pathologic consequences of an intronic mutation in the F9 gene and represents the first bona fide cellular model of HB allowing the

  10. NMD Microarray Analysis for Rapid Genome-Wide Screen of Mutated Genes in Cancer

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    Maija Wolf

    2005-01-01

    Full Text Available Gene mutations play a critical role in cancer development and progression, and their identification offers possibilities for accurate diagnostics and therapeutic targeting. Finding genes undergoing mutations is challenging and slow, even in the post-genomic era. A new approach was recently developed by Noensie and Dietz to prioritize and focus the search, making use of nonsense-mediated mRNA decay (NMD inhibition and microarray analysis (NMD microarrays in the identification of transcripts containing nonsense mutations. We combined NMD microarrays with array-based CGH (comparative genomic hybridization in order to identify inactivation of tumor suppressor genes in cancer. Such a “mutatomics” screening of prostate cancer cell lines led to the identification of inactivating mutations in the EPHB2 gene. Up to 8% of metastatic uncultured prostate cancers also showed mutations of this gene whose loss of function may confer loss of tissue architecture. NMD microarray analysis could turn out to be a powerful research method to identify novel mutated genes in cancer cell lines, providing targets that could then be further investigated for their clinical relevance and therapeutic potential.

  11. IRF6 mutation screening in non-syndromic orofacial clefting

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    Leslie, Elizabeth J; Koboldt, Daniel C; Kang, C. J.

    2016-01-01

    -syndromic OFCs. About 70% of causal VWS mutations occur in IRF6, a gene that is also associated with non-syndromic OFCs. Screening for IRF6 mutations in apparently non-syndromic cases has been performed in several modestly sized cohorts with mixed results. In this study, we screened 1521 trios with presumed non......-syndromic OFCs to determine the frequency of causal IRF6 mutations. We identified seven likely causal IRF6 mutations, although a posteriori review identified two misdiagnosed VWS families based on the presence of lip pits. We found no evidence for association between rare IRF6 polymorphisms and non......-syndromic OFCs. We combined our results with other similar studies (totaling 2472 families) and conclude that causal IRF6 mutations are found in 0.24–0.44% of apparently non-syndromic OFC families. We suggest that clinical mutation screening for IRF6 be considered for certain family patterns such as families...

  12. siMacro: A Fast and Easy Data Processing Tool for Cell-Based Genomewide siRNA Screens

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    Nitin Kumar Singh

    2013-03-01

    Full Text Available Growing numbers of studies employ cell line-based systematic short interfering RNA (siRNA screens to study gene functions and to identify drug targets. As multiple sources of variations that are unique to siRNA screens exist, there is a growing demand for a computational tool that generates normalized values and standardized scores. However, only a few tools have been available so far with limited usability. Here, we present siMacro, a fast and easy-to-use Microsoft Office Excel-based tool with a graphic user interface, designed to process single-condition or two-condition synthetic screen datasets. siMacro normalizes position and batch effects, censors outlier samples, and calculates Z-scores and robust Z-scores, with a spreadsheet output of >120,000 samples in under 1 minute.

  13. Systematic Prediction of the Impacts of Mutations in MicroRNA Seed Sequences

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    Bhattacharya Anindya

    2017-05-01

    Full Text Available MicroRNAs are a class of small non-coding RNAs that are involved in many important biological processes and the dysfunction of microRNA has been associated with many diseases. The seed region of a microRNA is of crucial importance to its target recognition. Mutations in microRNA seed regions may disrupt the binding of microRNAs to their original target genes and make them bind to new target genes. Here we use a knowledge-based computational method to systematically predict the functional effects of all the possible single nucleotide mutations in human microRNA seed regions. The result provides a comprehensive reference for the functional assessment of the impacts of possible natural and artificial single nucleotide mutations in microRNA seed regions.

  14. Classifications within molecular subtypes enables identification of BRCA1/BRCA2 mutation carriers by RNA tumor profiling

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    Larsen, Martin J; Kruse, Torben A; Tan, Qihua

    2013-01-01

    Pathogenic germline mutations in BRCA1 or BRCA2 are detected in less than one third of families with a strong history of breast cancer. It is therefore expected that mutations still remain undetected by currently used screening methods. In addition, a growing number of BRCA1/2 sequence variants...... of unclear pathogen significance are found in the families, constituting an increasing clinical challenge. New methods are therefore needed to improve the detection rate and aid the interpretation of the clinically uncertain variants. In this study we analyzed a series of 33 BRCA1, 22 BRCA2, and 128 sporadic...... tumors by RNA profiling to investigate the classification potential of RNA profiles to predict BRCA1/2 mutation status. We found that breast tumors from BRCA1 and BRCA2 mutation carriers display characteristic RNA expression patterns, allowing them to be distinguished from sporadic tumors. The majority...

  15. A whole mitochondrial genome screening in a MELAS patient: A novel mitochondrial tRNAVal mutation

    International Nuclear Information System (INIS)

    Mezghani, Najla; Mnif, Mouna; Kacem, Maha; Mkaouar-Rebai, Emna; Hadj Salem, Ikhlass; Kallel, Nozha; Charfi, Nadia; Abid, Mohamed; Fakhfakh, Faiza

    2011-01-01

    Highlights: → We report a young Tunisian patient with clinical features of MELAS syndrome. → Reported mitochondrial mutations were absent after a mutational screening of the whole mtDNA. → We described a novel m.1640A>G mutation in the tRNA Val gene which was absent in 150 controls. → Mitochondrial deletions and POLG1 gene mutations were absent. → The m.1640A>G mutation could be associated to MELAS syndrome. -- Abstract: Mitochondrial encephalopathy, lactic acidosis and strokelike episodes (MELAS) syndrome is a mitochondrial disorder characterized by a wide variety of clinical presentations and a multisystemic organ involvement. In this study, we report a Tunisian girl with clinical features of MELAS syndrome who was negative for the common m.3243A>G mutation, but also for the reported mitochondrial DNA (mtDNA) mutations and deletions. Screening of the entire mtDNA genome showed several known mitochondrial variants besides to a novel transition m.1640A>G affecting a wobble adenine in the anticodon stem region of the tRNA Val . This nucleotide was conserved and it was absent in 150 controls suggesting its pathogenicity. In addition, no mutations were found in the nuclear polymerase gamma-1 gene (POLG1). These results suggest further investigation nuclear genes encoding proteins responsible for stability and structural components of the mtDNA or to the oxidative phosphorylation machinery to explain the phenotypic variability in the studied family.

  16. Mutation analysis of pre-mRNA splicing genes in Chinese families with retinitis pigmentosa

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    Pan, Xinyuan; Chen, Xue; Liu, Xiaoxing; Gao, Xiang; Kang, Xiaoli; Xu, Qihua; Chen, Xuejuan; Zhao, Kanxing; Zhang, Xiumei; Chu, Qiaomei; Wang, Xiuying

    2014-01-01

    Purpose Seven genes involved in precursor mRNA (pre-mRNA) splicing have been implicated in autosomal dominant retinitis pigmentosa (adRP). We sought to detect mutations in all seven genes in Chinese families with RP, to characterize the relevant phenotypes, and to evaluate the prevalence of mutations in splicing genes in patients with adRP. Methods Six unrelated families from our adRP cohort (42 families) and two additional families with RP with uncertain inheritance mode were clinically characterized in the present study. Targeted sequence capture with next-generation massively parallel sequencing (NGS) was performed to screen mutations in 189 genes including all seven pre-mRNA splicing genes associated with adRP. Variants detected with NGS were filtered with bioinformatics analyses, validated with Sanger sequencing, and prioritized with pathogenicity analysis. Results Mutations in pre-mRNA splicing genes were identified in three individual families including one novel frameshift mutation in PRPF31 (p.Leu366fs*1) and two known mutations in SNRNP200 (p.Arg681His and p.Ser1087Leu). The patients carrying SNRNP200 p.R681H showed rapid disease progression, and the family carrying p.S1087L presented earlier onset ages and more severe phenotypes compared to another previously reported family with p.S1087L. In five other families, we identified mutations in other RP-related genes, including RP1 p. Ser781* (novel), RP2 p.Gln65* (novel) and p.Ile137del (novel), IMPDH1 p.Asp311Asn (recurrent), and RHO p.Pro347Leu (recurrent). Conclusions Mutations in splicing genes identified in the present and our previous study account for 9.5% in our adRP cohort, indicating the important role of pre-mRNA splicing deficiency in the etiology of adRP. Mutations in the same splicing gene, or even the same mutation, could correlate with different phenotypic severities, complicating the genotype–phenotype correlation and clinical prognosis. PMID:24940031

  17. RADIA: RNA and DNA integrated analysis for somatic mutation detection.

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    Amie J Radenbaugh

    Full Text Available The detection of somatic single nucleotide variants is a crucial component to the characterization of the cancer genome. Mutation calling algorithms thus far have focused on comparing the normal and tumor genomes from the same individual. In recent years, it has become routine for projects like The Cancer Genome Atlas (TCGA to also sequence the tumor RNA. Here we present RADIA (RNA and DNA Integrated Analysis, a novel computational method combining the patient-matched normal and tumor DNA with the tumor RNA to detect somatic mutations. The inclusion of the RNA increases the power to detect somatic mutations, especially at low DNA allelic frequencies. By integrating an individual's DNA and RNA, we are able to detect mutations that would otherwise be missed by traditional algorithms that examine only the DNA. We demonstrate high sensitivity (84% and very high precision (98% and 99% for RADIA in patient data from endometrial carcinoma and lung adenocarcinoma from TCGA. Mutations with both high DNA and RNA read support have the highest validation rate of over 99%. We also introduce a simulation package that spikes in artificial mutations to patient data, rather than simulating sequencing data from a reference genome. We evaluate sensitivity on the simulation data and demonstrate our ability to rescue back mutations at low DNA allelic frequencies by including the RNA. Finally, we highlight mutations in important cancer genes that were rescued due to the incorporation of the RNA.

  18. CORALINA: a universal method for the generation of gRNA libraries for CRISPR-based screening.

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    Köferle, Anna; Worf, Karolina; Breunig, Christopher; Baumann, Valentin; Herrero, Javier; Wiesbeck, Maximilian; Hutter, Lukas H; Götz, Magdalena; Fuchs, Christiane; Beck, Stephan; Stricker, Stefan H

    2016-11-14

    The bacterial CRISPR system is fast becoming the most popular genetic and epigenetic engineering tool due to its universal applicability and adaptability. The desire to deploy CRISPR-based methods in a large variety of species and contexts has created an urgent need for the development of easy, time- and cost-effective methods enabling large-scale screening approaches. Here we describe CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a method for the generation of comprehensive gRNA libraries for CRISPR-based screens. CORALINA gRNA libraries can be derived from any source of DNA without the need of complex oligonucleotide synthesis. We show the utility of CORALINA for human and mouse genomic DNA, its reproducibility in covering the most relevant genomic features including regulatory, coding and non-coding sequences and confirm the functionality of CORALINA generated gRNAs. The simplicity and cost-effectiveness make CORALINA suitable for any experimental system. The unprecedented sequence complexities obtainable with CORALINA libraries are a necessary pre-requisite for less biased large scale genomic and epigenomic screens.

  19. RNA-based mutation analysis identifies an unusual MSH6 splicing defect and circumvents PMS2 pseudogene interference.

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    Etzler, J; Peyrl, A; Zatkova, A; Schildhaus, H-U; Ficek, A; Merkelbach-Bruse, S; Kratz, C P; Attarbaschi, A; Hainfellner, J A; Yao, S; Messiaen, L; Slavc, I; Wimmer, K

    2008-02-01

    Heterozygous germline mutations in one of the mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2 cause hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome, a dominantly inherited cancer susceptibility syndrome. Recent reports provide evidence for a novel recessively inherited cancer syndrome with constitutive MMR deficiency due to biallelic germline mutations in one of the MMR genes. MMR-deficiency (MMR-D) syndrome is characterized by childhood brain tumors, hematological and/or gastrointestinal malignancies, and signs of neurofibromatosis type 1 (NF1). We established an RNA-based mutation detection assay for the four MMR genes, since 1) a number of splicing defects may escape detection by the analysis of genomic DNA, and 2) DNA-based mutation detection in the PMS2 gene is severely hampered by the presence of multiple highly similar pseudogenes, including PMS2CL. Using this assay, which is based on direct cDNA sequencing of RT-PCR products, we investigated two families with children suspected to suffer from MMR-D syndrome. We identified a homozygous complex MSH6 splicing alteration in the index patients of the first family and a novel homozygous PMS2 mutation (c.182delA) in the index patient of the second family. Furthermore, we demonstrate, by the analysis of a PMS2/PMS2CL "hybrid" allele carrier, that RNA-based PMS2 testing effectively avoids the caveats of genomic DNA amplification approaches; i.e., pseudogene coamplification as well as allelic dropout, and will, thus, allow more sensitive mutation analysis in MMR deficiency and in HNPCC patients with PMS2 defects. (c) 2007 Wiley-Liss, Inc.

  20. Mutations affecting RNA polymerase I-stimulated exchange and rDNA recombination in yeast

    International Nuclear Information System (INIS)

    Lin, Y.H.; Keil, R.L.

    1991-01-01

    HOT1 is a cis-acting recombination-stimulatory sequence isolated from the rDNA repeat unit of yeast. The ability of HOT1 to stimulate mitotic exchange appears to depend on its ability to promote high levels of RNA polymerase I transcription. A qualitative colony color sectoring assay was developed to screen for trans-acting mutations that alter the activity of HOT1. Both hypo-recombination and hyper-recombination mutants were isolated. Genetic analysis of seven HOT1 recombination mutants (hrm) that decrease HOT1 activity shows that they behave as recessive nuclear mutations and belong to five linkage groups. Three of these mutations, hrm1, hrm2, and hrm3, also decrease rDNA exchange but do not alter recombination in the absence of HOT1. Another mutation, hrm4, decreases HOT1-stimulated recombination but does not affect rDNA recombination or exchange in the absence of HOT1. Two new alleles of RAD52 were also isolated using this screen. With regard to HOT1 activity, rad52 is epistatic to all four hrm mutations indicating that the products of the HRM genes and of RAD52 mediate steps in the same recombination pathway. Finding mutations that decrease both the activity of HOT1 and exchange in the rDNA supports the hypothesis that HOT1 plays a role in rDNA recombination

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

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

    2014-10-01

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

  2. Nonsense mutations in the human β-globin gene affect mRNA metabolism

    International Nuclear Information System (INIS)

    Baserga, S.J.; Benz, E.J. Jr.

    1988-01-01

    A number of premature translation termination mutations (nonsense mutations) have been described in the human α- and β-globin genes. Studies on mRNA isolated from patients with β 0 -thalassemia have shown that for both the β-17 and the β-39 mutations less than normal levels of β-globin mRNA accumulate in peripheral blood cells. (The codon at which the mutation occurs designates the name of the mutation; there are 146 codons in human β-globin mRNA). In vitro studies using the cloned β-39 gene have reproduced this effect in a heterologous transfection system and have suggested that the defect resides in intranuclear metabolism. The authors have asked if this phenomenon of decreased mRNA accumulation is a general property of nonsense mutations and if the effect depends on the location or the type of mutation. Toward this end, they have studied the effect of five nonsense mutations and two missense mutations on the expression of human β-globin mRNA in a heterologous transfection system. In all cases studied, the presence of a translation termination codon correlates with a decrease in the steady-state level of mRNA. The data suggest that the metabolism of a mammalian mRNA is affected by the presence of a mutation that affects translation

  3. Mutation of miRNA target sequences during human evolution

    DEFF Research Database (Denmark)

    Gardner, Paul P; Vinther, Jeppe

    2008-01-01

    It has long-been hypothesized that changes in non-protein-coding genes and the regulatory sequences controlling expression could undergo positive selection. Here we identify 402 putative microRNA (miRNA) target sequences that have been mutated specifically in the human lineage and show that genes...... containing such deletions are more highly expressed than their mouse orthologs. Our findings indicate that some miRNA target mutations are fixed by positive selection and might have been involved in the evolution of human-specific traits....

  4. Implementation of the first worldwide quality assurance program for cystic fibrosis multiple mutation detection in population-based screening.

    Science.gov (United States)

    Earley, Marie C; Laxova, Anita; Farrell, Philip M; Driscoll-Dunn, Rena; Cordovado, Suzanne; Mogayzel, Peter J; Konstan, Michael W; Hannon, W Harry

    2011-07-15

    CDC's Newborn Screening Quality Assurance Program collaborated with several U.S. Cystic Fibrosis Care Centers to collect specimens for development of a molecular CFTR proficiency testing program using dried-blood spots for newborn screening laboratories. Adult and adolescent patients or carriers donated whole blood that was aliquoted onto filter paper cards. Five blind-coded specimens were sent to participating newborn screening laboratories quarterly. Proficiency testing results were evaluated based on presumptive clinical assessment. Individual evaluations and summary reports were sent to each participating laboratory and technical consultations were offered if incorrect assessments were reported. The current CDC repository contains specimens with 39 different CFTR mutations. Up to 45 laboratories have participated in the program. Three years of data showed that correct assessments were reported 97.7% of the time overall when both mutations could be determined. Incorrect assessments that could have lead to a missed case occurred 0.9% of the time, and no information was reported 1.1% of the time due to sample failure. Results show that laboratories using molecular assays to detect CFTR mutations are performing satisfactorily. The programmatic results presented demonstrate the importance and complexity of providing proficiency testing for DNA-based assays. Published by Elsevier B.V.

  5. Social Health Insurance-Based Simultaneous Screening for 154 Mutations in 19 Deafness Genes Efficiently Identified Causative Mutations in Japanese Hearing Loss Patients.

    Directory of Open Access Journals (Sweden)

    Kentaro Mori

    Full Text Available Sensorineural hearing loss is one of the most common neurosensory disorders in humans. The incidence of SNHL is estimated to be 1 in 500-1000 newborns. In more than half of these patients, the hearing loss is associated with genetic causes. In Japan, genetic testing for the patients with SNHL using the Invader assay to screen for 46 mutations in 13 deafness genes was approved by the Ministry of Health, Labour and Welfare for inclusion in social health insurance coverage in 2012. Furthermore, from August 2015, this genetic testing has been expanded to screen for 154 mutations in 19 deafness genes using targeted genomic enrichment with massively parallel DNA sequencing combined with the Invader assay and TaqMan genotyping. For this study we analyzed 717 unrelated Japanese hearing loss patients. The total allele frequency of 154 mutations in 19 deafness genes was 32.64% (468/1434 and the total numbers of cases associated with at least one mutation was 44.07% (316/717. Among these, we were able to diagnose 212 (30% patients, indicating that the present screening could efficiently identify causative mutations in hearing loss patients. It is noteworthy that 27 patients (3.8% had coexistent multiple mutations in different genes. Five of these 27 patients (0.7%, 5/717 overall were diagnosed with genetic hearing loss affected by concomitant with responsible mutations in more than two different genes. For patients identified with multiple mutations in different genes, it is necessary to consider that several genes might have an impact on their phenotypes.

  6. Germline PMS2 mutation screened by mismatch repair protein immunohistochemistry of colorectal cancer in Japan.

    Science.gov (United States)

    Sugano, Kokichi; Nakajima, Takeshi; Sekine, Shigeki; Taniguchi, Hirokazu; Saito, Shinya; Takahashi, Masahiro; Ushiama, Mineko; Sakamoto, Hiromi; Yoshida, Teruhiko

    2016-11-01

    Germline PMS2 gene mutations were detected by RT-PCR/direct sequencing of total RNA extracted from puromycin-treated peripheral blood lymphocytes (PBL) and multiplex ligation-dependent probe amplification (MLPA) analyses of Japanese patients with colorectal cancer (CRC) fulfilling either the revised Bethesda Guidelines or being an age at disease onset of younger than 70 years, and screened by mismatch repair protein immunohistochemistry of formalin-fixed paraffin embedded sections. Of the 501 subjects examined, 7 (1.40%) showed the downregulated expression of the PMS2 protein alone and were referred to the genetic counseling clinic. Germline PMS2 mutations were detected in 6 (85.7%), including 3 nonsense and 1 frameshift mutations by RT-PCR/direct sequencing and 2 genomic deletions by MLPA. No mutations were identified in the other MMR genes (i.e. MSH2, MLH1 and MSH6). The prevalence of the downregulated expression of the PMS2 protein alone was 1.40% among the subjects examined and IHC results predicted the presence of PMS2 germline mutations. RT-PCR from puromycin-treated PBL and MLPA may be employed as the first screening step to detect PMS2 mutations without pseudogene interference, followed by the long-range PCR/nested PCR validation using genomic DNA. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  7. Customized treatment in non-small-cell lung cancer based on EGFR mutations and BRCA1 mRNA expression.

    Directory of Open Access Journals (Sweden)

    Rafael Rosell

    Full Text Available BACKGROUND: Median survival is 10 months and 2-year survival is 20% in metastatic non-small-cell lung cancer (NSCLC treated with platinum-based chemotherapy. A small fraction of non-squamous cell lung cancers harbor EGFR mutations, with improved outcome to gefitinib and erlotinib. Experimental evidence suggests that BRCA1 overexpression enhances sensitivity to docetaxel and resistance to cisplatin. RAP80 and Abraxas are interacting proteins that form complexes with BRCA1 and could modulate the effect of BRCA1. In order to further examine the effect of EGFR mutations and BRCA1 mRNA levels on outcome in advanced NSCLC, we performed a prospective non-randomized phase II clinical trial, testing the hypothesis that customized therapy would confer improved outcome over non-customized therapy. In an exploratory analysis, we also examined the effect of RAP80 and Abraxas mRNA levels. METHODOLOGY/PRINCIPAL FINDINGS: We treated 123 metastatic non-squamous cell lung carcinoma patients using a customized approach. RNA and DNA were isolated from microdissected specimens from paraffin-embedded tumor tissue. Patients with EGFR mutations received erlotinib, and those without EGFR mutations received chemotherapy with or without cisplatin based on their BRCA1 mRNA levels: low, cisplatin plus gemcitabine; intermediate, cisplatin plus docetaxel; high, docetaxel alone. An exploratory analysis examined RAP80 and Abraxas expression. Median survival exceeded 28 months for 12 patients with EGFR mutations, and was 11 months for 38 patients with low BRCA1, 9 months for 40 patients with intermediate BRCA1, and 11 months for 33 patients with high BRCA1. Two-year survival was 73.3%, 41.2%, 15.6% and 0%, respectively. Median survival was influenced by RAP80 expression in the three BRCA1 groups. For example, for patients with both low BRCA1 and low RAP80, median survival exceeded 26 months. RAP80 was a significant factor for survival in patients treated according to BRCA1

  8. A mitochondrial tRNA(His) gene mutation causing pigmentary retinopathy and neurosensorial deafness.

    Science.gov (United States)

    Crimi, M; Galbiati, S; Perini, M P; Bordoni, A; Malferrari, G; Sciacco, M; Biunno, I; Strazzer, S; Moggio, M; Bresolin, N; Comi, G P

    2003-04-08

    We have identified a heteroplasmic G to A mutation at position 12,183 of the mitochondrial transfer RNA Histidine (tRNA(His)) gene in three related patients. These phenotypes varied according to mutation heteroplasmy: one had severe pigmentary retinopathy, neurosensorial deafness, testicular dysfunction, muscle hypotrophy, and ataxia; the other two had only retinal and inner ear involvement. The mutation is in a highly conserved region of the T(psi)C stem of the tRNA(His) gene and may alter secondary structure formation. This is the first described pathogenic, maternally inherited mutation of the mitochondrial tRNA(His) gene.

  9. Base substitutions at scissile bond sites are sufficient to alter RNA-binding and cleavage activity of RNase III.

    Science.gov (United States)

    Kim, Kyungsub; Sim, Se-Hoon; Jeon, Che Ok; Lee, Younghoon; Lee, Kangseok

    2011-02-01

    RNase III, a double-stranded RNA-specific endoribonuclease, degrades bdm mRNA via cleavage at specific sites. To better understand the mechanism of cleavage site selection by RNase III, we performed a genetic screen for sequences containing mutations at the bdm RNA cleavage sites that resulted in altered mRNA stability using a transcriptional bdm'-'cat fusion construct. While most of the isolated mutants showed the increased bdm'-'cat mRNA stability that resulted from the inability of RNase III to cleave the mutated sequences, one mutant sequence (wt-L) displayed in vivo RNA stability similar to that of the wild-type sequence. In vivo and in vitro analyses of the wt-L RNA substrate showed that it was cut only once on the RNA strand to the 5'-terminus by RNase III, while the binding constant of RNase III to this mutant substrate was moderately increased. A base substitution at the uncleaved RNase III cleavage site in wt-L mutant RNA found in another mutant lowered the RNA-binding affinity by 11-fold and abolished the hydrolysis of scissile bonds by RNase III. Our results show that base substitutions at sites forming the scissile bonds are sufficient to alter RNA cleavage as well as the binding activity of RNase III. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  10. Mutational analysis of Sep-tRNA:Cys-tRNA synthase reveals critical residues for tRNA-dependent cysteine formation.

    Science.gov (United States)

    Helgadóttir, Sunna; Sinapah, Sylvie; Söll, Dieter; Ling, Jiqiang

    2012-01-02

    In methanogenic archaea, Sep-tRNA:Cys-tRNA synthase (SepCysS) converts Sep-tRNA(Cys) to Cys-tRNA(Cys). The mechanism of tRNA-dependent cysteine formation remains unclear due to the lack of functional studies. In this work, we mutated 19 conserved residues in Methanocaldococcus jannaschii SepCysS, and employed an in vivo system to determine the activity of the resulting variants. Our results show that three active-site cysteines (Cys39, Cys42 and Cys247) are essential for SepCysS activity. In addition, combined with structural modeling, our mutational and functional analyses also reveal multiple residues that are important for the binding of PLP, Sep and tRNA. Our work thus represents the first systematic functional analysis of conserved residues in archaeal SepCysSs, providing insights into the catalytic and substrate binding mechanisms of this poorly characterized enzyme. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  11. Mutations in the human adenosine deaminase gene that affect protein structure and RNA splicing

    International Nuclear Information System (INIS)

    Akeson, A.L.; Wiginton, D.A.; States, C.J.; Perme, C.M.; Dusing, M.R.; Hutton, J.J.

    1987-01-01

    Adenosine deaminase deficiency is one cause of the genetic disease severe combined immunodeficiency. To identify mutations responsible for ADA deficiency, the authors synthesized cDNAs to ADA mRNAs from two cell lines, GM2756 and GM2825A, derived from ADA-deficient immunodeficient patients. Sequence analysis of GM2756 cDNA clones revealed a different point mutation in each allele that causes amino acid changes of alanine to valine and arginine to histidine. One allele of GM2825A also has a point mutation that causes an alanine to valine substitution. The other allele of GM2825A was found to produce an mRNA in which exon 4 had been spliced out but had no other detrimental mutations. S1 nuclease mapping of GM2825A mRNA showed equal abundance of the full-length ADA mRNA and the ADA mRNA that was missing exon 4. Several of the ADA cDNA clones extended 5' of the major initiation start site, indicating multiple start sites for ADA transcription. The point mutations in GM2756 and GM2825A and the absence of exon 4 in GM2825A appear to be directly responsible for the ADA deficiency. Comparison of a number of normal and mutant ADA cDNA sequences showed a number of changes in the third base of codons. These change do not affect the amino acid sequence. Analyses of ADA cDNAs from different cell lines detected aberrant RNA species that either included intron 7 or excluded exon 7. Their presence is a result of aberrant splicing of pre-mRNAs and is not related to mutations that cause ADA deficiency

  12. Disease-associated mutations that alter the RNA structural ensemble.

    Directory of Open Access Journals (Sweden)

    Matthew Halvorsen

    2010-08-01

    Full Text Available Genome-wide association studies (GWAS often identify disease-associated mutations in intergenic and non-coding regions of the genome. Given the high percentage of the human genome that is transcribed, we postulate that for some observed associations the disease phenotype is caused by a structural rearrangement in a regulatory region of the RNA transcript. To identify such mutations, we have performed a genome-wide analysis of all known disease-associated Single Nucleotide Polymorphisms (SNPs from the Human Gene Mutation Database (HGMD that map to the untranslated regions (UTRs of a gene. Rather than using minimum free energy approaches (e.g. mFold, we use a partition function calculation that takes into consideration the ensemble of possible RNA conformations for a given sequence. We identified in the human genome disease-associated SNPs that significantly alter the global conformation of the UTR to which they map. For six disease-states (Hyperferritinemia Cataract Syndrome, beta-Thalassemia, Cartilage-Hair Hypoplasia, Retinoblastoma, Chronic Obstructive Pulmonary Disease (COPD, and Hypertension, we identified multiple SNPs in UTRs that alter the mRNA structural ensemble of the associated genes. Using a Boltzmann sampling procedure for sub-optimal RNA structures, we are able to characterize and visualize the nature of the conformational changes induced by the disease-associated mutations in the structural ensemble. We observe in several cases (specifically the 5' UTRs of FTL and RB1 SNP-induced conformational changes analogous to those observed in bacterial regulatory Riboswitches when specific ligands bind. We propose that the UTR and SNP combinations we identify constitute a "RiboSNitch," that is a regulatory RNA in which a specific SNP has a structural consequence that results in a disease phenotype. Our SNPfold algorithm can help identify RiboSNitches by leveraging GWAS data and an analysis of the mRNA structural ensemble.

  13. A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish

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    Sergey V. Prykhozhij

    2017-06-01

    Full Text Available Clustered regularly interspaced palindromic repeats (CRISPR/Cas-based adaptive immunity against pathogens in bacteria has been adapted for genome editing and applied in zebrafish (Danio rerio to generate frameshift mutations in protein-coding genes. Although there are methods to detect, quantify and sequence CRISPR/Cas9-induced mutations, identifying mutations in F1 heterozygous fish remains challenging. Additionally, sequencing a mutation and assuming that it causes a frameshift does not prove causality because of possible alternative translation start sites and potential effects of mutations on splicing. This problem is compounded by the relatively few antibodies available for zebrafish proteins, limiting validation at the protein level. To address these issues, we developed a detailed protocol to screen F1 mutation carriers, and clone and sequence identified mutations. In order to verify that mutations actually cause frameshifts, we created a fluorescent reporter system that can detect frameshift efficiency based on the cloning of wild-type and mutant cDNA fragments and their expression levels. As proof of principle, we applied this strategy to three CRISPR/Cas9-induced mutations in pycr1a, chd7 and hace1 genes. An insertion of seven nucleotides in pycr1a resulted in the first reported observation of exon skipping by CRISPR/Cas9-induced mutations in zebrafish. However, of these three mutant genes, the fluorescent reporter revealed effective frameshifting exclusively in the case of a two-nucleotide deletion in chd7, suggesting activity of alternative translation sites in the other two mutants even though pycr1a exon-skipping deletion is likely to be deleterious. This article provides a protocol for characterizing frameshift mutations in zebrafish, and highlights the importance of checking mutations at the mRNA level and verifying their effects on translation by fluorescent reporters when antibody detection of protein loss is not possible.

  14. Screening for duplications, deletions and a common intronic mutation detects 35% of second mutations in patients with USH2A monoallelic mutations on Sanger sequencing.

    Science.gov (United States)

    Steele-Stallard, Heather B; Le Quesne Stabej, Polona; Lenassi, Eva; Luxon, Linda M; Claustres, Mireille; Roux, Anne-Francoise; Webster, Andrew R; Bitner-Glindzicz, Maria

    2013-08-08

    Usher Syndrome is the leading cause of inherited deaf-blindness. It is divided into three subtypes, of which the most common is Usher type 2, and the USH2A gene accounts for 75-80% of cases. Despite recent sequencing strategies, in our cohort a significant proportion of individuals with Usher type 2 have just one heterozygous disease-causing mutation in USH2A, or no convincing disease-causing mutations across nine Usher genes. The purpose of this study was to improve the molecular diagnosis in these families by screening USH2A for duplications, heterozygous deletions and a common pathogenic deep intronic variant USH2A: c.7595-2144A>G. Forty-nine Usher type 2 or atypical Usher families who had missing mutations (mono-allelic USH2A or no mutations following Sanger sequencing of nine Usher genes) were screened for duplications/deletions using the USH2A SALSA MLPA reagent kit (MRC-Holland). Identification of USH2A: c.7595-2144A>G was achieved by Sanger sequencing. Mutations were confirmed by a combination of reverse transcription PCR using RNA extracted from nasal epithelial cells or fibroblasts, and by array comparative genomic hybridisation with sequencing across the genomic breakpoints. Eight mutations were identified in 23 Usher type 2 families (35%) with one previously identified heterozygous disease-causing mutation in USH2A. These consisted of five heterozygous deletions, one duplication, and two heterozygous instances of the pathogenic variant USH2A: c.7595-2144A>G. No variants were found in the 15 Usher type 2 families with no previously identified disease-causing mutations. In 11 atypical families, none of whom had any previously identified convincing disease-causing mutations, the mutation USH2A: c.7595-2144A>G was identified in a heterozygous state in one family. All five deletions and the heterozygous duplication we report here are novel. This is the first time that a duplication in USH2A has been reported as a cause of Usher syndrome. We found that 8 of

  15. Variation in RNA virus mutation rates across host cells.

    Directory of Open Access Journals (Sweden)

    Marine Combe

    2014-01-01

    Full Text Available It is well established that RNA viruses exhibit higher rates of spontaneous mutation than DNA viruses and microorganisms. However, their mutation rates vary amply, from 10(-6 to 10(-4 substitutions per nucleotide per round of copying (s/n/r and the causes of this variability remain poorly understood. In addition to differences in intrinsic fidelity or error correction capability, viral mutation rates may be dependent on host factors. Here, we assessed the effect of the cellular environment on the rate of spontaneous mutation of the vesicular stomatitis virus (VSV, which has a broad host range and cell tropism. Luria-Delbrück fluctuation tests and sequencing showed that VSV mutated similarly in baby hamster kidney, murine embryonic fibroblasts, colon cancer, and neuroblastoma cells (approx. 10(-5 s/n/r. Cell immortalization through p53 inactivation and oxygen levels (1-21% did not have a significant impact on viral replication fidelity. This shows that previously published mutation rates can be considered reliable despite being based on a narrow and artificial set of laboratory conditions. Interestingly, we also found that VSV mutated approximately four times more slowly in various insect cells compared with mammalian cells. This may contribute to explaining the relatively slow evolution of VSV and other arthropod-borne viruses in nature.

  16. Fragment length analysis screening for detection of CEBPA mutations in intermediate-risk karyotype acute myeloid leukemia.

    Science.gov (United States)

    Fuster, Oscar; Barragán, Eva; Bolufer, Pascual; Such, Esperanza; Valencia, Ana; Ibáñez, Mariam; Dolz, Sandra; de Juan, Inmaculada; Jiménez, Antonio; Gómez, Maria Teresa; Buño, Ismael; Martínez, Joaquín; Cervera, José; Montesinos, Pau; Moscardó, Federico; Sanz, Miguel Ángel

    2012-01-01

    During last years, molecular markers have been increased as prognostic factors routinely screened in acute myeloid leukemia (AML). Recently, an increasing interest has been reported in introducing to clinical practice screening for mutations in the CCAAT/enhancer-binding protein α (CEBPA) gene in AML, as it seems to be a good prognostic factor. However, there is no reliable established method for assessing CEBPA mutations during the diagnostic work-up of AMLs. We describe here a straightforward and reliable fragment analysis method based in PCR capillary electrophoresis (PCR-CE) for screening of CEBPA mutations; moreover, we present the results obtained in 151 intermediate-risk karyotype AML patients (aged 16-80 years). The method gave a specificity of 100% and sensitivity of 93% with a lower detection limit of 1-5% for CEBPA mutations. The series found 19 mutations and four polymorphisms in 12 patients, seven of whom (58%) presented two mutations. The overall frequency of CEBPA mutations in AML was 8% (n = 12). CEBPA mutations showed no coincidence with FLT3-ITD or NPM1 mutations. CEBPA mutation predicted better disease-free survival in the group of patients without FLT3-ITD, NPM, or both genes mutated (HR 3.6, IC 95%; 1.0-13.2, p = 0.05) and better overall survival in patients younger than 65 of this group without molecular markers (HR 4.0, IC 95%; 1.0-17.4, p = 0.05). In conclusion, the fragment analysis method based in PCR-CE is a rapid, specific, and sensitive method for CEBPA mutation screening and our results confirm that CEBPA mutations can identify a subgroup of patients with favorable prognosis in AML with intermediate-risk karyotype.

  17. RNA-TVcurve: a Web server for RNA secondary structure comparison based on a multi-scale similarity of its triple vector curve representation.

    Science.gov (United States)

    Li, Ying; Shi, Xiaohu; Liang, Yanchun; Xie, Juan; Zhang, Yu; Ma, Qin

    2017-01-21

    RNAs have been found to carry diverse functionalities in nature. Inferring the similarity between two given RNAs is a fundamental step to understand and interpret their functional relationship. The majority of functional RNAs show conserved secondary structures, rather than sequence conservation. Those algorithms relying on sequence-based features usually have limitations in their prediction performance. Hence, integrating RNA structure features is very critical for RNA analysis. Existing algorithms mainly fall into two categories: alignment-based and alignment-free. The alignment-free algorithms of RNA comparison usually have lower time complexity than alignment-based algorithms. An alignment-free RNA comparison algorithm was proposed, in which novel numerical representations RNA-TVcurve (triple vector curve representation) of RNA sequence and corresponding secondary structure features are provided. Then a multi-scale similarity score of two given RNAs was designed based on wavelet decomposition of their numerical representation. In support of RNA mutation and phylogenetic analysis, a web server (RNA-TVcurve) was designed based on this alignment-free RNA comparison algorithm. It provides three functional modules: 1) visualization of numerical representation of RNA secondary structure; 2) detection of single-point mutation based on secondary structure; and 3) comparison of pairwise and multiple RNA secondary structures. The inputs of the web server require RNA primary sequences, while corresponding secondary structures are optional. For the primary sequences alone, the web server can compute the secondary structures using free energy minimization algorithm in terms of RNAfold tool from Vienna RNA package. RNA-TVcurve is the first integrated web server, based on an alignment-free method, to deliver a suite of RNA analysis functions, including visualization, mutation analysis and multiple RNAs structure comparison. The comparison results with two popular RNA

  18. 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

  19. In Vivo RNAi-Based Screens: Studies in Model Organisms

    Directory of Open Access Journals (Sweden)

    Miki Yamamoto-Hino

    2013-11-01

    Full Text Available RNA interference (RNAi is a technique widely used for gene silencing in organisms and cultured cells, and depends on sequence homology between double-stranded RNA (dsRNA and target mRNA molecules. Numerous cell-based genome-wide screens have successfully identified novel genes involved in various biological processes, including signal transduction, cell viability/death, and cell morphology. However, cell-based screens cannot address cellular processes such as development, behavior, and immunity. Drosophila and Caenorhabditis elegans are two model organisms whose whole bodies and individual body parts have been subjected to RNAi-based genome-wide screening. Moreover, Drosophila RNAi allows the manipulation of gene function in a spatiotemporal manner when it is implemented using the Gal4/UAS system. Using this inducible RNAi technique, various large-scale screens have been performed in Drosophila, demonstrating that the method is straightforward and valuable. However, accumulated results reveal that the results of RNAi-based screens have relatively high levels of error, such as false positives and negatives. Here, we review in vivo RNAi screens in Drosophila and the methods that could be used to remove ambiguity from screening results.

  20. Alanyl-tRNA synthetase mutation in a family with dominant distal hereditary motor neuropathy

    Science.gov (United States)

    Zhao, Z.; Hashiguchi, A.; Sakiyama, Y.; Okamoto, Y.; Tokunaga, S.; Zhu, L.; Shen, H.; Takashima, H.

    2012-01-01

    Objective: To identify a new genetic cause of distal hereditary motor neuropathy (dHMN), which is also known as a variant of Charcot-Marie-Tooth disease (CMT), in a Chinese family. Methods: We investigated a Chinese family with dHMN clinically, electrophysiologically, and genetically. We screened for the mutations of 28 CMT or related pathogenic genes using an originally designed microarray resequencing DNA chip. Results: Investigation of the family history revealed an autosomal dominant transmission pattern. The clinical features of the family included mild weakness and wasting of the distal muscles of the lower limb and foot deformity, without clinical sensory involvement. Electrophysiologic studies revealed motor neuropathy. MRI of the lower limbs showed accentuated fatty infiltration of the gastrocnemius and vastus lateralis muscles. All 4 affected family members had a heterozygous missense mutation c.2677G>A (p.D893N) of alanyl-tRNA synthetase (AARS), which was not found in the 4 unaffected members and control subjects. Conclusion: An AARS mutation caused dHMN in a Chinese family. AARS mutations result in not only a CMT phenotype but also a dHMN phenotype. PMID:22573628

  1. Risk modeling and screening for BRCA1 mutations among Filipino breast cancer patients

    International Nuclear Information System (INIS)

    Nato, Alejandro Q. Jr.

    2003-03-01

    Breast cancer susceptibility gene, type 1(BRCA1) has been thought to be responsible for ∼45% of families with multiple breast carcinomas and for ∼80% of breast and ovarian cancer families. In this study, we investigated 34 familial Filipino breast cancer (BC) patients to: (a) estimate breast cancer risks and BRCA1/2 mutation carrier probabilities using risk assessment and prior probability models, respectively; (b) screen for putative polymorphisms at selected smaller exons of BRCA1 by single-strand conformation polymorphism (SSCP) analysis; (c) screen for truncated mutations at BRCA1 exon 11 by radioactive protein truncation test (PTT); and (d) estimate posterior probabilities upon incorporation of screening results. SSCP analysis revealed 8 unique putative polymorphisms. Low prevalence of unique putative polymorphisms at exon 2, 5, 17, and 22 may indicate probable mutations. Contrastingly, high prevalence of unique putative polymorphisms at exons 13, 15, and 16 may suggest true polymorphisms which are biologically insignificant. PTT, DHPLC, and sequence analyses revealed a novel mutation in exon 11 involving GT insertion that resulted to a stop codon which generated a 29.7 kDa truncated protein product. This is the second documented mutation in BRCA1 exon 11 in a Filipino BC patient since 1998. Initial genotype-phenotype correlations in Filipino BC patients may be elucidated based on screening tests performed. Our results corroborate the findings of a study on unselected incident Filipino BC cases where the reported prevalence of BRCA1 mutation is low. The higher prevalence of putative polypmorphisms may be attributed to the increased stringency in patient prospecting. The Gail, Claus, and BRCAPRO models can be utilized to estimate BC risk in unaffected high-risk individuals but validation is needed. Most of the BRCAPRO and Myriad.com prior probability estimates coincide with the presence of BRCA1 mutation and/or putative polymorphisms. This pioneering

  2. Risk modeling and screening for BRCA1 mutations among Filipino breast cancer patients

    Energy Technology Data Exchange (ETDEWEB)

    Nato, Jr, Alejandro Q

    2003-03-01

    Breast cancer susceptibility gene, type 1(BRCA1) has been thought to be responsible for {approx}45% of families with multiple breast carcinomas and for {approx}80% of breast and ovarian cancer families. In this study, we investigated 34 familial Filipino breast cancer (BC) patients to: (a) estimate breast cancer risks and BRCA1/2 mutation carrier probabilities using risk assessment and prior probability models, respectively; (b) screen for putative polymorphisms at selected smaller exons of BRCA1 by single-strand conformation polymorphism (SSCP) analysis; (c) screen for truncated mutations at BRCA1 exon 11 by radioactive protein truncation test (PTT); and (d) estimate posterior probabilities upon incorporation of screening results. SSCP analysis revealed 8 unique putative polymorphisms. Low prevalence of unique putative polymorphisms at exon 2, 5, 17, and 22 may indicate probable mutations. Contrastingly, high prevalence of unique putative polymorphisms at exons 13, 15, and 16 may suggest true polymorphisms which are biologically insignificant. PTT, DHPLC, and sequence analyses revealed a novel mutation in exon 11 involving GT insertion that resulted to a stop codon which generated a 29.7 kDa truncated protein product. This is the second documented mutation in BRCA1 exon 11 in a Filipino BC patient since 1998. Initial genotype-phenotype correlations in Filipino BC patients may be elucidated based on screening tests performed. Our results corroborate the findings of a study on unselected incident Filipino BC cases where the reported prevalence of BRCA1 mutation is low. The higher prevalence of putative polypmorphisms may be attributed to the increased stringency in patient prospecting. The Gail, Claus, and BRCAPRO models can be utilized to estimate BC risk in unaffected high-risk individuals but validation is needed. Most of the BRCAPRO and Myriad.com prior probability estimates coincide with the presence of BRCA1 mutation and/or putative polymorphisms. This

  3. MicroRNA-142 is mutated in about 20% of diffuse large B-cell lymphoma

    International Nuclear Information System (INIS)

    Kwanhian, Wiyada; Lenze, Dido; Alles, Julia; Motsch, Natalie; Barth, Stephanie; Döll, Celina; Imig, Jochen; Hummel, Michael; Tinguely, Marianne; Trivedi, Pankaj; Lulitanond, Viraphong; Meister, Gunter; Renner, Christoph; Grässer, Friedrich A

    2012-01-01

    MicroRNAs (miRNAs) are short 18–23 nucleotide long noncoding RNAs that posttranscriptionally regulate gene expression by binding to mRNA. Our previous miRNA profiling of diffuse large B-cell lymphoma (DLBCL) revealed a mutation in the seed sequence of miR-142-3p. Further analysis now showed that miR-142 was mutated in 11 (19.64%) of the 56 DLBCL cases. Of these, one case had a mutation in both alleles, with the remainder being heterozygous. Four mutations were found in the mature miR-142-5p, four in the mature miR-142-3p, and three mutations affected the miR-142 precursor. Two mutations in the seed sequence redirected miR-142-3p to the mRNA of the transcriptional repressor ZEB2 and one of them also targeted the ZEB1 mRNA. However, the other mutations in the mature miR-142-3p did not influence either the ZEB1 or ZEB2 3′ untranslated region (3′ UTR). On the other hand, the mutations affecting the seed sequence of miR-142-3p resulted in a loss of responsiveness in the 3′ UTR of the known miR-142-3p targets RAC1 and ADCY9. In contrast to the mouse p300 gene, the human p300 gene was not found to be a target for miR-142-5p. In one case with a mutation of the precursor, we observed aberrant processing of the miR-142-5p. Our data suggest that the mutations in miR-142 probably lead to a loss rather than a gain of function. This is the first report describing mutations of a miRNA gene in a large percentage of a distinct lymphoma subtype

  4. UPF1 silenced cellular model systems for screening of read-through agents active on β039 thalassemia point mutation.

    Science.gov (United States)

    Salvatori, Francesca; Pappadà, Mariangela; Breveglieri, Giulia; D'Aversa, Elisabetta; Finotti, Alessia; Lampronti, Ilaria; Gambari, Roberto; Borgatti, Monica

    2018-05-15

    Nonsense mutations promote premature translational termination, introducing stop codons within the coding region of mRNAs and causing inherited diseases, including thalassemia. For instance, in β 0 39 thalassemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well described NMD (nonsense-mediated mRNA decay). In order to develop an approach facilitating translation and, therefore, protection from NMD, ribosomal read-through molecules, such as aminoglycoside antibiotics, have been tested on mRNAs carrying premature stop codons. These findings have introduced new hopes for the development of a pharmacological approach to the β 0 39 thalassemia therapy. While several strategies, designed to enhance translational read-through, have been reported to inhibit NMD efficiency concomitantly, experimental tools for systematic analysis of mammalian NMD inhibition by translational read-through are lacking. We developed a human cellular model of the β 0 39 thalassemia mutation with UPF-1 suppressed and showing a partial NMD suppression. This novel cellular model could be used for the screening of molecules exhibiting preferential read-through activity allowing a great rescue of the mutated transcripts.

  5. Rich RNA Structure Landscapes Revealed by Mutate-and-Map Analysis.

    Directory of Open Access Journals (Sweden)

    Pablo Cordero

    2015-11-01

    Full Text Available Landscapes exhibiting multiple secondary structures arise in natural RNA molecules that modulate gene expression, protein synthesis, and viral infection [corrected]. We report herein that high-throughput chemical experiments can isolate an RNA's multiple alternative secondary structures as they are stabilized by systematic mutagenesis (mutate-and-map, M2 and that a computational algorithm, REEFFIT, enables unbiased reconstruction of these states' structures and populations. In an in silico benchmark on non-coding RNAs with complex landscapes, M2-REEFFIT recovers 95% of RNA helices present with at least 25% population while maintaining a low false discovery rate (10% and conservative error estimates. In experimental benchmarks, M2-REEFFIT recovers the structure landscapes of a 35-nt MedLoop hairpin, a 110-nt 16S rRNA four-way junction with an excited state, a 25-nt bistable hairpin, and a 112-nt three-state adenine riboswitch with its expression platform, molecules whose characterization previously required expert mutational analysis and specialized NMR or chemical mapping experiments. With this validation, M2-REEFFIT enabled tests of whether artificial RNA sequences might exhibit complex landscapes in the absence of explicit design. An artificial flavin mononucleotide riboswitch and a randomly generated RNA sequence are found to interconvert between three or more states, including structures for which there was no design, but that could be stabilized through mutations. These results highlight the likely pervasiveness of rich landscapes with multiple secondary structures in both natural and artificial RNAs and demonstrate an automated chemical/computational route for their empirical characterization.

  6. Mutations in domain II of 23 S rRNA facilitate translation of a 23 S rRNA-encoded pentapeptide conferring erythromycin resistance

    DEFF Research Database (Denmark)

    Dam, M; Douthwaite, S; Tenson, T

    1996-01-01

    Mutations in domain II of Escherichia coli 23 S rRNA that cause resistance to erythromycin do so in a manner fundamentally different from mutations at the drug binding site in domain V of the 23 S rRNA. The domain II mutations are located in a hairpin structure between nucleotides 1198 and 1247...... this hypothesis, a range of point mutations was generated in domain II of 23 S rRNA in the vicinity of the E-peptide open reading frame. We find a correlation between erythromycin resistance of the mutant clones and increased accessibility of the ribosome binding site of the E-peptide gene. Furthermore......, the erythromycin resistance determinant in the mutants was shown to be confined to a small 23 S rRNA segment containing the coding region and the ribosome binding site of the E-peptide open reading frame. It thus appears that the domain II mutations mediate erythromycin resistance by increasing expression...

  7. Photodynamic antisense regulation of mRNA having a point mutation with psoralen-conjugated oligonucleotide.

    Science.gov (United States)

    Higuchi, Maiko; Yamayoshi, Asako; Kobori, Akio; Murakami, Akira

    2008-01-01

    Nucleic acid-based drugs, such as antisense oligonucleotide, ribozyme, and small interfering RNA, are specific compounds that inhibit gene expression at the post-transcriptional level. To develop more effective nucleic acid-based drugs, we focused on photo-reactive antisense oligonucleotides. We have optimized the structure of psoralen-conjugated oligonucleotide to improve their sequence selectivity and photo-crosslinking efficiency. Previously, we reported that photo reactive oligonucleotides containing 2'-O-psoralenyl-methoxyethyl adenosine (2'-Ps-eom) showed drastic photo-reactivity with a strictly sequence specific manner in vitro. In this report, we evaluated the binding ability toward intracellular target mRNA. The 2'-Ps-eom selectively photo-cross-linked to the target mRNA extracted from cells. The 2'-Ps-eom also cross-linked to target mRNA in cells. Furthermore, 2'-Ps-eom did not cross-link to mRNA having a mismatch base. These results suggest that 2'-Ps-eom is a powerful antisense molecule to inhibit the expression of mRNA having a point mutation.

  8. Altered Pre-mRNA Splicing Caused by a Novel Intronic Mutation c.1443+5G>A in the Dihydropyrimidinase (DPYS) Gene.

    Science.gov (United States)

    Nakajima, Yoko; Meijer, Judith; Zhang, Chunhua; Wang, Xu; Kondo, Tomomi; Ito, Tetsuya; Dobritzsch, Doreen; Van Kuilenburg, André B P

    2016-01-12

    Dihydropyrimidinase (DHP) deficiency is an autosomal recessive disease caused by mutations in the DPYS gene. Patients present with highly elevated levels of dihydrouracil and dihydrothymine in their urine, blood and cerebrospinal fluid. The analysis of the effect of mutations in DPYS on pre-mRNA splicing is hampered by the fact that DHP is primarily expressed in liver and kidney cells. The minigene approach can detect mRNA splicing aberrations using cells that do not express the endogenous mRNA. We have used a minigene-based approach to analyze the effects of a presumptive pre-mRNA splicing mutation in two newly identified Chinese pediatric patients with DHP deficiency. Mutation analysis of DPYS showed that both patients were compound heterozygous for a novel intronic mutation c.1443+5G>A in intron 8 and a previously described missense mutation c.1001A>G (p.Q334R) in exon 6. Wild-type and the mutated minigene constructs, containing exons 7, 8 and 9 of DPYS, yielded different splicing products after expression in HEK293 cells. The c.1443+5G>A mutation resulted in altered pre-mRNA splicing of the DPYS minigene construct with full skipping of exon 8. Analysis of the DHP crystal structure showed that the deletion of exon 8 severely affects folding, stability and homooligomerization of the enzyme as well as disruption of the catalytic site. Thus, the analysis suggests that the c.1443+5G>A mutation results in aberrant splicing of the pre-mRNA encoding DHP, underlying the DHP deficiency in two unrelated Chinese patients.

  9. Screening for sequence-specific RNA-BPs by comprehensive UV crosslinking

    Directory of Open Access Journals (Sweden)

    Le Meuth-Metzinger Valerie

    2002-06-01

    Full Text Available Abstract Background Specific cis-elements and the associated trans-acting factors have been implicated in the post-transcriptional regulation of gene expression. In the era of genome wide analyses identifying novel trans-acting factors and cis-regulatory elements is a step towards understanding coordinated gene expression. UV-crosslink analysis is a standard method used to identify RNA-binding proteins. Uridine is traditionally used to radiolabel substrate RNAs, however, proteins binding to cis-elments particularly uridine poor will be weakly or not detected. We evaluate here the possibility of using UV-crosslinking with RNA substrates radiolabeled with each of the four ribonucleotides as an approach for screening for novel sequence specific RNA-binding proteins. Results The radiolabeled RNA substrates were derived from the 3'UTRs of the cloned Eg and c-mos Xenopus laevis maternal mRNAs. Specific, but not identical, uv-crosslinking signals were obtained, some of which corresponded to already identified proteins. A signal for a novel 90 kDa protein was observed with the c-mos 3'UTR radiolabeled with both CTP and GTP but not with UTP. The binding site of the 90 kDa RNA-binding protein was localised to a 59-nucleotide portion of the c-mos 3'UTR. Conclusion That the 90 kDa signal was detected with RNAs radiolabeled with CTP or GTP but not UTP illustrates the advantage of radiolabeling all four nucleotides in a UV-crosslink based screen. This method can be used for both long and short RNAs and does not require knowledge of the cis-acting sequence. It should be amenable to high throughput screening for RNA binding proteins.

  10. An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes

    NARCIS (Netherlands)

    Wheway, Gabrielle; Schmidts, Miriam; Mans, Dorus A; Szymanska, Katarzyna; Nguyen, Thanh-Minh T; Racher, Hilary; Phelps, Ian G; Toedt, Grischa; Kennedy, Julie; Wunderlich, Kirsten A; Sorusch, Nasrin; Abdelhamed, Zakia A; Natarajan, Subaashini; Herridge, Warren; van Reeuwijk, Jeroen; Horn, Nicola; Boldt, Karsten; Parry, David A; Letteboer, Stef J F; Roosing, Susanne; Adams, Matthew; Bell, Sandra M; Bond, Jacquelyn; Higgins, Julie; Morrison, Ewan E; Tomlinson, Darren C; Slaats, Gisela G; van Dam, Teunis J P; Huang, Lijia; Kessler, Kristin; Giessl, Andreas; Logan, Clare V; Boyle, Evan A; Shendure, Jay; Anazi, Shamsa; Aldahmesh, Mohammed; Al Hazzaa, Selwa; Hegele, Robert A; Ober, Carole; Frosk, Patrick; Mhanni, Aizeddin A; Chodirker, Bernard N; Chudley, Albert E; Lamont, Ryan; Bernier, Francois P; Beaulieu, Chandree L; Gordon, Paul; Pon, Richard T; Donahue, Clem; Barkovich, A James; Wolf, Louis; Toomes, Carmel; Thiel, Christian T; Boycott, Kym M; McKibbin, Martin; Inglehearn, Chris F; Stewart, Fiona; Omran, Heymut; Huynen, Martijn A; Sergouniotis, Panagiotis I; Alkuraya, Fowzan S; Parboosingh, Jillian S; Innes, A Micheil; Willoughby, Colin E; Giles, Rachel H; Webster, Andrew R; Ueffing, Marius; Blacque, Oliver; Gleeson, Joseph G; Wolfrum, Uwe; Beales, Philip L; Gibson, Toby; Doherty, Dan; Mitchison, Hannah M; Roepman, Ronald; Johnson, Colin A

    Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis

  11. ALS Associated Mutations in Matrin 3 Alter Protein-Protein Interactions and Impede mRNA Nuclear Export.

    Science.gov (United States)

    Boehringer, Ashley; Garcia-Mansfield, Krystine; Singh, Gurkaran; Bakkar, Nadine; Pirrotte, Patrick; Bowser, Robert

    2017-11-06

    Mutations in Matrin 3 have recently been linked to ALS, though the mechanism that induces disease in these patients is unknown. To define the protein interactome of wild-type and ALS-linked MATR3 mutations, we performed immunoprecipitation followed by mass spectrometry using NSC-34 cells expressing human wild-type or mutant Matrin 3. Gene ontology analysis identified a novel role for Matrin 3 in mRNA transport centered on proteins in the TRanscription and EXport (TREX) complex, known to function in mRNA biogenesis and nuclear export. ALS-linked mutations in Matrin 3 led to its re-distribution within the nucleus, decreased co-localization with endogenous Matrin 3 and increased co-localization with specific TREX components. Expression of disease-causing Matrin 3 mutations led to nuclear mRNA export defects of both global mRNA and more specifically the mRNA of TDP-43 and FUS. Our findings identify a potential pathogenic mechanism attributable to MATR3 mutations and further link cellular transport defects to ALS.

  12. Mutations in 23S rRNA at the Peptidyl Transferase Center and Their Relationship to Linezolid Binding and Cross-Resistance

    DEFF Research Database (Denmark)

    Long, Katherine; Munck, Christian

    2010-01-01

    The oxazolidinone antibiotic linezolid targets the peptidyl transferase center (PTC) on the bacterial ribosome. Thirteen single and four double 23S rRNA mutations were introduced into a Mycobacterium smegmatis strain with a single rRNA operon. Converting bacterial base identity by single mutations...... at positions 2032, 2453, and 2499 to human cytosolic base identity did not confer significantly reduced susceptibility to linezolid. The largest decrease in linezolid susceptibility for any of the introduced single mutations was observed with the G2576U mutation at a position that is 7.9 Å from linezolid....... Smaller decreases were observed with the A2503G, U2504G, and G2505A mutations at nucleotides proximal to linezolid, showing that the degree of resistance conferred is not simply inversely proportional to the nucleotide-drug distance. The double mutations G2032A-C2499A, G2032A-U2504G, C2055A-U2504G, and C...

  13. Knowledge-based analysis of functional impacts of mutations in ...

    Indian Academy of Sciences (India)

    Knowledge-based analysis of functional impacts of mutations in microRNA seed regions. Supplementary figure 1. Summary of predicted miRNA targets from ... All naturally occurred SNPs in seed regions of human miRNAs. The information of the columns is given in the second sheet. Hihly expressed miRNAs are ...

  14. Altered Pre-mRNA Splicing Caused by a Novel Intronic Mutation c.1443+5G>A in the Dihydropyrimidinase (DPYS Gene

    Directory of Open Access Journals (Sweden)

    Yoko Nakajima

    2016-01-01

    Full Text Available Dihydropyrimidinase (DHP deficiency is an autosomal recessive disease caused by mutations in the DPYS gene. Patients present with highly elevated levels of dihydrouracil and dihydrothymine in their urine, blood and cerebrospinal fluid. The analysis of the effect of mutations in DPYS on pre-mRNA splicing is hampered by the fact that DHP is primarily expressed in liver and kidney cells. The minigene approach can detect mRNA splicing aberrations using cells that do not express the endogenous mRNA. We have used a minigene-based approach to analyze the effects of a presumptive pre-mRNA splicing mutation in two newly identified Chinese pediatric patients with DHP deficiency. Mutation analysis of DPYS showed that both patients were compound heterozygous for a novel intronic mutation c.1443+5G>A in intron 8 and a previously described missense mutation c.1001A>G (p.Q334R in exon 6. Wild-type and the mutated minigene constructs, containing exons 7, 8 and 9 of DPYS, yielded different splicing products after expression in HEK293 cells. The c.1443+5G>A mutation resulted in altered pre-mRNA splicing of the DPYS minigene construct with full skipping of exon 8. Analysis of the DHP crystal structure showed that the deletion of exon 8 severely affects folding, stability and homooligomerization of the enzyme as well as disruption of the catalytic site. Thus, the analysis suggests that the c.1443+5G>A mutation results in aberrant splicing of the pre-mRNA encoding DHP, underlying the DHP deficiency in two unrelated Chinese patients.

  15. 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

  16. Breast cancer screening, outside the population-screening program, of women from breast cancer families without proven BRCA1/BRCA2 mutations : a simulation study

    NARCIS (Netherlands)

    Jacobi, C.E.; Nagelkerke, N.J.D.; van Houwelingen, J.C.; de Bock, G.H.

    Purpose: We assessed the cost-effectiveness of mammography screening for women under the age of 50, from breast cancer families without proven BRCA1./BRCA2 mutations, because current criteria for screening healthy women from breast cancer families are not evidence-based. Methods: We did simulation

  17. Breast cancer screening, outside the population-screening program, of women from breast cancer families without proven BRCA1/BRCA2 mutations: a simulation study

    NARCIS (Netherlands)

    Jacobi, C.E.; Nagelkerke, N.J.D.; van Houwelingen, J.C.; de Bock, Truuske

    2006-01-01

    Purpose: We assessed the cost-effectiveness of mammography screening for women under the age of 50, from breast cancer families without proven BRCA1./BRCA2 mutations, because current criteria for screening healthy women from breast cancer families are not evidence-based. Methods: We did simulation

  18. A genome-wide siRNA screen to identify modulators of insulin sensitivity and gluconeogenesis.

    Directory of Open Access Journals (Sweden)

    Ruojing Yang

    Full Text Available BACKGROUND: Hepatic insulin resistance impairs insulin's ability to suppress hepatic glucose production (HGP and contributes to the development of type 2 diabetes (T2D. Although the interests to discover novel genes that modulate insulin sensitivity and HGP are high, it remains challenging to have a human cell based system to identify novel genes. METHODOLOGY/PRINCIPAL FINDINGS: To identify genes that modulate hepatic insulin signaling and HGP, we generated a human cell line stably expressing beta-lactamase under the control of the human glucose-6-phosphatase (G6PC promoter (AH-G6PC cells. Both beta-lactamase activity and endogenous G6PC mRNA were increased in AH-G6PC cells by a combination of dexamethasone and pCPT-cAMP, and reduced by insulin. A 4-gene High-Throughput-Genomics assay was developed to concomitantly measure G6PC and pyruvate-dehydrogenase-kinase-4 (PDK4 mRNA levels. Using this assay, we screened an siRNA library containing pooled siRNA targeting 6650 druggable genes and identified 614 hits that lowered G6PC expression without increasing PDK4 mRNA levels. Pathway analysis indicated that siRNA-mediated knockdown (KD of genes known to positively or negatively affect insulin signaling increased or decreased G6PC mRNA expression, respectively, thus validating our screening platform. A subset of 270 primary screen hits was selected and 149 hits were confirmed by target gene KD by pooled siRNA and 7 single siRNA for each gene to reduce G6PC expression in 4-gene HTG assay. Subsequently, pooled siRNA KD of 113 genes decreased PEPCK and/or PGC1alpha mRNA expression thereby demonstrating their role in regulating key gluconeogenic genes in addition to G6PC. Last, KD of 61 of the above 113 genes potentiated insulin-stimulated Akt phosphorylation, suggesting that they suppress gluconeogenic gene by enhancing insulin signaling. CONCLUSIONS/SIGNIFICANCE: These results support the proposition that the proteins encoded by the genes identified in

  19. Compensatory mutations cause excess of antagonistic epistasis in RNA secondary structure folding

    Directory of Open Access Journals (Sweden)

    Adami Christoph

    2003-02-01

    Full Text Available Background The rate at which fitness declines as an organism's genome accumulates random mutations is an important variable in several evolutionary theories. At an intuitive level, it might seem natural that random mutations should tend to interact synergistically, such that the rate of mean fitness decline accelerates as the number of random mutations is increased. However, in a number of recent studies, a prevalence of antagonistic epistasis (the tendency of multiple mutations to have a mitigating rather than reinforcing effect has been observed. Results We studied in silico the net amount and form of epistatic interactions in RNA secondary structure folding by measuring the fraction of neutral mutants as a function of mutational distance d. We found a clear prevalence of antagonistic epistasis in RNA secondary structure folding. By relating the fraction of neutral mutants at distance d to the average neutrality at distance d, we showed that this prevalence derives from the existence of many compensatory mutations at larger mutational distances. Conclusions Our findings imply that the average direction of epistasis in simple fitness landscapes is directly related to the density with which fitness peaks are distributed in these landscapes.

  20. Compensatory mutations cause excess of antagonistic epistasis in RNA secondary structure folding.

    Science.gov (United States)

    Wilke, Claus O; Lenski, Richard E; Adami, Christoph

    2003-02-05

    The rate at which fitness declines as an organism's genome accumulates random mutations is an important variable in several evolutionary theories. At an intuitive level, it might seem natural that random mutations should tend to interact synergistically, such that the rate of mean fitness decline accelerates as the number of random mutations is increased. However, in a number of recent studies, a prevalence of antagonistic epistasis (the tendency of multiple mutations to have a mitigating rather than reinforcing effect) has been observed. We studied in silico the net amount and form of epistatic interactions in RNA secondary structure folding by measuring the fraction of neutral mutants as a function of mutational distance d. We found a clear prevalence of antagonistic epistasis in RNA secondary structure folding. By relating the fraction of neutral mutants at distance d to the average neutrality at distance d, we showed that this prevalence derives from the existence of many compensatory mutations at larger mutational distances. Our findings imply that the average direction of epistasis in simple fitness landscapes is directly related to the density with which fitness peaks are distributed in these landscapes.

  1. Amelogenesis Imperfecta and Screening of Mutation in Amelogenin Gene

    Directory of Open Access Journals (Sweden)

    Fernanda Veronese Oliveira

    2014-01-01

    Full Text Available The aim of this study was to report the clinical findings and the screening of mutations of amelogenin gene of a 7-year-old boy with amelogenesis imperfecta (AI. The genomic DNA was extracted from saliva of patient and his family, followed by PCR and direct DNA sequencing. The c.261C>T mutation was found in samples of mother, father, and brother, but the mutation was not found in the sequence of the patient. This mutation is a silent mutation and a single-nucleotide polymorphism (rs2106416. Thus, it is suggested that the mutation found was not related to the clinical presence of AI. Further research is necessary to examine larger number of patients and genes related to AI.

  2. A dominant mutation in mediator of paramutation2, one of three second-largest subunits of a plant-specific RNA polymerase, disrupts multiple siRNA silencing processes.

    Science.gov (United States)

    Sidorenko, Lyudmila; Dorweiler, Jane E; Cigan, A Mark; Arteaga-Vazquez, Mario; Vyas, Meenal; Kermicle, Jerry; Jurcin, Diane; Brzeski, Jan; Cai, Yu; Chandler, Vicki L

    2009-11-01

    Paramutation involves homologous sequence communication that leads to meiotically heritable transcriptional silencing. We demonstrate that mop2 (mediator of paramutation2), which alters paramutation at multiple loci, encodes a gene similar to Arabidopsis NRPD2/E2, the second-largest subunit of plant-specific RNA polymerases IV and V. In Arabidopsis, Pol-IV and Pol-V play major roles in RNA-mediated silencing and a single second-largest subunit is shared between Pol-IV and Pol-V. Maize encodes three second-largest subunit genes: all three genes potentially encode full length proteins with highly conserved polymerase domains, and each are expressed in multiple overlapping tissues. The isolation of a recessive paramutation mutation in mop2 from a forward genetic screen suggests limited or no functional redundancy of these three genes. Potential alternative Pol-IV/Pol-V-like complexes could provide maize with a greater diversification of RNA-mediated transcriptional silencing machinery relative to Arabidopsis. Mop2-1 disrupts paramutation at multiple loci when heterozygous, whereas previously silenced alleles are only up-regulated when Mop2-1 is homozygous. The dramatic reduction in b1 tandem repeat siRNAs, but no disruption of silencing in Mop2-1 heterozygotes, suggests the major role for tandem repeat siRNAs is not to maintain silencing. Instead, we hypothesize the tandem repeat siRNAs mediate the establishment of the heritable silent state-a process fully disrupted in Mop2-1 heterozygotes. The dominant Mop2-1 mutation, which has a single nucleotide change in a domain highly conserved among all polymerases (E. coli to eukaryotes), disrupts both siRNA biogenesis (Pol-IV-like) and potentially processes downstream (Pol-V-like). These results suggest either the wild-type protein is a subunit in both complexes or the dominant mutant protein disrupts both complexes. Dominant mutations in the same domain in E. coli RNA polymerase suggest a model for Mop2-1 dominance

  3. Screening of Modified RNA duplexes

    DEFF Research Database (Denmark)

    Schyth, Brian Dall; Bramsen, Jesper Bertram; Kjems, Jørgen

    protection against a fish pathogenic virus. This protection corresponded with an interferon response in the fish. Here we use this fish model to screen siRNAs containing various chemical modifications of the RNA backbone for their antiviral activity, the overall aim being identification of an siRNA form......Because of sequence specific gene targeting activity siRNAs are regarded as promising active compounds in gene medicine. But one serious problem with delivering siRNAs as treatment is the now well-established non-specific activities of some RNA duplexes. Cellular reactions towards double stranded...... RNAs include the 2´-5´ oligoadenylate synthetase system, the protein kinase R, RIG-I and Toll-like receptor activated pathways all resulting in antiviral defence mechanism. We have previously shown that antiviral innate immune reactions against double stranded RNAs could be detected in vivo as partial...

  4. A double base change in alternate base pairs induced by ultraviolet irradiation in a glycine transfer RNA gene

    International Nuclear Information System (INIS)

    Coleman, R.D.; Dunst, R.W.; Hill, C.W.; Pennsylvania State Univ., Hershey

    1980-01-01

    The glyUsusub(AGA) mutation affects Escherichia coli tRNAsup(Gly)sub(GGG), changing it to an AGA missense suppressor tRNA. Sequence studies have shown that the mutation involves a double base substitution at the first and third positions of the tRNA anticodon, the result being a change in the anticodon from CCC to UCU. A system has been developed to facilitate the detection of this novel mutation, and we have shown that ultraviolet irradiation and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are effective in causing the double base change. A single observation of the mutation occuring spontaneously has been made also. The frequency of MNNG-induced glyUsusub(AGA) mutations is compatible with their being caused by two separate mutagenic events. The frequency of UV-induced glyUsusub(AGA) mutations, however, strongly suggests that the occurence of one base substitution strongly enhances the chance of finding the second substitution at the alternate position. In addition to the double change in the anticodon, the glyUsusub(AGA) tRNA differs from tRNAsup(gly)sub(GGG) in that it bears a modification of the A adjacent to the 3' position of the anticodon. Most likely, this modified base is N-[9-(β-D-ribofuranosyl)-purin-6-ylcarbamoyl] threonine. (orig.) 891 AJ/orig. 892 BRE [de

  5. Punctual mutations in 23S rRNA gene of clarithromycin-resistant Helicobacter pylori in Colombian populations.

    Science.gov (United States)

    Matta, Andrés Jenuer; Zambrano, Diana Carolina; Pazos, Alvaro Jairo

    2018-04-14

    To characterize punctual mutations in 23S rRNA gene of clarithromycin-resistant Helicobacter pylori ( H. pylori ) and determine their association with therapeutic failure. PCR products of 23S rRNA gene V domain of 74 H. pylori isolates; 34 resistant to clarithromycin (29 from a low-risk gastric cancer (GC) population: Tumaco-Colombia, and 5 from a high-risk population: Tuquerres-Colombia) and 40 from a susceptible population (28 from Tumaco and 12 from Túquerres) were sequenced using capillary electrophoresis. The concordance between mutations of V domain 23S rRNA gene of H. pylori and therapeutic failure was determined using the Kappa coefficient and McNemar's test was performed to determine the relationship between H. pylori mutations and clarithromycin resistance. 23S rRNA gene from H. pylori was amplified in 56/74 isolates, of which 25 were resistant to clarithromycin (20 from Tumaco and 5 from Túquerres, respectively). In 17 resistant isolates (13 from Tumaco and 4 from Túquerres) the following mutations were found: A1593T1, A1653G2, C1770T, C1954T1, and G1827C in isolates from Tumaco, and A2144G from Túquerres. The mutations T2183C, A2144G and C2196T in H. pylori isolates resistant to clarithromycin from Colombia are reported for the first time. No association between the H. pylori mutations and in vitro clarithromycin resistance was found. However, therapeutic failure of eradication treatment was associated with mutations of 23S rRNA gene in clarithromycin-resistant H. pylori ( κ = 0.71). The therapeutic failure of eradication treatment in the two populations from Colombia was associated with mutations of the 23S rRNA gene in clarithromycin-resistant H. pylori .

  6. High-throughput screening of effective siRNAs using luciferase-linked chimeric mRNA.

    Directory of Open Access Journals (Sweden)

    Shen Pang

    Full Text Available The use of siRNAs to knock down gene expression can potentially be an approach to treat various diseases. To avoid siRNA toxicity the less transcriptionally active H1 pol III promoter, rather than the U6 promoter, was proposed for siRNA expression. To identify highly efficacious siRNA sequences, extensive screening is required, since current computer programs may not render ideal results. Here, we used CCR5 gene silencing as a model to investigate a rapid and efficient screening approach. We constructed a chimeric luciferase-CCR5 gene for high-throughput screening of siRNA libraries. After screening approximately 900 shRNA clones, 12 siRNA sequences were identified. Sequence analysis demonstrated that most (11 of the 12 sequences of these siRNAs did not match those identified by available siRNA prediction algorithms. Significant inhibition of CCR5 in a T-lymphocyte cell line and primary T cells by these identified siRNAs was confirmed using the siRNA lentiviral vectors to infect these cells. The inhibition of CCR5 expression significantly protected cells from R5 HIV-1JRCSF infection. These results indicated that the high-throughput screening method allows efficient identification of siRNA sequences to inhibit the target genes at low levels of expression.

  7. Specific and Efficient Regression of Cancers Harboring KRAS Mutation by Targeted RNA Replacement.

    Science.gov (United States)

    Kim, Sung Jin; Kim, Ju Hyun; Yang, Bitna; Jeong, Jin-Sook; Lee, Seong-Wook

    2017-02-01

    Mutations in the KRAS gene, which persistently activate RAS function, are most frequently found in many types of human cancers. Here, we proposed and verified a new approach against cancers harboring the KRAS mutation with high cancer selectivity and efficient anti-cancer effects based on targeted RNA replacement. To this end, trans-splicing ribozymes from Tetrahymena group I intron were developed, which can specifically target and reprogram the mutant KRAS G12V transcript to induce therapeutic gene activity in cells. Adenoviral vectors containing the specific ribozymes with downstream suicide gene were constructed and then infection with the adenoviruses specifically downregulated KRAS G12V expression and killed KRAS G12V-harboring cancer cells additively upon pro-drug treatment, but it did not affect the growth of wild-type KRAS-expressing cells. Minimal liver toxicity was noted when the adenoviruses were administered systemically in vivo. Importantly, intratumoral injection of the adenoviruses with pro-drug treatment specifically and significantly impeded the growth of xenografted tumors harboring KRAS G12V through a trans-splicing reaction with the target RNA. In contrast, xenografted tumors harboring wild-type KRAS were not affected by the adenoviruses. Therefore, RNA replacement with a mutant KRAS-targeting trans-splicing ribozyme is a potentially useful therapeutic strategy to combat tumors harboring KRAS mutation. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

  8. Enzymic colorimetry-based DNA chip: a rapid and accurate assay for detecting mutations for clarithromycin resistance in the 23S rRNA gene of Helicobacter pylori.

    Science.gov (United States)

    Xuan, Shi-Hai; Zhou, Yu-Gui; Shao, Bo; Cui, Ya-Lin; Li, Jian; Yin, Hong-Bo; Song, Xiao-Ping; Cong, Hui; Jing, Feng-Xiang; Jin, Qing-Hui; Wang, Hui-Min; Zhou, Jie

    2009-11-01

    Macrolide drugs, such as clarithromycin (CAM), are a key component of many combination therapies used to eradicate Helicobacter pylori. However, resistance to CAM is increasing in H. pylori and is becoming a serious problem in H. pylori eradication therapy. CAM resistance in H. pylori is mostly due to point mutations (A2142G/C, A2143G) in the peptidyltransferase-encoding region of the 23S rRNA gene. In this study an enzymic colorimetry-based DNA chip was developed to analyse single-nucleotide polymorphisms of the 23S rRNA gene to determine the prevalence of mutations in CAM-related resistance in H. pylori-positive patients. The results of the colorimetric DNA chip were confirmed by direct DNA sequencing. In 63 samples, the incidence of the A2143G mutation was 17.46 % (11/63). The results of the colorimetric DNA chip were concordant with DNA sequencing in 96.83 % of results (61/63). The colorimetric DNA chip could detect wild-type and mutant signals at every site, even at a DNA concentration of 1.53 x 10(2) copies microl(-1). Thus, the colorimetric DNA chip is a reliable assay for rapid and accurate detection of mutations in the 23S rRNA gene of H. pylori that lead to CAM-related resistance, directly from gastric tissues.

  9. Mutational screening of the USH2A gene in Spanish USH patients reveals 23 novel pathogenic mutations

    Directory of Open Access Journals (Sweden)

    Diaz-Llopis Manuel

    2011-10-01

    Full Text Available Abstract Background Usher Syndrome type II (USH2 is an autosomal recessive disorder, characterized by moderate to severe hearing impairment and retinitis pigmentosa (RP. Among the three genes implicated, mutations in the USH2A gene account for 74-90% of the USH2 cases. Methods To identify the genetic cause of the disease and determine the frequency of USH2A mutations in a cohort of 88 unrelated USH Spanish patients, we carried out a mutation screening of the 72 coding exons of this gene by direct sequencing. Moreover, we performed functional minigene studies for those changes that were predicted to affect splicing. Results As a result, a total of 144 DNA sequence variants were identified. Based upon previous studies, allele frequencies, segregation analysis, bioinformatics' predictions and in vitro experiments, 37 variants (23 of them novel were classified as pathogenic mutations. Conclusions This report provide a wide spectrum of USH2A mutations and clinical features, including atypical Usher syndrome phenotypes resembling Usher syndrome type I. Considering only the patients clearly diagnosed with Usher syndrome type II, and results obtained in this and previous studies, we can state that mutations in USH2A are responsible for 76.1% of USH2 disease in patients of Spanish origin.

  10. MELAS syndrome associated with a new mitochondrial tRNA-Val gene mutation (m.1616A>G).

    Science.gov (United States)

    Toyoshima, Yuka; Tanaka, Yuji; Satomi, Kazuo

    2017-09-11

    We describe the case of a 40-year-old-man with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome, with cardiomyopathy and severe heart failure. He had a mitochondrial transfer RNA (tRNA) mutation (m.1616A>G) of the (tRNA-Val) gene, and it was not found in MELAS syndrome ever before. The presence of this newly observed tRNA-Val mutation (m.1616A>G) may induce multiple respiratory chain enzyme deficiencies and contribute to MELAS syndrome symptoms that are associated with mitochondrial DNA (mtDNA) mutations. We report that the pathognomonic symptom in MELAS syndrome caused by this newly observed mtDNA mutation may be rapid progression of cardiomyopathy and severe heart failure. © BMJ Publishing Group Ltd (unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  11. Mutation screening of the TP53 gene by temporal temperature gradient gel electrophoresis.

