BRAF, NRAS and C-KIT Advanced Melanoma: Clinico-pathological Features, Targeted-Therapy Strategies and Survival.

PubMed

Ponti, Giovanni; Manfredini, Marco; Greco, Stefano; Pellacani, Giovanni; Depenni, Roberta; Tomasi, Aldo; Maccaferri, Monia; Cascinu, Stefano

2017-12-01

The mutational status of stage III and IV melanomas should be recognized in order to allow for targeted therapies. The aim of our study was the characterization of BRAF, NRAS and C-KIT melanoma patients, in order to define their optimal management. Between 1991 and 2015, 63 mutated melanoma patients were treated and monitored during their diagnostic and therapeutic management at a single institution. BRAF-mutated melanoma patients were the most common, representing 70% of the study population, while NRAS- and C-KIT-mutated melanoma represented 19% and 11% respectively. BRAF-mutated melanomas were mostly located at sites of intermittent sun exposure, and were associated with higher Breslow thickness and an increased number of mitosis. NRAS mutated melanoma were mainly observed in chronic sun-damaged areas and had a negative prognostic value, with shorter time to progression and a high incidence of central nervous system involvement. C-KIT mutated melanoma were located at acral and mucosal sites. Overall survival observed in the three groups of patients revealed wide differences. BRAF, NRAS and C-KIT melanomas constitute distinct clinico-pathological entities. BRAF-mutated melanoma benefit from both anti-BRAF and anti-MEK targeted therapies while triple-negative melanomas could benefit from novel anti-CTLA-4 and anti-PD-L1 immunotherapeutic approaches. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Active RNAP pre-initiation sites are highly mutated by cytidine deaminases in yeast, with AID targeting small RNA genes

PubMed Central

Taylor, Benjamin JM; Wu, Yee Ling; Rada, Cristina

2014-01-01

Cytidine deaminases are single stranded DNA mutators diversifying antibodies and restricting viral infection. Improper access to the genome leads to translocations and mutations in B cells and contributes to the mutation landscape in cancer, such as kataegis. It remains unclear how deaminases access double stranded genomes and whether off-target mutations favor certain loci, although transcription and opportunistic access during DNA repair are thought to play a role. In yeast, AID and the catalytic domain of APOBEC3G preferentially mutate transcriptionally active genes within narrow regions, 110 base pairs in width, fixed at RNA polymerase initiation sites. Unlike APOBEC3G, AID shows enhanced mutational preference for small RNA genes (tRNAs, snoRNAs and snRNAs) suggesting a putative role for RNA in its recruitment. We uncover the high affinity of the deaminases for the single stranded DNA exposed by initiating RNA polymerases (a DNA configuration reproduced at stalled polymerases) without a requirement for specific cofactors. DOI: http://dx.doi.org/10.7554/eLife.03553.001 PMID:25237741

Broad Resistance to ACCase Inhibiting Herbicides in a Ryegrass Population Is Due Only to a Cysteine to Arginine Mutation in the Target Enzyme

PubMed Central

Kaundun, Shiv Shankhar; Hutchings, Sarah-Jane; Dale, Richard Paul; McIndoe, Eddie

2012-01-01

Background The design of sustainable weed management strategies requires a good understanding of the mechanisms by which weeds evolve resistance to herbicides. Here we have conducted a study on the mechanism of resistance to ACCase inhibiting herbicides in a Lolium multiflorum population (RG3) from the UK. Methodology/Principal Findings Analysis of plant phenotypes and genotypes showed that all the RG3 plants (72%) that contained the cysteine to arginine mutation at ACCase codon position 2088 were resistant to ACCase inhibiting herbicides. Whole plant dose response tests on predetermined wild and mutant 2088 genotypes from RG3 and a standard sensitive population indicated that the C2088R mutation is the only factor conferring resistance to all ten ACCase herbicides tested. The associated resistance indices ranged from 13 for clethodim to over 358 for diclofop-methyl. Clethodim, the most potent herbicide was significantly affected even when applied on small mutant plants at the peri-emergence and one leaf stages. Conclusion/Significance This study establishes the clear and unambiguous importance of the C2088R target site mutation in conferring broad resistance to ten commonly used ACCase inhibiting herbicides. It also demonstrates that low levels “creeping”, multigenic, non target site resistance, is not always selected before single gene target site resistance appears in grass weed populations subjected to herbicide selection pressure. PMID:22768118

A novel pathogenic splice acceptor site germline mutation in intron 14 of the APC gene in a Chinese family with familial adenomatous polyposis.

PubMed

Wang, Dan; Liang, Shengyun; Zhang, Zhao; Zhao, Guoru; Hu, Yuan; Liang, Shengran; Zhang, Xipeng; Banerjee, Santasree

2017-03-28

Familial adenomatous polyposis (FAP) is an autosomal dominant precancerous condition, clinically characterized by the presence of multiple colorectal adenomas or polyps. Patients with FAP has a high risk of developing colorectal cancer (CRC) from these colorectal adenomatous polyps by the mean age of diagnosis at 40 years. Germline mutations of the APC gene cause familial adenomatous polyposis (FAP). Colectomy has recommended for the FAP patients with significant polyposis. Here, we present a clinical molecular study of a four generation Chinese family with FAP. Clinical diagnosis of FAP has been done according to the phenotype, family history and medical records. Patient's blood samples were collected and genomic DNA was extracted. In order to identify the pathogenic mutation underlying the disease phenotype targeted next-generation sequencing and confirmatory sanger sequencing has undertaken. Targeted next generation sequencing identified a novel heterozygous splice-acceptor site mutation [c.1744-1G>A] in intron 14 of APC gene, which is co-segregated with the FAP phenotypes in the proband and amongst all the affected family members. This mutation is not present in unaffected family members and in normal healthy controls of same ethnic origin. According to the LOVD database for Chinese colorectal cancer patients, in Chinese population, 60% of the previously reported APC gene mutations causes FAP, are missense mutations. This novel splice-acceptor site mutation causing FAP in this Chinese family expands the germline mutation spectrum of the APC gene in the Chinese population.

CRISPR/Cas9-Mediated Insertion of loxP Sites in the Mouse Dock7 Gene Provides an Effective Alternative to Use of Targeted Embryonic Stem Cells.

PubMed

Bishop, Kathleen A; Harrington, Anne; Kouranova, Evguenia; Weinstein, Edward J; Rosen, Clifford J; Cui, Xiaoxia; Liaw, Lucy

2016-07-07

Targeted gene mutation in the mouse is a primary strategy to understand gene function and relation to phenotype. The Knockout Mouse Project (KOMP) had an initial goal to develop a public resource of mouse embryonic stem (ES) cell clones that carry null mutations in all genes. Indeed, many useful novel mouse models have been generated from publically accessible targeted mouse ES cell lines. However, there are limitations, including incorrect targeting or cassette structure, and difficulties with germline transmission of the allele from chimeric mice. In our experience, using a small sample of targeted ES cell clones, we were successful ∼50% of the time in generating germline transmission of a correctly targeted allele. With the advent of CRISPR/Cas9 as a mouse genome modification tool, we assessed the efficiency of creating a conditional targeted allele in one gene, dedicator of cytokinesis 7 (Dock7), for which we were unsuccessful in generating a null allele using a KOMP targeted ES cell clone. The strategy was to insert loxP sites to flank either exons 3 and 4, or exons 3 through 7. By coinjecting Cas9 mRNA, validated sgRNAs, and oligonucleotide donors into fertilized eggs from C57BL/6J mice, we obtained a variety of alleles, including mice homozygous for the null alleles mediated by nonhomologous end joining, alleles with one of the two desired loxP sites, and correctly targeted alleles with both loxP sites. We also found frequent mutations in the inserted loxP sequence, which is partly attributable to the heterogeneity in the original oligonucleotide preparation. Copyright © 2016 Bishop et al.

Rational modification of protein stability by targeting surface sites leads to complicated results

PubMed Central

Xiao, Shifeng; Patsalo, Vadim; Shan, Bing; Bi, Yuan; Green, David F.; Raleigh, Daniel P.

2013-01-01

The rational modification of protein stability is an important goal of protein design. Protein surface electrostatic interactions are not evolutionarily optimized for stability and are an attractive target for the rational redesign of proteins. We show that surface charge mutants can exert stabilizing effects in distinct and unanticipated ways, including ones that are not predicted by existing methods, even when only solvent-exposed sites are targeted. Individual mutation of three solvent-exposed lysines in the villin headpiece subdomain significantly stabilizes the protein, but the mechanism of stabilization is very different in each case. One mutation destabilizes native-state electrostatic interactions but has a larger destabilizing effect on the denatured state, a second removes the desolvation penalty paid by the charged residue, whereas the third introduces unanticipated native-state interactions but does not alter electrostatics. Our results show that even seemingly intuitive mutations can exert their effects through unforeseen and complex interactions. PMID:23798426

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

Yuchi, Zhiguang; Lau, Kelvin; Van Petegem, Filip

Ryanodine Receptors (RyRs) are huge Ca{sup 2+} release channels in the endoplasmic reticulum membrane and form targets for phosphorylation and disease mutations. We present crystal structures of a domain in three RyR isoforms, containing the Ser2843 (RyR1) and Ser2808/Ser2814 (RyR2) phosphorylation sites. The RyR1 domain is the target for 11 disease mutations. Several of these are clustered near the phosphorylation sites, suggesting that phosphorylation and disease mutations may affect the same interface. The L2867G mutation causes a drastic thermal destabilization and aggregation at room temperature. Crystal structures for other disease mutants show that they affect surface properties and intradomain saltmore » bridges. In vitro phosphorylation experiments show that up to five residues in one long loop of RyR2 can be phosphorylated by PKA or CaMKII. Docking into cryo-electron microscopy maps suggests a putative location in the clamp region, implying that mutations and phosphorylation may affect the allosteric motions within this area.« less

Targeted next-generation sequencing reveals that a compound heterozygous mutation in phosphodiesterase 6a gene leads to retinitis pigmentosa in a Chinese family.

PubMed

Zhang, Shanshan; Li, Jie; Li, Shujin; Yang, Yeming; Yang, Mu; Yang, Zhenglin; Zhu, Xianjun; Zhang, Lin

2018-04-25

Retinitis pigmentosa (RP) is a genetically heterogeneous disease with over 70 causative genes identified to date. However, approximately 40% of RP cases remain genetically unsolved, suggesting that many novel disease-causing mutations are yet to be identified. The purpose of this study is to identify the causative mutations of a Chinese RP family. Targeted next-generation sequencing (NGS) for a total of 163 genes which involved in inherited retinal disorders were used to screen the possible causative mutations. Sanger sequencing was used to verify the mutations. As results, we identified two heterozygous mutations: a splicing site mutation c.1407 + 1G>C and a nonsense mutation c. 1957C>T (p.R653X) in phosphodiesterase 6A (PDE6A) gene in the RP patient. These two mutations are inherited from his father and mother, respectively. Furthermore, these mutations are unique in our in-house database and are rare in human genome databases, implicating that these two mutations are pathological. By using targeted NGS method, we identified a compound heterozygous mutation in PDE6A gene that is associated with RP in a Chinese family.

Analogous pleiotropic effects of insecticide resistance genotypes in peach-potato aphids and houseflies.

PubMed

Foster, S P; Young, S; Williamson, M S; Duce, I; Denholm, I; Devine, G J

2003-08-01

We show that single-point mutations conferring target-site resistance (kdr) to pyrethroids and DDT in aphids and houseflies, and gene amplification conferring metabolic resistance (carboxylesterase) to organophosphates and carbamates in aphids, can have deleterious pleiotropic effects on fitness. Behavioural studies on peach-potato aphids showed that a reduced response to alarm pheromone was associated with both gene amplification and the kdr target-site mutation. In this species, gene amplification was also associated with a decreased propensity to move from senescing leaves to fresh leaves at low temperature. Housefly genotypes possessing the identical kdr mutation were also shown to exhibit behavioural differences in comparison with susceptible insects. In this species, resistant individuals showed no positional preference along a temperature gradient while susceptible genotypes exhibited a strong preference for warmer temperatures.

Deep mutational scanning identifies sites in influenza nucleoprotein that affect viral inhibition by MxA

PubMed Central

Ashenberg, Orr; Padmakumar, Jai

2017-01-01

The innate-immune restriction factor MxA inhibits influenza replication by targeting the viral nucleoprotein (NP). Human influenza virus is more resistant than avian influenza virus to inhibition by human MxA, and prior work has compared human and avian viral strains to identify amino-acid differences in NP that affect sensitivity to MxA. However, this strategy is limited to identifying sites in NP where mutations that affect MxA sensitivity have fixed during the small number of documented zoonotic transmissions of influenza to humans. Here we use an unbiased deep mutational scanning approach to quantify how all single amino-acid mutations to NP affect MxA sensitivity in the context of replication-competent virus. We both identify new sites in NP where mutations affect MxA resistance and re-identify mutations known to have increased MxA resistance during historical adaptations of influenza to humans. Most of the sites where mutations have the greatest effect are almost completely conserved across all influenza A viruses, and the amino acids at these sites confer relatively high resistance to MxA. These sites cluster in regions of NP that appear to be important for its recognition by MxA. Overall, our work systematically identifies the sites in influenza nucleoprotein where mutations affect sensitivity to MxA. We also demonstrate a powerful new strategy for identifying regions of viral proteins that affect inhibition by host factors. PMID:28346537

A novel W1999S mutation and non-target site resistance impact on acetyl-CoA carboxylase inhibiting herbicides to varying degrees in a UK Lolium multiflorum population.

PubMed

Kaundun, Shiv Shankhar; Bailly, Geraldine C; Dale, Richard P; Hutchings, Sarah-Jane; McIndoe, Eddie

2013-01-01

Acetyl-CoA carboxylase (ACCase) inhibiting herbicides are important products for the post-emergence control of grass weed species in small grain cereal crops. However, the appearance of resistance to ACCase herbicides over time has resulted in limited options for effective weed control of key species such as Lolium spp. In this study, we have used an integrated biological and molecular biology approach to investigate the mechanism of resistance to ACCase herbicides in a Lolium multiflorum Lam. from the UK (UK21). The study revealed a novel tryptophan to serine mutation at ACCase codon position 1999 impacting on ACCase inhibiting herbicides to varying degrees. The W1999S mutation confers dominant resistance to pinoxaden and partially recessive resistance to cycloxydim and sethoxydim. On the other hand, plants containing the W1999S mutation were sensitive to clethodim and tepraloxydim. Additionally population UK21 is characterised by other resistance mechanisms, very likely non non-target site based, affecting several aryloxyphenoxyproprionate (FOP) herbicides but not the practical field rate of pinoxaden. The positive identification of wild type tryptophan and mutant serine alleles at ACCase position 1999 could be readily achieved with an original DNA based derived cleaved amplified polymorphic sequence (dCAPS) assay that uses the same PCR product but two different enzymes for positively identifying the wild type tryptophan and mutant serine alleles identified here. This paper highlights intrinsic differences between ACCase inhibiting herbicides that could be exploited for controlling ryegrass populations such as UK21 characterised by compound-specific target site and non-target site resistance.

A Novel W1999S Mutation and Non-Target Site Resistance Impact on Acetyl-CoA Carboxylase Inhibiting Herbicides to Varying Degrees in a UK Lolium multiflorum Population

PubMed Central

Kaundun, Shiv Shankhar; Bailly, Geraldine C.; Dale, Richard P.; Hutchings, Sarah-Jane; McIndoe, Eddie

2013-01-01

Background Acetyl-CoA carboxylase (ACCase) inhibiting herbicides are important products for the post-emergence control of grass weed species in small grain cereal crops. However, the appearance of resistance to ACCase herbicides over time has resulted in limited options for effective weed control of key species such as Lolium spp. In this study, we have used an integrated biological and molecular biology approach to investigate the mechanism of resistance to ACCase herbicides in a Lolium multiflorum Lam. from the UK (UK21). Methodology/Principal Findings The study revealed a novel tryptophan to serine mutation at ACCase codon position 1999 impacting on ACCase inhibiting herbicides to varying degrees. The W1999S mutation confers dominant resistance to pinoxaden and partially recessive resistance to cycloxydim and sethoxydim. On the other hand, plants containing the W1999S mutation were sensitive to clethodim and tepraloxydim. Additionally population UK21 is characterised by other resistance mechanisms, very likely non non-target site based, affecting several aryloxyphenoxyproprionate (FOP) herbicides but not the practical field rate of pinoxaden. The positive identification of wild type tryptophan and mutant serine alleles at ACCase position 1999 could be readily achieved with an original DNA based derived cleaved amplified polymorphic sequence (dCAPS) assay that uses the same PCR product but two different enzymes for positively identifying the wild type tryptophan and mutant serine alleles identified here. Conclusion/Significance This paper highlights intrinsic differences between ACCase inhibiting herbicides that could be exploited for controlling ryegrass populations such as UK21 characterised by compound-specific target site and non-target site resistance. PMID:23469130

Cloning-free CRISPR

PubMed Central

Arbab, Mandana; Srinivasan, Sharanya; Hashimoto, Tatsunori; Geijsen, Niels; Sherwood, Richard I.

2015-01-01

Summary We present self-cloning CRISPR/Cas9 (scCRISPR), a technology that allows for CRISPR/Cas9-mediated genomic mutation and site-specific knockin transgene creation within several hours by circumventing the need to clone a site-specific single-guide RNA (sgRNA) or knockin homology construct for each target locus. We introduce a self-cleaving palindromic sgRNA plasmid and a short double-stranded DNA sequence encoding the desired locus-specific sgRNA into target cells, allowing them to produce a locus-specific sgRNA plasmid through homologous recombination. scCRISPR enables efficient generation of gene knockouts (∼88% mutation rate) at approximately one-sixth the cost of plasmid-based sgRNA construction with only 2 hr of preparation for each targeted site. Additionally, we demonstrate efficient site-specific knockin of GFP transgenes without any plasmid cloning or genome-integrated selection cassette in mouse and human embryonic stem cells (2%–4% knockin rate) through PCR-based addition of short homology arms. scCRISPR substantially lowers the bar on mouse and human transgenesis. PMID:26527385

Identification of a Non-Gatekeeper Hot Spot for Drug-Resistant Mutations in mTOR Kinase.

PubMed

Wu, Tzung-Ju; Wang, Xiaowen; Zhang, Yanjie; Meng, Linghua; Kerrigan, John E; Burley, Stephen K; Zheng, X F Steven

2015-04-21

Protein kinases are therapeutic targets for human cancer. However, "gatekeeper" mutations in tyrosine kinases cause acquired clinical resistance, limiting long-term treatment benefits. mTOR is a key cancer driver and drug target. Numerous small-molecule mTOR kinase inhibitors have been developed, with some already in human clinical trials. Given our clinical experience with targeted therapeutics, acquired drug resistance in mTOR is thought likely, but not yet documented. Herein, we describe identification of a hot spot (L2185) for drug-resistant mutations, which is distinct from the gatekeeper site, and a chemical scaffold refractory to drug-resistant mutations. We also provide new insights into mTOR kinase structure and function. The hot spot mutations are potentially useful as surrogate biomarkers for acquired drug resistance in ongoing clinical trials and future treatments and for the design of the next generation of mTOR-targeted drugs. Our study provides a foundation for further research into mTOR kinase function and targeting. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Efficient CRISPR/Cas9-mediated Targeted Mutagenesis in Populus in the First Generation

PubMed Central

Fan, Di; Liu, Tingting; Li, Chaofeng; Jiao, Bo; Li, Shuang; Hou, Yishu; Luo, Keming

2015-01-01

Recently, RNA-guided genome editing using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system has been applied to edit the plant genome in several herbaceous plant species. However, it remains unknown whether this system can be used for genome editing in woody plants. In this study, we describe the genome editing and targeted gene mutation in a woody species, Populus tomentosa Carr. via the CRISPR/Cas9 system. Four guide RNAs (gRNAs) were designed to target with distinct poplar genomic sites of the phytoene desaturase gene 8 (PtoPDS) which are followed by the protospacer-adjacent motif (PAM). After Agrobacterium-mediated transformation, obvious albino phenotype was observed in transgenic poplar plants. By analyzing the RNA-guided genome-editing events, 30 out of 59 PCR clones were homozygous mutants, 2 out of 59 were heterozygous mutants and the mutation efficiency at these target sites was estimated to be 51.7%. Our data demonstrate that the Cas9/sgRNA system can be exploited to precisely edit genomic sequence and effectively create knockout mutations in woody plants. PMID:26193631

Disruption of Ankyrin B and Caveolin-1 Interaction Sites Alters Na+,K+-ATPase Membrane Diffusion.

PubMed

Junghans, Cornelia; Vukojević, Vladana; Tavraz, Neslihan N; Maksimov, Eugene G; Zuschratter, Werner; Schmitt, Franz-Josef; Friedrich, Thomas

2017-11-21

The Na + ,K + -ATPase is a plasma membrane ion transporter of high physiological importance for ion homeostasis and cellular excitability in electrically active tissues. Mutations in the genes coding for Na + ,K + -ATPase α-subunit isoforms lead to severe human pathologies including Familial Hemiplegic Migraine type 2, Alternating Hemiplegia of Childhood, Rapid-onset Dystonia Parkinsonism, or epilepsy. Many of the reported mutations lead to change- or loss-of-function effects, whereas others do not alter the functional properties, but lead to, e.g., reduced protein stability, reduced protein expression, or defective plasma membrane targeting. Na + ,K + -ATPase frequently assembles with other membrane transporters or cellular matrix proteins in specialized plasma membrane microdomains, but the effects of these interactions on targeting or protein mobility are elusive so far. Mutation of established interaction motifs of the Na + ,K + -ATPase with ankyrin B and caveolin-1 are expected to result in changes in plasma membrane targeting, changes of the localization pattern, and of the diffusion behavior of the enzyme. We studied the consequences of mutations in these binding sites by monitoring diffusion of eGFP-labeled Na + ,K + -ATPase constructs in the plasma membrane of HEK293T cells by fluorescence correlation spectroscopy as well as fluorescence recovery after photobleaching or photoswitching, and observed significant differences compared to the wild-type enzyme, with synergistic effects for combinations of interaction site mutations. These measurements expand the possibilities to study the consequences of Na + ,K + -ATPase mutations and provide information about the interaction of Na + ,K + -ATPase α-isoforms with cellular matrix proteins, the cytoskeleton, or other membrane protein complexes. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Geographic spread, genetics and functional characteristics of ryanodine receptor based target-site resistance to diamide insecticides in diamondback moth, Plutella xylostella.

PubMed

Steinbach, Denise; Gutbrod, Oliver; Lümmen, Peter; Matthiesen, Svend; Schorn, Corinna; Nauen, Ralf

2015-08-01

Anthranilic diamides and flubendiamide belong to a new chemical class of insecticides acting as conformation sensitive activators of the insect ryanodine receptor (RyR). These compounds control a diverse range of different herbivorous insects including diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), a notorious global pest on cruciferous crops, which recently developed resistance due to target-site mutations located in the trans-membrane domain of the Plutella RyR. In the present study we further investigated the genetics and functional implications of a RyR G4946E target-site mutation we recently identified in a Philippine diamondback moth strain (Sudlon). Strain Sudlon is homozygous for the G4946E mutation and has been maintained under laboratory conditions without selection pressure for almost four years, and still exhibit stable resistance ratios of >2000-fold to all commercial diamides. Its F1 progeny resulting from reciprocal crosses with a susceptible strain (BCS-S) revealed no maternal effects and a diamide susceptible phenotype, suggesting an autosomally almost recessive mode of inheritance. Subsequent back-crosses indicate a near monogenic nature of the diamide resistance in strain Sudlon. Radioligand binding studies with Plutella thoracic microsomal membrane preparations provided direct evidence for the dramatic functional implications of the RyR G4946E mutation on both diamide specific binding and its concentration dependent modulation of [(3)H]ryanodine binding. Computational modelling based on a cryo-EM structure of rabbit RyR1 suggests that Plutella G4946E is located in trans-membrane helix S4 close to S4-S5 linker domain supposed to be involved in the modulation of the voltage sensor, and another recently described mutation, I4790M in helix S2 approx. 13 Å opposite of G4946E. Genotyping by pyrosequencing revealed the presence of the RyR G4946E mutation in larvae collected in 2013/14 in regions of ten different countries where diamide insecticides largely failed to control diamondback moth populations. Thus, our study highlights the global importance of the G4946E RyR target-site mutation, which as a mechanism on its own, confers high-level resistance to diamide insecticides in diamondback moth. Copyright © 2015 Elsevier Ltd. All rights reserved.

Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs.

PubMed

Gagnon, James A; Valen, Eivind; Thyme, Summer B; Huang, Peng; Akhmetova, Laila; Ahkmetova, Laila; Pauli, Andrea; Montague, Tessa G; Zimmerman, Steven; Richter, Constance; Schier, Alexander F

2014-01-01

The CRISPR/Cas9 system has been implemented in a variety of model organisms to mediate site-directed mutagenesis. A wide range of mutation rates has been reported, but at a limited number of genomic target sites. To uncover the rules that govern effective Cas9-mediated mutagenesis in zebrafish, we targeted over a hundred genomic loci for mutagenesis using a streamlined and cloning-free method. We generated mutations in 85% of target genes with mutation rates varying across several orders of magnitude, and identified sequence composition rules that influence mutagenesis. We increased rates of mutagenesis by implementing several novel approaches. The activities of poor or unsuccessful single-guide RNAs (sgRNAs) initiating with a 5' adenine were improved by rescuing 5' end homogeneity of the sgRNA. In some cases, direct injection of Cas9 protein/sgRNA complex further increased mutagenic activity. We also observed that low diversity of mutant alleles led to repeated failure to obtain frame-shift mutations. This limitation was overcome by knock-in of a stop codon cassette that ensured coding frame truncation. Our improved methods and detailed protocols make Cas9-mediated mutagenesis an attractive approach for labs of all sizes.

Mutated form (G52E) of inactive diphtheria toxin CRM197: molecular simulations clearly display effect of the mutation to NAD binding.

PubMed

Salmas, Ramin Ekhteiari; Mestanoglu, Mert; Unlu, Ayhan; Yurtsever, Mine; Durdagi, Serdar

2016-11-01

Mutated form (G52E) of diphtheria toxin (DT) CRM197 is an inactive and nontoxic enzyme. Here, we provided a molecular insight using comparative molecular dynamics (MD) simulations to clarify the influence of a single point mutation on overall protein and active-site loop. Post-processing MD analysis (i.e. stability, principal component analysis, hydrogen-bond occupancy, etc.) is carried out on both wild and mutated targets to investigate and to better understand the mechanistic differences of structural and dynamical properties on an atomic scale especially at nicotinamide adenine dinucleotide (NAD) binding site when a single mutation (G52E) happens at the DT. In addition, a docking simulation is performed for wild and mutated forms. The docking scoring analysis and docking poses results revealed that mutant form is not able to properly accommodate the NAD molecule.

Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system

PubMed Central

Chen, Xiugui; Lu, Xuke; Shu, Na; Wang, Shuai; Wang, Junjuan; Wang, Delong; Guo, Lixue; Ye, Wuwei

2017-01-01

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system has been widely used for genome editing in various plants because of its simplicity, high efficiency and design flexibility. However, to our knowledge, there is no report on the application of CRISPR/Cas9-mediated targeted mutagenesis in cotton. Here, we report the genome editing and targeted mutagenesis in upland cotton (Gossypium hirsutum L., hereafter cotton) using the CRISPR/Cas9 system. We designed two guide RNAs to target distinct sites of the cotton Cloroplastos alterados 1 (GhCLA1) and vacuolar H+-pyrophosphatase (GhVP) genes. Mutations in these two genes were detected in cotton protoplasts. Most of the mutations were nucleotide substitutions, with one nucleotide insertion and one substitution found in GhCLA1 and one deletion found in GhVP in cotton protoplasts. Subsequently, the two vectors were transformed into cotton shoot apexes through Agrobacterium-mediated transformation, resulting in efficient target gene editing. Most of the mutations were nucleotide deletions, and the mutation efficiencies were 47.6–81.8% in transgenic cotton plants. Evaluation using restriction-enzyme-PCR assay and sequence analysis detected no off-target mutations. Our results indicated that the CRISPR/Cas9 system was an efficient and specific tool for targeted mutagenesis of the cotton genome. PMID:28287154

Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system.

PubMed

Chen, Xiugui; Lu, Xuke; Shu, Na; Wang, Shuai; Wang, Junjuan; Wang, Delong; Guo, Lixue; Ye, Wuwei

2017-03-13

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system has been widely used for genome editing in various plants because of its simplicity, high efficiency and design flexibility. However, to our knowledge, there is no report on the application of CRISPR/Cas9-mediated targeted mutagenesis in cotton. Here, we report the genome editing and targeted mutagenesis in upland cotton (Gossypium hirsutum L., hereafter cotton) using the CRISPR/Cas9 system. We designed two guide RNAs to target distinct sites of the cotton Cloroplastos alterados 1 (GhCLA1) and vacuolar H + -pyrophosphatase (GhVP) genes. Mutations in these two genes were detected in cotton protoplasts. Most of the mutations were nucleotide substitutions, with one nucleotide insertion and one substitution found in GhCLA1 and one deletion found in GhVP in cotton protoplasts. Subsequently, the two vectors were transformed into cotton shoot apexes through Agrobacterium-mediated transformation, resulting in efficient target gene editing. Most of the mutations were nucleotide deletions, and the mutation efficiencies were 47.6-81.8% in transgenic cotton plants. Evaluation using restriction-enzyme-PCR assay and sequence analysis detected no off-target mutations. Our results indicated that the CRISPR/Cas9 system was an efficient and specific tool for targeted mutagenesis of the cotton genome.

Development and validation of a clinical trial patient stratification assay that interrogates 27 mutation sites in MAPK pathway genes.

PubMed

Chang, Ken C N; Galuska, Stefan; Weiner, Russell; Marton, Matthew J

2013-01-01

Somatic mutations identified on genes related to the cancer-developing signaling pathways have drawn attention in the field of personalized medicine in recent years. Treatments developed to target a specific signaling pathway may not be effective when tumor activating mutations occur downstream of the target and bypass the targeted mechanism. For instance, mutations detected in KRAS/BRAF/NRAS genes can lead to EGFR-independent intracellular signaling pathway activation. Most patients with these mutations do not respond well to anti-EGFR treatment. In an effort to detect various mutations in FFPE tissue samples among multiple solid tumor types for patient stratification many mutation assays were evaluated. Since there were more than 30 specific mutations among three targeted RAS/RAF oncogenes that could activate MAPK pathway genes, a custom designed Single Nucleotide Primer Extension (SNPE) multiplexing mutation assay was developed and analytically validated as a clinical trial assay. Throughout the process of developing and validating the assay we overcame many technical challenges which include: the designing of PCR primers for FFPE tumor tissue samples versus normal blood samples, designing of probes for detecting consecutive nucleotide double mutations, the kinetics and thermodynamics aspects of probes competition among themselves and against target PCR templates, as well as validating an assay when positive control tumor tissue or cell lines with specific mutations are not available. We used Next Generation sequencing to resolve discordant calls between the SNPE mutation assay and Sanger sequencing. We also applied a triplicate rule to reduce potential false positives and false negatives, and proposed special considerations including pre-define a cut-off percentage for detecting very low mutant copies in the wild-type DNA background.

Temporal Interplay between Efflux Pumps and Target Mutations in Development of Antibiotic Resistance in Escherichia coli

PubMed Central

Singh, Renu; Swick, Michelle C.; Ledesma, Kimberly R.; Yang, Zhen; Hu, Ming; Zechiedrich, Lynn

2012-01-01

The emergence of resistance presents a debilitating change in the management of infectious diseases. Currently, the temporal relationship and interplay between various mechanisms of drug resistance are not well understood. A thorough understanding of the resistance development process is needed to facilitate rational design of countermeasure strategies. Using an in vitro hollow-fiber infection model that simulates human drug treatment, we examined the appearance of efflux pump (acrAB) overexpression and target topoisomerase gene (gyrA and parC) mutations over time in the emergence of quinolone resistance in Escherichia coli. Drug-resistant isolates recovered early (24 h) had 2- to 8-fold elevation in the MIC due to acrAB overexpression, but no point mutations were noted. In contrast, high-level (≥64× MIC) resistant isolates with target site mutations (gyrA S83L with or without parC E84K) were selected more readily after 120 h, and regression of acrAB overexpression was observed at 240 h. Using a similar dosing selection pressure, the emergence of levofloxacin resistance was delayed in a strain with acrAB deleted compared to the isogenic parent. The role of efflux pumps in bacterial resistance development may have been underappreciated. Our data revealed the interplay between two mechanisms of quinolone resistance and provided a new mechanistic framework in the development of high-level resistance. Early low-level levofloxacin resistance conferred by acrAB overexpression preceded and facilitated high-level resistance development mediated by target site mutation(s). If this interpretation is correct, then these findings represent a paradigm shift in the way quinolone resistance is thought to develop. PMID:22232279

TALEN-mediated generation and genetic correction of disease-specific human induced pluripotent stem cells.

PubMed

Ramalingam, Sivaprakash; Annaluru, Narayana; Kandavelou, Karthikeyan; Chandrasegaran, Srinivasan

2014-01-01

Generation and precise genetic correction of patient-derived hiPSCs have great potential in regenerative medicine. Such targeted genetic manipulations can now be achieved using gene-editing nucleases. Here, we report generation of cystic fibrosis (CF) and Gaucher's disease (GD) hiPSCs respectively from CF (homozygous for CFTRΔF508 mutation) and Type II GD [homozygous for β-glucocerebrosidase (GBA) 1448T>C mutation] patient fibroblasts, using CCR5- specific TALENs. Site-specific addition of loxP-flanked Oct4/Sox2/Klf4/Lin28/Nanog/eGFP gene cassette at the endogenous CCR5 site of patient-derived disease-specific primary fibroblasts induced reprogramming, giving rise to both monoallele (heterozygous) and biallele CCR5-modified hiPSCs. Subsequent excision of the donor cassette was done by treating CCR5-modified CF and GD hiPSCs with Cre. We also demonstrate site-specific correction of sickle cell disease (SCD) mutations at the endogenous HBB locus of patient-specific hiPSCs [TNC1 line that is homozygous for mutated β- globin alleles (βS/βS)], using HBB-specific TALENs. SCD-corrected hiPSC lines showed gene conversion of the mutated βS to the wild-type βA in one of the HBB alleles, while the other allele remained a mutant phenotype. After excision of the loxP-flanked DNA cassette from the SCD-corrected hiPSC lines using Cre, we obtained secondary heterozygous βS/βA hiPSCs, which express the wild-type (βA) transcript to 30-40% level as compared to uncorrected (βS/βS) SCD hiPSCs when differentiated into erythroid cells. Furthermore, we also show that TALEN-mediated generation and genetic correction of disease-specific hiPSCs did not induce any off-target mutations at closely related sites.

Targeted DNA sequencing and in situ mutation analysis using mobile phone microscopy

NASA Astrophysics Data System (ADS)

Kühnemund, Malte; Wei, Qingshan; Darai, Evangelia; Wang, Yingjie; Hernández-Neuta, Iván; Yang, Zhao; Tseng, Derek; Ahlford, Annika; Mathot, Lucy; Sjöblom, Tobias; Ozcan, Aydogan; Nilsson, Mats

2017-01-01

Molecular diagnostics is typically outsourced to well-equipped centralized laboratories, often far from the patient. We developed molecular assays and portable optical imaging designs that permit on-site diagnostics with a cost-effective mobile-phone-based multimodal microscope. We demonstrate that targeted next-generation DNA sequencing reactions and in situ point mutation detection assays in preserved tumour samples can be imaged and analysed using mobile phone microscopy, achieving a new milestone for tele-medicine technologies.

Mutation site and context dependent effects of ESR1 mutation in genome-edited breast cancer cell models.

PubMed

Bahreini, Amir; Li, Zheqi; Wang, Peilu; Levine, Kevin M; Tasdemir, Nilgun; Cao, Lan; Weir, Hazel M; Puhalla, Shannon L; Davidson, Nancy E; Stern, Andrew M; Chu, David; Park, Ben Ho; Lee, Adrian V; Oesterreich, Steffi

2017-05-23

Mutations in the estrogen receptor alpha (ERα) 1 gene (ESR1) are frequently detected in ER+ metastatic breast cancer, and there is increasing evidence that these mutations confer endocrine resistance in breast cancer patients with advanced disease. However, their functional role is not well-understood, at least in part due to a lack of ESR1 mutant models. Here, we describe the generation and characterization of genome-edited T47D and MCF7 breast cancer cell lines with the two most common ESR1 mutations, Y537S and D538G. Genome editing was performed using CRISPR and adeno-associated virus (AAV) technologies to knock-in ESR1 mutations into T47D and MCF7 cell lines, respectively. Various techniques were utilized to assess the activity of mutant ER, including transactivation, growth and chromatin-immunoprecipitation (ChIP) assays. The level of endocrine resistance was tested in mutant cells using a number of selective estrogen receptor modulators (SERMs) and degraders (SERDs). RNA sequencing (RNA-seq) was employed to study gene targets of mutant ER. Cells with ESR1 mutations displayed ligand-independent ER activity, and were resistant to several SERMs and SERDs, with cell line and mutation-specific differences with respect to magnitude of effect. The SERD AZ9496 showed increased efficacy compared to other drugs tested. Wild-type and mutant cell co-cultures demonstrated a unique evolution of mutant cells under estrogen deprivation and tamoxifen treatment. Transcriptome analysis confirmed ligand-independent regulation of ERα target genes by mutant ERα, but also identified novel target genes, some of which are involved in metastasis-associated phenotypes. Despite significant overlap in the ligand-independent genes between Y537S and D538G, the number of mutant ERα-target genes shared between the two cell lines was limited, suggesting context-dependent activity of the mutant receptor. Some genes and phenotypes were unique to one mutation within a given cell line, suggesting a mutation-specific effect. Taken together, ESR1 mutations in genome-edited breast cancer cell lines confer ligand-independent growth and endocrine resistance. These biologically relevant models can be used for further mechanistic and translational studies, including context-specific and mutation site-specific analysis of the ESR1 mutations.

Maternally expressed PGK-Cre transgene as a tool for early and uniform activation of the Cre site-specific recombinase.

PubMed

Lallemand, Y; Luria, V; Haffner-Krausz, R; Lonai, P

1998-03-01

A transgenic mouse strain with early and uniform expression of the Cre site-specific recombinase is described. In this strain, PGK-Crem, Cre is driven by the early acting PGK-1 promoter, but, probably due to cis effects at the integration site, the recombinase is under dominant maternal control. When Cre is transmitted by PGK-Crem females mated to males that carry a reporter transgene flanked by loxP sites, even offspring that do not inherit PGK-Cre delete the target gene. It follows that in the PGK-Crem female Cre activity commences in the diploid phase of oogenesis. In PGK-Crem crosses complete recombination was observed in all organs, including testis and ovary. We prepared a mouse stock that is homozygous for PGK-Crem and at the albino (c) locus. This strain will be useful for the early and uniform induction of ectopic and dominant negative mutations, for the in vivo removal of selective elements from targeted mutations and in connection with the manipulation of targeted loci in 'knock in' and related technologies.