    Science.gov (United States)

    Sørlie, Therese; Johnsen, Hilde; Vu, Phuong; Lind, Guro Elisabeth; Lothe, Ragnhild; Børresen-Dale, Anne-Lise

    2005-01-01

    A protocol for detection of mutations in the TP53 gene using temporal temperature gradient gel electrophoresis (TTGE) is described. TTGE is a mutation detection technique that separates DNA fragments differing by single base pairs according to their melting properties in a denaturing gel. It is based on constant denaturing conditions in the gel combined with a temperature gradient during the electrophoretic run. This method combines some of the advantages of the related techniques denaturing gradient gel electrophoresis (DGGE) and constant denaturant gel electrophoresis (CDGE) and eliminates some of the problems. The result is a rapid and sensitive screening technique that is robust and easily set up in smaller laboratory environments.

  12. Mutation screening of the TP53 gene by temporal temperature gel electrophoresis (TTGE).

    Science.gov (United States)

    Sørlie, Therese; Johnsen, Hilde; Vu, Phuong; Lind, Guro Elisabeth; Lothe, Ragnhild; Børresen-Dale, Anne-Lise

    2014-01-01

    A protocol for detection of mutations in the TP53 gene using temporal temperature gradient electrophoresis (TTGE) is described. TTGE is a mutation detection technique that separates DNA fragments differing by single base pairs according to their melting properties in a denaturing gel. It is based on constant denaturing conditions in the gel combined with a temperature gradient during the electrophoretic run. This method combines some of the advantages of the related techniques, denaturing gradient gel electrophoresis and constant denaturant gel electrophoresis, and eliminates some of the problems. The result is a rapid and sensitive screening technique which is robust and easily set up in smaller laboratory environments.

  13. iScreen: Image-Based High-Content RNAi Screening Analysis Tools.

    Science.gov (United States)

    Zhong, Rui; Dong, Xiaonan; Levine, Beth; Xie, Yang; Xiao, Guanghua

    2015-09-01

    High-throughput RNA interference (RNAi) screening has opened up a path to investigating functional genomics in a genome-wide pattern. However, such studies are often restricted to assays that have a single readout format. Recently, advanced image technologies have been coupled with high-throughput RNAi screening to develop high-content screening, in which one or more cell image(s), instead of a single readout, were generated from each well. This image-based high-content screening technology has led to genome-wide functional annotation in a wider spectrum of biological research studies, as well as in drug and target discovery, so that complex cellular phenotypes can be measured in a multiparametric format. Despite these advances, data analysis and visualization tools are still largely lacking for these types of experiments. Therefore, we developed iScreen (image-Based High-content RNAi Screening Analysis Tool), an R package for the statistical modeling and visualization of image-based high-content RNAi screening. Two case studies were used to demonstrate the capability and efficiency of the iScreen package. iScreen is available for download on CRAN (http://cran.cnr.berkeley.edu/web/packages/iScreen/index.html). The user manual is also available as a supplementary document. © 2014 Society for Laboratory Automation and Screening.

  14. 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

  15. A fast, simple method for screening radiation susceptibility genes by RNA interference

    International Nuclear Information System (INIS)

    Tsuji, Atsushi B.; Sudo, Hitomi; Sugyo, Aya; Otsuki, Marika; Miyagishi, Makoto; Taira, Kazunari; Imai, Takashi; Harada, Yoshi-nobu

    2005-01-01

    Radiotherapy can cause unacceptable levels of damage to normal tissues in some cancer patients. To understand the molecular mechanisms underlying radiation-induced physiological responses, and to be able to predict the radiation susceptibility of normal tissues in individual patients, it is important to identify a comprehensive set of genes responsible for radiation susceptibility. We have developed a simple and rapid 96-well screening protocol using cell proliferation assays and RNA interference to identify genes associated with radiation susceptibility. We evaluated the performance of alamarBlue-, BrdU-, and sulforhodamine B-based cell proliferation assays using the 96-well format. Each proliferation assay detected the known radiation susceptibility gene, PRKDC. In a trial screen using 28 shRNA vectors, another known gene, CDKN1A, and one new radiation susceptibility gene, ATP5G3, were identified. Our results indicate that this method may be useful for large-scale screens designed to identify novel radiation susceptibility genes

  16. Carrier screening of RTEL1 mutations in the Ashkenazi Jewish population.

    Science.gov (United States)

    Fedick, A M; Shi, L; Jalas, C; Treff, N R; Ekstein, J; Kornreich, R; Edelmann, L; Mehta, L; Savage, S A

    2015-08-01

    Hoyeraal-Hreidarsson syndrome (HH) is a clinically severe variant of dyskeratosis congenita (DC), characterized by cerebellar hypoplasia, microcephaly, intrauterine growth retardation, and severe immunodeficiency in addition to features of DC. Germline mutations in the RTEL1 gene have recently been identified as causative of HH. In this study, the carrier frequency for five RTEL1 mutations that occurred in individuals of Ashkenazi Jewish descent was investigated in order to advise on including them in existing clinical mutation panels for this population. Our screening showed that the carrier frequency for c.3791G>A (p.R1264H) was higher than expected, 1% in the Ashkenazi Orthodox and 0.45% in the general Ashkenazi Jewish population. Haplotype analyses suggested the presence of a common founder. We recommend that the c.3791G>A RTEL1 mutation be considered for inclusion in carrier screening panels in the Ashkenazi population. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. Severe epilepsy as the major symptom of new mutations in the mitochondrial tRNA(Phe) gene.

    Science.gov (United States)

    Zsurka, G; Hampel, K G; Nelson, I; Jardel, C; Mirandola, S R; Sassen, R; Kornblum, C; Marcorelles, P; Lavoué, S; Lombès, A; Kunz, W S

    2010-02-09

    To present 2 families with maternally inherited severe epilepsy as the main symptom of mitochondrial disease due to point mutations at position 616 in the mitochondrial tRNA(Phe) (MT-TF) gene. Histologic stainings were performed on skeletal muscle slices from the 2 index patients. Oxidative phosphorylation activity was measured by oxygraphic and spectrophotometric methods. The patients' complete mitochondrial DNA (mtDNA) and the relevant mtDNA region in maternal relatives were sequenced. Muscle histology showed only decreased overall COX staining, while a combined respiratory chain defect, most severely affecting complex IV, was noted in both patients' skeletal muscle. Sequencing of the mtDNA revealed in both patients a mutation at position 616 in the MT-TF gene (T>C or T>G). These mutations disrupt a base pair in the anticodon stem at a highly conserved position. They were apparently homoplasmic in both patients, and had different heteroplasmy levels in the investigated maternal relatives. Deleterious mutations in the mitochondrial tRNA(Phe) may solely manifest with epilepsy when segregating to homoplasmy. They may be overlooked in the absence of lactate accumulation and typical mosaic mitochondrial defects in muscle.

  18. Screening women for cervical cancer carcinoma with a HPV mRNA test: first results from the Venice pilot program.

    Science.gov (United States)

    Maggino, Tiziano; Sciarrone, Rocco; Murer, Bruno; Dei Rossi, Maria Rosa; Fedato, Chiara; Maran, Michela; Lorio, Melania; Soldà, Marika; Zago, Fiorella; Giorgi Rossi, Paolo; Zorzi, Manuel

    2016-08-23

    HPV DNA-based screening is more effective than a Pap test in preventing cervical cancer, but the test is less specific. New HPV tests have been proposed for primary screening. The HPV mRNA test showed a similar or slightly lower sensitivity than the HPV DNA tests but with a higher specificity. We report the results of an organised HPV mRNA-based screening pilot program in Venice, Italy. From October 2011 to May 2014, women aged 25-64 years were invited to undergo a HPV mRNA test (Aptima). Those testing positive underwent cytological triage. Women with positive cytology were referred to colposcopy, whereas those with negative cytology were referred to repeat the HPV mRNA test 1 year later. The results of the HPV mRNA test program were compared with both the local historical cytology-based program and with four neighbouring DNA HPV-based pilot projects. Overall, 23 211 women underwent a HPV mRNA test. The age-standardised positivity rate was 7.0%, higher than in HPV DNA programs (6.8%; relative rate (RR) 1.11, 95% confidence interval (CI) 1.05-1.17). The total colposcopy referral was 5.1%, double than with cytology (2.6%; RR 2.02, 95% CI 1.82-2.25) but similar to the HPV DNA programs (4.8%; RR 1.02; 95% CI 0.96-1.08). The cervical intraepithelial neoplasia grade 2+ detection rate with HPV mRNA was greater than in the HPV DNA programs at baseline (RR 1.50; 95% CI 1.19-1.88) and not significantly lower at the 1-year repeat (RR 0.70; 95% CI 0.40-1.16). The overall RR was 1.29 (95% CI 1.05-1.59), which was much higher than with cytology (detection rate 5.5‰ vs 2.1‰; RR 2.50, 95% CI 1.76-3.62). A screening programme based on the HPV mRNA obtained results similar to those observed with the HPV DNA test. In routine screening programmes, even a limited increase in HPV prevalence may conceal the advantage represented by the higher specificity of HPV mRNA.

  19. A genetic screen for mutations affecting embryonic development in medaka fish (Oryzias latipes).

    Science.gov (United States)

    Loosli, F; Köster, R W; Carl, M; Kühnlein, R; Henrich, T; Mücke, M; Krone, A; Wittbrodt, J

    2000-10-01

    In a pilot screen, we assayed the efficiency of ethylnitrosourea (ENU) as a chemical mutagen to induce mutations that lead to early embryonic and larval lethal phenotypes in the Japanese medaka fish, Oryzias latipes. ENU acts as a very efficient mutagen inducing mutations at high rates in germ cells. Three repeated treatments of male fish in 3 mM ENU for 1 h results in locus specific mutation rates of 1.1-1.95 x10(-3). Mutagenized males were outcrossed to wild type females and the F1 offspring was used to establish F2 families. F2 siblings were intercrossed and the F3 progeny was scored 24, 48 and 72 h after fertilization for morphological alterations affecting eye development. The presented mutant phenotypes were identified using morphological criteria and occur during early developmental stages of medaka. They are stably inherited in a Mendelian fashion. The high efficiency of ENU to induce mutations in this pilot screen indicates that chemical mutagenesis and screening for morphologically visible phenotypes in medaka fish allows the genetic analysis of specific aspects of vertebrate development complementing the screens performed in other vertebrate model systems.

  20. A New Targeted CFTR Mutation Panel Based on Next-Generation Sequencing Technology.

    Science.gov (United States)

    Lucarelli, Marco; Porcaro, Luigi; Biffignandi, Alice; Costantino, Lucy; Giannone, Valentina; Alberti, Luisella; Bruno, Sabina Maria; Corbetta, Carlo; Torresani, Erminio; Colombo, Carla; Seia, Manuela

    2017-09-01

    Searching for mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) is a key step in the diagnosis of and neonatal and carrier screening for cystic fibrosis (CF), and it has implications for prognosis and personalized therapy. The large number of mutations and genetic and phenotypic variability make this search a complex task. Herein, we developed, validated, and tested a laboratory assay for an extended search for mutations in CFTR using a next-generation sequencing-based method, with a panel of 188 CFTR mutations customized for the Italian population. Overall, 1426 dried blood spots from neonatal screening, 402 genomic DNA samples from various origins, and 1138 genomic DNA samples from patients with CF were analyzed. The assay showed excellent analytical and diagnostic operative characteristics. We identified and experimentally validated 159 (of 188) CFTR mutations. The assay achieved detection rates of 95.0% and 95.6% in two large-scale case series of CF patients from central and northern Italy, respectively. These detection rates are among the highest reported so far with a genetic test for CF based on a mutation panel. This assay appears to be well suited for diagnostics, neonatal and carrier screening, and assisted reproduction, and it represents a considerable advantage in CF genetic counseling. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  1. Zebrafish usp39 mutation leads to rb1 mRNA splicing defect and pituitary lineage expansion.

    Directory of Open Access Journals (Sweden)

    Yesenia Ríos

    2011-01-01

    Full Text Available Loss of retinoblastoma (Rb tumor suppressor function is associated with human malignancies. Molecular and genetic mechanisms responsible for tumorigenic Rb downregulation are not fully defined. Through a forward genetic screen and positional cloning, we identified and characterized a zebrafish ubiquitin specific peptidase 39 (usp39 mutation, the yeast and human homolog of which encodes a component of RNA splicing machinery. Zebrafish usp39 mutants exhibit microcephaly and adenohypophyseal cell lineage expansion without apparent changes in major hypothalamic hormonal and regulatory signals. Gene expression profiling of usp39 mutants revealed decreased rb1 and increased e2f4, rbl2 (p130, and cdkn1a (p21 expression. Rb1 mRNA overexpression, or antisense morpholino knockdown of e2f4, partially reversed embryonic pituitary expansion in usp39 mutants. Analysis of pre-mRNA splicing status of critical cell cycle regulators showed misspliced Rb1 pre-mRNA resulting in a premature stop codon. These studies unravel a novel mechanism for rb1 regulation by a neuronal mRNA splicing factor, usp39. Zebrafish usp39 regulates embryonic pituitary homeostasis by targeting rb1 and e2f4 expression, respectively, contributing to increased adenohypophyseal sensitivity to these altered cell cycle regulators. These results provide a mechanism for dysregulated rb1 and e2f4 pathways that may result in pituitary tumorigenesis.

  2. Parkinson disease: α-synuclein mutational screening and new clinical insight into the p.E46K mutation.

    Science.gov (United States)

    Pimentel, Márcia M G; Rodrigues, Fabíola C; Leite, Marco Antônio A; Campos Júnior, Mário; Rosso, Ana Lucia; Nicaretta, Denise H; Pereira, João S; Silva, Delson José; Della Coletta, Marcus V; Vasconcellos, Luiz Felipe R; Abreu, Gabriella M; Dos Santos, Jussara M; Santos-Rebouças, Cíntia B

    2015-06-01

    Amongst Parkinson's disease-causing genetic factors, missense mutations and genomic multiplications in the gene encoding α-synuclein are well established causes of the disease, although genetic data in populations with a high degree of admixture, such as the Brazilian one, are still scarce. In this study, we conducted a molecular screening of α-synuclein point mutations and copy number variation in the largest cohort of Brazilian patients with Parkinson's disease (n = 549) and also in twelve Portuguese and one Bolivian immigrants. Genomic DNA was isolated from peripheral blood leukocytes or saliva, and the mutational screening was performed by quantitative and qualitative real-time PCR. The only alteration identified was the p.E46K mutation in a 60-year-old man, born in Bolivia, with a familial history of autosomal dominant Parkinson's disease. This is the second family ever reported, in which this rare pathogenic mutation is segregating. The same mutation was firstly described ten years ago in a Spanish family with a neurodegenerative syndrome combining parkinsonism, dementia and visual hallucinations. The clinical condition of our proband reveals a less aggressive phenotype than previously described and reinforces that marked phenotypic heterogeneity is common among patients with Parkinson's disease, even among those carriers sharing the same mutation. Our findings add new insight into the preexisting information about α-synuclein p.E46K, improving our understanding about the endophenotypes associated to this mutation and corroborate that missense alterations and multiplications in α-synuclein are uncommon among Brazilian patients with Parkinson's disease. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Mutation screening of the PCDH15 gene in Spanish patients with Usher syndrome type I.

    Science.gov (United States)

    Jaijo, Teresa; Oshima, Aki; Aller, Elena; Carney, Carol; Usami, Shin-ichi; Millán, José M; Kimberling, William J

    2012-01-01

    PCDH15 codes for protocadherin-15, a cell-cell adhesion protein essential in the morphogenesis and cohesion of stereocilia bundles and in the function or preservation of photoreceptor cells. Mutations in the PCDH15 gene are responsible for Usher syndrome type I (USH1F) and non-syndromic hearing loss (DFNB23). The purpose of this work was to perform PCDH15 mutation screening to identify the genetic cause of the disease in a cohort of Spanish patients with Usher syndrome type I and establish phenotype-genotype correlation. Mutation analysis of PCDH15 included additional exons recently identified and was performed by direct sequencing. The screening was performed in 19 probands with USH already screened for mutations in the most prevalent USH1 genes, myosin VIIA (MYO7A) and cadherin-23 (CDH23), and for copy number variants in PCDH15. Seven different point mutations, five novel, were detected. Including the large PCDH15 rearrangements previously reported in our cohort of patients, a total of seven of 19 patients (36.8%) were carriers of at least one pathogenic allele. Thirteen out of the 38 screened alleles carried pathogenic PCDH15 variants (34.2%). Five out of the seven point mutations reported in the present study are novel, supporting the idea that most PCDH15 mutations are private. Furthermore, no mutational hotspots have been identified. In most patients, detected mutations led to a truncated protein, reinforcing the hypothesis that severe mutations cause the Usher I phenotype and that missense variants are mainly responsible for non-syndromic hearing impairment.

  4. Mutations in conserved helix 69 of 23S rRNA of Thermus thermophilus that affect capreomycin resistance but not posttranscriptional modifications

    DEFF Research Database (Denmark)

    Monshupanee, Tanakarn; Gregory, Steven T; Douthwaite, Stephen

    2008-01-01

    of previously reported capreomycin resistance base substitutions. Capreomycin resistance in other bacteria has been shown to result from inactivation of the TlyA methyltransferase which 2'-O methylates C1920 of 23S rRNA. Inactivation of the tlyA gene in T. thermophilus does not affect its sensitivity...... for resistance to the tuberactinomycin antibiotic capreomycin. Two base substitutions, A1913U and mU1915G, and a single base deletion, DeltamU1915, were identified in helix 69 of 23S rRNA, a structural element that forms part of an interribosomal subunit bridge with the decoding center of 16S rRNA, the site...... to capreomycin. Finally, none of the mutations in helix 69 interferes with methylation at C1920 or with pseudouridylation at positions 1911 and 1917. We conclude that the resistance phenotype is a consequence of structural changes introduced by the mutations....

  5. Rapid screening for nuclear genes mutations in isolated respiratory chain complex I defects.

    Science.gov (United States)

    Pagniez-Mammeri, Hélène; Lombes, Anne; Brivet, Michèle; Ogier-de Baulny, Hélène; Landrieu, Pierre; Legrand, Alain; Slama, Abdelhamid

    2009-04-01

    Complex I or reduced nicotinamide adenine dinucleotide (NADH): ubiquinone oxydoreductase deficiency is the most common cause of respiratory chain defects. Molecular bases of complex I deficiencies are rarely identified because of the dual genetic origin of this multi-enzymatic complex (nuclear DNA and mitochondrial DNA) and the lack of phenotype-genotype correlation. We used a rapid method to screen patients with isolated complex I deficiencies for nuclear genes mutations by Surveyor nuclease digestion of cDNAs. Eight complex I nuclear genes, among the most frequently mutated (NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS7, NDUFS8, NDUFV1 and NDUFV2), were studied in 22 cDNA fragments spanning their coding sequences in 8 patients with a biochemically proved complex I deficiency. Single nucleotide polymorphisms and missense mutations were detected in 18.7% of the cDNA fragments by Surveyor nuclease treatment. Molecular defects were detected in 3 patients. Surveyor nuclease screening is a reliable method for genotyping nuclear complex I deficiencies, easy to interpret, and limits the number of sequence reactions. Its use will enhance the possibility of prenatal diagnosis and help us for a better understanding of complex I molecular defects.

  6. Screening of 99 Danish patients with congenital heart disease for GATA4 mutations

    DEFF Research Database (Denmark)

    Zhang, Litu; Tümer, Zeynep; Jacobsen, Joes Ramsøe

    2006-01-01

    stenosis. We have screened 99 unrelated Danish patients with different CHD phenotypes to evaluate the prevalence of GATA4 mutations in CHD. No pathogenic mutations were found among the patients, suggesting that GATA4 mutations are relatively rare among CHD patients. Thus, the diagnostic importance of GATA4...

  7. Plasma Screening for Progranulin Mutations in Patients with Progressive Supranuclear Palsy and Corticobasal Syndromes.

    Science.gov (United States)

    Galimberti, Daniela; Bertram, Kelly; Formica, Alessandra; Fenoglio, Chiara; Cioffi, Sara M G; Arighi, Andrea; Scarpini, Elio; Colosimo, Carlo

    2016-05-04

    Progranulin gene (GRN) mutations are characterized by heterogeneous presentations. Corticobasal syndrome (CBS) is often associated with GRN mutations, whereas association with progressive supranuclear palsy syndrome (PSPS) is rare. Plasma progranulin levels were evaluated in 34 patients, including 19 with PSPS, 12 with CBS, and 3 with mixed signs, with the purpose to screen for the presence of causal mutations, associated with low levels. We found undetectable levels in a patient with CBS. Sequencing confirmed the presence of the Thr272fs deletion. Progranulin mutation screening is suggested in cases of CBS, even in the absence of positive family history for dementia and/or movement disorders.

  8. High resolution melting curve analysis targeting the HBB gene mutational hot-spot offers a reliable screening approach for all common as well as most of the rare beta-globin gene mutations in Bangladesh.

    Science.gov (United States)

    Islam, Md Tarikul; Sarkar, Suprovath Kumar; Sultana, Nusrat; Begum, Mst Noorjahan; Bhuyan, Golam Sarower; Talukder, Shezote; Muraduzzaman, A K M; Alauddin, Md; Islam, Mohammad Sazzadul; Biswas, Pritha Promita; Biswas, Aparna; Qadri, Syeda Kashfi; Shirin, Tahmina; Banu, Bilquis; Sadya, Salma; Hussain, Manzoor; Sarwardi, Golam; Khan, Waqar Ahmed; Mannan, Mohammad Abdul; Shekhar, Hossain Uddin; Chowdhury, Emran Kabir; Sajib, Abu Ashfaqur; Akhteruzzaman, Sharif; Qadri, Syed Saleheen; Qadri, Firdausi; Mannoor, Kaiissar

    2018-01-02

    Bangladesh lies in the global thalassemia belt, which has a defined mutational hot-spot in the beta-globin gene. The high carrier frequencies of beta-thalassemia trait and hemoglobin E-trait in Bangladesh necessitate a reliable DNA-based carrier screening approach that could supplement the use of hematological and electrophoretic indices to overcome the barriers of carrier screening. With this view in mind, the study aimed to establish a high resolution melting (HRM) curve-based rapid and reliable mutation screening method targeting the mutational hot-spot of South Asian and Southeast Asian countries that encompasses exon-1 (c.1 - c.92), intron-1 (c.92 + 1 - c.92 + 130) and a portion of exon-2 (c.93 - c.217) of the HBB gene which harbors more than 95% of mutant alleles responsible for beta-thalassemia in Bangladesh. Our HRM approach could successfully differentiate ten beta-globin gene mutations, namely c.79G > A, c.92 + 5G > C, c.126_129delCTTT, c.27_28insG, c.46delT, c.47G > A, c.92G > C, c.92 + 130G > C, c.126delC and c.135delC in heterozygous states from the wild type alleles, implying the significance of the approach for carrier screening as the first three of these mutations account for ~85% of total mutant alleles in Bangladesh. Moreover, different combinations of compound heterozygous mutations were found to generate melt curves that were distinct from the wild type alleles and from one another. Based on the findings, sixteen reference samples were run in parallel to 41 unknown specimens to perform direct genotyping of the beta-thalassemia specimens using HRM. The HRM-based genotyping of the unknown specimens showed 100% consistency with the sequencing result. Targeting the mutational hot-spot, the HRM approach could be successfully applied for screening of beta-thalassemia carriers in Bangladesh as well as in other countries of South Asia and Southeast Asia. The approach could be a useful supplement of hematological and

  9. Benign and Deleterious Cystic Fibrosis Transmembrane Conductance Regulator Mutations Identified by Sequencing in Positive Cystic Fibrosis Newborn Screen Children from California.

    Science.gov (United States)

    Salinas, Danieli B; Sosnay, Patrick R; Azen, Colleen; Young, Suzanne; Raraigh, Karen S; Keens, Thomas G; Kharrazi, Martin

    2016-01-01

    Of the 2007 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) mutations, 202 have been assigned disease liability. California's racially diverse population, along with CFTR sequencing as part of newborn screening model, provides the opportunity to examine the phenotypes of children with uncategorized mutations to help inform disease liability and penetrance. We conducted a retrospective cohort study based on children screened from 2007 to 2011 and followed for two to six years. Newborns that screened positive were divided into three genotype groups: those with two CF-causing mutations (CF-C); those with one mutation of varying clinic consequence (VCC); and those with one mutation of unknown disease liability (Unknown). Sweat chloride tests, pancreatic sufficiency status, and Pseudomonas aeruginosa colonization were compared. Children with two CF-causing mutations had a classical CF phenotype, while 5% of VCC (4/78) and 11% of Unknown (27/244) met diagnostic criteria of CF. Children carrying Unknown mutations 2215insG with D836Y, and T1036N had early and classical CF phenotype, while others carrying 1525-42G>A, L320V, L967S, R170H, and 296+28A>G had a benign clinical presentation, suggesting that these are non-CF causing. While most infants with VCC and Unknown CFTR mutations do not meet diagnostic criteria for CF, a small proportion do. These findings highlight the range of genotypes and phenotypes in the first few years of life following CF newborn screening when CFTR sequencing is performed.

  10. Single base mutation in the proα2(I) collagen gene that causes efficient splicing of RNA from exon 27 to exon 29 and synthesis of a shortened but in-frame proα2(I) chain

    International Nuclear Information System (INIS)

    Tromp, G.; Prockop, D.J.

    1988-01-01

    Previous observations demonstrated that a lethal variant of osteogenesis imperfecta had two altered alleles for proα2(I) chains of type I procollagen. One mutation produced a nonfunctioning allele in that there was synthesis of mRNA but no detectable synthesis of proα2(I) chains from the allele. The mutation in the other allele caused synthesis of shortened proα2(I) chains that lacked most or all of the 18 amino acids encoded by exon 28. Subclones of the proα2(I) gene were prepared from the proband's DNA and the DNA sequence was determined for a 582-base-pair (bp) region that extended from the last 30 bp of intervening sequence 26 to the first 26 bp of intervening sequence 29. Data from six independent subclones demonstrated that all had the same sequence as a previously isolated normal clone for the proα2(I) gene except that four subclones had a single base mutation at the 3' end of intervening sequence 27. The mutation was a substitution of guanine for adenine that changed the universal consensus sequence for the 3' splicing site of RNA from -AG- to -GG-. S1 nuclease experiments demonstrated that about half the proα2(I) mRNA in the proband's fibroblasts was abnormally spliced and that the major species of abnormal proα2(I) mRNA was completely spliced from the last codon of exon 27 to the first codon of exon 29. The mutation is apparently unique among RNA splicing mutations of mammalian systems in producing a shortened polypeptide chain that is in-frame in terms of coding sequences, that is used in the subunit assembly of a protein, and that contributes to a lethal phenotype

  11. Mutation analysis for DJ-1 in sporadic and familial parkinsonism: screening strategy in parkinsonism.

    Science.gov (United States)

    Tomiyama, Hiroyuki; Li, Yuanzhe; Yoshino, Hiroyo; Mizuno, Yoshikuni; Kubo, Shin-Ichiro; Toda, Tatsushi; Hattori, Nobutaka

    2009-05-22

    DJ-1 mutations cause autosomal recessive parkinsonism (ARP). Although some reports of DJ-1 mutations have been published, there is lack of information on the prevalence of these mutations in large-scale studies of both familial and sporadic parkinsonism. In this genetic screening study, we analyzed the distribution and frequency of DJ-1 mutations by direct nucleotide sequencing of coding exons and exon-intron boundaries of DJ-1, in 386 parkin-negative parkinsonism patients (371 index cases: 67 probands of autosomal recessive parkinsonism families, 90 probands of autosomal dominant parkinsonism families, 201 patients with sporadic parkinsonism, and 13 with unknown family histories) from 12 countries (Japan 283, China 27, Taiwan 22, Korea 22, Israel 16, Turkey 5, Philippines 2, Bulgaria 2, Greece 2, Tunisia 1, USA 2, Ukraine 1, unknown 1). None had causative mutation in DJ-1, suggesting DJ-1 mutation is very rare among patients with familial and sporadic parkinsonism from Asian countries and those with other ethnic background. This is in contrast to the higher frequencies and worldwide distribution of parkin- and PINK1-related parkinsonism in ARP and sporadic parkinsonism. Thus, after obtaining clinical information, screening for mutations in (1) parkin, (2) PINK1, (3) DJ-1, (4) ATP13A2 should be conducted in that order, in ARP and sporadic parkinsonism, based on their reported frequencies. In addition, haplotype analysis should be employed to check for homozygosity of 1p36, which harbors a cluster of causative genes for ARP such as DJ-1, PINK1 and ATP13A2 in ARP and sporadic parkinsonism, especially in parkinsonism with consanguinity.

  12. A PCR-Based Method to Construct Lentiviral Vector Expressing Double Tough Decoy for miRNA Inhibition.

    Directory of Open Access Journals (Sweden)

    Huiling Qiu

    Full Text Available DNA vector-encoded Tough Decoy (TuD miRNA inhibitor is attracting increased attention due to its high efficiency in miRNA suppression. The current methods used to construct TuD vectors are based on synthesizing long oligonucleotides (~90 mer, which have been costly and problematic because of mutations during synthesis. In this study, we report a PCR-based method for the generation of double Tough Decoy (dTuD vector in which only two sets of shorter oligonucleotides (< 60 mer were used. Different approaches were employed to test the inhibitory potency of dTuDs. We demonstrated that dTuD is the most efficient method in miRNA inhibition in vitro and in vivo. Using this method, a mini dTuD library against 88 human miRNAs was constructed and used for a high-throughput screening (HTS of AP-1 pathway-related miRNAs. Seven miRNAs (miR-18b-5p, -101-3p, -148b-3p, -130b-3p, -186-3p, -187-3p and -1324 were identified as candidates involved in AP-1 pathway regulation. This novel method allows for an accurate and cost-effective generation of dTuD miRNA inhibitor, providing a powerful tool for efficient miRNA suppression in vitro and in vivo.

  13. Selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile, and accurate RNA structure analysis

    Science.gov (United States)

    Smola, Matthew J.; Rice, Greggory M.; Busan, Steven; Siegfried, Nathan A.; Weeks, Kevin M.

    2016-01-01

    SHAPE chemistries exploit small electrophilic reagents that react with the 2′-hydroxyl group to interrogate RNA structure at single-nucleotide resolution. Mutational profiling (MaP) identifies modified residues based on the ability of reverse transcriptase to misread a SHAPE-modified nucleotide and then counting the resulting mutations by massively parallel sequencing. The SHAPE-MaP approach measures the structure of large and transcriptome-wide systems as accurately as for simple model RNAs. This protocol describes the experimental steps, implemented over three days, required to perform SHAPE probing and construct multiplexed SHAPE-MaP libraries suitable for deep sequencing. These steps include RNA folding and SHAPE structure probing, mutational profiling by reverse transcription, library construction, and sequencing. Automated processing of MaP sequencing data is accomplished using two software packages. ShapeMapper converts raw sequencing files into mutational profiles, creates SHAPE reactivity plots, and provides useful troubleshooting information, often within an hour. SuperFold uses these data to model RNA secondary structures, identify regions with well-defined structures, and visualize probable and alternative helices, often in under a day. We illustrate these algorithms with the E. coli thiamine pyrophosphate riboswitch, E. coli 16S rRNA, and HIV-1 genomic RNAs. SHAPE-MaP can be used to make nucleotide-resolution biophysical measurements of individual RNA motifs, rare components of complex RNA ensembles, and entire transcriptomes. The straightforward MaP strategy greatly expands the number, length, and complexity of analyzable RNA structures. PMID:26426499

  14. Fluorescence-based high-throughput screening of dicer cleavage activity.

    Science.gov (United States)

    Podolska, Katerina; Sedlak, David; Bartunek, Petr; Svoboda, Petr

    2014-03-01

    Production of small RNAs by ribonuclease III Dicer is a key step in microRNA and RNA interference pathways, which employ Dicer-produced small RNAs as sequence-specific silencing guides. Further studies and manipulations of microRNA and RNA interference pathways would benefit from identification of small-molecule modulators. Here, we report a study of a fluorescence-based in vitro Dicer cleavage assay, which was adapted for high-throughput screening. The kinetic assay can be performed under single-turnover conditions (35 nM substrate and 70 nM Dicer) in a small volume (5 µL), which makes it suitable for high-throughput screening in a 1536-well format. As a proof of principle, a small library of bioactive compounds was analyzed, demonstrating potential of the assay.

  15. TMEM106B, the risk gene for frontotemporal dementia, is regulated by the miRNA-132/212 cluster and affects progranulin pathways

    Science.gov (United States)

    Chen-Plotkin, Alice S.; Unger, Travis L.; Gallagher, Michael D.; Bill, Emily; Kwong, Linda K.; Volpicelli-Daley, Laura; Busch, Johanna I.; Akle, Sebastian; Grossman, Murray; Van Deerlin, Vivianna; Trojanowski, John Q.; Lee, Virginia M.-Y.

    2012-01-01

    Frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) is a fatal neurodegenerative disease with no available treatments. Mutations in the progranulin gene (GRN) causing impaired production or secretion of progranulin are a common Mendelian cause of FTLD-TDP; additionally, common variants at chromosome 7p21 in the uncharacterized gene TMEM106B were recently linked by genome-wide association to FTLD-TDP with and without GRN mutations. Here we show that TMEM106B is neuronally expressed in postmortem human brain tissue, and that expression levels are increased in FTLD-TDP brain. Furthermore, using an unbiased, microarray-based screen of over 800 microRNAs, we identify microRNA-132 as the top microRNA differentiating FTLD-TDP and control brains, with progranulin in late endo-lysosomes, and TMEM106B over-expression increases intracellular levels of progranulin. Thus, TMEM106B is an FTLD-TDP risk gene, with microRNA-132/212 depression as an event which can lead to aberrant over-expression of TMEM106B, which in turn alters progranulin pathways. Evidence for this pathogenic cascade includes the striking convergence of two independent, genomic-scale screens on a microRNA:mRNA regulatory pair. Our findings open novel directions for elucidating miRNA-based therapies in FTLD-TDP. PMID:22895706

  16. Random small interfering RNA library screen identifies siRNAs that induce human erythroleukemia cell differentiation.

    Science.gov (United States)

    Fan, Cuiqing; Xiong, Yuan; Zhu, Ning; Lu, Yabin; Zhang, Jiewen; Wang, Song; Liang, Zicai; Shen, Yan; Chen, Meihong

    2011-03-01

    Cancers are characterized by poor differentiation. Differentiation therapy is a strategy to alleviate malignant phenotypes by inducing cancer cell differentiation. Here we carried out a combinatorial high-throughput screen with a random siRNA library on human erythroleukemia K-562 cell differentiation. Two siRNAs screened from the library were validated to be able to induce erythroid differentiation to varying degrees, determined by CD235 and globin up-regulation, GATA-2 down-regulation, and cell growth inhibition. The screen we performed here is the first trial of screening cancer differentiation-inducing agents from a random siRNA library, demonstrating that a random siRNA library can be considered as a new resource in efforts to seek new therapeutic agents for cancers. As a random siRNA library has a broad coverage for the entire genome, including known/unknown genes and protein coding/non-coding sequences, screening using a random siRNA library can be expected to greatly augment the repertoire of therapeutic siRNAs for cancers.

  17. Axonal Transport of TDP-43 mRNA Granules Is Impaired by ALS-Causing Mutations

    OpenAIRE

    Alami, Nael H.; Smith, Rebecca B.; 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

    2014-01-01

    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. ...

  18. A Mutation in the Bacillus subtilis rsbU Gene That Limits RNA Synthesis during Sporulation.

    Science.gov (United States)

    Rothstein, David M; Lazinski, David; Osburne, Marcia S; Sonenshein, Abraham L

    2017-07-15

    Mutants of Bacillis subtilis that are temperature sensitive for RNA synthesis during sporulation were isolated after selection with a 32 P suicide agent. Whole-genome sequencing revealed that two of the mutants carried an identical lesion in the rsbU gene, which encodes a phosphatase that indirectly activates SigB, the stress-responsive RNA polymerase sigma factor. The mutation appeared to cause RsbU to be hyperactive, because the mutants were more resistant than the parent strain to ethanol stress. In support of this hypothesis, pseudorevertants that regained wild-type levels of sporulation at high temperature had secondary mutations that prevented expression of the mutant rsbU gene. The properties of these RsbU mutants support the idea that activation of SigB diminishes the bacterium's ability to sporulate. IMPORTANCE Most bacterial species encode multiple RNA polymerase promoter recognition subunits (sigma factors). Each sigma factor directs RNA polymerase to different sets of genes; each gene set typically encodes proteins important for responses to specific environmental conditions, such as changes in temperature, salt concentration, and nutrient availability. A selection for mutants of Bacillus subtilis that are temperature sensitive for RNA synthesis during sporulation unexpectedly yielded strains with a point mutation in rsbU , a gene that encodes a protein that normally activates sigma factor B (SigB) under conditions of salt stress. The mutation appears to cause RsbU, and therefore SigB, to be active inappropriately, thereby inhibiting, directly or indirectly, the ability of the cells to transcribe sporulation genes. Copyright © 2017 American Society for Microbiology.

  19. Benign and Deleterious Cystic Fibrosis Transmembrane Conductance Regulator Mutations Identified by Sequencing in Positive Cystic Fibrosis Newborn Screen Children from California.

    Directory of Open Access Journals (Sweden)

    Danieli B Salinas

    Full Text Available Of the 2007 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR mutations, 202 have been assigned disease liability. California's racially diverse population, along with CFTR sequencing as part of newborn screening model, provides the opportunity to examine the phenotypes of children with uncategorized mutations to help inform disease liability and penetrance.We conducted a retrospective cohort study based on children screened from 2007 to 2011 and followed for two to six years. Newborns that screened positive were divided into three genotype groups: those with two CF-causing mutations (CF-C; those with one mutation of varying clinic consequence (VCC; and those with one mutation of unknown disease liability (Unknown. Sweat chloride tests, pancreatic sufficiency status, and Pseudomonas aeruginosa colonization were compared.Children with two CF-causing mutations had a classical CF phenotype, while 5% of VCC (4/78 and 11% of Unknown (27/244 met diagnostic criteria of CF. Children carrying Unknown mutations 2215insG with D836Y, and T1036N had early and classical CF phenotype, while others carrying 1525-42G>A, L320V, L967S, R170H, and 296+28A>G had a benign clinical presentation, suggesting that these are non-CF causing.While most infants with VCC and Unknown CFTR mutations do not meet diagnostic criteria for CF, a small proportion do. These findings highlight the range of genotypes and phenotypes in the first few years of life following CF newborn screening when CFTR sequencing is performed.

  20. Infantile presentation of the mtDNA A3243G tRNA(Leu (UUR)) mutation.

    NARCIS (Netherlands)

    Okhuijsen-Kroes, E.J.; Trijbels, J.M.F.; Sengers, R.C.A.; Mariman, E.C.M.; Heuvel, L.P.W.J. van den; Wendel, U.A.H.; Koch, G.; Smeitink, J.A.M.

    2001-01-01

    Mitochondrial DNA (mtDNA) disorders are clinically very heterogeneous, ranging from single organ involvement to severe multisystem disease. One of the most frequently observed mtDNA mutations is the A-to-G transition at position 3243 of the tRNA(Leu (UUR)) gene. This mutation is often related to

  1. Effect of mutations in the A site of 16 S rRNA on aminoglycoside antibiotic-ribosome interaction

    DEFF Research Database (Denmark)

    Recht, M I; Douthwaite, S; Dahlquist, K D

    1999-01-01

    antibiotics, which also interact with this region of rRNA. Mutations of certain nucleotides in rRNA reduce aminoglycoside binding affinity, as previously demonstrated using a model RNA oligonucleotide system. Here, predictions from the oligonucleotide system were tested in the ribosome by mutation...... for the aminoglycoside paromomycin, whereas no discernible reduction in affinity was observed with 1406 mutant ribosomes. These data are consistent with prior NMR structural determination of aminoglycoside interaction with the decoding region, and further our understanding of how aminoglycoside resistance can...

  2. Screening of 1331 Danish breast and/or ovarian cancer families identified 40 novel BRCA1 and BRCA2 mutations

    DEFF Research Database (Denmark)

    Hansen, Thomas V O; Jønson, Lars; Steffensen, Ane Y

    2011-01-01

    Germ-line mutations in the tumour suppressor genes BRCA1 and BRCA2 predispose to breast and ovarian cancer. Since 1999 we have performed mutational screening of breast and/or ovarian cancer patients in East Denmark. During this period we have identified 40 novel sequence variations in BRCA1...... and BRCA2 in high risk breast and/or ovarian cancer families. The mutations were detected via pre-screening using dHPLC or high-resolution melting and direct sequencing. We identified 16 variants in BRCA1, including 9 deleterious frame-shift mutations, 2 intronic variants, 4 missense mutations, and 1......, the presumed significance of the missense mutations was predicted in silico using the align GVGD algorithm. In conclusion, the mutation screening identified 40 novel variants in the BRCA1 and BRCA2 genes and thereby extends the knowledge of the BRCA1/BRCA2 mutation spectrum. Nineteen of the mutations were...

  3. Identification of ALK germline mutation (3605delG) in pediatric anaplastic medulloblastoma.

    Science.gov (United States)

    Coco, Simona; De Mariano, Marilena; Valdora, Francesca; Servidei, Tiziana; Ridola, Vita; Andolfo, Immacolata; Oberthuer, André; Tonini, Gian Paolo; Longo, Luca

    2012-10-01

    The anaplastic lymphoma kinase (ALK) gene has been found either rearranged or mutated in several neoplasms such as anaplastic large-cell lymphoma, non-small-cell lung cancer, neuroblastoma and anaplastic thyroid cancer. Medulloblastoma (MB) is an embryonic pediatric cancer arising from nervous system, a tissue in which ALK is expressed during embryonic development. We performed an ALK mutation screening in 52 MBs and we found a novel heterozygous germline deletion of a single base in exon 23 (3605delG) in a case with marked anaplasia. This G deletion results in a frameshift mutation producing a premature stop codon in exon 25 of ALK tyrosine kinase domain. We also screened three human MB cell lines without finding any mutation of ALK gene. Quantitative expression analysis of 16 out of 52 samples showed overexpression of ALK mRNA in three MBs. In the present study, we report the first mutation of ALK found in MB. Moreover, a deletion of ALK gene producing a stop codon has not been detected in human tumors up to now. Further investigations are now required to elucidate whether the truncated form of ALK may have a role in signal transduction.

  4. RNAmutants: a web server to explore the mutational landscape of RNA secondary structures

    Science.gov (United States)

    Waldispühl, Jerome; Devadas, Srinivas; Berger, Bonnie; Clote, Peter

    2009-01-01

    The history and mechanism of molecular evolution in DNA have been greatly elucidated by contributions from genetics, probability theory and bioinformatics—indeed, mathematical developments such as Kimura's neutral theory, Kingman's coalescent theory and efficient software such as BLAST, ClustalW, Phylip, etc., provide the foundation for modern population genetics. In contrast to DNA, the function of most noncoding RNA depends on tertiary structure, experimentally known to be largely determined by secondary structure, for which dynamic programming can efficiently compute the minimum free energy secondary structure. For this reason, understanding the effect of pointwise mutations in RNA secondary structure could reveal fundamental properties of structural RNA molecules and improve our understanding of molecular evolution of RNA. The web server RNAmutants provides several efficient tools to compute the ensemble of low-energy secondary structures for all k-mutants of a given RNA sequence, where k is bounded by a user-specified upper bound. As we have previously shown, these tools can be used to predict putative deleterious mutations and to analyze regulatory sequences from the hepatitis C and human immunodeficiency genomes. Web server is available at http://bioinformatics.bc.edu/clotelab/RNAmutants/, and downloadable binaries at http://rnamutants.csail.mit.edu/. PMID:19531740

  5. GUItars: a GUI tool for analysis of high-throughput RNA interference screening data.

    Directory of Open Access Journals (Sweden)

    Asli N Goktug

    Full Text Available High-throughput RNA interference (RNAi screening has become a widely used approach to elucidating gene functions. However, analysis and annotation of large data sets generated from these screens has been a challenge for researchers without a programming background. Over the years, numerous data analysis methods were produced for plate quality control and hit selection and implemented by a few open-access software packages. Recently, strictly standardized mean difference (SSMD has become a widely used method for RNAi screening analysis mainly due to its better control of false negative and false positive rates and its ability to quantify RNAi effects with a statistical basis. We have developed GUItars to enable researchers without a programming background to use SSMD as both a plate quality and a hit selection metric to analyze large data sets.The software is accompanied by an intuitive graphical user interface for easy and rapid analysis workflow. SSMD analysis methods have been provided to the users along with traditionally-used z-score, normalized percent activity, and t-test methods for hit selection. GUItars is capable of analyzing large-scale data sets from screens with or without replicates. The software is designed to automatically generate and save numerous graphical outputs known to be among the most informative high-throughput data visualization tools capturing plate-wise and screen-wise performances. Graphical outputs are also written in HTML format for easy access, and a comprehensive summary of screening results is written into tab-delimited output files.With GUItars, we demonstrated robust SSMD-based analysis workflow on a 3840-gene small interfering RNA (siRNA library and identified 200 siRNAs that increased and 150 siRNAs that decreased the assay activities with moderate to stronger effects. GUItars enables rapid analysis and illustration of data from large- or small-scale RNAi screens using SSMD and other traditional analysis

  6. Mitochondrial 12S rRNA A827G mutation is involved in the genetic susceptibility to aminoglycoside ototoxicity

    International Nuclear Information System (INIS)

    Xing Guangqian; Chen Zhibin; Wei Qinjun; Tian Huiqin; Li Xiaolu; Zhou Aidong; Bu Xingkuan; Cao Xin

    2006-01-01

    We have analyzed the clinical and molecular characterization of a Chinese family with aminoglycoside-induced and non-syndromic hearing impairment. Clinical evaluations revealed that only those family members who had a history of exposure to aminoglycoside antibiotics subsequently developed hearing loss, suggesting mitochondrial genome involvement. Sequence analysis of the mitochondrial 12S rRNA and tRNA Ser(UCN) genes led to the identification of a homoplasmic A827G mutation in all maternal relatives, a mutation that was identified previously in a few sporadic patients and in another Chinese family with non-syndromic deafness. The pathogenicity of the A827G mutation is strongly supported by the occurrence of the same mutation in two independent families and several genetically unrelated subjects. The A827G mutation is located at the A-site of the mitochondrial 12S rRNA gene which is highly conserved in mammals. It is possible that the alteration of the tertiary or quaternary structure of this rRNA by the A827G mutation may lead to mitochondrial dysfunction, thereby playing a role in the pathogenesis of hearing loss and aminoglycoside hypersensitivity. However, incomplete penetrance of hearing impairment indicates that the A827G mutation itself is not sufficient to produce clinical phenotype but requires the involvement of modifier factors for the phenotypic expression. Indeed, aminoglycosides may contribute to the phenotypic manifestation of the A827G mutation in this family. In contrast with the congenital or early-onset hearing impairment in another Chinese family carrying the A827G mutation, three patients in this pedigree developed hearing loss only after use of aminoglycosides. This discrepancy likely reflects the difference of genetic backgrounds, either mitochondrial haplotypes or nuclear modifier genes, between two families

  7. No efficacy of annual gynaecological screening in BRCA1/2 mutation carriers; an observational follow-up study

    NARCIS (Netherlands)

    Hermsen, B. B. J.; Olivier, R. I.; Verheijen, R. H. M.; van Beurden, M.; de Hullu, J. A.; Massuger, L. F.; Burger, C. W.; Brekelmans, C. T.; Mourits, M. J.; de Bock, G. H.; Gaarenstroom, K. N.; van Boven, H. H.; Mooij, T. M.; Rookus, M. A.

    2007-01-01

    BRCA1/ 2 mutation carriers are offered gynaecological screening with the intention to reduce mortality by detecting ovarian cancer at an early stage. We examined compliance and efficacy of gynaecological screening in BRCA1/ 2 mutation carriers. In this multicentre, observational, follow-up study we

  8. Mitochondrial phenylalanyl-tRNA synthetase mutations underlie fatal infantile Alpers encephalopathy

    DEFF Research Database (Denmark)

    Elo, Jenni M; Yadavalli, Srujana S; Euro, Liliya

    2012-01-01

    was impaired. Our results imply that the three FARS2 mutations directly impair aminoacylation function and stability of mtPheRS, leading to a decrease in overall tRNA charging capacity. This study establishes a new genetic cause of infantile mitochondrial Alpers encephalopathy and reports a new mitochondrial...

  9. Switching off small RNA regulation with trap-mRNA

    DEFF Research Database (Denmark)

    Overgaard, Martin; Johansen, Jesper; Møller-Jensen, Jakob

    2009-01-01

    to operate at the level of transcription initiation. By employing a highly sensitive genetic screen we uncovered a novel RNA-based regulatory principle in which induction of a trap-mRNA leads to selective degradation of a small regulatory RNA molecule, thereby abolishing the sRNA-based silencing of its...

  10. iDoRNA: An Interacting Domain-based Tool for Designing RNA-RNA Interaction Systems

    Directory of Open Access Journals (Sweden)

    Jittrawan Thaiprasit

    2016-03-01

    Full Text Available RNA-RNA interactions play a crucial role in gene regulation in living organisms. They have gained increasing interest in the field of synthetic biology because of their potential applications in medicine and biotechnology. However, few novel regulators based on RNA-RNA interactions with desired structures and functions have been developed due to the challenges of developing design tools. Recently, we proposed a novel tool, called iDoDe, for designing RNA-RNA interacting sequences by first decomposing RNA structures into interacting domains and then designing each domain using a stochastic algorithm. However, iDoDe did not provide an optimal solution because it still lacks a mechanism to optimize the design. In this work, we have further developed the tool by incorporating a genetic algorithm (GA to find an RNA solution with maximized structural similarity and minimized hybridized RNA energy, and renamed the tool iDoRNA. A set of suitable parameters for the genetic algorithm were determined and found to be a weighting factor of 0.7, a crossover rate of 0.9, a mutation rate of 0.1, and the number of individuals per population set to 8. We demonstrated the performance of iDoRNA in comparison with iDoDe by using six RNA-RNA interaction models. It was found that iDoRNA could efficiently generate all models of interacting RNAs with far more accuracy and required far less computational time than iDoDe. Moreover, we compared the design performance of our tool against existing design tools using forty-four RNA-RNA interaction models. The results showed that the performance of iDoRNA is better than RiboMaker when considering the ensemble defect, the fitness score and computation time usage. However, it appears that iDoRNA is outperformed by NUPACK and RNAiFold 2.0 when considering the ensemble defect. Nevertheless, iDoRNA can still be an useful alternative tool for designing novel RNA-RNA interactions in synthetic biology research. The source code of iDoRNA

  11. A Panel of High Resolution Melting (HRM Technology-Based Assays with Direct Sequencing Possibility for Effective Mutation Screening of EGFR and K-ras Genes

    Directory of Open Access Journals (Sweden)

    D. A. M. Heideman

    2009-01-01

    Full Text Available Background: Increasing data from clinical trials support EGFR and K-ras mutation status as predictive markers of tumour response to EGFR-targeted therapies. Consequently, rapid and reliable mutation screening assays are demanded to guide rational use of EGFR-targeted therapies.

  12. Mitochondrial DNA mutation screening of male patients with obstructive sleep apnea-hypopnea syndrome.

    Science.gov (United States)

    Huang, Xiao-Ying; Li, Hong; Xu, Xiao-Mei; Wang, Liang-Xing

    2014-08-01

    The aim of the present study was to analyze the differences between the genes of the mitochondrial DNA (mtDNA) displacement loop (D-loop) region and the Cambridge Reference sequence, in order to screen the mutation sites and investigate the correlation between mutations, clinical parameters and complications associated with obstructive sleep apnea-hypopnea syndrome (OSAHS). mtDNA was obtained from male patients with OSAHS in the Zhejiang Province. In total, 60 male patients with OSAHS and 102 healthy adults were assessed to determine the levels of fasting blood glucose, total cholesterol, triglyceride (TG) and high-density and low-density lipoproteins (LDL). Furthermore, peripheral mtDNA was extracted and bidirectional sequencing was conducted to enable mutation screening. In the mtDNA D-loop region, 178 mutation sites were identified, of which 115 sites were present in the two groups. The number of non-common sites in the OSAHS group was significantly higher compared with the control group (P0.05). A total of 21 cases in the severe OSAHS group exhibited mutation rates of >10%. In the control group, there were 24 cases where the np73A-G and np263A-G mutations were predominant. The np303-np315 region was identified to be the highly variable region and various mutation forms were observed. Statistically significant differences were observed in the neck perimeter, TG and LDL levels among the OSAHS-no-mutation subgroups (P<0.05) and LDL was shown to be associated with an mtDNA mutation in the OSAHS group. Numerous polymorphic mutation sites were identified in the mtDNA D-loop region of the OSAHS group. Therefore, mtDNA mutation sites may be closely associated with the clinical manifestations and complications of OSAHS.

  13. Mutation of the human mitochondrial phenylalanine-tRNA synthetase causes infantile-onset epilepsy and cytochrome c oxidase deficiency.

    Science.gov (United States)

    Almalki, Abdulraheem; Alston, Charlotte L; Parker, Alasdair; Simonic, Ingrid; Mehta, Sarju G; He, Langping; Reza, Mojgan; Oliveira, Jorge M A; Lightowlers, Robert N; McFarland, Robert; Taylor, Robert W; Chrzanowska-Lightowlers, Zofia M A

    2014-01-01

    Mitochondrial aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in protein synthesis since they charge tRNAs with their cognate amino acids. Mutations in the genes encoding mitochondrial aaRSs have been associated with a wide spectrum of human mitochondrial diseases. Here we report the identification of pathogenic mutations (a partial genomic deletion and a highly conserved p. Asp325Tyr missense variant) in FARS2, the gene encoding mitochondrial phenylalanyl-tRNA synthetase, in a patient with early-onset epilepsy and isolated complex IV deficiency in muscle. The biochemical defect was expressed in myoblasts but not in fibroblasts and associated with decreased steady state levels of COXI and COXII protein and reduced steady state levels of the mt-tRNA(Phe) transcript. Functional analysis of the recombinant mutant p. Asp325Tyr FARS2 protein showed an inability to bind ATP and consequently undetectable aminoacylation activity using either bacterial tRNA or human mt-tRNA(Phe) as substrates. Lentiviral transduction of cells with wildtype FARS2 restored complex IV protein levels, confirming that the p.Asp325Tyr mutation is pathogenic, causing respiratory chain deficiency and neurological deficits on account of defective aminoacylation of mt-tRNA(Phe). © 2013. Published by Elsevier B.V. All rights reserved.

  14. A Phenotypic Screen for Functional Mutants of Human Adenosine Deaminase Acting on RNA 1.

    Science.gov (United States)

    Wang, Yuru; Havel, Jocelyn; Beal, Peter A

    2015-11-20

    Adenosine deaminases acting on RNA (ADARs) are RNA-editing enzymes responsible for the conversion of adenosine to inosine at specific locations in cellular RNAs. ADAR1 and ADAR2 are two members of the family that have been shown to be catalytically active. Earlier, we reported a phenotypic screen for the study of human ADAR2 using budding yeast S. cerevisiae as the host system. While this screen has been successfully applied to the study of ADAR2, it failed with ADAR1. Here, we report a new reporter that uses a novel editing substrate and is suitable for the study of ADAR1. We screened plasmid libraries with randomized codons for two important residues in human ADAR1 (G1007 and E1008). The screening results combined with in vitro deamination assays led to the identification of mutants that are more active than the wild type protein. Furthermore, a screen of the ADAR1 E1008X library with a reporter construct bearing an A•G mismatch at the editing site suggests one role for the residue at position 1008 is to sense the identity of the base pairing partner for the editing site adenosine. This work has provided a starting point for future in vitro evolution studies of ADAR1 and led to new insight into ADAR's editing site selectivity.

  15. RPE65 gene: multiplex PCR and mutation screening in patients from ...

    Indian Academy of Sciences (India)

    Unknown

    The RPE65 protein is believed to play an important role in the metabolism of vitamin A in the ... PCR and mutation screening in patients from India with retinal degenerative diseases. ..... Bennett J. 2001 Gene therapy restores vision in a canine.

  16. RNA-dependent RNA polymerase: Addressing Zika outbreak by a phylogeny-based drug target study.

    Science.gov (United States)

    Stephen, Preyesh; Lin, Sheng-Xiang

    2018-01-01

    Since the first major outbreak of Zika virus (ZIKV) in 2007, ZIKV is spreading explosively through South and Central America, and recent reports in highly populated developing countries alarm the possibility of a more catastrophic outbreak. ZIKV infection in pregnant women leads to embryonic microcephaly and Guillain-Barré syndrome in adults. At present, there is limited understanding of the infectious mechanism, and no approved therapy has been reported. Despite the withdrawal of public health emergency, the WHO still considers the ZIKV as a highly significant and long-term public health challenge that the situation has to be addressed rapidly. Non-structural protein 5 is essential for capping and replication of viral RNA and comprises a methyltransferase and RNA-dependent RNA polymerase (RdRp) domain. We used molecular modeling to obtain the structure of ZIKV RdRp, and by molecular docking and phylogeny analysis, we here demonstrate the potential sites for drug screening. Two metal binding sites and an NS3-interacting region in ZIKV RdRp are demonstrated as potential drug screening sites. The docked structures reveal a remarkable degree of conservation at the substrate binding site and the potential drug screening sites. A phylogeny-based approach is provided for an emergency preparedness, where similar class of ligands could target phylogenetically related proteins. © 2017 John Wiley & Sons A/S.

  17. Detection of Deafness-Causing Mutations in the Greek Mitochondrial Genome

    Directory of Open Access Journals (Sweden)

    Haris Kokotas

    2011-01-01

    Full Text Available Mitochondrion harbors its own DNA, known as mtDNA, encoding certain essential components of the mitochondrial respiratory chain and protein synthesis apparatus. mtDNA mutations have an impact on cellular ATP production and many of them are undoubtedly a factor that contributes to sensorineural deafness, including both syndromic and non-syndromic forms. Hot spot regions for deafness mutations are the MTRNR1 gene, encoding the 12S rRNA, the MTTS1 gene, encoding the tRNA for Ser(UCN, and the MTTL1 gene, encoding the tRNA for Leu(UUR. We investigated the impact of mtDNA mutations in the Greek hearing impaired population, by testing a cohort of 513 patients suffering from childhood onset prelingual or postlingual, bilateral, sensorineural, syndromic or non-syndromic hearing loss of any degree for six mitochondrial variants previously associated with deafness. Screening involved the MTRNR1 961delT/insC and A1555G mutations, the MTTL1 A3243G mutation, and the MTTS1 A7445G, 7472insC and T7510C mutations. Although two patients were tested positive for the A1555G mutation, we failed to identify any subject carrying the 961delT/insC, A3243G, A7445G, 7472insC, or T7510C mutations. Our findings strongly support our previously raised conclusion that mtDNA mutations are not a major risk factor for sensorineural deafness in the Greek population.

  18. A novel glutamine–RNA interaction identified by screening libraries in mammalian cells

    OpenAIRE

    Tan, Ruoying; Frankel, Alan D.

    1998-01-01

    The arginine-rich motif provides a versatile framework for RNA recognition in which few amino acids other than arginine are needed to mediate specific binding. Using a mammalian screening system based on transcriptional activation by HIV Tat, we identified novel arginine-rich peptides from combinatorial libraries that bind tightly to the Rev response element of HIV. Remarkably, a single glutamine, but not asparagine, within a stretch of polyarginine can mediate high-affinity binding. These re...

  19. Screening for mutations in the androgen receptor gene (AR) causing infertility in Syrian men using real-time PCR

    International Nuclear Information System (INIS)

    Madania, A.; Ghouri, I.; Abou-Alshamat, Gh.; Issa, M.; Al-Halabi, M.

    2012-01-01

    14 known point mutations in the androgen receptor gene (AR) causing male infertility were screened by real time PCR and by DNA sequencing, in order to identify point mutations in the AR gene causing infertility in azoospermic men. We screened 110 Syrian patients suffering from non-obstructive azoospermia with no chromosomal aberrations or AZF micro deletions. We discovered a new AR mutation, del 57Leu, described for the first time as a possible cause of male infertility. Furthermore, we found two patients with the Ala474Val mutation and one patient bearing the Pro390Ser mutation. Our results indicate that these mutations are significant markers for idiopathic male infertility in the Syrian society and in Mediterranean populations in general. (author)

  20. Emergence of methicillin-resistant coagulase-negative staphylococci resistant to linezolid with rRNA gene C2190T and G2603T mutations.

    Science.gov (United States)

    Cidral, Thiago André; Carvalho, Maria Cícera; Figueiredo, Agnes Marie Sá; de Melo, Maria Celeste Nunes

    2015-10-01

    The aim of this article were to determinate the mechanism of linezolid resistance in coagulase-negative methicillin-resistant staphylococci from hospitals in the northeast of Brazil. We identified the isolates using VITEK(®) 2 and MALDI-TOF. Susceptibility to antibiotics was measured by the disk-diffusion method and by Etest(®) . Extraction of the whole genome DNA was performed, followed by screening of all the strains for the presence of mecA and cfr genes. The domain V region of 23S rRNA gene was sequenced and then aligned with a linezolid-susceptible reference strain. Pulsed-field gel electrophoresis (PFGE) macro-restriction analysis was performed. Three linezolid-resistant Staphylococcus hominis and two linezolid-resistant Staphylococcus epidermidis strains were analyzed. The isolates showed two point mutations in the V region of the 23S rRNA gene (C2190T and G2603T). We did not detect the cfr gene in any isolate by PCR. The S. hominis showed the same pulsotype, while the S. epidermidis did not present any genetic relation to each other. In conclusion, this study revealed three S. hominis and two S. epidermidis strains with resistance to linezolid due to a double mutation (C2190T and G2603T) in the domain V of the 23S rRNA gene. For the first time, the mutation of C2190T in S. epidermidis is described. This study also revealed the clonal spread of a S. hominis pulsotype between three public hospitals in the city of Natal, Brazil. These findings highlight the importance of continued vigilance of linezolid resistance in staphylococci. © 2015 APMIS. Published by John Wiley & Sons Ltd.

  1. Gold nanoparticle-based beacon to detect STAT5b mRNA expression in living cells: a case optimized by bioinformatics screen.

    Science.gov (United States)

    Deng, Dawei; Li, Yang; Xue, Jianpeng; Wang, Jie; Ai, Guanhua; Li, Xin; Gu, Yueqing

    2015-01-01

    Messenger RNA (mRNA), a single-strand ribonucleic acid with functional gene information is usually abnormally expressed in cancer cells and has become a promising biomarker for the study of tumor progress. Hairpin DNA-coated gold nanoparticle (hDAuNP) beacon containing a bare gold nanoparticle (AuNP) as fluorescence quencher and thiol-terminated fluorescently labeled stem-loop-stem oligonucleotide sequences attached by Au-S bond is currently a new nanoscale biodiagnostic platform capable of mRNA detection, in which the design of the loop region sequence is crucial for hybridizing with the target mRNA. Hence, in this study, to improve the sensitivity and selectivity of hDAuNP beacon simultaneously, the loop region of hairpin DNA was screened by bioinformatics strategy. Here, signal transducer and activator of transcription 5b (STAT5b) mRNA was selected and used as a practical example. The results from the combined characterizations using optical techniques, flow cytometry assay, and cell microscopic imaging showed that after optimization, the as-prepared hDAuNP beacon had higher selectivity and sensitivity for the detection of STAT5b mRNA in living cells, as compared with our previous beacon. Thus, the bioinformatics method may be a promising new strategy for assisting in the designing of the hDAuNP beacon, extending its application in the detection of mRNA expression and the resultant mRNA-based biological processes and disease pathogenesis.

  2. Screening for circulating RAS/RAF mutations by multiplex digital PCR

    DEFF Research Database (Denmark)

    Andersen, Rikke Fredslund; Jakobsen, Anders

    2016-01-01

    by technical challenges primarily due to the low levels of ctDNA in patients with localized disease and in patients responding to therapy. The approach presented here is a multiplex digital PCR method of screening for 31 mutations in the KRAS, NRAS, BRAF, and PIK3CA genes in the plasma. The upper level...

  3. Mutations of the aminoacyl-tRNA-synthetases SARS and WARS2 are implicated in the etiology of autosomal recessive intellectual disability.

    Science.gov (United States)

    Musante, Luciana; Püttmann, Lucia; Kahrizi, Kimia; Garshasbi, Masoud; Hu, Hao; Stehr, Henning; Lipkowitz, Bettina; Otto, Sabine; Jensen, Lars R; Tzschach, Andreas; Jamali, Payman; Wienker, Thomas; Najmabadi, Hossein; Ropers, Hans Hilger; Kuss, Andreas W

    2017-06-01

    Intellectual disability (ID) is the hallmark of an extremely heterogeneous group of disorders that comprises a wide variety of syndromic and non-syndromic phenotypes. Here, we report on mutations in two aminoacyl-tRNA synthetases that are associated with ID in two unrelated Iranian families. In the first family, we identified a homozygous missense mutation (c.514G>A, p.Asp172Asn) in the cytoplasmic seryl-tRNA synthetase (SARS) gene. The mutation affects the enzymatic core domain of the protein and impairs its enzymatic activity, probably leading to reduced cytoplasmic tRNA Ser concentrations. The mutant protein was predicted to be unstable, which could be substantiated by investigating ectopic mutant SARS in transfected HEK293T cells. In the second family, we found a compound heterozygous genotype of the mitochondrial tryptophanyl-tRNA synthetase (WARS2) gene, comprising a nonsense mutation (c.325delA, p.Ser109Alafs*15), which very likely entails nonsense-mediated mRNA decay and a missense mutation (c.37T>G, p.Trp13Gly). The latter affects the mitochondrial localization signal of WARS2, causing protein mislocalization. Including AIMP1, which we have recently implicated in the etiology of ID, three genes with a role in tRNA-aminoacylation are now associated with this condition. We therefore suggest that the functional integrity of tRNAs in general is an important factor in the development and maintenance of human cognitive functions. © 2017 Wiley Periodicals, Inc.

  4. Restriction digest screening facilitates efficient detection of site-directed mutations introduced by CRISPR in C. albicans UME6.

    Science.gov (United States)

    Evans, Ben A; Smith, Olivia L; Pickerill, Ethan S; York, Mary K; Buenconsejo, Kristen J P; Chambers, Antonio E; Bernstein, Douglas A

    2018-01-01

    Introduction of point mutations to a gene of interest is a powerful tool when determining protein function. CRISPR-mediated genome editing allows for more efficient transfer of a desired mutation into a wide range of model organisms. Traditionally, PCR amplification and DNA sequencing is used to determine if isolates contain the intended mutation. However, mutation efficiency is highly variable, potentially making sequencing costly and time consuming. To more efficiently screen for correct transformants, we have identified restriction enzymes sites that encode for two identical amino acids or one or two stop codons. We used CRISPR to introduce these restriction sites directly upstream of the Candida albicans UME6 Zn 2+ -binding domain, a known regulator of C. albicans filamentation. While repair templates coding for different restriction sites were not equally successful at introducing mutations, restriction digest screening enabled us to rapidly identify isolates with the intended mutation in a cost-efficient manner. In addition, mutated isolates have clear defects in filamentation and virulence compared to wild type C. albicans . Our data suggest restriction digestion screening efficiently identifies point mutations introduced by CRISPR and streamlines the process of identifying residues important for a phenotype of interest.

  5. Cell-Based Reporter System for High-Throughput Screening of MicroRNA Pathway Inhibitors and Its Limitations

    Czech Academy of Sciences Publication Activity Database

    Bruštíková, Kateřina; Sedlák, David; Kubíková, Jana; Škuta, Ctibor; Šolcová, Kateřina; Malík, Radek; Bartůněk, Petr; Svoboda, Petr

    2018-01-01

    Roč. 9 (2018), č. článku 45. ISSN 1664-8021 R&D Projects: GA ČR GA13-29531S; GA MŠk LO1220; GA MŠk LM2015063; GA MŠk LM2011022 Institutional support: RVO:68378050 Keywords : miRNA * high-throughput screening * miR-30 * let-7 * Argonaute Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.789, year: 2016

  6. Screening for mutations in the uroporphyrinogen decarboxylase gene using denaturing gradient gel electrophoresis

    DEFF Research Database (Denmark)

    Christiansen, L; Ged, C; Hombrados, I

    1999-01-01

    to exon skipping, and a 2-bp deletion (415-416delTA) resulting in a frameshift and the introduction of a premature stop codon. Heterologous expression and enzymatic studies of the mutant proteins demonstrate that the three mutations leading to shortening or truncation of the UROD protein have no residual......, confirming the heterogeneity of the underlying genetic defects of these diseases. We have established a denaturing gradient gel electrophoresis (DGGE) assay for mutation detection in the UROD gene, enabling the simultaneous screening for known and unknown mutations. The established assay has proved able...

  7. Limited diagnostic value of enzyme analysis in patients with mitochondrial tRNA mutations

    DEFF Research Database (Denmark)

    Wibrand, Flemming; Jeppesen, Tina Dysgaard; Frederiksen, Anja L

    2010-01-01

    We evaluated the diagnostic value of respiratory chain (RC) enzyme analysis of muscle in adult patients with mitochondrial myopathy (MM). RC enzyme activity was measured in muscle biopsies from 39 patients who carry either the 3243A>G mutation, other tRNA point mutations, or single, large......, respectively, in these three groups. The results indicate that RC enzyme analysis in muscle is not a sensitive test for MM in adults. In these patients, abnormal muscle histochemistry appears to be a better predictor ofMM....

  8. A simple and robust vector-based shRNA expression system used for RNA interference.

    Science.gov (United States)

    Wang, Xue-jun; Li, Ying; Huang, Hai; Zhang, Xiu-juan; Xie, Pei-wen; Hu, Wei; Li, Dan-dan; Wang, Sheng-qi

    2013-01-01

    RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful genetic tool for conducting functional studies. Previously, vector-based shRNA-expression strategies capable of inducing RNAi in viable cells have been developed, however, these vector systems have some disadvantages, either because they were error-prone or cost prohibitive. In this report we described the development of a simple, robust shRNA expression system utilizing 1 long oligonucleotide or 2 short oligonucleotides for half the cost of conventional shRNA construction methods and with a >95% cloning success rate. The shRNA loop sequence and stem structure were also compared and carefully selected for better RNAi efficiency. Furthermore, an easier strategy was developed based on isocaudomers which permit rapid combination of the most efficient promoter-shRNA cassettes. Finally, using this method, the conservative target sites for hepatitis B virus (HBV) knockdown were systemically screened and HBV antigen expression shown to be successfully suppressed in the presence of connected multiple shRNAs both in vitro and in vivo. This novel design describes an inexpensive and effective way to clone and express single or multiple shRNAs from the same vector with the capacity for potent and effective silencing of target genes.

  9. Triplet-Based Codon Organization Optimizes the Impact of Synonymous Mutation on Nucleic Acid Molecular Dynamics.

    Science.gov (United States)

    Babbitt, Gregory A; Coppola, Erin E; Mortensen, Jamie S; Ekeren, Patrick X; Viola, Cosmo; Goldblatt, Dallan; Hudson, André O

    2018-02-01

    Since the elucidation of the genetic code almost 50 years ago, many nonrandom aspects of its codon organization remain only partly resolved. Here, we investigate the recent hypothesis of 'dual-use' codons which proposes that in addition to allowing adjustment of codon optimization to tRNA abundance, the degeneracy in the triplet-based genetic code also multiplexes information regarding DNA's helical shape and protein-binding dynamics while avoiding interference with other protein-level characteristics determined by amino acid properties. How such structural optimization of the code within eukaryotic chromatin could have arisen from an RNA world is a mystery, but would imply some preadaptation in an RNA context. We analyzed synonymous (protein-silent) and nonsynonymous (protein-altering) mutational impacts on molecular dynamics in 13823 identically degenerate alternative codon reorganizations, defined by codon transitions in 7680 GPU-accelerated molecular dynamic simulations of implicitly and explicitly solvated double-stranded aRNA and bDNA structures. When compared to all possible alternative codon assignments, the standard genetic code minimized the impact of synonymous mutations on the random atomic fluctuations and correlations of carbon backbone vector trajectories while facilitating the specific movements that contribute to DNA polymer flexibility. This trend was notably stronger in the context of RNA supporting the idea that dual-use codon optimization and informational multiplexing in DNA resulted from the preadaptation of the RNA duplex to resist changes to thermostability. The nonrandom and divergent molecular dynamics of synonymous mutations also imply that the triplet-based code may have resulted from adaptive functional expansion enabling a primordial doublet code to multiplex gene regulatory information via the shape and charge of the minor groove.

  10. Altered microRNA expression in frontotemporal lobar degeneration with TDP-43 pathology caused by progranulin mutations

    Directory of Open Access Journals (Sweden)

    Kocerha Jannet

    2011-10-01

    Full Text Available Abstract Background Frontotemporal lobar degeneration (FTLD is a progressive neurodegenerative disorder that can be triggered through genetic or sporadic mechanisms. MicroRNAs (miRNAs have become a major therapeutic focus as their pervasive expression and powerful regulatory roles in disease pathogenesis become increasingly apparent. Here we examine the role of miRNAs in FTLD patients with TAR DNA-binding protein 43 pathology (FTLD-TDP caused by genetic mutations in the progranulin (PGRN gene. Results Using miRNA array profiling, we identified the 20 miRNAs that showed greatest evidence (unadjusted P PGRN mutations when compared to 32 FTLD-TDP patients with no apparent genetic abnormalities. Quantitative real-time PCR (qRT-PCR analyses provided technical validation of the differential expression for 9 of the 20 miRNAs in frontal cortex. Additional qRT-PCR analyses showed that 5 out of 9 miRNAs (miR-922, miR-516a-3p, miR-571, miR-548b-5p, and miR-548c-5p were also significantly dysregulated (unadjusted P PGRN mutation carriers, consistent with a systemic reduction in PGRN levels. We developed a list of gene targets for the 5 candidate miRNAs and found 18 genes dysregulated in a reported FTLD mRNA study to exhibit anti-correlated miRNA-mRNA patterns in affected cortex and cerebellar tissue. Among the targets is brain-specific angiogenesis inhibitor 3, which was recently identified as an important player in synapse biology. Conclusions Our study suggests that miRNAs may contribute to the pathogenesis of FTLD-TDP caused by PGRN mutations and provides new insight into potential future therapeutic options.

  11. Antisense Oligonucleotide (AON-based Therapy for Leber Congenital Amaurosis Caused by a Frequent Mutation in CEP290

    Directory of Open Access Journals (Sweden)

    Rob WJ Collin

    2012-01-01

    Full Text Available Leber congenital amaurosis (LCA is the most severe form of inherited retinal degeneration, with an onset in the first year of life. The most frequent mutation that causes LCA, present in at least 10% of individuals with LCA from North-American and Northern-European descent, is an intronic mutation in CEP290 that results in the inclusion of an aberrant exon in the CEP290 mRNA. Here, we describe a genetic therapy approach that is based on antisense oligonucleotides (AONs, small RNA molecules that are able to redirect normal splicing of aberrantly processed pre-mRNA. Immortalized lymphoblastoid cells of individuals with LCA homozygously carrying the intronic CEP290 mutation were transfected with several AONs that target the aberrant exon that is incorporated in the mutant CEP290 mRNA. Subsequent RNA isolation and reverse transcription-PCR analysis revealed that a number of AONs were capable of almost fully redirecting normal CEP290 splicing, in a dose-dependent manner. Other AONs however, displayed no effect on CEP290 splicing at all, indicating that the rescue of aberrant CEP290 splicing shows a high degree of sequence specificity. Together, our data show that AON-based therapy is a promising therapeutic approach for CEP290-associated LCA that warrants future research in animal models to develop a cure for this blinding disease.

  12. Discovering cancer vulnerabilities using high-throughput micro-RNA screening.

    Science.gov (United States)

    Nikolic, Iva; Elsworth, Benjamin; Dodson, Eoin; Wu, Sunny Z; Gould, Cathryn M; Mestdagh, Pieter; Marshall, Glenn M; Horvath, Lisa G; Simpson, Kaylene J; Swarbrick, Alexander

    2017-12-15

    Micro-RNAs (miRNAs) are potent regulators of gene expression and cellular phenotype. Each miRNA has the potential to target hundreds of transcripts within the cell thus controlling fundamental cellular processes such as survival and proliferation. Here, we exploit this important feature of miRNA networks to discover vulnerabilities in cancer phenotype, and map miRNA-target relationships across different cancer types. More specifically, we report the results of a functional genomics screen of 1280 miRNA mimics and inhibitors in eight cancer cell lines, and its presentation in a sophisticated interactive data portal. This resource represents the most comprehensive survey of miRNA function in oncology, incorporating breast cancer, prostate cancer and neuroblastoma. A user-friendly web portal couples this experimental data with multiple tools for miRNA target prediction, pathway enrichment analysis and visualization. In addition, the database integrates publicly available gene expression and perturbation data enabling tailored and context-specific analysis of miRNA function in a particular disease. As a proof-of-principle, we use the database and its innovative features to uncover novel determinants of the neuroblastoma malignant phenotype. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. In vitro base modification of Escherichia coli glutamate 2 transfer-RNA and phenylalanine transfer-RNA gene transcripts

    International Nuclear Information System (INIS)

    Shahan, M.N.

    1989-01-01

    Plasmids were constructed that contain an E. Coli tRNA 2 Glu or tRNA Phe gene in a system transcribable by T7 or SP6 RNA polymerase. Selectively 32 P-labeled transcripts of these plasmids were used to study tRNA base modification in vitro in crude extracts by nearest neighbor analysis. The synthesis of 5-methyl-aminomethyl-2-thiouridine (mnm 5 s 2 U) was studied. Complete synthesis of mnm 5 s 2 2U is not observed. Instead, 2-thiouridine (s 2 U) is synthesized. Synthesis requires ATP, cysteine, Mg + , and monovalent cation concentrations below 50 mM. The reaction has a pH optimum above 7.0. Sulfide ion will substitute for cysteine in the reaction but sulfate, sulfite, methionine, homocysteine, and β-mercaptopyruvate will not. Extracts from E. coli strains carrying either the asuE or asuF mutations have reduced s 2 U synthetic activity which supports in vivo evidence that the wild type genes are involved in 2-thiolation of uridine. The enzyme is shown to be unstable both upon storage at -80 degree C and during the modification reaction. A method was developed to study the synthesis of any one of four pseudouridines ψ found at different positions of the tRNA cloverleaf. Synthesis of ψ is observed at three of the four positions-positions 32, 39, and 55. The asuC mutation is shown to affect ψ synthesis only at position 39 confirming that it is an allele of hisT and that the hisT mutations do not affect ψ synthesis at position 32 in E. coli. Synthesis of ψ32, ψ39, and ψ55 does not require any prior modification. Synthesis of dihydrouridine, 7-methylguanosine, and 3(3-amino-3-carboxypropyl)uridine is also observed. Synthesis of 2-methyladenosine and ψ 13 is not seen. Removal of part of the aminoacyl stem reduces synthesis of all modifications examined by 3' fold or more

  14. 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

  15. All azoospermic males should be screened for cystic fibrosis mutations before intracytoplasmic sperm injection.

    LENUS (Irish Health Repository)

    Mocanu, Edgar

    2012-02-01

    We assessed the frequency of CFTR mutations in groups with varying degrees of sub-fertility and compared these groups to a fertile male group with proven paternity. Screening for CFTR mutations should be routine for all azoospermic males, irrespective of obstructive or non-obstructive etiology, prior to proposing ICSI treatment. CFTR testing has no value in the investigation of non-azoospermic infertile males.