Simultaneous site-directed mutagenesis of duplicated loci in soybean using a single guide RNA.

PubMed

Kanazashi, Yuhei; Hirose, Aya; Takahashi, Ippei; Mikami, Masafumi; Endo, Masaki; Hirose, Sakiko; Toki, Seiichi; Kaga, Akito; Naito, Ken; Ishimoto, Masao; Abe, Jun; Yamada, Tetsuya

2018-03-01

Using a gRNA and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two GmPPD loci in soybean. Mutations in GmPPD loci were confirmed in at least 33% of T 2 seeds. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is a powerful tool for site-directed mutagenesis in crops. Using a single guide RNA (gRNA) and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two homoeologous loci in soybean (Glycine max), GmPPD1 and GmPPD2, which encode the orthologs of Arabidopsis thaliana PEAPOD (PPD). Most of the T 1 plants had heterozygous and/or chimeric mutations for the targeted loci. The sequencing analysis of T 1 and T 2 generations indicates that putative mutation induced in the T 0 plant is transmitted to the T 1 generation. The inheritable mutation induced in the T 1 plant was also detected. This result indicates that continuous induction of mutations during T 1 plant development increases the occurrence of mutations in germ cells, which ensures the transmission of mutations to the next generation. Simultaneous site-directed mutagenesis in both GmPPD loci was confirmed in at least 33% of T 2 seeds examined. Approximately 19% of double mutants did not contain the Cas9/gRNA expression construct. Double mutants with frameshift mutations in both GmPPD1 and GmPPD2 had dome-shaped trifoliate leaves, extremely twisted pods, and produced few seeds. Taken together, our data indicate that continuous induction of mutations in the whole plant and advancing generations of transgenic plants enable efficient simultaneous site-directed mutagenesis in duplicated loci in soybean.

A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep.

PubMed

Clop, Alex; Marcq, Fabienne; Takeda, Haruko; Pirottin, Dimitri; Tordoir, Xavier; Bibé, Bernard; Bouix, Jacques; Caiment, Florian; Elsen, Jean-Michel; Eychenne, Francis; Larzul, Catherine; Laville, Elisabeth; Meish, Françoise; Milenkovic, Dragan; Tobin, James; Charlier, Carole; Georges, Michel

2006-07-01

Texel sheep are renowned for their exceptional meatiness. To identify the genes underlying this economically important feature, we performed a whole-genome scan in a Romanov x Texel F2 population. We mapped a quantitative trait locus with a major effect on muscle mass to chromosome 2 and subsequently fine-mapped it to a chromosome interval encompassing the myostatin (GDF8) gene. We herein demonstrate that the GDF8 allele of Texel sheep is characterized by a G to A transition in the 3' UTR that creates a target site for mir1 and mir206, microRNAs (miRNAs) that are highly expressed in skeletal muscle. This causes translational inhibition of the myostatin gene and hence contributes to the muscular hypertrophy of Texel sheep. Analysis of SNP databases for humans and mice demonstrates that mutations creating or destroying putative miRNA target sites are abundant and might be important effectors of phenotypic variation.

Structural signatures of DRD4 mutants revealed using molecular dynamics simulations: Implications for drug targeting.

PubMed

Jatana, Nidhi; Thukral, Lipi; Latha, N

2016-01-01

Human Dopamine Receptor D4 (DRD4) orchestrates several neurological functions and represents a target for many psychological disorders. Here, we examined two rare variants in DRD4; V194G and R237L, which elicit functional alterations leading to disruption of ligand binding and G protein coupling, respectively. Using atomistic molecular dynamics (MD) simulations, we provide in-depth analysis to reveal structural signatures of wild and mutant complexes with their bound agonist and antagonist ligands. We constructed intra-protein network graphs to discriminate the global conformational changes induced by mutations. The simulations also allowed us to elucidate the local side-chain dynamical variations in ligand-bound mutant receptors. The data suggest that the mutation in transmembrane V (V194G) drastically disrupts the organization of ligand binding site and causes disorder in the native helical arrangement. Interestingly, the R237L mutation leads to significant rewiring of side-chain contacts in the intracellular loop 3 (site of mutation) and also affects the distant transmembrane topology. Additionally, these mutations lead to compact ICL3 region compared to the wild type, indicating that the receptor would be inaccessible for G protein coupling. Our findings thus reveal unreported structural determinants of the mutated DRD4 receptor and provide a robust framework for design of effective novel drugs.

Modulation of Global Transcriptional Regulatory Networks as a Strategy for Increasing Kanamycin Resistance of the Translational Elongation Factor-G Mutants in Escherichia coli

PubMed Central

Mogre, Aalap; Veetil, Reshma T.; Seshasayee, Aswin Sai Narain

2017-01-01

Evolve and resequence experiments have provided us a tool to understand bacterial adaptation to antibiotics. In our previous work, we used short-term evolution to isolate mutants resistant to the ribosome targeting antibiotic kanamycin, and reported that Escherichia coli develops low cost resistance to kanamycin via different point mutations in the translation Elongation Factor-G (EF-G). Furthermore, we had shown that the resistance of EF-G mutants could be increased by second site mutations in the genes rpoD/cpxA/topA/cyaA. Mutations in three of these genes had been discovered in earlier screens for aminoglycoside resistance. In this work, we expand our understanding of these second site mutations, the goal being to understand how these mutations affect the activities of the mutated gene products to confer resistance. We show that the mutation in cpxA most likely results in an active Cpx stress response. Further evolution of an EF-G mutant in a higher concentration of kanamycin than what was used in our previous experiments identified the cpxA locus as a primary target for a significant increase in resistance. The mutation in cyaA results in a loss of catalytic activity and probably results in resistance via altered CRP function. Despite a reduction in cAMP levels, the CyaAN600Y mutant has a transcriptome indicative of increased CRP activity, pointing to an unknown role for CyaA and / or cAMP in gene expression. From the transcriptomes of double and single mutants, we describe the epistasis between the mutation in EF-G and these second site mutations. We show that the large scale transcriptomic changes in the topoisomerase I (FusAA608E-TopAS180L) mutant likely result from increased negative supercoiling in the cell. Finally, genes with known roles in aminoglycoside resistance were present among the misregulated genes in the mutants. PMID:29046437

Analysis of aberrant pre-messenger RNA splicing resulting from mutations in ATP8B1 and efficient in vitro rescue by adapted U1 small nuclear RNA.

PubMed

van der Woerd, Wendy L; Mulder, Johanna; Pagani, Franco; Beuers, Ulrich; Houwen, Roderick H J; van de Graaf, Stan F J

2015-04-01

ATP8B1 deficiency is a severe autosomal recessive liver disease resulting from mutations in the ATP8B1 gene characterized by a continuous phenotypical spectrum from intermittent (benign recurrent intrahepatic cholestasis; BRIC) to progressive familial intrahepatic cholestasis (PFIC). Current therapeutic options are insufficient, and elucidating the molecular consequences of mutations could lead to personalized mutation-specific therapies. We investigated the effect on pre-messenger RNA splicing of 14 ATP8B1 mutations at exon-intron boundaries using an in vitro minigene system. Eleven mutations, mostly associated with a PFIC phenotype, resulted in aberrant splicing and a complete absence of correctly spliced product. In contrast, three mutations led to partially correct splicing and were associated with a BRIC phenotype. These findings indicate an inverse correlation between the level of correctly spliced product and disease severity. Expression of modified U1 small nuclear RNAs (snRNA) complementary to the splice donor sites strongly improved or completely rescued splicing for several ATP8B1 mutations located at donor, as well as acceptor, splice sites. In one case, we also evaluated exon-specific U1 snRNAs that, by targeting nonconserved intronic sequences, might reduce possible off-target events. Although very effective in correcting exon skipping, they also induced retention of the short downstream intron. We systematically characterized the molecular consequences of 14 ATP8B1 mutations at exon-intron boundaries associated with ATP8B1 deficiency and found that the majority resulted in total exon skipping. The amount of correctly spliced product inversely correlated with disease severity. Compensatory modified U1 snRNAs, complementary to mutated donor splice sites, were able to improve exon definition very efficiently and could be a novel therapeutic strategy in ATP8B1 deficiency as well as other genetic diseases. © 2014 by the American Association for the Study of Liver Diseases.

Global connectivity of hub residues in Oncoprotein structures encodes genetic factors dictating personalized drug response to targeted Cancer therapy

NASA Astrophysics Data System (ADS)

Soundararajan, Venky; Aravamudan, Murali

2014-12-01

The efficacy and mechanisms of therapeutic action are largely described by atomic bonds and interactions local to drug binding sites. Here we introduce global connectivity analysis as a high-throughput computational assay of therapeutic action - inspired by the Google page rank algorithm that unearths most ``globally connected'' websites from the information-dense world wide web (WWW). We execute short timescale (30 ps) molecular dynamics simulations with high sampling frequency (0.01 ps), to identify amino acid residue hubs whose global connectivity dynamics are characteristic of the ligand or mutation associated with the target protein. We find that unexpected allosteric hubs - up to 20Å from the ATP binding site, but within 5Å of the phosphorylation site - encode the Gibbs free energy of inhibition (ΔGinhibition) for select protein kinase-targeted cancer therapeutics. We further find that clinically relevant somatic cancer mutations implicated in both drug resistance and personalized drug sensitivity can be predicted in a high-throughput fashion. Our results establish global connectivity analysis as a potent assay of protein functional modulation. This sets the stage for unearthing disease-causal exome mutations and motivates forecast of clinical drug response on a patient-by-patient basis. We suggest incorporation of structure-guided genetic inference assays into pharmaceutical and healthcare Oncology workflows.

Multicenter validation of cancer gene panel-based next-generation sequencing for translational research and molecular diagnostics.

PubMed

Hirsch, B; Endris, V; Lassmann, S; Weichert, W; Pfarr, N; Schirmacher, P; Kovaleva, V; Werner, M; Bonzheim, I; Fend, F; Sperveslage, J; Kaulich, K; Zacher, A; Reifenberger, G; Köhrer, K; Stepanow, S; Lerke, S; Mayr, T; Aust, D E; Baretton, G; Weidner, S; Jung, A; Kirchner, T; Hansmann, M L; Burbat, L; von der Wall, E; Dietel, M; Hummel, M

2018-04-01

The simultaneous detection of multiple somatic mutations in the context of molecular diagnostics of cancer is frequently performed by means of amplicon-based targeted next-generation sequencing (NGS). However, only few studies are available comparing multicenter testing of different NGS platforms and gene panels. Therefore, seven partner sites of the German Cancer Consortium (DKTK) performed a multicenter interlaboratory trial for targeted NGS using the same formalin-fixed, paraffin-embedded (FFPE) specimen of molecularly pre-characterized tumors (n = 15; each n = 5 cases of Breast, Lung, and Colon carcinoma) and a colorectal cancer cell line DNA dilution series. Detailed information regarding pre-characterized mutations was not disclosed to the partners. Commercially available and custom-designed cancer gene panels were used for library preparation and subsequent sequencing on several devices of two NGS different platforms. For every case, centrally extracted DNA and FFPE tissue sections for local processing were delivered to each partner site to be sequenced with the commercial gene panel and local bioinformatics. For cancer-specific panel-based sequencing, only centrally extracted DNA was analyzed at seven sequencing sites. Subsequently, local data were compiled and bioinformatics was performed centrally. We were able to demonstrate that all pre-characterized mutations were re-identified correctly, irrespective of NGS platform or gene panel used. However, locally processed FFPE tissue sections disclosed that the DNA extraction method can affect the detection of mutations with a trend in favor of magnetic bead-based DNA extraction methods. In conclusion, targeted NGS is a very robust method for simultaneous detection of various mutations in FFPE tissue specimens if certain pre-analytical conditions are carefully considered.

Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides.

PubMed

Rivera-Torres, Natalia; Banas, Kelly; Bialk, Pawel; Bloh, Kevin M; Kmiec, Eric B

2017-01-01

CRISPR/Cas9 and single-stranded DNA oligonucleotides (ssODNs) have been used to direct the repair of a single base mutation in human genes. Here, we examine a method designed to increase the precision of RNA guided genome editing in human cells by utilizing a CRISPR/Cas9 ribonucleoprotein (RNP) complex to initiate DNA cleavage. The RNP is assembled in vitro and induces a double stranded break at a specific site surrounding the mutant base designated for correction by the ssODN. We use an integrated mutant eGFP gene, bearing a single base change rendering the expressed protein nonfunctional, as a single copy target in HCT 116 cells. We observe significant gene correction activity of the mutant base, promoted by the RNP and single-stranded DNA oligonucleotide with validation through genotypic and phenotypic readout. We demonstrate that all individual components must be present to obtain successful gene editing. Importantly, we examine the genotype of individually sorted corrected and uncorrected clonally expanded cell populations for the mutagenic footprint left by the action of these gene editing tools. While the DNA sequence of the corrected population is exact with no adjacent sequence modification, the uncorrected population exhibits heterogeneous mutagenicity with a wide variety of deletions and insertions surrounding the target site. We designate this type of DNA aberration as on-site mutagenicity. Analyses of two clonal populations bearing specific DNA insertions surrounding the target site, indicate that point mutation repair has occurred at the level of the gene. The phenotype, however, is not rescued because a section of the single-stranded oligonucleotide has been inserted altering the reading frame and generating truncated proteins. These data illustrate the importance of analysing mutagenicity in uncorrected cells. Our results also form the basis of a simple model for point mutation repair directed by a short single-stranded DNA oligonucleotides and CRISPR/Cas9 ribonucleoprotein complex.

Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides

PubMed Central

Rivera-Torres, Natalia; Bialk, Pawel; Bloh, Kevin M.; Kmiec, Eric B.

2017-01-01

CRISPR/Cas9 and single-stranded DNA oligonucleotides (ssODNs) have been used to direct the repair of a single base mutation in human genes. Here, we examine a method designed to increase the precision of RNA guided genome editing in human cells by utilizing a CRISPR/Cas9 ribonucleoprotein (RNP) complex to initiate DNA cleavage. The RNP is assembled in vitro and induces a double stranded break at a specific site surrounding the mutant base designated for correction by the ssODN. We use an integrated mutant eGFP gene, bearing a single base change rendering the expressed protein nonfunctional, as a single copy target in HCT 116 cells. We observe significant gene correction activity of the mutant base, promoted by the RNP and single-stranded DNA oligonucleotide with validation through genotypic and phenotypic readout. We demonstrate that all individual components must be present to obtain successful gene editing. Importantly, we examine the genotype of individually sorted corrected and uncorrected clonally expanded cell populations for the mutagenic footprint left by the action of these gene editing tools. While the DNA sequence of the corrected population is exact with no adjacent sequence modification, the uncorrected population exhibits heterogeneous mutagenicity with a wide variety of deletions and insertions surrounding the target site. We designate this type of DNA aberration as on-site mutagenicity. Analyses of two clonal populations bearing specific DNA insertions surrounding the target site, indicate that point mutation repair has occurred at the level of the gene. The phenotype, however, is not rescued because a section of the single-stranded oligonucleotide has been inserted altering the reading frame and generating truncated proteins. These data illustrate the importance of analysing mutagenicity in uncorrected cells. Our results also form the basis of a simple model for point mutation repair directed by a short single-stranded DNA oligonucleotides and CRISPR/Cas9 ribonucleoprotein complex. PMID:28052104

Altering the spectrum of immunoglobulin V gene somatic hypermutation by modifying the active site of AID.

PubMed

Wang, Meng; Rada, Cristina; Neuberger, Michael S

2010-01-18

High-affinity antibodies are generated by somatic hypermutation with nucleotide substitutions introduced into the IgV in a semirandom fashion, but with intrinsic mutational hotspots strategically located to optimize antibody affinity maturation. The process is dependent on activation-induced deaminase (AID), an enzyme that can deaminate deoxycytidine in DNA in vitro, where its activity is sensitive to the identity of the 5'-flanking nucleotide. As a critical test of whether such DNA deamination activity underpins antibody diversification and to gain insight into the extent to which the antibody mutation spectrum is dependent on the intrinsic substrate specificity of AID, we investigated whether it is possible to change the IgV mutation spectrum by altering AID's active site such that it prefers a pyrimidine (rather than a purine) flanking the targeted deoxycytidine. Consistent with the DNA deamination mechanism, B cells expressing the modified AID proteins yield altered IgV mutation spectra (exhibiting a purine-->pyrimidine shift in flanking nucleotide preference) and altered hotspots. However, AID-catalyzed deamination of IgV targets in vitro does not yield the same degree of hotspot dominance to that observed in vivo, indicating the importance of features beyond AID's active site and DNA local sequence environment in determining in vivo hotspot dominance.

Targeted DNA sequencing and in situ mutation analysis using mobile phone microscopy

PubMed Central

Kühnemund, Malte; Wei, Qingshan; Darai, Evangelia; Wang, Yingjie; Hernández-Neuta, Iván; Yang, Zhao; Tseng, Derek; Ahlford, Annika; Mathot, Lucy; Sjöblom, Tobias; Ozcan, Aydogan; Nilsson, Mats

2017-01-01

Molecular diagnostics is typically outsourced to well-equipped centralized laboratories, often far from the patient. We developed molecular assays and portable optical imaging designs that permit on-site diagnostics with a cost-effective mobile-phone-based multimodal microscope. We demonstrate that targeted next-generation DNA sequencing reactions and in situ point mutation detection assays in preserved tumour samples can be imaged and analysed using mobile phone microscopy, achieving a new milestone for tele-medicine technologies. PMID:28094784

Effects of Point Mutations in the Major Capsid Protein of Beet Western Yellows Virus on Capsid Formation, Virus Accumulation, and Aphid Transmission

PubMed Central

Brault, V.; Bergdoll, M.; Mutterer, J.; Prasad, V.; Pfeffer, S.; Erdinger, M.; Richards, K. E.; Ziegler-Graff, V.

2003-01-01

Point mutations were introduced into the major capsid protein (P3) of cloned infectious cDNA of the polerovirus beet western yellows virus (BWYV) by manipulation of cloned infectious cDNA. Seven mutations targeted sites on the S domain predicted to lie on the capsid surface. An eighth mutation eliminated two arginine residues in the R domain, which is thought to extend into the capsid interior. The effects of the mutations on virus capsid formation, virus accumulation in protoplasts and plants, and aphid transmission were tested. All of the mutants replicated in protoplasts. The S-domain mutant W166R failed to protect viral RNA from RNase attack, suggesting that this particular mutation interfered with stable capsid formation. The R-domain mutant R7A/R8A protected ∼90% of the viral RNA strand from RNase, suggesting that lower positive-charge density in the mutant capsid interior interfered with stable packaging of the complete strand into virions. Neither of these mutants systemically infected plants. The six remaining mutants properly packaged viral RNA and could invade Nicotiana clevelandii systemically following agroinfection. Mutant Q121E/N122D was poorly transmitted by aphids, implicating one or both targeted residues in virus-vector interactions. Successful transmission of mutant D172N was accompanied either by reversion to the wild type or by appearance of a second-site mutation, N137D. This finding indicates that D172 is also important for transmission but that the D172N transmission defect can be compensated for by a “reverse” substitution at another site. The results have been used to evaluate possible structural models for the BWYV capsid. PMID:12584348

Resistance to AHAS inhibitor herbicides: current understanding.

PubMed

Yu, Qin; Powles, Stephen B

2014-09-01

Acetohydroxyacid synthase (AHAS) inhibitor herbicides currently comprise the largest site-of-action group (with 54 active ingredients across five chemical groups) and have been widely used in world agriculture since they were first introduced in 1982. Resistance evolution in weeds to AHAS inhibitors has been rapid and identified in populations of many weed species. Often, evolved resistance is associated with point mutations in the target AHAS gene; however non-target-site enhanced herbicide metabolism occurs as well. Many AHAS gene resistance mutations can occur and be rapidly enriched owing to a high initial resistance gene frequency, simple and dominant genetic inheritance and lack of major fitness cost of the resistance alleles. Major advances in the elucidation of the crystal structure of the AHAS (Arabidopsis thaliana) catalytic subunit in complex with various AHAS inhibitor herbicides have greatly improved current understanding of the detailed molecular interactions between AHAS, cofactors and herbicides. Compared with target-site resistance, non-target-site resistance to AHAS inhibitor herbicides is less studied and hence less understood. In a few well-studied cases, non-target-site resistance is due to enhanced rates of herbicide metabolism (metabolic resistance), mimicking that occurring in tolerant crop species and often involving cytochrome P450 monooxygenases. However, the specific herbicide-metabolising, resistance-endowing genes are yet to be identified in resistant weed species. The current state of mechanistic understanding of AHAS inhibitor herbicide resistance is reviewed, and outstanding research issues are outlined. © 2013 Society of Chemical Industry.

A target-unrelated peptide in an M13 phage display library traced to an advantageous mutation in the gene II ribosome-binding site.

PubMed

Brammer, Leighanne A; Bolduc, Benjamin; Kass, Jessica L; Felice, Kristin M; Noren, Christopher J; Hall, Marilena Fitzsimons

2008-02-01

Screening of the commercially available Ph.D.-7 phage-displayed heptapeptide library for peptides that bind immobilized Zn2+ resulted in the repeated selection of the peptide HAIYPRH, although binding assays indicated that HAIYPRH is not a zinc-binding peptide. HAIYPRH has also been selected in several other laboratories using completely different targets, and its ubiquity suggests that it is a target-unrelated peptide. We demonstrated that phage displaying HAIYPRH are enriched after serial amplification of the library without exposure to target. The amplification of phage displaying HAIYPRH was found to be dramatically faster than that of the library itself. DNA sequencing uncovered a mutation in the Shine-Dalgarno (SD) sequence for gIIp, a protein involved in phage replication, imparting to the SD sequence better complementarity to the 16S ribosomal RNA (rRNA). Introducing this mutation into phage lacking a displayed peptide resulted in accelerated propagation, whereas phage displaying HAIYPRH with a wild-type SD sequence were found to amplify normally. The SD mutation may alter gIIp expression and, consequently, the rate of propagation of phage. In the Ph.D.-7 library, the mutation is coincident with the displayed peptide HAIYPRH, accounting for the target-unrelated selection of this peptide in multiple reported panning experiments.

Longitudinal Antigenic Sequences and Sites from Intra-Host Evolution (LASSIE) identifies immune-selected HIV variants

DOE PAGES

Hraber, Peter; Korber, Bette; Wagh, Kshitij; ...

2015-10-21

Within-host genetic sequencing from samples collected over time provides a dynamic view of how viruses evade host immunity. Immune-driven mutations might stimulate neutralization breadth by selecting antibodies adapted to cycles of immune escape that generate within-subject epitope diversity. Comprehensive identification of immune-escape mutations is experimentally and computationally challenging. With current technology, many more viral sequences can readily be obtained than can be tested for binding and neutralization, making down-selection necessary. Typically, this is done manually, by picking variants that represent different time-points and branches on a phylogenetic tree. Such strategies are likely to miss many relevant mutations and combinations ofmore » mutations, and to be redundant for other mutations. Longitudinal Antigenic Sequences and Sites from Intrahost Evolution (LASSIE) uses transmitted founder loss to identify virus “hot-spots” under putative immune selection and chooses sequences that represent recurrent mutations in selected sites. LASSIE favors earliest sequences in which mutations arise. Here, with well-characterized longitudinal Env sequences, we confirmed selected sites were concentrated in antibody contacts and selected sequences represented diverse antigenic phenotypes. Finally, practical applications include rapidly identifying immune targets under selective pressure within a subject, selecting minimal sets of reagents for immunological assays that characterize evolving antibody responses, and for immunogens in polyvalent “cocktail” vaccines.« less

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

PubMed Central

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

2018-01-01

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

Investigating the Impact of Asp181 Point Mutations on Interactions between PTP1B and Phosphotyrosine Substrate

NASA Astrophysics Data System (ADS)

Liu, Mengyuan; Wang, Lushan; Sun, Xun; Zhao, Xian

2014-05-01

Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of insulin and leptin signaling, which suggests that it is an attractive therapeutic target in type II diabetes and obesity. The aim of this research is to explore residues which interact with phosphotyrosine substrate can be affected by D181 point mutations and lead to increased substrate binding. To achieve this goal, molecular dynamics simulations were performed on wild type (WT) and two mutated PTP1B/substrate complexes. The cross-correlation and principal component analyses show that point mutations can affect the motions of some residues in the active site of PTP1B. Moreover, the hydrogen bond and energy decomposition analyses indicate that apart from residue 181, point mutations have influence on the interactions of substrate with several residues in the active site of PTP1B.

Concerted action of target-site mutations and high EPSPS activity in glyphosate-resistant junglerice (Echinochloa colona) from California

USDA-ARS?s Scientific Manuscript database

Glyphosate is the most widely used non-selective herbicide and Echinochloa colona is an annual weed affecting field crops and orchards in California. A population carrying a glyphosate-resistance-endowing mutation in the EPSPS gene was found in the Northern Sacramento Valley. We used selfed lines ...

Landscape of somatic mutations and clonal evolution in mantle cell lymphoma.

PubMed

Beà, Sílvia; Valdés-Mas, Rafael; Navarro, Alba; Salaverria, Itziar; Martín-Garcia, David; Jares, Pedro; Giné, Eva; Pinyol, Magda; Royo, Cristina; Nadeu, Ferran; Conde, Laura; Juan, Manel; Clot, Guillem; Vizán, Pedro; Di Croce, Luciano; Puente, Diana A; López-Guerra, Mónica; Moros, Alexandra; Roue, Gael; Aymerich, Marta; Villamor, Neus; Colomo, Lluís; Martínez, Antonio; Valera, Alexandra; Martín-Subero, José I; Amador, Virginia; Hernández, Luis; Rozman, Maria; Enjuanes, Anna; Forcada, Pilar; Muntañola, Ana; Hartmann, Elena M; Calasanz, María J; Rosenwald, Andreas; Ott, German; Hernández-Rivas, Jesús M; Klapper, Wolfram; Siebert, Reiner; Wiestner, Adrian; Wilson, Wyndham H; Colomer, Dolors; López-Guillermo, Armando; López-Otín, Carlos; Puente, Xose S; Campo, Elías

2013-11-05

Mantle cell lymphoma (MCL) is an aggressive tumor, but a subset of patients may follow an indolent clinical course. To understand the mechanisms underlying this biological heterogeneity, we performed whole-genome and/or whole-exome sequencing on 29 MCL cases and their respective matched normal DNA, as well as 6 MCL cell lines. Recurrently mutated genes were investigated by targeted sequencing in an independent cohort of 172 MCL patients. We identified 25 significantly mutated genes, including known drivers such as ataxia-telangectasia mutated (ATM), cyclin D1 (CCND1), and the tumor suppressor TP53; mutated genes encoding the anti-apoptotic protein BIRC3 and Toll-like receptor 2 (TLR2); and the chromatin modifiers WHSC1, MLL2, and MEF2B. We also found NOTCH2 mutations as an alternative phenomenon to NOTCH1 mutations in aggressive tumors with a dismal prognosis. Analysis of two simultaneous or subsequent MCL samples by whole-genome/whole-exome (n = 8) or targeted (n = 19) sequencing revealed subclonal heterogeneity at diagnosis in samples from different topographic sites and modulation of the initial mutational profile at the progression of the disease. Some mutations were predominantly clonal or subclonal, indicating an early or late event in tumor evolution, respectively. Our study identifies molecular mechanisms contributing to MCL pathogenesis and offers potential targets for therapeutic intervention.

Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization.

PubMed

Sarraf, Shireen A; Raman, Malavika; Guarani-Pereira, Virginia; Sowa, Mathew E; Huttlin, Edward L; Gygi, Steven P; Harper, J Wade

2013-04-18

The PARKIN ubiquitin ligase (also known as PARK2) and its regulatory kinase PINK1 (also known as PARK6), often mutated in familial early-onset Parkinson's disease, have central roles in mitochondrial homeostasis and mitophagy. Whereas PARKIN is recruited to the mitochondrial outer membrane (MOM) upon depolarization via PINK1 action and can ubiquitylate porin, mitofusin and Miro proteins on the MOM, the full repertoire of PARKIN substrates--the PARKIN-dependent ubiquitylome--remains poorly defined. Here we use quantitative diGly capture proteomics (diGly) to elucidate the ubiquitylation site specificity and topology of PARKIN-dependent target modification in response to mitochondrial depolarization. Hundreds of dynamically regulated ubiquitylation sites in dozens of proteins were identified, with strong enrichment for MOM proteins, indicating that PARKIN dramatically alters the ubiquitylation status of the mitochondrial proteome. Using complementary interaction proteomics, we found depolarization-dependent PARKIN association with numerous MOM targets, autophagy receptors, and the proteasome. Mutation of the PARKIN active site residue C431, which has been found mutated in Parkinson's disease patients, largely disrupts these associations. Structural and topological analysis revealed extensive conservation of PARKIN-dependent ubiquitylation sites on cytoplasmic domains in vertebrate and Drosophila melanogaster MOM proteins. These studies provide a resource for understanding how the PINK1-PARKIN pathway re-sculpts the proteome to support mitochondrial homeostasis.

GeneChip{sup {trademark}} screening assay for cystic fibrosis mutations

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

Cronn, M.T.; Miyada, C.G.; Fucini, R.V.

1994-09-01

GeneChip{sup {trademark}} assays are based on high density, carefully designed arrays of short oligonucleotide probes (13-16 bases) built directly on derivatized silica substrates. DNA target sequence analysis is achieved by hybridizing fluorescently labeled amplification products to these arrays. Fluorescent hybridization signals located within the probe array are translated into target sequence information using the known probe sequence at each array feature. The mutation screening assay for cystic fibrosis includes sets of oligonucleotide probes designed to detect numerous different mutations that have been described in 14 exons and one intron of the CFTR gene. Each mutation site is addressed by amore » sub-array of at least 40 probe sequences, half designed to detect the wild type gene sequence and half designed to detect the reported mutant sequence. Hybridization with homozygous mutant, homozygous wild type or heterozygous targets results in distinctive hybridization patterns within a sub-array, permitting specific discrimination of each mutation. The GeneChip probe arrays are very small (approximately 1 cm{sup 2}). There miniature size coupled with their high information content make GeneChip probe arrays a useful and practical means for providing CF mutation analysis in a clinical setting.« less

BRAF/NRAS mutation frequencies among primary tumors and metastases in patients with melanoma.

PubMed

Colombino, Maria; Capone, Mariaelena; Lissia, Amelia; Cossu, Antonio; Rubino, Corrado; De Giorgi, Vincenzo; Massi, Daniela; Fonsatti, Ester; Staibano, Stefania; Nappi, Oscar; Pagani, Elena; Casula, Milena; Manca, Antonella; Sini, Mariacristina; Franco, Renato; Botti, Gerardo; Caracò, Corrado; Mozzillo, Nicola; Ascierto, Paolo A; Palmieri, Giuseppe

2012-07-10

The prevalence of BRAF, NRAS, and p16CDKN2A mutations during melanoma progression remains inconclusive. We investigated the prevalence and distribution of mutations in these genes in different melanoma tissues. In all, 291 tumor tissues from 132 patients with melanoma were screened. Paired samples of primary melanomas (n = 102) and synchronous or asynchronous metastases from the same patients (n = 165) were included. Tissue samples underwent mutation analysis (automated DNA sequencing). Secondary lesions included lymph nodes (n = 84), and skin (n = 36), visceral (n = 25), and brain (n = 44) sites. BRAF/NRAS mutations were identified in 58% of primary melanomas (43% BRAF; 15% NRAS); 62% in lymph nodes, 61% subcutaneous, 56% visceral, and 70% in brain sites. Mutations were observed in 63% of metastases (48% BRAF; 15% NRAS), a nonsignificant increase in mutation frequency after progression from primary melanoma. Of the paired samples, lymph nodes (93% consistency) and visceral metastases (96% consistency) presented a highly similar distribution of BRAF/NRAS mutations versus primary melanomas, with a significantly less consistent pattern in brain (80%) and skin metastases (75%). This suggests that independent subclones are generated in some patients. p16CDKN2A mutations were identified in 7% and 14% of primary melanomas and metastases, with a low consistency (31%) between secondary and primary tumor samples. In the era of targeted therapies, assessment of the spectrum and distribution of alterations in molecular targets among patients with melanoma is needed. Our findings about the prevalence of BRAF/NRAS/p16CDKN2A mutations in paired tumor lesions from patients with melanoma may be useful in the management of this disease.

Active site remodeling switches HIV specificity of antiretroviral TRIMCyp

PubMed Central

Price, Amanda J; Marzetta, Flavia; Lammers, Michael; Ylinen, Laura M J; Schaller, Torsten; Wilson, Sam J; Towers, Greg J; James, Leo C

2011-01-01

TRIMCyps are primate antiretroviral proteins that potently inhibit HIV replication. Here we describe how rhesus macaque TRIMCyp (RhTC) has evolved to target and restrict HIV-2. We show that the ancestral cyclophilin A (CypA) domain of RhTC targets HIV-2 capsid with weak affinity, which is strongly increased in RhTC by two mutations (D66N and R69H) at the expense of HIV-1 binding. These mutations disrupt a constraining intramolecular interaction in CypA, triggering the complete restructuring (>16 Å) of an active site loop. This new configuration discriminates between divergent HIV-1 and HIV-2 loop conformations mediated by capsid residue 88. Viral sensitivity to RhTC restriction can be conferred or abolished by mutating position 88. Furthermore, position 88 determines the susceptibility of naturally occurring HIV-1 sequences to restriction. Our results reveal the complex molecular, structural and thermodynamic changes that underlie the ongoing evolutionary race between virus and host. PMID:19767750

The clustered regularly interspaced short palindromic repeats/associated proteins system for the induction of gene mutations and phenotypic changes in Bombyx mori.

PubMed

Song, Jia; Che, Jiaqian; You, Zhengying; Ye, Xiaogang; Li, Jisheng; Ye, Lupeng; Zhang, Yuyu; Qian, Qiujie; Zhong, Boxiong

2016-12-01

To probe the general phenomena of gene mutations, Bombyx mori, the lepidopterous model organism, was chosen as the experimental model. To easily detect phenotypic variations, the piggyBac system was utilized to introduce two marker genes into the silkworm, and 23.4% transposition efficiency aided in easily breeding a new strain for the entire experiment. Then, the clustered regularly interspaced short palindromic repeats/an associated protein (Cas9) system was utilized. The results showed that the Cas9 system can induce efficient gene mutations and the base changes could be detected since the G 0 individuals in B. mori; and that the mutation rates on different target sites were diverse. Next, the gRNA2-targeted site that generated higher mutation rate was chosen, and the experimental results were enumerated. First, the mutation proportion in G 1 generation was 30.1%, and some gene mutations were not inherited from the G 0 generation; second, occasionally, base substitutions did not lead to variation in the amino-acid sequence, which decreased the efficiency of phenotypic changes compared with that of genotypic changes. These results laid the foundation for better use of the Cas9 system in silkworm gene editing. © The Author 2016. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Novel mutations in LRP6 highlight the role of WNT signaling in tooth agenesis

PubMed Central

Ludwig, Kerstin U.; Sullivan, Robert; van Rooij, Iris A.L.M.; Thonissen, Michelle; Swinnen, Steven; Phan, Milien; Conte, Federica; Ishorst, Nina; Gilissen, Christian; RoaFuentes, Laury; van de Vorst, Maartje; Henkes, Arjen; Steehouwer, Marloes; van Beusekom, Ellen; Bloemen, Marjon; Vankeirsbilck, Bruno; Bergé, Stefaan; Hens, Greet; Schoenaers, Joseph; Poorten, Vincent Vander; Roosenboom, Jasmien; Verdonck, An; Devriendt, Koen; Roeleveldt, Nel; Jhangiani, Shalini N.; Vissers, Lisenka E.L.M.; Lupski, James R.; de Ligt, Joep; Von den Hoff, Johannes W.; Pfundt, Rolph; Brunner, Han G.; Zhou, Huiqing; Dixon, Jill; Mangold, Elisabeth; van Bokhoven, Hans; Dixon, Michael J.; Kleefstra, Tjitske

2016-01-01

Purpose Here we aimed to identify a novel genetic cause of tooth agenesis (TA) and/or orofacial clefting (OFC) by combining whole exome sequencing (WES) and targeted re-sequencing in a large cohort of TA and OFC patients. Methods WES was performed in two unrelated patients, one with severe TA and OFC and another with severe TA only. After identifying deleterious mutations in a gene encoding the low density lipoprotein receptor-related protein 6 (LRP6), all its exons were re-sequenced with molecular inversion probes, in 67 patients with TA, 1,072 patients with OFC and in 706 controls. Results We identified a frameshift (c.4594delG, p.Cys1532fs) and a canonical splice site mutation (c.3398-2A>C, p.?) in LRP6 respectively in the patient with TA and OFC, and in the patient with severe TA only. The targeted re-sequencing showed significant enrichment of unique LRP6 variants in TA patients, but not in nonsyndromic OFC. From the 5 variants in patients with TA, 2 affect the canonical splice site and 3 were missense variants; all variants segregated with the dominant phenotype and in 1 case the missense mutation occurred de novo. Conclusion Mutations in LRP6 cause tooth agenesis in man. PMID:26963285

Accurate detection for a wide range of mutation and editing sites of microRNAs from small RNA high-throughput sequencing profiles

PubMed Central

Zheng, Yun; Ji, Bo; Song, Renhua; Wang, Shengpeng; Li, Ting; Zhang, Xiaotuo; Chen, Kun; Li, Tianqing; Li, Jinyan

2016-01-01

Various types of mutation and editing (M/E) events in microRNAs (miRNAs) can change the stabilities of pre-miRNAs and/or complementarities between miRNAs and their targets. Small RNA (sRNA) high-throughput sequencing (HTS) profiles can contain many mutated and edited miRNAs. Systematic detection of miRNA mutation and editing sites from the huge volume of sRNA HTS profiles is computationally difficult, as high sensitivity and low false positive rate (FPR) are both required. We propose a novel method (named MiRME) for an accurate and fast detection of miRNA M/E sites using a progressive sequence alignment approach which refines sensitivity and improves FPR step-by-step. From 70 sRNA HTS profiles with over 1.3 billion reads, MiRME has detected thousands of statistically significant M/E sites, including 3′-editing sites, 57 A-to-I editing sites (of which 32 are novel), as well as some putative non-canonical editing sites. We demonstrated that a few non-canonical editing sites were not resulted from mutations in genome by integrating the analysis of genome HTS profiles of two human cell lines, suggesting the existence of new editing types to further diversify the functions of miRNAs. Compared with six existing studies or methods, MiRME has shown much superior performance for the identification and visualization of the M/E sites of miRNAs from the ever-increasing sRNA HTS profiles. PMID:27229138

Off-target Effects in CRISPR/Cas9-mediated Genome Engineering

PubMed Central

Zhang, Xiao-Hui; Tee, Louis Y; Wang, Xiao-Gang; Huang, Qun-Shan; Yang, Shi-Hua

2015-01-01

CRISPR/Cas9 is a versatile genome-editing technology that is widely used for studying the functionality of genetic elements, creating genetically modified organisms as well as preclinical research of genetic disorders. However, the high frequency of off-target activity (≥50%)—RGEN (RNA-guided endonuclease)-induced mutations at sites other than the intended on-target site—is one major concern, especially for therapeutic and clinical applications. Here, we review the basic mechanisms underlying off-target cutting in the CRISPR/Cas9 system, methods for detecting off-target mutations, and strategies for minimizing off-target cleavage. The improvement off-target specificity in the CRISPR/Cas9 system will provide solid genotype–phenotype correlations, and thus enable faithful interpretation of genome-editing data, which will certainly facilitate the basic and clinical application of this technology. PMID:26575098

Global connectivity of hub residues in Oncoprotein structures encodes genetic factors dictating personalized drug response to targeted Cancer therapy

PubMed Central

Soundararajan, Venky; Aravamudan, Murali

2014-01-01

The efficacy and mechanisms of therapeutic action are largely described by atomic bonds and interactions local to drug binding sites. Here we introduce global connectivity analysis as a high-throughput computational assay of therapeutic action – inspired by the Google page rank algorithm that unearths most “globally connected” websites from the information-dense world wide web (WWW). We execute short timescale (30 ps) molecular dynamics simulations with high sampling frequency (0.01 ps), to identify amino acid residue hubs whose global connectivity dynamics are characteristic of the ligand or mutation associated with the target protein. We find that unexpected allosteric hubs – up to 20Å from the ATP binding site, but within 5Å of the phosphorylation site – encode the Gibbs free energy of inhibition (ΔGinhibition) for select protein kinase-targeted cancer therapeutics. We further find that clinically relevant somatic cancer mutations implicated in both drug resistance and personalized drug sensitivity can be predicted in a high-throughput fashion. Our results establish global connectivity analysis as a potent assay of protein functional modulation. This sets the stage for unearthing disease-causal exome mutations and motivates forecast of clinical drug response on a patient-by-patient basis. We suggest incorporation of structure-guided genetic inference assays into pharmaceutical and healthcare Oncology workflows. PMID:25465236

Reduction in mRNA and protein expression of a nicotinic acetylcholine receptor α8 subunit is associated with resistance to imidacloprid in the brown planthopper, Nilaparvata lugens.