  16. All azoospermic males should be screened for cystic fibrosis mutations before intracytoplasmic sperm injection.

    LENUS (Irish Health Repository)

    Mocanu, Edgar

    2010-11-01

    We assessed the frequency of CFTR mutations in groups with varying degrees of sub-fertility and compared these groups to a fertile male group with proven paternity. Screening for CFTR mutations should be routine for all azoospermic males, irrespective of obstructive or non-obstructive etiology, prior to proposing ICSI treatment. CFTR testing has no value in the investigation of non-azoospermic infertile males.

  17. Modification of mutation frequency in Saccharomyces Cerevisiae

    International Nuclear Information System (INIS)

    Vashishat, R.K.; Kakar, S.N.

    1976-01-01

    In a reverse mutation system, using haploid, histidine-requirinq strain of Saccharomyces cerevisiae, the frequency of uv-induced prototrophs increased if the post-irradiation minimal medium was supplemented with limited amounts of histidine. Addition of natural amino acids or RNA bases in the post-irradiation minimal medium, with or without histidine, also increased the uv-induced mutation frequency. Thus, post-irradiation conditions favouring protein and RNA synthesis, are effective in increasing uv-induced mutations in yeast. As compared to uv light, nitrous acid was more effective in inducing reversions in this strain and the frequency increased if the treated cells were plated on minimal medium supplemented with limited amounts of histidine. However, the addition of amino acids or RNA bases decreased the number of revertants. An additional inclusion of histidine reversed the suppressive effect of these metabolites. The mutation induction processes are thus different or differently modifiable in uv and nitrous acid. (author)

  18. A simple and robust vector-based shRNA expression system used for RNA interference.

    Directory of Open Access Journals (Sweden)

    Xue-jun Wang

    Full Text Available BACKGROUND: RNA interference (RNAi mediated by small interfering RNAs (siRNAs or short hairpin RNAs (shRNAs has become a powerful genetic tool for conducting functional studies. Previously, vector-based shRNA-expression strategies capable of inducing RNAi in viable cells have been developed, however, these vector systems have some disadvantages, either because they were error-prone or cost prohibitive. RESULTS: In this report we described the development of a simple, robust shRNA expression system utilizing 1 long oligonucleotide or 2 short oligonucleotides for half the cost of conventional shRNA construction methods and with a >95% cloning success rate. The shRNA loop sequence and stem structure were also compared and carefully selected for better RNAi efficiency. Furthermore, an easier strategy was developed based on isocaudomers which permit rapid combination of the most efficient promoter-shRNA cassettes. Finally, using this method, the conservative target sites for hepatitis B virus (HBV knockdown were systemically screened and HBV antigen expression shown to be successfully suppressed in the presence of connected multiple shRNAs both in vitro and in vivo. CONCLUSION: This novel design describes an inexpensive and effective way to clone and express single or multiple shRNAs from the same vector with the capacity for potent and effective silencing of target genes.

  19. University of Texas Southwestern Medical Center: High-Throughput siRNA Screening of a Non-Small Cell Lung Cancer (NSCLC) Cell Line Panel | Office of Cancer Genomics

    Science.gov (United States)

    The goal of this project is to use siRNA screens to identify NSCLC-selective siRNAs from two genome-wide libraries that will allow us to functionally define genetic dependencies of subtypes of NSCLC. Using bioinformatics tools, the CTD2 center at the University of Texas Southwestern Medical Center are discovering associations between this functional data (siRNAs) and NSCLC mutational status, methylation arrays, gene expression arrays, and copy number variation data that will help us identify new targets and enrollment biomarkers. 

  20. Mechanistic study on the nuclear modifier gene MSS1 mutation suppressing neomycin sensitivity of the mitochondrial 15S rRNA C1477G mutation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Zhou, Qiyin; Wang, Wei; He, Xiangyu; Zhu, Xiaoyu; Shen, Yaoyao; Yu, Zhe; Wang, Xuexiang; Qi, Xuchen; Zhang, Xuan; Fan, Mingjie; Dai, Yu; Yang, Shuxu; Yan, Qingfeng

    2014-01-01

    The phenotypic manifestation of mitochondrial DNA (mtDNA) mutations can be modulated by nuclear genes and environmental factors. However, neither the interaction among these factors nor their underlying mechanisms are well understood. The yeast Saccharomyces cerevisiae mtDNA 15S rRNA C1477G mutation (PR) corresponds to the human 12S rRNA A1555G mutation. Here we report that a nuclear modifier gene mss1 mutation suppresses the neomycin-sensitivity phenotype of a yeast C1477G mutant in fermentable YPD medium. Functional assays show that the mitochondrial function of the yeast C1477G mutant was impaired severely in YPD medium with neomycin. Moreover, the mss1 mutation led to a significant increase in the steady-state level of HAP5 (heme activated protein), which greatly up-regulated the expression of glycolytic transcription factors RAP1, GCR1, and GCR2 and thus stimulated glycolysis. Furthermore, the high expression of the key glycolytic enzyme genes HXK2, PFK1 and PYK1 indicated that enhanced glycolysis not only compensated for the ATP reduction from oxidative phosphorylation (OXPHOS) in mitochondria, but also ensured the growth of the mss1(PR) mutant in YPD medium with neomycin. This study advances our understanding of the phenotypic manifestation of mtDNA mutations.

  1. 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.

  2. Mutation screening of the CDKL5 gene in cryptogenic infantile intractable epilepsy and review of clinical sensitivity.

    Science.gov (United States)

    Intusoma, Utcharee; Hayeeduereh, Fadell; Plong-On, Oradawan; Sripo, Thanya; Vasiknanonte, Punnee; Janjindamai, Supachai; Lusawat, Apasri; Thammongkol, Sasipa; Visudtibhan, Anannit; Limprasert, Pornprot

    2011-09-01

    To perform CDKL5 mutation screening in Thai children with cryptogenic infantile intractable epilepsy and to determine the clinical sensitivity of CDKL5 screening when different inclusion criteria were applied. Children with cryptogenic infantile intractable epilepsy were screened for CDKL5 mutation using multiplex ligation-dependent probe amplification and DNA sequencing. The clinical sensitivity was reviewed by combining the results of studies using similar inclusion screening criteria. Thirty children (19 girls and 11 boys) with a median seizure onset of 7 months were screened. Almost a half had infantile spasms and one fifth had stereotypic hand movements. A novel c.2854C>T (p.R952X) was identified in an ambulatory girl who had severe mental retardation, multiple types of seizures without Rett-like features. Her mother had a mild intellectual disability, yet her grandmother and half sister were normal despite having the same genetic alteration (random X-inactivation patterns). The pathogenicity of p.R952X identified here was uncertain since healthy relatives and 6 female controls also harbor this alteration. The clinical sensitivity of CDKL5 mutation screening among females with Rett-like features and negative MECP2 screening was 7.8% while the clinical sensitivity among females having cryptogenic intractable seizures with an onset before the ages of 12, 6 and 3 months were 4.7, 11.6 and 14.3%, respectively. Copyright © 2011 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

  3. Gold nanoparticle-based beacon to detect STAT5b mRNA expression in living cells: a case optimized by bioinformatics screen

    Directory of Open Access Journals (Sweden)

    Deng D

    2015-04-01

    Full Text Available Dawei Deng,* Yang Li,* Jianpeng Xue, Jie Wang, Guanhua Ai, Xin Li, Yueqing GuDepartment of Biomedical Engineering, China Pharmaceutical University, Nanjing, People’s Republic of China*These authors contributed equally to this workAbstract: Messenger RNA (mRNA, a single-strand ribonucleic acid with functional gene information is usually abnormally expressed in cancer cells and has become a promising biomarker for the study of tumor progress. Hairpin DNA-coated gold nanoparticle (hDAuNP beacon containing a bare gold nanoparticle (AuNP as fluorescence quencher and thiol-terminated fluorescently labeled stem–loop–stem oligonucleotide sequences attached by Au–S bond is currently a new nanoscale biodiagnostic platform capable of mRNA detection, in which the design of the loop region sequence is crucial for hybridizing with the target mRNA. Hence, in this study, to improve the sensitivity and selectivity of hDAuNP beacon simultaneously, the loop region of hairpin DNA was screened by bioinformatics strategy. Here, signal transducer and activator of transcription 5b (STAT5b mRNA was selected and used as a practical example. The results from the combined characterizations using optical techniques, flow cytometry assay, and cell microscopic imaging showed that after optimization, the as-prepared hDAuNP beacon had higher selectivity and sensitivity for the detection of STAT5b mRNA in living cells, as compared with our previous beacon. Thus, the bioinformatics method may be a promising new strategy for assisting in the designing of the hDAuNP beacon, extending its application in the detection of mRNA expression and the resultant mRNA-based biological processes and disease pathogenesis.Keywords: molecular beacon, bioinformatics, gold nanoparticle, STAT5b mRNA, visual detection

  4. Transfer RNA and human disease

    Directory of Open Access Journals (Sweden)

    Jamie A Abbott

    2014-06-01

    Full Text Available Pathological mutations in tRNA genes and tRNA processing enzymes are numerous and result in very complicated clinical phenotypes. Mitochondrial tRNA (mt-tRNA genes are hotspots for pathological mutations and over 200 mt-tRNA mutations have been linked to various disease states. Often these mutations prevent tRNA aminoacylation. Disrupting this primary function affects protein synthesis and the expression, folding, and function of oxidative phosphorylation enzymes. Mitochondrial tRNA mutations manifest in a wide panoply of diseases related to cellular energetics, including COX deficiency (cytochrome C oxidase, mitochondrial myopathy, MERRF (Myoclonic Epilepsy with Ragged Red Fibers, and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes. Diseases caused by mt-tRNA mutations can also affect very specific tissue types, as in the case of neurosensory non-syndromic hearing loss and pigmentary retinopathy, diabetes mellitus, and hypertrophic cardiomyopathy. Importantly, mitochondrial heteroplasmy plays a role in disease severity and age of onset as well. Not surprisingly, mutations in enzymes that modify cytoplasmic and mitochondrial tRNAs are also linked to a diverse range of clinical phenotypes. In addition to compromised aminoacylation of the tRNAs, mutated modifying enzymes can also impact tRNA expression and abundance, tRNA modifications, tRNA folding, and even tRNA maturation (e.g., splicing. Some of these pathological mutations in tRNAs and processing enzymes are likely to affect non-canonical tRNA functions, and contribute to the diseases without significantly impacting on translation. This chapter will review recent literature on the relation of mitochondrial and cytoplasmic tRNA, and enzymes that process tRNAs, to human disease. We explore the mechanisms involved in the clinical presentation of these various diseases with an emphasis on neurological disease.

  5. Transfer RNA and human disease.

    Science.gov (United States)

    Abbott, Jamie A; Francklyn, Christopher S; Robey-Bond, Susan M

    2014-01-01

    Pathological mutations in tRNA genes and tRNA processing enzymes are numerous and result in very complicated clinical phenotypes. Mitochondrial tRNA (mt-tRNA) genes are "hotspots" for pathological mutations and over 200 mt-tRNA mutations have been linked to various disease states. Often these mutations prevent tRNA aminoacylation. Disrupting this primary function affects protein synthesis and the expression, folding, and function of oxidative phosphorylation enzymes. Mitochondrial tRNA mutations manifest in a wide panoply of diseases related to cellular energetics, including COX deficiency (cytochrome C oxidase), mitochondrial myopathy, MERRF (Myoclonic Epilepsy with Ragged Red Fibers), and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). Diseases caused by mt-tRNA mutations can also affect very specific tissue types, as in the case of neurosensory non-syndromic hearing loss and pigmentary retinopathy, diabetes mellitus, and hypertrophic cardiomyopathy. Importantly, mitochondrial heteroplasmy plays a role in disease severity and age of onset as well. Not surprisingly, mutations in enzymes that modify cytoplasmic and mitochondrial tRNAs are also linked to a diverse range of clinical phenotypes. In addition to compromised aminoacylation of the tRNAs, mutated modifying enzymes can also impact tRNA expression and abundance, tRNA modifications, tRNA folding, and even tRNA maturation (e.g., splicing). Some of these pathological mutations in tRNAs and processing enzymes are likely to affect non-canonical tRNA functions, and contribute to the diseases without significantly impacting on translation. This chapter will review recent literature on the relation of mitochondrial and cytoplasmic tRNA, and enzymes that process tRNAs, to human disease. We explore the mechanisms involved in the clinical presentation of these various diseases with an emphasis on neurological disease.

  6. 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.

  7. 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...

  8. Common pathological mutations in PQBP1 induce nonsense-mediated mRNA decay and enhance exclusion of the mutant exon.

    Science.gov (United States)

    Musante, Luciana; Kunde, Stella-Amrei; Sulistio, Tina O; Fischer, Ute; Grimme, Astrid; Frints, Suzanna G M; Schwartz, Charles E; Martínez, Francisco; Romano, Corrado; Ropers, Hans-Hilger; Kalscheuer, Vera M

    2010-01-01

    The polyglutamine binding protein 1 (PQBP1) gene plays an important role in X-linked mental retardation (XLMR). Nine of the thirteen PQBP1 mutations known to date affect the AG hexamer in exon 4 and cause frameshifts introducing premature termination codons (PTCs). However, the phenotype in this group of patients is variable. To investigate the pathology of these PQBP1 mutations, we evaluated their consequences on mRNA and protein expression. RT-PCRs revealed mutation-specific reduction of PQBP1 mRNAs carrying the PTCs that can be partially restored by blocking translation, thus indicating a role for the nonsense-mediated mRNA decay pathway. In addition, these mutations resulted in altered levels of PQBP1 transcripts that skipped exon 4, probably as a result of altering important splicing motifs via nonsense-associated altered splicing (NAS). This hypothesis is supported by transfection experiments using wild-type and mutant PQBP1 minigenes. Moreover, we show that a truncated PQBP1 protein is indeed present in the patients. Remarkably, patients with insertion/deletion mutations in the AG hexamer express significantly increased levels of a PQBP1 isoform, which is very likely encoded by the transcripts without exon 4, confirming the findings at the mRNA level. Our study provides significant insight into the early events contributing to the pathogenesis of the PQBP1 related XLMR disease.

  9. Screening for calreticulin mutations in a cohort of patients suspected ...

    African Journals Online (AJOL)

    Of the 36 types of insertions and deletions identified, type 1 (a. 52-base pair deletion) and type 2 (a 5-base pair insertion) mutations account for >80% of CALR mutations.[7] Phenotypic differences between type 1 and type 2 carriers have been implicated. [3] All recurrent mutations cause a frameshift in the region encoding.

  10. Mutations in 23S rRNA gene associated with decreased susceptibility to tiamulin and valnemulin in Mycoplasma gallisepticum.

    Science.gov (United States)

    Li, Bei-Bei; Shen, Jian-Zhong; Cao, Xing-Yuan; Wang, Yang; Dai, Lei; Huang, Si-Yang; Wu, Cong-Ming

    2010-07-01

    Mycoplasma gallisepticum is a major etiological agent of chronic respiratory disease (CRD) in chickens and sinusitis in turkeys. The pleuromutilin antibiotics tiamulin and valnemulin are currently used in the treatment of M. gallisepticum infection. We studied the in vitro development of pleuromutilin resistance in M. gallisepticum and investigated the molecular mechanisms involved in this process. Pleuromutilin-resistant mutants were selected by serial passages of M. gallisepticum strains PG31 and S6 in broth medium containing subinhibitory concentrations of tiamulin or valnemulin. A portion of the gene encoding 23S rRNA gene (domain V) and the gene encoding ribosome protein L3 were amplified and sequenced. No mutation could be detected in ribosome protein L3. Mutations were found at nucleotide positions 2058, 2059, 2061, 2447 and 2503 of 23S rRNA gene (Escherichia coli numbering). Although a single mutation could cause elevation of tiamulin and valnemulin MICs, combinations of two or three mutations were necessary to produce high-level resistance. All the mutants were cross-resistant to lincomycin, chloramphenicol and florfenicol. Mutants with the A2058G or the A2059G mutation exhibited cross-resistance to macrolide antibiotics erythromycin, tilmicosin and tylosin.

  11. Novel C16orf57 mutations in patients with Poikiloderma with Neutropenia: bioinformatic analysis of the protein and predicted effects of all reported mutations

    Directory of Open Access Journals (Sweden)

    Colombo Elisa A

    2012-01-01

    Full Text Available Abstract Background Poikiloderma with Neutropenia (PN is a rare autosomal recessive genodermatosis caused by C16orf57 mutations. To date 17 mutations have been identified in 31 PN patients. Results We characterize six PN patients expanding the clinical phenotype of the syndrome and the mutational repertoire of the gene. We detect the two novel C16orf57 mutations, c.232C>T and c.265+2T>G, as well as the already reported c.179delC, c.531delA and c.693+1G>T mutations. cDNA analysis evidences the presence of aberrant transcripts, and bioinformatic prediction of C16orf57 protein structure gauges the mutations effects on the folded protein chain. Computational analysis of the C16orf57 protein shows two conserved H-X-S/T-X tetrapeptide motifs marking the active site of a two-fold pseudosymmetric structure recalling the 2H phosphoesterase superfamily. Based on this model C16orf57 is likely a 2H-active site enzyme functioning in RNA processing, as a presumptive RNA ligase. According to bioinformatic prediction, all known C16orf57 mutations, including the novel mutations herein described, impair the protein structure by either removing one or both tetrapeptide motifs or by destroying the symmetry of the native folding. Finally, we analyse the geographical distribution of the recurrent mutations that depicts clusters featuring a founder effect. Conclusions In cohorts of patients clinically affected by genodermatoses with overlapping symptoms, the molecular screening of C16orf57 gene seems the proper way to address the correct diagnosis of PN, enabling the syndrome-specific oncosurveillance. The bioinformatic prediction of the C16orf57 protein structure denotes a very basic enzymatic function consistent with a housekeeping function. Detection of aberrant transcripts, also in cells from PN patients carrying early truncated mutations, suggests they might be translatable. Tissue-specific sensitivity to the lack of functionally correct protein accounts for the

  12. Missense mutation in GRN gene affecting RNA splicing and plasma progranulin level in a family affected by frontotemporal lobar degeneration.

    Science.gov (United States)

    Luzzi, Simona; Colleoni, Lara; Corbetta, Paola; Baldinelli, Sara; Fiori, Chiara; Girelli, Francesca; Silvestrini, Mauro; Caroppo, Paola; Giaccone, Giorgio; Tagliavini, Fabrizio; Rossi, Giacomina

    2017-06-01

    Gene coding for progranulin, GRN, is a major gene linked to frontotemporal lobar degeneration. While most of pathogenic GRN mutations are null mutations leading to haploinsufficiency, GRN missense mutations do not have an obvious pathogenicity, and only a few have been revealed to act through different pathogenetic mechanisms, such as cytoplasmic missorting, protein degradation, and abnormal cleavage by elastase. The aim of this study was to disclose the pathogenetic mechanisms of the GRN A199V missense mutation, which was previously reported not to alter physiological progranulin features but was associated with a reduced plasma progranulin level. After investigating the family pedigree, we performed genetic and biochemical analysis on its members and performed RNA expression studies. We found that the mutation segregates with the disease and discovered that its pathogenic feature is the alteration of GRN mRNA splicing, actually leading to haploinsufficiency. Thus, when facing with a missense GRN mutation, its pathogenetic effects should be investigated, especially if associated with low plasma progranulin levels, to determine its nature of either benign polymorphism or pathogenic mutation. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Effects of mutation on the downfield proton nuclear magnetic resonance spectrum of the 5S RNA of Escherichia coli

    International Nuclear Information System (INIS)

    Gewirth, D.T.; Moore, P.B.

    1987-01-01

    The imino proton spectra of several mutants of the 5S RNA of Escherichia coli are compared with that of the wild type. Three of the variants discussed are point mutations, and the fourth is a deletion mutant lacking bases 11-69 of the parent sequence, all obtained by site-directed mutagenesis techniques. The spectroscopic effects of mutation are limited in all cases, and the differences between normal and mutant spectra can be used to make or confirm the assignments of resonances. Several new assignments in the 5S spectrum are reported. Spectroscopic differences due to sequence differences permit the products of single genes within the 5S gene family to be distinguished and their fates followed by NMR

  14. Breast cancer screening in BRCA1 and BRCA2 mutation carriers after risk reducing salpingo-oophorectomy

    NARCIS (Netherlands)

    Fakkert, I.E.; Jansen, L.; Meijer, K.; Kok, Theo; Oosterwijk, J.C.; Mourits, M.J.E.; de Bock, G.H.

    Breast cancer screening is offered to BRCA1 and BRCA2 mutation carriers from the age of 25 years because of their increased risk of breast cancer. As ovarian cancer screening is not effective, risk-reducing salpingho-oophorectomy (RRSO) is offered after child bearing age. RRSO before menopause

  15. VLCAD deficiency: Pitfalls in newborn screening and confirmation of diagnosis by mutation analysis

    DEFF Research Database (Denmark)

    Boneh, A; Andresen, Brage Storstein; Gregersen, Niels

    2006-01-01

    samples taken at age 48-72 h were diagnostic whereas repeat samples at an older age were normal in 4/6 babies. Urine analysis was normal in 5/5. We conclude that the timing of blood sampling for newborn screening is important and that it is important to perform mutation analysis to avoid false......-negative diagnoses of VLCADD in asymptomatic newborn babies. In view of the emerging genotype-phenotype correlation in this disorder, the information derived from mutational analysis can be helpful in designing the appropriate follow-up and therapeutic regime for these patients....

  16. Four-channel asymmetric Real-Time PCR hybridization probe assay: a rapid pre-screening method for critical BCR-ABL kinase domain mutations.

    Science.gov (United States)

    Martinez-Serra, Jordi; Gutiérrez, Antonio; Marcús, Toni F; Soverini, Simona; Amat, Juan Carlos; Navarro-Palou, María; Ros, Teresa; Bex, Teresa; Ballester, Carmen; Bauça, Josep Miquel; SanFelix, Sara; Novo, Andrés; Vidal, Carmen; Santos, Carmen; Besalduch, Joan

    2012-03-01

    Within the laboratory protocols, used for the study of BCR-ABL resistance mutations in chronic myeloid leukemia patients treated with Imatinib, direct sequencing remains the reference method. Since the incidence of patients with a mutation-related loss of response is not very high, it is very useful in the routine laboratory to perform a fast pre-screening method. With this in mind, we have designed a new technique, based on a single Real-Time FRET-based PCR, followed by a study of melting peaks. This new tool, developed in a LightCycler 2.0, combines four different fluorescence channels for the simultaneous detection, in a single close tube, of critical mutations within the ABL kinase domain. Assay evaluation performed on 33 samples, previously genotyped by sequentiation, resulted in full concordance of results. This new methodology detects in a few steps the presence of critical mutations associated to Imatinib resistance. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. Cancer and Scleroderma: A Paraneoplastic Disease with Implications for Malignancy Screening

    Science.gov (United States)

    Shah, Ami A.; Rosen, Livia Casciola

    2015-01-01

    Purpose of review Recent data suggest a paraneoplastic mechanism of scleroderma pathogenesis in unique subsets of scleroderma patients. In this article, we review these data, explore potential links between cancer and scleroderma, and propose an approach to malignancy screening in scleroderma. Recent findings Emerging data have demonstrated that patients with scleroderma and RNA polymerase III autoantibodies have a significantly increased risk of cancer within a few years of scleroderma onset. Genetic alterations in the gene encoding RNA polymerase III (POLR3A) have been identified, and patients with somatic mutations in POLR3A have evidence of mutation specific T cell immune responses with generation of cross-reactive RNA polymerase III autoantibodies. These data strongly suggest that scleroderma is a by-product of anti-tumor immune responses in some patients. Additional epidemiologic data demonstrate that patients developing scleroderma at older ages may also have a short cancer-scleroderma interval, suggestive of paraneoplastic disease. Summary Scleroderma may be a paraneoplastic disease in unique patient subsets. Aggressive malignancy screening in these patients may aid in early cancer detection. Further study is required to determine whether cancer therapy could improve scleroderma outcomes in this patient population. PMID:26352736

  18. Mutations that alter a conserved element upstream of the potato virus X triple block and coat protein genes affect subgenomic RNA accumulation.

    Science.gov (United States)

    Kim, K H; Hemenway, C

    1997-05-26

    The putative subgenomic RNA (sgRNA) promoter regions upstream of the potato virus X (PVX) triple block and coat protein (CP) genes contain sequences common to other potexviruses. The importance of these sequences to PVX sgRNA accumulation was determined by inoculation of Nicotiana tabacum NT1 cell suspension protoplasts with transcripts derived from wild-type and modified PVX cDNA clones. Analyses of RNA accumulation by S1 nuclease digestion and primer extension indicated that a conserved octanucleotide sequence element and the spacing between this element and the start-site for sgRNA synthesis are critical for accumulation of the two major sgRNA species. The impact of mutations on CP sgRNA levels was also reflected in the accumulation of CP. In contrast, genomic minus- and plus-strand RNA accumulation were not significantly affected by mutations in these regions. Studies involving inoculation of tobacco plants with the modified transcripts suggested that the conserved octanucleotide element functions in sgRNA accumulation and some other aspect of the infection process.

  19. Utilization of gene mapping and candidate gene mutation screening for diagnosing clinically equivocal conditions: a Norrie disease case study.

    Science.gov (United States)

    Chini, Vasiliki; Stambouli, Danai; Nedelea, Florina Mihaela; Filipescu, George Alexandru; Mina, Diana; Kambouris, Marios; El-Shantil, Hatem

    2014-06-01

    Prenatal diagnosis was requested for an undiagnosed eye disease showing X-linked inheritance in a family. No medical records existed for the affected family members. Mapping of the X chromosome and candidate gene mutation screening identified a c.C267A[p.F89L] mutation in NPD previously described as possibly causing Norrie disease. The detection of the c.C267A[p.F89L] variant in another unrelated family confirms the pathogenic nature of the mutation for the Norrie disease phenotype. Gene mapping, haplotype analysis, and candidate gene screening have been previously utilized in research applications but were applied here in a diagnostic setting due to the scarcity of available clinical information. The clinical diagnosis and mutation identification were critical for providing proper genetic counseling and prenatal diagnosis for this family.

  20. HPV16 RNA patterns defined by novel high-throughput RT-qPCR as triage marker in HPV-based cervical cancer precursor screening.

    Science.gov (United States)

    Höfler, Daniela; Böhmer, Gerd; von Wasielewski, Reinhard; Neumann, Heinrich; Halec, Gordana; Holzinger, Dana; Dondog, Bolormaa; Gissmann, Lutz; Pawlita, Michael; Schmitt, Markus

    2015-09-01

    Cervical cancer precursor screening by HPV testing has a low positive predictive value for advanced lesion. HPV16 RNA patterns characteristic for HPV16-transformed cells but based on laborious, cost-intensive singleplex NASBA reactions promised high value in triaging HPV16 DNA-positive women. We developed two high-throughput reverse transcriptase quantitative (RT-q) PCR assays for the HPV16 transcripts E6*I, E1^E4 and E1C and the cellular transcript ubiquitin C and analysed RNA of 158 singly HPV16 DNA-positive cervical cell samples archived in PreservCyt buffer for the presence of transformation-associated HPV16 RNA patterns, i.e., upregulation of E6*I relative to E1^E4 and/or presence of E1C. HPV16 RNA pattern analyses classified 85% of 58 samples diagnosed ≤CIN1 (no cytologically and histologically detectable cervical lesion or CIN grade 1) as negative and 90% of 59 samples diagnosed as ≥CIN3 (CIN grade 3 or invasive cancer) as positive. Among 41 CIN grade 2 samples representing an intermediate lesion group, 49% were HPV16 RNA patterns-positive. Interestingly, 3 of 4 HPV16 RNA patterns-positive lesions initially diagnosed as ≤CIN1 at follow-up 5-24 months later had progressed to ≥CIN2. We successfully developed and validated a second generation of HPV16 RNA patterns assay by rapid RT-qPCR as triage marker for HPV16 DNA-positive women offering clinical utility to distinguish between the need for immediate colposcopy and continued observation. Limited follow-up data suggests that HPV16 RNA patterns-positivity in ≤CIN1 lesions can predict disease progression. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Psychosocial impact of undergoing prostate cancer screening for men with BRCA1 or BRCA2 mutations.

    Science.gov (United States)

    Bancroft, Elizabeth K; Saya, Sibel; Page, Elizabeth C; Myhill, Kathryn; Thomas, Sarah; Pope, Jennifer; Chamberlain, Anthony; Hart, Rachel; Glover, Wayne; Cook, Jackie; Rosario, Derek J; Helfand, Brian T; Hutten Selkirk, Christina; Davidson, Rosemarie; Longmuir, Mark; Eccles, Diana M; Gadea, Neus; Brewer, Carole; Barwell, Julian; Salinas, Monica; Greenhalgh, Lynn; Tischkowitz, Marc; Henderson, Alex; Evans, David Gareth; Buys, Saundra S; Eeles, Rosalind A; Aaronson, Neil K

    2018-05-26

    To report the baseline results of a longitudinal psychosocial study that forms part of the IMPACT study, a multi-national investigation of targeted prostate cancer (PCa) screening among men with a known pathogenic germline mutation in the BRCA1 or BRCA2 genes. Men enrolled in the IMPACT study were invited to complete a questionnaire at collaborating sites prior to each annual screening visit. The questionnaire included sociodemographic characteristics and the following measures: the Hospital Anxiety and Depression Scale (HADS), Impact of Event Scale (IES), 36-item short-form health survey (SF-36), Memorial Anxiety Scale for Prostate Cancer, Cancer Worry Scale-Revised, risk perception and knowledge. The results of the baseline questionnaire are presented. A total of 432 men completed questionnaires: 98 and 160 had mutations in BRCA1 and BRCA2 genes, respectively, and 174 were controls (familial mutation negative). Participants' perception of PCa risk was influenced by genetic status. Knowledge levels were high and unrelated to genetic status. Mean scores for the HADS and SF-36 were within reported general population norms and mean IES scores were within normal range. IES mean intrusion and avoidance scores were significantly higher in BRCA1/BRCA2 carriers than in controls and were higher in men with increased PCa risk perception. At the multivariate level, risk perception contributed more significantly to variance in IES scores than genetic status. This is the first study to report the psychosocial profile of men with BRCA1/BRCA2 mutations undergoing PCa screening. No clinically concerning levels of general or cancer-specific distress or poor quality of life were detected in the cohort as a whole. A small subset of participants reported higher levels of distress, suggesting the need for healthcare professionals offering PCa screening to identify these risk factors and offer additional information and support to men seeking PCa screening. © 2018 The Authors BJU

  2. Targeted prostate cancer screening in BRCA1 and BRCA2 mutation carriers

    DEFF Research Database (Denmark)

    Bancroft, Elizabeth K; Page, Elizabeth C; Castro, Elena

    2014-01-01

    AND PARTICIPANTS: We recruited men aged 40-69 yr with germline BRCA1/2 mutations and a control group of men who have tested negative for a pathogenic BRCA1 or BRCA2 mutation known to be present in their families. All men underwent prostate-specific antigen (PSA) testing at enrollment, and those men with PSA >3 ng......BACKGROUND: Men with germline breast cancer 1, early onset (BRCA1) or breast cancer 2, early onset (BRCA2) gene mutations have a higher risk of developing prostate cancer (PCa) than noncarriers. IMPACT (Identification of Men with a genetic predisposition to ProstAte Cancer: Targeted screening....../ml were offered prostate biopsy. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: PSA levels, PCa incidence, and tumour characteristics were evaluated. The Fisher exact test was used to compare the number of PCa cases among groups and the differences among disease types. RESULTS AND LIMITATIONS: We...

  3. APP, PSEN1, and PSEN2 mutations in early-onset Alzheimer disease: A genetic screening study of familial and sporadic cases.

    Science.gov (United States)

    Lanoiselée, Hélène-Marie; Nicolas, Gaël; Wallon, David; Rovelet-Lecrux, Anne; Lacour, Morgane; Rousseau, Stéphane; Richard, Anne-Claire; Pasquier, Florence; Rollin-Sillaire, Adeline; Martinaud, Olivier; Quillard-Muraine, Muriel; de la Sayette, Vincent; Boutoleau-Bretonniere, Claire; Etcharry-Bouyx, Frédérique; Chauviré, Valérie; Sarazin, Marie; le Ber, Isabelle; Epelbaum, Stéphane; Jonveaux, Thérèse; Rouaud, Olivier; Ceccaldi, Mathieu; Félician, Olivier; Godefroy, Olivier; Formaglio, Maite; Croisile, Bernard; Auriacombe, Sophie; Chamard, Ludivine; Vincent, Jean-Louis; Sauvée, Mathilde; Marelli-Tosi, Cecilia; Gabelle, Audrey; Ozsancak, Canan; Pariente, Jérémie; Paquet, Claire; Hannequin, Didier; Campion, Dominique

    2017-03-01

    Amyloid protein precursor (APP), presenilin-1 (PSEN1), and presenilin-2 (PSEN2) mutations cause autosomal dominant forms of early-onset Alzheimer disease (AD-EOAD). Although these genes were identified in the 1990s, variant classification remains a challenge, highlighting the need to colligate mutations from large series. We report here a novel update (2012-2016) of the genetic screening of the large AD-EOAD series ascertained across 28 French hospitals from 1993 onwards, bringing the total number of families with identified mutations to n = 170. Families were included when at least two first-degree relatives suffered from early-onset Alzheimer disease (EOAD) with an age of onset (AOO) ≤65 y in two generations. Furthermore, we also screened 129 sporadic cases of Alzheimer disease with an AOO below age 51 (44% males, mean AOO = 45 ± 2 y). APP, PSEN1, or PSEN2 mutations were identified in 53 novel AD-EOAD families. Of the 129 sporadic cases screened, 17 carried a PSEN1 mutation and 1 carried an APP duplication (13%). Parental DNA was available for 10 sporadic mutation carriers, allowing us to show that the mutation had occurred de novo in each case. Thirteen mutations (12 in PSEN1 and 1 in PSEN2) identified either in familial or in sporadic cases were previously unreported. Of the 53 mutation carriers with available cerebrospinal fluid (CSF) biomarkers, 46 (87%) had all three CSF biomarkers-total tau protein (Tau), phospho-tau protein (P-Tau), and amyloid β (Aβ)42-in abnormal ranges. No mutation carrier had the three biomarkers in normal ranges. One limitation of this study is the absence of functional assessment of the possibly and probably pathogenic variants, which should help their classification. Our findings suggest that a nonnegligible fraction of PSEN1 mutations occurs de novo, which is of high importance for genetic counseling, as PSEN1 mutational screening is currently performed in familial cases only. Among the 90 distinct mutations found in the whole

  4. APP, PSEN1, and PSEN2 mutations in early-onset Alzheimer disease: A genetic screening study of familial and sporadic cases.

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    Hélène-Marie Lanoiselée

    2017-03-01

    Full Text Available Amyloid protein precursor (APP, presenilin-1 (PSEN1, and presenilin-2 (PSEN2 mutations cause autosomal dominant forms of early-onset Alzheimer disease (AD-EOAD. Although these genes were identified in the 1990s, variant classification remains a challenge, highlighting the need to colligate mutations from large series.We report here a novel update (2012-2016 of the genetic screening of the large AD-EOAD series ascertained across 28 French hospitals from 1993 onwards, bringing the total number of families with identified mutations to n = 170. Families were included when at least two first-degree relatives suffered from early-onset Alzheimer disease (EOAD with an age of onset (AOO ≤65 y in two generations. Furthermore, we also screened 129 sporadic cases of Alzheimer disease with an AOO below age 51 (44% males, mean AOO = 45 ± 2 y. APP, PSEN1, or PSEN2 mutations were identified in 53 novel AD-EOAD families. Of the 129 sporadic cases screened, 17 carried a PSEN1 mutation and 1 carried an APP duplication (13%. Parental DNA was available for 10 sporadic mutation carriers, allowing us to show that the mutation had occurred de novo in each case. Thirteen mutations (12 in PSEN1 and 1 in PSEN2 identified either in familial or in sporadic cases were previously unreported. Of the 53 mutation carriers with available cerebrospinal fluid (CSF biomarkers, 46 (87% had all three CSF biomarkers-total tau protein (Tau, phospho-tau protein (P-Tau, and amyloid β (Aβ42-in abnormal ranges. No mutation carrier had the three biomarkers in normal ranges. One limitation of this study is the absence of functional assessment of the possibly and probably pathogenic variants, which should help their classification.Our findings suggest that a nonnegligible fraction of PSEN1 mutations occurs de novo, which is of high importance for genetic counseling, as PSEN1 mutational screening is currently performed in familial cases only. Among the 90 distinct mutations found in the

  5. The Accuracy of Seryl-tRNA Synthesis

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    Ita Gruic-Sovulj

    2002-01-01

    Full Text Available The high level of translational fidelity is ensured by various types of quality control mechanisms, which are adapted to prevent or correct naturally occurring mistakes. Accurate aminoacyl-tRNA synthesis is mostly dependent on the specificity of the aminoacyl-tRNA synthetases (aaRS, i.e. their ability to choose among competing structurally similar substrates. Our studies have revealed that accurate seryl-tRNA synthesis in yeast and plants is accomplished via tRNA-assisted optimization of amino acid binding to the active site of seryl-tRNA synthetase (SerRS. Based on our recent kinetic data, a mechanism is proposed by which transient protein : RNA complex activates the cognate amino acid more efficiently and more specifically than the apoenzyme alone. This may proceed via a tRNA induced conformational change in the enzyme’s active site. The influence of tRNASer, on the activation of serine by SerRS variants mutated in the active site, is much less pronounced. Although SerRS misactivates structurally similar threonine in vitro, the formation of such erroneous threonyl-adenylate is reduced in the presence of nonchargeable tRNASer analog. Thus, the sequence-specific tRNA : SerRS interactions enhance the accuracy of amino acid recognition. Another type of quality control mechanism in tRNA serylation is assumed to be based on the complex formation between SerRS and a nonsynthetase protein. Using in vivo interaction screen, yeast peroxin Pex21p was identified as SerRS interacting protein. This was confirmed by an in vitro binding assay. Kinetic experiments performed in the presence of Pex21p revealed that this peroxin acts as an activator of seryl-tRNA synthetase in the aminoacylation reaction.

  6. Mutations that alter a repeated ACCA element located at the 5' end of the Potato virus X genome affect RNA accumulation.

    Science.gov (United States)

    Park, Mi-Ri; Kwon, Sun-Jung; Choi, Hong-Soo; Hemenway, Cynthia L; Kim, Kook-Hyung

    2008-08-15

    The repeated ACCA or AC-rich sequence and structural (SL1) elements in the 5' non-translated region (NTR) of the Potato virus X (PVX) RNA play vital roles in the PVX life cycle by controlling translation, RNA replication, movement, and assembly. It has already been shown that the repeated ACCA or AC-rich sequence affect both gRNA and sgRNA accumulation, while not affecting minus-strand RNA accumulation, and are also required for host protein binding. The functional significance of the repeated ACCA sequence elements in the 5' NTR region was investigated by analyzing the effects of deletion and site-directed mutations on PVX replication in Nicotiana benthamiana plants and NT1 protoplasts. Substitution (ACCA into AAAA or UUUU) mutations introduced in the first (nt 10-13) element in the 5' NTR of the PVX RNA significantly affected viral replication, while mutations introduced in the second (nt 17-20) and third (nt 20-23) elements did not. The fourth (nt 29-32) ACCA element weakly affected virus replication, whereas mutations in the fifth (nt 38-41) significantly reduced virus replication due to the structure disruption of SL1 by AAAA and/or UUUU substitutions. Further characterization of the first ACCA element indicated that duplication of ACCA at nt 10-13 (nt 10-17, ACCAACCA) caused severe symptom development as compared to that of wild type, while deletion of the single element (nt 10-13), DeltaACCA) or tripling of this element caused reduced symptom development. Single- and double-nucleotide substitutions introduced into the first ACCA element revealed the importance of CC located at nt positions 11 and 12. Altogether, these results indicate that the first ACCA element is important for PVX replication.

  7. Inhibitors of MyD88-dependent proinflammatory cytokine production identified utilizing a novel RNA interference screening approach.

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    John S Cho

    2009-09-01

    Full Text Available The events required to initiate host defenses against invading pathogens involve complex signaling cascades comprised of numerous adaptor molecules, kinases, and transcriptional elements, ultimately leading to the production of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha. How these signaling cascades are regulated, and the proteins and regulatory elements participating are still poorly understood.We report here the development a completely random short-hairpin RNA (shRNA library coupled with a novel forward genetic screening strategy to identify inhibitors of Toll-like receptor (TLR dependent proinflammatory responses. We developed a murine macrophage reporter cell line stably transfected with a construct expressing diphtheria toxin-A (DT-A under the control of the TNF-alpha-promoter. Stimulation of the reporter cell line with the TLR ligand lipopolysaccharide (LPS resulted in DT-A induced cell death, which could be prevented by the addition of an shRNA targeting the TLR adaptor molecule MyD88. Utilizing this cell line, we screened a completely random lentiviral short hairpin RNA (shRNA library for sequences that inhibited TLR-mediated TNF-alpha production. Recovery of shRNA sequences from surviving cells led to the identification of unique shRNA sequences that significantly inhibited TLR4-dependent TNF-alpha gene expression. Furthermore, these shRNA sequences specifically blocked TLR2 but not TLR3-dependent TNF-alpha production.Thus, we describe the generation of novel tools to facilitate large-scale forward genetic screens in mammalian cells and the identification of potent shRNA inhibitors of TLR2 and TLR4- dependent proinflammatory responses.

  8. Gene mutation, quantitative mutagenesis, and mutagen screening in mammalian cells: study with the CHO/HGPRT system

    International Nuclear Information System (INIS)

    Hsie, A.W.

    1980-01-01

    We have employed CHO cells to develop and define a set of stringent conditions for studying mutation induction to TG resistance. Several lines of evidence support the CHO/HGPRT system as a specific-locus mutational assay. The system permits quantification of mutation at the HGPRT locus induced by various physical and chemical mutagens. The quantitative nature of the system provides a basis for the study of structure-function relationships of various classes of chemical mutagens. The intra- and interlaboratory reproducibility of this system suggests its potential for screening environmental agents for mutagenic activity

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

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    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.

  10. The somatic FAH C.1061C>A change counteracts the frequent FAH c.1062+5G>A mutation and permits U1snRNA-based splicing correction.

    Science.gov (United States)

    Scalet, Daniela; Sacchetto, Claudia; Bernardi, Francesco; Pinotti, Mirko; van de Graaf, Stan F J; Balestra, Dario

    2018-05-01

    In tyrosinaemia type 1(HT1), a mosaic pattern of fumarylacetoacetase (FAH) immunopositive or immunonegative nodules in liver tissue has been reported in many patients. This aspect is generally explained by a spontaneous reversion of the mutation into a normal genotype. In one HT1 patient carrying the frequent FAH c.1062+5G>A mutation, a second somatic change (c.1061C>A) has been reported in the same allele, and found in immunopositive nodules. Here, we demonstrated that the c.1062+5G>A prevents usage of the exon 12 5' splice site (ss), even when forced by an engineered U1snRNA specifically designed on the FAH 5'ss to strengthen its recognition. Noticeably the new somatic c.1061C>A change, in linkage with the c.1062+5G>A mutation, partially rescues the defective 5'ss and is associated to trace level (~5%) of correct transcripts. Interestingly, this combined genetic condition strongly favored the rescue by the engineered U1snRNA, with correct transcripts reaching up to 60%. Altogether, these findings elucidate the molecular basis of HT1 caused by the frequent FAH c.1062+5G>A mutation, and demonstrate the compensatory effect of the c.1061C>A change in promoting exon definition, thus unraveling a rare mechanism leading to FAH immune-reactive mosaicism.

  11. Clinical and biological characteristics of cervical neoplasias with FGFR3 mutation

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    Thiery Jean

    2005-05-01

    Full Text Available Abstract Background We have previously reported activating mutations of the gene coding for the fibroblast growth factor receptor 3 (FGFR3 in invasive cervical carcinoma. To further analyze the role of FGFR3 in cervical tumor progression, we extended our study to screen a total of 75 invasive tumors and 80 cervical intraepithelial neoplasias (40 low-grade and 40 high-grade lesions. Results Using single strand conformation polymorphism (SSCP followed by DNA sequencing, we found FGFR3 mutation (S249C in all cases in 5% of invasive cervical carcinomas and no mutation in intraepithelial lesions. These results suggest that, unlike in bladder carcinoma, FGFR3 mutation does not or rarely occur in non invasive lesions. Compared to patients with wildtype FGFR3 tumor, patients with S249C FGFR3 mutated tumors were older (mean age 64 vs. 49.4 years, P = 0.02, and were more likely to be associated with a non-16/18 HPV type in their tumor. Gene expression analysis demonstrated that FGFR3 mutated tumors were associated with higher FGFR3b mRNA expression levels compared to wildtype FGFR3 tumors. Supervised analysis of Affymetrix expression data identified a significant number of genes specifically differentially expressed in tumors with respect to FGFR3 mutation status. Conclusion This study suggest that tumors with FGFR3 mutation appear to have distinctive clinical and biological characteristics that may help in defining a population of patients for FGFR3 mutation screening.

  12. Peptide nucleic acid probe-based fluorescence melting curve analysis for rapid screening of common JAK2, MPL, and CALR mutations.

    Science.gov (United States)

    Park, Joonhong; Song, Minsik; Jang, Woori; Chae, Hyojin; Lee, Gun Dong; Kim, KyungTak; Park, Heekyung; Kim, Myungshin; Kim, Yonggoo

    2017-02-01

    We developed and evaluated the feasibility of peptide nucleic acid (PNA)-based fluorescence melting curve analysis (FMCA) to detect common mutations in myeloproliferative neoplasms (MPNs). We have set up two separate reactions of PNA-based FMCA: JAK2 V617F &CALR p.Leu367fs*46 (set A) and MPL W515L/K &CALR p.Lys385fs*47 (set B). Clinical usefulness was validated with allele-specific real-time PCR, fragment analysis, Sanger sequencing in 57 BCR-ABL1-negative MPNs. The limit of detection (LOD) of PNA-based FMCA was approximately 10% for each mutation and interference reactions using mixtures of different mutations were not observed. Non-specific amplification was not observed in normal control. PNA-based FMCA was able to detect all JAK2 V617F (n=20), CALR p.Leu367fs*46 (n=10) and p.Lys385fs*47 (n=8). Three of six MPL mutations were detected except three samples with low mutant concentration in out of LOD. JAK2 exon 12 mutations (n=7) were negative without influencing V617F results. Among six variant CALR exon 9 mutations, two were detected by this method owing to invading of probe binding site. PNA-based FMCA for detecting common JAK2, MPL, and CALR mutations is a rapid, simple, and sensitive technique in BCR-ABL1-negative MPNs with >10% mutant allele at the time of initial diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. G-Quadruplexes influence pri-microRNA processing.

    Science.gov (United States)

    Rouleau, Samuel G; Garant, Jean-Michel; Bolduc, François; Bisaillon, Martin; Perreault, Jean-Pierre

    2018-02-01

    RNA G-Quadruplexes (G4) have been shown to possess many biological functions, including the regulation of microRNA (miRNA) biogenesis and function. However, their impact on pri-miRNA processing remains unknown. We identified G4 located near the Drosha cleavage site in three distinct pri-miRNAs: pri-mir200c, pri-mir451a, and pri-mir497. The folding of the potential G4 motifs was determined in solution. Subsequently, mutations disrupting G4 folding led to important changes in the mature miRNAs levels in cells. Moreover, using small antisense oligonucleotides binding to the pri-miRNA, it was possible to modulate, either positively or negatively, the mature miRNA levels. Together, these data demonstrate that G4 motifs could contribute to the regulation of pri-mRNA processing, a novel role for G4. Considering that bio-informatics screening indicates that between 9% and 50% of all pri-miRNAs contain a putative G4, these structures possess interesting potential as future therapeutic targets.

  14. 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.

  15. Altered Pre-mRNA Splicing Caused by a Novel Intronic Mutation c.1443+5G>A in the Dihydropyrimidinase (DPYS) Gene

    NARCIS (Netherlands)

    Nakajima, Yoko; Meijer, Judith; Zhang, Chunhua; Wang, Xu; Kondo, Tomomi; Ito, Tetsuya; Dobritzsch, Doreen; van Kuilenburg, André B. P.

    2016-01-01

    Dihydropyrimidinase (DHP) deficiency is an autosomal recessive disease caused by mutations in the DPYS gene. Patients present with highly elevated levels of dihydrouracil and dihydrothymine in their urine, blood and cerebrospinal fluid. The analysis of the effect of mutations in DPYS on pre-mRNA

  16. Identification of Rift Valley fever virus nucleocapsid protein-RNA binding inhibitors using a high-throughput screening assay.

    Science.gov (United States)

    Ellenbecker, Mary; Lanchy, Jean-Marc; Lodmell, J Stephen

    2012-09-01

    Rift Valley fever virus (RVFV) is an emerging infectious pathogen that causes severe disease in humans and livestock and has the potential for global spread. Currently, there is no proven effective treatment for RVFV infection, and there is no licensed vaccine. Inhibition of RNA binding to the essential viral nucleocapsid (N) protein represents a potential antiviral therapeutic strategy because all of the functions performed by N during infection involve RNA binding. To target this interaction, we developed a fluorescence polarization-based high-throughput drug-screening assay and tested 26 424 chemical compounds for their ability to disrupt an N-RNA complex. From libraries of Food and Drug Administration-approved drugs, druglike molecules, and natural product extracts, we identified several lead compounds that are promising candidates for medicinal chemistry.

  17. Screening for Antifibrotic Compounds Using High Throughput System Based on Fluorescence Polarization

    Directory of Open Access Journals (Sweden)

    Branko Stefanovic

    2014-04-01

    Full Text Available Fibroproliferative diseases are one of the leading causes of death worldwide. They are characterized by reactive fibrosis caused by uncontrolled synthesis of type I collagen. There is no cure for fibrosis and development of therapeutics that can inhibit collagen synthesis is urgently needed. Collagen α1(I mRNA and α2(I mRNA encode for type I collagen and they have a unique 5' stem-loop structure in their 5' untranslated regions (5'SL. Collagen 5'SL binds protein LARP6 with high affinity and specificity. The interaction between LARP6 and the 5'SL is critical for biosynthesis of type I collagen and development of fibrosis in vivo. Therefore, this interaction represents is an ideal target to develop antifibrotic drugs. A high throughput system to screen for chemical compounds that can dissociate LARP6 from 5'SL has been developed. It is based on fluorescence polarization and can be adapted to screen for inhibitors of other protein-RNA interactions. Screening of 50,000 chemical compounds yielded a lead compound that can inhibit type I collagen synthesis at nanomolar concentrations. The development, characteristics, and critical appraisal of this assay are presented.

  18. Selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis.

    Science.gov (United States)

    Smola, Matthew J; Rice, Greggory M; Busan, Steven; Siegfried, Nathan A; Weeks, Kevin M

    2015-11-01

    Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) chemistries exploit small electrophilic reagents that react with 2'-hydroxyl groups to interrogate RNA structure at single-nucleotide resolution. Mutational profiling (MaP) identifies modified residues by using reverse transcriptase to misread a SHAPE-modified nucleotide and then counting the resulting mutations by massively parallel sequencing. The SHAPE-MaP approach measures the structure of large and transcriptome-wide systems as accurately as can be done for simple model RNAs. This protocol describes the experimental steps, implemented over 3 d, that are required to perform SHAPE probing and to construct multiplexed SHAPE-MaP libraries suitable for deep sequencing. Automated processing of MaP sequencing data is accomplished using two software packages. ShapeMapper converts raw sequencing files into mutational profiles, creates SHAPE reactivity plots and provides useful troubleshooting information. SuperFold uses these data to model RNA secondary structures, identify regions with well-defined structures and visualize probable and alternative helices, often in under 1 d. SHAPE-MaP can be used to make nucleotide-resolution biophysical measurements of individual RNA motifs, rare components of complex RNA ensembles and entire transcriptomes.

  19. Machine Learning Approaches Toward Building Predictive Models for Small Molecule Modulators of miRNA and Its Utility in Virtual Screening of Molecular Databases.

    Science.gov (United States)

    Periwal, Vinita; Scaria, Vinod

    2017-01-01

    The ubiquitous role of microRNAs (miRNAs) in a number of pathological processes has suggested that they could act as potential drug targets. RNA-binding small molecules offer an attractive means for modulating miRNA function. The availability of bioassay data sets for a variety of biological assays and molecules in public domain provides a new opportunity toward utilizing them to create models and further utilize them for in silico virtual screening approaches to prioritize or assign potential functions for small molecules. Here, we describe a computational strategy based on machine learning for creation of predictive models from high-throughput biological screens for virtual screening of small molecules with the potential to inhibit microRNAs. Such models could be potentially used for computational prioritization of small molecules before performing high-throughput biological assay.

  20. The 5'UTR-specific mutation in VEEV TC-83 genome has a strong effect on RNA replication and subgenomic RNA synthesis, but not on translation of the encoded proteins.

    Science.gov (United States)

    Kulasegaran-Shylini, Raghavendran; Thiviyanathan, Varatharasa; Gorenstein, David G; Frolov, Ilya

    2009-04-25

    Venezuelan equine encephalitis virus (VEEV) is one of the most pathogenic members of the Alphavirus genus in the Togaviridae family. Viruses in the VEEV serocomplex continuously circulate in the Central and South America. The only currently available attenuated strain VEEV TC-83 is being used only for vaccination of at-risk laboratory workers and military personnel. Its attenuated phenotype was shown to rely only on two point mutations, one of which, G3A, was found in the 5' untranslated region (5'UTR) of the viral genome. Our data demonstrate that the G3A mutation strongly affects the secondary structure of VEEV 5'UTR, but has only a minor effect on translation. The indicated mutation increases replication of the viral genome, downregulates transcription of the subgenomic RNA, and, thus, affects the ratio of genomic and subgenomic RNA synthesis. These findings and the previously reported G3A-induced, higher sensitivity of VEEV TC-83 to IFN-alpha/beta suggest a plausible explanation for its attenuated phenotype.

  1. A Method to Predict the Structure and Stability of RNA/RNA Complexes.

    Science.gov (United States)

    Xu, Xiaojun; Chen, Shi-Jie

    2016-01-01

    RNA/RNA interactions are essential for genomic RNA dimerization and regulation of gene expression. Intermolecular loop-loop base pairing is a widespread and functionally important tertiary structure motif in RNA machinery. However, computational prediction of intermolecular loop-loop base pairing is challenged by the entropy and free energy calculation due to the conformational constraint and the intermolecular interactions. In this chapter, we describe a recently developed statistical mechanics-based method for the prediction of RNA/RNA complex structures and stabilities. The method is based on the virtual bond RNA folding model (Vfold). The main emphasis in the method is placed on the evaluation of the entropy and free energy for the loops, especially tertiary kissing loops. The method also uses recursive partition function calculations and two-step screening algorithm for large, complicated structures of RNA/RNA complexes. As case studies, we use the HIV-1 Mal dimer and the siRNA/HIV-1 mutant (T4) to illustrate the method.

  2. A single mutation in the 15S rRNA gene confers nonsense suppressor activity and interacts with mRF1 the release factor in yeast mitochondria

    Directory of Open Access Journals (Sweden)

    Ali Gargouri

    2015-08-01

    Full Text Available We have determined the nucleotide sequence of the mim3-1 mitochondrial ribosomal suppressor, acting on ochre mitochondrial mutations and one frameshift mutation in Saccharomyces cerevisiae. The 15s rRNA suppressor gene contains a G633 to C transversion. Yeast mitochondrial G633 corresponds to G517 of the E.coli 15S rRNA, which is occupied by an invariant G in all known small rRNA sequences. Interestingly, this mutation has occurred at the same position as the known MSU1 mitochondrial suppressor which changes G633 to A. The suppressor mutation lies in a highly conserved region of the rRNA, known in E.coli as the 530-loop, interacting with the S4, S5 and S12 ribosomal proteins. We also show an interesting interaction between the mitochondrial mim3-1 and the nuclear nam3-1 suppressors, both of which have the same action spectrum on mitochondrial mutations: nam3-1 abolishes the suppressor effect when present with mim3-1 in the same haploid cell. We discuss these results in the light of the nature of Nam3, identified by [1] as the yeast mitochondrial translation release factor. A hypothetical mechanism of suppression by "ribosome shifting" is also discussed in view of the nature of mutations suppressed and not suppressed.

  3. Optimized Mitochondrial Targeting of Proteins Encoded by Modified mRNAs Rescues Cells Harboring Mutations in mtATP6

    Directory of Open Access Journals (Sweden)

    Randall Marcelo Chin

    2018-03-01

    Full Text Available Summary: Mitochondrial disease may be caused by mutations in the protein-coding genes of the mitochondrial genome. A promising strategy for treating such diseases is allotopic expression—the translation of wild-type copies of these proteins in the cytosol, with subsequent translocation into the mitochondria, resulting in rescue of mitochondrial function. In this paper, we develop an automated, quantitative, and unbiased screening platform to evaluate protein localization and mitochondrial morphology. This platform was used to compare 31 mitochondrial targeting sequences and 15 3′ UTRs in their ability to localize up to 9 allotopically expressed proteins to the mitochondria and their subsequent impact on mitochondrial morphology. Taking these two factors together, we synthesized chemically modified mRNAs that encode for an optimized allotopic expression construct for mtATP6. These mRNAs were able to functionally rescue a cell line harboring the 8993T > G point mutation in the mtATP6 gene. : Allotopic expression of proteins normally encoded by mtDNA is a promising therapy for mitochondrial disease. Chin et al. use an unbiased and high-content imaging-based screening platform to optimize allotopic expression. Modified mRNAs encoding for the optimized allotopic expression constructs rescued the respiration and growth of mtATP6-deficient cells. Keywords: mitochondria, mitochondrial disease, mRNA, modified mRNA, ATP6, allotopic expression, rare disease, gene therapy, screening, high content imaging

  4. Investigation of the Mitochondrial ATPase 6/8 and tRNA(Lys) Genes Mutations in Autism.

    Science.gov (United States)

    Piryaei, Fahimeh; Houshmand, Massoud; Aryani, Omid; Dadgar, Sepideh; Soheili, Zahra-Soheila

    2012-01-01

    Autism results from developmental factors that affect many or all functional brain systems. Brain is one of tissues which are crucially in need of adenosine triphosphate (ATP). Autism is noticeably affected by mitochondrial dysfunction which impairs energy metabolism. Considering mutations within ATPase 6, ATPase 8 and tRNA(Lys) genes, associated with different neural diseases, and the main role of ATPase 6/8 in energy generation, we decided to investigate mutations on these mtDNA-encoded genes to reveal their roles in autism pathogenesis. In this experimental study, mutation analysis for the mentioned genes were performed in a cohort of 24 unrelated patients with idiopathic autism by employing amplicon sequencing of mtDNA fragments. In this study, 12 patients (50%) showed point mutations that represent a significant correlation between autism and mtDNA variations. Most of the identified substitutions (55.55%) were observed on MT-ATP6, altering some conserved amino acids to other ones which could potentially affect ATPase 6 function. Mutations causing amino acid replacement denote involvement of mtDNA genes, especially ATPase 6 in autism pathogenesis. MtDNA mutations in relation with autism could be remarkable to realize an understandable mechanism of pathogenesis in order to achieve therapeutic solutions.

  5. Screening of siRNA nanoparticles for delivery to airway epithelial cells using high-content analysis

    LENUS (Irish Health Repository)

    Hibbitts, Alan

    2011-08-01

    Aims: Delivery of siRNA to the lungs via inhalation offers a unique opportunity to develop a new treatment paradigm for a range of respiratory conditions. However, progress has been greatly hindered by safety and delivery issues. This study developed a high-throughput method for screening novel nanotechnologies for pulmonary siRNA delivery. Methodology: Following physicochemical analysis, the ability of PEI–PEG–siRNA nanoparticles to facilitate siRNA delivery was determined using high-content analysis (HCA) in Calu-3 cells. Results obtained from HCA were validated using confocal microscopy. Finally, cytotoxicity of the PEI–PEG–siRNA particles was analyzed by HCA using the Cellomics® multiparameter cytotoxicity assay. Conclusion: PEI–PEG–siRNA nanoparticles facilitated increased siRNA uptake and luciferase knockdown in Calu-3 cells compared with PEI–siRNA.

  6. A new mitochondrial point mutation in the transfer RNA(Lys) gene associated with progressive external ophthalmoplegia with impaired respiratory regulation.

    Science.gov (United States)

    Wolf, Joachim; Obermaier-Kusser, Bert; Jacobs, Martina; Milles, Cornelia; Mörl, Mario; von Pein, Harald D; Grau, Armin J; Bauer, Matthias F

    2012-05-15

    We report a novel heteroplasmic point mutation G8299A in the gene for mitochondrial tRNA(Lys) in a patient with progressive external ophthalmoplegia complicated by recurrent respiratory insufficiency. Biochemical analysis of respiratory chain complexes in muscle homogenate showed a combined complex I and IV deficiency. The transition does not represent a known neutral polymorphism and affects a position in the tRNA acceptor stem which is conserved in primates, leading to a destabilization of this functionally important domain. In vitro analysis of an essential maturation step of the tRNA transcript indicates the probable pathogenicity of this mutation. We hypothesize that there is a causal relationship between the novel G8299A transition and progressive external ophthalmoplegia with recurrent respiratory failure due to a depressed respiratory drive. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Two-miRNA classifiers differentiate mutation-negative follicular thyroid carcinomas and follicular thyroid adenomas in fine needle aspirations with high specificity

    DEFF Research Database (Denmark)

    Stokowy, Tomasz; Wojtas, Bartosz; Jarzab, Barbara

    2016-01-01

    Diagnosis of thyroid by fine needle aspiration is challenging for the "indeterminate" category and can be supported by molecular testing. We set out to identify miRNA markers that could be used in a diagnostic setting to improve the discrimination of mutation-negative indeterminate fine needle...... aspirations. miRNA high-throughput sequencing was performed for freshly frozen tissue samples of 19 RAS and PAX8/PPARG mutation-negative follicular thyroid carcinomas, and 23 RAS and PAX8/PPARG mutation-negative follicular adenomas. Differentially expressed miRNAs were validated by quantitative polymerase...... chain reaction in a set of 44 fine needle aspiration samples representing 24 follicular thyroid carcinomas and 20 follicular adenomas. Twenty-six miRNAs characterized by a significant differential expression between follicular thyroid carcinomas and follicular adenomas were identified. Nevertheless...

  8. Mutations in MARS identified in a specific type of pulmonary alveolar proteinosis alter methionyl-tRNA synthetase activity.

    Science.gov (United States)

    Comisso, Martine; Hadchouel, Alice; de Blic, Jacques; Mirande, Marc

    2018-05-18

    Biallelic missense mutations in MARS are responsible for rare but severe cases of pulmonary alveolar proteinosis (PAP) prevalent on the island of La Réunion. MARS encodes cytosolic methionyl-tRNA synthetase (MetRS), an essential translation factor. The multisystemic effects observed in patients with this form of PAP are consistent with a loss-of-function defect in an ubiquitously expressed enzyme. The pathophysiological mechanisms involved in MARS-related PAP are currently unknown. In this work, we analyzed the effect of the PAP-related mutations in MARS on the thermal stability and on the catalytic parameters of the MetRS mutants, relative to wild-type. The effect of these mutations on the structural integrity of the enzyme as a member of the cytosolic multisynthetase complex was also investigated. Our results establish that the PAP-related substitutions in MetRS impact the tRNA Met -aminoacylation reaction especially at the level of methionine recognition, and suggest a direct link between the loss of activity of the enzyme and the pathological disorders in PAP. © 2018 Federation of European Biochemical Societies.

  9. Cardiac abnormalities in diabetic patients with mutation in the mitochondrial tRNA Leu(UUR)Gene

    International Nuclear Information System (INIS)

    Ueno, Hiroshi; Shiotani, Hideyuki

    1999-01-01

    An A-to-G transition at position 3243 of the mitochondrial DNA is known to be a pathogenic factor for mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS), diabetes and cardiomyopathy. This mutation causes dysfunction of the central nervous system in MELAS. Because the heart, as well as the brain and nervous system, is highly dependent on the energy produced by mitochondrial oxidation, these tissues are more vulnerable to mitochondrial defects. Cardiac abnormalities were assessed in 10 diabetic patients associated with this mutation using echocardiography and 123 I-metaiodobenzylguanidine (MIBG) scintigraphy, and compared with 19 diabetic patients without the mutation. Duration of diabetes, therapy, control of blood glucose and diabetic complications, such as diabetic retinopathy and nephropathy, were not different between the 2 groups. Diabetic patients with the mutation had a significantly thicker interventricular septum (16.8±3.7 vs 11.0±1.6 mm, p 0.05). In conclusion, left ventricular hypertrophy with or without abnormal wall motion and severely reduced MIBG uptake may be characteristic in diabetic patients with a mutation in the mitochondrial tRNA Leu(UUR) gene. (author)

  10. A novel homozygous stop-codon mutation in human HFE responsible for nonsense-mediated mRNA decay.

    Science.gov (United States)

    Padula, Maria Carmela; Martelli, Giuseppe; Larocca, Marilena; Rossano, Rocco; Olivieri, Attilio

    2014-09-01

    HFE-hemochromatosis (HH) is an autosomal disease characterized by excessive iron absorption. Homozygotes for H63D variant, and still less H63D heterozygotes, generally do not express HH phenotype. The data collected in our previous study in the province of Matera (Basilicata, Italy) underlined that some H63D carriers showed altered iron metabolism, without additional factors. In this study, we selected a cohort of 10/22 H63D carriers with severe biochemical iron overload (BIO). Additional analysis was performed for studying HFE exons, exon-intron boundaries, and untranslated regions (UTRs) by performing DNA extraction, PCR amplification and sequencing. The results showed a novel substitution (NM_000410.3:c.847C>T) in a patient exon 4 (GenBankJQ478433); it introduces a premature stop-codon (PTC). RNA extraction and reverse-transcription were also performed. Quantitative real-time PCR was carried out for verifying if our aberrant mRNA is targeted for nonsense-mediated mRNA decay (NMD); we observed that patient HFE mRNA was expressed much less than calibrator, suggesting that the mutated HFE protein cannot play its role in iron metabolism regulation, resulting in proband BIO. Our finding is the first evidence of a variation responsible for a PTC in iron cycle genes. The genotype-phenotype correlation observed in our cases could be related to the additional mutation. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. CRISPR/Cas9-mediated gene knockout is insensitive to target copy number but is dependent on guide RNA potency and Cas9/sgRNA threshold expression level.

    Science.gov (United States)

    Yuen, Garmen; Khan, Fehad J; Gao, Shaojian; Stommel, Jayne M; Batchelor, Eric; Wu, Xiaolin; Luo, Ji

    2017-11-16

    CRISPR/Cas9 is a powerful gene editing tool for gene knockout studies and functional genomic screens. Successful implementation of CRISPR often requires Cas9 to elicit efficient target knockout in a population of cells. In this study, we investigated the role of several key factors, including variation in target copy number, inherent potency of sgRNA guides, and expression level of Cas9 and sgRNA, in determining CRISPR knockout efficiency. Using isogenic, clonal cell lines with variable copy numbers of an EGFP transgene, we discovered that CRISPR knockout is relatively insensitive to target copy number, but is highly dependent on the potency of the sgRNA guide sequence. Kinetic analysis revealed that most target mutation occurs between 5 and 10 days following Cas9/sgRNA transduction, while sgRNAs with different potencies differ by their knockout time course and by their terminal-phase knockout efficiency. We showed that prolonged, low level expression of Cas9 and sgRNA often fails to elicit target mutation, particularly if the potency of the sgRNA is also low. Our findings provide new insights into the behavior of CRISPR/Cas9 in mammalian cells that could be used for future improvement of this platform. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

  12. HSJ1-related hereditary neuropathies: novel mutations and extended clinical spectrum.

    Science.gov (United States)

    Gess, Burkhard; Auer-Grumbach, Michaela; Schirmacher, Anja; Strom, Tim; Zitzelsberger, Manuela; Rudnik-Schöneborn, Sabine; Röhr, Dominik; Halfter, Hartmut; Young, Peter; Senderek, Jan

    2014-11-04

    To determine the nature and frequency of HSJ1 mutations in patients with hereditary motor and hereditary motor and sensory neuropathies. Patients were screened for mutations by genome-wide or targeted linkage and homozygosity studies, whole-exome sequencing, and Sanger sequencing. RNA and protein studies of skin fibroblasts were used for functional characterization. We describe 2 additional mutations in the HSJ1 gene in a cohort of 90 patients with autosomal recessive distal hereditary motor neuropathy (dHMN) and Charcot-Marie-Tooth disease type 2 (CMT2). One family with a dHMN phenotype showed the homozygous splice-site mutation c.229+1G>A, which leads to retention of intron 4 in the HSJ1 messenger RNA with a premature stop codon and loss of protein expression. Another family, presenting with a CMT2 phenotype, carried the homozygous missense mutation c.14A>G (p.Tyr5Cys). This mutation was classified as likely disease-related by several automatic algorithms for prediction of possible impact of an amino acid substitution on the structure and function of proteins. Both mutations cosegregated with autosomal recessive inheritance of the disease and were absent from the general population. Taken together, in our cohort of 90 probands, we confirm that HSJ1 mutations are a rare but detectable cause of autosomal recessive dHMN and CMT2. We provide clinical and functional information on an HSJ1 splice-site mutation and report the detailed phenotype of 2 patients with CMT2, broadening the phenotypic spectrum of HSJ1-related neuropathies. © 2014 American Academy of Neurology.

  13. Nuclear modifier MTO2 modulates the aminoglycoside-sensitivity of mitochondrial 15S rRNA C1477G mutation in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Xiangyu He

    Full Text Available The phenotypic manifestations of mitochondrial DNA (mtDNA mutations are modulated by mitochondrial DNA haplotypes, nuclear modifier genes and environmental factors. The yeast mitochondrial 15S rRNA C1477G (P(R or P(R 454 mutation corresponds to the human 12S rRNA C1494T and A1555G mutations, which are well known as primary factors for aminoglycoside-induced nonsyndromic deafness. Here we report that the deletion of the nuclear modifier gene MTO2 suppressed the aminoglycoside-sensitivity of mitochondrial 15S rRNA C1477G mutation in Saccharomyces cerevisiae. First, the strain with a single mtDNA C1477G mutation exhibited hypersensitivity to neomycin. Functional assays indicated that the steady-state transcription level of mitochondrial DNA, the mitochondrial respiratory rate, and the membrane potential decreased significantly after neomycin treatment. The impaired mitochondria could not produce sufficient energy to maintain cell viability. Second, when the mto2 null and the mitochondrial C1477G mutations co-existed (mto2(P(R, the oxygen consumption rate in the double mutant decreased markedly compared to that of the control strains (MTO2(P(S, mto2(P(S and MTO2(P(R. The expression levels of the key glycolytic genes HXK2, PFK1 and PYK1 in the mto2(P(R strain were stimulated by neomycin and up-regulated by 89%, 112% and 55%, respectively. The enhanced glycolysis compensated for the respiratory energy deficits, and could be inhibited by the glycolytic enzyme inhibitor. Our findings in yeast will provide a new insight into the pathogenesis of human deafness.

  14. A nonsense mutation causing decreased levels of insulin receptor mRNA: Detection by a simplified technique for direct sequencing of genomic DNA amplified by the polymerase chain reaction

    International Nuclear Information System (INIS)

    Kadowaki, T.; Kadowaki, H.; Taylor, S.I.

    1990-01-01

    Mutations in the insulin receptor gene can render the cell resistant to the biological action of insulin. The authors have studied a patient with leprechaunism (leprechaun/Minn-1), a genetic syndrome associated with intrauterine growth retardation and extreme insulin resistance. Genomic DNA from the patient was amplified by the polymerase chain reaction catalyzed by Thermus aquaticus (Taq) DNA polymerase, and the amplified DNA was directly sequenced. A nonsense mutations was identified at codon 897 in exon 14 in the paternal allele of the patient's insulin receptor gene. Levels of insulin receptor mRNA are decreased to <10% of normal in Epstein-Barr virus-transformed lymphoblasts and cultured skin fibroblasts from this patient. Thus, this nonsense mutation appears to cause a decrease in the levels of insulin receptor mRNA. In addition, they have obtained indirect evidence that the patient's maternal allele of the insulin receptor gene contains a cis-acting dominant mutation that also decreases the level of mRNA, but by a different mechanism. The nucleotide sequence of the entire protein-coding domain and the sequences of the intron-exon boundaries for all 22 exons of the maternal allele were normal. Presumably, the mutation in the maternal allele maps elsewhere in the insulin receptor gene. Thus, they conclude that the patient is a compound heterozygote for two cis-acting dominant mutations in the insulin receptor gene: (i) a nonsense mutation in the paternal allel that reduces the level of insulin receptor mRNA and (ii) an as yet unidentified mutation in the maternal allele that either decreases the rate of transcription or decreases the stability of the mRNA

  15. Structural and mutational analyses of cis-acting sequences in the 5'-untranslated region of satellite RNA of bamboo mosaic potexvirus

    International Nuclear Information System (INIS)

    Annamalai, Padmanaban; Hsu, Y.-H.; Liu, Y.-P.; Tsai, C.-H.; Lin, N.-S.

    2003-01-01

    The satellite RNA of Bamboo mosaic virus (satBaMV) contains on open reading frame for a 20-kDa protein that is flanked by a 5'-untranslated region (UTR) of 159 nucleotides (nt) and a 3'-UTR of 129 nt. A secondary structure was predicted for the 5'-UTR of satBaMV RNA, which folds into a large stem-loop (LSL) and a small stem-loop. Enzymatic probing confirmed the existence of LSL (nt 8-138) in the 5'-UTR. The essential cis-acting sequences in the 5'-UTR required for satBaMV RNA replication were determined by deletion and substitution mutagenesis. Their replication efficiencies were analyzed in Nicotiana benthamiana protoplasts and Chenopodium quinoa plants coinoculated with helper BaMV RNA. All deletion mutants abolished the replication of satBaMV RNA, whereas mutations introduced in most of the loop regions and stems showed either no replication or a decreased replication efficiency. Mutations that affected the positive-strand satBaMV RNA accumulation also affected the accumulation of negative-strand RNA; however, the accumulation of genomic and subgenomic RNAs of BaMV were not affected. Moreover, covariation analyses of natural satBaMV variants provide substantial evidence that the secondary structure in the 5'-UTR of satBaMV is necessary for efficient replication

  16. Screening for NDP mutations in 44 unrelated patients with familial exudative vitreoretinopathy or Norrie disease.

    Science.gov (United States)

    Yang, Huiqin; Li, Shiqiang; Xiao, Xueshan; Guo, Xiangming; Zhang, Qingjiong

    2012-08-01

    To screen mutations in the norrin (NDP) gene in 44 unrelated Chinese patients with familial exudative vitreoretinopathy (FEVR, 38 cases) or Norrie disease (6 cases) and to describe the associated phenotypes. Of the 44 patients, mutation in FZD4, LRP5, and TSPAN12 was excluded in 38 patients with FEVR in previous study. Sanger sequencing was used to analyze the 2 coding exons and their adjacent regions of NDP in the 44 patients. Clinical data were presented for patients with mutation. NDP variants in 5 of the 6 patients with Norrie disease were identified, including a novel missense mutation (c.164G>A, p.Cys55Phe) in one patient, two known missense mutations (c.122G>A, p.Arg41Lys; c.220C>T, p.Arg74Cys) in two patients, and a gross deletion encompassing the two coding exons in two patients. Of the 5 patients, 3 had a family history and 2 were singleton cases. No mutation in NDP was detected in the 38 patients with FEVR. NDP mutations are common cause of Norrie disease but might be rare cause for FEVR in Chinese.

  17. Targeted Prostate Cancer Screening in BRCA1 and BRCA2 Mutation Carriers: Results from the Initial Screening Round of the IMPACT Study

    NARCIS (Netherlands)

    Bancroft, Elizabeth K.; Page, Elizabeth C.; Castro, Elena; Lilja, Hans; Vickers, Andrew; Sjoberg, Daniel; Assel, Melissa; Foster, Christopher S.; Mitchell, Gillian; Drew, Kate; Mæhle, Lovise; Axcrona, Karol; Evans, D. Gareth; Bulman, Barbara; Eccles, Diana; McBride, Donna; van Asperen, Christi; Vasen, Hans; Kiemeney, Lambertus A.; Ringelberg, Janneke; Cybulski, Cezary; Wokolorczyk, Dominika; Selkirk, Christina; Hulick, Peter J.; Bojesen, Anders; Skytte, Anne-Bine; Lam, Jimmy; Taylor, Louise; Oldenburg, Rogier; Cremers, Ruben; Verhaegh, Gerald; van Zelst-Stams, Wendy A.; Oosterwijk, Jan C.; Blanco, Ignacio; Salinas, Monica; Cook, Jackie; Rosario, Derek J.; Buys, Saundra; Conner, Tom; Ausems, Margreet G.; Ong, Kai-Ren; Hoffman, Jonathan; Domchek, Susan; Powers, Jacquelyn; Teixeira, Manuel R.; Maia, Sofia; Foulkes, William D.; Taherian, Nassim; Ruijs, Marielle; van Os, Theo

    2014-01-01

    Background: Men with germline breast cancer 1, early onset (BRCA1) or breast cancer 2, early onset (BRCA2) gene mutations have a higher risk of developing prostate cancer (PCa) than noncarriers. IMPACT (Identification of Men with a genetic predisposition to ProstAte Cancer: Targeted screening in

  18. Targeted Prostate Cancer Screening in BRCA1 and BRCA2 Mutation Carriers : Results from the Initial Screening Round of the IMPACT Study

    NARCIS (Netherlands)

    Bancroft, Elizabeth K.; Page, Elizabeth C.; Castro, Elena; Lilja, Hans; Vickers, Andrew; Sjoberg, Daniel; Assel, Melissa; Foster, Christopher S.; Mitchell, Gillian; Drew, Kate; Maehle, Lovise; Axcrona, Karol; Evans, D. Gareth; Bulman, Barbara; Eccles, Diana; McBride, Donna; van Asperen, Christi; Vasen, Hans; Kiemeney, Lambertus A.; Ringelberg, Janneke; Cybulski, Cezary; Wokolorczyk, Dominika; Selkirk, Christina; Hulick, Peter J.; Bojesen, Anders; Skytte, Anne-Bine; Lam, Jimmy; Taylor, Louise; Oldenburg, Rogier; Cremers, Ruben; Verhaegh, Gerald; van Zelst-Stams, Wendy A.; Oosterwijk, Jan C.; Blanco, Ignacio; Salinas, Monica; Cook, Jackie; Rosario, Derek J.; Buys, Saundra; Conner, Tom; Ausems, Margreet G.; Ong, Kai-ren; Hoffman, Jonathan; Domchek, Susan; Powers, Jacquelyn; Teixeira, Manuel R.; Maia, Sofia; Foulkes, William D.; Taherian, Nassim; Ruijs, Marielle; Helderman-van den Enden, Apollonia T.

    Background: Men with germline breast cancer 1, early onset (BRCA1) or breast cancer 2, early onset (BRCA2) gene mutations have a higher risk of developing prostate cancer (PCa) than noncarriers. IMPACT (Identification of Men with a genetic predisposition to ProstAte Cancer: Targeted screening in

  19. Methylmethane-sulphonate and X-ray-induced mutations in the Chinese hamster hprt gene: mRNA phenotyping using polymerase chain reactions

    International Nuclear Information System (INIS)

    Chaudhry, M.A.; Fox, Margaret

    1990-01-01

    Alterations in the hprt gene of Chinese hamster cells were determined in 71 spontaneous, methylmethane sulphonate (MMS)-and X-ray induced mutants, using the Southern blot hybridization technique. To investigate the possibility of small deletions, MMS-induced mutants were studied with proves derived from exons 3 and 9 but no evidence of specific deletion of these two exons was found. The polymerase chain reaction (PCR) was used to phenotype hprt transcripts in 48 MMS, X-ray and spontaneous Chinese hamster mutants by amplifying the coding region of their cDNA. Among 22 MMS-induced mutants the message was present in 16 instances. An analysis of 20 X-ray-induced mutants showed the presence of hprt mRNA in 11 of them with five having low levels of transcription. Among six spontaneous mutants, four were negative for mRNA on standard Northern blots and in one the message was only detected after PCR amplification. Direct DNA sequencing of 10 mutants revealed the presence of base substitutions in five of them while a 7 bp deletion was found in another. No mutations were found in another four mutants, suggesting the presence of mutation outside the coding region. (author)

  20. Correction of the consequences of mitochondrial 3243A>G mutation in the MT-TL1 gene causing the MELAS syndrome by tRNA import into mitochondria.

    Science.gov (United States)

    Karicheva, Olga Z; Kolesnikova, Olga A; Schirtz, Tom; Vysokikh, Mikhail Y; Mager-Heckel, Anne-Marie; Lombès, Anne; Boucheham, Abdeldjalil; Krasheninnikov, Igor A; Martin, Robert P; Entelis, Nina; Tarassov, Ivan

    2011-10-01

    Mutations in human mitochondrial DNA are often associated with incurable human neuromuscular diseases. Among these mutations, an important number have been identified in tRNA genes, including 29 in the gene MT-TL1 coding for the tRNA(Leu(UUR)). The m.3243A>G mutation was described as the major cause of the MELAS syndrome (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes). This mutation was reported to reduce tRNA(Leu(UUR)) aminoacylation and modification of its anti-codon wobble position, which results in a defective mitochondrial protein synthesis and reduced activities of respiratory chain complexes. In the present study, we have tested whether the mitochondrial targeting of recombinant tRNAs bearing the identity elements for human mitochondrial leucyl-tRNA synthetase can rescue the phenotype caused by MELAS mutation in human transmitochondrial cybrid cells. We demonstrate that nuclear expression and mitochondrial targeting of specifically designed transgenic tRNAs results in an improvement of mitochondrial translation, increased levels of mitochondrial DNA-encoded respiratory complexes subunits, and significant rescue of respiration. These findings prove the possibility to direct tRNAs with changed aminoacylation specificities into mitochondria, thus extending the potential therapeutic strategy of allotopic expression to address mitochondrial disorders.

  1. A small interfering RNA screen of genes involved in DNA repair identifies tumor-specific radiosensitization by POLQ knockdown

    DEFF Research Database (Denmark)

    Higgins, Geoff S; Prevo, Remko; Lee, Yin-Fai

    2010-01-01

    The effectiveness of radiotherapy treatment could be significantly improved if tumor cells could be rendered more sensitive to ionizing radiation (IR) without altering the sensitivity of normal tissues. However, many of the key therapeutically exploitable mechanisms that determine intrinsic tumor...... radiosensitivity are largely unknown. We have conducted a small interfering RNA (siRNA) screen of 200 genes involved in DNA damage repair aimed at identifying genes whose knockdown increased tumor radiosensitivity. Parallel siRNA screens were conducted in irradiated and unirradiated tumor cells (SQ20B......) and irradiated normal tissue cells (MRC5). Using gammaH2AX foci at 24 hours after IR, we identified several genes, such as BRCA2, Lig IV, and XRCC5, whose knockdown is known to cause increased cell radiosensitivity, thereby validating the primary screening end point. In addition, we identified POLQ (DNA...

  2. Splicing Analysis of Exonic OCRL Mutations Causing Lowe Syndrome or Dent-2 Disease

    Directory of Open Access Journals (Sweden)

    Lorena Suarez-Artiles

    2018-01-01

    Full Text Available Mutations in the OCRL gene are associated with both Lowe syndrome and Dent-2 disease. Patients with Lowe syndrome present congenital cataracts, mental disabilities and a renal proximal tubulopathy, whereas patients with Dent-2 disease exhibit similar proximal tubule dysfunction but only mild, or no additional clinical defects. It is not yet understood why some OCRL mutations cause the phenotype of Lowe syndrome, while others develop the milder phenotype of Dent-2 disease. Our goal was to gain new insights into the consequences of OCRL exonic mutations on pre-mRNA splicing. Using predictive bioinformatics tools, we selected thirteen missense mutations and one synonymous mutation based on their potential effects on splicing regulatory elements or splice sites. These mutations were analyzed in a minigene splicing assay. Results of the RNA analysis showed that three presumed missense mutations caused alterations in pre-mRNA splicing. Mutation c.741G>T; p.(Trp247Cys generated splicing silencer sequences and disrupted splicing enhancer motifs that resulted in skipping of exon 9, while mutations c.2581G>A; p.(Ala861Thr and c.2581G>C; p.(Ala861Pro abolished a 5′ splice site leading to skipping of exon 23. Mutation c.741G>T represents the first OCRL exonic variant outside the conserved splice site dinucleotides that results in alteration of pre-mRNA splicing. Our results highlight the importance of evaluating the effects of OCRL exonic mutations at the mRNA level.

  3. Two-dimensional combinatorial screening enables the bottom-up design of a microRNA-10b inhibitor.

    Science.gov (United States)

    Velagapudi, Sai Pradeep; Disney, Matthew D

    2014-03-21

    The RNA motifs that bind guanidinylated kanamycin A (G Kan A) and guanidinylated neomycin B (G Neo B) were identified via two-dimensional combinatorial screening (2DCS). The results of these studies enabled the "bottom-up" design of a small molecule inhibitor of oncogenic microRNA-10b.

  4. Using answer set programming to integrate RNA expression with signalling pathway information to infer how mutations affect ageing.

    Science.gov (United States)

    Papatheodorou, Irene; Ziehm, Matthias; Wieser, Daniela; Alic, Nazif; Partridge, Linda; Thornton, Janet M

    2012-01-01

    A challenge of systems biology is to integrate incomplete knowledge on pathways with existing experimental data sets and relate these to measured phenotypes. Research on ageing often generates such incomplete data, creating difficulties in integrating RNA expression with information about biological processes and the phenotypes of ageing, including longevity. Here, we develop a logic-based method that employs Answer Set Programming, and use it to infer signalling effects of genetic perturbations, based on a model of the insulin signalling pathway. We apply our method to RNA expression data from Drosophila mutants in the insulin pathway that alter lifespan, in a foxo dependent fashion. We use this information to deduce how the pathway influences lifespan in the mutant animals. We also develop a method for inferring the largest common sub-paths within each of our signalling predictions. Our comparisons reveal consistent homeostatic mechanisms across both long- and short-lived mutants. The transcriptional changes observed in each mutation usually provide negative feedback to signalling predicted for that mutation. We also identify an S6K-mediated feedback in two long-lived mutants that suggests a crosstalk between these pathways in mutants of the insulin pathway, in vivo. By formulating the problem as a logic-based theory in a qualitative fashion, we are able to use the efficient search facilities of Answer Set Programming, allowing us to explore larger pathways, combine molecular changes with pathways and phenotype and infer effects on signalling in in vivo, whole-organism, mutants, where direct signalling stimulation assays are difficult to perform. Our methods are available in the web-service NetEffects: http://www.ebi.ac.uk/thornton-srv/software/NetEffects.

  5. Creation of targeted inversion mutations in plants using an RNA-guided endonuclease

    Institute of Scientific and Technical Information of China (English)

    Congsheng Zhang; Changlin Liu; Jianfeng Weng; Beijiu Cheng; Fang Liu; Xinhai Li; Chuanxiao Xie

    2017-01-01

    Inversions are DNA rearrangements that are essential for plant gene evolution and adaptation to environmental changes. We demonstrate the creation of targeted inversions and previously reported targeted deletion mutations via delivery of a pair of RNA-guided endonucleases (RGENs) of CRISPR/Cas9. The efficiencies of the targeted inversions were 2.6%and 2.2%in the Arabidopsis FLOWERING TIME (AtFT) and TERMINAL FLOWER 1 (AtTFL1) loci, respectively. Thus, we successfully established an approach that can potentially be used to introduce targeted DNA inversions of interest for functional studies and crop improvement.

  6. BeeDoctor, a versatile MLPA-based diagnostic tool for screening bee viruses.

    Science.gov (United States)

    De Smet, Lina; Ravoet, Jorgen; de Miranda, Joachim R; Wenseleers, Tom; Mueller, Matthias Y; Moritz, Robin F A; de Graaf, Dirk C

    2012-01-01

    The long-term decline of managed honeybee hives in the world has drawn significant attention to the scientific community and bee-keeping industry. A high pathogen load is believed to play a crucial role in this phenomenon, with the bee viruses being key players. Most of the currently characterized honeybee viruses (around twenty) are positive stranded RNA viruses. Techniques based on RNA signatures are widely used to determine the viral load in honeybee colonies. High throughput screening for viral loads necessitates the development of a multiplex polymerase chain reaction approach in which different viruses can be targeted simultaneously. A new multiparameter assay, called "BeeDoctor", was developed based on multiplex-ligation probe dependent amplification (MLPA) technology. This assay detects 10 honeybee viruses in one reaction. "BeeDoctor" is also able to screen selectively for either the positive strand of the targeted RNA bee viruses or the negative strand, which is indicative for active viral replication. Due to its sensitivity and specificity, the MLPA assay is a useful tool for rapid diagnosis, pathogen characterization, and epidemiology of viruses in honeybee populations. "BeeDoctor" was used for screening 363 samples from apiaries located throughout Flanders; the northern half of Belgium. Using the "BeeDoctor", virus infections were detected in almost eighty percent of the colonies, with deformed wing virus by far the most frequently detected virus and multiple virus infections were found in 26 percent of the colonies.

  7. BeeDoctor, a versatile MLPA-based diagnostic tool for screening bee viruses.

    Directory of Open Access Journals (Sweden)

    Lina De Smet

    Full Text Available The long-term decline of managed honeybee hives in the world has drawn significant attention to the scientific community and bee-keeping industry. A high pathogen load is believed to play a crucial role in this phenomenon, with the bee viruses being key players. Most of the currently characterized honeybee viruses (around twenty are positive stranded RNA viruses. Techniques based on RNA signatures are widely used to determine the viral load in honeybee colonies. High throughput screening for viral loads necessitates the development of a multiplex polymerase chain reaction approach in which different viruses can be targeted simultaneously. A new multiparameter assay, called "BeeDoctor", was developed based on multiplex-ligation probe dependent amplification (MLPA technology. This assay detects 10 honeybee viruses in one reaction. "BeeDoctor" is also able to screen selectively for either the positive strand of the targeted RNA bee viruses or the negative strand, which is indicative for active viral replication. Due to its sensitivity and specificity, the MLPA assay is a useful tool for rapid diagnosis, pathogen characterization, and epidemiology of viruses in honeybee populations. "BeeDoctor" was used for screening 363 samples from apiaries located throughout Flanders; the northern half of Belgium. Using the "BeeDoctor", virus infections were detected in almost eighty percent of the colonies, with deformed wing virus by far the most frequently detected virus and multiple virus infections were found in 26 percent of the colonies.

  8. Modeling the Mutational and Phenotypic Landscapes of Pelizaeus-Merzbacher Disease with Human iPSC-Derived Oligodendrocytes

    DEFF Research Database (Denmark)

    Nevin, Zachary S.; Factor, Daniel C.; Karl, Robert T.

    2017-01-01

    in humans. Attempts to identify a common pathogenic process underlying PMD have been complicated by an incomplete understanding of PLP1 dysfunction and limited access to primary human oligodendrocytes. To address this, we generated panels of human induced pluripotent stem cells (hiPSCs) and hi...... individual and shared defects in PLP1 mRNA expression and splicing, oligodendrocyte progenitor development, and oligodendrocyte morphology and capacity for myelination. These observations enabled classification of PMD subgroups by cell-intrinsic phenotypes and identified a subset of mutations for targeted...... treatment approaches for subsets of individuals. More broadly, this study demonstrates the versatility of a hiPSC-based panel spanning the mutational heterogeneity within a single disease and establishes a widely applicable platform for genotype-phenotype correlation and drug screening in any human myelin...

  9. eRNA: a graphic user interface-based tool optimized for large data analysis from high-throughput RNA sequencing.

    Science.gov (United States)

    Yuan, Tiezheng; Huang, Xiaoyi; Dittmar, Rachel L; Du, Meijun; Kohli, Manish; Boardman, Lisa; Thibodeau, Stephen N; Wang, Liang

    2014-03-05

    RNA sequencing (RNA-seq) is emerging as a critical approach in biological research. However, its high-throughput advantage is significantly limited by the capacity of bioinformatics tools. The research community urgently needs user-friendly tools to efficiently analyze the complicated data generated by high throughput sequencers. We developed a standalone tool with graphic user interface (GUI)-based analytic modules, known as eRNA. The capacity of performing parallel processing and sample management facilitates large data analyses by maximizing hardware usage and freeing users from tediously handling sequencing data. The module miRNA identification" includes GUIs for raw data reading, adapter removal, sequence alignment, and read counting. The module "mRNA identification" includes GUIs for reference sequences, genome mapping, transcript assembling, and differential expression. The module "Target screening" provides expression profiling analyses and graphic visualization. The module "Self-testing" offers the directory setups, sample management, and a check for third-party package dependency. Integration of other GUIs including Bowtie, miRDeep2, and miRspring extend the program's functionality. eRNA focuses on the common tools required for the mapping and quantification analysis of miRNA-seq and mRNA-seq data. The software package provides an additional choice for scientists who require a user-friendly computing environment and high-throughput capacity for large data analysis. eRNA is available for free download at https://sourceforge.net/projects/erna/?source=directory.

  10. Frequency of Fanconi anemia in Brazil and efficacy of screening for the FANCA 3788-3790del mutation

    Directory of Open Access Journals (Sweden)

    N. Magdalena

    2005-05-01

    Full Text Available Fanconi anemia (FA is an autosomal recessive genetic disease characterized by progressive bone marrow failure, susceptibility to cancer and multiple congenital anomalies. There is important clinical variability among patients and the knowledge of factors which might predict outcome would greatly help the decision making regarding the choices of treatment and the appropriate time to start it. Future studies of the possible correlation between specific mutations with specific clinical presentations will provide the answer to one of these factors. At our Center we standardized a rapid and precise screening test using a mismatch PCR assay for a specific mutation (3788-3790del in exon 38 of gene FANCA in Brazilian FA patients. We present the results obtained after screening 80 non-consanguineous FA patients referred from all regions of Brazil with a clinical diagnosis of FA supported by cellular hypersensitivity to diepoxybutane. We were able to detect the 3788-3790del allele in 24 of the 80 (30% FA patients studied. Thirteen of the 80 (16.25% were homozygotes and 11 of the 80 (13.75% were compound heterozygotes, thus confirming the high frequency of the FANCA 3788-3790del mutation in Brazilian FA patients. The identification of patients with specific mutations in the FA genes may lead to a better clinical description of this condition, also providing data for genotype-phenotype correlations, to a better understanding of the interaction of this specific mutation with other mutations in compound heterozygote patients, and ultimately to the right choices of treatment for each patient with improvement of the prognosis on future studies.

  11. Frequency of Fanconi anemia in Brazil and efficacy of screening for the FANCA 3788-3790del mutation.

    Science.gov (United States)

    Magdalena, N; Pilonetto, D V; Bitencourt, M A; Pereira, N F; Ribeiro, R C; Jeng, M; Pasquini, R

    2005-05-01

    Fanconi anemia (FA) is an autosomal recessive genetic disease characterized by progressive bone marrow failure, susceptibility to cancer and multiple congenital anomalies. There is important clinical variability among patients and the knowledge of factors which might predict outcome would greatly help the decision making regarding the choices of treatment and the appropriate time to start it. Future studies of the possible correlation between specific mutations with specific clinical presentations will provide the answer to one of these factors. At our Center we standardized a rapid and precise screening test using a mismatch PCR assay for a specific mutation (3788-3790del in exon 38 of gene FANCA) in Brazilian FA patients. We present the results obtained after screening 80 non-consanguineous FA patients referred from all regions of Brazil with a clinical diagnosis of FA supported by cellular hypersensitivity to diepoxybutane. We were able to detect the 3788-3790del allele in 24 of the 80 (30%) FA patients studied. Thirteen of the 80 (16.25%) were homozygotes and 11 of the 80 (13.75%) were compound heterozygotes, thus confirming the high frequency of the FANCA 3788-3790del mutation in Brazilian FA patients. The identification of patients with specific mutations in the FA genes may lead to a better clinical description of this condition, also providing data for genotype-phenotype correlations, to a better understanding of the interaction of this specific mutation with other mutations in compound heterozygote patients, and ultimately to the right choices of treatment for each patient with improvement of the prognosis on future studies.

  12. Mutation in WDR4 impairs tRNA m(7)G46 methylation and causes a distinct form of microcephalic primordial dwarfism.

    Science.gov (United States)

    Shaheen, Ranad; Abdel-Salam, Ghada M H; Guy, Michael P; Alomar, Rana; Abdel-Hamid, Mohamed S; Afifi, Hanan H; Ismail, Samira I; Emam, Bayoumi A; Phizicky, Eric M; Alkuraya, Fowzan S

    2015-09-28

    Primordial dwarfism is a state of extreme prenatal and postnatal growth deficiency, and is characterized by marked clinical and genetic heterogeneity. Two presumably unrelated consanguineous families presented with an apparently novel form of primordial dwarfism in which severe growth deficiency is accompanied by distinct facial dysmorphism, brain malformation (microcephaly, agenesis of corpus callosum, and simplified gyration), and severe encephalopathy with seizures. Combined autozygome/exome analysis revealed a novel missense mutation in WDR4 as the likely causal variant. WDR4 is the human ortholog of the yeast Trm82, an essential component of the Trm8/Trm82 holoenzyme that effects a highly conserved and specific (m(7)G46) methylation of tRNA. The human mutation and the corresponding yeast mutation result in a significant reduction of m(7)G46 methylation of specific tRNA species, which provides a potential mechanism for primordial dwarfism associated with this lesion, since reduced m(7)G46 modification causes a growth deficiency phenotype in yeast. Our study expands the number of biological pathways underlying primordial dwarfism and adds to a growing list of human diseases linked to abnormal tRNA modification.

  13. Glucokinase gene mutations (MODY 2) in Asian Indians.

    Science.gov (United States)

    Kanthimathi, Sekar; Jahnavi, Suresh; Balamurugan, Kandasamy; Ranjani, Harish; Sonya, Jagadesan; Goswami, Soumik; Chowdhury, Subhankar; Mohan, Viswanathan; Radha, Venkatesan

    2014-03-01

    Heterozygous inactivating mutations in the glucokinase (GCK) gene cause a hyperglycemic condition termed maturity-onset diabetes of the young (MODY) 2 or GCK-MODY. This is characterized by mild, stable, usually asymptomatic, fasting hyperglycemia that rarely requires pharmacological intervention. The aim of the present study was to screen for GCK gene mutations in Asian Indian subjects with mild hyperglycemia. Of the 1,517 children and adolescents of the population-based ORANGE study in Chennai, India, 49 were found to have hyperglycemia. These children along with the six patients referred to our center with mild hyperglycemia were screened for MODY 2 mutations. The GCK gene was bidirectionally sequenced using BigDye(®) Terminator v3.1 (Applied Biosystems, Foster City, CA) chemistry. In silico predictions of the pathogenicity were carried out using the online tools SIFT, Polyphen-2, and I-Mutant 2.0 software programs. Direct sequencing of the GCK gene in the patients referred to our Centre revealed one novel mutation, Thr206Ala (c.616A>G), in exon 6 and one previously described mutation, Met251Thr (c.752T>C), in exon 7. In silico analysis predicted the novel mutation to be pathogenic. The highly conserved nature and critical location of the residue Thr206 along with the clinical course suggests that the Thr206Ala is a MODY 2 mutation. However, we did not find any MODY 2 mutations in the 49 children selected from the population-based study. Hence prevalence of GCK mutations in Chennai is MODY 2 mutations from India and confirms the importance of considering GCK gene mutation screening in patients with mild early-onset hyperglycemia who are negative for β-cell antibodies.

  14. A mutation in the glutamate-rich region of RNA-binding motif protein 20 causes dilated cardiomyopathy through missplicing of titin and impaired Frank-Starling mechanism

    DEFF Research Database (Denmark)

    Beqqali, Abdelaziz; Bollen, I. A. E.; Rasmussen, T. B.

    2016-01-01

    Mutations in the RS-domain of RNA-binding motif protein 20 (RBM20) have recently been identified to segregate with aggressive forms of familial dilated cardiomyopathy (DCM). Loss of RBM20 in rats results in missplicing of the sarcomeric gene titin (TTN). The functional and physiological consequen......Mutations in the RS-domain of RNA-binding motif protein 20 (RBM20) have recently been identified to segregate with aggressive forms of familial dilated cardiomyopathy (DCM). Loss of RBM20 in rats results in missplicing of the sarcomeric gene titin (TTN). The functional and physiological...... consequences of RBM20 mutations outside the mutational hotspot of RBM20 have not been explored to date. In this study, we investigated the pathomechanism of DCM caused by a novel RBM20 mutation in human cardiomyocytes. We identified a family with DCM carrying a mutation (RBM20(E913K/+)) in a glutamate...... to the early onset, and malignant course of DCM caused by RBM20 mutations. Altogether, our results demonstrate that heterozygous loss of RBM20 suffices to profoundly impair myocyte biomechanics by its disturbance of TTN splicing....

  15. An RNA polymerase II-driven Ebola virus minigenome system as an advanced tool for antiviral drug screening.

    Science.gov (United States)

    Nelson, Emily V; Pacheco, Jennifer R; Hume, Adam J; Cressey, Tessa N; Deflubé, Laure R; Ruedas, John B; Connor, John H; Ebihara, Hideki; Mühlberger, Elke

    2017-10-01

    Ebola virus (EBOV) causes a severe disease in humans with the potential for significant international public health consequences. Currently, treatments are limited to experimental vaccines and therapeutics. Therefore, research into prophylaxis and antiviral strategies to combat EBOV infections is of utmost importance. The requirement for high containment laboratories to study EBOV infection is a limiting factor for conducting EBOV research. To overcome this issue, minigenome systems have been used as valuable tools to study EBOV replication and transcription mechanisms and to screen for antiviral compounds at biosafety level 2. The most commonly used EBOV minigenome system relies on the ectopic expression of the T7 RNA polymerase (T7), which can be limiting for certain cell types. We have established an improved EBOV minigenome system that utilizes endogenous RNA polymerase II (pol II) as a driver for the synthesis of minigenome RNA. We show here that this system is as efficient as the T7-based minigenome system, but works in a wider range of cell types, including biologically relevant cell types such as bat cells. Importantly, we were also able to adapt this system to a reliable and cost-effective 96-well format antiviral screening assay with a Z-factor of 0.74, indicative of a robust assay. Using this format, we identified JG40, an inhibitor of Hsp70, as an inhibitor of EBOV replication, highlighting the potential for this system as a tool for antiviral drug screening. In summary, this updated EBOV minigenome system provides a convenient and effective means of advancing the field of EBOV research. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Evolutionary patterns of RNA-based duplication in non-mammalian chordates.

    Directory of Open Access Journals (Sweden)

    Ming Chen

    Full Text Available The role of RNA-based duplication, or retroposition, in the evolution of new gene functions in mammals, plants, and Drosophila has been widely reported. However, little is known about RNA-based duplication in non-mammalian chordates. In this study, we screened ten non-mammalian chordate genomes for retrocopies and investigated their evolutionary patterns. We identified numerous retrocopies in these species. Examination of the age distribution of these retrocopies revealed no burst of young retrocopies in ancient chordate species. Upon comparing these non-mammalian chordate species to the mammalian species, we observed that a larger fraction of the non-mammalian retrocopies was under strong evolutionary constraints than mammalian retrocopies are, as evidenced by signals of purifying selection and expression profiles. For the Western clawed frog, Medaka, and Sea squirt, many retrogenes have evolved gonad and brain expression patterns, similar to what was observed in human. Testing of retrogene movement in the Medaka genome, where the nascent sex chrosomes have been well assembled, did not reveal any significant gene movement. Taken together, our analyses demonstrate that RNA-based duplication generates many functional genes and can make a significant contribution to the evolution of non-mammalian genomes.

  17. Fabry Disease: prevalence of affected males and heterozygotes with pathogenic GLA mutations identified by screening renal, cardiac and stroke clinics, 1995-2017.

    Science.gov (United States)

    Doheny, Dana; Srinivasan, Ram; Pagant, Silvere; Chen, Brenden; Yasuda, Makiko; Desnick, Robert J

    2018-04-01

    Fabry Disease (FD), an X linked lysosomal storage disease due to pathogenic α-galactosidase A ( GLA ) mutations, results in two major subtypes, the early-onset Type 1 'Classic' and the Type 2 'Later-Onset' phenotypes. To identify previously unrecognised patients, investigators screened cardiac, renal and stroke clinics by enzyme assays. However, some screening studies did not perform confirmatory GLA mutation analyses, and many included recently recognised 'benign/likely-benign' variants, thereby inflating prevalence estimates. Online databases were searched for all FD screening studies in high-risk clinics (1995-2017). Studies reporting GLA mutations were re-analysed for pathogenic mutations, sex and phenotype. Phenotype-specific and sex-specific prevalence rates were determined. Of 67 studies, 63 that screened 51363patients (33943M and 17420F) and provided GLA mutations were reanalysed for disease-causing mutations. Of reported GLA mutations, benign variants occurred in 47.9% of males and 74.1% of females. The following were the revised prevalence estimates: among 36820 (23954M and 12866F) haemodialysis screenees, 0.21% males and 0.15% females; among 3074 (2031M and 1043F) renal transplant screenees, 0.25% males and no females; among 5491 (4054M and 1437F) cardiac screenees, 0.94% males and 0.90% females; and among 5978 (3904M and 2074F) stroke screenees, 0.13% males and 0.14% females. Among male and female screenees with pathogenic mutations, the type 1 Classic phenotype was predominant (~60%), except more male cardiac patients (75%) had type 2 Later-Onset phenotype. Compared with previous findings, reanalysis of 63 studies increased the screenee numbers (~3.4-fold), eliminated 20 benign/likely benign variants, and provided more accurate sex-specific and phenotype-specific prevalence estimates, ranging from ~0.13% of stroke to ~0.9% of cardiac male or female screenees. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article

  18. A genetic screen of the mutations in the Korean patients with early-onset Alzheimer’s disease

    Directory of Open Access Journals (Sweden)

    An SS

    2016-12-01

    Full Text Available Seong Soo An,1,* Sun Ah Park,2,* Eva Bagyinszky,1 Sun Oh Bae,1 Yoon-Jeong Kim,2 Ji Young Im,2 Kyung Won Park,3 Kee Hyung Park,4 Eun-Joo Kim,5 Jee Hyang Jeong,6 Jong Hun Kim,7 Hyun Jeong Han,8 Seong Hye Choi,9 SangYun Kim10 1Department of Bionano Technology, Gachon University, Seongnam-si, 2Department of Neurology, Soonchunhyang University Bucheon Hospital, Bucheon, 3Department of Neurology, Dong-A University College of Medicine and Institute of Convergence Bio-Health, Busan, 4Department of Neurology, Gachon University Gil Medical Center, Incheon, 5Department of Neurology, Pusan National University Hospital, Busan, 6Department of Neurology, Ewha Womans University Mokdong Hospital, Seoul, 7Department of Neurology, Ilsan Hospital, National Health Insurance Corporation, 8Department of Neurology, Myongii Hospital, Goyang, 9Department of Neurology, Inha University School of Medicine, Incheon, 10Department of Neurology, Seoul National University College of Medicine & Neurocognitive Behavior Center, Seoul National University Bundang Hospital, Seongnam-si, Republic of Korea *These authors contributed equally to this work Abstract: Early-onset Alzheimer’s disease (EOAD has distinct clinical characteristics in comparison to late-onset Alzheimer’s disease (LOAD. The genetic contribution is suggested to be more potent in EOAD. However, the frequency of causative mutations in EOAD could be variable depending on studies. Moreover, no mutation screening study has been performed yet employing large population in Korea. Previously, we reported that the rate of family history of dementia in EOAD patients was 18.7% in a nationwide hospital-based cohort study, the Clinical Research Center for Dementia of South Korea (CREDOS study. This rate is much lower than in other countries and is even comparable to the frequency of LOAD patients in our country. To understand the genetic characteristics of EOAD in Korea, we screened the common Alzheimer’s disease (AD

  19. Mutation-Guided Unbiased Modeling of the Fat Sensor GPR119 for High-Yield Agonist Screening

    DEFF Research Database (Denmark)

    Norn, Christoffer; Pedersen, Maria Hauge; Engelstoft, Maja S.

    2015-01-01

    and is consistent with the structure-activity relationship for a large no. of AR231453 analogs. Another key property of the refined models is their success in sepg. active ligands from decoys in a large-scale virtual screening. These results demonstrate that mutation-guided receptor modeling can provide predictions...

  20. A negative screen for mutations in calstabin 1 and 2 genes in patients with dilated cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Biagi Diogo G

    2012-01-01

    Full Text Available Abstract Background Calstabins 1 and 2 bind to Ryanodine receptors regulating muscle excitation-contraction coupling. Mutations in Ryanodine receptors affecting their interaction with calstabins lead to different cardiac pathologies. Animal studies suggest the involvement of calstabins with dilated cardiomyopathy. Results We tested the hypothesis that calstabins mutations may cause dilated cardiomyopathy in humans screening 186 patients with idiopathic dilated cardiomyopathy for genetic alterations in calstabins 1 and 2 genes (FKBP12 and FKBP12.6. No missense variant was found. Five no-coding variations were found but not related to the disease. Conclusions These data corroborate other studies suggesting that mutations in FKBP12 and FKBP12.6 genes are not commonly related to cardiac diseases.

  1. Cardiac abnormalities in diabetic patients with mutation in the mitochondrial tRNA {sup Leu(UUR)}Gene

    Energy Technology Data Exchange (ETDEWEB)

    Ueno, Hiroshi [Hyogo Medical Center for Adults, Akashi (Japan); Shiotani, Hideyuki

    1999-11-01

    An A-to-G transition at position 3243 of the mitochondrial DNA is known to be a pathogenic factor for mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS), diabetes and cardiomyopathy. This mutation causes dysfunction of the central nervous system in MELAS. Because the heart, as well as the brain and nervous system, is highly dependent on the energy produced by mitochondrial oxidation, these tissues are more vulnerable to mitochondrial defects. Cardiac abnormalities were assessed in 10 diabetic patients associated with this mutation using echocardiography and {sup 123}I-metaiodobenzylguanidine (MIBG) scintigraphy, and compared with 19 diabetic patients without the mutation. Duration of diabetes, therapy, control of blood glucose and diabetic complications, such as diabetic retinopathy and nephropathy, were not different between the 2 groups. Diabetic patients with the mutation had a significantly thicker interventricular septum (16.8{+-}3.7 vs 11.0{+-}1.6 mm, p<0.001) than those without the mutation. Fractional shortening was lower in diabetic patients with the mutation than those without it (30.7{+-}7.0 vs 42.5{+-}6.6, p<0.001). MIBG uptake on the delayed MIBG image was significantly lower in diabetic patients with the mutation than in those without the mutation (mean value of the heart to mediastinum ratio: 1.6{+-}0.2 vs 2.0{+-}0.4, p>0.05). In conclusion, left ventricular hypertrophy with or without abnormal wall motion and severely reduced MIBG uptake may be characteristic in diabetic patients with a mutation in the mitochondrial tRNA {sup Leu(UUR)} gene. (author)

  2. Functional characterization of the Drosophila MRP (mitochondrial RNA processing) RNA gene.

    Science.gov (United States)

    Schneider, Mary D; Bains, Anupinder K; Rajendra, T K; Dominski, Zbigniew; Matera, A Gregory; Simmonds, Andrew J

    2010-11-01

    MRP RNA is a noncoding RNA component of RNase mitochondrial RNA processing (MRP), a multi-protein eukaryotic endoribonuclease reported to function in multiple cellular processes, including ribosomal RNA processing, mitochondrial DNA replication, and cell cycle regulation. A recent study predicted a potential Drosophila ortholog of MRP RNA (CR33682) by computer-based genome analysis. We have confirmed the expression of this gene and characterized the phenotype associated with this locus. Flies with mutations that specifically affect MRP RNA show defects in growth and development that begin in the early larval period and end in larval death during the second instar stage. We present several lines of evidence demonstrating a role for Drosophila MRP RNA in rRNA processing. The nuclear fraction of Drosophila MRP RNA localizes to the nucleolus. Further, a mutant strain shows defects in rRNA processing that include a defect in 5.8S rRNA processing, typical of MRP RNA mutants in other species, as well as defects in early stages of rRNA processing.

  3. The clinical value of HPV E6/E7 and STAT3 mRNA detection in cervical cancer screening.

    Science.gov (United States)

    Fan, Yibing; Shen, Zongji

    2018-02-11

    To explore the value of human papillomavirus (HPV) E6/E7 and signal transducer and activator of transcription 3 (STAT3) mRNA detection in the screening of cervical lesions. 192 patients with abnormal ThinPrep cytology test (TCT) results and/or high-risk HPV infection were screened to identify possible cervical lesions in cases. Diagnoses were confirmed by histopathology. Fluorescence in situ hybridization (FISH) was performed to detect and qualify the mRNAs of HPV E6/E7, STAT3, and Survivin in cervical exfoliated cells. In addition, the performance of separate and combined mRNA detection methods were compared with TCT, HR-HPV DNA schemes respectively. 1. Compared with HPVE6/E7 and STAT3 mRNA methods, Survivin mRNA assay had poor specificity (Sp), Youden index (YI) and concordance rate. 2. HPV E6/E7, STAT3, and STAT3 + HR-HPV methods had the best Sp, concordance rate and positive predictive value (PPV) for cervical lesions screening and atypical squamous cells of undetermined significance (ASCUS) triage. For screening of high grade squamous intraepithelial lesions or greater (HSILs+), no difference was observed in the Se of mRNA detection methods in comparison with that of TCT, HR-HPV and TCT + HR-HPV, whereas the false positive rate (FPR) decreased by 41.48%/55.99%/17.19% and the colposcopy referral rate reduced by about 20.00%/25.00%/11.17%. For triage of women with ASCUS, no difference was observed in the Se of mRNA detection methods as compared to that of HR-HPV (χ 2  = 1.05, P > 0.75), while the FPR decreased by 45.83%/37.50%/41.66% and the colposcopy referral rate reduced by 32.42%/22.60%/25.28%, respectively. The Se, YI, and PPV of the combined methods increased in comparison to each method alone. 3. Compared with the TCT + HR-HPV method, HPV E6/E7 + STAT3 method had perfect Sp (95.92%) and PPV (95.40%) for screening HSILs+, the FPR and colposcopy referral rate decreased by 31.06% and 22.48% respectively. 1. The expression of HPV E6/E

  4. Screening of three Mediterranean phenylketonuria mutations in ...

    Indian Academy of Sciences (India)

    as the most frequent mutation (Dahri et al. 2010). The. E280K mutation was also reported in Mediterranean popu- lations (Guldberg et al. 1993). Since Tunisia is a Mediter- ranean country, patients with PKU are presumed to have these mutations. The aim of this study was to assess prevalence of the three above mutations ...

  5. OBSCN Mutations Associated with Dilated Cardiomyopathy and Haploinsufficiency.

    Directory of Open Access Journals (Sweden)

    Steven Marston

    Full Text Available Studies of the functional consequences of DCM-causing mutations have been limited to a few cases where patients with known mutations had heart transplants. To increase the number of potential tissue samples for direct investigation we performed whole exon sequencing of explanted heart muscle samples from 30 patients that had a diagnosis of familial dilated cardiomyopathy and screened for potentially disease-causing mutations in 58 HCM or DCM-related genes.We identified 5 potentially disease-causing OBSCN mutations in 4 samples; one sample had two OBSCN mutations and one mutation was judged to be not disease-related. Also identified were 6 truncating mutations in TTN, 3 mutations in MYH7, 2 in DSP and one each in TNNC1, TNNI3, MYOM1, VCL, GLA, PLB, TCAP, PKP2 and LAMA4. The mean level of obscurin mRNA was significantly greater and more variable in healthy donor samples than the DCM samples but did not correlate with OBSCN mutations. A single obscurin protein band was observed in human heart myofibrils with apparent mass 960 ± 60 kDa. The three samples with OBSCN mutations had significantly lower levels of obscurin immunoreactive material than DCM samples without OBSCN mutations (45±7, 48±3, and 72±6% of control level.Obscurin levels in DCM controls, donor heart and myectomy samples were the same.OBSCN mutations may result in the development of a DCM phenotype via haploinsufficiency. Mutations in the obscurin gene should be considered as a significant causal factor of DCM, alone or in concert with other mutations.

  6. A comprehensive platform for highly multiplexed mammalian functional genetic screens

    Directory of Open Access Journals (Sweden)

    Cheung-Ong Kahlin

    2011-05-01

    Full Text Available Abstract Background Genome-wide screening in human and mouse cells using RNA interference and open reading frame over-expression libraries is rapidly becoming a viable experimental approach for many research labs. There are a variety of gene expression modulation libraries commercially available, however, detailed and validated protocols as well as the reagents necessary for deconvolving genome-scale gene screens using these libraries are lacking. As a solution, we designed a comprehensive platform for highly multiplexed functional genetic screens in human, mouse and yeast cells using popular, commercially available gene modulation libraries. The Gene Modulation Array Platform (GMAP is a single microarray-based detection solution for deconvolution of loss and gain-of-function pooled screens. Results Experiments with specially constructed lentiviral-based plasmid pools containing ~78,000 shRNAs demonstrated that the GMAP is capable of deconvolving genome-wide shRNA "dropout" screens. Further experiments with a larger, ~90,000 shRNA pool demonstrate that equivalent results are obtained from plasmid pools and from genomic DNA derived from lentivirus infected cells. Parallel testing of large shRNA pools using GMAP and next-generation sequencing methods revealed that the two methods provide valid and complementary approaches to deconvolution of genome-wide shRNA screens. Additional experiments demonstrated that GMAP is equivalent to similar microarray-based products when used for deconvolution of open reading frame over-expression screens. Conclusion Herein, we demonstrate four major applications for the GMAP resource, including deconvolution of pooled RNAi screens in cells with at least 90,000 distinct shRNAs. We also provide detailed methodologies for pooled shRNA screen readout using GMAP and compare next-generation sequencing to GMAP (i.e. microarray based deconvolution methods.

  7. Combined mutation and rearrangement screening by quantitative PCR high-resolution melting: is it relevant for hereditary recurrent Fever genes?

    Directory of Open Access Journals (Sweden)

    Nathalie Pallares-Ruiz

    2010-11-01

    Full Text Available The recent identification of genes implicated in hereditary recurrent fevers has allowed their specific diagnosis. So far however, only punctual mutations have been identified and a significant number of patients remain with no genetic confirmation of their disease after routine molecular approaches such as sequencing. The possible involvement of sequence rearrangements in these patients has only been examined in familial Mediterranean fever and was found to be unlikely. To assess the existence of larger genetic alterations in 3 other concerned genes, MVK (Mevalonate kinase, NLRP3 (Nod like receptor family, pyrin domain containing 3 and TNFRSF1A (TNF receptor superfamily 1A, we adapted the qPCR-HRM method to study possible intragenic deletions and duplications. This single-tube approach, combining both qualitative (mutations and quantitative (rearrangement screening, has proven effective in Lynch syndrome diagnosis. Using this approach, we studied 113 unselected (prospective group and 88 selected (retrospective group patients and identified no intragenic rearrangements in the 3 genes. Only qualitative alterations were found with a sensitivity similar to that obtained using classical molecular techniques for screening punctual mutations. Our results support that deleterious copy number alterations in MVK, NLRP3 and TNFRSF1A are rare or absent from the mutational spectrum of hereditary recurrent fevers, and demonstrate that a routine combined method such as qPCR-HRM provides no further help in genetic diagnosis. However, quantitative approaches such as qPCR or SQF-PCR did prove to be quick and effective and could still be useful after non contributory punctual mutation screening in the presence of clinically evocative signs.

  8. Studying the evolutionary relationships and phylogenetic trees of 21 groups of tRNA sequences based on complex networks.

    Science.gov (United States)

    Wei, Fangping; Chen, Bowen

    2012-03-01

    To find out the evolutionary relationships among different tRNA sequences of 21 amino acids, 22 networks are constructed. One is constructed from whole tRNAs, and the other 21 networks are constructed from the tRNAs which carry the same amino acids. A new method is proposed such that the alignment scores of any two amino acids groups are determined by the average degree and the average clustering coefficient of their networks. The anticodon feature of isolated tRNA and the phylogenetic trees of 21 group networks are discussed. We find that some isolated tRNA sequences in 21 networks still connect with other tRNAs outside their group, which reflects the fact that those tRNAs might evolve by intercrossing among these 21 groups. We also find that most anticodons among the same cluster are only one base different in the same sites when S ≥ 70, and they stay in the same rank in the ladder of evolutionary relationships. Those observations seem to agree on that some tRNAs might mutate from the same ancestor sequences based on point mutation mechanisms.

  9. Mutations in 23S rRNA Confer Resistance against Azithromycin in Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Marvig, Rasmus Lykke; Søndergaard, Mette S. R.; Pedersen, Søren Damkiær

    2012-01-01

    The emergence of antibiotic-resistant Pseudomonas aeruginosa is an important concern in the treatment of long-term airway infections in cystic fibrosis patients. In this study, we report the occurrence of azithromycin resistance among clinical P. aeruginosa DK2 isolates. We demonstrate that resis...... that resistance is associated with specific mutations (A2058G, A2059G, and C2611T in Escherichia coli numbering) in domain V of 23S rRNA and that introduction of A2058G and C2611T into strain PAO1 results in azithromycin resistance....

  10. Mitochondrial DNA triplication and punctual mutations in patients with mitochondrial neuromuscular disorders

    Energy Technology Data Exchange (ETDEWEB)

    Mkaouar-Rebai, Emna, E-mail: emna.mkaouar@gmail.com [Département des Sciences de la Vie, Faculté des Sciences de Sfax, Université de Sfax (Tunisia); Felhi, Rahma; Tabebi, Mouna [Laboratoire de Génétique Moléculaire Humaine, Faculté de Médecine de Sfax, Université de Sfax (Tunisia); Alila-Fersi, Olfa; Chamkha, Imen [Département des Sciences de la Vie, Faculté des Sciences de Sfax, Université de Sfax (Tunisia); Maalej, Marwa; Ammar, Marwa [Laboratoire de Génétique Moléculaire Humaine, Faculté de Médecine de Sfax, Université de Sfax (Tunisia); Kammoun, Fatma [Service de pédiatrie, C.H.U. Hedi Chaker de Sfax (Tunisia); Keskes, Leila [Laboratoire de Génétique Moléculaire Humaine, Faculté de Médecine de Sfax, Université de Sfax (Tunisia); Hachicha, Mongia [Service de pédiatrie, C.H.U. Hedi Chaker de Sfax (Tunisia); Fakhfakh, Faiza, E-mail: faiza.fakhfakh02@gmail.com [Département des Sciences de la Vie, Faculté des Sciences de Sfax, Université de Sfax (Tunisia)

    2016-04-29

    Mitochondrial diseases are a heterogeneous group of disorders caused by the impairment of the mitochondrial oxidative phosphorylation system which have been associated with various mutations of the mitochondrial DNA (mtDNA) and nuclear gene mutations. The clinical phenotypes are very diverse and the spectrum is still expanding. As brain and muscle are highly dependent on OXPHOS, consequently, neurological disorders and myopathy are common features of mtDNA mutations. Mutations in mtDNA can be classified into three categories: large-scale rearrangements, point mutations in tRNA or rRNA genes and point mutations in protein coding genes. In the present report, we screened mitochondrial genes of complex I, III, IV and V in 2 patients with mitochondrial neuromuscular disorders. The results showed the presence the pathogenic heteroplasmic m.9157G>A variation (A211T) in the MT-ATP6 gene in the first patient. We also reported the first case of triplication of 9 bp in the mitochondrial NC7 region in Africa and Tunisia, in association with the novel m.14924T>C in the MT-CYB gene in the second patient with mitochondrial neuromuscular disorder. - Highlights: • We reported 2 patients with mitochondrial neuromuscular disorders. • The heteroplasmic MT-ATP6 9157G>A variation was reported. • A triplication of 9 bp in the mitochondrial NC7 region was detected. • The m.14924T>C transition (S60P) in the MT-CYB gene was found.

  11. Microarray-based mutation detection and phenotypic characterization in Korean patients with retinitis pigmentosa

    Science.gov (United States)

    Kim, Cinoo; Kim, Kwang Joong; Bok, Jeong; Lee, Eun-Ju; Kim, Dong-Joon; Oh, Ji Hee; Park, Sung Pyo; Shin, Joo Young; Lee, Jong-Young

    2012-01-01

    Purpose To evaluate microarray-based genotyping technology for the detection of mutations responsible for retinitis pigmentosa (RP) and to perform phenotypic characterization of patients with pathogenic mutations. Methods DNA from 336 patients with RP and 360 controls was analyzed using the GoldenGate assay with microbeads containing 95 previously reported disease-associated mutations from 28 RP genes. Mutations identified by microarray-based genotyping were confirmed by direct sequencing. Segregation analysis and phenotypic characterization were performed in patients with mutations. The disease severity was assessed by visual acuity, electroretinography, optical coherence tomography, and kinetic perimetry. Results Ten RP-related mutations of five RP genes (PRP3 pre-mRNA processing factor 3 homolog [PRPF3], rhodopsin [RHO], phosphodiesterase 6B [PDE6B], peripherin 2 [PRPH2], and retinitis pigmentosa 1 [RP1]) were identified in 26 of the 336 patients (7.7%) and in six of the 360 controls (1.7%). The p.H557Y mutation in PDE6B, which was homozygous in four patients and heterozygous in nine patients, was the most frequent mutation (2.5%). Mutation segregation was assessed in four families. Among the patients with missense mutations, the most severe phenotype occurred in patients with p.D984G in RP1; less severe phenotypes occurred in patients with p.R135W in RHO; a relatively moderate phenotype occurred in patients with p.T494M in PRPF3, p.H557Y in PDE6B, or p.W316G in PRPH2; and a mild phenotype was seen in a patient with p.D190N in RHO. Conclusions The results reveal that the GoldenGate assay may not be an efficient method for molecular diagnosis in RP patients with rare mutations, although it has proven to be reliable and efficient for high-throughput genotyping of single-nucleotide polymorphisms. The clinical features varied according to the mutations. Continuous effort to identify novel RP genes and mutations in a population is needed to improve the efficiency and

  12. BRCA Genetic Screening in Middle Eastern and North African: Mutational Spectrum and Founder BRCA1 Mutation (c.798_799delTT in North African

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    Abdelilah Laraqui

    2015-01-01

    Full Text Available Background. The contribution of BRCA1 mutations to both hereditary and sporadic breast and ovarian cancer (HBOC has not yet been thoroughly investigated in MENA. Methods. To establish the knowledge about BRCA1 mutations and their correlation with the clinical aspect in diagnosed cases of HBOC in MENA populations. A systematic review of studies examining BRCA1 in BC women in Cyprus, Jordan, Egypt, Lebanon, Morocco, Algeria, and Tunisia was conducted. Results. Thirteen relevant references were identified, including ten studies which performed DNA sequencing of all BRCA1 exons. For the latter, 31 mutations were detected in 57 of the 547 patients ascertained. Familial history of BC was present in 388 (71% patients, of whom 50 were mutation carriers. c.798_799delTT was identified in 11 North African families, accounting for 22% of total identified BRCA1 mutations, suggesting a founder allele. A broad spectrum of other mutations including c.68_69delAG, c.181T>G, c.5095C>T, and c.5266dupC, as well as sequence of unclassified variants and polymorphisms, was also detected. Conclusion. The knowledge of genetic structure of BRCA1 in MENA should contribute to the assessment of the necessity of preventive programs for mutation carriers and clinical management. The high prevalence of BC and the presence of frequent mutations of the BRCA1 gene emphasize the need for improving screening programs and individual testing/counseling.

  13. Biomarker MicroRNAs for Diagnosis of Oral Squamous Cell Carcinoma Identified Based on Gene Expression Data and MicroRNA-mRNA Network Analysis

    Science.gov (United States)

    Zhang, Hui; Li, Tangxin; Zheng, Linqing

    2017-01-01

    Oral squamous cell carcinoma is one of the most malignant tumors with high mortality rate worldwide. Biomarker discovery is critical for early diagnosis and precision treatment of this disease. MicroRNAs are small noncoding RNA molecules which often regulate essential biological processes and are good candidates for biomarkers. By integrative analysis of both the cancer-associated gene expression data and microRNA-mRNA network, miR-148b-3p, miR-629-3p, miR-27a-3p, and miR-142-3p were screened as novel diagnostic biomarkers for oral squamous cell carcinoma based on their unique regulatory abilities in the network structure of the conditional microRNA-mRNA network and their important functions. These findings were confirmed by literature verification and functional enrichment analysis. Future experimental validation is expected for the further investigation of their molecular mechanisms. PMID:29098014

  14. antaRNA: ant colony-based RNA sequence design.

    Science.gov (United States)

    Kleinkauf, Robert; Mann, Martin; Backofen, Rolf

    2015-10-01

    RNA sequence design is studied at least as long as the classical folding problem. Although for the latter the functional fold of an RNA molecule is to be found ,: inverse folding tries to identify RNA sequences that fold into a function-specific target structure. In combination with RNA-based biotechnology and synthetic biology ,: reliable RNA sequence design becomes a crucial step to generate novel biochemical components. In this article ,: the computational tool antaRNA is presented. It is capable of compiling RNA sequences for a given structure that comply in addition with an adjustable full range objective GC-content distribution ,: specific sequence constraints and additional fuzzy structure constraints. antaRNA applies ant colony optimization meta-heuristics and its superior performance is shown on a biological datasets. http://www.bioinf.uni-freiburg.de/Software/antaRNA CONTACT: backofen@informatik.uni-freiburg.de Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press.

  15. The different faces of the p. A53T alpha-synuclein mutation: A screening of Greek patients with parkinsonism and/or dementia.

    Science.gov (United States)

    Breza, Marianthi; Koutsis, Georgios; Karadima, Georgia; Potagas, Constantin; Kartanou, Chrisoula; Papageorgiou, Sokratis G; Paraskevas, George P; Kapaki, Elisabeth; Stefanis, Leonidas; Panas, Marios

    2018-04-13

    The p. A53T mutation in the alpha-synuclein (SNCA) gene is a rare cause of autosomal dominant Parkinson's disease (PD). Although generally rare, it is particularly common in the Greek population due to a founder effect. A53T-positive PD patients often develop dementia during disease course and may very rarely present with dementia. We screened for the p. A53T SNCA mutation a total of 347 cases of Greek origin with parkinsonism and/or dementia, collected over 15 years at the Neurogenetics Unit, Eginition Hospital, University of Athens. Cases were classified into: "pure parkinsonism", "pure dementia" and "parkinsonism plus dementia". In total, 4 p. A53T SNCA mutation carriers were identified. All had autosomal dominant family history and early onset. Screening of the "pure parkinsonism" category revealed 2 cases with typical PD. The other two mutation carriers were identified in the "parkinsonism plus dementia" category. One had a diagnosis of PD dementia and the other of behavioral variant frontotemporal dementia. Screening of patients with "pure dementia" failed to identify any further A53T-positive cases. Our results confirm that the p. A53T SNCA mutation is relatively common in Greek patients with PD or PD plus dementia, particularly in cases with early onset and/or autosomal dominant family history. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Mutation of mapped TIA-1/TIAR binding sites in the 3' terminal stem-loop of West Nile virus minus-strand RNA in an infectious clone negatively affects genomic RNA amplification.

    Science.gov (United States)

    Emara, Mohamed M; Liu, Hsuan; Davis, William G; Brinton, Margo A

    2008-11-01

    Previous data showed that the cellular proteins TIA-1 and TIAR bound specifically to the West Nile virus 3' minus-strand stem-loop [WNV3'(-)SL] RNA (37) and colocalized with flavivirus replication complexes in WNV- and dengue virus-infected cells (21). In the present study, the sites on the WNV3'(-)SL RNA required for efficient in vitro T-cell intracellular antigen-related (TIAR) and T-cell intracellular antigen-1 (TIA-1) protein binding were mapped to short AU sequences (UAAUU) located in two internal loops of the WNV3'(-)SL RNA structure. Infectious clone RNAs with all or most of the binding site nucleotides in one of the 3' (-)SL loops deleted or substituted did not produce detectable virus after transfection or subsequent passage. With one exception, deletion/mutation of a single terminal nucleotide in one of the binding sequences had little effect on the efficiency of protein binding or virus production, but mutation of a nucleotide in the middle of a binding sequence reduced both the in vitro protein binding efficiency and virus production. Plaque size, intracellular genomic RNA levels, and virus production progressively decreased with decreasing in vitro TIAR/TIA-1 binding activity, but the translation efficiency of the various mutant RNAs was similar to that of the parental RNA. Several of the mutant RNAs that inefficiently interacted with TIAR/TIA-1 in vitro rapidly reverted in vivo, indicating that they could replicate at a low level and suggesting that an interaction between TIAR/TIA-1 and the viral 3'(-)SL RNA is not required for initial low-level symmetric RNA replication but instead facilitates the subsequent asymmetric amplification of genome RNA from the minus-strand template.

  17. DHPLC-based mutation analysis of ENG and ALK-1 genes in HHT Italian population.

    Science.gov (United States)

    Lenato, Gennaro M; Lastella, Patrizia; Di Giacomo, Marilena C; Resta, Nicoletta; Suppressa, Patrizia; Pasculli, Giovanna; Sabbà, Carlo; Guanti, Ginevra

    2006-02-01

    Hereditary haemorrhagic telangiectasia (HHT or Rendu-Osler-Weber syndrome) is an autosomal dominant disorder characterized by localized angiodysplasia due to mutations in endoglin, ALK-1 gene, and a still unidentified locus. The lack of highly recurrent mutations, locus heterogeneity, and the presence of mutations in almost all coding exons of the two genes makes the screening for mutations time-consuming and costly. In the present study, we developed a DHPLC-based protocol for mutation detection in ALK1 and ENG genes through retrospective analysis of known sequence variants, 20 causative mutations and 11 polymorphisms, and a prospective analysis on 47 probands with unknown mutation. Overall DHPLC analysis identified the causative mutation in 61 out 66 DNA samples (92.4%). We found 31 different mutations in the ALK1 gene, of which 15 are novel, and 20, of which 12 are novel, in the ENG gene, thus providing for the first time the mutational spectrum in a cohort of Italian HHT patients. In addition, we characterized the splicing pattern of ALK1 gene in lymphoblastoid cells, both in normal controls and in two individuals carrying a mutation in the non-invariant -3 position of the acceptor splice site upstream exon 6 (c.626-3C>G). Functional essay demonstrated the existence, also in normal individuals, of a small proportion of ALK1 alternative splicing, due to exon 5 skipping, and the presence of further aberrant splicing isoforms in the individuals carrying the c.626-3C>G mutation. 2006 Wiley-Liss, Inc.

  18. Oxazolidinone resistance mutations in 23S rRNA of Escherichia coli reveal the central region of domain V as the primary site of drug action

    DEFF Research Database (Denmark)

    Xiong, L; Kloss, P; Douthwaite, S

    2000-01-01

    Oxazolidinone antibiotics inhibit bacterial protein synthesis by interacting with the large ribosomal subunit. The structure and exact location of the oxazolidinone binding site remain obscure, as does the manner in which these drugs inhibit translation. To investigate the drug-ribosome interaction......, we selected Escherichia coli oxazolidinone-resistant mutants, which contained a randomly mutagenized plasmid-borne rRNA operon. The same mutation, G2032 to A, was identified in the 23S rRNA genes of several independent resistant isolates. Engineering of this mutation by site-directed mutagenesis...

  19. CodonShuffle: a tool for generating and analyzing synonymously mutated sequences

    OpenAIRE

    Jorge, Daniel Macedo de Melo; Mills, Ryan E.; Lauring, Adam S.

    2015-01-01

    Because synonymous mutations do not change the amino acid sequence of a protein, they are generally considered to be selectively neutral. Empiric data suggest, however, that a significant fraction of viral mutational fitness effects may be attributable to synonymous mutation. Bias in synonymous codon usage in viruses may result from selection for translational efficiency, mutational bias, base pairing requirements in RNA structures, or even selection against specific dinucleotides by innate i...

  20. Targeted prostate cancer screening in men with mutations in BRCA1 and BRCA2 detects aggressive prostate cancer: preliminary analysis of the results of the IMPACT study

    DEFF Research Database (Denmark)

    Mitra, Anita V; Bancroft, Elizabeth K; Barbachano, Yolanda

    2011-01-01

    mutations were offered annual prostate specific antigen (PSA) testing, and those with PSA >3 ng/mL, were offered a prostate biopsy. Controls were men age-matched (± 5 years) who were negative for the familial mutation. RESULTS: In total, 300 men were recruited (205 mutation carriers; 89 BRCA1, 116 BRCA2......Study Type - Diagnostic (validating cohort)
Level of Evidence 1b OBJECTIVES: To evaluate the role of targeted prostate cancer screening in men with BRCA1 or BRCA2 mutations, an international study, IMPACT (Identification of Men with a genetic predisposition to ProstAte Cancer: Targeted screening...... in BRCA1/2 mutation carriers and controls), was established. This is the first multicentre screening study targeted at men with a known genetic predisposition to prostate cancer. A preliminary analysis of the data is reported. MATERIALS AND METHODS: Men aged 40-69 years from families with BRCA1 or BRCA2...

  1. Gene editing by co-transformation of TALEN and chimeric RNA/DNA oligonucleotides on the rice OsEPSPS gene and the inheritance of mutations.

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    Mugui Wang

    Full Text Available Although several site-specific nucleases (SSNs, such as zinc-finger nucleases (ZFNs, transcription activator-like effector nucleases (TALENs, and the clustered regularly interspaced short palindromic repeat (CRISPR/Cas, have emerged as powerful tools for targeted gene editing in many organisms, to date, gene targeting (GT in plants remains a formidable challenge. In the present study, we attempted to substitute a single base in situ on the rice OsEPSPS gene by co-transformation of TALEN with chimeric RNA/DNA oligonucleotides (COs, including different strand composition such as RNA/DNA (C1 or DNA/RNA (C2 but contained the same target base to be substituted. In contrast to zero GT event obtained by the co-transformation of TALEN with homologous recombination plasmid (HRP, we obtained one mutant showing target base substitution although accompanied by undesired deletion of 12 bases downstream the target site from the co-transformation of TALEN and C1. In addition to this typical event, we also obtained 16 mutants with different length of base deletions around the target site among 105 calli lines derived from transformation of TALEN alone (4/19 as well as co-transformation of TELAN with either HRP (5/30 or C1 (2/25 or C2 (5/31. Further analysis demonstrated that the homozygous gene-edited mutants without foreign gene insertion could be obtained in one generation. The induced mutations in transgenic generation were also capable to pass to the next generation stably. However, the genotypes of mutants did not segregate normally in T1 population, probably due to lethal mutations. Phenotypic assessments in T1 generation showed that the heterozygous plants with either one or three bases deletion on target sequence, called d1 and d3, were more sensitive to glyphosate and the heterozygous d1 plants had significantly lower seed-setting rate than wild-type.

  2. A single nucleotide mutation in Nppc is associated with a long bone abnormality in lbab mice.

    Science.gov (United States)

    Jiao, Yan; Yan, Jian; Jiao, Feng; Yang, Hongbin; Donahue, Leah Rae; Li, Xinmin; Roe, Bruce A; Stuart, John; Gu, Weikuan

    2007-04-17

    The long bone abnormality (lbab) mouse is a new autosomal recessive mutant characterized by overall smaller body size with proportionate dwarfing of all organs and shorter long bones. Previous linkage analysis has located the lbab mutation on chromosome 1 between the markers D1Mit9 and D1Mit488. A genome-based positional approach was used to identify a mutation associated with lbab disease. A total of 122 genes and expressed sequence tags at the lbab region were screened for possible mutation by using genomic DNA from lbabl/lbab, lbab/+, and +/+ B6 mice and high throughput temperature gradient capillary electrophoresis. A sequence difference was identified in one of the amplicons of gene Nppc between lbab/lbab and +/+ mice. One-step reverse transcriptase polymerase chain reaction was performed to validate the difference of Nppc in different types of mice at the mRNA level. The mutation of Nppc was unique in lbab/lbab mice among multiple mouse inbred strains. The mutation of Nppc is co-segregated with lbab disease in 200 progenies produced from heterozygous lbab/+ parents. A single nucleotide mutation of Nppc is associated with dwarfism in lbab/lbab mice. Current genome information and technology allow us to efficiently identify single nucleotide mutations from roughly mapped disease loci. The lbab mouse is a useful model for hereditary human achondroplasia.

  3. A single nucleotide mutation in Nppc is associated with a long bone abnormality in lbab mice

    Directory of Open Access Journals (Sweden)

    Roe Bruce A

    2007-04-01

    Full Text Available Abstract Background The long bone abnormality (lbab mouse is a new autosomal recessive mutant characterized by overall smaller body size with proportionate dwarfing of all organs and shorter long bones. Previous linkage analysis has located the lbab mutation on chromosome 1 between the markers D1Mit9 and D1Mit488. Results A genome-based positional approach was used to identify a mutation associated with lbab disease. A total of 122 genes and expressed sequence tags at the lbab region were screened for possible mutation by using genomic DNA from lbabl/lbab, lbab/+, and +/+ B6 mice and high throughput temperature gradient capillary electrophoresis. A sequence difference was identified in one of the amplicons of gene Nppc between lbab/lbab and +/+ mice. One-step reverse transcriptase polymerase chain reaction was performed to validate the difference of Nppc in different types of mice at the mRNA level. The mutation of Nppc was unique in lbab/lbab mice among multiple mouse inbred strains. The mutation of Nppc is co-segregated with lbab disease in 200 progenies produced from heterozygous lbab/+ parents. Conclusion A single nucleotide mutation of Nppc is associated with dwarfism in lbab/lbab mice. Current genome information and technology allow us to efficiently identify single nucleotide mutations from roughly mapped disease loci. The lbab mouse is a useful model for hereditary human achondroplasia.

  4. Mutations in Cytosine-5 tRNA Methyltransferases Impact Mobile Element Expression and Genome Stability at Specific DNA Repeats

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    Bianca Genenncher

    2018-02-01

    Full Text Available The maintenance of eukaryotic genome stability is ensured by the interplay of transcriptional as well as post-transcriptional mechanisms that control recombination of repeat regions and the expression and mobility of transposable elements. We report here that mutations in two (cytosine-5 RNA methyltransferases, Dnmt2 and NSun2, impact the accumulation of mobile element-derived sequences and DNA repeat integrity in Drosophila. Loss of Dnmt2 function caused moderate effects under standard conditions, while heat shock exacerbated these effects. In contrast, NSun2 function affected mobile element expression and genome integrity in a heat shock-independent fashion. Reduced tRNA stability in both RCMT mutants indicated that tRNA-dependent processes affected mobile element expression and DNA repeat stability. Importantly, further experiments indicated that complex formation with RNA could also contribute to the impact of RCMT function on gene expression control. These results thus uncover a link between tRNA modification enzymes, the expression of repeat DNA, and genomic integrity.

  5. Screening individuals with intellectual disability, autism and Tourette's syndrome for KCNK9 mutations and aberrant DNA methylation within the 8q24 imprinted cluster

    DEFF Research Database (Denmark)

    Delgado, Marta Sánchez; Camprubí, Cristina; Tümer, Zeynep

    2014-01-01

    The phenotype overlap between autism spectrum disorders (ASD) & intellectual disabilities (ID) is mirrored at the genetic level, with common genes being reported mutated in variety of developmental disabilities. However despite widespread genetic screening for mutations, in approximately 40......-60% of childhood developmental disorders the genetic cause remains unknown. Several genome-wide linkage screens in ASD have identified a locus mapping to distal 8q. We have recently identified a novel brain-specific imprinted cluster at this location, which contains the reciprocally expressed maternal KCNK9...... and paternally expressed non-coding PEG13 transcripts, the latter located within an intron of TRAPPC9. Interestingly, mutations of KCNK9 and TRAPPC9 have been reported in Birk-Barel mental retardation and non-syndromic familial forms of ID, respectively. Here, we report a genetic screen for KCNK9 coding...

  6. BRCA1 and BRCA2 Gene Mutations Screening In Sporadic Breast Cancer Patients In Kazakhstan.

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    Ainur R. Akilzhanova

    2013-05-01

    Full Text Available Background: A large number of distinct mutations in the BRCA1 and BRCA2 genes have been reported worldwide, but little is known regarding the role of these inherited susceptibility genes in breast cancer risk among Kazakhstan women. Aim: To evaluate the role of BRCA1/2 mutations in Kazakhstan women presenting with sporadic breast cancer. Methods: We investigated the distribution and nature of polymorphisms in BRCA1 and BRCA2 entire coding regions in 156 Kazakhstan sporadic breast cancer cases and 112 age-matched controls using automatic direct sequencing. Results: We identified 22 distinct variants, including 16 missense mutations and 6 polymorphisms in BRCA1/2 genes. In BRCA1, 9 missense mutations and 3 synonymous polymorphisms were observed. In BRCA2, 7 missense mutations and 3 polymorphisms were detected. There was a higher prevalence of observed mutations in Caucasian breast cancer cases compared to Asian cases (p<0.05; higher frequencies of sequence variants were observed in Asian controls. No recurrent or founder mutations were observed in BRCA1/2 genes. There were no statistically significant differences in age at diagnosis, tumor histology, size of tumor, and lymph node involvement between women with breast cancer with or without the BRCA sequence alterations. Conclusions: Considering the majority of breast cancer cases are sporadic, the present study will be helpful in the evaluation of the need for the genetic screening of BRCA1/2 mutations and reliable genetic counseling for Kazakhstan sporadic breast cancer patients. Evaluation of common polymorphisms and mutations and breast cancer risk in families with genetic predisposition to breast cancer is ongoing in another current investigation. 

  7. The Usher Syndrome Type IIIB Histidyl-tRNA Synthetase Mutation Confers Temperature Sensitivity.

    Science.gov (United States)

    Abbott, Jamie A; Guth, Ethan; Kim, Cindy; Regan, Cathy; Siu, Victoria M; Rupar, C Anthony; Demeler, Borries; Francklyn, Christopher S; Robey-Bond, Susan M

    2017-07-18

    Histidyl-tRNA synthetase (HARS) is a highly conserved translation factor that plays an essential role in protein synthesis. HARS has been implicated in the human syndromes Charcot-Marie-Tooth (CMT) Type 2W and Type IIIB Usher (USH3B). The USH3B mutation, which encodes a Y454S substitution in HARS, is inherited in an autosomal recessive fashion and associated with childhood deafness, blindness, and episodic hallucinations during acute illness. The biochemical basis of the pathophysiologies linked to USH3B is currently unknown. Here, we present a detailed functional comparison of wild-type (WT) and Y454S HARS enzymes. Kinetic parameters for enzymes and canonical substrates were determined using both steady state and rapid kinetics. Enzyme stability was examined using differential scanning fluorimetry. Finally, enzyme functionality in a primary cell culture was assessed. Our results demonstrate that the Y454S substitution leaves HARS amino acid activation, aminoacylation, and tRNA His binding functions largely intact compared with those of WT HARS, and the mutant enzyme dimerizes like the wild type does. Interestingly, during our investigation, it was revealed that the kinetics of amino acid activation differs from that of the previously characterized bacterial HisRS. Despite the similar kinetics, differential scanning fluorimetry revealed that Y454S is less thermally stable than WT HARS, and cells from Y454S patients grown at elevated temperatures demonstrate diminished levels of protein synthesis compared to those of WT cells. The thermal sensitivity associated with the Y454S mutation represents a biochemical basis for understanding USH3B.

  8. Analysis of hepatitis C virus RNA dimerization and core–RNA interactions

    Science.gov (United States)

    Ivanyi-Nagy, Roland; Kanevsky, Igor; Gabus, Caroline; Lavergne, Jean-Pierre; Ficheux, Damien; Penin, François; Fossé, Philippe; Darlix, Jean-Luc

    2006-01-01

    The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3′-untranslated region (3′-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623–2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3′-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus. PMID:16707664

  9. Analysis of hepatitis C virus RNA dimerization and core-RNA interactions.

    Science.gov (United States)

    Ivanyi-Nagy, Roland; Kanevsky, Igor; Gabus, Caroline; Lavergne, Jean-Pierre; Ficheux, Damien; Penin, François; Fossé, Philippe; Darlix, Jean-Luc

    2006-01-01

    The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3'-untranslated region (3'-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623-2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3'-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus.

  10. Molecular screening of 980 cases of suspected hereditary optic neuropathy with a report on 77 novel OPA1 mutations

    DEFF Research Database (Denmark)

    Ferré, Marc; Bonneau, Dominique; Milea, Dan

    2009-01-01

    We report the results of molecular screening in 980 patients carried out as part of their work-up for suspected hereditary optic neuropathies. All the patients were investigated for Leber's hereditary optic neuropathy (LHON) and autosomal dominant optic atrophy (ADOA), by searching for the ten...... and OPA3 mutations in cases of suspected hereditary optic neuropathy, even in absence of a family history of the disease....... novel OPA1 mutations reported here. The statistical analysis of this large set of mutations has led us to propose a diagnostic strategy that should help with the molecular work-up of optic neuropathies. Our results highlight the importance of investigating LHON-causing mtDNA mutations as well as OPA1...

  11. Refining the role of PMS2 in Lynch syndrome: germline mutational analysis improved by comprehensive assessment of variants.

    Science.gov (United States)

    Borràs, Ester; Pineda, Marta; Cadiñanos, Juan; Del Valle, Jesús; Brieger, Angela; Hinrichsen, Inga; Cabanillas, Ruben; Navarro, Matilde; Brunet, Joan; Sanjuan, Xavier; Musulen, Eva; van der Klift, Helen; Lázaro, Conxi; Plotz, Guido; Blanco, Ignacio; Capellá, Gabriel

    2013-08-01

    The majority of mismatch repair (MMR) gene mutations causing Lynch syndrome (LS) occur either in MLH1 or MSH2. However, the relative contribution of PMS2 is less well defined. The aim of this study was to evaluate the role of PMS2 in LS by assessing the pathogenicity of variants of unknown significance (VUS) detected in the mutational analysis of PMS2 in a series of Spanish patients. From a cohort of 202 LS suspected patients, 13 patients showing loss of PMS2 expression in tumours were screened for germline mutations in PMS2, using a long range PCR based strategy and multiplex ligation dependent probe amplification (MLPA). Pathogenicity assessment of PMS2 VUS was performed evaluating clinicopathological data, frequency in control population and in silico and in vitro analyses at the RNA and protein level. Overall 25 different PMS2 DNA variants were detected. Fourteen were classified as polymorphisms. Nine variants were classified as pathogenic: seven alterations based on their molecular nature and two after demonstrating a functional defect (c.538-3C>G affected mRNA processing and c.137G>T impaired MMR activity). The c.1569C>G variant was classified as likely neutral while the c.384G>A remained as a VUS. We have also shown that the polymorphic variant c.59G>A is MMR proficient. Pathogenic PMS2 mutations were detected in 69% of patients harbouring LS associated tumours with loss of PMS2 expression. In all, PMS2 mutations account for 6% of the LS cases identified. The comprehensive functional analysis shown here has been useful in the classification of PMS2 VUS and contributes to refining the role of PMS2 in LS.

  12. 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

  13. Identification of nonviable genes affecting touch sensitivity in Caenorhabditis elegans using neuronally enhanced feeding RNA interference.

    Science.gov (United States)

    Chen, Xiaoyin; Cuadros, Margarete Diaz; Chalfie, Martin

    2015-01-09

    Caenorhabditis elegans senses gentle touch along the body via six touch receptor neurons. Although genetic screens and microarray analyses have identified several genes needed for touch sensitivity, these methods miss pleiotropic genes that are essential for the viability, movement, or fertility of the animals. We used neuronally enhanced feeding RNA interference to screen genes that cause lethality or paralysis when mutated, and we identified 61 such genes affecting touch sensitivity, including five positive controls. We confirmed 18 genes by using available alleles, and further studied one of them, tag-170, now renamed txdc-9. txdc-9 preferentially affects anterior touch response but is needed for tubulin acetylation and microtubule formation in both the anterior and posterior touch receptor neurons. Our results indicate that neuronally enhanced feeding RNA interference screens complement traditional mutageneses by identifying additional nonviable genes needed for specific neuronal functions. Copyright © 2015 Chen et al.

  14. Screening for acyanogenic somatic mutations in cassava (Manihot esculenta Crantz)

    International Nuclear Information System (INIS)

    Moh, C.C.

    1976-01-01

    By irradiating the young stem cuttings (6-8 months old wood) of a cassava cultivar, Japonesa, (Manihot esculenta Crantz) with an acute dose of 4 kR from a 60 CO source, it was found that in a number of cases, the induced mutant characters appeared in the whole R 1 plants or in large chimeric sectors. This result suggested that a cassava plant could develop from one or two initial cells in the shoot apex of a bud. This unusual biological response to radiation provides a great advantage for selection in mutation breeding. By using the sodium picrate method, 2676 leaves from 1338 R 1 plants irradiated with 4 kR were screened for hydrocyanic acid content (HCN). As compared with the control, some leaves had higher and some had lower HCN level, indicating that the radiation broadened the variability. Whether or not those R 1 plants producing a lower level of HCN in the leaves are truly a genetic mutant cannot be ascertained at present. Further screening of the selected R 1 plants in the subsequent vegetative propagation generations will help to distinguish whether they are genetic mutants. (author)

  15. RNA Interference Screen to Identify Pathways That Enhance or Reduce Nonviral Gene Transfer During Lipofection

    OpenAIRE

    Barker, Gregory A; Diamond, Scott L

    2008-01-01

    Some barriers to DNA lipofection are well characterized; however, there is as yet no method of finding unknown pathways that impact the process. A druggable genome small-interfering RNA (siRNA) screen against 5,520 genes was tested for its effect on lipofection of human aortic endothelial cells (HAECs). We found 130 gene targets which, when silenced by pooled siRNAs (three siRNAs per gene), resulted in enhanced luminescence after lipofection (86 gene targets showed reduced expression). In con...

  16. NMD and microRNA expression profiling of the HPCX1 locus reveal MAGEC1 as a candidate prostate cancer predisposition gene

    International Nuclear Information System (INIS)

    Mattila, Henna; Schindler, Martin; Isotalo, Jarkko; Ikonen, Tarja; Vihinen, Mauno; Oja, Hannu; Tammela, Teuvo LJ; Wahlfors, Tiina; Schleutker, Johanna

    2011-01-01

    Several predisposition loci for hereditary prostate cancer (HPC) have been suggested, including HPCX1 at Xq27-q28, but due to the complex structure of the region, the susceptibility gene has not yet been identified. In this study, nonsense-mediated mRNA decay (NMD) inhibition was used for the discovery of truncating mutations. Six prostate cancer (PC) patients and their healthy brothers were selected from a group of HPCX1-linked families. Expression analyses were done using Agilent 44 K oligoarrays, and selected genes were screened for mutations by direct sequencing. In addition, microRNA expression levels in the lymphoblastic cells were analyzed to trace variants that might alter miRNA expression and explain partly an inherited genetic predisposion to PC. Seventeen genes were selected for resequencing based on the NMD array, but no truncating mutations were found. The most interesting variant was MAGEC1 p.Met1?. An association was seen between the variant and unselected PC (OR = 2.35, 95% CI = 1.10-5.02) and HPC (OR = 3.38, 95% CI = 1.10-10.40). miRNA analysis revealed altogether 29 miRNAs with altered expression between the PC cases and controls. miRNA target analysis revealed that 12 of them also had possible target sites in the MAGEC1 gene. These miRNAs were selected for validation process including four miRNAs located in the X chromosome. The expressions of 14 miRNAs were validated in families that contributed to the significant signal differences in Agilent arrays. Further functional studies are needed to fully understand the possible contribution of these miRNAs and MAGEC1 start codon variant to PC

  17. Diagnostic yield, interpretation, and clinical utility of mutation screening of sarcomere encoding genes in Danish hypertrophic cardiomyopathy patients and relatives

    DEFF Research Database (Denmark)

    Andersen, Paal Skytt; Havndrup, Ole; Hougs, Lotte

    2008-01-01

    persons. Index patients were screened for mutations in all coding regions of 10 sarcomere genes (MYH7, MYL3, MYBPC3, TNNI3, TNNT2, TPM1, ACTC, CSRP3, TCAP, and TNNC1) and five exons of TTN. Relatives were screened for presence of minor or major diagnostic criteria for HCM and tracking of DNA variants...

  18. SCREEN FOR DOMINANT BEHAVIORAL MUTATIONS CAUSED BY GENOMIC INSERTION OF P-ELEMENT TRANSPOSONS IN DROSOPHILA: AN EXAMINATION OF THE INTEGRATION OF VIRAL VECTOR SEQUENCES

    OpenAIRE

    FOX, LYLE E.; GREEN, DAVID; YAN, ZIYING; ENGELHARDT, JOHN F.; WU, CHUN-FANG

    2007-01-01

    Here we report the development of a high-throughput screen to assess dominant mutation rates caused by P-element transposition within the Drosophila genome that is suitable for assessing the undesirable effects of integrating foreign regulatory sequences (viral cargo) into a host genome. Three different behavioral paradigms were used: sensitivity to mechanical stress, response to heat stress, and ability to fly. The results, from our screen of 35,000 flies, indicate that mutations caused by t...

  19. In-vivo quantification of primary microRNA processing by Drosha with a luciferase based system

    International Nuclear Information System (INIS)

    Allegra, Danilo; Mertens, Daniel

    2011-01-01

    Research highlights: → Posttranscriptional regulation of miRNA processing is difficult to quantify. → Our in-vivo processing assay can quantify Drosha cleavage in live cells. → It is based on luciferase reporters fused with pri-miRNAs. → The assay validates the processing defect caused by a mutation in pri-16-1. → It is a sensitive method to quantify pri-miRNA cleavage by Drosha in live cells. -- Abstract: The RNAse III Drosha is responsible for the first step of microRNA maturation, the cleavage of primary miRNA to produce the precursor miRNA. Processing by Drosha is finely regulated and influences the amount of mature microRNA in a cell. We describe in the present work a method to quantify Drosha processing activity in-vivo, which is applicable to any microRNA. With respect to other methods for measuring Drosha activity, our system is faster and scalable, can be used with any cellular system and does not require cell sorting or use of radioactive isotopes. This system is useful to study regulation of Drosha activity in physiological and pathological conditions.

  20. A novel AMELX mutation causes hypoplastic amelogenesis imperfecta.

    Science.gov (United States)

    Kim, Young-Jae; Kim, Youn Jung; Kang, Jenny; Shin, Teo Jeon; Hyun, Hong-Keun; Lee, Sang-Hoon; Lee, Zang Hee; Kim, Jung-Wook

    2017-04-01

    Amelogenesis imperfecta (AI) is a hereditary genetic defect affecting tooth enamel. AI is heterogeneous in clinical phenotype as well as in genetic etiology. To date, more than 10 genes have been associated with the etiology of AI. Amelogenin is the most abundant enamel matrix protein, most of which is encoded by the amelogenin gene in the X-chromosome (AMELX). More than 16 alternative splicing transcripts have been identified in the murine Amelx gene. The purpose of this study was to identify the genetic cause of an AI family. We recruited a family with hypoplastic AI and performed mutational analysis on the candidate gene based on the clinical phenotype. Mutational analysis revealed a missense mutation in exon 6 (NM_182680.1; c.242C > T), which changes a sequence in a highly conserved amino acid (NP_872621.1; p.Pro81Leu). Furthermore, a splicing assay using a minigene displayed that the mutation changed the mRNA splicing repertory. In this study, we identified a novel AMELX missense mutation causing hypoplastic AI, and this mutation also resulted in altered mRNA splicing. These results will not only expand the mutation spectrum causing AI but also broaden our understanding of the biological mechanism of enamel formation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. First Report of the 23S rRNA Gene A2058G Point Mutation Associated With Macrolide Resistance in Treponema pallidum From Syphilis Patients in Cuba.