PubMed

Zhang, Yixi; Wang, Xin; Yang, Baojun; Hu, Yuanyuan; Huang, Lixin; Bass, Chris; Liu, Zewen

2015-11-01

Target-site resistance is commonly caused by qualitative changes in insecticide target-receptors and few studies have implicated quantitative changes in insecticide targets in resistance. Here we show that resistance to imidacloprid in a selected strain of Nilaparvata lugens is associated with a reduction in expression levels of the nicotinic acetylcholine receptor (nAChR) subunit Nlα8. Synergism bioassays of the selected strain suggested resistance was conferred, in part, by a target-site mechanism. Sequencing of N. lugens nAChR subunit genes identified no mutations associated with resistance, however, a decrease in mRNA and protein levels of Nlα8 was observed during selection. RNA interference knockdown of Nlα8 decreased the sensitivity of N. lugens to imidacloprid, demonstrating that a decrease in Nlα8 expression is sufficient to confer resistance in vivo. Radioligand binding assays revealed that the affinity of the high-affinity imidacloprid-binding site of native nAChRs was reduced by selection, and reducing the amount of Nlα8 cRNA injected into Xenopus oocytes significantly decreased imidacloprid potency on recombinant receptors. Taken together, these results provide strong evidence that a decrease in Nlα8 levels confers resistance to imidacloprid in N. lugens, and thus provides a rare example of target-site resistance associated with a quantitative rather than qualitative change. In insects, target-site mutations often cause high resistance to insecticides, such as neonicotinoids acting on nicotinic acetylcholine receptors (nAChRs). Here we found that a quantitative change in target-protein level, decrease in mRNA and protein levels of Nlα8, contributed importantly to imidacloprid resistance in Nilaparvata lugens. This finding provides a new target-site mechanism of insecticide resistance. © 2015 International Society for Neurochemistry.

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

Hraber, Peter; Korber, Bette; Wagh, Kshitij

Within-host genetic sequencing from samples collected over time provides a dynamic view of how viruses evade host immunity. Immune-driven mutations might stimulate neutralization breadth by selecting antibodies adapted to cycles of immune escape that generate within-subject epitope diversity. Comprehensive identification of immune-escape mutations is experimentally and computationally challenging. With current technology, many more viral sequences can readily be obtained than can be tested for binding and neutralization, making down-selection necessary. Typically, this is done manually, by picking variants that represent different time-points and branches on a phylogenetic tree. Such strategies are likely to miss many relevant mutations and combinations ofmore » mutations, and to be redundant for other mutations. Longitudinal Antigenic Sequences and Sites from Intrahost Evolution (LASSIE) uses transmitted founder loss to identify virus “hot-spots” under putative immune selection and chooses sequences that represent recurrent mutations in selected sites. LASSIE favors earliest sequences in which mutations arise. Here, with well-characterized longitudinal Env sequences, we confirmed selected sites were concentrated in antibody contacts and selected sequences represented diverse antigenic phenotypes. Finally, practical applications include rapidly identifying immune targets under selective pressure within a subject, selecting minimal sets of reagents for immunological assays that characterize evolving antibody responses, and for immunogens in polyvalent “cocktail” vaccines.« less

Molecular methods for the detection of mutations.

PubMed

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.

High-Resolution Analysis of the Efficiency, Heritability, and Editing Outcomes of CRISPR/Cas9-Induced Modifications of NCED4 in Lettuce (Lactuca sativa).

PubMed

Bertier, Lien D; Ron, Mily; Huo, Heqiang; Bradford, Kent J; Britt, Anne B; Michelmore, Richard W

2018-05-04

CRISPR/Cas9 is a transformative tool for making targeted genetic alterations. In plants, high mutation efficiencies have been reported in primary transformants. However, many of the mutations analyzed were somatic and therefore not heritable. To provide more insights into the efficiency of creating stable homozygous mutants using CRISPR/Cas9, we targeted LsNCED4 ( 9-cis-EPOXYCAROTENOID DIOXYGENASE4) , a gene conditioning thermoinhibition of seed germination in lettuce. Three constructs, each capable of expressing Cas9 and a single gRNA targeting different sites in LsNCED4 , were stably transformed into lettuce (Lactuca sativa) cvs. Salinas and Cobham Green. Analysis of 47 primary transformants (T 1 ) and 368 T 2 plants by deep amplicon sequencing revealed that 57% of T 1 plants contained events at the target site: 28% of plants had germline mutations in one allele indicative of an early editing event (mono-allelic), 8% of plants had germline mutations in both alleles indicative of two early editing events (bi-allelic), and the remaining 21% of plants had multiple low frequency mutations indicative of late events (chimeric plants). Editing efficiency was similar in both genotypes, while the different gRNAs varied in efficiency. Amplicon sequencing of 20 T 1 and more than 100 T 2 plants for each of the three gRNAs showed that repair outcomes were not random, but reproducible and characteristic for each gRNA. Knockouts of NCED4 resulted in large increases in the maximum temperature for seed germination, with seeds of both cultivars capable of germinating >70% at 37°. Knockouts of NCED4 provide a whole-plant selectable phenotype that has minimal pleiotropic consequences. Targeting NCED4 in a co-editing strategy could therefore be used to enrich for germline-edited events simply by germinating seeds at high temperature. Copyright © 2018 Bertier et al.

High-Resolution Analysis of the Efficiency, Heritability, and Editing Outcomes of CRISPR/Cas9-Induced Modifications of NCED4 in Lettuce (Lactuca sativa)

PubMed Central

Bertier, Lien D.; Ron, Mily; Huo, Heqiang; Bradford, Kent J.; Britt, Anne B.; Michelmore, Richard W.

2018-01-01

CRISPR/Cas9 is a transformative tool for making targeted genetic alterations. In plants, high mutation efficiencies have been reported in primary transformants. However, many of the mutations analyzed were somatic and therefore not heritable. To provide more insights into the efficiency of creating stable homozygous mutants using CRISPR/Cas9, we targeted LsNCED4 (9-cis-EPOXYCAROTENOID DIOXYGENASE4), a gene conditioning thermoinhibition of seed germination in lettuce. Three constructs, each capable of expressing Cas9 and a single gRNA targeting different sites in LsNCED4, were stably transformed into lettuce (Lactuca sativa) cvs. Salinas and Cobham Green. Analysis of 47 primary transformants (T1) and 368 T2 plants by deep amplicon sequencing revealed that 57% of T1 plants contained events at the target site: 28% of plants had germline mutations in one allele indicative of an early editing event (mono-allelic), 8% of plants had germline mutations in both alleles indicative of two early editing events (bi-allelic), and the remaining 21% of plants had multiple low frequency mutations indicative of late events (chimeric plants). Editing efficiency was similar in both genotypes, while the different gRNAs varied in efficiency. Amplicon sequencing of 20 T1 and more than 100 T2 plants for each of the three gRNAs showed that repair outcomes were not random, but reproducible and characteristic for each gRNA. Knockouts of NCED4 resulted in large increases in the maximum temperature for seed germination, with seeds of both cultivars capable of germinating >70% at 37°. Knockouts of NCED4 provide a whole-plant selectable phenotype that has minimal pleiotropic consequences. Targeting NCED4 in a co-editing strategy could therefore be used to enrich for germline-edited events simply by germinating seeds at high temperature. PMID:29511025

Activity loss by H46A mutation in Mycobacterium tuberculosis isocitrate lyase is due to decrease in structural plasticity and collective motions of the active site.

PubMed

Shukla, Rohit; Shukla, Harish; Tripathi, Timir

2018-01-01

Mycobacterium tuberculosis isocitrate lyase (MtbICL) is a crucial enzyme of the glyoxylate cycle and is a validated anti-tuberculosis drug target. Structurally distant, non-active site mutation (H46A) in MtbICL has been found to cause loss of enzyme activity. The aim of the present work was to explore the structural alterations induced by H46A mutation that caused the loss of enzyme activity. The structural and dynamic consequences of H46A mutation were studied using multiple computational methods such as docking, molecular dynamics simulation and residue interaction network analysis (RIN). Principal component analysis and cross correlation analysis revealed the difference in conformational flexibility and collective modes of motions between the wild-type and mutant enzyme, particularly in the active site region. RIN analysis revealed that the active site geometry was disturbed in the mutant enzyme. Thus, the dynamic perturbation of the active site led to enzyme transition from its active form to inactive form upon mutation. The computational analyses elucidated the mutant-specific conformational alterations, differential dominant motions, and anomalous residue level interactions that contributed to the abrogated function of mutant MtbICL. An understanding of interactions of mutant enzymes may help in modifying the existing drugs and designing improved drugs for successful control of tuberculosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Deep comparative genomics among Chlamydia trachomatis lymphogranuloma venereum isolates highlights genes potentially involved in pathoadaptation.

PubMed

Borges, Vítor; Gomes, João Paulo

2015-06-01

Lymphogranuloma venereum (LGV) is a human sexually transmitted disease caused by the obligate intracellular bacterium Chlamydia trachomatis (serovars L1-L3). LGV clinical manifestations range from severe ulcerative proctitis (anorectal syndrome), primarily caused by the epidemic L2b strains, to painful inguinal lymphadenopathy (the typical LGV bubonic form). Besides potential host-related factors, the differential disease severity and tissue tropism among LGV strains is likely a function of the genetic backbone of the strains. We aimed to characterize the genetic variability among LGV strains as strain- or serovar-specific mutations may underlie phenotypic signatures, and to investigate the mutational events that occurred throughout the pathoadaptation of the epidemic L2b lineage. By analyzing 20 previously published genomes from L1, L2, L2b and L3 strains and two new genomes from L2b strains, we detected 1497 variant sites and about 100 indels, affecting 453 genes and 144 intergenic regions, with 34 genes displaying a clear overrepresentation of nonsynonymous mutations. Effectors and/or type III secretion substrates (almost all of those described in the literature) and inclusion membrane proteins showed amino acid changes that were about fivefold more frequent than silent changes. More than 120 variant sites occurred in plasmid-regulated virulence genes, and 66% yielded amino acid changes. The identified serovar-specific variant sites revealed that the L2b-specific mutations are likely associated with higher fitness and pointed out potential targets for future highly discriminatory diagnostic/typing tests. By evaluating the evolutionary pathway beyond the L2b clonal radiation, we observed that 90.2% of the intra-L2b variant sites occurring in coding regions involve nonsynonymous mutations, where CT456/tarp has been the main target. Considering the progress on C. trachomatis genetic manipulation, this study may constitute an important contribution for prioritizing study targets for functional genomics aiming to dissect the impact of the identified intra-LGV polymorphisms on virulence or tropism dissimilarities among LGV strains. Copyright © 2015 Elsevier B.V. All rights reserved.

Adjacent DNA sequences modulate Sox9 transcriptional activation at paired Sox sites in three chondrocyte-specific enhancer elements

PubMed Central

Bridgewater, Laura C.; Walker, Marlan D.; Miller, Gwen C.; Ellison, Trevor A.; Holsinger, L. Daniel; Potter, Jennifer L.; Jackson, Todd L.; Chen, Reuben K.; Winkel, Vicki L.; Zhang, Zhaoping; McKinney, Sandra; de Crombrugghe, Benoit

2003-01-01

Expression of the type XI collagen gene Col11a2 is directed to cartilage by at least three chondrocyte-specific enhancer elements, two in the 5′ region and one in the first intron of the gene. The three enhancers each contain two heptameric sites with homology to the Sox protein-binding consensus sequence. The two sites are separated by 3 or 4 bp and arranged in opposite orientation to each other. Targeted mutational analyses of these three enhancers showed that in the intronic enhancer, as in the other two enhancers, both Sox sites in a pair are essential for enhancer activity. The transcription factor Sox9 binds as a dimer at the paired sites, and the introduction of insertion mutations between the sites demonstrated that physical interactions between the adjacently bound proteins are essential for enhancer activity. Additional mutational analyses demonstrated that although Sox9 binding at the paired Sox sites is necessary for enhancer activity, it alone is not sufficient. Adjacent DNA sequences in each enhancer are also required, and mutation of those sequences can eliminate enhancer activity without preventing Sox9 binding. The data suggest a new model in which adjacently bound proteins affect the DNA bend angle produced by Sox9, which in turn determines whether an active transcriptional enhancer complex is assembled. PMID:12595563

Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

PubMed Central

Cozens, Christopher

2018-01-01

Abstract Engineering proteins for designer functions and biotechnological applications almost invariably requires (or at least benefits from) multiple mutations to non-contiguous residues. Several methods for multiple site-directed mutagenesis exist, but there remains a need for fast and simple methods to efficiently introduce such mutations – particularly for generating large, high quality libraries for directed evolution. Here, we present Darwin Assembly, which can deliver high quality libraries of >108 transformants, targeting multiple (>10) distal sites with minimal wild-type contamination (<0.25% of total population) and which takes a single working day from purified plasmid to library transformation. We demonstrate its efficacy with whole gene codon reassignment of chloramphenicol acetyl transferase, mutating 19 codons in a single reaction in KOD DNA polymerase and generating high quality, multiple-site libraries in T7 RNA polymerase and Tgo DNA polymerase. Darwin Assembly uses commercially available enzymes, can be readily automated, and offers a cost-effective route to highly complex and customizable library generation. PMID:29409059

Small-scale high-throughput sequencing-based identification of new therapeutic tools in cystic fibrosis.

PubMed

Bonini, Jennifer; Varilh, Jessica; Raynal, Caroline; Thèze, Corinne; Beyne, Emmanuelle; Audrezet, Marie-Pierre; Ferec, Claude; Bienvenu, Thierry; Girodon, Emmanuelle; Tuffery-Giraud, Sylvie; Des Georges, Marie; Claustres, Mireille; Taulan-Cadars, Magali

2015-10-01

Although 97-99% of CFTR mutations have been identified, great efforts must be made to detect yet-unidentified mutations. We developed a small-scale next-generation sequencing approach for reliably and quickly scanning the entire gene, including noncoding regions, to identify new mutations. We applied this approach to 18 samples from patients suffering from cystic fibrosis (CF) in whom only one mutation had hitherto been identified. Using an in-house bioinformatics pipeline, we could rapidly identify a second disease-causing CFTR mutation for 16 of 18 samples. Of them, c.1680-883A>G was found in three unrelated CF patients. Analysis of minigenes and patients' transcripts showed that this mutation results in aberrantly spliced transcripts because of the inclusion of a pseudoexon. It is located only three base pairs from the c.1680-886A>G mutation (1811+1.6kbA>G), the fourth most frequent mutation in southwestern Europe. We next tested the effect of antisense oligonucleotides targeting splice sites on these two mutations on pseudoexon skipping. Oligonucleotide transfection resulted in the restoration of the full-length, in-frame CFTR transcript, demonstrating the effect of antisense oligonucleotide-induced pseudoexon skipping in CF. Our data confirm the importance of analyzing noncoding regions to find unidentified mutations, which is essential to designing targeted therapeutic approaches.

Jittery, a Mutator Distant Relative with a Paradoxical Mobile Behavior: Excision without Reinsertion

PubMed Central

Xu, Zhennan; Yan, Xianghe; Maurais, Steve; Fu, Huihua; O'Brien, David G.; Mottinger, John; Dooner, Hugo K.