    Science.gov (United States)

    Noda, Angel A; Matos, Nelvis; Blanco, Orestes; Rodríguez, Islay; Stamm, Lola Virginia

    2016-05-01

    This study aimed to assess the presence of macrolide-resistant Treponema pallidum subtypes in Havana, Cuba. Samples from 41 syphilis patients were tested for T. pallidum 23S rRNA gene mutations. Twenty-five patients (61%) harbored T. pallidum with the A2058G mutation, which was present in all 8 subtypes that were identified. The A2059G mutation was not detected.

  2. Genomic sequencing in cystic fibrosis newborn screening: what works best, two-tier predefined CFTR mutation panels or second-tier CFTR panel followed by third-tier sequencing?

    Science.gov (United States)

    Currier, Robert J; Sciortino, Stan; Liu, Ruiling; Bishop, Tracey; Alikhani Koupaei, Rasoul; Feuchtbaum, Lisa

    2017-10-01

    PurposeThe purpose of this study was to model the performance of several known two-tier, predefined mutation panels and three-tier algorithms for cystic fibrosis (CF) screening utilizing the ethnically diverse California population.MethodsThe cystic fibrosis transmembrane conductance regulator (CFTR) mutations identified among the 317 CF cases in California screened between 12 August 2008 and 18 December 2012 were used to compare the expected CF detection rates for several two- and three-tier screening approaches, including the current California approach, which consists of a population-specific 40-mutation panel followed by third-tier sequencing when indicated.ResultsThe data show that the strategy of using third-tier sequencing improves CF detection following an initial elevated immunoreactive trypsinogen and detection of only one mutation on a second-tier panel.ConclusionIn a diverse population, the use of a second-tier panel followed by third-tier CFTR gene sequencing provides a better detection rate for CF, compared with the use of a second-tier approach alone, and is an effective way to minimize the referrals of CF carriers for sweat testing. Restricting screening to a second-tier testing to predefined mutation panels, even broad ones, results in some missed CF cases and demonstrates the limited utility of this approach in states that have diverse multiethnic populations.

  3. Mitochondrial 12S ribosomal RNA A1555G mutation associated with cardiomyopathy and hearing loss following high-dose chemotherapy and repeated aminoglycoside exposure

    DEFF Research Database (Denmark)

    Skou, Anne-Sofie; Tranebjærg, Lisbeth; Jensen, Tim

    2014-01-01

    A 19-month-old girl with the A1555G mitochondrial mutation in the 12S ribosomal RNA gene and acute myelogenous leukemia developed dilated cardiomyopathy and bilateral sensorineural hearing loss before undergoing allogeneic stem cell transplantation. She had received gentamicin during episodes of ...... of febrile neutropenia. Testing for the A1555G mutation is recommended in patients frequently treated with aminoglycosides....

  4. The identification and functional annotation of RNA structures conserved in vertebrates

    DEFF Research Database (Denmark)

    Seemann, Ernst Stefan; Mirza, Aashiq Hussain; Hansen, Claus

    2017-01-01

    Structured elements of RNA molecules are essential in, e.g., RNA stabilization, localization and protein interaction, and their conservation across species suggests a common functional role. We computationally screened vertebrate genomes for Conserved RNA Structures (CRSs), leveraging structure-b......-structured counterparts. Our findings of transcribed uncharacterized regulatory regions that contain CRSs support their RNA-mediated functionality.......Structured elements of RNA molecules are essential in, e.g., RNA stabilization, localization and protein interaction, and their conservation across species suggests a common functional role. We computationally screened vertebrate genomes for Conserved RNA Structures (CRSs), leveraging structure......-based, rather than sequence-based, alignments. After careful correction for sequence identity and GC content, we predict ~516k human genomic regions containing CRSs. We find that a substantial fraction of human-mouse CRS regions (i) co-localize consistently with binding sites of the same RNA binding proteins...

  5. Enriched whole genome sequencing identified compensatory mutations in the RNA polymerase gene of rifampicin-resistant Mycobacterium leprae strains.

    Science.gov (United States)

    Lavania, Mallika; Singh, Itu; Turankar, Ravindra P; Gupta, Anuj Kumar; Ahuja, Madhvi; Pathak, Vinay; Sengupta, Utpal

    2018-01-01

    Despite more than three decades of multidrug therapy (MDT), leprosy remains a major public health issue in several endemic countries, including India. The emergence of drug resistance in Mycobacterium leprae (M. leprae) is a cause of concern and poses a threat to the leprosy-control program, which might ultimately dampen the achievement of the elimination program of the country. Rifampicin resistance in clinical strains of M. leprae are supposed to arise from harboring bacterial strains with mutations in the 81-bp rifampicin resistance determining region (RRDR) of the rpoB gene. However, complete dynamics of rifampicin resistance are not explained only by this mutation in leprosy strains. To understand the role of other compensatory mutations and transmission dynamics of drug-resistant leprosy, a genome-wide sequencing of 11 M. leprae strains - comprising five rifampicin-resistant strains, five sensitive strains, and one reference strain - was done in this study. We observed the presence of compensatory mutations in two rifampicin-resistant strains in rpoC and mmpL7 genes, along with rpoB , that may additionally be responsible for conferring resistance in those strains. Our findings support the role for compensatory mutation(s) in RNA polymerase gene(s), resulting in rifampicin resistance in relapsed leprosy patients.

  6. Asynchronous accumulation of lettuce infectious yellows virus RNAs 1 and 2 and identification of an RNA 1 trans enhancer of RNA 2 accumulation.

    Science.gov (United States)

    Yeh, H H; Tian, T; Rubio, L; Crawford, B; Falk, B W

    2000-07-01

    Time course and mutational analyses were used to examine the accumulation in protoplasts of progeny RNAs of the bipartite Crinivirus, Lettuce infectious yellow virus (LIYV; family Closteroviridae). Hybridization analyses showed that simultaneous inoculation of LIYV RNAs 1 and 2 resulted in asynchronous accumulation of progeny LIYV RNAs. LIYV RNA 1 progeny genomic and subgenomic RNAs could be detected in protoplasts as early as 12 h postinoculation (p.i.) and accumulated to high levels by 24 h p.i. The LIYV RNA 1 open reading frame 2 (ORF 2) subgenomic RNA was the most abundant of all LIYV RNAs detected. In contrast, RNA 2 progeny were not readily detected until ca. 36 h p.i. Mutational analyses showed that in-frame stop codons introduced into five of seven RNA 2 ORFs did not affect accumulation of progeny LIYV RNA 1 or RNA 2, confirming that RNA 2 does not encode proteins necessary for LIYV RNA replication. Mutational analyses also supported that LIYV RNA 1 encodes proteins necessary for replication of LIYV RNAs 1 and 2. A mutation introduced into the LIYV RNA 1 region encoding the overlapping ORF 1B and ORF 2 was lethal. However, mutations introduced into only LIYV RNA 1 ORF 2 resulted in accumulation of progeny RNA 1 near or equal to wild-type RNA 1. In contrast, the RNA 1 ORF 2 mutants did not efficiently support the trans accumulation of LIYV RNA 2. Three distinct RNA 1 ORF 2 mutants were analyzed and all exhibited a similar phenotype for progeny LIYV RNA accumulation. These data suggest that the LIYV RNA 1 ORF 2 encodes a trans enhancer for RNA 2 accumulation.

  7. The Arabidopsis thaliana F-box gene HAWAIIAN SKIRT is a new player in the microRNA pathway.

    Directory of Open Access Journals (Sweden)

    Xuebin Zhang

    Full Text Available In Arabidopsis, the F-box HAWAIIAN SKIRT (HWS protein is important for organ growth. Loss of function of HWS exhibits pleiotropic phenotypes including sepal fusion. To dissect the HWS role, we EMS-mutagenized hws-1 seeds and screened for mutations that suppress hws-1 associated phenotypes. We identified shs-2 and shs-3 (suppressor of hws-2 and 3 mutants in which the sepal fusion phenotype of hws-1 was suppressed. shs-2 and shs-3 (renamed hst-23/hws-1 and hst-24/hws-1 carry transition mutations that result in premature terminations in the plant homolog of Exportin-5 HASTY (HST, known to be important in miRNA biogenesis, function and transport. Genetic crosses between hws-1 and mutant lines for genes in the miRNA pathway also suppress the phenotypes associated with HWS loss of function, corroborating epistatic relations between the miRNA pathway genes and HWS. In agreement with these data, accumulation of miRNA is modified in HWS loss or gain of function mutants. Our data propose HWS as a new player in the miRNA pathway, important for plant growth.

  8. Plasmon based biosensor for distinguishing different peptides mutation states

    KAUST Repository

    Das, Gobind

    2013-05-08

    Periodic and reproducible gold nanocuboids with various matrix dimensions and with different inter-particle gaps were fabricated by means of top-down technique. Rhodamine 6G was used as a probe molecule to optimize the design and the fabrication of the cuboid nanostructures. The electric field distribution for the nanocuboids with varying matrix dimensions/inter-particle gap was also investigated. These SERS devices were employed as biosensors through the investigation of both myoglobin and wild/mutated peptides. The results demonstrate the probing and the screening of wild/mutated BRCA1 peptides, thus opening a path for the fabrication of simple and cheap SERS device capable of early detection of several diseases.

  9. Distinct roles for RDE-1 and RDE-4 during RNA interference in Caenorhabditis elegans.

    Science.gov (United States)

    Parrish, S; Fire, A

    2001-10-01

    RNA interference (RNAi) is a cellular defense mechanism that uses double-stranded RNA (dsRNA) as a sequence-specific trigger to guide the degradation of homologous single-stranded RNAs. RNAi is a multistep process involving several proteins and at least one type of RNA intermediate, a population of small 21-25 nt RNAs (called siRNAs) that are initially derived from cleavage of the dsRNA trigger. Genetic screens in Caenorhabditis elegans have identified numerous mutations that cause partial or complete loss of RNAi. In this work, we analyzed cleavage of injected dsRNA to produce the initial siRNA population in animals mutant for rde-1 and rde-4, two genes that are essential for RNAi but that are not required for organismal viability or fertility. Our results suggest distinct roles for RDE-1 and RDE-4 in the interference process. Although null mutants lacking rde-1 show no phenotypic response to dsRNA, the amount of siRNAs generated from an injected dsRNA trigger was comparable to that of wild-type. By contrast, mutations in rde-4 substantially reduced the population of siRNAs derived from an injected dsRNA trigger. Injection of chemically synthesized 24- or 25-nt siRNAs could circumvent RNAi resistance in rde-4 mutants, whereas no bypass was observed in rde-1 mutants. These results support a model in which RDE-4 is involved before or during production of siRNAs, whereas RDE-1 acts after the siRNAs have been formed.

  10. 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

  11. [Genetic diagnostics of pathogenic splicing abnormalities in the clinical laboratory--pitfalls and screening approaches].

    Science.gov (United States)

    Niimi, Hideki; Ogawa, Tomomi; Note, Rhougou; Hayashi, Shirou; Ueno, Tomohiro; Harada, Kenu; Uji, Yoshinori; Kitajima, Isao

    2010-12-01

    In recent years, genetic diagnostics of pathogenic splicing abnormalities are increasingly recognized as critically important in the clinical genetic diagnostics. It is reported that approximately 10% of pathogenic mutations causing human inherited diseases are splicing mutations. Nonetheless, it is still difficult to identify splicing abnormalities in routine genetic diagnostic settings. Here, we studied two different kinds of cases with splicing abnormalities. The first case is a protein S deficiency. Nucleotide analyses revealed that the proband had a previously reported G to C substitution in the invariant AG dinucleotide at the splicing acceptor site of intronl/exon2, which produces multiple splicing abnormalities resulting in protein S deficiency. The second case is an antithrombin (AT) deficiency. This proband had a previously reported G to A substitution, at nucleotide position 9788 in intron 4, 14 bp in front of exon 5, which created a de novo exon 5 splice site and resulted in AT deficiency. From a practical standpoint, we discussed the pitfalls, attentions, and screening approaches in genetic diagnostics of pathogenic splicing abnormalities. Due to the difficulty with full-length sequence analysis of introns, and the lack of RNA samples, splicing mutations may escape identification. Although current genetic testing remains to be improved, to screen for splicing abnormalities more efficiently, it is significant to use an appropriate combination of various approaches such as DNA and/or RNA samples, splicing mutation databases, bioinformatic tools to detect splice sites and cis-regulatory elements, and in vitro and/or in vivo experimentally methods as needed.

  12. Telomerase reverse transcriptase promoter mutations in bladder cancer

    DEFF Research Database (Denmark)

    Allory, Yves; Beukers, Willemien; Sagrera, Ana

    2014-01-01

    for detection of recurrences in urine in patients with urothelial bladder cancer (UBC). DESIGN, SETTING, AND PARTICIPANTS: A set of 111 UBCs of different stages was used to assess TERT promoter mutations by Sanger sequencing and TERT messenger RNA (mRNA) expression by reverse transcription...... surveillance after diagnosis of non-muscle-invasive UBC (n=194), was tested using a SNaPshot assay. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Association of mutation status with age, sex, tobacco, stage, grade, fibroblast growth factor receptor 3 (FGFR3) mutation, progression-free survival, disease...... frequent among FGFR3 mutant tumors (p=0.0002). There was no association between TERT mutations and mRNA expression (p=0.3). Mutations were not associated with clinical outcome. In urine, TERT mutations had 90% specificity in subjects with hematuria but no bladder tumor, and 73% in recurrence-free UBC...

  13. Biallelic mutations in the 3' exonuclease TOE1 cause pontocerebellar hypoplasia and uncover a role in snRNA processing

    DEFF Research Database (Denmark)

    Lardelli, Rea M.; Schaffer, Ashleigh E.; Eggens, Veerle R C

    2017-01-01

    ) is a unique recessive syndrome characterized by neurodegeneration and ambiguous genitalia. We studied 12 human families with PCH7, uncovering biallelic, loss-of-function mutations in TOE1, which encodes an unconventional deadenylase. toe1-morphant zebrafish displayed midbrain and hindbrain degeneration...... of TOE1 accumulated 3'-end-extended pre-snRNAs, and the immunoisolated TOE1 complex was sufficient for 3'-end maturation of snRNAs. Our findings identify the cause of a neurodegenerative syndrome linked to snRNA maturation and uncover a key factor involved in the processing of snRNA 3' ends....

  14. Molecular insights into the specific recognition between the RNA binding domain qRRM2 of hnRNP F and G-tract RNA: A molecular dynamics study.

    Science.gov (United States)

    Wang, Lingyun; Yan, Feng

    2017-12-09

    Heterogeneous nuclear ribonucleoprotein F (hnRNP F) controls the expression of various genes through regulating the alternative splicing of pre-mRNAs in the nucleus. It uses three quasi-RNA recognition motifs (qRRMs) to recognize G-tract RNA which contains at least three consecutive guanines. The structures containing qRRMs of hnRNP F in complex with G-tract RNA have been determined by nuclear magnetic resonance (NMR) spectroscopy, shedding light on the recognition mechanism of qRRMs with G-tract RNA. However, knowledge of the recognition details is still lacking. To investigate how qRRMs specifically bind with G-tract RNA and how the mutations of any guanine to an adenine in the G-tract affect the binding, molecular dynamics simulations with binding free energy analysis were performed based on the NMR structure of qRRM2 in complex with G-tract RNA. Simulation results demonstrate that qRRM2 binds strongly with G-tract RNA, but any mutation of the G-tract leads to a drastic reduction of the binding free energy. Further comparisons of the energetic components reveal that van der Waals and non-polar interactions play essential roles in the binding between qRRM2 and G-tract RNA, but the interactions are weakened by the effect of RNA mutations. Structural and dynamical analyses indicate that when qRRM2 binds with G-tract RNA, both qRRM2 and G-tract maintain stabilized structures and dynamics; however, the stability is disrupted by the mutations of the G-tract. These results provide novel insights into the recognition mechanism of qRRM2 with G-tract RNA that are not elucidated by the NMR technique. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Mpn1, Mutated in Poikiloderma with Neutropenia Protein 1, Is a Conserved 3′-to-5′ RNA Exonuclease Processing U6 Small Nuclear RNA

    Directory of Open Access Journals (Sweden)

    Vadim Shchepachev

    2012-10-01

    Full Text Available Clericuzio-type poikiloderma with neutropenia (PN is a rare genodermatosis associated with mutations in the C16orf57 gene, which codes for the uncharacterized protein hMpn1. We show here that, in both fission yeasts and humans, Mpn1 processes the spliceosomal U6 small nuclear RNA (snRNA posttranscriptionally. In Mpn1-deficient cells, U6 molecules carry 3′ end polyuridine tails that are longer than those in normal cells and lack a terminal 2′,3′ cyclic phosphate group. In mpn1Δ yeast cells, U6 snRNA and U4/U6 di-small nuclear RNA protein complex levels are diminished, leading to precursor messenger RNA splicing defects, which are reverted by expression of either yeast or human Mpn1 and by overexpression of U6. Recombinant hMpn1 is a 3′-to-5′ RNA exonuclease that removes uridines from U6 3′ ends, generating terminal 2′,3′ cyclic phosphates in vitro. Finally, U6 degradation rates increase in mpn1Δ yeasts and in lymphoblasts established from individuals affected by PN. Our data indicate that Mpn1 promotes U6 stability through 3′ end posttranscriptional processing and implicate altered U6 metabolism as a potential mechanism for PN pathogenesis.

  16. Quantitative PCR high-resolution melting (qPCR-HRM) curve analysis, a new approach to simultaneously screen point mutations and large rearrangements: application to MLH1 germline mutations in Lynch syndrome.

    Science.gov (United States)

    Rouleau, Etienne; Lefol, Cédrick; Bourdon, Violaine; Coulet, Florence; Noguchi, Tetsuro; Soubrier, Florent; Bièche, Ivan; Olschwang, Sylviane; Sobol, Hagay; Lidereau, Rosette

    2009-06-01

    Several techniques have been developed to screen mismatch repair (MMR) genes for deleterious mutations. Until now, two different techniques were required to screen for both point mutations and large rearrangements. For the first time, we propose a new approach, called "quantitative PCR (qPCR) high-resolution melting (HRM) curve analysis (qPCR-HRM)," which combines qPCR and HRM to obtain a rapid and cost-effective method suitable for testing a large series of samples. We designed PCR amplicons to scan the MLH1 gene using qPCR HRM. Seventy-six patients were fully scanned in replicate, including 14 wild-type patients and 62 patients with known mutations (57 point mutations and five rearrangements). To validate the detected mutations, we used sequencing and/or hybridization on a dedicated MLH1 array-comparative genomic hybridization (array-CGH). All point mutations and rearrangements detected by denaturing high-performance liquid chromatography (dHPLC)+multiplex ligation-dependent probe amplification (MLPA) were successfully detected by qPCR HRM. Three large rearrangements were characterized with the dedicated MLH1 array-CGH. One variant was detected with qPCR HRM in a wild-type patient and was located within the reverse primer. One variant was not detected with qPCR HRM or with dHPLC due to its proximity to a T-stretch. With qPCR HRM, prescreening for point mutations and large rearrangements are performed in one tube and in one step with a single machine, without the need for any automated sequencer in the prescreening process. In replicate, its reagent cost, sensitivity, and specificity are comparable to those of dHPLC+MLPA techniques. However, qPCR HRM outperformed the other techniques in terms of its rapidity and amount of data provided.

  17. RNA interference screen to identify pathways that enhance or reduce nonviral gene transfer during lipofection.

    Science.gov (United States)

    Barker, Gregory A; Diamond, Scott L

    2008-09-01

    Some barriers to DNA lipofection are well characterized; however, there is as yet no method of finding unknown pathways that impact the process. A druggable genome small-interfering RNA (siRNA) screen against 5,520 genes was tested for its effect on lipofection of human aortic endothelial cells (HAECs). We found 130 gene targets which, when silenced by pooled siRNAs (three siRNAs per gene), resulted in enhanced luminescence after lipofection (86 gene targets showed reduced expression). In confirmation tests with single siRNAs, 18 of the 130 hits showed enhanced lipofection with two or more individual siRNAs in the absence of cytotoxicity. Of these confirmed gene targets, we identified five leading candidates, two of which are isoforms of the regulatory subunit of protein phosphatase 2A (PP2A). The best candidate siRNA targeted the PPP2R2C gene and produced a 65% increase in luminescence from lipofection, with a quantitative PCR-validated knockdown of approximately 76%. Flow cytometric analysis confirmed that the silencing of the PPP2R2C gene resulted in an improvement of 10% in transfection efficiency, thereby demonstrating an increase in the number of transfected cells. These results show that an RNA interference (RNAi) high-throughput screen (HTS) can be applied to nonviral gene transfer. We have also demonstrated that siRNAs can be co-delivered with lipofected DNA to increase the transfection efficiency in vitro.

  18. Structural Implications of Mutations Conferring Rifampin Resistance in Mycobacterium leprae.

    Science.gov (United States)

    Vedithi, Sundeep Chaitanya; Malhotra, Sony; Das, Madhusmita; Daniel, Sheela; Kishore, Nanda; George, Anuja; Arumugam, Shantha; Rajan, Lakshmi; Ebenezer, Mannam; Ascher, David B; Arnold, Eddy; Blundell, Tom L

    2018-03-22

    The rpoB gene encodes the β subunit of RNA polymerase holoenzyme in Mycobacterium leprae (M. leprae). Missense mutations in the rpoB gene were identified as etiological factors for rifampin resistance in leprosy. In the present study, we identified mutations corresponding to rifampin resistance in relapsed leprosy cases from three hospitals in southern India which treat leprosy patients. DNA was extracted from skin biopsies of 35 relapse/multidrug therapy non-respondent leprosy cases, and PCR was performed to amplify the 276 bp rifampin resistance-determining region of the rpoB gene. PCR products were sequenced, and mutations were identified in four out of the 35 cases at codon positions D441Y, D441V, S437L and H476R. The structural and functional effects of these mutations were assessed in the context of three-dimensional comparative models of wild-type and mutant M. leprae RNA polymerase holoenzyme (RNAP), based on the recently solved crystal structures of RNAP of Mycobacterium tuberculosis, containing a synthetic nucleic acid scaffold and rifampin. The resistance mutations were observed to alter the hydrogen-bonding and hydrophobic interactions of rifampin and the 5' ribonucleotide of the growing RNA transcript. This study demonstrates that rifampin-resistant strains of M. leprae among leprosy patients in southern India are likely to arise from mutations that affect the drug-binding site and stability of RNAP.

  19. BRAFV600 mutations in solid tumors, other than metastatic melanoma and papillary thyroid cancer, or multiple myeloma: a screening study

    Directory of Open Access Journals (Sweden)

    Cohn AL

    2017-02-01

    Full Text Available Allen L Cohn,1 Bann-Mo Day,2 Sarang Abhyankar,3 Edward McKenna,2 Todd Riehl,4 Igor Puzanov5 1Medical Research, Rocky Mountain Cancer Centers, Denver, CO, 2US Medical Affairs, 3Global Safety and Risk Management, 4Product Development Oncology, Genentech, Inc., South San Francisco, CA, 5Melanoma Section, Division of Hematology-Oncology, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA Background: Mutations in the BRAF gene have been implicated in several human cancers. The objective of this screening study was to identify patients with solid tumors (other than metastatic melanoma or papillary thyroid cancer or multiple myeloma harboring activating BRAFV600 mutations for enrollment in a vemurafenib clinical study.Methods: Formalin-fixed, paraffin-embedded tumor samples were collected and sent to a central laboratory to identify activating BRAFV600 mutations by bidirectional direct Sanger sequencing.Results: Overall incidence of BRAFV600E mutation in evaluable patients (n=548 was 3% (95% confidence interval [CI], 1.7–4.7: 11% in colorectal tumors (n=75, 6% in biliary tract tumors (n=16, 3% in non-small cell lung cancers (n=71, 2% in other types of solid tumors (n=180, and 3% in multiple myeloma (n=31. There were no BRAFV600 mutations in this cohort of patients with ovarian tumors (n=68, breast cancer (n=86, or prostate cancer (n=21.Conclusion: This multicenter, national screening study confirms previously reported incidences of BRAFV600 mutations from single-center studies. Patients identified with BRAFV600 mutations were potentially eligible for enrollment in the VE-BASKET study. Keywords: genetic testing, proto-oncogene proteins B-raf, PLX4032

  20. [Hot spot mutation screening of RYR1 gene in diagnosis of congenital myopathies].

    Science.gov (United States)

    Chang, Xing-zhi; Jin, Yi-wen; Wang, Jing-min; Yuan, Yun; Xiong, Hui; Wang, Shuang; Qin, Jiong

    2014-10-18

    the different subtype have the similar clinical manifestations, signs, enzyme detection and electromyography changes. Muscle biopsy plays an important role in the selection of genes to be detected. Hot spot mutation in C-terminal of the RYR1 gene can only be identified in patients with central core disease, so we suggest this hot spot gene mutation screening apply to the suspicious patient with central core disease only.

  1. siRNA-mediated Allele-specific Silencing of a COL6A3 Mutation in a Cellular Model of Dominant Ullrich Muscular Dystrophy

    Directory of Open Access Journals (Sweden)

    Véronique Bolduc

    2014-01-01

    Full Text Available Congenital muscular dystrophy type Ullrich (UCMD is a severe disorder of early childhood onset for which currently there is no effective treatment. UCMD commonly is caused by dominant-negative mutations in the genes coding for collagen type VI, a major microfibrillar component of the extracellular matrix surrounding the muscle fibers. To explore RNA interference (RNAi as a potential therapy for UCMD, we designed a series of small interfering RNA (siRNA oligos that specifically target the most common mutations resulting in skipping of exon 16 in the COL6A3 gene and tested them in UCMD-derived dermal fibroblasts. Transcript analysis by semiquantitative and quantitative reverse transcriptase PCR showed that two of these siRNAs were the most allele-specific, i.e., they efficiently knocked down the expression from the mutant allele, without affecting the normal allele. In HEK293T cells, these siRNAs selectively suppressed protein expression from a reporter construct carrying the mutation, with no or minimal suppression of the wild-type (WT construct, suggesting that collagen VI protein levels are as also reduced in an allele-specific manner. Furthermore, we found that treating UCMD fibroblasts with these siRNAs considerably improved the quantity and quality of the collagen VI matrix, as assessed by confocal microscopy. Our current study establishes RNAi as a promising molecular approach for treating dominant COL6-related dystrophies.

  2. Coexistence of mitochondrial 12S rRNA C1494T and CO1/tRNASer(UCN) G7444A mutations in two Han Chinese pedigrees with aminoglycoside-induced and non-syndromic hearing loss

    International Nuclear Information System (INIS)

    Yuan Huijun; Chen Jing; Liu Xin; Cheng Jing; Wang Xinjian; Yang Li; Yang Shuzhi; Cao Juyang; Kang Dongyang; Dai Pu; Zha, Suoqiang; Han Dongyi; Young Wieyen; Guan Minxin

    2007-01-01

    Mutations in mitochondrial DNA are one of the important causes of hearing loss. We report here the clinical, genetic, and molecular characterization of two Han Chinese pedigrees with maternally transmitted aminoglycoside-induced and nonsyndromic bilateral hearing loss. Clinical evaluation revealed the wide range of severity, age-at-onset, and audiometric configuration of hearing impairment in matrilineal relatives in these families. The penetrances of hearing loss in these pedigrees were 20% and 18%, when aminoglycoside-induced deafness was included. When the effect of aminoglycosides was excluded, the penetrances of hearing loss in these seven pedigrees were 10% and 15%. Sequence analysis of the complete mitochondrial genomes in these pedigrees showed the presence of the deafness-associated 12S rRNA C1494T and CO1/tRNA Ser(UCN) G7444A mutations. Their distinct sets of mtDNA polymorphism belonged to Eastern Asian haplogroup C4a1, while other previously identified six Chinese mitochondrial genomes harboring the C1494T mutation belong to haplogroups D5a2, D, R, and F1, respectively. This suggested that the C1494T or G7444A mutation occurred sporadically and multiplied through evolution of the mitochondrial DNA (mtDNA). The absence of functionally significant mutations in tRNA and rRNAs or secondary LHON mutations in their mtDNA suggest that these mtDNA haplogroup-specific variants may not play an important role in the phenotypic expression of the 12S rRNA C1494T and CO1/tRNA Ser(UCN) G7444A mutations in those Chinese families. However, aminoglycosides and other nuclear modifier genes play a modifying role in the phenotypic manifestation of the C1494T mutation in these Chinese families

  3. Defective i6A37 modification of mitochondrial and cytosolic tRNAs results from pathogenic mutations in TRIT1 and its substrate tRNA.

    Directory of Open Access Journals (Sweden)

    John W Yarham

    2014-06-01

    Full Text Available Identifying the genetic basis for mitochondrial diseases is technically challenging given the size of the mitochondrial proteome and the heterogeneity of disease presentations. Using next-generation exome sequencing, we identified in a patient with severe combined mitochondrial respiratory chain defects and corresponding perturbation in mitochondrial protein synthesis, a homozygous p.Arg323Gln mutation in TRIT1. This gene encodes human tRNA isopentenyltransferase, which is responsible for i6A37 modification of the anticodon loops of a small subset of cytosolic and mitochondrial tRNAs. Deficiency of i6A37 was previously shown in yeast to decrease translational efficiency and fidelity in a codon-specific manner. Modelling of the p.Arg323Gln mutation on the co-crystal structure of the homologous yeast isopentenyltransferase bound to a substrate tRNA, indicates that it is one of a series of adjacent basic side chains that interact with the tRNA backbone of the anticodon stem, somewhat removed from the catalytic center. We show that patient cells bearing the p.Arg323Gln TRIT1 mutation are severely deficient in i6A37 in both cytosolic and mitochondrial tRNAs. Complete complementation of the i6A37 deficiency of both cytosolic and mitochondrial tRNAs was achieved by transduction of patient fibroblasts with wild-type TRIT1. Moreover, we show that a previously-reported pathogenic m.7480A>G mt-tRNASer(UCN mutation in the anticodon loop sequence A36A37A38 recognised by TRIT1 causes a loss of i6A37 modification. These data demonstrate that deficiencies of i6A37 tRNA modification should be considered a potential mechanism of human disease caused by both nuclear gene and mitochondrial DNA mutations while providing insight into the structure and function of TRIT1 in the modification of cytosolic and mitochondrial tRNAs.

  4. Rank-Based miRNA Signatures for Early Cancer Detection

    Directory of Open Access Journals (Sweden)

    Mario Lauria

    2014-01-01

    Full Text Available We describe a new signature definition and analysis method to be used as biomarker for early cancer detection. Our new approach is based on the construction of a reference map of transcriptional signatures of both healthy and cancer affected individuals using circulating miRNA from a large number of subjects. Once such a map is available, the diagnosis for a new patient can be performed by observing the relative position on the map of his/her transcriptional signature. To demonstrate its efficacy for this specific application we report the results of the application of our method to published datasets of circulating miRNA, and we quantify its performance compared to current state-of-the-art methods. A number of additional features make this method an ideal candidate for large-scale use, for example, as a mass screening tool for early cancer detection or for at-home diagnostics. Specifically, our method is minimally invasive (because it works well with circulating miRNA, it is robust with respect to lab-to-lab protocol variability and batch effects (it requires that only the relative ranking of expression value of miRNA in a profile be accurate not their absolute values, and it is scalable to a large number of subjects. Finally we discuss the need for HPC capability in a widespread application of our or similar methods.

  5. The conformation of 23S rRNA nucleotide A2058 determines its recognition by the ErmE methyltransferase

    DEFF Research Database (Denmark)

    Vester, B; Hansen, L H; Douthwaite, S

    1995-01-01

    the effects of mutations around position A2058 on methylation. Mutagenizing A2058 (to G or U) completely abolishes methylation of 23S rRNA by ErmE. No methylation occurred at other sites in the rRNA, demonstrating the fidelity of ErmE for A2058. Breaking the neighboring G2057-C2611 Watson-Crick base pair...... by introducing either an A2057 or a U2611 mutation, greatly reduces the rate of methylation at A2058. Methylation remains impaired after these mutations have been combined to create a new A2057-U2611 Watson-Crick base interaction. The conformation of this region in 23S rRNA was probed with chemical reagents...

  6. RNA interference gene therapy in dominant retinitis pigmentosa and cone-rod dystrophy mouse models caused by GCAP1 mutations

    Directory of Open Access Journals (Sweden)

    Li eJiang

    2014-04-01

    Full Text Available RNA interference (RNAi knockdown is an efficacious therapeutic strategy for silencing genes causative for dominant retinal dystrophies. To test this, we used self-complementary (sc AAV2/8 vector to develop an RNAi-based therapy in two dominant retinal degeneration mouse models. The allele-specific model expresses transgenic bovine GCAP1(Y99C establishing a rapid RP-like phenotype, whereas the nonallele-specific model expresses mouse GCAP1(L151F producing a slowly progressing cone/rod dystrophy (CORD. The late onset GCAP1(L151F-CORD mimics the dystrophy observed in human GCAP1-CORD patients. Subretinal injection of scAAV2/8 carrying shRNA expression cassettes specific for bovine or mouse GCAP1 showed strong expression at one week post-injection. In both allele-specific (GCAP1(Y99C-RP and nonallele-specific (GCAP1(L151F-CORD models of dominant retinal dystrophy, RNAi-mediated gene silencing enhanced photoreceptor survival, delayed onset of degeneration and improved visual function. Such results provide a proof of concept toward effective RNAi-based gene therapy mediated by scAAV2/8 for dominant retinal disease based on GCAP1 mutation. Further, nonallele-specific RNAi knockdown of GCAP1 may prove generally applicable toward the rescue of any human GCAP1-based dominant cone-rod dystrophy.

  7. Mutanome Engineered RNA Immunotherapy: Towards Patient-Centered Tumor Vaccination

    Directory of Open Access Journals (Sweden)

    Mathias Vormehr

    2015-01-01

    Full Text Available Advances in nucleic acid sequencing technologies have revolutionized the field of genomics, allowing the efficient targeting of mutated neoantigens for personalized cancer vaccination. Due to their absence during negative selection of T cells and their lack of expression in healthy tissue, tumor mutations are considered as optimal targets for cancer immunotherapy. Preclinical and early clinical data suggest that synthetic mRNA can serve as potent drug format allowing the cost efficient production of highly efficient vaccines in a timely manner. In this review, we describe a process, which integrates next generation sequencing based cancer mutanome mapping, in silico target selection and prioritization approaches, and mRNA vaccine manufacturing and delivery into a process we refer to as MERIT (mutanome engineered RNA immunotherapy.

  8. RBC Antibody Screen

    Science.gov (United States)

    ... C Cystic Fibrosis (CF) Gene Mutations Testing Cytomegalovirus (CMV) Tests D-dimer Dengue Fever Testing Des-gamma- ... Index of Screening Recommendations Not Listed? Not Listed? Newborn Screening Screening Tests for Infants Screening Tests for ...

  9. From structure prediction to genomic screens for novel non-coding RNAs.

    Science.gov (United States)

    Gorodkin, Jan; Hofacker, Ivo L

    2011-08-01

    Non-coding RNAs (ncRNAs) are receiving more and more attention not only as an abundant class of genes, but also as regulatory structural elements (some located in mRNAs). A key feature of RNA function is its structure. Computational methods were developed early for folding and prediction of RNA structure with the aim of assisting in functional analysis. With the discovery of more and more ncRNAs, it has become clear that a large fraction of these are highly structured. Interestingly, a large part of the structure is comprised of regular Watson-Crick and GU wobble base pairs. This and the increased amount of available genomes have made it possible to employ structure-based methods for genomic screens. The field has moved from folding prediction of single sequences to computational screens for ncRNAs in genomic sequence using the RNA structure as the main characteristic feature. Whereas early methods focused on energy-directed folding of single sequences, comparative analysis based on structure preserving changes of base pairs has been efficient in improving accuracy, and today this constitutes a key component in genomic screens. Here, we cover the basic principles of RNA folding and touch upon some of the concepts in current methods that have been applied in genomic screens for de novo RNA structures in searches for novel ncRNA genes and regulatory RNA structure on mRNAs. We discuss the strengths and weaknesses of the different strategies and how they can complement each other.

  10. [TYPING OF LEPTOSPIRA SPP. STRAINS BASED ON 16S rRNA].

    Science.gov (United States)

    Ostankova, Yu V; Semenov, A V; Stoyanova, N A; Tokarevich, N K; Lyubimova, N E; Petrova, O A; Ananina, Yu V; Petrov, E M

    2016-01-01

    Comparative typing of Leptospira spp. strain collection based on analysis of 16S RNA fragment. 2 pairs of primers were used for PCR, that jointly flank 1423b.p. sized fragment. Sequences of Leptospira spp. strain 16S rRNA, presented in the international database, were used for phylogenetic analysis. A high similarity, including interspecies, of the 16S fragment in Leptospira spp. strains was shown independently of the source, serovar and serogroup. Heterogeneity of the primary matrix, spontaneous mutations of hotspots and erroneous nucleotide couplings, characteristic for 16S sequence of pathogenic Leptospira spp. strains, are discussed. Molecular-genetic characteristic of certain reference Leptospira spp. strains by 16S sequence is obtained. Results of the studies give evidence on expedience of introduction into clinical practice of identification of Leptospira spp. by 16S sequence directly from the clinical material, that would allow to significantly reduce identification time, dismiss complex type-specific sera and other labor-intensive methods.

  11. A stable RNA virus-based vector for citrus trees

    International Nuclear Information System (INIS)

    Folimonov, Alexey S.; Folimonova, Svetlana Y.; Bar-Joseph, Moshe; Dawson, William O.

    2007-01-01

    Virus-based vectors are important tools in plant molecular biology and plant genomics. A number of vectors based on viruses that infect herbaceous plants are in use for expression or silencing of genes in plants as well as screening unknown sequences for function. Yet there is a need for useful virus-based vectors for woody plants, which demand much greater stability because of the longer time required for systemic infection and analysis. We examined several strategies to develop a Citrus tristeza virus (CTV)-based vector for transient expression of foreign genes in citrus trees using a green fluorescent protein (GFP) as a reporter. These strategies included substitution of the p13 open reading frame (ORF) by the ORF of GFP, construction of a self-processing fusion of GFP in-frame with the major coat protein (CP), or expression of the GFP ORF as an extra gene from a subgenomic (sg) mRNA controlled either by a duplicated CTV CP sgRNA controller element (CE) or an introduced heterologous CE of Beet yellows virus. Engineered vector constructs were examined for replication, encapsidation, GFP expression during multiple passages in protoplasts, and for their ability to infect, move, express GFP, and be maintained in citrus plants. The most successful vectors based on the 'add-a-gene' strategy have been unusually stable, continuing to produce GFP fluorescence after more than 4 years in citrus trees

  12. Rapid recombination mapping for high-throughput genetic screens in Drosophila.

    Science.gov (United States)

    Sapiro, Anne L; Ihry, Robert J; Buhr, Derek L; Konieczko, Kevin M; Ives, Sarah M; Engstrom, Anna K; Wleklinski, Nicholas P; Kopish, Kristin J; Bashirullah, Arash

    2013-12-09

    Mutagenesis screens are a staple of classical genetics. Chemical-induced mutations, however, are often difficult and time-consuming to identify. Here, we report that recombination analysis with pairs of dominant visible markers provides a rapid and reliable strategy to map mutations in Drosophila melanogaster. This method requires only two generations and a total of six crosses in vials to estimate the genetic map position of the responsible lesion with high accuracy. This genetic map position can then be reliably used to identify the mutated gene through complementation testing with an average of nine deficiencies and Sanger sequencing. We have used this approach to successfully map a collection of mutations from an ethyl methanesulfonate-based mutagenesis screen on the third chromosome. We propose that this method also may be used in conjunction with whole-genome sequencing, particularly when multiple independent alleles of the mutated locus are not available. By facilitating the rapid identification of mutated genes, our mapping strategy removes a primary obstacle to the widespread use of powerful chemical mutagenesis screens to understand fundamental biological phenomena.

  13. Screening Yield of HIV Antigen/Antibody Combination and Pooled HIV RNA Testing for Acute HIV Infection in a High-Prevalence Population.

    Science.gov (United States)

    Peters, Philip J; Westheimer, Emily; Cohen, Stephanie; Hightow-Weidman, Lisa B; Moss, Nicholas; Tsoi, Benjamin; Hall, Laura; Fann, Charles; Daskalakis, Demetre C; Beagle, Steve; Patel, Pragna; Radix, Asa; Foust, Evelyn; Kohn, Robert P; Marmorino, Jenni; Pandori, Mark; Fu, Jie; Samandari, Taraz; Gay, Cynthia L

    2016-02-16

    Although acute HIV infection contributes disproportionately to onward HIV transmission, HIV testing has not routinely included screening for acute HIV infection. To evaluate the performance of an HIV antigen/antibody (Ag/Ab) combination assay to detect acute HIV infection compared with pooled HIV RNA testing. Multisite, prospective, within-individual comparison study conducted between September 2011 and October 2013 in 7 sexually transmitted infection clinics and 5 community-based programs in New York, California, and North Carolina. Participants were 12 years or older and seeking HIV testing, without known HIV infection. All participants with a negative rapid HIV test result were screened for acute HIV infection with an HIV Ag/Ab combination assay (index test) and pooled human immunodeficiency virus 1 (HIV-1) RNA testing. HIV RNA testing was the reference standard, with positive reference standard result defined as detectable HIV-1 RNA on an individual RNA test. Number and proportion with acute HIV infections detected. Among 86,836 participants with complete test results (median age, 29 years; 75.0% men; 51.8% men who have sex with men), established HIV infection was diagnosed in 1158 participants (1.33%) and acute HIV infection was diagnosed in 168 participants (0.19%). Acute HIV infection was detected in 134 participants with HIV Ag/Ab combination testing (0.15% [95% CI, 0.13%-0.18%]; sensitivity, 79.8% [95% CI, 72.9%-85.6%]; specificity, 99.9% [95% CI, 99.9%-99.9%]; positive predictive value, 59.0% [95% CI, 52.3%-65.5%]) and in 164 participants with pooled HIV RNA testing (0.19% [95% CI, 0.16%-0.22%]; sensitivity, 97.6% [95% CI, 94.0%-99.4%]; specificity, 100% [95% CI, 100%-100%]; positive predictive value, 96.5% [95% CI, 92.5%-98.7%]; sensitivity comparison, P testing detected 82% of acute HIV infections detectable by pooled HIV RNA testing. Compared with rapid HIV testing alone, HIV Ag/Ab combination testing increased the relative HIV diagnostic yield (both

  14. Screens

    OpenAIRE

    2016-01-01

    This Sixth volume in the series The Key Debates. Mutations and Appropriations in European Film Studies investigates the question of screens in the context both of the dematerialization due to digitalization and the multiplication of media screens. Scholars offer various infomations and theories of topics such as the archeology of screen, film and media theories, contemporary art, pragmatics of new ways of screening (from home video to street screening).

  15. Protein truncation test: analysis of two novel point mutations at the carboxy-terminus of the human dystrophin gene associated with mental retardation.

    Science.gov (United States)

    Tuffery, S; Lenk, U; Roberts, R G; Coubes, C; Demaille, J; Claustres, M

    1995-01-01

    Approximately one-third of the mutations responsible for Duchenne muscular dytrophy (DMD) do not involve gross rearrangements of the dystrophin gene. Methods for intensive mutation screening have recently been applied to this immense gene, which resulted in the identification of a number of point mutations in DMD patients, mostly translation-terminating mutations. A number of data raised the possibility that the C-terminal region of dystrophin might be involved in some cases of mental retardation associated with DMD. Using single-strand conformation analysis of products amplified by polymerase chain reaction (PCR-SSCA) to screen the terminal domains of the dystrophin gene (exons 60-79) of 20 unrelated patients with DMD or BMD, we detected two novel point mutations in two mentally retarded DMD patients: a 1-bp deletion in exon 70 (10334delC) and a 5' splice donor site alteration in intron 69 (10294 + 1G-->T). Both mutations should result in a premature translation termination of dystrophin. The possible effects on the reading frame were analyzed by the study of reverse transcripts amplified from peripheral blood lymphocytes mRNA and by the protein truncation test.

  16. Drug discovery for Duchenne muscular dystrophy via utrophin promoter activation screening.

    Directory of Open Access Journals (Sweden)

    Catherine Moorwood

    Full Text Available Duchenne muscular dystrophy (DMD is a devastating muscle wasting disease caused by mutations in dystrophin, a muscle cytoskeletal protein. Utrophin is a homologue of dystrophin that can functionally compensate for its absence when expressed at increased levels in the myofibre, as shown by studies in dystrophin-deficient mice. Utrophin upregulation is therefore a promising therapeutic approach for DMD. The use of a small, drug-like molecule to achieve utrophin upregulation offers obvious advantages in terms of delivery and bioavailability. Furthermore, much of the time and expense involved in the development of a new drug can be eliminated by screening molecules that are already approved for clinical use.We developed and validated a cell-based, high-throughput screening assay for utrophin promoter activation, and used it to screen the Prestwick Chemical Library of marketed drugs and natural compounds. Initial screening produced 20 hit molecules, 14 of which exhibited dose-dependent activation of the utrophin promoter and were confirmed as hits. Independent validation demonstrated that one of these compounds, nabumetone, is able to upregulate endogenous utrophin mRNA and protein, in C2C12 muscle cells.We have developed a cell-based, high-throughput screening utrophin promoter assay. Using this assay, we identified and validated a utrophin promoter-activating drug, nabumetone, for which pharmacokinetics and safety in humans are already well described, and which represents a lead compound for utrophin upregulation as a therapy for DMD.

  17. Mutations in matrix and SP1 repair the packaging specificity of a Human Immunodeficiency Virus Type 1 mutant by reducing the association of Gag with spliced viral RNA

    Directory of Open Access Journals (Sweden)

    Ristic Natalia

    2010-09-01

    Full Text Available Abstract Background The viral genome of HIV-1 contains several secondary structures that are important for regulating viral replication. The stem-loop 1 (SL1 sequence in the 5' untranslated region directs HIV-1 genomic RNA dimerization and packaging into the virion. Without SL1, HIV-1 cannot replicate in human T cell lines. The replication restriction phenotype in the SL1 deletion mutant appears to be multifactorial, with defects in viral RNA dimerization and packaging in producer cells as well as in reverse transcription of the viral RNA in infected cells. In this study, we sought to characterize SL1 mutant replication restrictions and provide insights into the underlying mechanisms of compensation in revertants. Results HIV-1 lacking SL1 (NLΔSL1 did not replicate in PM-1 cells until two independent non-synonymous mutations emerged: G913A in the matrix domain (E42K on day 18 postinfection and C1907T in the SP1 domain (P10L on day 11 postinfection. NLΔSL1 revertants carrying either compensatory mutation showed enhanced infectivity in PM-1 cells. The SL1 revertants produced significantly more infectious particles per nanogram of p24 than did NLΔSL1. The SL1 deletion mutant packaged less HIV-1 genomic RNA and more cellular RNA, particularly signal recognition particle RNA, in the virion than the wild-type. NLΔSL1 also packaged 3- to 4-fold more spliced HIV mRNA into the virion, potentially interfering with infectious virus production. In contrast, both revertants encapsidated 2.5- to 5-fold less of these HIV-1 mRNA species. Quantitative RT-PCR analysis of RNA cross-linked with Gag in formaldehyde-fixed cells demonstrated that the compensatory mutations reduced the association between Gag and spliced HIV-1 RNA, thereby effectively preventing these RNAs from being packaged into the virion. The reduction of spliced viral RNA in the virion may have a major role in facilitating infectious virus production, thus restoring the infectivity of NLΔSL1

  18. A forward genetic screen reveals essential and non-essential RNAi factors in Paramecium tetraurelia

    Science.gov (United States)

    Marker, Simone; Carradec, Quentin; Tanty, Véronique; Arnaiz, Olivier; Meyer, Eric

    2014-01-01

    In most eukaryotes, small RNA-mediated gene silencing pathways form complex interacting networks. In the ciliate Paramecium tetraurelia, at least two RNA interference (RNAi) mechanisms coexist, involving distinct but overlapping sets of protein factors and producing different types of short interfering RNAs (siRNAs). One is specifically triggered by high-copy transgenes, and the other by feeding cells with double-stranded RNA (dsRNA)-producing bacteria. In this study, we designed a forward genetic screen for mutants deficient in dsRNA-induced silencing, and a powerful method to identify the relevant mutations by whole-genome sequencing. We present a set of 47 mutant alleles for five genes, revealing two previously unknown RNAi factors: a novel Paramecium-specific protein (Pds1) and a Cid1-like nucleotidyl transferase. Analyses of allelic diversity distinguish non-essential and essential genes and suggest that the screen is saturated for non-essential, single-copy genes. We show that non-essential genes are specifically involved in dsRNA-induced RNAi while essential ones are also involved in transgene-induced RNAi. One of the latter, the RNA-dependent RNA polymerase RDR2, is further shown to be required for all known types of siRNAs, as well as for sexual reproduction. These results open the way for the dissection of the genetic complexity, interconnection, mechanisms and natural functions of RNAi pathways in P. tetraurelia. PMID:24860163

  19. Dissection of specific binding of HIV-1 Gag to the 'packaging signal' in viral RNA.

    Science.gov (United States)

    Comas-Garcia, Mauricio; Datta, Siddhartha Ak; Baker, Laura; Varma, Rajat; Gudla, Prabhakar R; Rein, Alan

    2017-07-20

    Selective packaging of HIV-1 genomic RNA (gRNA) requires the presence of a cis -acting RNA element called the 'packaging signal' (Ψ). However, the mechanism by which Ψ promotes selective packaging of the gRNA is not well understood. We used fluorescence correlation spectroscopy and quenching data to monitor the binding of recombinant HIV-1 Gag protein to Cy5-tagged 190-base RNAs. At physiological ionic strength, Gag binds with very similar, nanomolar affinities to both Ψ-containing and control RNAs. We challenged these interactions by adding excess competing tRNA; introducing mutations in Gag; or raising the ionic strength. These modifications all revealed high specificity for Ψ. This specificity is evidently obscured in physiological salt by non-specific, predominantly electrostatic interactions. This nonspecific activity was attenuated by mutations in the MA, CA, and NC domains, including CA mutations disrupting Gag-Gag interaction. We propose that gRNA is selectively packaged because binding to Ψ nucleates virion assembly with particular efficiency.

  20. The frequency of Tay-Sachs disease causing mutations in the Brazilian Jewish population justifies a carrier screening program.

    Science.gov (United States)

    Rozenberg, R; Pereira, L da V

    2001-07-05

    Tay-Sachs disease is an autosomal recessive disease characterized by progressive neurologic degeneration, fatal in early childhood. In the Ashkenazi Jewish population the disease incidence is about 1 in every 3,500 newborns and the carrier frequency is 1 in every 29 individuals. Carrier screening programs for Tay-Sachs disease have reduced disease incidence by 90% in high-risk populations in several countries. The Brazilian Jewish population is estimated at 90,000 individuals. Currently, there is no screening program for Tay-Sachs disease in this population. To evaluate the importance of a Tay-Sachs disease carrier screening program in the Brazilian Jewish population by determining the frequency of heterozygotes and the acceptance of the program by the community. Laboratory of Molecular Genetics--Institute of Biosciences--Universidade de São Paulo. 581 senior students from selected Jewish high schools. Molecular analysis of Tay-Sachs disease causing mutations by PCR amplification of genomic DNA, followed by restriction enzyme digestion. Among 581 students that attended educational classes, 404 (70%) elected to be tested for Tay-Sachs disease mutations. Of these, approximately 65% were of Ashkenazi Jewish origin. Eight carriers were detected corresponding to a carrier frequency of 1 in every 33 individuals in the Ashkenazi Jewish fraction of the sample. The frequency of Tay-Sachs disease carriers among the Ashkenazi Jewish population of Brazil is similar to that of other countries where carrier screening programs have led to a significant decrease in disease incidence. Therefore, it is justifiable to implement a Tay-Sachs disease carrier screening program for the Brazilian Jewish population.

  1. Screening for mutations in human alpha-globin genes by nonradioactive single-strand conformation polymorphism

    Directory of Open Access Journals (Sweden)

    Jorge S.B.

    2003-01-01

    Full Text Available Point mutations and small insertions or deletions in the human alpha-globin genes may produce alpha-chain structural variants and alpha-thalassemia. Mutations can be detected either by direct DNA sequencing or by screening methods, which select the mutated exon for sequencing. Although small (about 1 kb, 3 exons and 2 introns, the alpha-globin genes are duplicate (alpha2 and alpha1 and highy G-C rich, which makes them difficult to denature, reducing sequencing efficiency and causing frequent artifacts. We modified some conditions for PCR and electrophoresis in order to detect mutations in these genes employing nonradioactive single-strand conformation polymorphism (SSCP. Primers previously described by other authors for radioactive SSCP and phast-SSCP plus denaturing gradient gel electrophoresis were here combined and the resultant fragments (6 new besides 6 original per alpha-gene submitted to silver staining SSCP. Nine structural and one thalassemic mutations were tested, under different conditions including two electrophoretic apparatus (PhastSystem(TM and GenePhor(TM, Amersham Biosciences, different polyacrylamide gel concentrations, run temperatures and denaturing agents, and entire and restriction enzyme cut fragments. One hundred percent of sensitivity was achieved with four of the new fragments formed, using the PhastSystem(TM and 20% gels at 15ºC, without the need of restriction enzymes. This nonradioactive PCR-SSCP approach showed to be simple, rapid and sensitive, reducing the costs involved in frequent sequencing repetitions and increasing the reliability of the results. It can be especially useful for laboratories which do not have an automated sequencer.

  2. Structure-Based Mutational Analysis of the Hepatitis C Virus NS3 Helicase

    Science.gov (United States)

    Tai, Chun-Ling; Pan, Wen-Ching; Liaw, Shwu-Huey; Yang, Ueng-Cheng; Hwang, Lih-Hwa; Chen, Ding-Shinn

    2001-01-01

    The carboxyl terminus of the hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses ATP-dependent RNA helicase activity. Based on the conserved sequence motifs and the crystal structures of the helicase domain, 17 mutants of the HCV NS3 helicase were generated. The ATP hydrolysis, RNA binding, and RNA unwinding activities of the mutant proteins were examined in vitro to determine the functional role of the mutated residues. The data revealed that Lys-210 in the Walker A motif and Asp-290, Glu-291, and His-293 in the Walker B motif were crucial to ATPase activity and that Thr-322 and Thr-324 in motif III and Arg-461 in motif VI significantly influenced ATPase activity. When the pairing between His-293 and Gln-460, referred to as gatekeepers, was replaced with the Asp-293/His-460 pair, which makes the NS3 helicase more like the DEAD helicase subgroup, ATPase activity was not restored. It thus indicated that the whole microenvironment surrounding the gatekeepers, rather than the residues per se, was important to the enzymatic activities. Arg-461 and Trp-501 are important residues for RNA binding, while Val-432 may only play a coadjutant role. The data demonstrated that RNA helicase activity was possibly abolished by the loss of ATPase activity or by reduced RNA binding activity. Nevertheless, a low threshold level of ATPase activity was found sufficient for helicase activity. Results in this study provide a valuable reference for efforts under way to develop anti-HCV therapeutic drugs targeting NS3. PMID:11483774

  3. 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

  4. Resistance mechanisms of linezolid-nonsusceptible enterococci in Korea: low rate of 23S rRNA mutations in Enterococcus faecium.

    Science.gov (United States)

    Lee, Sae-Mi; Huh, Hee Jae; Song, Dong Joon; Shim, Hyang Jin; Park, Kyung Sun; Kang, Cheol-In; Ki, Chang-Seok; Lee, Nam Yong

    2017-12-01

    To investigate linezolid-resistance mechanisms in linezolid-nonsusceptible enterococci (LNSE) isolated from a tertiary hospital in Korea. Enterococcal isolates exhibiting linezolid MICs ≥4 mg l -1 that were isolated between December 2011 and May 2016 were investigated by PCR and sequencing for mutations in 23S rRNA or ribosomal proteins (L3, L4 and L22) and for the presence of cfr, cfr(B) and optrA genes.Results/Key findings. Among 135 LNSE (87 Enterococcus faecium and 48 Enterococcus faecalis isolates), 39.1 % (34/87) of E. faecium and 18.8 % (9/48) of E. faecalis isolates were linezolid-resistant. The optrA carriage was the dominant mechanism in E. faecalis: 13 isolates, including 10 E. faecalis [70 % (7/10) linezolid-resistant and 30 % (3/10) linezolid-intermediate] and three E. faecium [33.3 % (1/3) linezolid-resistant and 66.7 % (2/3) linezolid-intermediate], contained the optrA gene. G2576T mutations in the 23S rRNA gene were detected only in E. faecium [14 isolates; 71.4 % (10/14) linezolid-resistant and 28.6 % (4/14) linezolid-intermediate]. One linezolid-intermediate E. faecium harboured a L22 protein alteration (Ser77Thr). No isolates contained cfr or cfr(B) genes and any L3 or L4 protein alterations. No genetic mechanism of resistance was identified for 67.6 % (23/34) of linezolid-resistant E. faecium. A low rate of 23S rRNA mutations and the absence of known linezolid-resistance mechanisms in the majority of E. faecium isolates suggest regional differences in the mechanisms of linezolid resistance and the possibility of additional mechanisms.

  5. MicroRNA-Mediated Myostatin Silencing in Caprine Fetal Fibroblasts

    Science.gov (United States)

    Zhong, Bushuai; Zhang, Yanli; Yan, Yibo; Wang, Ziyu; Ying, Shijia; Huang, Mingrui; Wang, Feng

    2014-01-01

    Myostatin functions as a negative regulator of skeletal muscle growth by suppressing proliferation and differentiation of myoblasts. Dysfunction of the myostatin gene, either due to natural mutation or genetic manipulations such as knockout or knockdown, has been reported to increase muscle mass in mammalian species. RNA interference (RNAi) mediated by microRNAs (miRNAs) is a promising method for gene knockdown studies. In the present study, transient and stable silencing of the myostatin gene in caprine fetal fibroblasts (CFF) was evaluated using the two most effective constructs selected from four different miRNA expression constructs screened in 293FT cells. Using these two miRNA constructs, we achieved up to 84% silencing of myostatin mRNA in transiently transfected CFF cells and up to 31% silencing in stably transfected CFF cells. Moreover, off-target effects due to induction of interferon (IFN) response genes, such as interferon beta (IFN-β) and 2′-5′-oligoadenylate synthetase 2 (OAS2), were markedly fewer in stably transfected CFF cells than in transiently transfected cells. Stable expression of anti-myostatin miRNA with minimal induction of interferon shows great promise for increasing muscle mass in transgenic goats. PMID:25244645

  6. MicroRNA-mediated myostatin silencing in caprine fetal fibroblasts.

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    Bushuai Zhong

    Full Text Available Myostatin functions as a negative regulator of skeletal muscle growth by suppressing proliferation and differentiation of myoblasts. Dysfunction of the myostatin gene, either due to natural mutation or genetic manipulations such as knockout or knockdown, has been reported to increase muscle mass in mammalian species. RNA interference (RNAi mediated by microRNAs (miRNAs is a promising method for gene knockdown studies. In the present study, transient and stable silencing of the myostatin gene in caprine fetal fibroblasts (CFF was evaluated using the two most effective constructs selected from four different miRNA expression constructs screened in 293FT cells. Using these two miRNA constructs, we achieved up to 84% silencing of myostatin mRNA in transiently transfected CFF cells and up to 31% silencing in stably transfected CFF cells. Moreover, off-target effects due to induction of interferon (IFN response genes, such as interferon beta (IFN-β and 2'-5'-oligoadenylate synthetase 2 (OAS2, were markedly fewer in stably transfected CFF cells than in transiently transfected cells. Stable expression of anti-myostatin miRNA with minimal induction of interferon shows great promise for increasing muscle mass in transgenic goats.

  7. Usher syndrome type 1 due to missense mutations on both CDH23 alleles: investigation of mRNA splicing.

    Science.gov (United States)

    Becirovic, Elvir; Ebermann, Inga; Nagy, Ditta; Zrenner, Eberhart; Seeliger, Mathias Wolfgang; Bolz, Hanno Jörn

    2008-03-01

    Usher syndrome (USH) is an autosomal recessive condition characterized by sensorineural hearing loss, vestibular dysfunction, and visual impairment due to retinitis pigmentosa. Truncating mutations in the cadherin-23 gene (CDH23) result in Usher syndrome type 1D (USH1D), whereas missense mutations affecting strongly conserved motifs of the CDH23 protein cause non-syndromic deafness (DFNB12). Four missense mutations constitute an exception from this genotype-phenotype correlation: they have been described in USH1 patients in homozygous state. Using a minigene assay, we have investigated these changes (c.1450G>C, p.A484P; c.3625A>G, p.T1209A; c.4520G>A, p.R1507Q; and c.5237G>A, p.R1746Q) for a possible impact on mRNA splicing which could explain the syndromic phenotype. While in silico analysis suggested impairment of splicing in all four cases, we found aberrant splicing for only one mutation, p.R1746Q. However, splicing was normal in case of p.A484P, p.T1209A and p.R1507Q. These three latter CDH23 missense mutations could interfere with functions of both, the auditory and the visual system. Alternatively, they could represent rare non-pathogenic polymorphisms.

  8. VHL Frameshift Mutation as Target of Nonsense-Mediated mRNA Decay in Drosophila melanogaster and Human HEK293 Cell Line

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    Lucia Micale

    2009-01-01

    Full Text Available There are many well-studied examples of human phenotypes resulting from nonsense or frameshift mutations that are modulated by Nonsense-Mediated mRNA Decay (NMD, a process that typically degrades transcripts containing premature termination codons (PTCs in order to prevent translation of unnecessary or aberrant transcripts. Different types of germline mutations in the VHL gene cause the von Hippel-Lindau disease, a dominantly inherited familial cancer syndrome with a marked phenotypic variability and age-dependent penetrance. By generating the Drosophila UAS:Upf1D45B line we showed the possible involvement of NMD mechanism in the modulation of the c.172delG frameshift mutation located in the exon 1 of Vhl gene. Further, by Quantitative Real-time PCR (QPCR we demonstrated that the corresponding c.163delG human mutation is targeted by NMD in human HEK 293 cells. The UAS:Upf1D45B line represents a useful system to identify novel substrates of NMD pathway in Drosophila melanogaster. Finally, we suggest the possible role of NMD on the regulation of VHL mutations.

  9. Transcript specificity in yeast pre-mRNA splicing revealed by mutations in core spliceosomal components.

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    Jeffrey A Pleiss

    2007-04-01

    Full Text Available Appropriate expression of most eukaryotic genes requires the removal of introns from their pre-messenger RNAs (pre-mRNAs, a process catalyzed by the spliceosome. In higher eukaryotes a large family of auxiliary factors known as SR proteins can improve the splicing efficiency of transcripts containing suboptimal splice sites by interacting with distinct sequences present in those pre-mRNAs. The yeast Saccharomyces cerevisiae lacks functional equivalents of most of these factors; thus, it has been unclear whether the spliceosome could effectively distinguish among transcripts. To address this question, we have used a microarray-based approach to examine the effects of mutations in 18 highly conserved core components of the spliceosomal machinery. The kinetic profiles reveal clear differences in the splicing defects of particular pre-mRNA substrates. Most notably, the behaviors of ribosomal protein gene transcripts are generally distinct from other intron-containing transcripts in response to several spliceosomal mutations. However, dramatically different behaviors can be seen for some pairs of transcripts encoding ribosomal protein gene paralogs, suggesting that the spliceosome can readily distinguish between otherwise highly similar pre-mRNAs. The ability of the spliceosome to distinguish among its different substrates may therefore offer an important opportunity for yeast to regulate gene expression in a transcript-dependent fashion. Given the high level of conservation of core spliceosomal components across eukaryotes, we expect that these results will significantly impact our understanding of how regulated splicing is controlled in higher eukaryotes as well.

  10. Expedited quantification of mutant ribosomal RNA by binary deoxyribozyme (BiDz) sensors.

    Science.gov (United States)

    Gerasimova, Yulia V; Yakovchuk, Petro; Dedkova, Larisa M; Hecht, Sidney M; Kolpashchikov, Dmitry M

    2015-10-01

    Mutations in ribosomal RNA (rRNA) have traditionally been detected by the primer extension assay, which is a tedious and multistage procedure. Here, we describe a simple and straightforward fluorescence assay based on binary deoxyribozyme (BiDz) sensors. The assay uses two short DNA oligonucleotides that hybridize specifically to adjacent fragments of rRNA, one of which contains a mutation site. This hybridization results in the formation of a deoxyribozyme catalytic core that produces the fluorescent signal and amplifies it due to multiple rounds of catalytic action. This assay enables us to expedite semi-quantification of mutant rRNA content in cell cultures starting from whole cells, which provides information useful for optimization of culture preparation prior to ribosome isolation. The method requires less than a microliter of a standard Escherichia coli cell culture and decreases analysis time from several days (for primer extension assay) to 1.5 h with hands-on time of ∼10 min. It is sensitive to single-nucleotide mutations. The new assay simplifies the preliminary analysis of RNA samples and cells in molecular biology and cloning experiments and is promising in other applications where fast detection/quantification of specific RNA is required. © 2015 Gerasimova et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  11. Temperature-sensitive mutations for live-attenuated Rift Valley fever vaccines: Implications from other RNA viruses

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    Shoko eNishiyama

    2015-08-01

    Full Text Available Rift Valley fever (RVF is a mosquito-borne zoonotic disease endemic to the African continent. RVF is characterized by high rate of abortions in ruminants and hemorrhagic fever, encephalitis or blindness in humans. RVF is caused by the Rift Valley fever virus (RVFV: genus Phlebovirus, family Bunyaviridae. Vaccination is the only known effective strategy to prevent the disease, but there are no licensed RVF vaccines available for humans. A live-attenuated vaccine candidate derived from the wild-type pathogenic Egyptian ZH548 strain, MP-12, has been conditionally licensed for veterinary use in the United States. MP-12 displays a temperature-sensitive (ts phenotype and does not replicate at 41oC. The ts mutation limits viral replication at a specific body temperature and may lead to an attenuation of the virus. Here we will review well-characterized ts mutations for RNA viruses, and further discuss the potential in designing novel live-attenuated vaccines for RVF.

  12. Development of RNA-FISH Assay for Detection of Oncogenic FGFR3-TACC3 Fusion Genes in FFPE Samples.

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    Masahiro Kurobe

    Full Text Available Oncogenic FGFR3-TACC3 fusions and FGFR3 mutations are target candidates for small molecule inhibitors in bladder cancer (BC. Because FGFR3 and TACC3 genes are located very closely on chromosome 4p16.3, detection of the fusion by DNA-FISH (fluorescent in situ hybridization is not a feasible option. In this study, we developed a novel RNA-FISH assay using branched DNA probe to detect FGFR3-TACC3 fusions in formaldehyde-fixed paraffin-embedded (FFPE human BC samples.The RNA-FISH assay was developed and validated using a mouse xenograft model with human BC cell lines. Next, we assessed the consistency of the RNA-FISH assay using 104 human BC samples. In this study, primary BC tissues were stored as frozen and FFPE tissues. FGFR3-TACC3 fusions were independently detected in FFPE sections by the RNA-FISH assay and in frozen tissues by RT-PCR. We also analyzed the presence of FGFR3 mutations by targeted sequencing of genomic DNA extracted from deparaffinized FFPE sections.FGFR3-TACC3 fusion transcripts were identified by RNA-FISH and RT-PCR in mouse xenograft FFPE tissues using the human BC cell lines RT112 and RT4. These cell lines have been reported to be fusion-positive. Signals for FGFR3-TACC3 fusions by RNA-FISH were positive in 2/60 (3% of non-muscle-invasive BC (NMIBC and 2/44 (5% muscle-invasive BC (MIBC patients. The results of RT-PCR of all 104 patients were identical to those of RNA-FISH. FGFR3 mutations were detected in 27/60 (45% NMIBC and 8/44 (18% MIBC patients. Except for one NMIBC patient, FGFR3 mutation and FGFR3-TACC3 fusion were mutually exclusive.We developed an RNA-FISH assay for detection of the FGFR3-TACC3 fusion in FFPE samples of human BC tissues. Screening for not only FGFR3 mutations, but also for FGFR3-TACC3 fusion transcripts has the potential to identify additional patients that can be treated with FGFR inhibitors.

  13. Fast selection of miRNA candidates based on large-scale pre-computed MFE sets of randomized sequences.

    Science.gov (United States)

    Warris, Sven; Boymans, Sander; Muiser, Iwe; Noback, Michiel; Krijnen, Wim; Nap, Jan-Peter

    2014-01-13

    Small RNAs are important regulators of genome function, yet their prediction in genomes is still a major computational challenge. Statistical analyses of pre-miRNA sequences indicated that their 2D structure tends to have a minimal free energy (MFE) significantly lower than MFE values of equivalently randomized sequences with the same nucleotide composition, in contrast to other classes of non-coding RNA. The computation of many MFEs is, however, too intensive to allow for genome-wide screenings. Using a local grid infrastructure, MFE distributions of random sequences were pre-calculated on a large scale. These distributions follow a normal distribution and can be used to determine the MFE distribution for any given sequence composition by interpolation. It allows on-the-fly calculation of the normal distribution for any candidate sequence composition. The speedup achieved makes genome-wide screening with this characteristic of a pre-miRNA sequence practical. Although this particular property alone will not be able to distinguish miRNAs from other sequences sufficiently discriminative, the MFE-based P-value should be added to the parameters of choice to be included in the selection of potential miRNA candidates for experimental verification.

  14. From structure prediction to genomic screens for novel non-coding RNAs.

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    Jan Gorodkin

    2011-08-01

    Full Text Available Non-coding RNAs (ncRNAs are receiving more and more attention not only as an abundant class of genes, but also as regulatory structural elements (some located in mRNAs. A key feature of RNA function is its structure. Computational methods were developed early for folding and prediction of RNA structure with the aim of assisting in functional analysis. With the discovery of more and more ncRNAs, it has become clear that a large fraction of these are highly structured. Interestingly, a large part of the structure is comprised of regular Watson-Crick and GU wobble base pairs. This and the increased amount of available genomes have made it possible to employ structure-based methods for genomic screens. The field has moved from folding prediction of single sequences to computational screens for ncRNAs in genomic sequence using the RNA structure as the main characteristic feature. Whereas early methods focused on energy-directed folding of single sequences, comparative analysis based on structure preserving changes of base pairs has been efficient in improving accuracy, and today this constitutes a key component in genomic screens. Here, we cover the basic principles of RNA folding and touch upon some of the concepts in current methods that have been applied in genomic screens for de novo RNA structures in searches for novel ncRNA genes and regulatory RNA structure on mRNAs. We discuss the strengths and weaknesses of the different strategies and how they can complement each other.

  15. Analysis of 30 putative BRCA1 splicing mutations in hereditary breast and ovarian cancer families identifies exonic splice site mutations that escape in silico prediction.

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    Barbara Wappenschmidt

    Full Text Available Screening for pathogenic mutations in breast and ovarian cancer genes such as BRCA1/2, CHEK2 and RAD51C is common practice for individuals from high-risk families. However, test results may be ambiguous due to the presence of unclassified variants (UCV in the concurrent absence of clearly cancer-predisposing mutations. Especially the presence of intronic or exonic variants within these genes that possibly affect proper pre-mRNA processing poses a challenge as their functional implications are not immediately apparent. Therefore, it appears necessary to characterize potential splicing UCV and to develop appropriate classification tools. We investigated 30 distinct BRCA1 variants, both intronic and exonic, regarding their spliceogenic potential by commonly used in silico prediction algorithms (HSF, MaxEntScan along with in vitro transcript analyses. A total of 25 variants were identified spliceogenic, either causing/enhancing exon skipping or activation of cryptic splice sites, or both. Except from a single intronic variant causing minor effects on BRCA1 pre-mRNA processing in our analyses, 23 out of 24 intronic variants were correctly predicted by MaxEntScan, while HSF was less accurate in this cohort. Among the 6 exonic variants analyzed, 4 severely impair correct pre-mRNA processing, while the remaining two have partial effects. In contrast to the intronic alterations investigated, only half of the spliceogenic exonic variants were correctly predicted by HSF and/or MaxEntScan. These data support the idea that exonic splicing mutations are commonly disease-causing and concurrently prone to escape in silico prediction, hence necessitating experimental in vitro splicing analysis.

  16. The European Regulatory Environment of RNA-Based Vaccines.

    Science.gov (United States)

    Hinz, Thomas; Kallen, Kajo; Britten, Cedrik M; Flamion, Bruno; Granzer, Ulrich; Hoos, Axel; Huber, Christoph; Khleif, Samir; Kreiter, Sebastian; Rammensee, Hans-Georg; Sahin, Ugur; Singh-Jasuja, Harpreet; Türeci, Özlem; Kalinke, Ulrich

    2017-01-01

    A variety of different mRNA-based drugs are currently in development. This became possible, since major breakthroughs in RNA research during the last decades allowed impressive improvements of translation, stability and delivery of mRNA. This article focuses on antigen-encoding RNA-based vaccines that are either directed against tumors or pathogens. mRNA-encoded vaccines are developed both for preventive or therapeutic purposes. Most mRNA-based vaccines are directly administered to patients. Alternatively, primary autologous cells from cancer patients are modified ex vivo by the use of mRNA and then are adoptively transferred to patients. In the EU no regulatory guidelines presently exist that specifically address mRNA-based vaccines. The existing regulatory framework, however, clearly defines that mRNA-based vaccines in most cases have to be centrally approved. Interestingly, depending on whether RNA-based vaccines are directed against tumors or infectious disease, they are formally considered gene therapy products or not, respectively. Besides an overview on the current clinical use of mRNA vaccines in various therapeutic areas a detailed discussion of the current regulatory situation is provided and regulatory perspectives are discussed.

  17. Screening individuals with intellectual disability, autism and Tourette's syndrome for KCNK9 mutations and aberrant DNA methylation within the 8q24 imprinted cluster.

    Science.gov (United States)

    Sánchez Delgado, Marta; Camprubí, Cristina; Tümer, Zeynep; Martínez, Francisco; Milà, Montserrat; Monk, David

    2014-09-01

    The phenotype overlap between autism spectrum disorders (ASD) & intellectual disabilities (ID) is mirrored at the genetic level, with common genes being reported mutated in variety of developmental disabilities. However despite widespread genetic screening for mutations, in approximately 40-60% of childhood developmental disorders the genetic cause remains unknown. Several genome-wide linkage screens in ASD have identified a locus mapping to distal 8q. We have recently identified a novel brain-specific imprinted cluster at this location, which contains the reciprocally expressed maternal KCNK9 and paternally expressed non-coding PEG13 transcripts, the latter located within an intron of TRAPPC9. Interestingly, mutations of KCNK9 and TRAPPC9 have been reported in Birk-Barel mental retardation and non-syndromic familial forms of ID, respectively. Here, we report a genetic screen for KCNK9 coding mutations and potential epigenetic aberrations that could result in deregulated imprinting in a cohort of 120 ID, 86 ASD and 86 Tourette syndrome patients. Fifteen of the ID patients had clinical characteristics overlapping with Birk-Barel syndrome. Sequencing of the two coding exons of KCNK9 failed to identify pathologic mutations, with only one variant, rs2615374, being present with allele frequencies similar to those described in dbSNP database. DNA methylation profiling of the KCNK9 and TRAPPC9 promoters, the maternally methylated PEG13 DMR and a long-range enhancer region were normal in all patients. Our findings suggest that mutations of KCNK9 or epigenetic disturbances within the PEG13 imprinted cluster do not significantly contribute to the cause of the developmental disabilities tested in this study. © 2014 Wiley Periodicals, Inc.

  18. Changes in screening behaviors and attitudes toward screening from pre-test genetic counseling to post-disclosure in Lynch syndrome families

    Science.gov (United States)

    Burton-Chase, Allison M.; Hovick, Shelly R.; Peterson, Susan K.; Marani, Salma K.; Vernon, Sally W.; Amos, Christopher I.; Frazier, Marsha L.; Lynch, Patrick M.; Gritz, Ellen R.

    2013-01-01

    Purpose This study examined colonoscopy adherence and attitudes towards colorectal cancer (CRC) screening in individuals who underwent Lynch syndrome genetic counseling and testing. Methods We evaluated changes in colonoscopy adherence and CRC screening attitudes in 78 cancer-unaffected relatives of Lynch syndrome mutation carriers before pre-test genetic counseling (baseline) and at 6 and 12 months post-disclosure of test results (52 mutation-negative, 26 mutation-positive). Results While both groups were similar at baseline, at 12 months post-disclosure, a greater number of mutation-positive individuals had had a colonoscopy compared with mutation-negative individuals. From baseline to 12 months post-disclosure, the mutation-positive group demonstrated an increase in mean scores on measures of colonoscopy commitment, self-efficacy, and perceived benefits of CRC screening, and a decrease in mean scores for perceived barriers to CRC screening. Mean scores on colonoscopy commitment decreased from baseline to 6 months in the mutation-negative group. Conclusion Adherence to risk-appropriate guidelines for CRC surveillance improved after genetic counseling and testing for Lynch syndrome. Mutation-positive individuals reported increasingly positive attitudes toward CRC screening after receiving genetic test results, potentially reinforcing longer term colonoscopy adherence. PMID:23414081

  19. snoSeeker: an advanced computational package for screening of guide and orphan snoRNA genes in the human genome

    OpenAIRE

    Yang, Jian-Hua; Zhang, Xiao-Chen; Huang, Zhan-Peng; Zhou, Hui; Huang, Mian-Bo; Zhang, Shu; Chen, Yue-Qin; Qu, Liang-Hu

    2006-01-01

    Small nucleolar RNAs (snoRNAs) represent an abundant group of non-coding RNAs in eukaryotes. They can be divided into guide and orphan snoRNAs according to the presence or absence of antisense sequence to rRNAs or snRNAs. Current snoRNA-searching programs, which are essentially based on sequence complementarity to rRNAs or snRNAs, exist only for the screening of guide snoRNAs. In this study, we have developed an advanced computational package, snoSeeker, which includes CDseeker and ACAseeker ...

  20. The frequency of Tay-Sachs disease causing mutations in the Brazilian Jewish population justifies a carrier screening program

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    Roberto Rozenberg

    Full Text Available CONTEXT: Tay-Sachs disease is an autosomal recessive disease characterized by progressive neurologic degeneration, fatal in early childhood. In the Ashkenazi Jewish population the disease incidence is about 1 in every 3,500 newborns and the carrier frequency is 1 in every 29 individuals. Carrier screening programs for Tay-Sachs disease have reduced disease incidence by 90% in high-risk populations in several countries. The Brazilian Jewish population is estimated at 90,000 individuals. Currently, there is no screening program for Tay-Sachs disease in this population. OBJECTIVE: To evaluate the importance of a Tay-Sachs disease carrier screening program in the Brazilian Jewish population by determining the frequency of heterozygotes and the acceptance of the program by the community. SETTING: Laboratory of Molecular Genetics - Institute of Biosciences - Universidade de São Paulo. PARTICIPANTS: 581 senior students from selected Jewish high schools. PROCEDURE: Molecular analysis of Tay-Sachs disease causing mutations by PCR amplification of genomic DNA, followed by restriction enzyme digestion. RESULTS: Among 581 students that attended educational classes, 404 (70% elected to be tested for Tay-Sachs disease mutations. Of these, approximately 65% were of Ashkenazi Jewish origin. Eight carriers were detected corresponding to a carrier frequency of 1 in every 33 individuals in the Ashkenazi Jewish fraction of the sample. CONCLUSION: The frequency of Tay-Sachs disease carriers among the Ashkenazi Jewish population of Brazil is similar to that of other countries where carrier screening programs have led to a significant decrease in disease incidence. Therefore, it is justifiable to implement a Tay-Sachs disease carrier screening program for the Brazilian Jewish population.