2004-01-01

The unstable mutation bz-m039 arose in a maize (Zea mays) stock that originated from a plant infected with barley stripe mosaic virus. The instability of the mutation is caused by a 3.9-kb mobile element that has been named Jittery (Jit). Jit has terminal inverted repeats (TIRs) of 181 bp, causes a 9-bp direct duplication of the target site, and appears to excise autonomously. It is predicted to encode a single 709–amino acid protein, JITA, which is distantly related to the MURA transposase protein of the Mutator system but is more closely related to the MURA protein of Mutator-like elements (MULEs) from Arabidopsis thaliana and rice (Oryza sativa). Like MULEs, Jit resembles Mutator in the length of the element's TIRs, the size of the target site duplication, and in the makeup of its transposase but differs from the autonomous element Mutator–Don Robertson in that it encodes a single protein. Jit also differs from Mutator elements in the high frequency with which it excises to produce germinal revertants and in its copy number in the maize genome: Jit-like TIRs are present at low copy number in all maize lines and teosinte accessions examined, and JITA sequences occur in only a few maize inbreds. However, Jit cannot be considered a bona fide transposon in its present host line because it does not leave footprints upon excision and does not reinsert in the genome. These unusual mobile element properties are discussed in light of the structure and gene organization of Jit and related elements. PMID:15075398

Genetic diagnosis of familial hypercholesterolaemia by targeted next-generation sequencing

PubMed Central

Maglio, C; Mancina, R M; Motta, B M; Stef, M; Pirazzi, C; Palacios, L; Askaryar, N; Borén, J; Wiklund, O; Romeo, S

2014-01-01

Maglio C., Mancina R. M., Motta B. M., Stef M., Pirazzi C., Palacios L., Askaryar N., Borén J., Wiklund O., Romeo S. (University of Gothenburg, Gothenburg, Sweden; University Magna Graecia of Catanzaro, Italy; University of Milan, Italy; Progenika Biopharma SA, Derio, Spain). Genetic diagnosis of familial hypercholesterolaemia by targeted next-generation sequencing. Objectives The aim of this study was to combine clinical criteria and next-generation sequencing (pyrosequencing) to establish a diagnosis of familial hypercholesterolaemia (FH). Design, setting and subjects A total of 77 subjects with a Dutch Lipid Clinic Network score of ≥3 (possible, probable or definite FH clinical diagnosis) were recruited from the Lipid Clinic at Sahlgrenska Hospital, Gothenburg, Sweden. Next-generation sequencing was performed in all subjects using SEQPRO LIPO RS, a kit that detects mutations in the low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), proprotein convertase subtilisin/kexin type 9 (PCSK9) and LDLR adapter protein 1 (LDLRAP1) genes; copy-number variations in the LDLR gene were also examined. Results A total of 26 mutations were detected in 50 subjects (65% success rate). Amongst these, 23 mutations were in the LDLR gene, two in the APOB gene and one in the PCSK9 gene. Four mutations with unknown pathogenicity were detected in LDLR. Of these, three mutations (Gly505Asp, Ile585Thr and Gln660Arg) have been previously reported in subjects with FH, but their pathogenicity has not been proved. The fourth, a mutation in LDLR affecting a splicing site (exon 6–intron 6) has not previously been reported; it was found to segregate with high cholesterol levels in the family of the proband. Conclusions Using a combination of clinical criteria and targeted next-generation sequencing, we have achieved FH diagnosis with a high success rate. Furthermore, we identified a new splicing-site mutation in the LDLR gene. PMID:24785115

Mutations on M3 helix of Plutella xylostella glutamate-gated chloride channel confer unequal resistance to abamectin by two different mechanisms.

PubMed

Wang, Xingliang; Puinean, Alin M; O Reilly, Andrias O; Williamson, Martin S; Smelt, Charles L C; Millar, Neil S; Wu, Yidong

2017-07-01

Abamectin is one of the most widely used avermectins for agricultural pests control, but the emergence of resistance around the world is proving a major threat to its sustained application. Abamectin acts by directly activating glutamate-gated chloride channels (GluCls) and modulating other Cys-loop ion channels. To date, three mutations occurring in the transmembrane domain of arthropod GluCls are associated with target-site resistance to abamectin: A309V in Plutella xylostella GluCl (PxGluCl), G323D in Tetranychus urticae GluCl1 (TuGluCl1) and G326E in TuGluCl3. To compare the effects of these mutations in a single system, A309V/I/G and G315E (corresponding to G323 in TuGluCl1 and G326 in TuGluCl3) substitutions were introduced individually into the PxGluCl channel. Functional analysis using Xenopus oocytes showed that the A309V and G315E mutations reduced the sensitivity to abamectin by 4.8- and 493-fold, respectively. In contrast, the substitutions A309I/G show no significant effects on the response to abamectin. Interestingly, the A309I substitution increased the channel sensitivity to glutamate by one order of magnitude (∼12-fold). Analysis of PxGluCl homology models indicates that the G315E mutation interferes with abamectin binding through a steric hindrance mechanism. In contrast, the structural consequences of the A309 mutations are not so clear and an allosteric modification of the binding site is the most likely mechanism. Overall the results show that both A309V and G315E mutations may contribute to target-site resistance to abamectin and may be important for the future prediction and monitoring of abamectin resistance in P. xylostella and other arthropod pests. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification of a novel MYO7A mutation in Usher syndrome type 1.

PubMed

Cheng, Ling; Yu, Hongsong; Jiang, Yan; He, Juan; Pu, Sisi; Li, Xin; Zhang, Li

2018-01-05

Usher syndrome (USH) is an autosomal recessive disease characterized by deafness and retinitis pigmentosa. In view of the high phenotypic and genetic heterogeneity in USH, performing genetic screening with traditional methods is impractical. In the present study, we carried out targeted next-generation sequencing (NGS) to uncover the underlying gene in an USH family (2 USH patients and 15 unaffected relatives). One hundred and thirty-five genes associated with inherited retinal degeneration were selected for deep exome sequencing. Subsequently, variant analysis, Sanger validation and segregation tests were utilized to identify the disease-causing mutations in this family. All affected individuals had a classic USH type I (USH1) phenotype which included deafness, vestibular dysfunction and retinitis pigmentosa. Targeted NGS and Sanger sequencing validation suggested that USH1 patients carried an unreported splice site mutation, c.5168+1G>A, as a compound heterozygous mutation with c.6070C>T (p.R2024X) in the MYO7A gene. A functional study revealed decreased expression of the MYO7A gene in the individuals carrying heterozygous mutations. In conclusion, targeted next-generation sequencing provided a comprehensive and efficient diagnosis for USH1. This study revealed the genetic defects in the MYO7A gene and expanded the spectrum of clinical phenotypes associated with USH1 mutations.

Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization

PubMed Central

Sarraf, Shireen A.; Raman, Malavika; Guarani-Pereira, Virginia; Sowa, Mathew E.; Huttlin, Edward L.; Gygi, Steven P.; Harper, J. Wade

2013-01-01

The PARKIN (PARK2) ubiquitin ligase and its regulatory kinase PINK1 (PARK6), often mutated in familial early onset Parkinson’s Disease (PD), play central roles in mitochondrial homeostasis and mitophagy.1–3 While PARKIN is recruited to the mitochondrial outer membrane (MOM) upon depolarization via PINK1 action and can ubiquitylate Porin, Mitofusin, and Miro proteins on the MOM,1,4–11 the full repertoire of PARKIN substrates – the PARKIN-dependent ubiquitylome - remains poorly defined. Here we employ quantitative diGLY capture proteomics12,13 to elucidate the ubiquitylation site-specificity and topology of PARKIN-dependent target modification in response to mitochondrial depolarization. Hundreds of dynamically regulated ubiquitylation sites in dozens of proteins were identified, with strong enrichment for MOM proteins, indicating that PARKIN dramatically alters the ubiquitylation status of the mitochondrial proteome. Using complementary interaction proteomics, we found depolarization-dependent PARKIN association with numerous MOM targets, autophagy receptors, and the proteasome. Mutation of PARKIN’s active site residue C431, which has been found mutated in PD patients, largely disrupts these associations. Structural and topological analysis revealed extensive conservation of PARKIN-dependent ubiquitylation sites on cytoplasmic domains in vertebrate and D. melanogaster MOM proteins. These studies provide a resource for understanding how the PINK1-PARKIN pathway re-sculpts the proteome to support mitochondrial homeostasis. PMID:23503661

Modeling the full length HIV-1 Gag polyprotein reveals the role of its p6 subunit in viral maturation and the effect of non-cleavage site mutations in protease drug resistance.

PubMed

Su, Chinh Tran-To; Kwoh, Chee-Keong; Verma, Chandra Shekhar; Gan, Samuel Ken-En

2017-12-27

HIV polyprotein Gag is increasingly found to contribute to protease inhibitor resistance. Despite its role in viral maturation and in developing drug resistance, there remain gaps in the knowledge of the role of certain Gag subunits (e.g. p6), and that of non-cleavage mutations in drug resistance. As p6 is flexible, it poses a problem for structural experiments, and is hence often omitted in experimental Gag structural studies. Nonetheless, as p6 is an indispensable component for viral assembly and maturation, we have modeled the full length Gag structure based on several experimentally determined constraints and studied its structural dynamics. Our findings suggest that p6 can mechanistically modulate Gag conformations. In addition, the full length Gag model reveals that allosteric communication between the non-cleavage site mutations and the first Gag cleavage site could possibly result in protease drug resistance, particularly in the absence of mutations in Gag cleavage sites. Our study provides a mechanistic understanding to the structural dynamics of HIV-1 Gag, and also proposes p6 as a possible drug target in anti-HIV therapy.

GNAQ and GNA11 mutations occur in 9.5% of mucosal melanoma and are associated with poor prognosis.

PubMed

Sheng, Xinan; Kong, Yan; Li, Yiqian; Zhang, Qiannan; Si, Lu; Cui, Chuanliang; Chi, Zhihong; Tang, Bixia; Mao, Lili; Lian, Bin; Wang, Xuan; Yan, Xieqiao; Li, Siming; Dai, Jie; Guo, Jun

2016-09-01

Mucosal melanoma (MM) is a rare subtype of melanoma in Caucasians with extremely poor prognosis, and therapy strategy has not been clearly established for MM. We aimed to investigate the genetic aberrations possibly applicable in targeted therapy of MM. We examined the somatic mutations of GNAQ and GNA11 (GNAQ/11, encoding the guanine nucleotide-binding alpha subunits) in MM and evaluated their correlation to clinicopathologic features of MM. This study collected samples from primary lesions of 284 MM patients. Tissue samples were analysed for mutations in exons 4 and 5 of GNAQ/11 in genomic DNA by polymerase chain reaction amplification and Sanger sequencing. Correlations of GNAQ/11 mutations to clinicopathologic features and prognosis of MM were evaluated. The overall mutation frequency of GNAQ/11 in MM was 9.5% (27 in 284), with a frequency of 4.6% and 4.9% for GNAQ and GNA11 mutations, respectively. The mutations in exon 5 of GNAQ/11 occurred exclusively in codon 209. GNAQ(Q209L) was the most prevalent variation (92.3% of missense GNAQ mutations). GNAQ/11 mutations were not significantly associated with age, gender, ulceration, and primary anatomic site. The median overall survival for MM patients with GNAQ mutations (16.0 versus 26.0 months, P = 0.031) or GNA11 mutations (15.0 versus 26.0 months, P = 0.039) were significantly shorter than those for patients with wild-type GNAQ and GNA11, respectively. Our study suggests that GNAQ and GNA11 mutations occur frequently in MM and may be a prognostic factor for MM. Our data implicate that GNAQ/11 may be potential targets for targeted therapy of MM. Copyright © 2016 Elsevier Ltd. All rights reserved.

Steric and thermodynamic limits of design for the incorporation of large unnatural amino acids in aminoacyl-tRNA synthetase enzymes.

PubMed

Armen, Roger S; Schiller, Stefan M; Brooks, Charles L

2010-06-01

Orthogonal aminoacyl-tRNA synthetase/tRNA pairs from archaea have been evolved to facilitate site specific in vivo incorporation of unnatural amino acids into proteins in Escherichia coli. Using this approach, unnatural amino acids have been successfully incorporated with high translational efficiency and fidelity. In this study, CHARMM-based molecular docking and free energy calculations were used to evaluate rational design of specific protein-ligand interactions for aminoacyl-tRNA synthetases. A series of novel unnatural amino acid ligands were docked into the p-benzoyl-L-phenylalanine tRNA synthetase, which revealed that the binding pocket of the enzyme does not provide sufficient space for significantly larger ligands. Specific binding site residues were mutated to alanine to create additional space to accommodate larger target ligands, and then mutations were introduced to improve binding free energy. This approach was used to redesign binding sites for several different target ligands, which were then tested against the standard 20 amino acids to verify target specificity. Only the synthetase designed to bind Man-alpha-O-Tyr was predicted to be sufficiently selective for the target ligand and also thermodynamically stable. Our study suggests that extensive redesign of the tRNA synthatase binding pocket for large bulky ligands may be quite thermodynamically unfavorable.

Inactivation of Hepatitis B Virus Replication in Cultured Cells and In Vivo with Engineered Transcription Activator-Like Effector Nucleases

PubMed Central

Bloom, Kristie; Ely, Abdullah; Mussolino, Claudio; Cathomen, Toni; Arbuthnot, Patrick

2013-01-01

Chronic hepatitis B virus (HBV) infection remains an important global health problem. Stability of the episomal covalently closed circular HBV DNA (cccDNA) is largely responsible for the modest curative efficacy of available therapy. Since licensed anti-HBV drugs have a post-transcriptional mechanism of action, disabling cccDNA is potentially of therapeutic benefit. To develop this approach, we engineered mutagenic transcription activator-like effector nucleases (TALENs) that target four HBV-specific sites within the viral genome. TALENs with cognate sequences in the S or C open-reading frames (ORFs) efficiently disrupted sequences at the intended sites and suppressed markers of viral replication. Following triple transfection of cultured HepG2.2.15 cells under mildly hypothermic conditions, the S TALEN caused targeted mutation in ~35% of cccDNA molecules. Markers of viral replication were also inhibited in vivo in a murine hydrodynamic injection model of HBV replication. HBV target sites within S and C ORFs of the injected HBV DNA were mutated without evidence of toxicity. These findings are the first to demonstrate a targeted nuclease-mediated disruption of HBV cccDNA. Efficacy in vivo also indicates that these engineered nucleases have potential for use in treatment of chronic HBV infection. PMID:23883864

Highly efficient targeted mutagenesis in axolotl using Cas9 RNA-guided nuclease

PubMed Central

Flowers, G. Parker; Timberlake, Andrew T.; Mclean, Kaitlin C.; Monaghan, James R.; Crews, Craig M.

2014-01-01

Among tetrapods, only urodele salamanders, such as the axolotl Ambystoma mexicanum, can completely regenerate limbs as adults. The mystery of why salamanders, but not other animals, possess this ability has for generations captivated scientists seeking to induce this phenomenon in other vertebrates. Although many recent advances in molecular biology have allowed limb regeneration and tissue repair in the axolotl to be investigated in increasing detail, the molecular toolkit for the study of this process has been limited. Here, we report that the CRISPR-Cas9 RNA-guided nuclease system can efficiently create mutations at targeted sites within the axolotl genome. We identify individual animals treated with RNA-guided nucleases that have mutation frequencies close to 100% at targeted sites. We employ this technique to completely functionally ablate EGFP expression in transgenic animals and recapitulate developmental phenotypes produced by loss of the conserved gene brachyury. Thus, this advance allows a reverse genetic approach in the axolotl and will undoubtedly provide invaluable insight into the mechanisms of salamanders' unique regenerative ability. PMID:24764077

Target-site mutations conferring resistance to glyphosate in feathertop Rhodes grass (Chloris virgata) populations in Australia.

PubMed

Ngo, The D; Krishnan, Mahima; Boutsalis, Peter; Gill, Gurjeet; Preston, Christopher

2018-05-01

Chloris virgata is a warm-season, C 4 , annual grass weed affecting field crops in northern Australia that has become an emerging weed in southern Australia. Four populations with suspected resistance to glyphosate were collected in South Australia, Queensland and New South Wales, Australia, and compared with one susceptible (S) population to confirm glyphosate resistance and elucidate possible mechanisms of resistance. Based on the rate of glyphosate required to kill 50% of treated plants (LD 50 ), glyphosate resistance (GR) was confirmed in four populations of C. virgata (V12, V14.2, V14.16 and V15). GR plants were 2-9.7-fold more resistant and accumulated less shikimate after glyphosate treatment than S plants. GR and S plants did not differ in glyphosate absorption and translocation. Target-site EPSPS mutations corresponding to Pro-106-Leu (V14.2) and Pro-106-Ser (V15, V14.16 and V12) substitutions were found in GR populations. The population with Pro-106-Leu substitution was 2.9-4.9-fold more resistant than the three other populations with Pro-106-Ser substitution. This report confirms glyphosate resistance in C. virgata and shows that target-site EPSPS mutations confer resistance to glyphosate in this species. The evolution of glyphosate resistance in C. virgata highlights the need to identify alternative control tactics. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

A Bioinformatic Pipeline for Monitoring of the Mutational Stability of Viral Drug Targets with Deep-Sequencing Technology.

PubMed

Kravatsky, Yuri; Chechetkin, Vladimir; Fedoseeva, Daria; Gorbacheva, Maria; Kravatskaya, Galina; Kretova, Olga; Tchurikov, Nickolai

2017-11-23

The efficient development of antiviral drugs, including efficient antiviral small interfering RNAs (siRNAs), requires continuous monitoring of the strict correspondence between a drug and the related highly variable viral DNA/RNA target(s). Deep sequencing is able to provide an assessment of both the general target conservation and the frequency of particular mutations in the different target sites. The aim of this study was to develop a reliable bioinformatic pipeline for the analysis of millions of short, deep sequencing reads corresponding to selected highly variable viral sequences that are drug target(s). The suggested bioinformatic pipeline combines the available programs and the ad hoc scripts based on an original algorithm of the search for the conserved targets in the deep sequencing data. We also present the statistical criteria for the threshold of reliable mutation detection and for the assessment of variations between corresponding data sets. These criteria are robust against the possible sequencing errors in the reads. As an example, the bioinformatic pipeline is applied to the study of the conservation of RNA interference (RNAi) targets in human immunodeficiency virus 1 (HIV-1) subtype A. The developed pipeline is freely available to download at the website http://virmut.eimb.ru/. Brief comments and comparisons between VirMut and other pipelines are also presented.

Molecular investigation of active binding site of isoniazid (INH) and insight into resistance mechanism of S315T-MtKatG in Mycobacterium tuberculosis.

PubMed

Srivastava, Gaurava; Tripathi, Shubhandra; Kumar, Akhil; Sharma, Ashok

2017-07-01

Multi drug resistant tuberculosis is a major threat for mankind. Resistance against Isoniazid (INH), targeting MtKatG protein, is one of the most commonly occurring resistances in MDR TB strains. S315T-MtKatG mutation is widely reported for INH resistance. Despite having knowledge about the mechanism of INH, exact binding site of INH to MtKatG is still uncertain and proposed to have three presumable binding sites (site-1, site-2, and site-3). In the current study docking, molecular dynamics simulation, binding free energy estimation, principal component analysis and free energy landscape analysis were performed to get molecular level details of INH binding site on MtKatG, and to probe the effect of S315T mutation on INH binding. Molecular docking and MD analysis suggested site-1 as active binding site of INH, where the effects of S315T mutation were observed on both access tunnel as well as molecular interaction between INH and its neighboring residues. MMPBSA also supported site-1 as potential binding site with lowest binding energy of -44.201 kJ/mol. Moreover, PCA and FEL revealed that S315T mutation not only reduces the dimension of heme access tunnel but also showed that extra methyl group at 315 position altered heme cavity, enforcing heme group distantly from INH, and thus preventing INH activation. The present study not only investigated the active binding site of INH but also provides a new insight about the conformational changes in the binding site of S315T-MtKatG. Copyright © 2017 Elsevier Ltd. All rights reserved.

Single-Step qPCR and dPCR Detection of Diverse CRISPR-Cas9 Gene Editing Events In Vivo.

PubMed

Falabella, Micol; Sun, Linqing; Barr, Justin; Pena, Andressa Z; Kershaw, Erin E; Gingras, Sebastien; Goncharova, Elena A; Kaufman, Brett A

2017-10-05

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-based technology is currently the most flexible means to create targeted mutations by recombination or indel mutations by nonhomologous end joining. During mouse transgenesis, recombinant and indel alleles are often pursued simultaneously. Multiple alleles can be formed in each animal to create significant genetic complexity that complicates the CRISPR-Cas9 approach and analysis. Currently, there are no rapid methods to measure the extent of on-site editing with broad mutation sensitivity. In this study, we demonstrate the allelic diversity arising from targeted CRISPR editing in founder mice. Using this DNA sample collection, we validated specific quantitative and digital PCR methods (qPCR and dPCR, respectively) for measuring the frequency of on-target editing in founder mice. We found that locked nucleic acid (LNA) probes combined with an internal reference probe (Drop-Off Assay) provide accurate measurements of editing rates. The Drop-Off LNA Assay also detected on-target CRISPR-Cas9 gene editing in blastocysts with a sensitivity comparable to PCR-clone sequencing. Lastly, we demonstrate that the allele-specific LNA probes used in qPCR competitor assays can accurately detect recombinant mutations in founder mice. In summary, we show that LNA-based qPCR and dPCR assays provide a rapid method for quantifying the extent of on-target genome editing in vivo , testing RNA guides, and detecting recombinant mutations. Copyright © 2017 Falabella et al.