  1. Human TRMU encoding the mitochondrial 5-methylaminomethyl-2-thiouridylate-methyltransferase is a putative nuclear modifier gene for the phenotypic expression of the deafness-associated 12S rRNA mutations

    International Nuclear Information System (INIS)

    Yan Qingfeng; Bykhovskaya, Yelena; Li Ronghua; Mengesha, Emebet; Shohat, Mordechai; Estivill, Xavier; Fischel-Ghodsian, Nathan; Guan Minxin

    2006-01-01

    Nuclear modifier genes have been proposed to modulate the phenotypic manifestation of human mitochondrial 12S rRNA A1491G mutation associated with deafness in many families world-wide. Here we identified and characterized the putative nuclear modifier gene TRMU encoding a highly conserved mitochondrial protein related to tRNA modification. A 1937 bp TRMU cDNA has been isolated and the genomic organization of TRMU has been elucidated. The human TRMU gene containing 11 exons encodes a 421 residue protein with a strong homology to the TRMU-like proteins of bacteria and other homologs. TRMU is ubiquitously expressed in various tissues, but abundantly in tissues with high metabolic rates including heart, liver, kidney, and brain. Immunofluorescence analysis of human 143B cells expressing TRMU-GFP fusion protein demonstrated that the human Trmu localizes and functions in mitochondrion. Furthermore, we show that in families with the deafness-associated 12S rRNA A1491G mutation there is highly suggestive linkage and linkage disequilibrium between microsatellite markers adjacent to TRMU and the presence of deafness. These observations suggest that human TRMU may modulate the phenotypic manifestation of the deafness-associated mitochondrial 12S rRNA mutations

  2. Mutations in ribosomal protein L3 and 23S ribosomal RNA at the peptidyl transferase centre are associated with reduced susceptibility to tiamulin in Brachyspira spp. isolates.

    Science.gov (United States)

    Pringle, Märit; Poehlsgaard, Jacob; Vester, Birte; Long, Katherine S

    2004-12-01

    The pleuromutilin antibiotic tiamulin binds to the ribosomal peptidyl transferase centre. Three groups of Brachyspira spp. isolates with reduced tiamulin susceptibility were analysed to define resistance mechanisms to the drug. Mutations were identified in genes encoding ribosomal protein L3 and 23S rRNA at positions proximal to the peptidyl transferase centre. In two groups of laboratory-selected mutants, mutations were found at nucleotide positions 2032, 2055, 2447, 2499, 2504 and 2572 of 23S rRNA (Escherichia coli numbering) and at amino acid positions 148 and 149 of ribosomal protein L3 (Brachyspira pilosicoli numbering). In a third group of clinical B. hyodysenteriae isolates, only a single mutation at amino acid 148 of ribosomal protein L3 was detected. Chemical footprinting experiments show a reduced binding of tiamulin to ribosomal subunits from mutants with decreased susceptibility to the drug. This reduction in drug binding is likely the resistance mechanism for these strains. Hence, the identified mutations located near the tiamulin binding site are predicted to be responsible for the resistance phenotype. The positions of the mutated residues relative to the bound drug advocate a model where the mutations affect tiamulin binding indirectly through perturbation of nucleotide U2504.

  3. High-throughput Screening for Protein-based Inheritance in S. cerevisiae.

    Science.gov (United States)

    Byers, James S; Jarosz, Daniel F

    2017-08-08

    The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrP Sc ) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that >1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

  4. Mutation spectrum of RB1 mutations in retinoblastoma cases from Singapore with implications for genetic management and counselling.

    Directory of Open Access Journals (Sweden)

    Swati Tomar

    Full Text Available Retinoblastoma (RB is a rare childhood malignant disorder caused by the biallelic inactivation of RB1 gene. Early diagnosis and identification of carriers of heritable RB1 mutations can improve disease outcome and management. In this study, mutational analysis was conducted on fifty-nine matched tumor and peripheral blood samples from 18 bilateral and 41 unilateral unrelated RB cases by a combinatorial approach of Multiplex Ligation-dependent Probe Amplification (MLPA assay, deletion screening, direct sequencing, copy number gene dosage analysis and methylation assays. Screening of both blood and tumor samples yielded a mutation detection rate of 94.9% (56/59 while only 42.4% (25/59 of mutations were detected if blood samples alone were analyzed. Biallelic mutations were observed in 43/59 (72.9% of tumors screened. There were 3 cases (5.1% in which no mutations could be detected and germline mutations were detected in 19.5% (8/41 of unilateral cases. A total of 61 point mutations were identified, of which 10 were novel. There was a high incidence of previously reported recurrent mutations, occurring at 38.98% (23/59 of all cases. Of interest were three cases of mosaic RB1 mutations detected in the blood from patients with unilateral retinoblastoma. Additionally, two germline mutations previously reported to be associated with low-penetrance phenotypes: missense-c.1981C>T and splice variant-c.607+1G>T, were observed in a bilateral and a unilateral proband, respectively. These findings have implications for genetic counselling and risk prediction for the affected families. This is the first published report on the spectrum of mutations in RB patients from Singapore and shows that further improved mutation screening strategies are required in order to provide a definitive molecular diagnosis for every case of RB. Our findings also underscore the importance of genetic testing in supporting individualized disease management plans for patients and

  5. HNF1 alpha gene coding regions mutations screening, in a Caucasian population clinically characterized as MODY from Argentina.

    Science.gov (United States)

    Lopez, Ariel Pablo; Foscaldi, Sabrina Andrea; Perez, Maria Silvia; Rodriguez, Martín; Traversa, Mercedes; Puchulu, Félix Miguel; Bergada, Ignacio; Frechtel, Gustavo Daniel

    2011-02-01

    There are at least six subtypes of Maturity Onset Diabetes of the Young (MODY) with distinctive genetic causes. MODY 3 is caused by mutations in HNF1A gene, an insulin transcription factor, so mutations in this gene are associated with impaired insulin secretion. MODY 3 prevalence differs according to the population analyzed, but it is one of the most frequent subtypes. Therefore, our aims in this work were to find mutations present in the HNF1A gene and provide information on their prevalence. Mutations screening was done in a group of 80 unrelated patients (average age 17.1 years) selected by clinical characterization of MODY, by SSCP electrophoresis followed by sequenciation. We found eight mutations, of which six were novel and four sequence variants, which were all novel. Therefore the prevalence of MODY 3 in this group was 10%. Compared clinical data between the non-MODY 3 patients and the MODY 3 diagnosed patients did not show any significant difference. Eight patients were diagnosed as MODY 3 and new data about the prevalence of that subtype is provided. Our results contribute to reveal novel mutations, providing new data about the prevalence of that subtype. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  6. Mutations Abrogating VP35 Interaction with Double-Stranded RNA Render Ebola Virus Avirulent in Guinea Pigs

    Energy Technology Data Exchange (ETDEWEB)

    Prins, Kathleen C.; Delpeut, Sebastien; Leung, Daisy W.; Reynard, Olivier; Volchkova, Valentina A.; Reid, St. Patrick; Ramanan, Parameshwaran; Cárdenas, Washington B.; Amarasinghe, Gaya K.; Volchkov, Viktor E.; Basler, Christopher F. (CNRS-INSERM); (Mount Sinai Hospital); (LB-Ecuador); (Iowa State)

    2010-10-11

    Ebola virus (EBOV) protein VP35 is a double-stranded RNA (dsRNA) binding inhibitor of host interferon (IFN)-{alpha}/{beta} responses that also functions as a viral polymerase cofactor. Recent structural studies identified key features, including a central basic patch, required for VP35 dsRNA binding activity. To address the functional significance of these VP35 structural features for EBOV replication and pathogenesis, two point mutations, K319A/R322A, that abrogate VP35 dsRNA binding activity and severely impair its suppression of IFN-{alpha}/{beta} production were identified. Solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography reveal minimal structural perturbations in the K319A/R322A VP35 double mutant and suggest that loss of basic charge leads to altered function. Recombinant EBOVs encoding the mutant VP35 exhibit, relative to wild-type VP35 viruses, minimal growth attenuation in IFN-defective Vero cells but severe impairment in IFN-competent cells. In guinea pigs, the VP35 mutant virus revealed a complete loss of virulence. Strikingly, the VP35 mutant virus effectively immunized animals against subsequent wild-type EBOV challenge. These in vivo studies, using recombinant EBOV viruses, combined with the accompanying biochemical and structural analyses directly correlate VP35 dsRNA binding and IFN inhibition functions with viral pathogenesis. Moreover, these studies provide a framework for the development of antivirals targeting this critical EBOV virulence factor.

  7. Fragment-based modelling of single stranded RNA bound to RNA recognition motif containing proteins

    Science.gov (United States)

    de Beauchene, Isaure Chauvot; de Vries, Sjoerd J.; Zacharias, Martin

    2016-01-01

    Abstract Protein-RNA complexes are important for many biological processes. However, structural modeling of such complexes is hampered by the high flexibility of RNA. Particularly challenging is the docking of single-stranded RNA (ssRNA). We have developed a fragment-based approach to model the structure of ssRNA bound to a protein, based on only the protein structure, the RNA sequence and conserved contacts. The conformational diversity of each RNA fragment is sampled by an exhaustive library of trinucleotides extracted from all known experimental protein–RNA complexes. The method was applied to ssRNA with up to 12 nucleotides which bind to dimers of the RNA recognition motifs (RRMs), a highly abundant eukaryotic RNA-binding domain. The fragment based docking allows a precise de novo atomic modeling of protein-bound ssRNA chains. On a benchmark of seven experimental ssRNA–RRM complexes, near-native models (with a mean heavy-atom deviation of <3 Å from experiment) were generated for six out of seven bound RNA chains, and even more precise models (deviation < 2 Å) were obtained for five out of seven cases, a significant improvement compared to the state of the art. The method is not restricted to RRMs but was also successfully applied to Pumilio RNA binding proteins. PMID:27131381

  8. Ensemble-based prediction of RNA secondary structures.

    Science.gov (United States)

    Aghaeepour, Nima; Hoos, Holger H

    2013-04-24

    Accurate structure prediction methods play an important role for the understanding of RNA function. Energy-based, pseudoknot-free secondary structure prediction is one of the most widely used and versatile approaches, and improved methods for this task have received much attention over the past five years. Despite the impressive progress that as been achieved in this area, existing evaluations of the prediction accuracy achieved by various algorithms do not provide a comprehensive, statistically sound assessment. Furthermore, while there is increasing evidence that no prediction algorithm consistently outperforms all others, no work has been done to exploit the complementary strengths of multiple approaches. In this work, we present two contributions to the area of RNA secondary structure prediction. Firstly, we use state-of-the-art, resampling-based statistical methods together with a previously published and increasingly widely used dataset of high-quality RNA structures to conduct a comprehensive evaluation of existing RNA secondary structure prediction procedures. The results from this evaluation clarify the performance relationship between ten well-known existing energy-based pseudoknot-free RNA secondary structure prediction methods and clearly demonstrate the progress that has been achieved in recent years. Secondly, we introduce AveRNA, a generic and powerful method for combining a set of existing secondary structure prediction procedures into an ensemble-based method that achieves significantly higher prediction accuracies than obtained from any of its component procedures. Our new, ensemble-based method, AveRNA, improves the state of the art for energy-based, pseudoknot-free RNA secondary structure prediction by exploiting the complementary strengths of multiple existing prediction procedures, as demonstrated using a state-of-the-art statistical resampling approach. In addition, AveRNA allows an intuitive and effective control of the trade-off between

  9. Baseline results from the UK SIGNIFY study: a whole-body MRI screening study in TP53 mutation carriers and matched controls.

    Science.gov (United States)

    Saya, Sibel; Killick, Emma; Thomas, Sarah; Taylor, Natalie; Bancroft, Elizabeth K; Rothwell, Jeanette; Benafif, Sarah; Dias, Alexander; Mikropoulos, Christos; Pope, Jenny; Chamberlain, Anthony; Gunapala, Ranga; Izatt, Louise; Side, Lucy; Walker, Lisa; Tomkins, Susan; Cook, Jackie; Barwell, Julian; Wiles, Vicki; Limb, Lauren; Eccles, Diana; Leach, Martin O; Shanley, Susan; Gilbert, Fiona J; Hanson, Helen; Gallagher, David; Rajashanker, Bala; Whitehouse, Richard W; Koh, Dow-Mu; Sohaib, S Aslam; Evans, D Gareth; Eeles, Rosalind A

    2017-07-01

    In the United Kingdom, current screening guidelines for TP53 germline mutation carriers solely recommends annual breast MRI, despite the wide spectrum of malignancies typically seen in this group. This study sought to investigate the role of one-off non-contrast whole-body MRI (WB MRI) in the screening of asymptomatic TP53 mutation carriers. 44 TP53 mutation carriers and 44 population controls were recruited. Scans were read by radiologists blinded to participant carrier status. The incidence of malignancies diagnosed in TP53 mutation carriers against general population controls was calculated. The incidences of non-malignant relevant disease and irrelevant disease were measured, as well as the number of investigations required to determine relevance of findings. In TP53 mutation carriers, 6 of 44 (13.6, 95% CI 5.2-27.4%) participants were diagnosed with cancer during the study, all of which would be considered life threatening if untreated. Two were found to have two primary cancers. Two participants with cancer had abnormalities on the MRI which were initially thought to be benign (a pericardial cyst and a uterine fibroid) but transpired to be sarcomas. No controls were diagnosed with cancer. Fifteen carriers (34.1, 95% CI 20.5-49.9%) and seven controls (15.9, 95% CI 6.7-30.1%) underwent further investigations following the WB MRI for abnormalities that transpired to be benign (p = 0.049). The cancer detection rate in this group justifies a minimum baseline non-contrast WB MRI in germline TP53 mutation carriers. This should be adopted into national guidelines for management of adult TP53 mutation carriers in addition to the current practice of contrast enhanced breast MRI imaging.

  10. Identification of a novel ZIC3 isoform and mutation screening in patients with heterotaxy and congenital heart disease.

    Directory of Open Access Journals (Sweden)

    James E J Bedard

    Full Text Available Patients with heterotaxy have characteristic cardiovascular malformations, abnormal arrangement of their visceral organs, and midline patterning defects that result from abnormal left-right patterning during embryogenesis. Loss of function of the transcription factor ZIC3 causes X-linked heterotaxy and isolated congenital heart malformations and represents one of the few known monogenic causes of congenital heart disease. The birth incidence of heterotaxy-spectrum malformations is significantly higher in males, but our previous work indicated that mutations within ZIC3 did not account for the male over-representation. Therefore, cross species comparative sequence alignment was used to identify a putative novel fourth exon, and the existence of a novel alternatively spliced transcript was confirmed by amplification from murine embryonic RNA and subsequent sequencing. This transcript, termed Zic3-B, encompasses exons 1, 2, and 4 whereas Zic3-A encompasses exons 1, 2, and 3. The resulting protein isoforms are 466 and 456 amino acid residues respectively, sharing the first 407 residues. Importantly, the last two amino acids in the fifth zinc finger DNA binding domain are altered in the Zic3-B isoform, indicating a potential functional difference that was further evaluated by expression, subcellular localization, and transactivation analyses. The temporo-spatial expression pattern of Zic3-B overlaps with Zic3-A in vivo, and both isoforms are localized to the nucleus in vitro. Both isoforms can transcriptionally activate a Gli binding site reporter, but only ZIC3-A synergistically activates upon co-transfection with Gli3, suggesting that the isoforms are functionally distinct. Screening 109 familial and sporadic male heterotaxy cases did not identify pathogenic mutations in the newly identified fourth exon and larger studies are necessary to establish the importance of the novel isoform in human disease.

  11. TOX3 mutations in breast cancer.

    Directory of Open Access Journals (Sweden)

    James Owain Jones

    Full Text Available TOX3 maps to 16q12, a region commonly lost in breast cancers and recently implicated in the risk of developing breast cancer. However, not much is known of the role of TOX3 itself in breast cancer biology. This is the first study to determine the importance of TOX3 mutations in breast cancers. We screened TOX3 for mutations in 133 breast tumours and identified four mutations (three missense, one in-frame deletion of 30 base pairs in six primary tumours, corresponding to an overall mutation frequency of 4.5%. One potentially deleterious missense mutation in exon 3 (Leu129Phe was identified in one tumour (genomic DNA and cDNA. Whilst copy number changes of 16q12 are common in breast cancer, our data show that mutations of TOX3 are present at low frequency in tumours. Our results support that TOX3 should be further investigated to elucidate its role in breast cancer biology.

  12. An albumin-mediated cholesterol design-based strategy for tuning siRNA pharmacokinetics and gene silencing.

    Science.gov (United States)

    Bienk, Konrad; Hvam, Michael Lykke; Pakula, Malgorzata Maria; Dagnæs-Hansen, Frederik; Wengel, Jesper; Malle, Birgitte Mølholm; Kragh-Hansen, Ulrich; Cameron, Jason; Bukrinski, Jens Thostrup; Howard, Kenneth A

    2016-06-28

    Major challenges for the clinical translation of small interfering RNA (siRNA) include overcoming the poor plasma half-life, site-specific delivery and modulation of gene silencing. In this work, we exploit the intrinsic transport properties of human serum albumin to tune the blood circulatory half-life, hepatic accumulation and gene silencing; based on the number of siRNA cholesteryl modifications. We demonstrate by a gel shift assay a strong and specific affinity of recombinant human serum albumin (rHSA) towards cholesteryl-modified siRNA (Kd>1×10(-7)M) dependent on number of modifications. The rHSA/siRNA complex exhibited reduced nuclease degradation and reduced induction of TNF-α production by human peripheral blood mononuclear cells. The increased solubility of heavily cholesteryl modified siRNA in the presence of rHSA facilitated duplex annealing and consequent interaction that allowed in vivo studies using multiple cholesteryl modifications. A structural-activity-based screen of in vitro EGFP-silencing was used to select optimal siRNA designs containing cholesteryl modifications within the sense strand that were used for in vivo studies. We demonstrate plasma half-life extension in NMRI mice from t1/2 12min (naked) to t1/2 45min (single cholesteryl) and t1/2 71min (double cholesteryl) using fluorescent live bioimaging. The biodistribution showed increased accumulation in the liver for the double cholesteryl modified siRNA that correlated with an increase in hepatic Factor VII gene silencing of 28% (rHSA/siRNA) compared to 4% (naked siRNA) 6days post-injection. This work presents a novel albumin-mediated cholesteryl design-based strategy for tuning pharmacokinetics and systemic gene silencing. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. High-throughput screening of filamentous fungi using nanoliter-range droplet-based microfluidics

    Science.gov (United States)

    Beneyton, Thomas; Wijaya, I. Putu Mahendra; Postros, Prexilia; Najah, Majdi; Leblond, Pascal; Couvent, Angélique; Mayot, Estelle; Griffiths, Andrew D.; Drevelle, Antoine

    2016-06-01

    Filamentous fungi are an extremely important source of industrial enzymes because of their capacity to secrete large quantities of proteins. Currently, functional screening of fungi is associated with low throughput and high costs, which severely limits the discovery of novel enzymatic activities and better production strains. Here, we describe a nanoliter-range droplet-based microfluidic system specially adapted for the high-throughput sceening (HTS) of large filamentous fungi libraries for secreted enzyme activities. The platform allowed (i) compartmentalization of single spores in ~10 nl droplets, (ii) germination and mycelium growth and (iii) high-throughput sorting of fungi based on enzymatic activity. A 104 clone UV-mutated library of Aspergillus niger was screened based on α-amylase activity in just 90 minutes. Active clones were enriched 196-fold after a single round of microfluidic HTS. The platform is a powerful tool for the development of new production strains with low cost, space and time footprint and should bring enormous benefit for improving the viability of biotechnological processes.

  14. Adaptive evolutionary walks require neutral intermediates in RNA fitness landscapes.

    Science.gov (United States)

    Rendel, Mark D

    2011-01-01

    In RNA fitness landscapes with interconnected networks of neutral mutations, neutral precursor mutations can play an important role in facilitating the accessibility of epistatic adaptive mutant combinations. I use an exhaustively surveyed fitness landscape model based on short sequence RNA genotypes (and their secondary structure phenotypes) to calculate the minimum rate at which mutants initially appearing as neutral are incorporated into an adaptive evolutionary walk. I show first, that incorporating neutral mutations significantly increases the number of point mutations in a given evolutionary walk when compared to estimates from previous adaptive walk models. Second, that incorporating neutral mutants into such a walk significantly increases the final fitness encountered on that walk - indeed evolutionary walks including neutral steps often reach the global optimum in this model. Third, and perhaps most importantly, evolutionary paths of this kind are often extremely winding in their nature and have the potential to undergo multiple mutations at a given sequence position within a single walk; the potential of these winding paths to mislead phylogenetic reconstruction is briefly considered. Copyright © 2010 Elsevier Inc. All rights reserved.

  15. Interaction between the cellular protein eEF1A and the 3'-terminal stem-loop of West Nile virus genomic RNA facilitates viral minus-strand RNA synthesis.

    Science.gov (United States)

    Davis, William G; Blackwell, Jerry L; Shi, Pei-Yong; Brinton, Margo A

    2007-09-01

    RNase footprinting and nitrocellulose filter binding assays were previously used to map one major and two minor binding sites for the cell protein eEF1A on the 3'(+) stem-loop (SL) RNA of West Nile virus (WNV) (3). Base substitutions in the major eEF1A binding site or adjacent areas of the 3'(+) SL were engineered into a WNV infectious clone. Mutations that decreased, as well as ones that increased, eEF1A binding in in vitro assays had a negative effect on viral growth. None of these mutations affected the efficiency of translation of the viral polyprotein from the genomic RNA, but all of the mutations that decreased in vitro eEF1A binding to the 3' SL RNA also decreased viral minus-strand RNA synthesis in transfected cells. Also, a mutation that increased the efficiency of eEF1A binding to the 3' SL RNA increased minus-strand RNA synthesis in transfected cells, which resulted in decreased synthesis of genomic RNA. These results strongly suggest that the interaction between eEF1A and the WNV 3' SL facilitates viral minus-strand synthesis. eEF1A colocalized with viral replication complexes (RC) in infected cells and antibody to eEF1A coimmunoprecipitated viral RC proteins, suggesting that eEF1A facilitates an interaction between the 3' end of the genome and the RC. eEF1A bound with similar efficiencies to the 3'-terminal SL RNAs of four divergent flaviviruses, including a tick-borne flavivirus, and colocalized with dengue virus RC in infected cells. These results suggest that eEF1A plays a similar role in RNA replication for all flaviviruses.

  16. A functional microRNA library screen reveals miR-410 as a novel anti-apoptotic regulator of cholangiocarcinoma

    International Nuclear Information System (INIS)

    Palumbo, Tiziana; Poultsides, George A.; Kouraklis, Grigorios; Liakakos, Theodore; Drakaki, Alexandra; Peros, George; Hatziapostolou, Maria; Iliopoulos, Dimitrios

    2016-01-01

    Cholangiocarcinoma is characterized by late diagnosis and a poor survival rate. MicroRNAs have been involved in the pathogenesis of different cancer types, including cholangiocarcinoma. Our aim was to identify novel microRNAs regulating cholangiocarcinoma cell growth in vitro and in vivo. A functional microRNA library screen was performed in human cholangiocarcinoma cells to identify microRNAs that regulate cholangiocarcinoma cell growth. Real-time PCR analysis evaluated miR-9 and XIAP mRNA levels in cholangiocarcinoma cells and tumors. The screen identified 21 microRNAs that regulated >50 % cholangiocarcinoma cell growth. MiR-410 was identified as the top suppressor of growth, while its overexpression significantly inhibited the invasion and colony formation ability of cholangiocarcinoma cells. Bioinformatics analysis revealed that microRNA-410 exerts its effects through the direct regulation of the X-linked inhibitor of apoptosis protein (XIAP). Furthermore, overexpression of miR-410 significantly reduced cholangiocarcinoma tumor growth in a xenograft mouse model through induction of apoptosis. In addition, we identified an inverse relationship between miR-410 and XIAP mRNA levels in human cholangiocarcinomas. Taken together, our study revealed a novel microRNA signaling pathway involved in cholangiocarcinoma and suggests that manipulation of the miR-410/XIAP pathway could have a therapeutic potential for cholangiocarcinoma. The online version of this article (doi:10.1186/s12885-016-2384-0) contains supplementary material, which is available to authorized users

  17. Screening of potential biomarkers in uterine leiomyomas disease via gene expression profiling analysis.

    Science.gov (United States)

    Liu, Xuhui; Liu, Yanfei; Zhao, Jingrong; Liu, Yan

    2018-05-01

    The present study aimed to screen potential biomarkers for uterine leiomyomas disease, particularly target genes associated with the mediator of RNA polymerase II transcription subunit 12 (MED12) mutation. The microarray data of GSE30673, including 10 MED12 wild-type myometrium, 8 MED12 mutation leiomyoma and 2 MED12 wild-type leiomyoma samples, were downloaded from the Gene Expression Omnibus database. Compared with myometrium samples, differently-expressed genes (DEGs) in the MED12 mutation and wild-type leiomyoma samples were identified using the Limma package. The two sets of DEGs obtained were intersected to screen common DEGs. The DEGs in the MED12 mutation and wild-type leiomyoma samples, and common DEGs were defined as group A, B and C. Gene Ontology (GO) and pathway enrichment analyses were performed using the Database for Annotation, Visualization and Integrated Discovery online tool. Based on the Kyoto Encyclopedia of Genes and Genomes database, pathway relation networks were constructed. DEGs in GO terms and pathways were intersected to screen important DEGs. Subsequently, a gene co‑expression network was constructed and visualized using Cytoscape software. Reverse transcription‑quantitative polymerase chain reaction was used to detect the expression levels of important DEGs. A total of 1,258 DEGs in group A were screened, and enriched for extracellular matrix (ECM) organization and ECM‑receptor interaction. In addition, a total of 1,571 DEGs in group B were enriched for cell adhesion. Furthermore, 391 DEGs were involved in extracellular matrix organization. Pathway relation networks of group A, B and C were constructed with nodes of 48, 39, and 28, respectively. Finally, 135 important DEGs were obtained, including Acyl‑CoA synthetase medium‑chain family member 3, protein S (α) (PROS1) and F11 receptor. A gene co‑expression network with 68 nodes was constructed. The expression of caspase 1 (CASP1) and aldehyde dehydrogenase 1 family member

  18. Screening for colorectal cancer

    DEFF Research Database (Denmark)

    Nielsen, Hans J.; Jakobsen, Karen V.; Christensen, Ib J.

    2011-01-01

    Emerging results indicate that screening improves survival of patients with colorectal cancer. Therefore, screening programs are already implemented or are being considered for implementation in Asia, Europe and North America. At present, a great variety of screening methods are available including...... into improvements of screening for colorectal cancer includes blood-based biological markers, such as proteins, DNA and RNA in combination with various demographically and clinically parameters into a "risk assessment evaluation" (RAE) test. It is assumed that such a test may lead to higher acceptance among...... procedures for colorectal cancer. Therefore, results of present research, validating RAE tests, are awaited with interest....

  19. Enriched whole genome sequencing identified compensatory mutations in the RNA polymerase gene of rifampicin-resistant Mycobacterium leprae strains

    Directory of Open Access Journals (Sweden)

    Lavania M

    2018-01-01

    Full Text Available Mallika Lavania,1 Itu Singh,1 Ravindra P Turankar,1 Anuj Kumar Gupta,2 Madhvi Ahuja,1 Vinay Pathak,1 Utpal Sengupta1 1Stanley Browne Laboratory, The Leprosy Mission Trust India, TLM Community Hospital Nand Nagari, 2Agilent Technologies India Pvt Ltd, Jasola District Centre, New Delhi, India Abstract: Despite more than three decades of multidrug therapy (MDT, leprosy remains a major public health issue in several endemic countries, including India. The emergence of drug resistance in Mycobacterium leprae (M. leprae is a cause of concern and poses a threat to the leprosy-control program, which might ultimately dampen the achievement of the elimination program of the country. Rifampicin resistance in clinical strains of M. leprae are supposed to arise from harboring bacterial strains with mutations in the 81-bp rifampicin resistance determining region (RRDR of the rpoB gene. However, complete dynamics of rifampicin resistance are not explained only by this mutation in leprosy strains. To understand the role of other compensatory mutations and transmission dynamics of drug-resistant leprosy, a genome-wide sequencing of 11 M. leprae strains – comprising five rifampicin-resistant strains, five sensitive strains, and one reference strain – was done in this study. We observed the presence of compensatory mutations in two rifampicin-resistant strains in rpoC and mmpL7 genes, along with rpoB, that may additionally be responsible for conferring resistance in those strains. Our findings support the role for compensatory mutation(s in RNA polymerase gene(s, resulting in rifampicin resistance in relapsed leprosy patients. Keywords: leprosy, rifampicin resistance, compensatory mutations, next generation sequencing, relapsed, MDT, India

  20. A screen of a large Czech cohort of oligodontia patients implicates a novel mutation in the PAX9 gene

    Czech Academy of Sciences Publication Activity Database

    Šerý, Omar; Bonczek, Ondřej; Hloušková, A.; Černochová, P.; Vaněk, J.; Míšek, Ivan; Krejčí, J.; Izakovičová Hollá, L.

    2015-01-01

    Roč. 123, č. 2 (2015), s. 65-71 ISSN 0909-8836 R&D Projects: GA ČR GB14-37368G; GA MZd(CZ) NT11420 Institutional support: RVO:67985904 Keywords : monozygotic twins * mutation screening * oligodontia Subject RIV: FF - HEENT, Dentistry Impact factor: 1.607, year: 2015

  1. Screening for germline BRCA1, BRCA2, TP53 and CHEK2 mutations in families at-risk for hereditary breast cancer identified in a population-based study from Southern Brazil

    Directory of Open Access Journals (Sweden)

    Edenir Inêz Palmero

    2016-01-01

    Full Text Available Abstract In Brazil, breast cancer is a public health care problem due to its high incidence and mortality rates. In this study, we investigated the prevalence of hereditary breast cancer syndromes (HBCS in a population-based cohort in Brazils southernmost capital, Porto Alegre. All participants answered a questionnaire about family history (FH of breast, ovarian and colorectal cancer and those with a positive FH were invited for genetic cancer risk assessment (GCRA. If pedigree analysis was suggestive of HBCS, genetic testing of the BRCA1, BRCA2, TP53, and CHEK2 genes was offered. Of 902 women submitted to GCRA, 214 had pedigrees suggestive of HBCS. Fifty of them underwent genetic testing: 18 and 40 for BRCA1/BRCA2 and TP53 mutation screening, respectively, and 7 for CHEK2 1100delC testing. A deleterious BRCA2 mutation was identified in one of the HBOC probands and the CHEK2 1100delC mutation occurred in one of the HBCC families. No deleterious germline alterations were identified in BRCA1 or TP53. Although strict inclusion criteria and a comprehensive testing approach were used, the suspected genetic risk in these families remains unexplained. Further studies in a larger cohort are necessary to better understand the genetic component of hereditary breast cancer in Southern Brazil.

  2. 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

  3. A novel RT-PCR for the detection of Helicobacter pylori and identification of clarithromycin resistance mediated by mutations in the 23S rRNA gene.

    Science.gov (United States)

    Redondo, Javier Jareño; Keller, Peter M; Zbinden, Reinhard; Wagner, Karoline

    2018-01-01

    In this study we evaluated the commercially available LightMix® RT-PCR assay for Helicobacter pylori detection and identification of clarithromycin (CLR) resistance in culture and clinical specimens (gastric biopsies and stool). The H. pylori LightMix® RT-PCR detects a 97bp long fragment of the 23S rRNA gene and allows the identification of 3 distinct point mutations conferring CLR resistance via melting curve analysis. The performance of the H. pylori LightMix® RT-PCR was evaluated using a set of 60 H. pylori strains showing phenotypical CLR susceptibility or CLR resistance (Minimum inhibitory concentrations from 0.016 to 256mg/L). We found high concordance (95%) between phenotypical CLR resistance screening by E-Test® and the Lightmix® RT-PCR. Discrepant results were verified by sequencing of the 23S rRNA gene that always confirmed the results obtained by Lightmix® RT-PCR. Furthermore, H. pylori was detected in clinical biopsy and stool specimens by Lightmix® RT-PCR that identified the correct H. pylori genotype. The LightMix® RT-PCR is an accurate, sensitive and easy to use test for H. pylori and CLR resistance detection and can therefore be readily implemented in any diagnostic laboratory. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Mutational analysis of the RNA-binding domain of the Prunus necrotic ringspot virus (PNRSV) movement protein reveals its requirement for cell-to-cell movement.

    Science.gov (United States)

    Carmen Herranz, Ma; Sanchez-Navarro, Jesús-Angel; Saurí, Ana; Mingarro, Ismael; Pallás, Vicente

    2005-08-15

    The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is required for cell-to-cell movement. MP subcellular localization studies using a GFP fusion protein revealed highly punctate structures between neighboring cells, believed to represent plasmodesmata. Deletion of the RNA-binding domain (RBD) of PNRSV MP abolishes the cell-to-cell movement. A mutational analysis on this RBD was performed in order to identify in vivo the features that govern viral transport. Loss of positive charges prevented the cell-to-cell movement even though all mutants showed a similar accumulation level in protoplasts to those observed with the wild-type (wt) MP. Synthetic peptides representing the mutants and wild-type RBDs were used to study RNA-binding affinities by EMSA assays being approximately 20-fold lower in the mutants. Circular dichroism analyses revealed that the secondary structure of the peptides was not significantly affected by mutations. The involvement of the affinity changes between the viral RNA and the MP in the viral cell-to-cell movement is discussed.

  5. Mutational analysis of the RNA-binding domain of the Prunus necrotic ringspot virus (PNRSV) movement protein reveals its requirement for cell-to-cell movement

    International Nuclear Information System (INIS)

    Carmen Herranz, Ma; Sanchez-Navarro, Jesus-Angel; Sauri, Ana; Mingarro, Ismael; Pallas, Vicente

    2005-01-01

    The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is required for cell-to-cell movement. MP subcellular localization studies using a GFP fusion protein revealed highly punctate structures between neighboring cells, believed to represent plasmodesmata. Deletion of the RNA-binding domain (RBD) of PNRSV MP abolishes the cell-to-cell movement. A mutational analysis on this RBD was performed in order to identify in vivo the features that govern viral transport. Loss of positive charges prevented the cell-to-cell movement even though all mutants showed a similar accumulation level in protoplasts to those observed with the wild-type (wt) MP. Synthetic peptides representing the mutants and wild-type RBDs were used to study RNA-binding affinities by EMSA assays being approximately 20-fold lower in the mutants. Circular dichroism analyses revealed that the secondary structure of the peptides was not significantly affected by mutations. The involvement of the affinity changes between the viral RNA and the MP in the viral cell-to-cell movement is discussed

  6. A path-based measurement for human miRNA functional similarities using miRNA-disease associations

    Science.gov (United States)

    Ding, Pingjian; Luo, Jiawei; Xiao, Qiu; Chen, Xiangtao

    2016-09-01

    Compared with the sequence and expression similarity, miRNA functional similarity is so important for biology researches and many applications such as miRNA clustering, miRNA function prediction, miRNA synergism identification and disease miRNA prioritization. However, the existing methods always utilized the predicted miRNA target which has high false positive and false negative to calculate the miRNA functional similarity. Meanwhile, it is difficult to achieve high reliability of miRNA functional similarity with miRNA-disease associations. Therefore, it is increasingly needed to improve the measurement of miRNA functional similarity. In this study, we develop a novel path-based calculation method of miRNA functional similarity based on miRNA-disease associations, called MFSP. Compared with other methods, our method obtains higher average functional similarity of intra-family and intra-cluster selected groups. Meanwhile, the lower average functional similarity of inter-family and inter-cluster miRNA pair is obtained. In addition, the smaller p-value is achieved, while applying Wilcoxon rank-sum test and Kruskal-Wallis test to different miRNA groups. The relationship between miRNA functional similarity and other information sources is exhibited. Furthermore, the constructed miRNA functional network based on MFSP is a scale-free and small-world network. Moreover, the higher AUC for miRNA-disease prediction indicates the ability of MFSP uncovering miRNA functional similarity.

  7. Association of BRAFV600E Mutation and MicroRNA Expression with Central Lymph Node Metastases in Papillary Thyroid Cancer: A Prospective Study from Four Endocrine Surgery Centers

    OpenAIRE

    Aragon Han, Patricia; Kim, Hyun-seok; Cho, Soonweng; Fazeli, Roghayeh; Najafian, Alireza; Khawaja, Hunain; McAlexander, Melissa; Dy, Benzon; Sorensen, Meredith; Aronova, Anna; Sebo, Thomas J.; Giordano, Thomas J.; Fahey, Thomas J.; Thompson, Geoffrey B.; Gauger, Paul G.

    2016-01-01

    Background: Studies have demonstrated an association of the BRAFV600E mutation and microRNA (miR) expression with aggressive clinicopathologic features in papillary thyroid cancer (PTC). Analysis of BRAFV600E mutations with miR expression data may improve perioperative decision making for patients with PTC, specifically in identifying patients harboring central lymph node metastases (CLNM).

  8. Probing of RNA structures in a positive sense RNA virus reveals selection pressures for structural elements

    Science.gov (United States)

    Watters, Kyle E; Choudhary, Krishna; Aviran, Sharon; Perry, Keith L

    2018-01-01

    Abstract In single stranded (+)-sense RNA viruses, RNA structural elements (SEs) play essential roles in the infection process from replication to encapsidation. Using selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) and covariation analysis, we explore the structural features of the third genome segment of cucumber mosaic virus (CMV), RNA3 (2216 nt), both in vitro and in plant cell lysates. Comparing SHAPE-Seq and covariation analysis results revealed multiple SEs in the coat protein open reading frame and 3′ untranslated region. Four of these SEs were mutated and serially passaged in Nicotiana tabacum plants to identify biologically selected changes to the original mutated sequences. After passaging, loop mutants showed partial reversion to their wild-type sequence and SEs that were structurally disrupted by mutations were restored to wild-type-like structures via synonymous mutations in planta. These results support the existence and selection of virus open reading frame SEs in the host organism and provide a framework for further studies on the role of RNA structure in viral infection. Additionally, this work demonstrates the applicability of high-throughput chemical probing in plant cell lysates and presents a new method for calculating SHAPE reactivities from overlapping reverse transcriptase priming sites. PMID:29294088

  9. Requirement for a conserved, tertiary interaction in the core of 23S ribosomal RNA

    DEFF Research Database (Denmark)

    Aagaard, C; Douthwaite, S

    1994-01-01

    RNA. Every substitution that disrupts the potential for Watson-Crick base pairing between these positions reduces or abolishes the participation of 23S rRNA in protein synthesis. All mutant 23S rRNAs are assembled into 50S subunits, but the mutant subunits are less able to stably interact with 30S subunits...... is nonfunctional. In contrast to the considerable effect the mutations have on function, they impart only slight structural changes on the naked rRNA, and these are limited to the immediate vicinity of the mutations. The data show that positions 1262 and 2017 pair in a Watson-Crick manner, but the data also...

  10. RNA-based therapies for genodermatoses

    NARCIS (Netherlands)

    Bornert, Olivier; Peking, Patricia; Bremer, Jeroen; Koller, Ulrich; van den Akker, Peter C.; Aartsma-Rus, Annemieke; Pasmooij, Anna M. G.; Murauer, Eva M.; Nystroem, Alexander

    Genetic disorders affecting the skin, genodermatoses, constitute a large and heterogeneous group of diseases, for which treatment is generally limited to management of symptoms. RNA-based therapies are emerging as a powerful tool to treat genodermatoses. In this review, we discuss in detail RNA

  11. [Mutation screening of MITF gene in patients with Waardenburg syndrome type 2].

    Science.gov (United States)

    Chen, Jing; Yang, Shu-Zhi; Liu, Jun; Han, Bing; Wang, Guo-Jian; Zhang, Xin; Kang, Dong-Yang; Dai, Pu; Young, Wie-Yen; Yuan, Hui-Jun

    2008-04-01

    Warrgenburg syndrome type 2 (WS2) is the most common autosomal dominantly-inherited syndrome with hearing loss. MITF (microphthalmia associated transcription factor)is a basic-helix-loop-helix-luecine zipper (bHLHZip) factor which regulates expression of tyrosinase, and is involved in melanocyte differentiation. Mutations in MITF associated with WS2 have been identified in some but not all affected families. Here, we report a three-generation Chinese family with a point mutation in the MITF gene causing WS2. The proband exhibits congenital severe sensorineural hearing loss, heterochromia iridis and facial freckles. One of family members manifests sensorineural deafness, and the other patients show premature greying or/and freckles. This mutation, heterozygous deletion c.639delA, creates a stop codon in exon 7 and is predicted to result in a truncated protein lacking normal interaction with its target DNA motif. This mutation is a novel mutation and the third case identified in exon 7 of MITF in WS2. Though there is only one base pair distance between this novel mutation and the other two documented cases and similar amino acids change, significant difference is seen in clinical phenotype, which suggests genetic background may play an important role.

  12. A multi-step strategy to obtain crystals of the dengue virus RNA-dependent RNA polymerase that diffract to high resolution

    International Nuclear Information System (INIS)

    Yap, Thai Leong; Chen, Yen Liang; Xu, Ting; Wen, Daying; Vasudevan, Subhash G.; Lescar, Julien

    2007-01-01

    Crystals of the RNA-dependent RNA polymerase catalytic domain from the dengue virus NS5 protein have been obtained using a strategy that included expression screening of naturally occurring serotype variants of the protein, the addition of divalent metal ions and crystal dehydration. These crystals diffract to 1.85 Å resolution and are thus suitable for a structure-based drug-design program. Dengue virus, a member of the Flaviviridae genus, causes dengue fever, an important emerging disease with several million infections occurring annually for which no effective therapy exists. The viral RNA-dependent RNA polymerase NS5 plays an important role in virus replication and represents an interesting target for the development of specific antiviral compounds. Crystals that diffract to 1.85 Å resolution that are suitable for three-dimensional structure determination and thus for a structure-based drug-design program have been obtained using a strategy that included expression screening of naturally occurring serotype variants of the protein, the addition of divalent metal ions and crystal dehydration

  13. A multi-step strategy to obtain crystals of the dengue virus RNA-dependent RNA polymerase that diffract to high resolution

    Energy Technology Data Exchange (ETDEWEB)

    Yap, Thai Leong [Novartis Institute for Tropical Diseases, 10 Biopolis Road, Chromos Building, Singapore 138670 (Singapore); School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Chen, Yen Liang; Xu, Ting; Wen, Daying; Vasudevan, Subhash G. [Novartis Institute for Tropical Diseases, 10 Biopolis Road, Chromos Building, Singapore 138670 (Singapore); Lescar, Julien, E-mail: julien@ntu.edu.sg [Novartis Institute for Tropical Diseases, 10 Biopolis Road, Chromos Building, Singapore 138670 (Singapore); School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore)

    2007-02-01

    Crystals of the RNA-dependent RNA polymerase catalytic domain from the dengue virus NS5 protein have been obtained using a strategy that included expression screening of naturally occurring serotype variants of the protein, the addition of divalent metal ions and crystal dehydration. These crystals diffract to 1.85 Å resolution and are thus suitable for a structure-based drug-design program. Dengue virus, a member of the Flaviviridae genus, causes dengue fever, an important emerging disease with several million infections occurring annually for which no effective therapy exists. The viral RNA-dependent RNA polymerase NS5 plays an important role in virus replication and represents an interesting target for the development of specific antiviral compounds. Crystals that diffract to 1.85 Å resolution that are suitable for three-dimensional structure determination and thus for a structure-based drug-design program have been obtained using a strategy that included expression screening of naturally occurring serotype variants of the protein, the addition of divalent metal ions and crystal dehydration.

  14. Pathway-based analysis of genome-wide siRNA screens reveals the regulatory landscape of APP processing.

    Directory of Open Access Journals (Sweden)

    Luiz Miguel Camargo

    Full Text Available The progressive aggregation of Amyloid-β (Aβ in the brain is a major trait of Alzheimer's Disease (AD. Aβ is produced as a result of proteolytic processing of the β-amyloid precursor protein (APP. Processing of APP is mediated by multiple enzymes, resulting in the production of distinct peptide products: the non-amyloidogenic peptide sAPPα and the amyloidogenic peptides sAPPβ, Aβ40, and Aβ42. Using a pathway-based approach, we analyzed a large-scale siRNA screen that measured the production of different APP proteolytic products. Our analysis identified many of the biological processes/pathways that are known to regulate APP processing and have been implicated in AD pathogenesis, as well as revealing novel regulatory mechanisms. Furthermore, we also demonstrate that some of these processes differentially regulate APP processing, with some mechanisms favouring production of certain peptide species over others. For example, synaptic transmission having a bias towards regulating Aβ40 production over Aβ42 as well as processes involved in insulin and pancreatic biology having a bias for sAPPβ production over sAPPα. In addition, some of the pathways identified as regulators of APP processing contain genes (CLU, BIN1, CR1, PICALM, TREM2, SORL1, MEF2C, DSG2, EPH1A recently implicated with AD through genome wide association studies (GWAS and associated meta-analysis. In addition, we provide supporting evidence and a deeper mechanistic understanding of the role of diabetes in AD. The identification of these processes/pathways, their differential impact on APP processing, and their relationships to each other, provide a comprehensive systems biology view of the "regulatory landscape" of APP.

  15. Two novel exonic point mutations in HEXA identified in a juvenile Tay-Sachs patient: role of alternative splicing and nonsense-mediated mRNA decay.

    Science.gov (United States)

    Levit, A; Nutman, D; Osher, E; Kamhi, E; Navon, R

    2010-06-01

    We have identified three mutations in the beta-hexoseaminidase A (HEXA) gene in a juvenile Tay-Sachs disease (TSD) patient, which exhibited a reduced level of HEXA mRNA. Two mutations are novel, c.814G>A (p.Gly272Arg) and c.1305C>T (p.=), located in exon 8 and in exon 11, respectively. The third mutation, c.1195A>G (p.Asn399Asp) in exon 11, has been previously characterized as a common polymorphism in African-Americans. Hex A activity measured in TSD Glial cells, transfected with HEXA cDNA constructs bearing these mutations, was unaltered from the activity level measured in normal HEXA cDNA. Analysis of RT-PCR products revealed three aberrant transcripts in the patient, one where exon 8 was absent, one where exon 11 was absent and a third lacking both exons 10 and 11. All three novel transcripts contain frameshifts resulting in premature termination codons (PTCs). Transfection of mini-gene constructs carrying the c.814G>A and c.1305C>T mutations proved that the two mutations result in exon skipping. mRNAs that harbor a PTC are detected and degraded by the nonsense-mediated mRNA decay (NMD) pathway to prevent synthesis of abnormal proteins. However, although NMD is functional in the patient's fibroblasts, aberrant transcripts are still present. We suggest that the level of correctly spliced transcripts as well as the efficiency in which NMD degrade the PTC-containing transcripts, apparently plays an important role in the phenotype severity of the unique patient and thus should be considered as a potential target for drug therapy.

  16. Estimating the Per-Base-Pair Mutation Rate in the Yeast Saccharomyces cerevisiae

    OpenAIRE

    Lang, Gregory I.; Murray, Andrew W.

    2008-01-01

    Although mutation rates are a key determinant of the rate of evolution they are difficult to measure precisely and global mutations rates (mutations per genome per generation) are often extrapolated from the per-base-pair mutation rate assuming that mutation rate is uniform across the genome. Using budding yeast, we describe an improved method for the accurate calculation of mutation rates based on the fluctuation assay. Our analysis suggests that the per-base-pair mutation rates at two genes...

  17. SOURCE OF THE FITNESS DEFECT IN RIFAMYCIN-RESISTANT M. TUBERCULOSIS RNA POLYMERASE AND THE MECHANISM OF COMPENSATION BY MUTATIONS IN THE β'-SUBUNIT.

    Science.gov (United States)

    Stefan, Maxwell A; Ugur, Fatima S; Garcia, George A

    2018-04-16

    Mycobacterium tuberculosis (MTB) is a critical threat to human health due to the increased prevalence of rifampin resistance (RMP R ). Fitness defects have been observed in RMP R mutants having amino acid substitutions in the β-subunit of RNA polymerase (RNAP). In clinical isolates, this fitness defect can be ameliorated by the presence of secondary mutations in the double-psi β-barrel (DPBB) domain of the β'-subunit of RNAP. To identify factors contributing to the fitness defects observed in vivo, several in vitro RNA transcription assays were utilized to probe initiation, elongation, termination, and 3' -RNA hydrolysis with the wild-type and RMP R MTB RNAPs. We found that the less prevalent RMP R mutants exhibit significantly poorer termination efficiencies relative to wild-type, an important factor for proper gene expression. We also found that several mechanistic aspects of transcription of the RMP R mutant RNAPs are impacted relative to wild-type. For the clinically most prevalent, βS450L mutant, these defects are mitigated by the presence of secondary/compensatory mutations in the DPBB domain of the β'-subunit. Copyright © 2018 American Society for Microbiology.

  18. Mutations in DCPS and EDC3 in autosomal recessive intellectual disability indicate a crucial role for mRNA decapping in neurodevelopment.

    Science.gov (United States)

    Ahmed, Iltaf; Buchert, Rebecca; Zhou, Mi; Jiao, Xinfu; Mittal, Kirti; Sheikh, Taimoor I; Scheller, Ute; Vasli, Nasim; Rafiq, Muhammad Arshad; Brohi, M Qasim; Mikhailov, Anna; Ayaz, Muhammad; Bhatti, Attya; Sticht, Heinrich; Nasr, Tanveer; Carter, Melissa T; Uebe, Steffen; Reis, André; Ayub, Muhammad; John, Peter; Kiledjian, Megerditch; Vincent, John B; Jamra, Rami Abou

    2015-06-01

    There are two known mRNA degradation pathways, 3' to 5' and 5' to 3'. We identified likely pathogenic variants in two genes involved in these two pathways in individuals with intellectual disability. In a large family with multiple branches, we identified biallelic variants in DCPS in three affected individuals; a splice site variant (c.636+1G>A) that results in an in-frame insertion of 45 nucleotides and a missense variant (c.947C>T; p.Thr316Met). DCPS decaps the cap structure generated by 3' to 5' exonucleolytic degradation of mRNA. In vitro decapping assays showed an ablation of decapping function for both variants in DCPS. In another family, we identified a homozygous mutation (c.161T>C; p.Phe54Ser) in EDC3 in two affected children. EDC3 stimulates DCP2, which decaps mRNAs at the beginning of the 5' to 3' degradation pathway. In vitro decapping assays showed that altered EDC3 is unable to enhance DCP2 decapping at low concentrations and even inhibits DCP2 decapping at high concentration. We show that individuals with biallelic mutations in these genes of seemingly central functions are viable and that these possibly lead to impairment of neurological functions linking mRNA decapping to normal cognition. Our results further affirm an emerging theme linking aberrant mRNA metabolism to neurological defects. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  19. Effects of the umuC36 mutation on ultraviolet-radiation-induced base-change and frameshift mutations in Escherichia coli

    International Nuclear Information System (INIS)

    Kato, T.; Nakano, E.

    1981-01-01

    The effects of the umuC36 mutation on the induction of base-change and frameshift mutations were studied. An active umuC gene was necessary in either the uvr + or uvr - strains of Escherichia coli K12 for UV- and X-ray-induced mutations to His + , ColE and Spc, which are presumably base-change mutations, but it was not essential for ethyl methanesulphonate or N-methyl-N'-nitro-N-nitrosoguanidine-induced His + mutations. In contrast, only 1 out of 13 trp - frameshift mutations examined was UV reversible, and the process of mutagenesis was umuC + -dependent, whereas a potent frameshift mutagen, ICR191, effectively induced Trp + mutations in most of the strains regardless of the umu + or umuC genetic background. These results suggest that base substitutions are a major mutational type derived from the umuC + -dependent pathway of error-prone repair. (orig.)

  20. The effect of dys-1 mutation on miRNA expression profile in Caenorhabditis elegans during Shenzhou-8 mission

    Science.gov (United States)

    Xu, Dan; Sun, Yeqing; Gao, Ying; Xing, Yanfang

    microRNAs (miRNAs) is reported to be sensitive to radiation exposure and altered gravity, involved in a variety of biological processes through negative regulation of gene expression. Dystrophin-like dys-1 gene is expressed and required in muscle tissue, which plays a vital role in mechanical transduction when gravity varies. In the present study, we investigated the effect of dys-1 mutation on miRNA expression profile in Caenorhabditis elegans (C. elegans) under space radiation associated with microgravity (R+M) and radiation alone (R) environment during Shenzhou-8 mission. We performed miRNA microarray analysis in dys-1 mutant and wide-type (WT) of dauer larvae and found that 27 miRNAs changed in abundance after spaceflight. Compared with WT, there was different miRNA expression pattern in different treatments in dys-1 mutant. Cel-miR-796 and miR-124 were reversely expressed under R+M and R environment in WT and dys-1 mutant, respectively, indicating they might be affected by microgravity. Mutation of dys-1 remarkably reduced the number of altered miRNAs under space environment, resulting in the decrease of genes in biological categories of “body morphogenesis”, “behavior”, “cell adhesion” and so on. Particularly, we found that those genes controlling regulation of locomotion in WT were lost in dys-1 mutant, while genes in positive regulation of developmental process only existed in dys-1 mutant. miR-796 was predicted to target genes ace-1 and dyc-1 that are functionally linked to dys-1. Integration analysis of miRNA and mRNA expression profile revealed that miR-56 and miR-124 were involved in behavior and locomotion by regulating different target genes under space environment, among which nep-11, deb-1, C07H4.1 and F11H8.2 might be associated with neuromuscular system. Our findings suggest that dys-1 could cause alteration of miRNAs and target genes, involved in regulating the response of C. elegans to space microgravity in neuromuscular system. This

  1. Population-based screening versus case detection.

    Directory of Open Access Journals (Sweden)

    Thomas Ravi

    2002-01-01

    Full Text Available India has a large burden of blindness and population-based screening is a strategy commonly employed to detect disease and prevent morbidity. However, not all diseases are amenable to screening. This communication examines the issue of "population-based screening" versus "case detection" in the Indian scenario. Using the example of glaucoma, it demonstrates that given the poor infrastructure, for a "rare" disease, case detection is more effective than population-based screening.

  2. Universal Versus Targeted Screening for Lynch Syndrome: Comparing Ascertainment and Costs Based on Clinical Experience.

    Science.gov (United States)

    Erten, Mujde Z; Fernandez, Luca P; Ng, Hank K; McKinnon, Wendy C; Heald, Brandie; Koliba, Christopher J; Greenblatt, Marc S

    2016-10-01

    Strategies to screen colorectal cancers (CRCs) for Lynch syndrome are evolving rapidly; the optimal strategy remains uncertain. We compared targeted versus universal screening of CRCs for Lynch syndrome. In 2010-2011, we employed targeted screening (age Lynch syndrome and estimated the 5-year costs of preventing CRC by colonoscopy screening, using a system dynamics model. Using targeted screening, 51/175 (29 %) cancers fit criteria and were tested by immunohistochemistry; 15/51 (29 %, or 8.6 % of all CRCs) showed suspicious loss of ≥1 mismatch repair protein. Germline mismatch repair gene mutations were found in 4/4 cases sequenced (11 suspected cases did not have germline testing). Using universal screening, 17/292 (5.8 %) screened cancers had abnormal immunohistochemistry suspicious for Lynch syndrome. Germline mismatch repair mutations were found in only 3/10 cases sequenced (7 suspected cases did not have germline testing). The mean cost to identify Lynch syndrome probands was ~$23,333/case for targeted screening and ~$175,916/case for universal screening at our institution. Estimated costs to identify and screen probands and relatives were: targeted, $9798/case and universal, $38,452/case. In real-world Lynch syndrome management, incomplete clinical follow-up was the major barrier to do genetic testing. Targeted screening costs 2- to 7.5-fold less than universal and rarely misses Lynch syndrome cases. Future changes in testing costs will likely change the optimal algorithm.

  3. Detection of MPL mutations by a novel allele-specific PCR-based strategy.

    Science.gov (United States)

    Furtado, Larissa V; Weigelin, Helmut C; Elenitoba-Johnson, Kojo S J; Betz, Bryan L

    2013-11-01

    MPL mutation testing is recommended in patients with suspected primary myelofibrosis or essential thrombocythemia who lack the JAK2 V617F mutation. MPL mutations can occur at allelic levels below 15%, which may escape detection by commonly used mutation screening methods such as Sanger sequencing. We developed a novel multiplexed allele-specific PCR assay capable of detecting most recurrent MPL exon 10 mutations associated with primary myelofibrosis and essential thrombocythemia (W515L, W515K, W515A, and S505N) down to a sensitivity of 2.5% mutant allele. Test results were reviewed from 15 reference cases and 1380 consecutive specimens referred to our laboratory for testing. Assay performance was compared to Sanger sequencing across a series of 58 specimens with MPL mutations. Positive cases consisted of 45 with W515L, 6 with S505N, 5 with W515K, 1 with W515A, and 1 with both W515L and S505N. Seven cases had mutations below 5% that were undetected by Sanger sequencing. Ten additional cases had mutation levels between 5% and 15% that were not consistently detected by sequencing. All results were easily interpreted in the allele-specific test. This assay offers a sensitive and reliable solution for MPL mutation testing. Sanger sequencing appears insufficiently sensitive for robust MPL mutation detection. Our data also suggest the relative frequency of S505N mutations may be underestimated, highlighting the necessity for inclusion of this mutation in MPL test platforms. Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  4. A Constant Rate of Spontaneous Mutation in DNA-Based Microbes

    Science.gov (United States)

    Drake, John W.

    1991-08-01

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

  5. Screening of point mutations by multiple SSCP analysis in the dystrophin gene

    Energy Technology Data Exchange (ETDEWEB)

    Lasa, A.; Baiget, M.; Gallano, P. [Hospital Sant Pau, Barcelona (Spain)

    1994-09-01

    Duchenne muscular dystrophy (DMD) is a lethal, X-linked neuromuscular disorder. The population frequency of DMD is one in approximately 3500 boys, of which one third is thought to be a new mutant. The DMD gene is the largest known to date, spanning over 2,3 Mb in band Xp21.2; 79 exons are transcribed into a 14 Kb mRNA coding for a protein of 427 kD which has been named dystrophin. It has been shown that about 65% of affected boys have a gene deletion with a wide variation in localization and size. The remaining affected individuals who have no detectable deletions or duplications would probably carry more subtle mutations that are difficult to detect. These mutations occur in several different exons and seem to be unique to single patients. Their identification represents a formidable goal because of the large size and complexity of the dystrophin gene. SSCP is a very efficient method for the detection of point mutations if the parameters that affect the separation of the strands are optimized for a particular DNA fragment. The multiple SSCP allows the simultaneous study of several exons, and implies the use of different conditions because no single set of conditions will be optimal for all fragments. Seventy-eight DMD patients with no deletion or duplication in the dystrophin gene were selected for the multiple SSCP analysis. Genomic DNA from these patients was amplified using the primers described for the diagnosis procedure (muscle promoter and exons 3, 8, 12, 16, 17, 19, 32, 45, 48 and 51). We have observed different mobility shifts in bands corresponding to exons 8, 12, 43 and 51. In exons 17 and 45, altered electrophoretic patterns were found in different samples identifying polymorphisms already described.

  6. Resistance to linezolid in Staphylococcus spp. clinical isolates associated with ribosomal binding site modifications: novel mutation in domain V of 23S rRNA.

    Science.gov (United States)

    Musumeci, Rosario; Calaresu, Enrico; Gerosa, Jolanda; Oggioni, Davide; Bramati, Simone; Morelli, Patrizia; Mura, Ida; Piana, Andrea; Are, Bianca Maria; Cocuzza, Clementina Elvezia

    2016-10-01

    Linezolid is the main representative of the oxazolidinones, introduced in 2000 in clinical practice to treat severe Gram-positive infections. This compound inhibits protein synthesis by binding to the peptidyl transferase centre of the 50S bacterial ribosomal subunit. The aim of this study was to characterize 12 clinical strains of linezolid-resistant Staphylococcus spp. isolated in Northern Italy. All isolates of Staphylococcus spp. studied showed a multi-antibiotic resistance phenotype. In particular, all isolates showed the presence of the mecA gene associated with SSCmec types IVa, V or I. Mutations in domain V of 23S rRNA were shown to be the most prevalent mechanism of linezolid resistance: among these a new C2551T mutation was found in S. aureus, whilst the G2576T mutation was shown to be the most prevalent overall. Moreover, three S. epidermidis isolates were shown to have linezolid resistance associated only with alterations in both L3 and L4 ribosomal proteins. No strain was shown to harbor the previously described cfr gene. These results have shown how the clinical use of linezolid in Northern Italy has resulted in the selection of multiple antibiotic-resistant clinical isolates of Staphylococcus spp., with linezolid resistance in these strains being associated with mutations in 23S rRNA or ribosomal proteins L3 and L4.

  7. Targeted CRISPR disruption reveals a role for RNase MRP RNA in human preribosomal RNA processing.

    Science.gov (United States)

    Goldfarb, Katherine C; Cech, Thomas R

    2017-01-01

    MRP RNA is an abundant, essential noncoding RNA whose functions have been proposed in yeast but are incompletely understood in humans. Mutations in the genomic locus for MRP RNA cause pleiotropic human diseases, including cartilage hair hypoplasia (CHH). Here we applied CRISPR-Cas9 genome editing to disrupt the endogenous human MRP RNA locus, thereby attaining what has eluded RNAi and RNase H experiments: elimination of MRP RNA in the majority of cells. The resulting accumulation of ribosomal RNA (rRNA) precursor-analyzed by RNA fluorescent in situ hybridization (FISH), Northern blots, and RNA sequencing-implicates MRP RNA in pre-rRNA processing. Amelioration of pre-rRNA imbalance is achieved through rescue of MRP RNA levels by ectopic expression. Furthermore, affinity-purified MRP ribonucleoprotein (RNP) from HeLa cells cleaves the human pre-rRNA in vitro at at least one site used in cells, while RNP isolated from cells with CRISPR-edited MRP loci loses this activity, and ectopic MRP RNA expression restores cleavage activity. Thus, a role for RNase MRP in human pre-rRNA processing is established. As demonstrated here, targeted CRISPR disruption is a valuable tool for functional studies of essential noncoding RNAs that are resistant to RNAi and RNase H-based degradation. © 2017 Goldfarb and Cech; Published by Cold Spring Harbor Laboratory Press.

  8. Minisequencing mitochondrial DNA pathogenic mutations

    Directory of Open Access Journals (Sweden)

    Carracedo Ángel

    2008-04-01

    Full Text Available Abstract Background There are a number of well-known mutations responsible of common mitochondrial DNA (mtDNA diseases. In order to overcome technical problems related to the analysis of complete mtDNA genomes, a variety of different techniques have been proposed that allow the screening of coding region pathogenic mutations. Methods We here propose a minisequencing assay for the analysis of mtDNA mutations. In a single reaction, we interrogate a total of 25 pathogenic mutations distributed all around the whole mtDNA genome in a sample of patients suspected for mtDNA disease. Results We have detected 11 causal homoplasmic mutations in patients suspected for Leber disease, which were further confirmed by standard automatic sequencing. Mutations m.11778G>A and m.14484T>C occur at higher frequency than expected by change in the Galician (northwest Spain patients carrying haplogroup J lineages (Fisher's Exact test, P-value Conclusion We here developed a minisequencing genotyping method for the screening of the most common pathogenic mtDNA mutations which is simple, fast, and low-cost. The technique is robust and reproducible and can easily be implemented in standard clinical laboratories.

  9. Seryl-tRNA Synthetases from Methanogenic Archaea: Suppression of Bacterial Amber Mutation and Heterologous Toxicity

    Directory of Open Access Journals (Sweden)

    Drasko Boko

    2010-01-01

    Full Text Available Methanogenic archaea possess unusual seryl-tRNA synthetases (SerRS, evolutionarily distinct from the SerRSs found in other archaea, eucaryotes and bacteria. Our recent X-ray structural analysis of Methanosarcina barkeri SerRS revealed an idiosyncratic N-terminal domain and catalytic zinc ion in the active site. To shed further light on substrate discrimination by methanogenic-type SerRS, we set up to explore in vivo the interaction of methanogenic-type SerRSs with their cognate tRNAs in Escherichia coli or Saccharomyces cerevisiae. The expression of various methanogenic-type SerRSs was toxic for E. coli, resulting in the synthesis of erroneous proteins, as revealed by β-galactosidase stability assay. Although SerRSs from methanogenic archaea recognize tRNAsSer from all three domains of life in vitro, the toxicity presumably precluded the complementation of endogenous SerRS function in both, E. coli and S. cerevisiae. However, despite the observed toxicity, coexpression of methanogenic-type SerRS with its cognate tRNA suppressed bacterial amber mutation.

  10. Effects of mutations in the VP2/VP4 cleavage site of Swine vesicular disease virus on RNA encapsidation and viral infectivity

    NARCIS (Netherlands)

    Rebel, J.M.J.; Leendertse, C.H.; Dekker, A.; Moormann, R.J.M.

    2003-01-01

    We studied VP0 cleavage of Swine vesicular disease virus (SVDV), a member of the Picornaviridae using a full-length cDNA copy of the Dutch SVDV isolate. The influences of mutations, introduced at the cleavage site of SVDV, on VP0 cleavage, RNA encapsidation and viral infection were studied. Double

  11. Genetic screens to identify new Notch pathway mutants in Drosophila.

    Science.gov (United States)

    Giagtzoglou, Nikolaos

    2014-01-01

    Notch signaling controls a wide range of developmental processes, including proliferation, apoptosis, and cell fate specification during both development and adult tissue homeostasis. The functional versatility of the Notch signaling pathway is tightly linked with the complexity of its regulation in different cellular contexts. To unravel the complexity of Notch signaling, it is important to identify the different components of the Notch signaling pathway. A powerful strategy to accomplish this task is based on genetic screens. Given that the developmental context of signaling is important, these screens should be customized to specific cell populations or tissues. Here, I describe how to perform F1 clonal forward genetic screens in Drosophila to identify novel components of the Notch signaling pathway. These screens combine a classical EMS (ethyl methanesulfonate) chemical mutagenesis protocol along with clonal analysis via FRT-mediated mitotic recombination. These F1 clonal screens allow rapid phenotypic screening within clones of mutant cells induced at specific developmental stages and in tissues of interest, bypassing the pleiotropic effects of isolated mutations. More importantly, since EMS mutations have been notoriously difficult to map to specific genes in the past, I briefly discuss mapping methods that allow rapid identification of the causative mutations.

  12. From structure prediction to genomic screens for novel non-coding RNAs

    DEFF Research Database (Denmark)

    Gorodkin, Jan; Hofacker, Ivo L.

    2011-01-01

    Abstract: Non-coding RNAs (ncRNAs) are receiving more and more attention not only as an abundant class of genes, but also as regulatory structural elements (some located in mRNAs). A key feature of RNA function is its structure. Computational methods were developed early for folding and prediction....... This and the increased amount of available genomes have made it possible to employ structure-based methods for genomic screens. The field has moved from folding prediction of single sequences to computational screens for ncRNAs in genomic sequence using the RNA structure as the main characteristic feature. Whereas early...... upon some of the concepts in current methods that have been applied in genomic screens for de novo RNA structures in searches for novel ncRNA genes and regulatory RNA structure on mRNAs. We discuss the strengths and weaknesses of the different strategies and how they can complement each other....

  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. Truncating mutation in the NHS gene: phenotypic heterogeneity of Nance-Horan syndrome in an asian Indian family.

    Science.gov (United States)

    Ramprasad, Vedam Lakshmi; Thool, Alka; Murugan, Sakthivel; Nancarrow, Derek; Vyas, Prateep; Rao, Srinivas Kamalakar; Vidhya, Authiappan; Ravishankar, Krishnamoorthy; Kumaramanickavel, Govindasamy

    2005-01-01

    A four-generation family containing eight affected males who inherited X-linked developmental lens opacity and microcornea was studied. Some members in the family had mild to moderate nonocular clinical features suggestive of Nance-Horan syndrome. The purpose of the study was to map genetically the gene in the large 57-live-member Asian-Indian pedigree. PCR-based genotyping was performed on the X-chromosome, by using fluorescent microsatellite markers (10-cM intervals). Parametric linkage analysis was performed by using two disease models, assuming either recessive or dominant X-linked transmission by the MLINK/ILINK and FASTLINK (version 4.1P) programs (http:www.hgmp.mrc.ac.uk/; provided in the public domain by the Human Genome Mapping Project Resources Centre, Cambridge, UK). The NHS gene at the linked region was screened for mutation. By fine mapping, the disease gene was localized to Xp22.13. Multipoint analysis placed the peak LOD of 4.46 at DSX987. The NHS gene mapped to this region. Mutational screening in all the affected males and carrier females (heterozygous form) revealed a truncating mutation 115C-->T in exon 1, resulting in conversion of glutamine to stop codon (Q39X), but was not observed in unaffected individuals and control subjects. conclusions. A family with X-linked Nance-Horan syndrome had severe ocular, but mild to moderate nonocular, features. The clinical phenotype of the truncating mutation (Q39X) in the NHS gene suggests allelic heterogeneity at the NHS locus or the presence of modifier genes. X-linked families with cataract should be carefully examined for both ocular and nonocular features, to exclude Nance-Horan syndrome. RT-PCR analysis did not suggest nonsense-mediated mRNA decay as the possible mechanism for clinical heterogeneity.

  15. Sensitive and fast mutation detection by solid phase chemical cleavage

    DEFF Research Database (Denmark)

    Hansen, Lise Lotte; Justesen, Just; Kruse, Torben A

    1996-01-01

    We have developed a solid phase chemical cleavage method (SpCCM) for screening large DNA fragments for mutations. All reactions can be carried out in microtiterwells from the first amplification of the patient (or test) DNA through the search for mutations. The reaction time is significantly...... reduced compared to the conventional chemical cleavage method (CCM), and even by using a uniformly labelled probe, the exact position and nature of the mutation can be revealed. The SpCCM is suitable for automatization using a workstation to carry out the reactions and a fluorescent detection-based DNA...

  16. Efficient fractal-based mutation in evolutionary algorithms from iterated function systems

    Science.gov (United States)

    Salcedo-Sanz, S.; Aybar-Ruíz, A.; Camacho-Gómez, C.; Pereira, E.

    2018-03-01

    In this paper we present a new mutation procedure for Evolutionary Programming (EP) approaches, based on Iterated Function Systems (IFSs). The new mutation procedure proposed consists of considering a set of IFS which are able to generate fractal structures in a two-dimensional phase space, and use them to modify a current individual of the EP algorithm, instead of using random numbers from different probability density functions. We test this new proposal in a set of benchmark functions for continuous optimization problems. In this case, we compare the proposed mutation against classical Evolutionary Programming approaches, with mutations based on Gaussian, Cauchy and chaotic maps. We also include a discussion on the IFS-based mutation in a real application of Tuned Mass Dumper (TMD) location and optimization for vibration cancellation in buildings. In both practical cases, the proposed EP with the IFS-based mutation obtained extremely competitive results compared to alternative classical mutation operators.

  17. A large-scale RNA interference screen identifies genes that regulate autophagy at different stages.

    Science.gov (United States)

    Guo, Sujuan; Pridham, Kevin J; Virbasius, Ching-Man; He, Bin; Zhang, Liqing; Varmark, Hanne; Green, Michael R; Sheng, Zhi

    2018-02-12

    Dysregulated autophagy is central to the pathogenesis and therapeutic development of cancer. However, how autophagy is regulated in cancer is not well understood and genes that modulate cancer autophagy are not fully defined. To gain more insights into autophagy regulation in cancer, we performed a large-scale RNA interference screen in K562 human chronic myeloid leukemia cells using monodansylcadaverine staining, an autophagy-detecting approach equivalent to immunoblotting of the autophagy marker LC3B or fluorescence microscopy of GFP-LC3B. By coupling monodansylcadaverine staining with fluorescence-activated cell sorting, we successfully isolated autophagic K562 cells where we identified 336 short hairpin RNAs. After candidate validation using Cyto-ID fluorescence spectrophotometry, LC3B immunoblotting, and quantitative RT-PCR, 82 genes were identified as autophagy-regulating genes. 20 genes have been reported previously and the remaining 62 candidates are novel autophagy mediators. Bioinformatic analyses revealed that most candidate genes were involved in molecular pathways regulating autophagy, rather than directly participating in the autophagy process. Further autophagy flux assays revealed that 57 autophagy-regulating genes suppressed autophagy initiation, whereas 21 candidates promoted autophagy maturation. Our RNA interference screen identifies identified genes that regulate autophagy at different stages, which helps decode autophagy regulation in cancer and offers novel avenues to develop autophagy-related therapies for cancer.

  18. PMS2 monoallelic mutation carriers: the known unknown.

    Science.gov (United States)

    Goodenberger, McKinsey L; Thomas, Brittany C; Riegert-Johnson, Douglas; Boland, C Richard; Plon, Sharon E; Clendenning, Mark; Win, Aung Ko; Senter, Leigha; Lipkin, Steven M; Stadler, Zsofia K; Macrae, Finlay A; Lynch, Henry T; Weitzel, Jeffrey N; de la Chapelle, Albert; Syngal, Sapna; Lynch, Patrick; Parry, Susan; Jenkins, Mark A; Gallinger, Steven; Holter, Spring; Aronson, Melyssa; Newcomb, Polly A; Burnett, Terrilea; Le Marchand, Loïc; Pichurin, Pavel; Hampel, Heather; Terdiman, Jonathan P; Lu, Karen H; Thibodeau, Stephen; Lindor, Noralane M

    2016-01-01

    Germ-line mutations in MLH1, MSH2, MSH6, and PMS2 have been shown to cause Lynch syndrome. The penetrance of the cancer and tumor spectrum has been repeatedly studied, and multiple professional societies have proposed clinical management guidelines for affected individuals. Several studies have demonstrated a reduced penetrance for monoallelic carriers of PMS2 mutations compared with the other mismatch repair (MMR) genes, but clinical management guidelines have largely proposed the same screening recommendations for all MMR gene carriers. The authors considered whether enough evidence existed to propose new screening guidelines specific to PMS2 mutation carriers with regard to age at onset and frequency of colonic screening. Published reports of PMS2 germ-line mutations were combined with unpublished cases from the authors' research registries and clinical practices, and a discussion of potential modification of cancer screening guidelines was pursued. A total of 234 monoallelic PMS2 mutation carriers from 170 families were included. Approximately 8% of those with colorectal cancer (CRC) were diagnosed before age 30, and each of these tumors presented on the left side of the colon. As it is currently unknown what causes the early onset of CRC in some families with monoallelic PMS2 germline mutations, the authors recommend against reducing cancer surveillance guidelines in families found having monoallelic PMS2 mutations in spite of the reduced penetrance.Genet Med 18 1, 13-19.

  19. Identification of Six Novel PTH1R Mutations in Families with a History of Primary Failure of Tooth Eruption

    DEFF Research Database (Denmark)

    Risom, Lotte; Christoffersen, Line Borck; Daugaard-Jensen, Jette

    2013-01-01

    Primary Failure of tooth Eruption (PFE) is a non-syndromic disorder which can be caused by mutations in the parathyroid hormone receptor 1 gene (PTH1R). Traditionally, the disorder has been identified clinically based on post-emergent failure of eruption of permanent molars. However, patients...... undergone surgical and/or orthodontic interventions, and identified novel PTH1R mutations in all. Four of the six mutations were predicted to abolish correct mRNA maturation either through introduction of premature stop codons (c.947C>A and c.1082G>A), or by altering correct mRNA splicing (c.544......-26_544-23del and c.989G>T). The latter was validated by transfection of minigenes. The six novel mutations expand the mutation spectrum for PFE from eight to 14 pathogenic mutations. Loss-of-function mutations in PTH1R are also associated with recessively inherited Blomstrand chondrodysplasia. We compiled all...

  20. The use of optical markers for mutation breeding

    International Nuclear Information System (INIS)

    Makino, Takahiro

    2003-01-01

    The use of radiation for mutation breeding has produced many kinds of practical varieties in crops and ornamental plants over the last several decades. Cold-tolerant rice and disease-resistant apple and pears are well-known varieties resulting from radiation breeding in Japan, and X-ray mutations were used routinely for the expansion of petal color in the chrysanthemum. Recently, the use of ion-beams for mutation induction was investigated as an effective source for producing varieties in cereal crops and flowers in Japan and China (Harten, 1998). Although we have not produced many varieties through radiation breeding, the success rate could increase with the addition of more resources. The success of mutation breeding greatly depends on the rate of mutation, the number of screened plants, and the mutation efficiency. The mutation rate is mainly a function of the total dose of the mutagen employed, although it can be modified by physical and biological factors. A large number of reports have been produced and effective methods of mutation treatments, such as gamma rays, established. Using higher doses inevitably brings about mortality, high pollen and seed sterility, and deleterious mutations. A practical useful dosage is usually found in the range much less than the maximum dose that can be applied. To increase the efficiency of mutation breeding, improvement of screening methods is more important than trials used for raising mutation probabilities. For this reason, we began studies to develop non-destructive and non-invasive optical high-throughput screening systems to increase the efficiency of mutation breeding. (author)

  1. Recent advances in mechanism-based chemotherapy drug-siRNA pairs in co-delivery systems for cancer: A review.

    Science.gov (United States)

    Wang, Mingfang; Wang, Jinyu; Li, Bingcheng; Meng, Lingxin; Tian, Zhaoxing

    2017-09-01

    Co-delivery of chemotherapy drugs and siRNA for cancer therapy has achieved remarkable results according to synergistic/combined antitumor effects, and is recognized as a promising therapeutic modality. However, little attention has been paid to the extremely complex mechanisms of chemotherapy drug-siRNA pairs during co-delivery process. Proper selection of chemotherapy drug-siRNA pairs is beneficial for achieving desirable cancer therapeutic effects. Exploring the inherent principles during chemotherapy drug-siRNA pair selection for co-delivery would greatly enhanced therapeutic efficiency. To achieve ideal results, this article will systematically review current different mechanism-based chemotherapy drug-siRNA pairs for co-delivery in cancer treatment. Large-scale library screening of recent different chemotherapy drug-siRNA pairs for co-delivery would help to establish the chemotherapy drug-siRNA pair selection principle, which could pave the way for co-delivery of chemotherapy drugs and siRNA for cancer treatment in clinic. Following the inherent principle of chemotherapy drug-siRNA pair, more effective co-delivery vectors can be designed in the future. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Inactivating Mutation screening of Exon 6 and Exon 10E of FSHR gene in women with Polycystic Ovarian Syndrome in Vellore population

    Science.gov (United States)

    Sekar, Nishu; Sapre, Madhura; Kale, Vaikhari; Prabhu, Yogamaya D.; Renu, Kaviyarasi; Ramgir, Shalaka S.; Abilash, V. G.

    2017-11-01

    Polycystic Ovarian syndrome (PCOS) is a major cause of infertility in females of reproducing age and is typified by oligo-anovulation, hyperandrogenism, hirsutism and polycystic ovaries. FSHR gene located on chromosome 2 p21 is responsible for the normal follicular development and any deletion or mutation in the gene affects the interaction of FSH with its receptor. Thus, it becomes the candidate gene for PCOS study. Inactivating mutation in FSHR gene limits the receptor’s function by creating a complete block, changing the receptor-ligand complex or the basic hormone signal transduction.To screen the inactivating mutations in Exon 6 and Exon 10E of FSHR gene in women diagnosed with PCOS.PCR-RFLP analysis indicated that there were no inactivating mutations found in Exon 6 and Exon 10E. Variations in hormone levels were seen amongst the PCOS patients. There were no inactivating mutations found in FSHR gene of the women diagnosed with PCOS according to the Rotterdam criteria in Vellore population.

  3. Screening Phosphorylation Site Mutations in Yeast Acetyl-CoA Carboxylase Using Malonyl-CoA Sensor to Improve Malonyl-CoA-Derived Product.

    Science.gov (United States)

    Chen, Xiaoxu; Yang, Xiaoyu; Shen, Yu; Hou, Jin; Bao, Xiaoming

    2018-01-01

    Malonyl-coenzyme A (malonyl-CoA) is a critical precursor for the biosynthesis of a variety of biochemicals. It is synthesized by the catalysis of acetyl-CoA carboxylase (Acc1p), which was demonstrated to be deactivated by the phosphorylation of Snf1 protein kinase in yeast. In this study, we designed a synthetic malonyl-CoA biosensor and used it to screen phosphorylation site mutations of Acc1p in Saccharomyces cerevisiae . Thirteen phosphorylation sites were mutated, and a combination of three site mutations in Acc1p, S686A, S659A, and S1157A, was found to increase malonyl-CoA availability. ACC1 S686AS659AS1157A expression also improved the production of 3-hydroxypropionic acid, a malonyl-CoA-derived chemical, compared to both wild type and the previously reported ACC1 S659AS1157A mutation. This mutation will also be beneficial for other malonyl-CoA-derived products.