Osimertinib making a breakthrough in lung cancer targeted therapy

PubMed Central

Zhang, Haijun

2016-01-01

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are the evidence-based first-line treatment for advanced non-small-cell lung cancer that harbors sensitizing EGFR mutations (EGFRm+) such as exon 19 deletions and L858R substitutions in exon 21. However, acquired resistance to EGFR TKIs is mostly driven by a second-site EGFR T790M mutation, which negates their inhibitory activity. Osimertinib (AZD9291, Tagrisso™), an oral, third-generation EGFR TKI, has been designed to target the EGFR T790M mutation, while sparing wild-type EGFR. In this up-to-date review, focus is not only on the structure, mechanisms, and pharmacokinetics of osimertinib but also on summarizing clinical trials and making recommendations of osimertinib for patients with non-small-cell lung cancer. PMID:27660466

Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9.

PubMed

Prykhozhij, Sergey V; Fuller, Charlotte; Steele, Shelby L; Veinotte, Chansey J; Razaghi, Babak; Robitaille, Johane M; McMaster, Christopher R; Shlien, Adam; Malkin, David; Berman, Jason N

2018-06-14

We have optimized point mutation knock-ins into zebrafish genomic sites using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 reagents and single-stranded oligodeoxynucleotides. The efficiency of knock-ins was assessed by a novel application of allele-specific polymerase chain reaction and confirmed by high-throughput sequencing. Anti-sense asymmetric oligo design was found to be the most successful optimization strategy. However, cut site proximity to the mutation and phosphorothioate oligo modifications also greatly improved knock-in efficiency. A previously unrecognized risk of off-target trans knock-ins was identified that we obviated through the development of a workflow for correct knock-in detection. Together these strategies greatly facilitate the study of human genetic diseases in zebrafish, with additional applicability to enhance CRISPR-based approaches in other animal model systems.

Evolutionary transitions to new DNA methyltransferases through target site expansion and shrinkage.

PubMed

Rockah-Shmuel, Liat; Tawfik, Dan S

2012-12-01

DNA-binding and modifying proteins show high specificity but also exhibit a certain level of promiscuity. Such latent promiscuous activities comprise the starting points for new protein functions, but this hypothesis presents a paradox: a new activity can only evolve if it already exists. How then, do novel activities evolve? DNA methyltransferases, for example, are highly divergent in their target sites, but how transitions toward novel sites occur remains unknown. We performed laboratory evolution of the DNA methyltransferase M.HaeIII. We found that new target sites emerged primarily through expansion of the original site, GGCC, and the subsequent shrinkage of evolved expanded sites. Variants evolved for sites that are promiscuously methylated by M.HaeIII [GG((A)/(T))CC and GGCGCC] carried mutations in 'gate-keeper' residues. They could thereby methylate novel target sites such as GCGC and GGATCC that were neither selected for nor present in M.HaeIII. These 'generalist' intermediates were further evolved to obtain variants with novel target specificities. Our results demonstrate the ease by which new DNA-binding and modifying specificities evolve and the mechanism by which they occur at both the protein and DNA levels.

RNA splicing factors as oncoproteins and tumor suppressors

PubMed Central

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

2016-01-01

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

A mouse model for the cystic fibrosis delta F508 mutation.

PubMed Central

van Doorninck, J H; French, P J; Verbeek, E; Peters, R H; Morreau, H; Bijman, J; Scholte, B J

1995-01-01

Most cystic fibrosis (CF) patients produce a mutant form (delta F508) of the cystic fibrosis transmembrane conductance regulator (CFTR), which is not properly processed in normal cells but is active as a chloride channel in several experimental systems. We used a double homologous recombination ('Hit and Run') procedure to generate a mouse model for the delta F508 mutation. Targeted embryonic stem (ES) cells (Hit clones) were found; of these either 80 or 20% of the clones had lost the delta F508 mutation, depending on the distance between the linearization site in the targeting construct and the delta F508 mutation. Correctly targeted clones underwent a second selection step resulting in ES cell clones (Run clones) heterozygous for the delta F508 mutation with an efficiency of 2-7%. Chimeric mice were generated and offspring homozygous for the delta F508 mutation showed electrophysiological abnormalities in nasal epithelium, gallbladder and in the intestine, and histological abnormalities in the intestine, typical of CF. Our data suggest that the delta F508 mice have residual delta F508 CFTR activity which would explain the mild pathology of the delta F508 mice. The delta F508 mouse may provide a useful model for the study of the processing defect of delta F508 CFTR and for the development of novel therapeutic approaches based on circumvention of the processing block. Images PMID:7556083

Antibiotic resistance in Staphylococcus aureus. Current status and future prospects.

PubMed

Foster, Timothy J

2017-05-01

The major targets for antibiotics in staphylococci are (i) the cell envelope, (ii) the ribosome and (iii) nucleic acids. Several novel targets emerged from recent targeted drug discovery programmes including the ClpP protease and FtsZ from the cell division machinery. Resistance can either develop by horizontal transfer of resistance determinants encoded by mobile genetic elements viz plasmids, transposons and the staphylococcal cassette chromosome or by mutations in chromosomal genes. Horizontally acquired resistance can occur by one of the following mechanisms: (i) enzymatic drug modification and inactivation, (ii) enzymatic modification of the drug binding site, (iii) drug efflux, (iv) bypass mechanisms involving acquisition of a novel drug-resistant target, (v) displacement of the drug to protect the target. Acquisition of resistance by mutation can result from (i) alteration of the drug target that prevents the inhibitor from binding, (ii) derepression of chromosomally encoded multidrug resistance efflux pumps and (iii) multiple stepwise mutations that alter the structure and composition of the cell wall and/or membrane to reduce drug access to its target. This review focuses on development of resistance to currently used antibiotics and examines future prospects for new antibiotics and informed use of drug combinations. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Target-site resistance to neonicotinoids.

PubMed

Crossthwaite, Andrew J; Rendine, Stefano; Stenta, Marco; Slater, Russell

2014-10-01

Neonicotinoid insecticides selectively target the invertebrate nicotinic acetylcholine receptor and disrupt excitatory cholinergic neurotransmission. First launched over 20 years ago, their broad pest spectrum, variety of application methods and relatively low risk to nontarget organisms have resulted in this class dominating the insecticide market with global annual sales in excess of $3.5 bn. This remarkable commercial success brings with it conditions in the field that favour selection of resistant phenotypes. A number of important pest species have been identified with mutations at the nicotinic acetylcholine receptor associated with insensitivity to neonicotinoids. The detailed characterization of these mutations has facilitated a greater understanding of the invertebrate nicotinic acetylcholine receptor.

Phosphorylation of ORF1p is required for L1 retrotransposition.

PubMed

Cook, Pamela R; Jones, Charles E; Furano, Anthony V

2015-04-07

Although members of the L1 (LINE-1) clade of non-LTR retrotransposons can be deleterious, the L1 clade has remained active in most mammals for ∼100 million years and generated almost 40% of the human genome. The details of L1-host interaction are largely unknown, however. Here we report that L1 activity requires phosphorylation of the protein encoded by the L1 ORF1 (ORF1p). Critical phospho-acceptor residues (two serines and two threonines) reside in four conserved proline-directed protein kinase (PDPK) target sites. The PDPK family includes mitogen-activated protein kinases and cyclin-dependent kinases. Mutation of any PDPK phospho-acceptor inhibits L1 retrotransposition. The phosphomimetic aspartic acid can restore activity at the two serine sites, but not at either threonine site, where it is strongly inhibitory. ORF1p also contains conserved PDPK docking sites, which promote specific interaction of PDPKs with their targets. As expected, mutations in these sites also inhibit L1 activity. PDPK mutations in ORF1p that inactivate L1 have no significant effect on the ability of ORF1p to anneal RNA in vitro, an important biochemical property of the protein. We show that phosphorylated PDPK sites in ORF1p are required for an interaction with the peptidyl prolyl isomerase 1 (Pin1), a critical component of PDPK-mediated regulation. Pin1 acts via isomerization of proline side chains at phosphorylated PDPK motifs, thereby affecting substrate conformation and activity. Our demonstration that L1 activity is dependent on and integrated with cellular phosphorylation regulatory cascades significantly increases our understanding of interactions between L1 and its host.

Protospacer Adjacent Motif (PAM)-Distal Sequences Engage CRISPR Cas9 DNA Target Cleavage

PubMed Central

Ethier, Sylvain; Schmeing, T. Martin; Dostie, Josée; Pelletier, Jerry

2014-01-01

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme Cas9 is an RNA-guided nuclease that has been widely adapted for genome editing in eukaryotic cells. However, the in vivo target specificity of Cas9 is poorly understood and most studies rely on in silico predictions to define the potential off-target editing spectrum. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq), we delineate the genome-wide binding panorama of catalytically inactive Cas9 directed by two different single guide (sg) RNAs targeting the Trp53 locus. Cas9:sgRNA complexes are able to load onto multiple sites with short seed regions adjacent to 5′NGG3′ protospacer adjacent motifs (PAM). Yet among 43 ChIP-seq sites harboring seed regions analyzed for mutational status, we find editing only at the intended on-target locus and one off-target site. In vitro analysis of target site recognition revealed that interactions between the 5′ end of the guide and PAM-distal target sequences are necessary to efficiently engage Cas9 nucleolytic activity, providing an explanation for why off-target editing is significantly lower than expected from ChIP-seq data. PMID:25275497

Steric and Thermodynamic Limits of Design for the Incorporation of Large UnNatural Amino Acids in Aminoacyl-tRNA Synthetase Enzymes

PubMed Central

Armen, Roger S.; Schiller, Stefan M.; Brooks, Charles L.

2015-01-01

Orthogonal aminoacyl-tRNA synthetase/tRNA pairs from archaea have been evolved to facilitate site specific in vivo incorporation of unnatural amino acids into proteins in Escherichia coli. Using this approach, unnatural amino acids have been successfully incorporated with high translational efficiency and fidelity. In this study, CHARMM-based molecular docking and free energy calculations were used to evaluate rational design of specific protein-ligand interactions for aminoacyl-tRNA synthetases. A series of novel unnatural amino acid ligands were docked into the p-benzoyl-L-phenylalanine tRNA synthetase, which revealed that the binding pocket of the enzyme does not provide sufficient space for significantly larger ligands. Specific binding site residues were mutated to alanine to create additional space to accommodate larger target ligands, and then mutations were introduced to improve binding free energy. This approach was used to redesign binding sites for several different target ligands, which were then tested against the standard 20 amino acids to verify target specificity. Only the synthetase designed to bind Man-α-O-Tyr was predicted to be sufficiently selective for the target ligand and also thermodynamically stable. Our study suggests that extensive redesign of the tRNA synthatase binding pocket for large bulky ligands may be quite thermodynamically unfavorable. PMID:20310065

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

PubMed Central

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

2017-01-01

Cholangiocarcinoma (CCA) is a hepatobiliary malignancy exhibiting high incidence in countries with endemic liver-fluke infection. We analysed 489 CCAs from 10 countries, combining whole-genome (71 cases), targeted/exome, copy-number, gene expression, and DNA methylation information. Integrative clustering defined four CCA clusters – Fluke-Positive CCAs (Clusters 1/2) are enriched in ERBB2 amplifications and TP53 mutations, conversely Fluke-Negative CCAs (Clusters 3/4) exhibit high copy-number alterations and PD-1/PD-L2 expression, or epigenetic mutations (IDH1/2, BAP1) and FGFR/PRKA-related gene rearrangements. Whole-genome analysis highlighted FGFR2 3′UTR deletion as a mechanism of FGFR2 upregulation. Integration of non-coding promoter mutations with protein-DNA binding profiles demonstrates pervasive modulation of H3K27me3-associated sites in CCA. Clusters 1 and 4 exhibit distinct DNA hypermethylation patterns targeting either CpG islands or shores – mutation signature and subclonality analysis suggests that these reflect different mutational pathways. Our results exemplify how genetics, epigenetics and environmental carcinogens can interplay across different geographies to generate distinct molecular subtypes of cancer. PMID:28667006

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

Jusakul, Apinya; Cutcutache, Ioana; Yong, Chern Han

Cholangiocarcinoma (CCA) is a hepatobiliary malignancy exhibiting high incidence in countries with endemic liver-fluke infection. We analysed 489 CCAs from 10 countries, combining whole-genome (71 cases), targeted/exome, copy-number, gene expression, and DNA methylation information. Integrative clustering defined four CCA clusters - Fluke- Positive CCAs (Clusters 1/2) are enriched in ERBB2 amplifications and TP53 mutations, conversely Fluke-Negative CCAs (Clusters 3/4) exhibit high copy-number alterations and PD-1/PD-L2 expression, or epigenetic mutations (IDH1/2, BAP1) and FGFR/PRKA-related gene rearrangements. Whole-genome analysis highlighted FGFR2 3’UTR deletion as a mechanism of FGFR2 upregulation. Integration of non-coding promoter mutations with protein-DNA binding profiles demonstrates pervasive modulation ofmore » H3K27me3-associated sites in CCA. Clusters 1 and 4 exhibit distinct DNA hypermethylation patterns targeting either CpG islands or shores - mutation signature and subclonality analysis suggests that these reflect different mutational pathways. Lastly, our results exemplify how genetics, epigenetics and environmental carcinogens can interplay across different geographies to generate distinct molecular subtypes of cancer.« less

Engineered Cpf1 variants with altered PAM specificities increase genome targeting range

PubMed Central

Gao, Linyi; Cox, David B.T.; Yan, Winston X.; Manteiga, John C.; Schneider, Martin W.; Yamano, Takashi; Nishimasu, Hiroshi; Nureki, Osamu; Crosetto, Nicola; Zhang, Feng

2017-01-01

The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells1–7. However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, we performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. We engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity using BLISS7 assay indicated that these variants retain high DNA targeting specificity, which we further improved by introducing an additional non-PAM-interacting mutation. Introducing the identified mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. Together, these variants increase the targeting range of Cpf1 by approximately three-fold in human coding sequences to one cleavage site per ~11 bp. PMID:28581492

Correction of the sickle cell disease mutation in human hematopoietic stem/progenitor cells.

PubMed

Hoban, Megan D; Cost, Gregory J; Mendel, Matthew C; Romero, Zulema; Kaufman, Michael L; Joglekar, Alok V; Ho, Michelle; Lumaquin, Dianne; Gray, David; Lill, Georgia R; Cooper, Aaron R; Urbinati, Fabrizia; Senadheera, Shantha; Zhu, Allen; Liu, Pei-Qi; Paschon, David E; Zhang, Lei; Rebar, Edward J; Wilber, Andrew; Wang, Xiaoyan; Gregory, Philip D; Holmes, Michael C; Reik, Andreas; Hollis, Roger P; Kohn, Donald B

2015-04-23

Sickle cell disease (SCD) is characterized by a single point mutation in the seventh codon of the β-globin gene. Site-specific correction of the sickle mutation in hematopoietic stem cells would allow for permanent production of normal red blood cells. Using zinc-finger nucleases (ZFNs) designed to flank the sickle mutation, we demonstrate efficient targeted cleavage at the β-globin locus with minimal off-target modification. By co-delivering a homologous donor template (either an integrase-defective lentiviral vector or a DNA oligonucleotide), high levels of gene modification were achieved in CD34(+) hematopoietic stem and progenitor cells. Modified cells maintained their ability to engraft NOD/SCID/IL2rγ(null) mice and to produce cells from multiple lineages, although with a reduction in the modification levels relative to the in vitro samples. Importantly, ZFN-driven gene correction in CD34(+) cells from the bone marrow of patients with SCD resulted in the production of wild-type hemoglobin tetramers. © 2015 by The American Society of Hematology.

SUMOylation target sites at the C terminus protect Axin from ubiquitination and confer protein stability

PubMed Central

Kim, Min Jung; Chia, Ian V.; Costantini, Frank

2008-01-01

Axin is a scaffold protein for the β-catenin destruction complex, and a negative regulator of canonical Wnt signaling. Previous studies implicated the six C-terminal amino acids (C6 motif) in the ability of Axin to activate c-Jun N-terminal kinase, and identified them as a SUMOylation target. Deletion of the C6 motif of mouse Axin in vivo reduced the steady-state protein level, which caused embryonic lethality. Here, we report that this deletion (Axin-ΔC6) causes a reduced half-life in mouse embryonic fibroblasts and an increased susceptibility to ubiquitination in HEK 293T cells. We confirmed the C6 motif as a SUMOylation target in vitro, and found that mutating the C-terminal SUMOylation target residues increased the susceptibility of Axin to polyubiquitination and reduced its steady-state level. Heterologous SUMOylation target sites could replace C6 in providing this protective effect. These findings suggest that SUMOylation of the C6 motif may prevent polyubiquitination, thus increasing the stability of Axin. Although C6 deletion also caused increased association of Axin with Dvl-1, this interaction was not altered by mutating the lysine residues in C6, nor could heterologous SUMOylation motifs replace the C6 motif in this assay. Therefore, some other specific property of the C6 motif seems to reduce the interaction of Axin with Dvl-1.—Kim, M. J., Chia, I. V., Costantini, F. SUMOylation target sites at the C terminus protect Axin from ubiquitination and confer protein stability. PMID:18632848

Massively Parallel Sequencing of Patients with Intellectual Disability, Congenital Anomalies and/or Autism Spectrum Disorders with a Targeted Gene Panel

PubMed Central

Brett, Maggie; McPherson, John; Zang, Zhi Jiang; Lai, Angeline; Tan, Ee-Shien; Ng, Ivy; Ong, Lai-Choo; Cham, Breana; Tan, Patrick; Rozen, Steve; Tan, Ene-Choo

2014-01-01

Developmental delay and/or intellectual disability (DD/ID) affects 1–3% of all children. At least half of these are thought to have a genetic etiology. Recent studies have shown that massively parallel sequencing (MPS) using a targeted gene panel is particularly suited for diagnostic testing for genetically heterogeneous conditions. We report on our experiences with using massively parallel sequencing of a targeted gene panel of 355 genes for investigating the genetic etiology of eight patients with a wide range of phenotypes including DD/ID, congenital anomalies and/or autism spectrum disorder. Targeted sequence enrichment was performed using the Agilent SureSelect Target Enrichment Kit and sequenced on the Illumina HiSeq2000 using paired-end reads. For all eight patients, 81–84% of the targeted regions achieved read depths of at least 20×, with average read depths overlapping targets ranging from 322× to 798×. Causative variants were successfully identified in two of the eight patients: a nonsense mutation in the ATRX gene and a canonical splice site mutation in the L1CAM gene. In a third patient, a canonical splice site variant in the USP9X gene could likely explain all or some of her clinical phenotypes. These results confirm the value of targeted MPS for investigating DD/ID in children for diagnostic purposes. However, targeted gene MPS was less likely to provide a genetic diagnosis for children whose phenotype includes autism. PMID:24690944

Chemical and UV Mutagenesis.