  4. Combinatorial RNA Interference Therapy Prevents Selection of Pre-existing HBV Variants in Human Liver Chimeric Mice

    Science.gov (United States)

    Shih, Yao-Ming; Sun, Cheng-Pu; Chou, Hui-Hsien; Wu, Tzu-Hui; Chen, Chun-Chi; Wu, Ping-Yi; Enya Chen, Yu-Chen; Bissig, Karl-Dimiter; Tao, Mi-Hua

    2015-01-01

    Selection of escape mutants with mutations within the target sequence could abolish the antiviral RNA interference activity. Here, we investigated the impact of a pre-existing shRNA-resistant HBV variant on the efficacy of shRNA therapy. We previously identified a highly potent shRNA, S1, which, when delivered by an adeno-associated viral vector, effectively inhibits HBV replication in HBV transgenic mice. We applied the “PICKY” software to systemically screen the HBV genome, then used hydrodynamic transfection and HBV transgenic mice to identify additional six highly potent shRNAs. Human liver chimeric mice were infected with a mixture of wild-type and T472C HBV, a S1-resistant HBV variant, and then treated with a single or combined shRNAs. The presence of T472C mutant compromised the therapeutic efficacy of S1 and resulted in replacement of serum wild-type HBV by T472C HBV. In contrast, combinatorial therapy using S1 and P28, one of six potent shRNAs, markedly reduced titers for both wild-type and T472C HBV. Interestingly, treatment with P28 alone led to the emergence of escape mutants with mutations in the P28 target region. Our results demonstrate that combinatorial RNAi therapy can minimize the escape of resistant viral mutants in chronic HBV patients. PMID:26482836

  5. Feature-Based and String-Based Models for Predicting RNA-Protein Interaction

    Directory of Open Access Journals (Sweden)

    Donald Adjeroh

    2018-03-01

    Full Text Available In this work, we study two approaches for the problem of RNA-Protein Interaction (RPI. In the first approach, we use a feature-based technique by combining extracted features from both sequences and secondary structures. The feature-based approach enhanced the prediction accuracy as it included much more available information about the RNA-protein pairs. In the second approach, we apply search algorithms and data structures to extract effective string patterns for prediction of RPI, using both sequence information (protein and RNA sequences, and structure information (protein and RNA secondary structures. This led to different string-based models for predicting interacting RNA-protein pairs. We show results that demonstrate the effectiveness of the proposed approaches, including comparative results against leading state-of-the-art methods.

  6. siRNA delivery with lipid-based systems

    DEFF Research Database (Denmark)

    Foged, Camilla

    2012-01-01

    A key hurdle for the further development of RNA interference (RNAi) therapeutics like small interfering RNA (siRNA) is their safe and effective delivery. Lipids are promising and versatile carriers because they are based on Nature's own building blocks and can be provided with properties which......RNA into more hydrophobic lipoplexes, which promote passage of the siRNA across cellular membrane barriers, especially when lipids are added that facilitate membrane fusion. Despite these attractive features, siRNA delivery vehicles are facing a number of challenges such as the limited delivery efficiency...

  7. 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.

  8. A BRCA2 mutation incorrectly mapped in the original BRCA2 reference sequence, is a common West Danish founder mutation disrupting mRNA splicing

    DEFF Research Database (Denmark)

    Thomassen, Mads; Pedersen, Inge Søkilde; Vogel, Ida

    2011-01-01

    Inherited mutations in the tumor suppressor genes BRCA1 and BRCA2 predispose carriers to breast and ovarian cancer. The authors have identified a mutation in BRCA2, 7845+1G>A (c.7617+1G>A), not previously regarded as deleterious because of incorrect mapping of the splice junction in the originally...... published genomic reference sequence. This reference sequence is generally used in many laboratories and it maps the mutation 16 base pairs inside intron 15. However, according to the recent reference sequences the mutation is located in the consensus donor splice sequence. By reverse transcriptase analysis......, loss of exon 15 in the final transcript interrupting the open reading frame was demonstrated. Furthermore, the mutation segregates with a cancer phenotype in 18 Danish families. By genetic analysis of more than 3,500 Danish breast/ovarian cancer risk families, the mutation was identified as the most...

  9. Structure of RDE-4 dsRBDs and mutational studies provide insights into dsRNA recognition in the Caenorhabditis elegans RNAi pathway.

    Science.gov (United States)

    Chiliveri, Sai Chaitanya; Deshmukh, Mandar V

    2014-02-15

    The association of RDE-4 (RNAi defective 4), a protein containing two dsRBDs (dsRNA-binding domains), with long dsRNA and Dcr-1 (Dicer1 homologue) initiates the siRNA pathway in Caenorhabditis elegans. Unlike its homologues in higher eukaryotes, RDE-4 dsRBDs possess weak (micromolar) affinity for short dsRNA. With increasing length of dsRNA, RDE-4 exhibits enhanced affinity due to co-operativity. The linker and dsRBD2 are indispensable for RDE-4's simultaneous interaction with dsRNA and Dcr-1. In the present study, we have determined the solution structures of RDE-4 constructs that contain both dsRBDs and the linker region. In addition to the canonical dsRBD fold, both dsRBDs of RDE-4 show modified structural features such as truncation in the β1-β2 loop that rationalize RDE-4's relatively weak dsRNA affinity. Structure and binding studies demonstrate that dsRBD2 plays a decisive role in the RDE-4-dsRNA interaction; however, in contrast with previous findings, we found ephemeral interaction of RDE-4 dsRBD1 with dsRNA. More importantly, mutations in two tandem lysine residues (Lys217 and Lys218) in dsRBD2 impair RDE-4's dsRNA-binding ability and could obliterate RNAi initiation in C. elegans. Additionally, we postulate a structural basis for the minimal requirement of linker and dsRBD2 for RDE-4's association with dsRNA and Dcr-1.

  10. Direct detection of hemophilia B F9 gene mutation using multiplex PCR and conformation sensitive gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Ki Young Yoo

    2010-03-01

    Full Text Available Purpose : The F9 gene is known to be the causative gene for hemophilia B, but unfortunately the detection rate for restriction fragment length polymorphism-based linkage analysis is only 55.6%. Direct DNA sequencing can detect 98% of mutations, but this alternative procedure is very costly. Here, we conducted multiplex polymerase chain reactions (PCRs and conformation sensitive gel electrophoresis (CSGE to perform a screened DNA sequencing for the F9 gene, and we compared the results with direct sequencing in terms of accuracy, cost, simplicity, and time consumption. Methods : A total of 27 unrelated hemophilia B patients were enrolled. Direct DNA sequencing was performed for 27 patients by a separate institute, and multiplex PCR-CSGE screened sequencing was done in our laboratory. Results of the direct DNA sequencing were used as a reference, to which the results of the multiplex PCR-CSGE screened sequencing were compared. For the patients whose mutation was not detected by the 2 methods, multiplex ligation-dependent probe amplification (MLPA was conducted. Results : With direct sequencing, the mutations could be identified from 26 patients (96.3%, whereas for multiplex PCR- CSGE screened sequencing, the mutations could be detected in 23 (85.2%. One patient’s mutation was identified by MLPA. A total of 21 different mutations were found among the 27 patients. Conclusion : Multiplex PCR-CSGE screened DNA sequencing detected 88.9% of mutations and reduced costs by 55.7% compared with direct DNA sequencing. However, it was more labor-intensive and time-consuming.

  11. MicroRNA-31 expression in relation to BRAF mutation, CpG island methylation and colorectal continuum in serrated lesions.

    Science.gov (United States)

    Ito, Miki; Mitsuhashi, Kei; Igarashi, Hisayoshi; Nosho, Katsuhiko; Naito, Takafumi; Yoshii, Shinji; Takahashi, Hiroaki; Fujita, Masahiro; Sukawa, Yasutaka; Yamamoto, Eiichiro; Takahashi, Taiga; Adachi, Yasushi; Nojima, Masanori; Sasaki, Yasushi; Tokino, Takashi; Baba, Yoshifumi; Maruyama, Reo; Suzuki, Hiromu; Imai, Kohzoh; Yamamoto, Hiroyuki; Shinomura, Yasuhisa

    2014-12-01

    The CpG island methylator phenotype (CIMP) is a distinct form of epigenomic instability. Many CIMP-high colorectal cancers (CRCs) with BRAF mutation are considered to arise from serrated pathway. We recently reported that microRNA-31 (miR-31) is associated with BRAF mutation in colorectal tumors. Emerging new approaches have revealed gradual changes in BRAF mutation and CIMP-high throughout the colorectum in CRCs. Here, we attempted to identify a possible association between miR-31 and epigenetic features in serrated pathway, and hypothesized that miR-31 supports the "colorectal continuum" concept. We evaluated miR-31 expression, BRAF mutation and epigenetic features including CIMP status in 381 serrated lesions and 222 non-serrated adenomas and examined associations between them and tumor location (rectum; sigmoid, descending, transverse and ascending colon and cecum). A significant association was observed between high miR-31 expression and CIMP-high status in serrated lesions with BRAF mutation (p = 0.0001). In contrast, miR-31 was slightly but insignificantly associated with CIMP status in the cases with wild-type BRAF. miR-31 expression in sessile serrated adenomas (SSAs) with cytological dysplasia was higher than that in SSAs, whereas, no significant difference was observed between traditional serrated adenomas (TSAs) and TSAs with high-grade dysplasia. The frequency of miR-31, BRAF mutation CIMP-high and MLH1 methylation increased gradually from the rectum to cecum in serrated lesions. In conclusion, miR-31 expression was associated with CIMP-high status in serrated lesions with BRAF mutation. Our data also suggested that miR-31 plays an important role in SSA evolution and may be a molecule supporting the colorectal continuum. © 2014 UICC.

  12. Interplay between DMD point mutations and splicing signals in Dystrophinopathy phenotypes.

    Directory of Open Access Journals (Sweden)

    Jonàs Juan-Mateu

    Full Text Available DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements.

  13. Literature-based condition-specific miRNA-mRNA target prediction.

    Directory of Open Access Journals (Sweden)

    Minsik Oh

    Full Text Available miRNAs are small non-coding RNAs that regulate gene expression by binding to the 3'-UTR of genes. Many recent studies have reported that miRNAs play important biological roles by regulating specific mRNAs or genes. Many sequence-based target prediction algorithms have been developed to predict miRNA targets. However, these methods are not designed for condition-specific target predictions and produce many false positives; thus, expression-based target prediction algorithms have been developed for condition-specific target predictions. A typical strategy to utilize expression data is to leverage the negative control roles of miRNAs on genes. To control false positives, a stringent cutoff value is typically set, but in this case, these methods tend to reject many true target relationships, i.e., false negatives. To overcome these limitations, additional information should be utilized. The literature is probably the best resource that we can utilize. Recent literature mining systems compile millions of articles with experiments designed for specific biological questions, and the systems provide a function to search for specific information. To utilize the literature information, we used a literature mining system, BEST, that automatically extracts information from the literature in PubMed and that allows the user to perform searches of the literature with any English words. By integrating omics data analysis methods and BEST, we developed Context-MMIA, a miRNA-mRNA target prediction method that combines expression data analysis results and the literature information extracted based on the user-specified context. In the pathway enrichment analysis using genes included in the top 200 miRNA-targets, Context-MMIA outperformed the four existing target prediction methods that we tested. In another test on whether prediction methods can re-produce experimentally validated target relationships, Context-MMIA outperformed the four existing target prediction

  14. Clinical experience in the screening and management of a large kindred with familial isolated pituitary adenoma due to an aryl hydrocarbon receptor interacting protein (AIP) mutation.

    Science.gov (United States)

    Williams, Fred; Hunter, Steven; Bradley, Lisa; Chahal, Harvinder S; Storr, Helen L; Akker, Scott A; Kumar, Ajith V; Orme, Stephen M; Evanson, Jane; Abid, Noina; Morrison, Patrick J; Korbonits, Márta; Atkinson, A Brew

    2014-04-01

    Germline AIP mutations usually cause young-onset acromegaly with low penetrance in a subset of familial isolated pituitary adenoma families. We describe our experience with a large family with R304* AIP mutation and discuss some of the diagnostic dilemmas and management issues. The aim of the study was to identify and screen mutation carriers in the family. Forty-three family members participated in the study. The study was performed in university hospitals. We conducted genetic and endocrine screening of family members. We identified 18 carriers of the R304* mutation, three family members with an AIP-variant A299V, and two family members who harbored both changes. One of the two index cases presented with gigantism and pituitary apoplexy, the other presented with young-onset acromegaly, and both had surgery and radiotherapy. After genetic and clinical screening of the family, two R304* carriers were diagnosed with acromegaly. They underwent transsphenoidal surgery after a short period of somatostatin analog treatment. One of these two patients is in remission; the other achieved successful pregnancy despite suboptimal control of acromegaly. One of the A299V carrier family members was previously diagnosed with a microprolactinoma; we consider this case to be a phenocopy. Height of the unaffected R304* carrier family members is not different compared to noncarrier relatives. Families with AIP mutations present particular problems such as the occurrence of large invasive tumors, poor response to medical treatment, difficulties with fertility and management of pregnancy, and the finding of AIP sequence variants of unknown significance. Because disease mostly develops at a younger age and penetrance is low, the timing and duration of the follow-up of carriers without overt disease requires further study. The psychological and financial impact of prolonged clinical screening must be considered. Excellent relationships between the family, endocrinologists, and

  15. SETD1A modulates cell cycle progression through a miRNA network that regulates p53 target genes

    OpenAIRE

    Tajima, Ken; Yae, Toshifumi; Javaid, Sarah; Tam, Oliver; Comaills, Valentine; Morris, Robert; Wittner, Ben S.; Liu, Mingzhu; Engstrom, Amanda; Takahashi, Fumiyuki; Black, Joshua C.; Ramaswamy, Sridhar; Shioda, Toshihiro; Hammell, Molly; Haber, Daniel A.

    2015-01-01

    Expression of the p53-inducible antiproliferative gene BTG2 is suppressed in many cancers in the absence of inactivating gene mutations, suggesting alternative mechanisms of silencing. Using a shRNA screen targeting 43 histone lysine methyltransferases (KMTs), we show that SETD1A suppresses BTG2 expression through its induction of several BTG2-targeting miRNAs. This indirect but highly specific mechanism, by which a chromatin regulator that mediates transcriptional activating marks can lead t...

  16. In Vivo RNA Interference Screening Identifies a Leukemia-Specific Dependence on Integrin Beta 3 Signaling

    Science.gov (United States)

    Miller, Peter G.; Al-Shahrour, Fatima; Hartwell, Kimberly A.; Chu, Lisa P.; Järås, Marcus; Puram, Rishi V.; Puissant, Alexandre; Callahan, Kevin P.; Ashton, John; McConkey, Marie E.; Poveromo, Luke P.; Cowley, Glenn S.; Kharas, Michael G.; Labelle, Myriam; Shterental, Sebastian; Fujisaki, Joji; Silberstein, Lev; Alexe, Gabriela; Al-Hajj, Muhammad A.; Shelton, Christopher A.; Armstrong, Scott A.; Root, David E.; Scadden, David T.; Hynes, Richard O.; Mukherjee, Siddhartha; Stegmaier, Kimberly; Jordan, Craig T.; Ebert, Benjamin L.

    2013-01-01

    SUMMARY We used an in vivo short hairpin RNA (shRNA) screening approach to identify genes that are essential for MLL-AF9 acute myeloid leukemia (AML). We found that Integrin Beta 3 (Itgb3) is essential for murine leukemia cells in vivo, and for human leukemia cells in xenotransplantation studies. In leukemia cells, Itgb3 knockdown impaired homing, downregulated LSC transcriptional programs, and induced differentiation via the intracellular kinase, Syk. In contrast, loss of Itgb3 in normal HSPCs did not affect engraftment, reconstitution, or differentiation. Finally, we confirmed that Itgb3 is dispensable for normal hematopoiesis and required for leukemogenesis using an Itgb3 knockout mouse model. Our results establish the significance of the Itgb3 signaling pathway as a potential therapeutic target in AML. PMID:23770013

  17. A cell-based high-throughput screening assay for radiation susceptibility using automated cell counting

    International Nuclear Information System (INIS)

    Hodzic, Jasmina; Dingjan, Ilse; Maas, Mariëlle JP; Meulen-Muileman, Ida H van der; Menezes, Renee X de; Heukelom, Stan; Verheij, Marcel; Gerritsen, Winald R; Geldof, Albert A; Triest, Baukelien van; Beusechem, Victor W van

    2015-01-01

    Radiotherapy is one of the mainstays in the treatment for cancer, but its success can be limited due to inherent or acquired resistance. Mechanisms underlying radioresistance in various cancers are poorly understood and available radiosensitizers have shown only modest clinical benefit. There is thus a need to identify new targets and drugs for more effective sensitization of cancer cells to irradiation. Compound and RNA interference high-throughput screening technologies allow comprehensive enterprises to identify new agents and targets for radiosensitization. However, the gold standard assay to investigate radiosensitivity of cancer cells in vitro, the colony formation assay (CFA), is unsuitable for high-throughput screening. We developed a new high-throughput screening method for determining radiation susceptibility. Fast and uniform irradiation of batches up to 30 microplates was achieved using a Perspex container and a clinically employed linear accelerator. The readout was done by automated counting of fluorescently stained nuclei using the Acumen eX3 laser scanning cytometer. Assay performance was compared to that of the CFA and the CellTiter-Blue homogeneous uniform-well cell viability assay. The assay was validated in a whole-genome siRNA library screening setting using PC-3 prostate cancer cells. On 4 different cancer cell lines, the automated cell counting assay produced radiation dose response curves that followed a linear-quadratic equation and that exhibited a better correlation to the results of the CFA than did the cell viability assay. Moreover, the cell counting assay could be used to detect radiosensitization by silencing DNA-PKcs or by adding caffeine. In a high-throughput screening setting, using 4 Gy irradiated and control PC-3 cells, the effects of DNA-PKcs siRNA and non-targeting control siRNA could be clearly discriminated. We developed a simple assay for radiation susceptibility that can be used for high-throughput screening. This will aid

  18. RNA surveillance via nonsense-mediated mRNA decay is crucial for longevity in daf-2/insulin/IGF-1 mutant C. elegans.

    Science.gov (United States)

    Son, Heehwa G; Seo, Mihwa; Ham, Seokjin; Hwang, Wooseon; Lee, Dongyeop; An, Seon Woo A; Artan, Murat; Seo, Keunhee; Kaletsky, Rachel; Arey, Rachel N; Ryu, Youngjae; Ha, Chang Man; Kim, Yoon Ki; Murphy, Coleen T; Roh, Tae-Young; Nam, Hong Gil; Lee, Seung-Jae V

    2017-03-09

    Long-lived organisms often feature more stringent protein and DNA quality control. However, whether RNA quality control mechanisms, such as nonsense-mediated mRNA decay (NMD), which degrades both abnormal as well as some normal transcripts, have a role in organismal aging remains unexplored. Here we show that NMD mediates longevity in C. elegans strains with mutations in daf-2/insulin/insulin-like growth factor 1 receptor. We find that daf-2 mutants display enhanced NMD activity and reduced levels of potentially aberrant transcripts. NMD components, including smg-2/UPF1, are required to achieve the longevity of several long-lived mutants, including daf-2 mutant worms. NMD in the nervous system of the animals is particularly important for RNA quality control to promote longevity. Furthermore, we find that downregulation of yars-2/tyrosyl-tRNA synthetase, an NMD target transcript, by daf-2 mutations contributes to longevity. We propose that NMD-mediated RNA surveillance is a crucial quality control process that contributes to longevity conferred by daf-2 mutations.

  19. Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity.

    Science.gov (United States)

    Fajardo, Teodoro; Sung, Po-Yu; Roy, Polly

    2015-12-01

    Bluetongue virus (BTV) causes hemorrhagic disease in economically important livestock. The BTV genome is organized into ten discrete double-stranded RNA molecules (S1-S10) which have been suggested to follow a sequential packaging pathway from smallest to largest segment during virus capsid assembly. To substantiate and extend these studies, we have investigated the RNA sorting and packaging mechanisms with a new experimental approach using inhibitory oligonucleotides. Putative packaging signals present in the 3'untranslated regions of BTV segments were targeted by a number of nuclease resistant oligoribonucleotides (ORNs) and their effects on virus replication in cell culture were assessed. ORNs complementary to the 3' UTR of BTV RNAs significantly inhibited virus replication without affecting protein synthesis. Same ORNs were found to inhibit complex formation when added to a novel RNA-RNA interaction assay which measured the formation of supramolecular complexes between and among different RNA segments. ORNs targeting the 3'UTR of BTV segment 10, the smallest RNA segment, were shown to be the most potent and deletions or substitution mutations of the targeted sequences diminished the RNA complexes and abolished the recovery of viable viruses using reverse genetics. Cell-free capsid assembly/RNA packaging assay also confirmed that the inhibitory ORNs could interfere with RNA packaging and further substitution mutations within the putative RNA packaging sequence have identified the recognition sequence concerned. Exchange of 3'UTR between segments have further demonstrated that RNA recognition was segment specific, most likely acting as part of the secondary structure of the entire genomic segment. Our data confirm that genome packaging in this segmented dsRNA virus occurs via the formation of supramolecular complexes formed by the interaction of specific sequences located in the 3' UTRs. Additionally, the inhibition of packaging in-trans with inhibitory ORNs

  20. [Analysis of mitochondrial 12S rRNA and tRNA(Ser(UCN)) genes in patients with nonsyndromic sensorineural hearing loss from various regions of Russia].

    Science.gov (United States)

    Dzhemileva, L U; Posukh, O L; Tazetdinov, A M; Barashkov, N A; Zhuravskiĭ, S G; Ponidelko, S N; Markova, T G; Tadinova, V N; Fedorova, S A; Maksimova, N R; Khusnutdinova, E K

    2009-07-01

    Mitochondrial DNA (mtDNA) mutations play an important role in etiology of hereditary hearing loss. In various regions of the world, patients suffer from nonsyndromic sensorineural hearing loss initiated by aminoglycoside antibiotics. Mutations that had been shown as pathogenetically important for hearing function disturbance were identified in mitochondrial 12S rRNA and tRNA(Ser(UCN)) genes while pathogenic role of several DNA sequences requires additional studies. This work presents the results of studying the spectrum of mutations and polymorphic variations in mtDNA genes 12S rRNA and tRNA(Ser(UGN)) in 410 patients with nonsyndromal sensoneural hearing impairment/loss from the Volga Ural region, St Petersburg, Yakutia, and Altai and in 520 individuals with normal hearing, which represent several ethnic groups (Russians, Tatars, Bashkirs, Yakuts, Altaians) residing in the Russian Federation. Pathogenetically significant mutation A1555G (12S rRNA) was found in two families (from Yakutia and St Peresburg) with hearing loss, probably caused by treatment with aminoglucosides, and in the population sample of Yakuts with a frequency of 0.83%. Further research is needed to confirm the role in hearing impairment of mutations 961insC, 961insC(n), 961delTinsC(n), T961G, T1095C (12S rRNA) and G7444A, A7445C (tRNA(Ser(UGN revealed in the patients. In addition, in the patients and the population groups, polymorphic mt DNA variants were detected, which are characteristic also of other Eurasian populations both in spectrum and frequency.

  1. Poliovirus Polymerase Leu420 Facilitates RNA Recombination and Ribavirin Resistance

    Science.gov (United States)

    Kempf, Brian J.; Peersen, Olve B.

    2016-01-01

    ABSTRACT RNA recombination is important in the formation of picornavirus species groups and the ongoing evolution of viruses within species groups. In this study, we examined the structure and function of poliovirus polymerase, 3Dpol, as it relates to RNA recombination. Recombination occurs when nascent RNA products exchange one viral RNA template for another during RNA replication. Because recombination is a natural aspect of picornavirus replication, we hypothesized that some features of 3Dpol may exist, in part, to facilitate RNA recombination. Furthermore, we reasoned that alanine substitution mutations that disrupt 3Dpol-RNA interactions within the polymerase elongation complex might increase and/or decrease the magnitudes of recombination. We found that an L420A mutation in 3Dpol decreased the frequency of RNA recombination, whereas alanine substitutions at other sites in 3Dpol increased the frequency of recombination. The 3Dpol Leu420 side chain interacts with a ribose in the nascent RNA product 3 nucleotides from the active site of the polymerase. Notably, the L420A mutation that reduced recombination also rendered the virus more susceptible to inhibition by ribavirin, coincident with the accumulation of ribavirin-induced G→A and C→U mutations in viral RNA. We conclude that 3Dpol Leu420 is critically important for RNA recombination and that RNA recombination contributes to ribavirin resistance. IMPORTANCE Recombination contributes to the formation of picornavirus species groups and the emergence of circulating vaccine-derived polioviruses (cVDPVs). The recombinant viruses that arise in nature are occasionally more fit than either parental strain, especially when the two partners in recombination are closely related, i.e., members of characteristic species groups, such as enterovirus species groups A to H or rhinovirus species groups A to C. Our study shows that RNA recombination requires conserved features of the viral polymerase. Furthermore, a

  2. Is high pressure liquid chromatography an effective screening tool for characterization of molecular defects in hemoglobinopathies?

    Directory of Open Access Journals (Sweden)

    Nikhil Moorchung

    2013-01-01

    Full Text Available Introduction: Hemoglobinopathies constitute entities that are generated by either abnormal hemoglobin or thalassemias. high pressure liquid chromatography (HPLC is one of the best methods for screening and detection of various hemoglobinopathies but it has intrinsic interpretive problems. The study was designed to evaluate the different mutations seen in cases of hemoglobinopathies and compare the same with screening tests. Materials and Methods: 68 patients of hemoglobinopathies were screened by HPLC. Mutation studies in the beta globin gene was performed using the polymerase chain reaction (PCR-based allele-specific Amplification Refractory Mutation System (ARMS. Molecular analysis for the sickle cell mutation was done by standard methods. Results: The IVS 1/5 mutation was the commonest mutation seen and it was seen in 26 (38.23% of the cases. This was followed by the IVS 1/1, codon 41/42, codon 8/9, del 22 mutation, codon 15 mutation and the -619 bp deletion. No mutation was seen in eight cases. There was a 100% concordance between the sickle cell trait as diagnosed by HPLC and genetic testing. Discussion and Conclusion: Our study underlies the importance of molecular testing in all cases of hemoglobinopathies. Although HPLC is a useful screening tool, molecular testing is very useful in accurately diagnosing the mutations. Molecular testing is especially applicable in cases with an abnormal hemoglobin (HbD, HbE and HbS because there may be a concomitant inheritance of a beta thalassemia mutation. Molecular testing is the gold standard when it comes to the diagnosis of hemoglobinopathies.

  3. Mutation screening and association analysis of six candidate genes for autism on chromosome 7q

    DEFF Research Database (Denmark)

    Bonora, E.; Lamb, J.A.; Barnby, G.

    2005-01-01

    in the genes CUTL1, LAMB1 and PTPRZ1. Analysis of genetic variants provided evidence for association with autism for one of the new missense changes identified in LAMB1; this effect was stronger in a subgroup of affected male sibling pair families, implying a possible specific sex-related effect......Genetic studies have provided evidence for an autism susceptibility locus (AUTS1) on chromosome 7q. Screening for mutations in six genes mapping to 7q, CUTL1, SRPK2, SYPL, LAMB1, NRCAM and PTPRZ1 in 48 unrelated individuals with autism led to the identification of several new coding variants...

  4. The evolution of RNA viruses: A population genetics view

    Science.gov (United States)

    Moya, Andrés; Elena, Santiago F.; Bracho, Alma; Miralles, Rosario; Barrio, Eladio

    2000-01-01

    RNA viruses are excellent experimental models for studying evolution under the theoretical framework of population genetics. For a proper justification of this thesis we have introduced some properties of RNA viruses that are relevant for studying evolution. On the other hand, population genetics is a reductionistic theory of evolution. It does not consider or make simplistic assumptions on the transformation laws within and between genotypic and phenotypic spaces. However, such laws are minimized in the case of RNA viruses because the phenotypic space maps onto the genotypic space in a much more linear way than on higher DNA-based organisms. Under experimental conditions, we have tested the role of deleterious and beneficial mutations in the degree of adaptation of vesicular stomatitis virus (VSV), a nonsegmented virus of negative strand. We also have studied how effective population size, initial genetic variability in populations, and environmental heterogeneity shapes the impact of mutations in the evolution of vesicular stomatitis virus. Finally, in an integrative attempt, we discuss pros and cons of the quasispecies theory compared with classic population genetics models for haploid organisms to explain the evolution of RNA viruses. PMID:10860958

  5. Rapid NMR screening of RNA secondary structure and binding

    International Nuclear Information System (INIS)

    Helmling, Christina; Keyhani, Sara; Sochor, Florian; Fürtig, Boris; Hengesbach, Martin; Schwalbe, Harald

    2015-01-01

    Determination of RNA secondary structures by NMR spectroscopy is a useful tool e.g. to elucidate RNA folding space or functional aspects of regulatory RNA elements. However, current approaches of RNA synthesis and preparation are usually time-consuming and do not provide analysis with single nucleotide precision when applied for a large number of different RNA sequences. Here, we significantly improve the yield and 3′ end homogeneity of RNA preparation by in vitro transcription. Further, by establishing a native purification procedure with increased throughput, we provide a shortcut to study several RNA constructs simultaneously. We show that this approach yields μmol quantities of RNA with purities comparable to PAGE purification, while avoiding denaturation of the RNA

  6. Rapid NMR screening of RNA secondary structure and binding

    Energy Technology Data Exchange (ETDEWEB)

    Helmling, Christina; Keyhani, Sara; Sochor, Florian; Fürtig, Boris; Hengesbach, Martin; Schwalbe, Harald, E-mail: schwalbe@nmr.uni-frankfurt.de [Johann Wolfgang Goethe-Universität, Institut für Organische Chemie und Chemische Biologie, Center for Biomolecular Magnetic Resonance (BMRZ) (Germany)

    2015-09-15

    Determination of RNA secondary structures by NMR spectroscopy is a useful tool e.g. to elucidate RNA folding space or functional aspects of regulatory RNA elements. However, current approaches of RNA synthesis and preparation are usually time-consuming and do not provide analysis with single nucleotide precision when applied for a large number of different RNA sequences. Here, we significantly improve the yield and 3′ end homogeneity of RNA preparation by in vitro transcription. Further, by establishing a native purification procedure with increased throughput, we provide a shortcut to study several RNA constructs simultaneously. We show that this approach yields μmol quantities of RNA with purities comparable to PAGE purification, while avoiding denaturation of the RNA.

  7. The prevalence of BRCA1 mutations among young women with triple-negative breast cancer

    International Nuclear Information System (INIS)

    Young, SR; DeSai, Damini; Zandvakili, Inuk; Royer, Robert; Li, Song; Narod, Steven A; Pilarski, Robert T; Donenberg, Talia; Shapiro, Charles; Hammond, Lyn S; Miller, Judith; Brooks, Karen A; Cohen, Stephanie; Tenenholz, Beverly

    2009-01-01

    Molecular screening for BRCA1 and BRCA2 mutations is now an established component of risk evaluation and management of familial breast cancer. Features of hereditary breast cancer include an early age-of-onset and over-representation of the 'triple-negative' phenotype (negative for estrogen-receptor, progesterone-receptor and HER2). The decision to offer genetic testing to a breast cancer patient is usually based on her family history, but in the absence of a family history of cancer, some women may qualify for testing based on the age-of-onset and/or the pathologic features of the breast cancer. We studied 54 women who were diagnosed with high-grade, triple-negative invasive breast cancer at or before age 40. These women were selected for study because they had little or no family history of breast or ovarian cancer and they did not qualify for genetic testing using conventional family history criteria. BRCA1 screening was performed using a combination of fluorescent multiplexed-PCR analysis, BRCA1 exon-13 6 kb duplication screening, the protein truncation test (PTT) and fluorescent multiplexed denaturing gradient gel electrophoresis (DGGE). All coding exons of BRCA1 were screened. The two large exons of BRCA2 were also screened using PTT. All mutations were confirmed with direct sequencing. Five deleterious BRCA1 mutations and one deleterious BRCA2 mutation were identified in the 54 patients with early-onset, triple-negative breast cancer (11%). Women with early-onset triple-negative breast cancer are candidates for genetic testing for BRCA1, even in the absence of a family history of breast or ovarian cancer

  8. Should all patients with hyperparathyroidism be screened for a CDC73 mutation?

    Directory of Open Access Journals (Sweden)

    Caroline Bachmeier

    2018-03-01

    Full Text Available Primary hyperparathyroidism (PH is a common endocrine abnormality and may occur as part of a genetic syndrome. Inactivating mutations of the tumour suppressor gene CDC73 have been identified as accounting for a large percentage of hyperparathyroidism-jaw tumour syndrome (HPT-JT cases and to a lesser degree account for familial isolated hyperparathyroidism (FIHP cases. Reports of CDC73 whole gene deletions are exceedingly rare. We report the case of a 39 year-old woman with PH secondary to a parathyroid adenoma associated with a large chromosomal deletion (2.5 Mb encompassing the entire CDC73 gene detected years after parathyroidectomy. This case highlights the necessity to screen young patients with hyperparathyroidism for an underlying genetic aetiology. It also demonstrates that molecular testing for this disorder should contain techniques that can detect large deletions.

  9. A discontinuous RNA platform mediates RNA virus replication: building an integrated model for RNA-based regulation of viral processes.

    Directory of Open Access Journals (Sweden)

    Baodong Wu

    2009-03-01

    Full Text Available Plus-strand RNA viruses contain RNA elements within their genomes that mediate a variety of fundamental viral processes. The traditional view of these elements is that of local RNA structures. This perspective, however, is changing due to increasing discoveries of functional viral RNA elements that are formed by long-range RNA-RNA interactions, often spanning thousands of nucleotides. The plus-strand RNA genomes of tombusviruses exemplify this concept by possessing different long-range RNA-RNA interactions that regulate both viral translation and transcription. Here we report that a third fundamental tombusvirus process, viral genome replication, requires a long-range RNA-based interaction spanning approximately 3000 nts. In vivo and in vitro analyses suggest that the discontinuous RNA platform formed by the interaction facilitates efficient assembly of the viral RNA replicase. This finding has allowed us to build an integrated model for the role of global RNA structure in regulating the reproduction of a eukaryotic RNA virus, and the insights gained have extended our understanding of the multifunctional nature of viral RNA genomes.

  10. Novel somatic mutations in large granular lymphocytic leukemia affecting the STAT-pathway and T-cell activation

    International Nuclear Information System (INIS)

    Andersson, E I; Rajala, H L M; Eldfors, S; Ellonen, P; Olson, T; Jerez, A; Clemente, M J; Kallioniemi, O; Porkka, K; Heckman, C; Loughran, T P Jr; Maciejewski, J P; Mustjoki, S

    2013-01-01

    T-cell large granular lymphocytic (T-LGL) leukemia is a clonal disease characterized by the expansion of mature CD3+CD8+ cytotoxic T cells. It is often associated with autoimmune disorders and immune-mediated cytopenias. Our recent findings suggest that up to 40% of T-LGL patients harbor mutations in the STAT3 gene, whereas STAT5 mutations are present in 2% of patients. In order to identify putative disease-causing genetic alterations in the remaining T-LGL patients, we performed exome sequencing from three STAT mutation-negative patients and validated the findings in 113 large granular lymphocytic (LGL) leukemia patients. On average, 11 CD8+ LGL leukemia cell-specific high-confidence nonsynonymous somatic mutations were discovered in each patient. Interestingly, all patients had at least one mutation that affects either directly the STAT3-pathway (such as PTPRT) or T-cell activation (BCL11B, SLIT2 and NRP1). In all three patients, the STAT3 pathway was activated when studied by RNA expression or pSTAT3 analysis. Screening of the remaining 113 LGL leukemia patients did not reveal additional patients with same mutations. These novel mutations are potentially biologically relevant and represent rare genetic triggers for T-LGL leukemia, and are associated with similar disease phenotype as observed in patients with mutations in the STAT3 gene

  11. [Generalized neonatal screening based on laboratory tests].

    Science.gov (United States)

    Ardaillou, Raymond; Le Gall, Jean-Yves

    2006-11-01

    Implementation of a generalized screening program for neonatal diseases must obey precise rules. The disease must be severe, recognizable at an early stage, amenable to an effective treatment, detectable with a non expensive and widely applicable test; it must also be a significant public health problem. Subjects with positive results must be offered immediate treatment or prevention. All screening programs must be regularly evaluated. In France, since 1978, a national screening program has been organized by a private association ("Association française pour le dépistage et la prévention des handicaps de l'enfant") and supervised by the "Caisse nationale d'assurance maladie" and "Direction Générale de la Sante". Five diseases are now included in the screening program: phenylketonuria, hypothyroidism, congenital adrenal hyperplasia, cystic fibrosis and sickle cell disease (the latter only in at-risk newborns). Toxoplasmosis is a particular problem because only the children of mothers who were not tested during the pregnancy or who seroconverted are screened. Neonatal screening for phenylketonuria and hypothyrodism is unanimously recommended. Screening for congenital adrenal hyperplasia is approved in most countries. Cases of sickle cell disease and cystic fibrosis are more complex because--not all children who carry the mutations develop severe forms;--there is no curative treatment;--parents may become anxious, even though the phenotype is sometimes mild or even asymptomatic. Supporters of screening stress the benefits of early diagnosis (which extends the life expectancy of these children, particularly in the case of sickle cell disease), the fact that it opens up the possibility of prenatal screening of future pregnancies, and the utility of informing heterozygous carriers identified by familial screening. Neonatal screening for other diseases is under discussion. Indeed, technical advances such as tandem mass spectrometry make it possible to detect about 50

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

    Science.gov (United States)

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

    2017-01-22

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

  13. Identification of luteolin as enterovirus 71 and coxsackievirus A16 inhibitors through reporter viruses and cell viability-based screening.

    Science.gov (United States)

    Xu, Lin; Su, Weiheng; Jin, Jun; Chen, Jiawen; Li, Xiaojun; Zhang, Xuyuan; Sun, Meiyan; Sun, Shiyang; Fan, Peihu; An, Dong; Zhang, Huafei; Zhang, Xiguang; Kong, Wei; Ma, Tonghui; Jiang, Chunlai

    2014-07-17

    Hand, foot and mouth disease (HFMD) is a common pediatric illness mainly caused by infection with enterovirus 71 (EV71) and coxsackievirus A16 (CA16). The frequent HFMD outbreaks have become a serious public health problem. Currently, no vaccine or antiviral drug for EV71/CA16 infections has been approved. In this study, a two-step screening platform consisting of reporter virus-based assays and cell viability‑based assays was developed to identify potential inhibitors of EV71/CA16 infection. Two types of reporter viruses, a pseudovirus containing luciferase-encoding RNA replicons encapsidated by viral capsid proteins and a full-length reporter virus containing enhanced green fluorescent protein, were used for primary screening of 400 highly purified natural compounds. Thereafter, a cell viability-based secondary screen was performed for the identified hits to confirm their antiviral activities. Three compounds (luteolin, galangin, and quercetin) were identified, among which luteolin exhibited the most potent inhibition of viral infection. In the cell viability assay and plaque reduction assay, luteolin showed similar 50% effective concentration (EC50) values of about 10 μM. Luteolin targeted the post-attachment stage of EV71 and CA16 infection by inhibiting viral RNA replication. This study suggests that luteolin may serve as a lead compound to develop potent anti-EV71 and CA16 drugs.

  14. A web-based platform for virtual screening.

    Science.gov (United States)

    Watson, Paul; Verdonk, Marcel; Hartshorn, Michael J

    2003-09-01

    A fully integrated, web-based, virtual screening platform has been developed to allow rapid virtual screening of large numbers of compounds. ORACLE is used to store information at all stages of the process. The system includes a large database of historical compounds from high throughput screenings (HTS) chemical suppliers, ATLAS, containing over 3.1 million unique compounds with their associated physiochemical properties (ClogP, MW, etc.). The database can be screened using a web-based interface to produce compound subsets for virtual screening or virtual library (VL) enumeration. In order to carry out the latter task within ORACLE a reaction data cartridge has been developed. Virtual libraries can be enumerated rapidly using the web-based interface to the cartridge. The compound subsets can be seamlessly submitted for virtual screening experiments, and the results can be viewed via another web-based interface allowing ad hoc querying of the virtual screening data stored in ORACLE.

  15. An efficient method for the prediction of deleterious multiple-point mutations in the secondary structure of RNAs using suboptimal folding solutions

    Directory of Open Access Journals (Sweden)

    Barash Danny

    2008-04-01

    Full Text Available Abstract Background RNAmute is an interactive Java application which, given an RNA sequence, calculates the secondary structure of all single point mutations and organizes them into categories according to their similarity to the predicted structure of the wild type. The secondary structure predictions are performed using the Vienna RNA package. A more efficient implementation of RNAmute is needed, however, to extend from the case of single point mutations to the general case of multiple point mutations, which may often be desired for computational predictions alongside mutagenesis experiments. But analyzing multiple point mutations, a process that requires traversing all possible mutations, becomes highly expensive since the running time is O(nm for a sequence of length n with m-point mutations. Using Vienna's RNAsubopt, we present a method that selects only those mutations, based on stability considerations, which are likely to be conformational rearranging. The approach is best examined using the dot plot representation for RNA secondary structure. Results Using RNAsubopt, the suboptimal solutions for a given wild-type sequence are calculated once. Then, specific mutations are selected that are most likely to cause a conformational rearrangement. For an RNA sequence of about 100 nts and 3-point mutations (n = 100, m = 3, for example, the proposed method reduces the running time from several hours or even days to several minutes, thus enabling the practical application of RNAmute to the analysis of multiple-point mutations. Conclusion A highly efficient addition to RNAmute that is as user friendly as the original application but that facilitates the practical analysis of multiple-point mutations is presented. Such an extension can now be exploited prior to site-directed mutagenesis experiments by virologists, for example, who investigate the change of function in an RNA virus via mutations that disrupt important motifs in its secondary

  16. Screening of the USH1G gene among Spanish patients with Usher syndrome. Lack of mutations and evidence of a minor role in the pathogenesis of the syndrome.

    Science.gov (United States)

    Aller, Elena; Jaijo, Teresa; Beneyto, Magdalena; Nájera, Carmen; Morera, Constantino; Pérez-Garrigues, Herminio; Ayuso, Carmen; Millán, Jose

    2007-09-01

    The Usher syndrome (USH) is an autosomal recessive hereditary disorder characterized by the association of sensorineural hearing loss, retinitis pigmentosa (RP) and, in some cases, vestibular dysfunction. The USH1G gene, encoding SANS, has been found to cause both Usher syndrome type I and atypical Usher syndrome. 109 Spanish unrelated patients suffering from Usher syndrome type I, type II, type III and unclassified Usher syndrome were screened for mutations in this gene, but only eight different changes without a clear pathogenic effect have been detected. Based on these results as well as previous studies in other populations where mutational analysis of this gene has been carried out, one can conclude that USH1G has a minor involvement in Usher syndrome pathogenesis.

  17. Targeting miRNA-based medicines to cystic fibrosis airway epithelial cells using nanotechnology

    Directory of Open Access Journals (Sweden)

    McKiernan PJ

    2013-10-01

    Full Text Available Paul J McKiernan,2 Orla Cunninghamm,1,2 Catherine M Greenem,2 Sally-Ann Cryan1,31School of Pharmacy, Royal College of Surgeons in Ireland, 2Respiratory Research Division, Department of Medicine, Royal College of Surgeons in Ireland Education and Research Centre, Beaumont Hospital, 3Trinity Centre for Bioengineering, Trinity College Dublin, Dublin, IrelandAbstract: Cystic fibrosis (CF is an inherited disorder characterized by chronic airway inflammation. microRNAs (miRNAs are endogenous small RNAs which act on messenger (mRNA at a post transcriptional level, and there is a growing understanding that altered expression of miRNA is involved in the CF phenotype. Modulation of miRNA by replacement using miRNA mimics (premiRs presents a new therapeutic paradigm for CF, but effective and safe methods of delivery to the CF epithelium are limiting clinical translation. Herein, polymeric nanoparticles are investigated for delivery of miRNA mimics into CF airway epithelial cells, using miR-126 as a proof-of-concept premiR cargo to determine efficiency. Two polymers, polyethyleneimine (PEI and chitosan, were used to prepare miRNA nanomedicines, characterized for their size, surface (zeta potential, and RNA complexation efficiency, and screened for delivery and cytotoxicity in CFBE41o- (human F508del cystic fibrosis transmembrane conductance regulator bronchial epithelial cells using a novel high content analysis method. RNA extraction was carried out 24 hours post transfection, and miR-126 and TOM1 (target of Myb1 expression (a validated miR-126 target was assessed. Manufacture was optimized to produce small nanoparticles that effectively complexed miRNA. Using high content analysis, PEI-based nanoparticles were more effective than chitosan-based nanoparticles in facilitating uptake of miRNA into CFBE41o- cells and this was confirmed in miR-126 assays. PEI-premiR-126 nanoparticles at low nitrogen/phosphate (N/P ratios resulted in significant knockdown of

  18. High prevalence of exon 8 G533C mutation in apparently sporadic medullary thyroid carcinoma in Greece.

    Science.gov (United States)

    Sarika, H L; Papathoma, A; Garofalaki, M; Vasileiou, V; Vlassopoulou, B; Anastasiou, E; Alevizaki, M

    2012-12-01

    Genetic screening for ret mutation has become routine practice in the evaluation of medullary thyroid carcinoma (MTC). Approximately 25% of these tumours are familial, and they occur as components of the multiple endocrine neoplasia type 2 syndromes (MEN 2A and 2B) or familial MTC. In familial cases, the majority of mutations are found in exons 10, 11, 13, 14 or 15 of the ret gene. A rare mutation involving exon 8 (G533C) has recently been reported in familial cases of MTC in Brazil and Greece; some of these cases were originally thought to be sporadic. The aim of this study was to re-evaluate a series of sporadic cases of MTC, with negative family history, and screen them for germline mutations in exon 8. Genomic DNA was extracted from peripheral lymphocytes in 129 unrelated individuals who had previously been characterized as 'sporadic' based on the negative family history and negative screening for ret gene mutations. Samples were analysed in Applied Biosystems 7500 real-time PCR and confirmed by sequencing. The G533C exon 8 mutation was identified in 10 of 129 patients with sporadic MTC. Asymptomatic gene carriers were subsequently identified in other family members. In our study, we found that 7·75% patients with apparently sporadic MTC do carry G533C mutation involving exon 8 of ret. We feel that there is now a need to include exon 8 mutation screening in all patients diagnosed as sporadic MTC, in Greece. © 2012 Blackwell Publishing Ltd.

  19. High content image-based screening of a protease inhibitor library reveals compounds broadly active against Rift Valley fever virus and other highly pathogenic RNA viruses.

    Directory of Open Access Journals (Sweden)

    Rajini Mudhasani

    2014-08-01

    Full Text Available High content image-based screening was developed as an approach to test a protease inhibitor small molecule library for antiviral activity against Rift Valley fever virus (RVFV and to determine their mechanism of action. RVFV is the causative agent of severe disease of humans and animals throughout Africa and the Arabian Peninsula. Of the 849 compounds screened, 34 compounds exhibited ≥ 50% inhibition against RVFV. All of the hit compounds could be classified into 4 distinct groups based on their unique chemical backbone. Some of the compounds also showed broad antiviral activity against several highly pathogenic RNA viruses including Ebola, Marburg, Venezuela equine encephalitis, and Lassa viruses. Four hit compounds (C795-0925, D011-2120, F694-1532 and G202-0362, which were most active against RVFV and showed broad-spectrum antiviral activity, were selected for further evaluation for their cytotoxicity, dose response profile, and mode of action using classical virological methods and high-content imaging analysis. Time-of-addition assays in RVFV infections suggested that D011-2120 and G202-0362 targeted virus egress, while C795-0925 and F694-1532 inhibited virus replication. We showed that D011-2120 exhibited its antiviral effects by blocking microtubule polymerization, thereby disrupting the Golgi complex and inhibiting viral trafficking to the plasma membrane during virus egress. While G202-0362 also affected virus egress, it appears to do so by a different mechanism, namely by blocking virus budding from the trans Golgi. F694-1532 inhibited viral replication, but also appeared to inhibit overall cellular gene expression. However, G202-0362 and C795-0925 did not alter any of the morphological features that we examined and thus may prove to be good candidates for antiviral drug development. Overall this work demonstrates that high-content image analysis can be used to screen chemical libraries for new antivirals and to determine their

  20. 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.

  1. [In silico CRISPR-based sgRNA design].

    Science.gov (United States)

    Wang, Yuanli; Chuai, Guohui; Yan, Jifang; Shi, Lei; Liu, Qi

    2017-10-25

    CRISPR-based genome editing has been widely implemented in various cell types. In-silico single guide RNA (sgRNA) design is a key step for successful gene editing using CRISPR system. Continuing efforts are made to refine in-silico sgRNA design with high on-target efficacy and reduced off-target effects. In this paper, we summarize the present sgRNA design tools, and show that efficient in-silico models can be built that integrate current heterogeneous genome-editing data to derive unbiased sgRNA design rules and identify key features for improving sgRNA design. Our review shows that systematic comparisons and evaluation of on-target and off-target effects of sgRNA will allow more precise genome editing and gene therapies using the CRISPR system.

  2. Immunofluorescence-based screening identifies germ cell associated microRNA 302 as an antagonist to p63 expression

    DEFF Research Database (Denmark)

    Scheel, Andreas Hans Joachim; Beyer, Ulrike; Agami, Reuven

    2009-01-01

    The tumor suppressor homologue p63 is required for proper skin and limb development, but specific isoforms of it also act as a "guardian of the germline." To gain insight into the regulation of p63 expression, we performed immunofluorescence-based screening assays. Using a large collection of micro...

  3. 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/ .

  4. Mutations in ALDH1A3 represent a frequent cause of microphthalmia/anophthalmia in consanguineous families.

    Science.gov (United States)

    Abouzeid, Hana; Favez, Tatiana; Schmid, Angélique; Agosti, Céline; Youssef, Mohammed; Marzouk, Iman; El Shakankiry, Nihal; Bayoumi, Nader; Munier, Francis L; Schorderet, Daniel F

    2014-08-01

    Anophthalmia or microphthalmia (A/M), characterized by absent or small eye, can be unilateral or bilateral and represent developmental anomalies due to the mutations in several genes. Recently, mutations in aldehyde dehydrogenase family 1, member A3 (ALDH1A3) also known as retinaldehyde dehydrogenase 3, have been reported to cause A/M. Here, we screened a cohort of 75 patients with A/M and showed that mutations in ALDH1A3 occurred in six families. Based on this series, we estimate that mutations in ALDH1A3 represent a major cause of A/M in consanguineous families, and may be responsible for approximately 10% of the cases. Screening of this gene should be performed in a first line of investigation, together with SOX2. © 2014 WILEY PERIODICALS, INC.

  5. A novel point mutation within the EDA gene causes an exon dropping in mature RNA in Holstein Friesian cattle breed affected by X-linked anhidrotic ectodermal dysplasia

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    Pariset Lorraine

    2011-07-01

    Full Text Available Abstract Background X-linked anhidrotic ectodermal dysplasia is a disorder characterized by abnormal development of tissues and organs of ectodermal origin caused by mutations in the EDA gene. The bovine EDA gene encodes the ectodysplasin A, a membrane protein expressed in keratinocytes, hair follicles and sweat glands, which is involved in the interactions between cell and cell and/or cell and matrix. Four mutations causing ectodermal dysplasia in cattle have been described so far. Results We identified a new single nucleotide polymorphism (SNP at the 9th base of exon 8 in the EDA gene in two calves of Holstein Friesian cattle breed affected by ectodermal dysplasia. This SNP is located in the exonic splicing enhancer (ESEs recognized by SRp40 protein. As a consequence, the spliceosome machinery is no longer able to recognize the sequence as exonic and causes exon skipping. The mutation determines the deletion of the entire exon (131 bp in the RNA processing, causing a severe alteration of the protein structure and thus the disease. Conclusion We identified a mutation, never described before, that changes the regulation of alternative splicing in the EDA gene and causes ectodermal dysplasia in cattle. The analysis of the SNP allows the identification of carriers that can transmit the disease to the offspring. This mutation can thus be exploited for a rational and efficient selection of unequivocally healthy cows for breeding.

  6. Prevalence of human immunodeficiency virus RNA and antibody in first-time, lapsed, and repeat blood donations across five international regions and relative efficacy of alternative screening scenarios.

    Science.gov (United States)

    Bruhn, Roberta; Lelie, Nico; Custer, Brian; Busch, Michael; Kleinman, Steven

    2013-10-01

    Twenty-one blood organizations from five geographical regions provided HIV individual donation (ID)-NAT and serology data on 11,787,610 donations. Infections were classified as anti-HIV-/RNA+ window period (WP), anti-HIV+/RNA+ concordant positive (CP) or anti-HIV+/RNA- elite controller (EC). Residual risk and efficacy of several screening scenarios were estimated for first time, lapsed and repeat donations. WP residual risk estimates assumed a 50% infectious dose of 3.16 virions and a 50% detection limit of 2.7 HIV RNA copies/mL for ID-NAT and 10,000 copies/mL for p24Ag. Infectivity for CP (100%) and EC (2.2%) donations was estimated based on viral load distributions and 100-fold reduced infectivity by antibody neutralization as reported elsewhere. Efficacy was calculated as proportion of transmission risk removed from baseline (i.e. in absence of any screening). There was no significant difference in transmission risk between lapsed and repeat donations in any region. Risk was 3.8-fold higher in first time than combined lapsed/repeat donations in South Africa but not in other regions. Screening strategies were most efficacious at interdicting infectious transfusions in first time (98.7-99.8%) followed by lapsed (97.6-99.7%) and repeat (86.8-97.7%) donations in all regions combined. In each donor category the efficacy of ID-NAT alone (97.7-99.8%) was superior to that of minipool (MP)-NAT/anti-HIV (95.0-99.6%) and p24 Ag/anti-HIV (89.8-99.1%). Efficacy patterns were similar by donor/donation status in each region despite large differences in HIV prevalence and transmission risk. As similar data become available for HBV and HCV, this modeling may be useful in cost effectiveness analyses of alternative testing scenarios. © 2013 American Association of Blood Banks.

  7. The effect of genotypes and parent of origin on cancer risk and age of cancer development in PMS2 mutation carriers.

    Science.gov (United States)

    Suerink, Manon; van der Klift, Heleen M; Ten Broeke, Sanne W; Dekkers, Olaf M; Bernstein, Inge; Capellá Munar, Gabriel; Gomez Garcia, Encarna; Hoogerbrugge, Nicoline; Letteboer, Tom G W; Menko, Fred H; Lindblom, Annika; Mensenkamp, Arjen; Moller, Pal; van Os, Theo A; Rahner, Nils; Redeker, Bert J W; Olderode-Berends, M J W; Olderode, Maran; Spruijt, Liesbeth; Vos, Yvonne J; Wagner, Anja; Morreau, Hans; Hes, Frederik J; Vasen, Hans F A; Tops, Carli M; Wijnen, Juul T; Nielsen, Maartje

    2016-04-01

    Lynch syndrome (LS), a heritable disorder with an increased risk of primarily colorectal cancer (CRC) and endometrial cancer (EC), can be caused by mutations in the PMS2 gene. We wished to establish whether genotype and/or parent-of-origin effects (POE) explain (part of) the reported variability in severity of the phenotype. European PMS2 mutation carriers (n = 381) were grouped and compared based on RNA expression and whether the mutation was inherited paternally or maternally. Mutation carriers with loss of RNA expression (group 1) had a significantly lower age at CRC diagnosis (51.1 years vs. 60.0 years, P = 0.035) and a lower age at EC diagnosis (55.8 years vs. 61.0 years, P = 0.2, nonsignificant) compared with group 2 (retention of RNA expression). Furthermore, group 1 showed slightly higher, but nonsignificant, hazard ratios (HRs) for both CRC (HR: 1.31, P = 0.38) and EC (HR: 1.22, P = 0.72). No evidence for a significant parent-of-origin effect was found for either CRC or EC. PMS2 mutation carriers with retention of RNA expression developed CRC 9 years later than those with loss of RNA expression. If confirmed, this finding would justify a delay in surveillance for these cases. Cancer risk was not influenced by a parent-of-origin effect.Genet Med 18 4, 405-409.

  8. Differential Regulation of rRNA and tRNA Transcription from the rRNA-tRNA Composite Operon in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Hiraku Takada

    Full Text Available Escherichia coli contains seven rRNA operons, each consisting of the genes for three rRNAs (16S, 23S and 5S rRNA in this order and one or two tRNA genes in the spacer between 16S and 23S rRNA genes and one or two tRNA genes in the 3' proximal region. All of these rRNA and tRNA genes are transcribed from two promoters, P1 and P2, into single large precursors that are afterward processed to individual rRNAs and tRNAs by a set of RNases. In the course of Genomic SELEX screening of promoters recognized by RNA polymerase (RNAP holoenzyme containing RpoD sigma, a strong binding site was identified within 16S rRNA gene in each of all seven rRNA operons. The binding in vitro of RNAP RpoD holoenzyme to an internal promoter, referred to the promoter of riRNA (an internal RNA of the rRNA operon, within each 16S rRNA gene was confirmed by gel shift assay and AFM observation. Using this riRNA promoter within the rrnD operon as a representative, transcription in vitro was detected with use of the purified RpoD holoenzyme, confirming the presence of a constitutive promoter in this region. LacZ reporter assay indicated that this riRNA promoter is functional in vivo. The location of riRNA promoter in vivo as identified using a set of reporter plasmids agrees well with that identified in vitro. Based on transcription profile in vitro and Northern blot analysis in vivo, the majority of transcript initiated from this riRNA promoter was estimated to terminate near the beginning of 23S rRNA gene, indicating that riRNA leads to produce the spacer-coded tRNA. Under starved conditions, transcription of the rRNA operon is markedly repressed to reduce the intracellular level of ribosomes, but the levels of both riRNA and its processed tRNAGlu stayed unaffected, implying that riRNA plays a role in the continued steady-state synthesis of tRNAs from the spacers of rRNA operons. We then propose that the tRNA genes organized within the spacers of rRNA-tRNA composite operons

  9. Label-Free Platform for MicroRNA Detection Based on the Fluorescence Quenching of Positively Charged Gold Nanoparticles to Silver Nanoclusters.

    Science.gov (United States)

    Miao, Xiangmin; Cheng, Zhiyuan; Ma, Haiyan; Li, Zongbing; Xue, Ning; Wang, Po

    2018-01-16

    A novel strategy was developed for microRNA-155 (miRNA-155) detection based on the fluorescence quenching of positively charged gold nanoparticles [(+)AuNPs] to Ag nanoclusters (AgNCs). In the designed system, DNA-stabilized Ag nanoclusters (DNA/AgNCs) were introduced as fluorescent probes, and DNA-RNA heteroduplexes were formed upon the addition of target miRNA-155. Meanwhile, the (+)AuNPs could be electrostatically adsorbed on the negatively charged single-stranded DNA (ssDNA) or DNA-RNA heteroduplexes to quench the fluorescence signal. In the presence of duplex-specific nuclease (DSN), DNA-RNA heteroduplexes became a substrate for the enzymatic hydrolysis of the DNA strand to yield a fluorescence signal due to the diffusion of AgNCs away from (+)AuNPs. Under the optimal conditions, (+)AuNPs displayed very high quenching efficiency to AgNCs, which paved the way for ultrasensitive detection with a low detection limit of 33.4 fM. In particular, the present strategy demonstrated excellent specificity and selectivity toward the detection of target miRNA against control miRNAs, including mutated miRNA-155, miRNA-21, miRNA-141, let-7a, and miRNA-182. Moreover, the practical application value of the system was confirmed by the evaluation of the expression levels of miRNA-155 in clinical serum samples with satisfactory results, suggesting that the proposed sensing platform is promising for applications in disease diagnosis as well as the fundamental research of biochemistry.

  10. Progranulin mutation causes frontotemporal dementia in the Swedish Karolinska family.

    Science.gov (United States)

    Chiang, Huei-Hsin; Rosvall, Lina; Brohede, Jesper; Axelman, Karin; Björk, Behnosh F; Nennesmo, Inger; Robins, Tiina; Graff, Caroline

    2008-11-01

    Frontotemporal dementia (FTD) is a neurodegenerative disease characterized by cognitive impairment, language dysfunction, and/or changes in personality. Recently it has been shown that progranulin (GRN) mutations can cause FTD as well as other neurodegenerative phenotypes. DNA from 30 family members, of whom seven were diagnosed with FTD, in the Karolinska family was available for GRN sequencing. Fibroblast cell mRNA from one affected family member and six control individuals was available for relative quantitative real-time polymerase chain reaction to investigate the effect of the mutation. Furthermore, the cDNA of an affected individual was sequenced. Clinical and neuropathologic findings of a previously undescribed family branch are presented. A frameshift mutation in GRN (g.102delC) was detected in all affected family members and absent in four unaffected family members older than 70 years. Real-time polymerase chain reaction data showed an approximately 50% reduction of GRN fibroblast mRNA in an affected individual. The mutated mRNA transcripts were undetectable by cDNA sequencing. Segregation and RNA analyses showed that the g.102delC mutation, previously reported, causes FTD in the Karolinska family. Our findings add further support to the significance of GRN in FTD etiology and the presence of modifying genes, which emphasize the need for further studies into the mechanisms of clinical heterogeneity. However, the results already call for attention to the complexity of predictive genetic testing of GRN mutations.

  11. A streptomycin resistance marker in H. parasuis based on site-directed mutations in rpsL gene to perform unmarked in-frame mutations and to verify natural transformation

    Directory of Open Access Journals (Sweden)

    Ke Dai

    2018-01-01

    Full Text Available Haemophilus parasuis is a member of the family Pasteurellaceae and a major causative agent of Glässer’s disease. This bacterium is normally a benign swine commensal but may become a deadly pathogen upon penetration into multiple tissues, contributing to severe lesions in swine. We have established a successive natural transformation-based markerless mutation system in this species. However, the two-step mutation system requires screening of natural competent cells, and cannot delete genes which regulate natural competence per se. In this study, we successfully obtained streptomycin-resistant derivatives from H. parasuis wild type strain SC1401 by using ethyl methane sulfonate (EMS, CH3SO2OC2H5. Upon sequencing and site-directed mutations, we uncovered that the EMS-induced point mutation in rpsL at codon 43rd (AAA → AGA; K43R or at 88th (AAA → AGA; K88R confers a much higher streptomycin resistance than clinical isolates. We have applied the streptomycin resistance marker as a positive selection marker to perform homologous recombination through conjugation and successfully generated a double unmarked in-frame targeted mutant 1401D88△tfox△arcA. Combined with a natural transformation-based knockout system and this genetic technique, multiple deletion mutants or attenuated strains of H. parasuis can be easily constructed. Moreover, the mutant genetic marker rpsL and streptomycin resistant phenotypes can serve as an effective tool to select naturally competent strains, and to verify natural transformation quantitatively.

  12. Novel NTRK1 mutations cause hereditary sensory and autonomic neuropathy type IV: demonstration of a founder mutation in the Turkish population.

    Science.gov (United States)

    Tüysüz, Beyhan; Bayrakli, Fatih; DiLuna, Michael L; Bilguvar, Kaya; Bayri, Yasar; Yalcinkaya, Cengiz; Bursali, Aysegul; Ozdamar, Elif; Korkmaz, Baris; Mason, Christopher E; Ozturk, Ali K; Lifton, Richard P; State, Matthew W; Gunel, Murat

    2008-05-01

    Hereditary sensory and autonomic neuropathy type IV (HSAN IV), or congenital insensitivity to pain with anhidrosis, is an autosomal recessive disorder characterized by insensitivity to noxious stimuli, anhidrosis from deinnervated sweat glands, and delayed mental and motor development. Mutations in the neurotrophic tyrosine kinase receptor type 1 (NTRK1), a receptor in the neurotrophin signaling pathway phosphorylated in response to nerve growth factor, are associated with this disorder. We identified six families from Northern Central Turkey with HSAN IV. We screened the NTRK1 gene for mutations in these families. Microsatellite and single nucleotide polymorphism (SNP) markers on the Affymetrix 250K chip platform were used to determine the haplotypes for three families harboring the same mutation. Screening for mutations in the NTRK1 gene demonstrated one novel frameshift mutation, two novel nonsense mutations, and three unrelated kindreds with the same splice-site mutation. Genotyping of the three families with the identical splice-site mutation revealed that they share the same haplotype. This report broadens the spectrum of mutations in NTRK1 that cause HSAN IV and demonstrates a founder mutation in the Turkish population.

  13. Fluorescence-based high-throughput screening of dicer cleavage activity

    Czech Academy of Sciences Publication Activity Database

    Podolská, Kateřina; Sedlák, David; Bartůněk, Petr; Svoboda, Petr

    2014-01-01

    Roč. 19, č. 3 (2014), s. 417-426 ISSN 1087-0571 R&D Projects: GA ČR GA13-29531S; GA MŠk(CZ) LC06077; GA MŠk LM2011022 Grant - others:EMBO(DE) 1483 Institutional support: RVO:68378050 Keywords : Dicer * siRNA * high-throughput screening Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.423, year: 2014

  14. Sequence-specific inhibition of Dicer measured with a force-based microarray for RNA ligands.

    Science.gov (United States)

    Limmer, Katja; Aschenbrenner, Daniela; Gaub, Hermann E

    2013-04-01

    Malfunction of protein translation causes many severe diseases, and suitable correction strategies may become the basis of effective therapies. One major regulatory element of protein translation is the nuclease Dicer that cuts double-stranded RNA independently of the sequence into pieces of 19-22 base pairs starting the RNA interference pathway and activating miRNAs. Inhibiting Dicer is not desirable owing to its multifunctional influence on the cell's gene regulation. Blocking specific RNA sequences by small-molecule binding, however, is a promising approach to affect the cell's condition in a controlled manner. A label-free assay for the screening of site-specific interference of small molecules with Dicer activity is thus needed. We used the Molecular Force Assay (MFA), recently developed in our lab, to measure the activity of Dicer. As a model system, we used an RNA sequence that forms an aptamer-binding site for paromomycin, a 615-dalton aminoglycoside. We show that Dicer activity is modulated as a function of concentration and incubation time: the addition of paromomycin leads to a decrease of Dicer activity according to the amount of ligand. The measured dissociation constant of paromomycin to its aptamer was found to agree well with literature values. The parallel format of the MFA allows a large-scale search and analysis for ligands for any RNA sequence.

  15. MicroRNA-181a* Targets Nanog in a Subpopulation of CD34+ Cells Isolated From Peripheral Blood

    Directory of Open Access Journals (Sweden)

    Paul J Mintz

    2012-01-01

    Full Text Available Exploiting the properties of stem cells by microRNA (miRNA profiling offers an attractive approach to identify new regulators of stem cell fate. Although numerous miRNA have been screened from hematopoietic stem cells (HSC, the targets corresponding to many of these miRNA have not yet been fully elucidated. By miRNA profiling in a subpopulation of CD34+ cells isolated from peripheral blood, we have identified eight clusters of miRNA that were differentially expressed. Further analysis of one of the clusters by bioinformatics revealed that a miRNA, miR-181a*, which is highly expressed in the adherent CD34+ cells, affects the expression levels of Nanog, a stem cell surrogate marker. We show specifically by reporter assay and mutational analysis that miR-181a* targets a seedless 3′ compensatory site in the 3′UTR of Nanog and affects gene expression. We demonstrate that inhibiting miR-181a* upregulates the Nanog expression level, in addition to an increase in alkaline phosphatase activity. Our studies suggest that miR-181a* may be important in controlling the expression level of Nanog in a subpopulation of CD34+ cells.

  16. A Conserved Target Site in HIV-1 Gag RNA is Accessible to Inhibition by Both an HDV Ribozyme and a Short Hairpin RNA

    Directory of Open Access Journals (Sweden)

    Robert J Scarborough

    2014-01-01

    Full Text Available Antisense-based molecules targeting HIV-1 RNA have the potential to be used as part of gene or drug therapy to treat HIV-1 infection. In this study, HIV-1 RNA was screened to identify more conserved and accessible target sites for ribozymes based on the hepatitis delta virus motif. Using a quantitative screen for effects on HIV-1 production, we identified a ribozyme targeting a highly conserved site in the Gag coding sequence with improved inhibitory potential compared to our previously described candidates targeting the overlapping Tat/Rev coding sequence. We also demonstrate that this target site is highly accessible to short hairpin directed RNA interference, suggesting that it may be available for the binding of antisense RNAs with different modes of action. We provide evidence that this target site is structurally conserved in diverse viral strains and that it is sufficiently different from the human transcriptome to limit off-target effects from antisense therapies. We also show that the modified hepatitis delta virus ribozyme is more sensitive to a mismatch in its target site compared to the short hairpin RNA. Overall, our results validate the potential of a new target site in HIV-1 RNA to be used for the development of antisense therapies.

  17. Hybridisation-based resequencing of 17 X-linked intellectual disability genes in 135 patients reveals novel mutations in ATRX, SLC6A8 and PQBP1

    Science.gov (United States)

    Jensen, Lars R; Chen, Wei; Moser, Bettina; Lipkowitz, Bettina; Schroeder, Christopher; Musante, Luciana; Tzschach, Andreas; Kalscheuer, Vera M; Meloni, Ilaria; Raynaud, Martine; van Esch, Hilde; Chelly, Jamel; de Brouwer, Arjan P M; Hackett, Anna; van der Haar, Sigrun; Henn, Wolfram; Gecz, Jozef; Riess, Olaf; Bonin, Michael; Reinhardt, Richard; Ropers, Hans-Hilger; Kuss, Andreas W

    2011-01-01

    X-linked intellectual disability (XLID), also known as X-linked mental retardation, is a highly genetically heterogeneous condition for which mutations in >90 different genes have been identified. In this study, we used a custom-made sequencing array based on the Affymetrix 50k platform for mutation screening in 17 known XLID genes in patients from 135 families and found eight single-nucleotide changes that were absent in controls. For four mutations affecting ATRX (p.1761M>T), PQBP1 (p.155R>X) and SLC6A8 (p.390P>L and p.477S>L), we provide evidence for a functional involvement of these changes in the aetiology of intellectual disability. PMID:21267006

  18. Molecular methods for the detection of mutations.

    Science.gov (United States)

    Monteiro, C; Marcelino, L A; Conde, A R; Saraiva, C; Giphart-Gassler, M; De Nooij-van Dalen, A G; Van Buuren-van Seggelen, V; Van der Keur, M; May, C A; Cole, J; Lehmann, A R; Steinsgrimsdottir, H; Beare, D; Capulas, E; Armour, J A

    2000-01-01

    We report the results of a collaborative study aimed at developing reliable, direct assays for mutation in human cells. The project used common lymphoblastoid cell lines, both with and without mutagen treatment, as a shared resource to validate the development of new molecular methods for the detection of low-level mutations in the presence of a large excess of normal alleles. As the "gold standard, " hprt mutation frequencies were also measured on the same samples. The methods under development included i) the restriction site mutation (RSM) assay, in which mutations lead to the destruction of a restriction site; ii) minisatellite length-change mutation, in which mutations lead to alleles containing new numbers of tandem repeat units; iii) loss of heterozygosity for HLA epitopes, in which antibodies can be used to direct selection for mutant cells; iv) multiple fluorescence-based long linker arm nucleotides assay (mf-LLA) technology, for the detection of substitutional mutations; v) detection of alterations in the TP53 locus using a (CA) array as the target for the screening; and vi) PCR analysis of lymphocytes for the presence of the BCL2 t(14:18) translocation. The relative merits of these molecular methods are discussed, and a comparison made with more "traditional" methods.

  19. Differential evolution enhanced with multiobjective sorting-based mutation operators.

    Science.gov (United States)

    Wang, Jiahai; Liao, Jianjun; Zhou, Ying; Cai, Yiqiao

    2014-12-01

    Differential evolution (DE) is a simple and powerful population-based evolutionary algorithm. The salient feature of DE lies in its mutation mechanism. Generally, the parents in the mutation operator of DE are randomly selected from the population. Hence, all vectors are equally likely to be selected as parents without selective pressure at all. Additionally, the diversity information is always ignored. In order to fully exploit the fitness and diversity information of the population, this paper presents a DE framework with multiobjective sorting-based mutation operator. In the proposed mutation operator, individuals in the current population are firstly sorted according to their fitness and diversity contribution by nondominated sorting. Then parents in the mutation operators are proportionally selected according to their rankings based on fitness and diversity, thus, the promising individuals with better fitness and diversity have more opportunity to be selected as parents. Since fitness and diversity information is simultaneously considered for parent selection, a good balance between exploration and exploitation can be achieved. The proposed operator is applied to original DE algorithms, as well as several advanced DE variants. Experimental results on 48 benchmark functions and 12 real-world application problems show that the proposed operator is an effective approach to enhance the performance of most DE algorithms studied.

  20. Exonic Splicing Mutations Are More Prevalent than Currently Estimated and Can Be Predicted by Using In Silico Tools

    Science.gov (United States)

    Soukarieh, Omar; Gaildrat, Pascaline; Hamieh, Mohamad; Drouet, Aurélie; Baert-Desurmont, Stéphanie; Frébourg, Thierry; Tosi, Mario; Martins, Alexandra

    2016-01-01

    The identification of a causal mutation is essential for molecular diagnosis and clinical management of many genetic disorders. However, even if next-generation exome sequencing has greatly improved the detection of nucleotide changes, the biological interpretation of most exonic variants remains challenging. Moreover, particular attention is typically given to protein-coding changes often neglecting the potential impact of exonic variants on RNA splicing. Here, we used the exon 10 of MLH1, a gene implicated in hereditary cancer, as a model system to assess the prevalence of RNA splicing mutations among all single-nucleotide variants identified in a given exon. We performed comprehensive minigene assays and analyzed patient’s RNA when available. Our study revealed a staggering number of splicing mutations in MLH1 exon 10 (77% of the 22 analyzed variants), including mutations directly affecting splice sites and, particularly, mutations altering potential splicing regulatory elements (ESRs). We then used this thoroughly characterized dataset, together with experimental data derived from previous studies on BRCA1, BRCA2, CFTR and NF1, to evaluate the predictive power of 3 in silico approaches recently described as promising tools for pinpointing ESR-mutations. Our results indicate that ΔtESRseq and ΔHZEI-based approaches not only discriminate which variants affect splicing, but also predict the direction and severity of the induced splicing defects. In contrast, the ΔΨ-based approach did not show a compelling predictive power. Our data indicates that exonic splicing mutations are more prevalent than currently appreciated and that they can now be predicted by using bioinformatics methods. These findings have implications for all genetically-caused diseases. PMID:26761715

  1. Exonic Splicing Mutations Are More Prevalent than Currently Estimated and Can Be Predicted by Using In Silico Tools.

    Directory of Open Access Journals (Sweden)

    Omar Soukarieh

    2016-01-01

    Full Text Available The identification of a causal mutation is essential for molecular diagnosis and clinical management of many genetic disorders. However, even if next-generation exome sequencing has greatly improved the detection of nucleotide changes, the biological interpretation of most exonic variants remains challenging. Moreover, particular attention is typically given to protein-coding changes often neglecting the potential impact of exonic variants on RNA splicing. Here, we used the exon 10 of MLH1, a gene implicated in hereditary cancer, as a model system to assess the prevalence of RNA splicing mutations among all single-nucleotide variants identified in a given exon. We performed comprehensive minigene assays and analyzed patient's RNA when available. Our study revealed a staggering number of splicing mutations in MLH1 exon 10 (77% of the 22 analyzed variants, including mutations directly affecting splice sites and, particularly, mutations altering potential splicing regulatory elements (ESRs. We then used this thoroughly characterized dataset, together with experimental data derived from previous studies on BRCA1, BRCA2, CFTR and NF1, to evaluate the predictive power of 3 in silico approaches recently described as promising tools for pinpointing ESR-mutations. Our results indicate that ΔtESRseq and ΔHZEI-based approaches not only discriminate which variants affect splicing, but also predict the direction and severity of the induced splicing defects. In contrast, the ΔΨ-based approach did not show a compelling predictive power. Our data indicates that exonic splicing mutations are more prevalent than currently appreciated and that they can now be predicted by using bioinformatics methods. These findings have implications for all genetically-caused diseases.

  2. Murine knockin model for progranulin-deficient frontotemporal dementia with nonsense-mediated mRNA decay.

    Science.gov (United States)

    Nguyen, Andrew D; Nguyen, Thi A; Zhang, Jiasheng; Devireddy, Swathi; Zhou, Ping; Karydas, Anna M; Xu, Xialian; Miller, Bruce L; Rigo, Frank; Ferguson, Shawn M; Huang, Eric J; Walther, Tobias C; Farese, Robert V

    2018-03-20

    Frontotemporal dementia (FTD) is the most common neurodegenerative disorder in individuals under age 60 and has no treatment or cure. Because many cases of FTD result from GRN nonsense mutations, an animal model for this type of mutation is highly desirable for understanding pathogenesis and testing therapies. Here, we generated and characterized Grn R493X knockin mice, which model the most common human GRN mutation, a premature stop codon at arginine 493 (R493X). Homozygous Grn R493X mice have markedly reduced Grn mRNA levels, lack detectable progranulin protein, and phenocopy Grn knockout mice, with CNS microgliosis, cytoplasmic TDP-43 accumulation, reduced synaptic density, lipofuscinosis, hyperinflammatory macrophages, excessive grooming behavior, and reduced survival. Inhibition of nonsense-mediated mRNA decay (NMD) by genetic, pharmacological, or antisense oligonucleotide-based approaches showed that NMD contributes to the reduced mRNA levels in Grn R493X mice and cell lines and in fibroblasts from patients containing the GRN R493X mutation. Moreover, the expressed truncated R493X mutant protein was functional in several assays in progranulin-deficient cells. Together, these findings establish a murine model for in vivo testing of NMD inhibition or other therapies as potential approaches for treating progranulin deficiency caused by the R493X mutation. Copyright © 2018 the Author(s). Published by PNAS.

  3. An integrated miRNA functional screening and target validation method for organ morphogenesis.

    Science.gov (United States)

    Rebustini, Ivan T; Vlahos, Maryann; Packer, Trevor; Kukuruzinska, Maria A; Maas, Richard L

    2016-03-16

    The relative ease of identifying microRNAs and their increasing recognition as important regulators of organogenesis motivate the development of methods to efficiently assess microRNA function during organ morphogenesis. In this context, embryonic organ explants provide a reliable and reproducible system that recapitulates some of the important early morphogenetic processes during organ development. Here we present a method to target microRNA function in explanted mouse embryonic organs. Our method combines the use of peptide-based nanoparticles to transfect specific microRNA inhibitors or activators into embryonic organ explants, with a microRNA pulldown assay that allows direct identification of microRNA targets. This method provides effective assessment of microRNA function during organ morphogenesis, allows prioritization of multiple microRNAs in parallel for subsequent genetic approaches, and can be applied to a variety of embryonic organs.

  4. Extremely low penetrance of hearing loss in four Chinese families with the mitochondrial 12S rRNA A1555G mutation

    International Nuclear Information System (INIS)

    Young Wieyen; Zhao Lidong; Qian Yaping; Wang Qiuju; Li Ning; Greinwald, John H.; Guan Minxin

    2005-01-01

    Mutations in mitochondrial DNA (mtDNA) have been found to be associated with sensorineural hearing loss. We report here the clinical, genetic, and molecular characterization of four Chinese pedigrees with aminoglycoside-induced and nonsyndromic hearing impairment. Clinical evaluation revealed the variable phenotype of hearing impairment including audiometric configuration in these subjects, although these subjects share some common features: bilateral and sensorineural hearing impairment. Strikingly, these Chinese pedigrees exhibited extremely low penetrance of hearing loss (5.2%, 4.8%, 4.2%, and 13.3%, respectively, and with an average 8% penetrance). In particular, four of all five affected matrilineal relatives of these pedigrees had aminoglycoside-induced hearing loss. Sequence analysis of the complete mitochondrial genomes in these pedigrees showed the distinct sets of mtDNA polymorphism, in addition to the identical homoplasmic A1555G mutation, associated with hearing impairment in many families from different genetic backgrounds. The fact that mtDNA of those pedigrees belonged to different haplogroups R9a, N9a, D4a, and D4 suggested that the A1555G mutation occurred sporadically and multiplied through evolution of the mtDNA in China. However, there was the absence of functionally significant mutations in tRNA and rRNAs or secondary LHON mutations in these Chinese families. These data imply that the nuclear background or/and mitochondrial haplotype may not play a significant role in the phenotypic expression of the A1555G mutation in these Chinese pedigrees. However, aminoglycoside appears to be a major modifier factor for the phenotypic manifestation of the A1555G mutation in these Chinese families

  5. Structural basis underlying CAC RNA recognition by the RRM domain of dimeric RNA-binding protein RBPMS

    Energy Technology Data Exchange (ETDEWEB)

    Teplova, Marianna; Farazi, Thalia A.; Tuschl, Thomas; Patel, Dinshaw J.

    2015-09-08

    Abstract

    RNA-binding protein with multiple splicing (designated RBPMS) is a higher vertebrate mRNA-binding protein containing a single RNA recognition motif (RRM). RBPMS has been shown to be involved in mRNA transport, localization and stability, with key roles in axon guidance, smooth muscle plasticity, as well as regulation of cancer cell proliferation and migration. We report on structure-function studies of the RRM domain of RBPMS bound to a CAC-containing single-stranded RNA. These results provide insights into potential topologies of complexes formed by the RBPMS RRM domain and the tandem CAC repeat binding sites as detected by photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation. These studies establish that the RRM domain of RBPMS forms a symmetrical dimer in the free state, with each monomer binding sequence-specifically to all three nucleotides of a CAC segment in the RNA bound state. Structure-guided mutations within the dimerization and RNA-binding interfaces of RBPMS RRM on RNA complex formation resulted in both disruption of dimerization and a decrease in RNA-binding affinity as observed by size exclusion chromatography and isothermal titration calorimetry. As anticipated from biochemical binding studies, over-expression of dimerization or RNA-binding mutants of Flag-HA-tagged RBPMS were no longer able to track with stress granules in HEK293 cells, thereby documenting the deleterious effects of such mutationsin vivo.

  6. Developments in SPR Fragment Screening.

    Science.gov (United States)

    Chavanieu, Alain; Pugnière, Martine

    2016-01-01

    Fragment-based approaches have played an increasing role alongside high-throughput screening in drug discovery for 15 years. The label-free biosensor technology based on surface plasmon resonance (SPR) is now sensitive and informative enough to serve during primary screens and validation steps. In this review, the authors discuss the role of SPR in fragment screening. After a brief description of the underlying principles of the technique and main device developments, they evaluate the advantages and adaptations of SPR for fragment-based drug discovery. SPR can also be applied to challenging targets such as membrane receptors and enzymes. The high-level of immobilization of the protein target and its stability are key points for a relevant screening that can be optimized using oriented immobilized proteins and regenerable sensors. Furthermore, to decrease the rate of false negatives, a selectivity test may be performed in parallel on the main target bearing the binding site mutated or blocked with a low-off-rate ligand. Fragment-based drug design, integrated in a rational workflow led by SPR, will thus have a predominant role for the next wave of drug discovery which could be greatly enhanced by new improvements in SPR devices.

  7. Yeast screens identify the RNA polymerase II CTD and SPT5 as relevant targets of BRCA1 interaction.

    Directory of Open Access Journals (Sweden)

    Craig B Bennett

    2008-01-01

    Full Text Available BRCA1 has been implicated in numerous DNA repair pathways that maintain genome integrity, however the function responsible for its tumor suppressor activity in breast cancer remains obscure. To identify the most highly conserved of the many BRCA1 functions, we screened the evolutionarily distant eukaryote Saccharomyces cerevisiae for mutants that suppressed the G1 checkpoint arrest and lethality induced following heterologous BRCA1 expression. A genome-wide screen in the diploid deletion collection combined with a screen of ionizing radiation sensitive gene deletions identified mutants that permit growth in the presence of BRCA1. These genes delineate a metabolic mRNA pathway that temporally links transcription elongation (SPT4, SPT5, CTK1, DEF1 to nucleopore-mediated mRNA export (ASM4, MLP1, MLP2, NUP2, NUP53, NUP120, NUP133, NUP170, NUP188, POM34 and cytoplasmic mRNA decay at P-bodies (CCR4, DHH1. Strikingly, BRCA1 interacted with the phosphorylated RNA polymerase II (RNAPII carboxy terminal domain (P-CTD, phosphorylated in the pattern specified by the CTDK-I kinase, to induce DEF1-dependent cleavage and accumulation of a RNAPII fragment containing the P-CTD. Significantly, breast cancer associated BRCT domain defects in BRCA1 that suppressed P-CTD cleavage and lethality in yeast also suppressed the physical interaction of BRCA1 with human SPT5 in breast epithelial cells, thus confirming SPT5 as a relevant target of BRCA1 interaction. Furthermore, enhanced P-CTD cleavage was observed in both yeast and human breast cells following UV-irradiation indicating a conserved eukaryotic damage response. Moreover, P-CTD cleavage in breast epithelial cells was BRCA1-dependent since damage-induced P-CTD cleavage was only observed in the mutant BRCA1 cell line HCC1937 following ectopic expression of wild type BRCA1. Finally, BRCA1, SPT5 and hyperphosphorylated RPB1 form a complex that was rapidly degraded following MMS treatment in wild type but not BRCA1

  8. Nonsense and sense suppression abilities of original and derivative Methanosarcina mazei pyrrolysyl-tRNA synthetase-tRNA(Pyl pairs in the Escherichia coli BL21(DE3 cell strain.

    Directory of Open Access Journals (Sweden)

    Keturah A Odoi

    Full Text Available Systematic studies of nonsense and sense suppression of the original and three derivative Methanosarcina mazei PylRS-tRNA(Pyl pairs and cross recognition between nonsense codons and various tRNA(Pyl anticodons in the Escherichia coli BL21(DE3 cell strain are reported. tRNA(CUA(Pyl is orthogonal in E. coli and able to induce strong amber suppression when it is co-expressed with pyrrolysyl-tRNA synthetase (PylRS and charged with a PylRS substrate, N(ε-tert-butoxycarbonyl-L-lysine (BocK. Similar to tRNA(CUA(Pyl, tRNA(UUA(Pyl is also orthogonal in E. coli and can be coupled with PylRS to genetically incorporate BocK at an ochre mutation site. Although tRNA(UUA(Pyl is expected to recognize a UAG codon based on the wobble hypothesis, the PylRS-tRNA(UUA(Pyl pair does not give rise to amber suppression that surpasses the basal amber suppression level in E. coli. E. coli itself displays a relatively high opal suppression level and tryptophan (Trp is incorporated at an opal mutation site. Although the PylRS-tRNA(UCA(Pyl pair can be used to encode BocK at an opal codon, the pair fails to suppress the incorporation of Trp at the same site. tRNA(CCU(Pyl fails to deliver BocK at an AGG codon when co-expressed with PylRS in E. coli.

  9. Reprogramming the Dynamin 2 mRNA by Spliceosome-mediated RNA Trans-splicing

    Directory of Open Access Journals (Sweden)

    Delphine Trochet

    2016-01-01

    Full Text Available Dynamin 2 (DNM2 is a large GTPase, ubiquitously expressed, involved in membrane trafficking and regulation of actin and microtubule cytoskeletons. DNM2 mutations cause autosomal dominant centronuclear myopathy which is a rare congenital myopathy characterized by skeletal muscle weakness and histopathological features including nuclear centralization in absence of regeneration. No curative treatment is currently available for the DNM2-related autosomal dominant centronuclear myopathy. In order to develop therapeutic strategy, we evaluated here the potential of Spliceosome-Mediated RNA Trans-splicing technology to reprogram the Dnm2-mRNA in vitro and in vivo in mice. We show that classical 3′-trans-splicing strategy cannot be considered as accurate therapeutic strategy regarding toxicity of the pre-trans-splicing molecules leading to low rate of trans-splicing in vivo. Thus, we tested alternative strategies devoted to prevent this toxicity and enhance frequency of trans-splicing events. We succeeded to overcome the toxicity through a 5′-trans-splicing strategy which also allows detection of trans-splicing events at mRNA and protein levels in vitro and in vivo. These results suggest that the Spliceosome-Mediated RNA Trans-splicing strategy may be used to reprogram mutated Dnm2-mRNA but highlight the potential toxicity linked to the molecular tools which have to be carefully investigated during preclinical development.

  10. Family-based cardiac screening in relatives of victims of sudden arrhythmic death syndrome.

    LENUS (Irish Health Repository)

    McGorrian, Catherine

    2013-02-03

    AIMS: Sudden arrhythmic death syndrome (SADS) occurs when a person suffers a sudden, unexpected death, with no cause found at postmortem examination. We aimed to describe the cardiac screening outcomes in a population of relatives of SADS victimsMETHODS AND RESULTS: Prospective and retrospective cohort study of consecutive families attending the Family Heart Screening clinic at the Mater Misericordiae Hospital in Dublin, Ireland, from January 2007 to September 2011. Family members of SADS victims underwent a standard screening protocol. Adjunct clinical and postmortem information was sought on the proband. Families who had an existing diagnosis, or where the proband had epilepsy, were excluded. Of 115 families identified, 73 were found to fit inclusion criteria and were retained for analysis, with data available on 262 relatives. Over half of the screened family members were female, and the mean age was 38.6 years (standard deviation 15.6). In 22 of 73 families (30%), and 36 of 262 family members (13.7%), a potentially inheritable cause of SADS was detected. Of the population screened, 32 patients (12.2%) were treated with medication, and 5 (1.9%) have received implantable cardiac defibrillators. Of the five families with long QT syndrome (LQTS) who had a pathogenic gene mutation identified, three carried two such mutations.CONCLUSION: In keeping with international estimates, 30% of families of SADS victims were found to have a potentially inherited cardiac disease. The most common positive finding was LQTS. Advances in postmortem standards and genetic studies may assist in achieving more diagnoses in these families.

  11. Mutation breeding in pepper

    International Nuclear Information System (INIS)

    Daskalov, S.

    1986-01-01

    Pepper (Capsicum sp.) is an important vegetable and spice crop widely grown in tropical as well as in temperate regions. Until recently the improvement programmes were based mainly on using natural sources of germ plasma, crossbreeding and exploiting the heterosis of F 1 hybrids. However, interest in using induced mutations is growing. A great number of agronomically useful mutants as well as mutants valuable for genetic, cytological and physiological studies have been induced and described. In this review information is presented about suitable mutagen treatment procedures with radiation as well as chemicals, M 1 effects, handling the treated material in M 1 , M 2 and subsequent generations, and mutant screening procedures. This is supplemented by a description of reported useful mutants and released cultivars. Finally, general advice is given on when and how to incorporate mutation induction in Capsicum improvement programmes. (author)

  12. RFLP analysis of rice semi-dwarf mutation induced by high energy argon ion radiation

    International Nuclear Information System (INIS)

    Zhuang Chuxiong; Hu Weimin; Mei Mantong

    1997-01-01

    Two Indica rice varieties, Bianpizhan and Xiangzhan, and their semi-dwarf mutants induced by high energy argon ion radiation, Ar-10, and Xiang-Ar-1, were examined with restriction fragment length polymorphism (RFLP) analysis by using 97 rice single copy genomic clones mapped on 12 chromosomes of molecular genetic map, combined with 5 restriction enzymes. Among the markers screened, 9 detected polymorphism were between Bianpizhen and Ar-10, and 11 detected polymorphism were between Xiangzhan and Xiang-Ar-1. Moreover, two or more restriction enzymes could generate RFLP patterns when screened with a given marker for several polymorphic markers. Based on the polymorphic allelic loci, the mutation frequencies were estimated as 5.15% and 6.39% for Ar-10 and Xiang-Ar-1 respectively. These results suggested that the nature of mutation on the DNA level was probably large genetic changes rather than point mutation. Genetic analysis and gene tagging of semi-dwarf mutation in one of the mutant line, Ar-10, indicated that this mutation was controlled by a major recessive gene, which was preliminary located on chromosome 4

  13. RFLP Analysis of rice semi dwarf mutation induced by high energy argon ion radiation

    International Nuclear Information System (INIS)

    Zhuang Chuxiong; Hu Weimin; Mei Mantong

    1997-01-01

    Two Indica rice varieties, Bianpizhan and Xiangzhan, and their semi dwarf mutants induced by high energy argon ion radiation, Ar 10, and Xiang Ar 1, were examined with restriction fragment length polymorphism(RFLP)analysis by using 97 rice single copy genomic clones mapped on 12 chromosomes of molecular genetic map, combined with 5 restriction enzymes.Among the markers screened, 9 detected polymorphism were between Bianpizhan and Ar 10, and 11 detected polymorphism were between Xiangzhan and Xiang Ar 1.Moreover, two or more restriction enzymes could generate RFLP patterns when screened with a given marker for several polymorphic markers. Based on the polymorphic allelic loci, the mutation frequencies were estimated as 5 15% and 6 39% for Ar 10 and Xiang Ar 1 respectively.These results suggested that the nature of mutation on the DNA level was probably large genetic changes rather than point mutation.Genetic analysis and gene tagging of semi dwarf mutation in one of the mutant line, Ar 10, indicated that this mutation was controlled by a major recessive gene, which was preliminary located on chromosome 4. (author)

  14. Comprehensive Mutation Analysis of PMS2 in a Large Cohort of Probands Suspected of Lynch Syndrome or Constitutional Mismatch Repair Deficiency Syndrome

    NARCIS (Netherlands)

    Klift, H.M. van der; Mensenkamp, A.R.; Drost, M.; Bik, E.C.; Vos, Y.J.; Gille, H.J.; Redeker, B.E.; Tiersma, Y.; Zonneveld, J.B.; Garcia, E.G.; Letteboer, T.G.; Olderode-Berends, M.J.; Hest, L.P. van; Os, T.A. van; Verhoef, S.; Wagner, A.; Asperen, C.J. van; Broeke, S.W. ten; Hes, F.J.; Wind, N. de; Nielsen, M.; Devilee, P.; Ligtenberg, M.J.L.; Wijnen, J.T.; Tops, C.M.

    2016-01-01

    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

  15. Comprehensive Mutation Analysis of PMS2 in a Large Cohort of Probands Suspected of Lynch Syndrome or Constitutional Mismatch Repair Deficiency Syndrome

    NARCIS (Netherlands)

    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, Jose B. M.; Garcia, Encarna Gomez; 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.

    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

  16. Mutation scanning of peach floral genes

    Directory of Open Access Journals (Sweden)

    Wilde H Dayton

    2011-05-01

    Full Text Available Abstract Background Mutation scanning technology has been used to develop crop species with improved traits. Modifications that improve screening throughput and sensitivity would facilitate the targeted mutation breeding of crops. Technical innovations for high-resolution melting (HRM analysis are enabling the clinic-based screening for human disease gene polymorphism. We examined the application of two HRM modifications, COLD-PCR and QMC-PCR, to the mutation scanning of genes in peach, Prunus persica. The targeted genes were the putative floral regulators PpAGAMOUS and PpTERMINAL FLOWER I. Results HRM analysis of PpAG and PpTFL1 coding regions in 36 peach cultivars found one polymorphic site in each gene. PpTFL1 and PpAG SNPs were used to examine approaches to increase HRM throughput. Cultivars with SNPs could be reliably detected in pools of twelve genotypes. COLD-PCR was found to increase the sensitivity of HRM analysis of pooled samples, but worked best with small amplicons. Examination of QMC-PCR demonstrated that primary PCR products for further analysis could be produced from variable levels of genomic DNA. Conclusions Natural SNPs in exons of target peach genes were discovered by HRM analysis of cultivars from a southeastern US breeding program. For detecting natural or induced SNPs in larger populations, HRM efficiency can be improved by increasing sample pooling and template production through approaches such as COLD-PCR and QMC-PCR. Technical advances developed to improve clinical diagnostics can play a role in the targeted mutation breeding of crops.

  17. Epidermal growth factor receptor mutation in gastric cancer.

    Science.gov (United States)

    Liu, Zhimin; Liu, Lina; Li, Mei; Wang, Zhaohui; Feng, Lu; Zhang, Qiuping; Cheng, Shihua; Lu, Shen

    2011-04-01

    Epidermal growth factor receptor (EGFR) and Kirsten-RAS (KRAS) mutations have been identified as predictors of response to EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer. We aimed to screen the mutations of both genes in gastric carcinoma to detect the suitability of EGFR TKIs for patients with gastric carcinoma. We screened EGFR mutation in exons 19-21 and KRAS mutation in exon 2 in 58 gastric adenocarcinomas from China using high resolution melting analysis (HRMA). Positive samples were confirmed by DNA sequencing. Three EGFR missense mutations (5.2%) and 22 single nucleotide polymorphisms (SNP, Q787Q, 37.9%) were identified. To our knowledge, we report for the first time three mutation patterns of EGFR, Y801C, L858R and G863D, in gastric carcinoma. Two samples with EGFR mutation were mucinous adenocarcinoma. These three samples were collected from male patients aged over 75 years old. The frequency of KRAS mutation was 10.3% (6/58). The exclusiveness of EGFR and KRAS mutations was proven for the first time in gastric cancer. Gastric carcinoma of the mucinous adenocarcinoma type collected from older male patients may harbour EGFR mutations. The small subset of gastric adenocarcinoma patients may respond to EGFR TKIs.

  18. Prostate screening uptake in Australian BRCA1 and BRCA2 carriers

    Directory of Open Access Journals (Sweden)

    McKinley Joanne M

    2007-09-01

    Full Text Available Abstract Men who carry mutations in BRCA1 or BRCA2 are at increased risk for prostate cancer. However the efficacy of prostate screening in this setting is uncertain and limited data exists on the uptake of prostate screening by mutation carriers. This study prospectively evaluated uptake of prostate cancer screening in a multi-institutional cohort of mutation carriers. Subjects were unaffected male BRCA1 and BRCA2 mutation carriers, aged 40–69 years, enrolled in the Kathleen Cuningham Consortium for Research into Familial Breast Cancer (kConFab and who had completed a mailed, self-report follow-up questionnaire 3 yearly after study entry. Of the 75 male carriers in this study, only 26 (35% had elected to receive their mutation result. Overall, 51 (68% did not recall having received a recommendation to have prostate screening because of their family history, but 41 (55% had undergone a prostate specific antigen (PSA test and 32 (43% a digital rectal examination (DRE in the previous 3 years. Those who were aware of their mutation result were more likely to have received a recommendation for prostate screening (43 vs. 6%, p = 0.0001, and to have had a PSA test (77 vs. 43%, p = 0.005 and a DRE (69 vs. 29%, p = 0.001 in the previous 3 years. The majority of unaffected males enrolled in kConFab with a BRCA1/2 mutation have not sought out their mutation result. However, of those aware of their positive mutation status, most have undergone at least one round of prostate screening in the previous 3 years.

  19. Restricted ultraviolet mutational spectrum in a shuttle vector propagated in xeroderma pigmentosum cells

    International Nuclear Information System (INIS)

    Bredberg, A.; Kraemer, K.H.; Seidman, M.M.

    1986-01-01

    A shuttle vector plasmid, pZ189, carrying a bacterial suppressor tRNA marker gene, was treated with ultraviolet radiation and propagated in cultured skin cells from a patient with the skin-cancer-prone, DNA repair-deficient disease xeroderma pigmentosum and in repair-proficient cells. After replication in the human cells, progeny plasmids were purified. Plasmid survival and mutations inactivating the marker gene were scored by transforming an indicator strain of Escherichia coli carrying a suppressible amber mutation in the beta-galactosidase gene. Plasmid survival in the xeroderma pigmentosum cells was less than that of pZ189 harvested from repair-proficient human cells. The point-mutation frequency in the 150-base-pair tRNA marker gene increased up to 100-fold with ultraviolet dose. Sequence analysis of 150 mutant plasmids revealed that mutations were infrequent at potential thymine-thymine dimer sites. Ninety-three percent of the mutant plasmids from the xeroderma pigmentosum cells showed G X C----A X T transitions, compared to 73% in the normal cells (P less than 0.002). There were significantly fewer transversions (P less than 0.002) (especially G X C----T X A) and multiple base substitutions (P less than 0.00001) than when pZ189 was passaged in repair-proficient cells. The subset of mutational changes that are common to ultraviolet-treated plasmids propagated in both repair-proficient and xeroderma pigmentosum skin cells may be associated with the development of ultraviolet-induced skin cancer in humans

  20. AIP mutations and gigantism.

    Science.gov (United States)

    Rostomyan, Liliya; Potorac, Iulia; Beckers, Pablo; Daly, Adrian F; Beckers, Albert

    2017-06-01

    AIP mutations are rare in sporadic acromegaly but they are seen at a higher frequency among certain specific populations of pituitary adenoma patients (pituitary gigantism cases, familial isolated pituitary adenoma (FIPA) kindreds, and patients with macroadenomas who are diagnosed ≤30 years). AIP mutations are most prevalent in patients with pituitary gigantism (29% of this group were found to have mutations in AIP gene). These data support targeted genetic screening for AIP mutations/deletions in these groups of pituitary adenoma patients. Earlier diagnosis of AIP-related acromegaly-gigantism cases enables timely clinical evaluation and treatment, thereby improving outcomes in terms of excessive linear growth and acromegaly comorbidities. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  1. Structure-Function Model for Kissing Loop Interactions That Initiate Dimerization of Ty1 RNA

    Directory of Open Access Journals (Sweden)

    Eric R. Gamache

    2017-04-01

    Full Text Available The genomic RNA of the retrotransposon Ty1 is packaged as a dimer into virus-like particles. The 5′ terminus of Ty1 RNA harbors cis-acting sequences required for translation initiation, packaging and initiation of reverse transcription (TIPIRT. To identify RNA motifs involved in dimerization and packaging, a structural model of the TIPIRT domain in vitro was developed from single-nucleotide resolution RNA structural data. In general agreement with previous models, the first 326 nucleotides of Ty1 RNA form a pseudoknot with a 7-bp stem (S1, a 1-nucleotide interhelical loop and an 8-bp stem (S2 that delineate two long, structured loops. Nucleotide substitutions that disrupt either pseudoknot stem greatly reduced helper-Ty1-mediated retrotransposition of a mini-Ty1, but only mutations in S2 destabilized mini-Ty1 RNA in cis and helper-Ty1 RNA in trans. Nested in different loops of the pseudoknot are two hairpins with complementary 7-nucleotide motifs at their apices. Nucleotide substitutions in either motif also reduced retrotransposition and destabilized mini- and helper-Ty1 RNA. Compensatory mutations that restore base-pairing in the S2 stem or between the hairpins rescued retrotransposition and RNA stability in cis and trans. These data inform a model whereby a Ty1 RNA kissing complex with two intermolecular kissing-loop interactions initiates dimerization and packaging.

  2. 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.

  3. New mutations affecting induced mutagenesis in yeast.

    Science.gov (United States)

    Lawrence, C W; Krauss, B R; Christensen, R B

    1985-01-01

    Previously isolated mutations in baker's yeast, Saccharomyces cerevisiae, that impair induced mutagenesis were all identified with the aid of tests that either exclusively or predominantly detect base-pair substitutions. To avoid this bias, we have screened 11 366 potentially mutant clones for UV-induced reversion of the frameshift allele, his4-38, and have identified 10 mutants that give much reduced yields of revertants. Complementation and recombination tests show that 6 of these carry mutations at the previously known REV1, REV1 and REV3 loci, while the remaining 4 define 3 new genes, REV4 (2 mutations), REV5 and REV6. The rev4 mutations are readily suppressed in many genetic backgrounds and, like the rev5 mutation, impart only a limited deficiency for induced mutagenesis: it is likely, therefore that the REV4+ and REV5+ gene functions are only remotely concerned with this process. The rev6 mutants have a more general deficiency, however, as well as marked sensitivity to UV and an increased spontaneous mutation rate, properties that suggest the REV6 gene is directly involved in mutation induction. The REV5 gene is located about 1 cM proximal to CYC1 on chromosome X.

  4. ENU mutagenesis screening for dominant behavioral mutations based on normal control data obtained in home-cage activity, open-field, and passive avoidance tests.

    Science.gov (United States)

    Wada, Yumiko; Furuse, Tamio; Yamada, Ikuko; Masuya, Hiroshi; Kushida, Tomoko; Shibukawa, Yoko; Nakai, Yuji; Kobayashi, Kimio; Kaneda, Hideki; Gondo, Yoichi; Noda, Tetsuo; Shiroishi, Toshihiko; Wakana, Shigeharu

    2010-01-01

    To establish the cutoff values for screening ENU-induced behavioral mutations, normal variations in mouse behavioral data were examined in home-cage activity (HA), open-field (OF), and passive-avoidance (PA) tests. We defined the normal range as one that included more than 95% of the normal control values. The cutoffs were defined to identify outliers yielding values that deviated from the normal by less than 5% for C57BL/6J, DBA/2J, DBF(1), and N(2) (DXDB) progenies. Cutoff values for G1-phenodeviant (DBF(1)) identification were defined based on values over +/- 3.0 SD from the mean of DBF(1) for all parameters assessed in the HA and OF tests. For the PA test, the cutoff values were defined based on whether the mice met the learning criterion during the 2nd (at a shock intensity of 0.3 mA) or the 3rd (at a shock intensity of 0.15 mA) retention test. For several parameters, the lower outliers were undetectable as the calculated cutoffs were negative values. Based on the cutoff criteria, we identified 275 behavioral phenodeviants among 2,646 G1 progeny. Of these, 64 were crossed with wild-type DBA/2J individuals, and the phenotype transmission was examined in the G2 progeny using the cutoffs defined for N(2) mice. In the G2 mice, we identified 15 novel dominant mutants exhibiting behavioral abnormalities, including hyperactivity in the HA or OF tests, hypoactivity in the OF test, and PA deficits. Genetic and detailed behavioral analysis of these ENU-induced mutants will provide novel insights into the molecular mechanisms underlying behavior.

  5. Beet Necrotic Yellow Vein Virus Noncoding RNA Production Depends on a 5′→3′ Xrn Exoribonuclease Activity

    Directory of Open Access Journals (Sweden)

    Alyssa Flobinus

    2018-03-01

    Full Text Available The RNA3 species of the beet necrotic yellow vein virus (BNYVV, a multipartite positive-stranded RNA phytovirus, contains the ‘core’ nucleotide sequence required for its systemic movement in Beta macrocarpa. Within this ‘core’ sequence resides a conserved “coremin” motif of 20 nucleotides that is absolutely essential for long-distance movement. RNA3 undergoes processing steps to yield a noncoding RNA3 (ncRNA3 possessing “coremin” at its 5′ end, a mandatory element for ncRNA3 accumulation. Expression of wild-type (wt or mutated RNA3 in Saccharomyces cerevisiae allows for the accumulation of ncRNA3 species. Screening of S. cerevisiae ribonuclease mutants identified the 5′-to-3′ exoribonuclease Xrn1 as a key enzyme in RNA3 processing that was recapitulated both in vitro and in insect cell extracts. Xrn1 stalled on ncRNA3-containing RNA substrates in these decay assays in a similar fashion as the flavivirus Xrn1-resistant structure (sfRNA. Substitution of the BNYVV-RNA3 ‘core’ sequence by the sfRNA sequence led to the accumulation of an ncRNA species in yeast in vitro but not in planta and no viral long distance occurred. Interestingly, XRN4 knockdown reduced BNYVV RNA accumulation suggesting a dual role for the ribonuclease in the viral cycle.

  6. Fragment-based screening in tandem with phenotypic screening provides novel antiparasitic hits.

    Science.gov (United States)

    Blaazer, Antoni R; Orrling, Kristina M; Shanmugham, Anitha; Jansen, Chimed; Maes, Louis; Edink, Ewald; Sterk, Geert Jan; Siderius, Marco; England, Paul; Bailey, David; de Esch, Iwan J P; Leurs, Rob

    2015-01-01

    Methods to discover biologically active small molecules include target-based and phenotypic screening approaches. One of the main difficulties in drug discovery is elucidating and exploiting the relationship between drug activity at the protein target and disease modification, a phenotypic endpoint. Fragment-based drug discovery is a target-based approach that typically involves the screening of a relatively small number of fragment-like (molecular weight <300) molecules that efficiently cover chemical space. Here, we report a fragment screening on TbrPDEB1, an essential cyclic nucleotide phosphodiesterase (PDE) from Trypanosoma brucei, and human PDE4D, an off-target, in a workflow in which fragment hits and a series of close analogs are subsequently screened for antiparasitic activity in a phenotypic panel. The phenotypic panel contained T. brucei, Trypanosoma cruzi, Leishmania infantum, and Plasmodium falciparum, the causative agents of human African trypanosomiasis (sleeping sickness), Chagas disease, leishmaniasis, and malaria, respectively, as well as MRC-5 human lung cells. This hybrid screening workflow has resulted in the discovery of various benzhydryl ethers with antiprotozoal activity and low toxicity, representing interesting starting points for further antiparasitic optimization. © 2014 Society for Laboratory Automation and Screening.

  7. Autosomal dominant pseudohypoaldosteronism type 1 with a novel splice site mutation in MR gene

    Directory of Open Access Journals (Sweden)

    Kaito Hiroshi

    2009-11-01

    Full Text Available Abstract Background Autosomal dominant pseudohypoaldosteronism type 1 (PHA1 is a rare inherited condition that is characterized by renal resistance to aldosterone as well as salt wasting, hyperkalemia, and metabolic acidosis. Renal PHA1 is caused by mutations of the human mineralcorticoid receptor gene (MR, but it is a matter of debate whether MR mutations cause mineralcorticoid resistance via haploinsufficiency or dominant negative mechanism. It was previously reported that in a case with nonsense mutation the mutant mRNA was absent in lymphocytes because of nonsense mediated mRNA decay (NMD and therefore postulated that haploinsufficiency alone can give rise to the PHA1 phenotype in patients with truncated mutations. Methods and Results We conducted genomic DNA analysis and mRNA analysis for familial PHA1 patients extracted from lymphocytes and urinary sediments and could detect one novel splice site mutation which leads to exon skipping and frame shift result in premature termination at the transcript level. The mRNA analysis showed evidence of wild type and exon-skipped RT-PCR products. Conclusion mRNA analysis have been rarely conducted for PHA1 because kidney tissues are unavailable for this disease. However, we conducted RT-PCR analysis using mRNA extracted from urinary sediments. We could demonstrate that NMD does not fully function in kidney cells and that haploinsufficiency due to NMD with premature termination is not sufficient to give rise to the PHA1 phenotype at least in this mutation of our patient. Additional studies including mRNA analysis will be needed to identify the exact mechanism of the phenotype of PHA.

  8. CRISPR/Cas9-mediated noncoding RNA editing in human cancers.

    Science.gov (United States)

    Yang, Jie; Meng, Xiaodan; Pan, Jinchang; Jiang, Nan; Zhou, Chengwei; Wu, Zhenhua; Gong, Zhaohui

    2018-01-02

    Cancer is characterized by multiple genetic and epigenetic alterations, including a higher prevalence of mutations of oncogenes and/or tumor suppressors. Mounting evidences have shown that noncoding RNAs (ncRNAs) are involved in the epigenetic regulation of cancer genes and their associated pathways. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease 9 (CRISPR/Cas9) system, a revolutionary genome-editing technology, has shed light on ncRNA-based cancer therapy. Here, we briefly introduce the classifications and mechanisms of CRISPR/Cas9 system. Importantly, we mainly focused on the applications of CRISPR/Cas9 system as a molecular tool for ncRNA (microRNA, long noncoding RNA and circular RNA, etc.) editing in human cancers, and the novel techniques that are based on CRISPR/Cas9 system. Additionally, the off-target effects and the corresponding solutions as well as the challenges toward CRISPR/Cas9 were also evaluated and discussed. Long- and short-ncRNAs have been employed as targets in precision oncology, and CRISPR/Cas9-mediated ncRNA editing may provide an excellent way to cure cancer.

  9. Messenger RNA surveillance: neutralizing natural nonsense

    DEFF Research Database (Denmark)

    Weischelfeldt, Joachim Lütken; Lykke-Andersen, Jens; Porse, Bo

    2005-01-01

    Messenger RNA transcripts that contain premature stop codons are degraded by a process termed nonsense-mediated mRNA decay (NMD). Although previously thought of as a pathway that rids the cell of non-functional mRNAs arising from mutations and processing errors, new research suggests a more general...

  10. POLR2C Mutations Are Associated With Primary Ovarian Insufficiency in Women.

    Science.gov (United States)

    Moriwaki, Mika; Moore, Barry; Mosbruger, Timothy; Neklason, Deborah W; Yandell, Mark; Jorde, Lynn B; Welt, Corrine K

    2017-03-01

    Primary ovarian insufficiency (POI) results from a premature loss of oocytes, causing infertility and early menopause. The etiology of POI remains unknown in a majority of cases. To identify candidate genes in families affected by POI. This was a family-based genetic study. The study was performed at two academic institutions. A family with four generations of women affected by POI (n = 5). Four of these women, three with an associated autoimmune diagnosis, were studied. The controls (n = 387) were recruited for health in old age. Whole-genome sequencing was performed. Candidate genes were identified by comparing gene mutations in three family members and 387 control subjects analyzed simultaneously using the pedigree Variant Annotation, Analysis and Search Tool. Data were also compared with that in publicly available databases. We identified a heterozygous nonsense mutation in a subunit of RNA polymerase II ( POLR2C ) that synthesizes messenger RNA. A rare sequence variant in POLR2C was also identified in one of 96 women with sporadic POI. POLR2C expression was decreased in the proband compared with women with POI from another cause. Knockdown in an embryonic carcinoma cell line resulted in decreased protein production and impaired cell proliferation. These data support a role for RNA polymerase II mutations as candidates in the etiology of POI. The current data also support results from genome-wide association studies that hypothesize a role for RNA polymerase II subunits in age at menopause in the population.

  11. Diversity of ARSACS mutations in French-Canadians.

    Science.gov (United States)

    Thiffault, I; Dicaire, M J; Tetreault, M; Huang, K N; Demers-Lamarche, J; Bernard, G; Duquette, A; Larivière, R; Gehring, K; Montpetit, A; McPherson, P S; Richter, A; Montermini, L; Mercier, J; Mitchell, G A; Dupré, N; Prévost, C; Bouchard, J P; Mathieu, J; Brais, B

    2013-01-01

    The growing number of spastic ataxia of Charlevoix-Saguenay (SACS) gene mutations reported worldwide has broadened the clinical phenotype of autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS). The identification of Quebec ARSACS cases without two known SACS mutation led to the development of a multi-modal genomic strategy to uncover mutations in this large gene and explore phenotype variability. Search for SACS mutations by combining various methods on 20 cases with a classical French-Canadian ARSACS phenotype without two mutations and a group of 104 sporadic or recessive spastic ataxia cases of unknown cause. Western blot on lymphoblast protein from cases with different genotypes was probed to establish if they still expressed sacsin. A total of 12 mutations, including 7 novels, were uncovered in Quebec ARSACS cases. The screening of 104 spastic ataxia cases of unknown cause for 98 SACS mutations did not uncover carriers of two mutations. Compounds heterozygotes for one missense SACS mutation were found to minimally express sacsin. The large number of SACS mutations present even in Quebec suggests that the size of the gene alone may explain the great genotypic diversity. This study does not support an expanding ARSACS phenotype in the French-Canadian population. Most mutations lead to loss of function, though phenotypic variability in other populations may reflect partial loss of function with preservation of some sacsin expression. Our results also highlight the challenge of SACS mutation screening and the necessity to develop new generation sequencing methods to ensure low cost complete gene sequencing.

  12. The RNA template channel of the RNA-dependent RNA polymerase as a target for development of antiviral therapy of multiple genera within a virus family.

    Directory of Open Access Journals (Sweden)

    Lonneke van der Linden

    2015-03-01

    Full Text Available The genus Enterovirus of the family Picornaviridae contains many important human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and enterovirus 71 for which no antiviral drugs are available. The viral RNA-dependent RNA polymerase is an attractive target for antiviral therapy. Nucleoside-based inhibitors have broad-spectrum activity but often exhibit off-target effects. Most non-nucleoside inhibitors (NNIs target surface cavities, which are structurally more flexible than the nucleotide-binding pocket, and hence have a more narrow spectrum of activity and are more prone to resistance development. Here, we report a novel NNI, GPC-N114 (2,2'-[(4-chloro-1,2-phenylenebis(oxy]bis(5-nitro-benzonitrile with broad-spectrum activity against enteroviruses and cardioviruses (another genus in the picornavirus family. Surprisingly, coxsackievirus B3 (CVB3 and poliovirus displayed a high genetic barrier to resistance against GPC-N114. By contrast, EMCV, a cardiovirus, rapidly acquired resistance due to mutations in 3Dpol. In vitro polymerase activity assays showed that GPC-N114 i inhibited the elongation activity of recombinant CVB3 and EMCV 3Dpol, (ii had reduced activity against EMCV 3Dpol with the resistance mutations, and (iii was most efficient in inhibiting 3Dpol when added before the RNA template-primer duplex. Elucidation of a crystal structure of the inhibitor bound to CVB3 3Dpol confirmed the RNA-binding channel as the target for GPC-N114. Docking studies of the compound into the crystal structures of the compound-resistant EMCV 3Dpol mutants suggested that the resistant phenotype is due to subtle changes that interfere with the binding of GPC-N114 but not of the RNA template-primer. In conclusion, this study presents the first NNI that targets the RNA template channel of the picornavirus polymerase and identifies a new pocket that can be used for the design of broad-spectrum inhibitors. Moreover, this study provides important new insight

  13. Common FLG mutation K4671X not associated with atopic dermatitis in Han Chinese in a family association study.

    Directory of Open Access Journals (Sweden)

    Ruhong Cheng

    Full Text Available BACKGROUND: Filaggrin gene (FLG mutations have been identified as the cause of ichthyosis vulgaris (IV and major predisposing factors for atopic dermatitis (AD. The relationship among AD, IV and FLG mutations has not been clarified yet. Mutations 3321delA and K4671X, two of the most common mutations in Chinese patients, were both statistically associated with AD in case-control studies. MATERIALS AND METHODS: A group of 100 family trios (a total of 300 members with one affected AD proband and both parents were recruited and screened for three filaggrin null mutations (3222del4, 3321delA and K4671X. The subjects' manifestations of AD and IV were assessed by two experienced dermatologists and recorded in detail. The relationship of common mutations to AD were assessed using both case-control and family-based tests of association. Filaggrin expression was measured in skin of 3 subjects with K4671X heterozygote and the normal control using quantitative real-time RT-PCR and immunohistochemistry. RESULTS: Of 100 probands for AD, 22 were carriers for common FLG mutations and only 2 of them were from 40 none-IV family trios (5.00%, consistent with that of the healthy control group (3.99%, P>0.05. Significant statistical associations were revealed between AD and 3321delA (P<0.001, odds ratio 12.28, 95% confidence interval 3.35-44.98 as well as K4671X (P = 0.002, odds ratio 4.53, 95% confidence interval 1.77-11.60. The family-based approach revealed that 3321delA was over-transmitted to AD offspring from parents (T:U = 12∶1, P = 0.003 but failed to demonstrate transmission disequilibrium between K4671X and AD (T:U = 10∶8, P = 0.815. Moreover, compared to the normal control, filaggrin expression at both mRNA and protein levels in epidermis of subjects with K4671X(heter was not reduced. CONCLUSIONS: AD patients from none-IV family trios have low probability of carrying FLG mutations. The present family samples confirmed the

  14. Clinical, pathologic, and biologic features associated with BRAF mutations in non-small cell lung cancer.

    Science.gov (United States)

    Cardarella, Stephanie; Ogino, Atsuko; Nishino, Mizuki; Butaney, Mohit; Shen, Jeanne; Lydon, Christine; Yeap, Beow Y; Sholl, Lynette M; Johnson, Bruce E; Jänne, Pasi A

    2013-08-15

    BRAF mutations are found in a subset of non-small cell lung cancers (NSCLC). We examined the clinical characteristics and treatment outcomes of patients with NSCLC harboring BRAF mutations. Using DNA sequencing, we successfully screened 883 patients with NSCLC for BRAF mutations between July 1, 2009 and July 16, 2012. Baseline characteristics and treatment outcomes were compared between patients with and without BRAF mutations. Wild-type controls consisted of patients with NSCLC without a somatic alteration in BRAF, KRAS, EGFR, and ALK. In vitro studies assessed the biologic properties of selected non-V600E BRAF mutations identified from patients with NSCLC. Of 883 tumors screened, 36 (4%) harbored BRAF mutations (V600E, 18; non-V600E, 18) and 257 were wild-type for BRAF, EGFR, KRAS, and ALK negative. Twenty-nine of 36 patients with BRAF mutations were smokers. There were no distinguishing clinical features between BRAF-mutant and wild-type patients. Patients with advanced NSCLC with BRAF mutations and wild-type tumors showed similar response rates and progression-free survival (PFS) to platinum-based combination chemotherapy and no difference in overall survival. Within the BRAF cohort, patients with V600E-mutated tumors had a shorter PFS to platinum-based chemotherapy compared with those with non-V600E mutations, although this did not reach statistical significance (4.1 vs. 8.9 months; P = 0.297). We identified five BRAF mutations not previously reported in NSCLC; two of five were associated with increased BRAF kinase activity. BRAF mutations occur in 4% of NSCLCs and half are non-V600E. Prospective trials are ongoing to validate BRAF as a therapeutic target in NSCLC. ©2013 AACR.

  15. Comprehensive Mutation Analysis of PMS2 in a Large Cohort of Probands Suspected of Lynch Syndrome or Constitutional Mismatch Repair Deficiency (CMMRD) Syndrome

    NARCIS (Netherlands)

    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

    2016-01-01

    Monoallelic PMS2 germline mutations cause 5-15% of Lynch syndrome, a midlife cancer predisposition, whereas biallelic PMS2 mutations cause approximately 60% of constitutional MMR deficiency (CMMRD), a rare childhood cancer syndrome. Recently improved DNA and RNA-based strategies are applied to

  16. Translation of the flavivirus kunjin NS3 gene in cis but not its RNA sequence or secondary structure is essential for efficient RNA packaging.

    Science.gov (United States)

    Pijlman, Gorben P; Kondratieva, Natasha; Khromykh, Alexander A

    2006-11-01

    Our previous studies using trans-complementation analysis of Kunjin virus (KUN) full-length cDNA clones harboring in-frame deletions in the NS3 gene demonstrated the inability of these defective complemented RNAs to be packaged into virus particles (W. J. Liu, P. L. Sedlak, N. Kondratieva, and A. A. Khromykh, J. Virol. 76:10766-10775). In this study we aimed to establish whether this requirement for NS3 in RNA packaging is determined by the secondary RNA structure of the NS3 gene or by the essential role of the translated NS3 gene product. Multiple silent mutations of three computer-predicted stable RNA structures in the NS3 coding region of KUN replicon RNA aimed at disrupting RNA secondary structure without affecting amino acid sequence did not affect RNA replication and packaging into virus-like particles in the packaging cell line, thus demonstrating that the predicted conserved RNA structures in the NS3 gene do not play a role in RNA replication and/or packaging. In contrast, double frameshift mutations in the NS3 coding region of full-length KUN RNA, producing scrambled NS3 protein but retaining secondary RNA structure, resulted in the loss of ability of these defective RNAs to be packaged into virus particles in complementation experiments in KUN replicon-expressing cells. Furthermore, the more robust complementation-packaging system based on established stable cell lines producing large amounts of complemented replicating NS3-deficient replicon RNAs and infection with KUN virus to provide structural proteins also failed to detect any secreted virus-like particles containing packaged NS3-deficient replicon RNAs. These results have now firmly established the requirement of KUN NS3 protein translated in cis for genome packaging into virus particles.

  17. A genome-wide shRNA screen identifies GAS1 as a novel melanoma metastasis suppressor gene.

    Science.gov (United States)

    Gobeil, Stephane; Zhu, Xiaochun; Doillon, Charles J; Green, Michael R

    2008-11-01

    Metastasis suppressor genes inhibit one or more steps required for metastasis without affecting primary tumor formation. Due to the complexity of the metastatic process, the development of experimental approaches for identifying genes involved in metastasis prevention has been challenging. Here we describe a genome-wide RNAi screening strategy to identify candidate metastasis suppressor genes. Following expression in weakly metastatic B16-F0 mouse melanoma cells, shRNAs were selected based upon enhanced satellite colony formation in a three-dimensional cell culture system and confirmed in a mouse experimental metastasis assay. Using this approach we discovered 22 genes whose knockdown increased metastasis without affecting primary tumor growth. We focused on one of these genes, Gas1 (Growth arrest-specific 1), because we found that it was substantially down-regulated in highly metastatic B16-F10 melanoma cells, which contributed to the high metastatic potential of this mouse cell line. We further demonstrated that Gas1 has all the expected properties of a melanoma tumor suppressor including: suppression of metastasis in a spontaneous metastasis assay, promotion of apoptosis following dissemination of cells to secondary sites, and frequent down-regulation in human melanoma metastasis-derived cell lines and metastatic tumor samples. Thus, we developed a genome-wide shRNA screening strategy that enables the discovery of new metastasis suppressor genes.

  18. cDNA sequencing improves the detection of P53 missense mutations in colorectal cancer

    International Nuclear Information System (INIS)

    Szybka, Malgorzata; Kordek, Radzislaw; Zakrzewska, Magdalena; Rieske, Piotr; Pasz-Walczak, Grazyna; Kulczycka-Wojdala, Dominika; Zawlik, Izabela; Stawski, Robert; Jesionek-Kupnicka, Dorota; Liberski, Pawel P

    2009-01-01

    Recently published data showed discrepancies beteween P53 cDNA and DNA sequencing in glioblastomas. We hypothesised that similar discrepancies may be observed in other human cancers. To this end, we analyzed 23 colorectal cancers for P53 mutations and gene expression using both DNA and cDNA sequencing, real-time PCR and immunohistochemistry. We found P53 gene mutations in 16 cases (15 missense and 1 nonsense). Two of the 15 cases with missense mutations showed alterations based only on cDNA, and not DNA sequencing. Moreover, in 6 of the 15 cases with a cDNA mutation those mutations were difficult to detect in the DNA sequencing, so the results of DNA analysis alone could be misinterpreted if the cDNA sequencing results had not also been available. In all those 15 cases, we observed a higher ratio of the mutated to the wild type template by cDNA analysis, but not by the DNA analysis. Interestingly, a similar overexpression of P53 mRNA was present in samples with and without P53 mutations. In terms of colorectal cancer, those discrepancies might be explained under three conditions: 1, overexpression of mutated P53 mRNA in cancer cells as compared with normal cells; 2, a higher content of cells without P53 mutation (normal cells and cells showing K-RAS and/or APC but not P53 mutation) in samples presenting P53 mutation; 3, heterozygous or hemizygous mutations of P53 gene. Additionally, for heterozygous mutations unknown mechanism(s) causing selective overproduction of mutated allele should also be considered. Our data offer new clues for studying discrepancy in P53 cDNA and DNA sequencing analysis

  19. Delineation of the Marfan phenotype associated with mutations in exons 23-32 of the FBN1 gene

    Energy Technology Data Exchange (ETDEWEB)

    Putnam, E.A.; Cho, M.; Milewicz, D.M. [Univ. of Texas-Houston Medical School, Houston, TX (United States)] [and others

    1996-03-29

    Marfan syndrome is a dominantly inherited connective tissue disorder with a wide range of phenotypic severity. The condition is the result of mutations in FBN1, a large gene composed of 65 exons encoding the fibrillin-1 protein. While mutations causing classic manifestations of Marfan syndrome have been identified throughout the FBN1 gene, the six previously characterized mutations resulting in the severe, perinatal lethal form of Marfan syndrome have clustered in exons 24-32 of the gene. We screened 8 patients with either neonatal Marfan syndrome or severe cardiovascular complications of Marfan syndrome for mutations in this region of the gene. Using intron-based exon-specific primers, we amplified exons 23-32 from genomic DNAs, screened these fragments by single-stranded conformational polymorphism analysis, and sequenced indicated exons. This analysis documented mutations in exons 25-27 of the FBN1 mutations in 6 of these patients. These results, taken together with previously published FBN1 mutations in this region, further define the phenotype associated with mutations in exons 24-32 of the FBN1 gene, information important for the development of possible diagnostic tests and genetic counseling. 49 refs., 4 figs., 2 tabs.

  20. Role of RNase MRP in viral RNA degradation and RNA recombination.

    Science.gov (United States)

    Jaag, Hannah M; Lu, Qiasheng; Schmitt, Mark E; Nagy, Peter D

    2011-01-01

    RNA degradation, together with RNA synthesis, controls the steady-state level of viral RNAs in infected cells. The endoribonucleolytic cleavage of viral RNA is important not only for viral RNA degradation but for RNA recombination as well, due to the participation of some RNA degradation products in the RNA recombination process. To identify host endoribonucleases involved in degradation of Tomato bushy stunt virus (TBSV) in a Saccharomyces cerevisiae model host, we tested eight known endoribonucleases. Here we report that downregulation of SNM1, encoding a component of the RNase MRP, and a temperature-sensitive mutation in the NME1 gene, coding for the RNA component of RNase MRP, lead to reduced production of the endoribonucleolytically cleaved TBSV RNA in yeast. We also show that the highly purified yeast RNase MRP cleaves the TBSV RNA in vitro, resulting in TBSV RNA degradation products similar in size to those observed in yeast cells. Knocking down the NME1 homolog in Nicotiana benthamiana also led to decreased production of the cleaved TBSV RNA, suggesting that in plants, RNase MRP is involved in TBSV RNA degradation. Altogether, this work suggests a role for the host endoribonuclease RNase MRP in viral RNA degradation and recombination.

  1. Bulldog dwarfism in Dexter cattle is caused by mutations in ACAN.

    Science.gov (United States)

    Cavanagh, Julie A L; Tammen, Imke; Windsor, Peter A; Bateman, John F; Savarirayan, Ravi; Nicholas, Frank W; Raadsma, Herman W

    2007-11-01

    Bulldog dwarfism in Dexter cattle is one of the earliest single-locus disorders described in animals. Affected fetuses display extreme disproportionate dwarfism, reflecting abnormal cartilage development (chondrodysplasia). Typically, they die around the seventh month of gestation, precipitating a natural abortion. Heterozygotes show a milder form of dwarfism, most noticeably having shorter legs. Homozygosity mapping in candidate regions in a small Dexter pedigree suggested aggrecan (ACAN) as the most likely candidate gene. Mutation screening revealed a 4-bp insertion in exon 11 (2266_2267insGGCA) (called BD1 for diagnostic testing) and a second, rarer transition in exon 1 (-198C>T) (called BD2) that cosegregate with the disorder. In chondrocytes from cattle heterozygous for the insertion, mutant mRNA is subject to nonsense-mediated decay, showing only 8% of normal expression. Genotyping in Dexter families throughout the world shows a one-to-one correspondence between genotype and phenotype at this locus. The heterozygous and homozygous-affected Dexter cattle could prove invaluable as a model for human disorders caused by mutations in ACAN.

  2. A Phenotype-Driven Approach to Generate Mouse Models with Pathogenic mtDNA Mutations Causing Mitochondrial Disease

    Directory of Open Access Journals (Sweden)

    Johanna H.K. Kauppila

    2016-09-01

    Full Text Available Mutations of mtDNA are an important cause of human disease, but few animal models exist. Because mammalian mitochondria cannot be transfected, the development of mice with pathogenic mtDNA mutations has been challenging, and the main strategy has therefore been to introduce mutations found in cell lines into mouse embryos. Here, we describe a phenotype-driven strategy that is based on detecting clonal expansion of pathogenic mtDNA mutations in colonic crypts of founder mice derived from heterozygous mtDNA mutator mice. As proof of concept, we report the generation of a mouse line transmitting a heteroplasmic pathogenic mutation in the alanine tRNA gene of mtDNA displaying typical characteristics of classic mitochondrial disease. In summary, we describe a straightforward and technically simple strategy based on mouse breeding and histology to generate animal models of mtDNA-mutation disease, which will be of great importance for studies of disease pathophysiology and preclinical treatment trials.

  3. Characterization of the ptr5+ gene involved in nuclear mRNA export in fission yeast

    International Nuclear Information System (INIS)

    Watanabe, Nobuyoshi; Ikeda, Terumasa; Mizuki, Fumitaka; Tani, Tokio

    2012-01-01

    Highlights: ► We cloned the ptr5 + gene involved in nuclear mRNA export in fission yeast. ► The ptr5 + gene was found to encode nucleoporin 85 (Nup85). ► Seh1p and Mlo3p are multi-copy suppressors for the ptr5 mutation. ► Ptr5p/Nup85p functions in nuclear mRNA export through the mRNA export factor Rae1p. ► Ptr5p/Nup85p interacts genetically with pre-mRNA splicing factors. -- Abstract: To analyze the mechanisms of mRNA export from the nucleus to the cytoplasm, we have isolated eleven mutants, ptr [poly(A) + RNA transport] 1 to 11, which accumulate poly(A) + RNA in the nucleus at a nonpermissive temperature in Schizosaccharomyces pombe. Of those, the ptr5–1 mutant shows dots- or a ring-like accumulation of poly(A) + RNA at the nuclear periphery after shifting to the nonpermissive temperature. We cloned the ptr5 + gene and found that it encodes a component of the nuclear pore complex (NPC), nucleoporin 85 (Nup85). The ptr5–1 mutant shows no defects in protein transport, suggesting the specific involvement of Ptr5p/Nup85p in nuclear mRNA export in S. pombe. We identified Seh1p, a nucleoporin interacting with Nup85p, an mRNA-binding protein Mlo3p, and Sac3p, a component of the TREX-2 complex involved in coupling of nuclear mRNA export with transcription, as multi-copy suppressors for the ptr5–1 mutation. In addition, we found that the ptr5–1 mutation is synthetically lethal with a mutation of the mRNA export factor Rae1p, and that the double mutant exaggerates defective nuclear mRNA export, suggesting that Ptr5p/Nup85p is involved in nuclear mRNA export through Rae1p. Interestingly, the ptr5–1 mutation also showed synthetic effects with several prp pre-mRNA splicing mutations, suggesting a functional linkage between the NPCs and the splicing apparatus in the yeast nucleus.

  4. A Viral RNA Structural Element Alters Host Recognition of Nonself RNA

    Energy Technology Data Exchange (ETDEWEB)

    Hyde, J. L.; Gardner, C. L.; Kimura, T.; White, J. P.; Liu, G.; Trobaugh, D. W.; Huang, C.; Tonelli, M.; Paessler, S.; Takeda, K.; Klimstra, W. B.; Amarasinghe, G. K.; Diamond, M. S.

    2014-01-30

    Although interferon (IFN) signaling induces genes that limit viral infection, many pathogenic viruses overcome this host response. As an example, 2'-O methylation of the 5' cap of viral RNA subverts mammalian antiviral responses by evading restriction of Ifit1, an IFN-stimulated gene that regulates protein synthesis. However, alphaviruses replicate efficiently in cells expressing Ifit1 even though their genomic RNA has a 5' cap lacking 2'-O methylation. We show that pathogenic alphaviruses use secondary structural motifs within the 5' untranslated region (UTR) of their RNA to alter Ifit1 binding and function. Mutations within the 5'-UTR affecting RNA structural elements enabled restriction by or antagonism of Ifit1 in vitro and in vivo. These results identify an evasion mechanism by which viruses use RNA structural motifs to avoid immune restriction.

  5. High-resolution melting curve analysis for rapid detection of mutations in a Medaka TILLING library

    Directory of Open Access Journals (Sweden)

    Deguchi Tomonori

    2010-09-01

    Full Text Available Abstract Background During the last two decades, DNA sequencing has led to the identification of numerous genes in key species; however, in most cases, their functions are still unknown. In this situation, reverse genetics is the most suitable method to assign function to a gene. TILLING (Targeting Induced Local Lesions IN Genomes is a reverse-genetic strategy that combines random chemical mutagenesis with high-throughput discovery of the induced mutations in target genes. The method has been applied to a variety of plant and animal species. Screening of the induced mutations is the most important step in TILLING. Currently, direct sequencing or nuclease-mediated screening of heteroduplexes is widely used for detection of mutations in TILLING. Both methods are useful, but the costs are substantial and turnaround times are relatively long. Thus, there is a need for an alternative method that is of higher throughput and more cost effective. Results In this study, we developed a high resolution melting (HRM assay and evaluated its effectiveness for screening ENU-induced mutations in a medaka TILLING library. We had previously screened mutations in the p53 gene by direct sequencing. Therefore, we first tested the efficiency of the HRM assay by screening mutations in p53, which indicated that the HRM assay is as useful as direct sequencing. Next, we screened mutations in the atr and atm genes with the HRM assay. Nonsense mutations were identified in each gene, and the phenotypes of these nonsense mutants confirmed their loss-of-function nature. Conclusions These results demonstrate that the HRM assay is useful for screening mutations in TILLING. Furthermore, the phenotype of the obtained mutants indicates that medaka is an excellent animal model for investigating genome stability and gene function, especially when combined with TILLING.

  6. Mutation analysis of breast cancer gene BRCA among breast cancer Jordanian females

    International Nuclear Information System (INIS)

    Atoum, Manar F.; Al-Kayed, Sameer A.

    2004-01-01

    To screen mutations of the tumor suppressor breast cancer susceptibility gene 1 (BRCA1) within 3 exons among Jordanian breast cancer females. A total of 135 Jordanian breast cancer females were genetically analyzed by denaturing gradient electrophoresis (DGGE) for mutation detection in 3 BRCA1 exons (2, 11 and 20) between 2000-2002 in Al-Basheer Hospital, Amman, Jordan. Of the studied patients 50 had a family history of breast cancer, 28 had a family history of cancer other than breast cancer, and 57 had no family history of any cancer. Five germline mutations were detected among breast cancer females with a family history of breast cancers (one in exon 2 and 4 mutations in exon 11). Another germline mutation (within exon 11) was detected among breast cancer females with family history of cancer other than breast cancer, and no mutation was detected among breast cancer females with no family history of any cancer or among normal control females. Screening mutations within exon 2, exon 11 and exon 20 showed that most screened mutations were within BRCA1 exon 11 among breast cancer Jordanian families with a family history of breast cancer. (author)

  7. Exploiting Pre-rRNA Processing in Diamond Blackfan Anemia Gene Discovery and Diagnosis

    Science.gov (United States)

    Farrar, Jason E.; Quarello, Paola; Fisher, Ross; O’Brien, Kelly A.; Aspesi, Anna; Parrella, Sara; Henson, Adrianna L.; Seidel, Nancy E.; Atsidaftos, Eva; Prakash, Supraja; Bari, Shahla; Garelli, Emanuela; Arceci, Robert J.; Dianzani, Irma; Ramenghi, Ugo; Vlachos, Adrianna; Lipton, Jeffrey M.; Bodine, David M.; Ellis, Steven R.

    2014-01-01

    Diamond Blackfan anemia (DBA), a syndrome primarily characterized by anemia and physical abnormalities, is one among a group of related inherited bone marrow failure syndromes (IBMFS) which share overlapping clinical features. Heterozygous mutations or single-copy deletions have been identified in 12 ribosomal protein genes in approximately 60% of DBA cases, with the genetic etiology unexplained in most remaining patients. Unlike many IBMFS, for which functional screening assays complement clinical and genetic findings, suspected DBA in the absence of typical alterations of the known genes must frequently be diagnosed after exclusion of other IBMFS. We report here a novel deletion in a child that presented such a diagnostic challenge and prompted development of a novel functional assay that can assist in the diagnosis of a significant fraction of patients with DBA. The ribosomal proteins affected in DBA are required for pre-rRNA processing, a process which can be interrogated to monitor steps in the maturation of 40S and 60S ribosomal subunits. In contrast to prior methods used to assess pre-rRNA processing, the assay reported here, based on capillary electrophoresis measurement of the maturation of rRNA in pre-60S ribosomal subunits, would be readily amenable to use in diagnostic laboratories. In addition to utility as a diagnostic tool, we applied this technique to gene discovery in DBA, resulting in the identification of RPL31 as a novel DBA gene. PMID:25042156

  8. Rapid screening of ASXL1, IDH1, IDH2, and c-CBL mutations in de novo acute myeloid leukemia by high-resolution melting.

    Science.gov (United States)

    Ibáñez, Mariam; Such, Esperanza; Cervera, José; Luna, Irene; Gómez-Seguí, Inés; López-Pavía, María; Dolz, Sandra; Barragán, Eva; Fuster, Oscar; Llop, Marta; Rodríguez-Veiga, Rebeca; Avaria, Amparo; Oltra, Silvestre; Senent, M Leonor; Moscardó, Federico; Montesinos, Pau; Martínez-Cuadrón, David; Martín, Guillermo; Sanz, Miguel A

    2012-11-01

    Recently, many novel molecular abnormalities were found to be distinctly associated with acute myeloid leukemia (AML). However, their clinical relevance and prognostic implications are not well established. We developed a new combination of high-resolution melting assays on a LightCycler 480 and direct sequencing to detect somatic mutations of ASXL1 (exon 12), IDH1 (exon 4), IDH2 (exon 4), and c-CBL (exons 8 and 9) genes to know their incidence and prognostic effect in a cohort of 175 patients with de novo AML: 16 patients (9%) carried ASXL1 mutations, 16 patients had IDH variations (3% with IDH1(R132) and 6% with IDH2(R140)), and none had c-CBL mutations. Patients with ASXL1 mutations did not harbor IDH1, [corrected] or CEBPA mutations, and a combination of ASXL1 and IDH2 mutations was found only in one patient. In addition, we did not find IDH1 and FLT3 or CEBPA mutations concurrently or IDH2 with CEBPA. IDH1 and IDH2 mutations were mutually exclusive. Alternatively, NPM1 mutations were concurrently found with ASXL1, IDH1, or IDH2 with a variable incidence. Mutations were not significantly correlated with any of the clinical and biological features studied. High-resolution melting is a reliable, rapid, and efficient screening technique for mutation detection in AML. The incidence for the studied genes was in the range of those previously reported. We were unable to find an effect on the outcome. Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  9. Mutation breeding in pepper

    Energy Technology Data Exchange (ETDEWEB)

    Daskalov, S [Plant Breeding Unit, Joint FAO/IAEA Division of Isotope and Radiation Applications of Atomic Energy for Food and Agricultural Development, Seibersdorf Laboratory, International Atomic Energy Agency, Vienna (Austria)

    1986-03-01

    Pepper (Capsicum sp.) is an important vegetable and spice crop widely grown in tropical as well as in temperate regions. Until recently the improvement programmes were based mainly on using natural sources of germ plasma, crossbreeding and exploiting the heterosis of F{sub 1} hybrids. However, interest in using induced mutations is growing. A great number of agronomically useful mutants as well as mutants valuable for genetic, cytological and physiological studies have been induced and described. In this review information is presented about suitable mutagen treatment procedures with radiation as well as chemicals, M{sub 1} effects, handling the treated material in M{sub 1}, M{sub 2} and subsequent generations, and mutant screening procedures. This is supplemented by a description of reported useful mutants and released cultivars. Finally, general advice is given on when and how to incorporate mutation induction in Capsicum improvement programmes. (author)

  10. Association of mutations in the hemochromatosis gene with shorter life expectancy

    DEFF Research Database (Denmark)

    Bathum, L; Christiansen, L; Nybo, H

    2001-01-01

    BACKGROUND: To investigate whether the frequency of carriers of mutations in the HFE gene associated with hereditary hemochromatosis diminishes with age as an indication that HFE mutations are associated with increased mortality. It is of value in the debate concerning screening for hereditary...... hemochromatosis to determine the significance of heterozygosity. METHODS: Genotyping for mutations in exons 2 and 4 of the HFE gene using denaturing gradient gel electrophoresis in 1784 participants aged 45 to 100 years from 4 population-based studies: all 183 centenarians from the Danish Centenarian Study, 601...... in the distribution of mutations in exon 2 in the different age groups. CONCLUSIONS: In a high-carrier frequency population like Denmark, mutations in HFE show an age-related reduction in the frequency of heterozygotes for C282Y, which suggests that carrier status is associated with shorter life expectancy....

  11. Molecular genetic approach for screening of hereditary non-polyposis colorectal cancer

    Directory of Open Access Journals (Sweden)

    Metka Ravnik-Glavač

    2005-07-01

    Full Text Available Background: The main goal of knowledge concerning human diseases is to transfer as much as possible useful information into clinical applications. Hereditary non-polyposis colorectal cancer (HNPCC is the most common autosomal dominant inherited predisposition for colorectal cancer, accounting for 1–2% of all bowel cancer. The only way to diagnose HNPCC is by a family history consistent with the disease defined by International Collaborative Group on HNPCC (Amsterdam criteria I and II. The main molecular cause of HNPCC is a constitutional mutation in one of the mismatch repair (MMR genes. Since HNPCC mutations have been detected also in families that did not fulfil the Amsterdam criteria, molecular genetic characteristics of HNPCC cancers have been proposed as valuable first step in HNPCC identification. Microsatellite instability is present in about 90% of cancers of HNPCC patients. However, of all MSI colorectal cancers 80– 90% are sporadic. Several molecular mechanisms have been uncovered that enable distinguishing to some extent between sporadic and HNPCC cancers with MSI including hypermethylation of hMLH1 promoter and frequent mutations in BAX and TGFBR2 in sporadic CRC with MSI-H.Conclusions: The determination of MSI status and careful separation of MSI positive colorectal cancer into sporadic MSIL, sporadic MSI-H, and HNPCC MSI-H followed by mutation detection in MMR genes is important for prevention, screening and management of colorectal cancer. In some studies we and others have already shown that large-scale molecular genetic analysis for HNPCC can be done and is sensitive enough to approve population screening. Population screening includes also colonoscopy which is restricted only to the obligate carriers of the mutation. This enables that the disease is detected in earlier stages which would greatly decrease medical treatment costs and most importantly decrease mortality. In Slovenia we have started population screening based

  12. Molecular Beacon-Based MicroRNA Imaging During Neurogenesis.

    Science.gov (United States)

    Lee, Jonghwan; Kim, Soonhag

    2016-01-01

    The fluorescence monitoring system for examining endogenous microRNA (miRNA) activity in cellular level provides crucial information on not only understanding a critical role of miRNA involving a variety of biological processes, but also evaluating miRNA expression patterns in a noninvasive manner. In this protocol, we report the details of a new procedure for a molecular beacon-based miRNA monitoring system, which includes the illustration scheme for miRNA detection strategy, exogenous miRNA detection, and measurement of endogenous miRNA expression level during neurogenesis. The fluorescence signal of miR-124a beacon quenched by BHQ2 was gradually recovered as increasing concentration of the miR-124a in tube. The functional work of miR-124a beacon was examined in intracellular environment, allowing for the internalization of the miR-124a beacon by lipofectamine, which resulted in activated fluorescent signals of the miR-124a beacon in the HeLa cells after the addition of synthetic miR-124a. The endogenous miR-124a expression level was detected by miR-124a beacon system during neurogenesis, showing brighter fluorescence intensity in cytoplasmic area of P19 cells after induction of neuronal differentiation by retinoic acid. The molecular beacon based-miRNA detection technique could be applicable to the simultaneous visualization of a variety of miRNA expression patterns using different fluorescence dyes. For the study of examining endogenous miRNA expression level using miRNA-beacon system, if cellular differentiation step is already prepared, transfection step of miR-124a beacon into P19 cells, and acquisition of activated fluorescence signal measured by confocal microscope can be conducted approximately within 6 h.

  13. Validation of high-resolution DNA melting analysis for mutation scanning of the CDKL5 gene: identification of novel mutations.

    Science.gov (United States)

    Raymond, Laure; Diebold, Bertrand; Leroux, Céline; Maurey, Hélène; Drouin-Garraud, Valérie; Delahaye, Andre; Dulac, Olivier; Metreau, Julia; Melikishvili, Gia; Toutain, Annick; Rivier, François; Bahi-Buisson, Nadia; Bienvenu, Thierry

    2013-01-01

    Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) have been predominantly described in epileptic encephalopathies of female, including infantile spasms with Rett-like features. Up to now, detection of mutations in this gene was made by laborious, expensive and/or time consuming methods. Here, we decided to validate high-resolution melting analysis (HRMA) for mutation scanning of the CDKL5 gene. Firstly, using a large DNA bank consisting to 34 samples carrying different mutations and polymorphisms, we validated our analytical conditions to analyse the different exons and flanking intronic sequences of the CDKL5 gene by HRMA. Secondly, we screened CDKL5 by both HRMA and denaturing high performance liquid chromatography (dHPLC) in a cohort of 135 patients with early-onset seizures. Our results showed that point mutations and small insertions and deletions can be reliably detected by HRMA. Compared to dHPLC, HRMA profiles are more discriminated, thereby decreasing unnecessary sequencing. In this study, we identified eleven novel sequence variations including four pathogenic mutations (2.96% prevalence). HRMA appears cost-effective, easy to set up, highly sensitive, non-toxic and rapid for mutation screening, ideally suited for large genes with heterogeneous mutations located along the whole coding sequence, such as the CDKL5 gene. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. A Short History and Description of Drosophila melanogaster Classical Genetics: Chromosome Aberrations, Forward Genetic Screens, and the Nature of Mutations.

    Science.gov (United States)

    Kaufman, Thomas C

    2017-06-01

    The purpose of this chapter in FlyBook is to acquaint the reader with the Drosophila genome and the ways in which it can be altered by mutation. Much of what follows will be familiar to the experienced Fly Pusher but hopefully will be useful to those just entering the field and are thus unfamiliar with the genome, the history of how it has been and can be altered, and the consequences of those alterations. I will begin with the structure, content, and organization of the genome, followed by the kinds of structural alterations (karyotypic aberrations), how they affect the behavior of chromosomes in meiotic cell division, and how that behavior can be used. Finally, screens for mutations as they have been performed will be discussed. There are several excellent sources of detailed information on Drosophila husbandry and screening that are recommended for those interested in further expanding their familiarity with Drosophila as a research tool and model organism. These are a book by Ralph Greenspan and a review article by John Roote and Andreas Prokop, which should be required reading for any new student entering a fly lab for the first time. Copyright © 2017 by the Genetics Society of America.

  15. Screening for mutations in two exons of FANCG gene in Pakistani population.

    Science.gov (United States)

    Aymun, Ujala; Iram, Saima; Aftab, Iram; Khaliq, Saba; Nadir, Ali; Nisar, Ahmed; Mohsin, Shahida

    2017-06-01

    Fanconi anemia is a rare autosomal recessive disorder of genetic instability. It is both molecularly and clinically, a heterogeneous disorder. Its incidence is 1 in 129,000 births and relatively high in some ethnic groups. Sixteen genes have been identified among them mutations in FANCG gene are most common after FANCA and FANCC gene mutations. To study mutations in exon 3 and 4 of FANCG gene in Pakistani population. Thirty five patients with positive Diepoxybutane test were included in the study. DNA was extracted and amplified for exons 3 and 4. Thereafter Sequencing was done and analyzed for the presence of mutations. No mutation was detected in exon 3 whereas a carrier of known mutation c.307+1 G>T was found in exon 4 of the FANCG gene. Absence of any mutation in exon 3 and only one heterozygous mutation in exon 4 of FANCG gene points to a different spectrum of FA gene pool in Pakistan that needs extensive research in this area.

  16. A mutation in a functional Sp1 binding site of the telomerase RNA gene (hTERC promoter in a patient with Paroxysmal Nocturnal Haemoglobinuria

    Directory of Open Access Journals (Sweden)

    Mason Philip J

    2004-06-01

    Full Text Available Abstract Background Mutations in the gene coding for the RNA component of telomerase, hTERC, have been found in autosomal dominant dyskeratosis congenita (DC and aplastic anemia. Paroxysmal nocturnal hemoglobinuria (PNH is a clonal blood disorder associated with aplastic anemia and characterized by the presence of one or more clones of blood cells lacking glycosylphosphatidylinositol (GPI anchored proteins due to a somatic mutation in the PIGA gene. Methods We searched for mutations in DNA extracted from PNH patients by amplification of the hTERC gene and denaturing high performance liquid chromatography (dHPLC. After a mutation was found in a potential transcription factor binding site in one patient electrophoretic mobility shift assays were used to detect binding of transcription factors to that site. The effect of the mutation on the function of the promoter was tested by transient transfection constructs in which the promoter is used to drive a reporter gene. Results Here we report the finding of a novel promoter mutation (-99C->G in the hTERC gene in a patient with PNH. The mutation disrupts an Sp1 binding site and destroys its ability to bind Sp1. Transient transfection assays show that mutations in this hTERC site including C-99G cause either up- or down-regulation of promoter activity and suggest that the site regulates core promoter activity in a context dependent manner in cancer cells. Conclusions These data are the first report of an hTERC promoter mutation from a patient sample which can modulate core promoter activity in vitro, raising the possibility that the mutation may affect the transcription of the gene in hematopoietic stem cells in vivo, and that dysregulation of telomerase may play a role in the development of bone marrow failure and the evolution of PNH clones.

  17. DES-ncRNA: A knowledgebase for exploring information about human micro and long noncoding RNAs based on literature-mining

    KAUST Repository

    Salhi, Adil

    2017-04-07

    Noncoding RNAs (ncRNAs), particularly microRNAs (miRNAs) and long ncRNAs (lncRNAs), are important players in diseases and emerge as novel drug targets. Thus, unraveling the relationships between ncRNAs and other biomedical entities in cells are critical for better understanding ncRNA roles that may eventually help develop their use in medicine. To support ncRNA research and facilitate retrieval of relevant information regarding miRNAs and lncRNAs from the plethora of published ncRNA-related research, we developed DES-ncRNA ( www.cbrc.kaust.edu.sa/des_ncrna ). DES-ncRNA is a knowledgebase containing text- and data-mined information from public scientific literature and other public resources. Exploration of mined information is enabled through terms and pairs of terms from 19 topic-specific dictionaries including, for example, antibiotics, toxins, drugs, enzymes, mutations, pathways, human genes and proteins, drug indications and side effects, mutations, diseases, etc. DES-ncRNA contains approximately 878,000 associations of terms from these dictionaries of which 36,222 (5,373) are with regards to miRNAs (lncRNAs). We provide several ways to explore information regarding ncRNAs to users including controlled generation of association networks as well as hypotheses generation. We show an example how DES-ncRNA can aid research on Alzheimer\\'s disease and suggest potential therapeutic role for Fasudil. DES-ncRNA is a powerful tool that can be used on its own or as a complement to the existing resources, to support research in human ncRNA. To our knowledge, this is the only knowledgebase dedicated to human miRNAs and lncRNAs derived primarily through literature-mining enabling exploration of a broad spectrum of associated biomedical entities, not paralleled by any other resource.

  18. DES-ncRNA: A knowledgebase for exploring information about human micro and long noncoding RNAs based on literature-mining.

    Science.gov (United States)

    Salhi, Adil; Essack, Magbubah; Alam, Tanvir; Bajic, Vladan P; Ma, Lina; Radovanovic, Aleksandar; Marchand, Benoit; Schmeier, Sebastian; Zhang, Zhang; Bajic, Vladimir B

    2017-07-03

    Noncoding RNAs (ncRNAs), particularly microRNAs (miRNAs) and long ncRNAs (lncRNAs), are important players in diseases and emerge as novel drug targets. Thus, unraveling the relationships between ncRNAs and other biomedical entities in cells are critical for better understanding ncRNA roles that may eventually help develop their use in medicine. To support ncRNA research and facilitate retrieval of relevant information regarding miRNAs and lncRNAs from the plethora of published ncRNA-related research, we developed DES-ncRNA ( www.cbrc.kaust.edu.sa/des_ncrna ). DES-ncRNA is a knowledgebase containing text- and data-mined information from public scientific literature and other public resources. Exploration of mined information is enabled through terms and pairs of terms from 19 topic-specific dictionaries including, for example, antibiotics, toxins, drugs, enzymes, mutations, pathways, human genes and proteins, drug indications and side effects, mutations, diseases, etc. DES-ncRNA contains approximately 878,000 associations of terms from these dictionaries of which 36,222 (5,373) are with regards to miRNAs (lncRNAs). We provide several ways to explore information regarding ncRNAs to users including controlled generation of association networks as well as hypotheses generation. We show an example how DES-ncRNA can aid research on Alzheimer disease and suggest potential therapeutic role for Fasudil. DES-ncRNA is a powerful tool that can be used on its own or as a complement to the existing resources, to support research in human ncRNA. To our knowledge, this is the only knowledgebase dedicated to human miRNAs and lncRNAs derived primarily through literature-mining enabling exploration of a broad spectrum of associated biomedical entities, not paralleled by any other resource.

  19. DES-ncRNA: A knowledgebase for exploring information about human micro and long noncoding RNAs based on literature-mining

    KAUST Repository

    Salhi, Adil; Essack, Magbubah; Alam, Tanvir; Bajic, Vladan P.; Ma, Lina; Radovanovic, Aleksandar; Marchand, Benoit; Schmeier, Sebastian; Zhang, Zhang; Bajic, Vladimir B.

    2017-01-01

    Noncoding RNAs (ncRNAs), particularly microRNAs (miRNAs) and long ncRNAs (lncRNAs), are important players in diseases and emerge as novel drug targets. Thus, unraveling the relationships between ncRNAs and other biomedical entities in cells are critical for better understanding ncRNA roles that may eventually help develop their use in medicine. To support ncRNA research and facilitate retrieval of relevant information regarding miRNAs and lncRNAs from the plethora of published ncRNA-related research, we developed DES-ncRNA ( www.cbrc.kaust.edu.sa/des_ncrna ). DES-ncRNA is a knowledgebase containing text- and data-mined information from public scientific literature and other public resources. Exploration of mined information is enabled through terms and pairs of terms from 19 topic-specific dictionaries including, for example, antibiotics, toxins, drugs, enzymes, mutations, pathways, human genes and proteins, drug indications and side effects, mutations, diseases, etc. DES-ncRNA contains approximately 878,000 associations of terms from these dictionaries of which 36,222 (5,373) are with regards to miRNAs (lncRNAs). We provide several ways to explore information regarding ncRNAs to users including controlled generation of association networks as well as hypotheses generation. We show an example how DES-ncRNA can aid research on Alzheimer's disease and suggest potential therapeutic role for Fasudil. DES-ncRNA is a powerful tool that can be used on its own or as a complement to the existing resources, to support research in human ncRNA. To our knowledge, this is the only knowledgebase dedicated to human miRNAs and lncRNAs derived primarily through literature-mining enabling exploration of a broad spectrum of associated biomedical entities, not paralleled by any other resource.

  20. Biosensor-based microRNA detection: techniques, design, performance, and challenges.

    Science.gov (United States)

    Johnson, Blake N; Mutharasan, Raj

    2014-04-07

    The current state of biosensor-based techniques for amplification-free microRNA (miRNA) detection is critically reviewed. Comparison with non-sensor and amplification-based molecular techniques (MTs), such as polymerase-based methods, is made in terms of transduction mechanism, associated protocol, and sensitivity. Challenges associated with miRNA hybridization thermodynamics which affect assay selectivity and amplification bias are briefly discussed. Electrochemical, electromechanical, and optical classes of miRNA biosensors are reviewed in terms of transduction mechanism, limit of detection (LOD), time-to-results (TTR), multiplexing potential, and measurement robustness. Current trends suggest that biosensor-based techniques (BTs) for miRNA assay will complement MTs due to the advantages of amplification-free detection, LOD being femtomolar (fM)-attomolar (aM), short TTR, multiplexing capability, and minimal sample preparation requirement. Areas of future importance in miRNA BT development are presented which include focus on achieving high measurement confidence and multiplexing capabilities.