PubMed

Bose, Jeffrey L

2016-01-01

The ability to create mutations is an important step towards understanding bacterial physiology and virulence. While targeted approaches are invaluable, the ability to produce genome-wide random mutations can lead to crucial discoveries. Transposon mutagenesis is a useful approach, but many interesting mutations can be missed by these insertions that interrupt coding and noncoding sequences due to the integration of an entire transposon. Chemical mutagenesis and UV-based random mutagenesis are alternate approaches to isolate mutations of interest with the potential of only single nucleotide changes. Once a standard method, difficulty in identifying mutation sites had decreased the popularity of this technique. However, thanks to the recent emergence of economical whole-genome sequencing, this approach to making mutations can once again become a viable option. Therefore, this chapter provides an overview protocol for random mutagenesis using UV light or DNA-damaging chemicals.

Mutations in the voltage-gated sodium channel gene of anophelines and their association with resistance to pyrethroids - a review.

PubMed

Silva, Ana Paula B; Santos, Joselita Maria M; Martins, Ademir J

2014-10-07

Constant and extensive use of chemical insecticides has created a selection pressure and favored resistance development in many insect species worldwide. One of the most important pyrethroid resistance mechanisms is classified as target site insensitivity, due to conformational changes in the target site that impair a proper binding of the insecticide molecule. The voltage-gated sodium channel (NaV) is the target of pyrethroids and DDT insecticides, used to control insects of medical, agricultural and veterinary importance, such as anophelines. It has been reported that the presence of a few non-silent point mutations in the NaV gene are associated with pyrethroid resistance, termed as 'kdr' (knockdown resistance) for preventing the knockdown effect of these insecticides. The presence of these mutations, as well as their effects, has been thoroughly studied in Anopheles mosquitoes. So far, kdr mutations have already been detected in at least 13 species (Anopheles gambiae, Anopheles arabiensis, Anopheles sinensis, Anopheles stephensi, Anopheles subpictus, Anopheles sacharovi, Anopheles culicifacies, Anopheles sundaicus, Anopheles aconitus, Anopheles vagus, Anopheles paraliae, Anopheles peditaeniatus and Anopheles albimanus) from populations of African, Asian and, more recently, American continents. Seven mutational variants (L1014F, L1014S, L1014C, L1014W, N1013S, N1575Y and V1010L) were described, with the highest prevalence of L1014F, which occurs at the 1014 site in NaV IIS6 domain. The increase of frequency and distribution of kdr mutations clearly shows the importance of this mechanism in the process of pyrethroid resistance. In this sense, several species-specific and highly sensitive methods have been designed in order to genotype individual mosquitoes for kdr in large scale, which may serve as important tolls for monitoring the dynamics of pyrethroid resistance in natural populations. We also briefly discuss investigations concerning the course of Plasmodium infection in kdr individuals. Considering the limitation of insecticides available for employment in public health campaigns and the absence of a vaccine able to brake the life cycle of the malaria parasites, the use of pyrethroids is likely to remain as the main strategy against mosquitoes by either indoor residual spraying (IR) and insecticide treated nets (ITN). Therefore, monitoring insecticide resistance programs is a crucial need in malaria endemic countries.

A site-directed mutagenesis method particularly useful for creating otherwise difficult-to-make mutants and alanine scanning.

PubMed

Wan, Haisu; Li, Yongwen; Fan, Yu; Meng, Fanrong; Chen, Chen; Zhou, Qinghua

2012-01-15

Site-directed mutagenesis has become routine in molecular biology. However, many mutants can still be very difficult to create. Complicated chimerical mutations, tandem repeats, inverted sequences, GC-rich regions, and/or heavy secondary structures can cause inefficient or incorrect binding of the mutagenic primer to the target sequence and affect the subsequent amplification. In theory, these problems can be avoided by introducing the mutations into the target sequence using mutagenic fragments and so removing the need for primer-template annealing. The cassette mutagenesis uses the mutagenic fragment in its protocol; however, in most cases it needs to perform two rounds of mutagenic primer-based mutagenesis to introduce suitable restriction enzyme sites into templates and is not suitable for routine mutagenesis. Here we describe a highly efficient method in which the template except the region to be mutated is amplified by polymerase chain reaction (PCR) and the type IIs restriction enzyme-digested PCR product is directly ligated with the mutagenic fragment. Our method requires no assistance of mutagenic primers. We have used this method to create various types of difficult-to-make mutants with mutagenic frequencies of nearly 100%. Our protocol has many advantages over the prevalent QuikChange method and is a valuable tool for studies on gene structure and function. Copyright © 2011 Elsevier Inc. All rights reserved.

Both TALENs and CRISPR/Cas9 directly target the HBB IVS2–654 (C > T) mutation in β-thalassemia-derived iPSCs

PubMed Central

Xu, Peng; Tong, Ying; Liu, Xiu-zhen; Wang, Ting-ting; Cheng, Li; Wang, Bo-yu; Lv, Xiang; Huang, Yue; Liu, De-pei

2015-01-01

β-Thalassemia is one of the most common genetic blood diseases and is caused by either point mutations or deletions in the β-globin (HBB) gene. The generation of patient-specific induced pluripotent stem cells (iPSCs) and subsequent correction of the disease-causing mutations may be a potential therapeutic strategy for this disease. Due to the low efficiency of typical homologous recombination, endonucleases, including TALENs and CRISPR/Cas9, have been widely used to enhance the gene correction efficiency in patient-derived iPSCs. Here, we designed TALENs and CRISPR/Cas9 to directly target the intron2 mutation site IVS2-654 in the globin gene. We observed different frequencies of double-strand breaks (DSBs) at IVS2-654 loci using TALENs and CRISPR/Cas9, and TALENs mediated a higher homologous gene targeting efficiency compared to CRISPR/Cas9 when combined with the piggyBac transposon donor. In addition, more obvious off-target events were observed for CRISPR/Cas9 compared to TALENs. Finally, TALENs-corrected iPSC clones were selected for erythroblast differentiation using the OP9 co-culture system and detected relatively higher transcription of HBB than the uncorrected cells. This comparison of using TALENs or CRISPR/Cas9 to correct specific HBB mutations in patient-derived iPSCs will guide future applications of TALENs- or CRISPR/Cas9-based gene therapies in monogenic diseases. PMID:26156589

Both TALENs and CRISPR/Cas9 directly target the HBB IVS2-654 (C > T) mutation in β-thalassemia-derived iPSCs.

PubMed

Xu, Peng; Tong, Ying; Liu, Xiu-zhen; Wang, Ting-ting; Cheng, Li; Wang, Bo-yu; Lv, Xiang; Huang, Yue; Liu, De-pei

2015-07-09

β-Thalassemia is one of the most common genetic blood diseases and is caused by either point mutations or deletions in the β-globin (HBB) gene. The generation of patient-specific induced pluripotent stem cells (iPSCs) and subsequent correction of the disease-causing mutations may be a potential therapeutic strategy for this disease. Due to the low efficiency of typical homologous recombination, endonucleases, including TALENs and CRISPR/Cas9, have been widely used to enhance the gene correction efficiency in patient-derived iPSCs. Here, we designed TALENs and CRISPR/Cas9 to directly target the intron2 mutation site IVS2-654 in the globin gene. We observed different frequencies of double-strand breaks (DSBs) at IVS2-654 loci using TALENs and CRISPR/Cas9, and TALENs mediated a higher homologous gene targeting efficiency compared to CRISPR/Cas9 when combined with the piggyBac transposon donor. In addition, more obvious off-target events were observed for CRISPR/Cas9 compared to TALENs. Finally, TALENs-corrected iPSC clones were selected for erythroblast differentiation using the OP9 co-culture system and detected relatively higher transcription of HBB than the uncorrected cells. This comparison of using TALENs or CRISPR/Cas9 to correct specific HBB mutations in patient-derived iPSCs will guide future applications of TALENs- or CRISPR/Cas9-based gene therapies in monogenic diseases.

High prevalence of mutations affecting the splicing process in a Spanish cohort with autosomal dominant retinitis pigmentosa

PubMed Central

Ezquerra-Inchausti, Maitane; Barandika, Olatz; Anasagasti, Ander; Irigoyen, Cristina; López de Munain, Adolfo; Ruiz-Ederra, Javier

2017-01-01

Retinitis pigmentosa is the most frequent group of inherited retinal dystrophies. It is highly heterogeneous, with more than 80 disease-causing genes 27 of which are known to cause autosomal dominant RP (adRP), having been identified. In this study a total of 29 index cases were ascertained based on a family tree compatible with adRP. A custom panel of 31 adRP genes was analysed by targeted next-generation sequencing using the Ion PGM platform in combination with Sanger sequencing. This allowed us to detect putative disease-causing mutations in 14 out of the 29 (48.28%) families analysed. Remarkably, around 38% of all adRP cases analysed showed mutations affecting the splicing process, mainly due to mutations in genes coding for spliceosome factors (SNRNP200 and PRPF8) but also due to splice-site mutations in RHO. Twelve of the 14 mutations found had been reported previously and two were novel mutations found in PRPF8 in two unrelated patients. In conclusion, our results will lead to more accurate genetic counselling and will contribute to a better characterisation of the disease. In addition, they may have a therapeutic impact in the future given the large number of studies currently underway based on targeted RNA splicing for therapeutic purposes. PMID:28045043

Occurrence of target-site resistance to neonicotinoids in the aphid Myzus persicae in Tunisia, and its status on different host plants.

PubMed

Charaabi, Kamel; Boukhris-Bouhachem, Sonia; Makni, Mohamed; Denholm, Ian

2018-06-01

The R81T mutation conferring target-site resistance to neonicotinoid insecticides in Myzus persicae was first detected in France and has since spread across much of southern Europe. In response to recent claims of control failure with neonicotinoids in Tunisia, we have used a molecular assay to investigate the presence and distribution of this target-site mutation in samples collected from six locations and six crops attacked by M. persicae. The resistance allele containing R81T was present at substantial frequencies (32-55%) in aphids collected between 2014 and 2016 from northern Tunisia but was much rarer further south. It occurred in aphids collected from the aphid's primary host (peach) and four secondary crop hosts (potato, pepper, tomato and melon). Its absence in aphids from tobacco highlights complexities in the systematics of M. persicae that require further investigation. This first report of R81T from North Africa reflects a continuing expansion of its range around the Mediterranean Basin, although it remains unrecorded elsewhere in the world. Loss of efficacy of neonicotinoids presents a serious threat to the sustainability of aphid control. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Mutations in POLR3A and POLR3B Encoding RNA Polymerase III Subunits Cause an Autosomal-Recessive Hypomyelinating Leukoencephalopathy

PubMed Central

Saitsu, Hirotomo; Osaka, Hitoshi; Sasaki, Masayuki; Takanashi, Jun-ichi; Hamada, Keisuke; Yamashita, Akio; Shibayama, Hidehiro; Shiina, Masaaki; Kondo, Yukiko; Nishiyama, Kiyomi; Tsurusaki, Yoshinori; Miyake, Noriko; Doi, Hiroshi; Ogata, Kazuhiro; Inoue, Ken; Matsumoto, Naomichi

2011-01-01

Congenital hypomyelinating disorders are a heterogeneous group of inherited leukoencephalopathies characterized by abnormal myelin formation. We have recently reported a hypomyelinating syndrome characterized by diffuse cerebral hypomyelination with cerebellar atrophy and hypoplasia of the corpus callosum (HCAHC). We performed whole-exome sequencing of three unrelated individuals with HCAHC and identified compound heterozygous mutations in POLR3B in two individuals. The mutations include a nonsense mutation, a splice-site mutation, and two missense mutations at evolutionally conserved amino acids. Using reverse transcription-PCR and sequencing, we demonstrated that the splice-site mutation caused deletion of exon 18 from POLR3B mRNA and that the transcript harboring the nonsense mutation underwent nonsense-mediated mRNA decay. We also identified compound heterozygous missense mutations in POLR3A in the remaining individual. POLR3A and POLR3B encode the largest and second largest subunits of RNA Polymerase III (Pol III), RPC1 and RPC2, respectively. RPC1 and RPC2 together form the active center of the polymerase and contribute to the catalytic activity of the polymerase. Pol III is involved in the transcription of small noncoding RNAs, such as 5S ribosomal RNA and all transfer RNAs (tRNA). We hypothesize that perturbation of Pol III target transcription, especially of tRNAs, could be a common pathological mechanism underlying POLR3A and POLR3B mutations. PMID:22036171

Erythrocytosis and Pulmonary Hypertension in a Mouse Model of Human HIF2A Gain of Function Mutation*

PubMed Central

Tan, Qiulin; Kerestes, Heddy; Percy, Melanie J.; Pietrofesa, Ralph; Chen, Li; Khurana, Tejvir S.; Christofidou-Solomidou, Melpo; Lappin, Terence R. J.; Lee, Frank S.

2013-01-01

The central pathway for oxygen-dependent control of red cell mass is the prolyl hydroxylase domain protein (PHD):hypoxia inducible factor (HIF) pathway. PHD site specifically prolyl hydroxylates the transcription factor HIF-α, thereby targeting the latter for degradation. Under hypoxia, this modification is attenuated, allowing stabilized HIF-α to activate target genes, including that for erythropoietin (EPO). Studies employing genetically modified mice point to Hif-2α, one of two main Hif-α isoforms, as being the critical regulator of Epo in the adult mouse. More recently, erythrocytosis patients with heterozygous point mutations in the HIF2A gene have been identified; whether these mutations were polymorphisms unrelated to the phenotype could not be ruled out. In the present report, we characterize a mouse line bearing a G536W missense mutation in the Hif2a gene that corresponds to the first such human mutation identified (G537W). We obtained mice bearing both heterozygous and homozygous mutations at this locus. We find that these mice display, in a mutation dose-dependent manner, erythrocytosis and pulmonary hypertension with a high degree of penetrance. These findings firmly establish missense mutations in HIF-2α as a cause of erythrocytosis, highlight the importance of this HIF-α isoform in erythropoiesis, and point to physiologic consequences of HIF-2α dysregulation. PMID:23640890

Structural insight into the role of Gln293Met mutation on the Peloruside A/Laulimalide association with αβ-tubulin from molecular dynamics simulations, binding free energy calculations and weak interactions analysis.

PubMed

Zúñiga, Matías A; Alderete, Joel B; Jaña, Gonzalo A; Jiménez, Verónica A

2017-07-01

Peloruside A (PLA) and Laulimalide (LAU) are novel microtubule-stabilizing agents with promising properties against different cancer types. These ligands share a non-taxoid binding site at the outer surface of β-tubulin and promote microtubule stabilization by bridging two adjacent αβ-tubulin dimers from parallel protofilaments. Recent site-directed mutagenesis experiments confirmed the existence of a unique β-tubulin site mutation (Gln293Met) that specifically increased the activity of PLA and caused resistance to LAU, without affecting the stability of microtubules in the absence of the ligands. In this work, fully atomistic molecular dynamics simulations were carried out to examine the PLA and LAU association with native and mutated αβ-tubulin in the search for structural and energetic evidence to explain the role of Gln293Met mutation on determining the activity of these ligands. Our results revealed that Gln293Met mutation induced the loss of relevant LAU-tubulin contacts but exerted negligible changes in the interaction networks responsible for PLA-tubulin association. Binding free energy calculations (MM/GBSA and MM/PBSA), and weak interaction analysis (aNCI) predicted an increased affinity for PLA, and a weakened association for LAU after mutation, thus suggesting that Gln293Met mutation exerts its action by a modulation of drug-tubulin interactions. These results are valuable to increase understanding about PLA and LAU activity and to assist the future design of novel agents targeting the PLA/LAU binding pocket.

Structural insight into the role of Gln293Met mutation on the Peloruside A/Laulimalide association with αβ-tubulin from molecular dynamics simulations, binding free energy calculations and weak interactions analysis

NASA Astrophysics Data System (ADS)

Zúñiga, Matías A.; Alderete, Joel B.; Jaña, Gonzalo A.; Jiménez, Verónica A.

2017-07-01

Peloruside A (PLA) and Laulimalide (LAU) are novel microtubule-stabilizing agents with promising properties against different cancer types. These ligands share a non-taxoid binding site at the outer surface of β-tubulin and promote microtubule stabilization by bridging two adjacent αβ-tubulin dimers from parallel protofilaments. Recent site-directed mutagenesis experiments confirmed the existence of a unique β-tubulin site mutation (Gln293Met) that specifically increased the activity of PLA and caused resistance to LAU, without affecting the stability of microtubules in the absence of the ligands. In this work, fully atomistic molecular dynamics simulations were carried out to examine the PLA and LAU association with native and mutated αβ-tubulin in the search for structural and energetic evidence to explain the role of Gln293Met mutation on determining the activity of these ligands. Our results revealed that Gln293Met mutation induced the loss of relevant LAU-tubulin contacts but exerted negligible changes in the interaction networks responsible for PLA-tubulin association. Binding free energy calculations (MM/GBSA and MM/PBSA), and weak interaction analysis (aNCI) predicted an increased affinity for PLA, and a weakened association for LAU after mutation, thus suggesting that Gln293Met mutation exerts its action by a modulation of drug-tubulin interactions. These results are valuable to increase understanding about PLA and LAU activity and to assist the future design of novel agents targeting the PLA/LAU binding pocket.

Understanding and Improving the Activity of Flavin Dependent Halogenases via Random and Targeted Mutagenesis

PubMed Central

Andorfer, Mary C.

2018-01-01

Flavin dependent halogenases (FDHs) catalyze the halogenation of organic substrates by coordinating reactions of reduced flavin, molecular oxygen, and chloride. Targeted and random mutagenesis of these enzymes has been used to both understand and alter their reactivity. These studies have led to insights into residues essential for catalysis and FDH variants with improved stability, expanded substrate scope, and altered site selectivity. Mutations throughout FDH structures have contributed to all of these advances. More recent studies have sought to rationalize the impact of these mutations on FDH function and to identify new FDHs to deepen our understanding of this enzyme class and to expand their utility for biocatalytic applications. PMID:29589959

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

PubMed

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

2017-07-25

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

Classical and atypical agonists activate M1 muscarinic acetylcholine receptors through common mechanisms.

PubMed

Randáková, Alena; Dolejší, Eva; Rudajev, Vladimír; Zimčík, Pavel; Doležal, Vladimír; El-Fakahany, Esam E; Jakubík, Jan

2015-07-01

We mutated key amino acids of the human variant of the M1 muscarinic receptor that target ligand binding, receptor activation, and receptor-G protein interaction. We compared the effects of these mutations on the action of two atypical M1 functionally preferring agonists (N-desmethylclozapine and xanomeline) and two classical non-selective orthosteric agonists (carbachol and oxotremorine). Mutations of D105 in the orthosteric binding site and mutation of D99 located out of the orthosteric binding site decreased affinity of all tested agonists that was translated as a decrease in potency in accumulation of inositol phosphates and intracellular calcium mobilization. Mutation of D105 decreased the potency of the atypical agonist xanomeline more than that of the classical agonists carbachol and oxotremorine. Mutation of the residues involved in receptor activation (D71) and coupling to G-proteins (R123) completely abolished the functional responses to both classical and atypical agonists. Our data show that both classical and atypical agonists activate hM1 receptors by the same molecular switch that involves D71 in the second transmembrane helix. The principal difference among the studied agonists is rather in the way they interact with D105 in the orthosteric binding site. Furthermore, our data demonstrate a key role of D105 in xanomeline wash-resistant binding and persistent activation of hM1 by wash-resistant xanomeline. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Impact of germline and somatic missense variations on drug binding sites.

PubMed

Yan, C; Pattabiraman, N; Goecks, J; Lam, P; Nayak, A; Pan, Y; Torcivia-Rodriguez, J; Voskanian, A; Wan, Q; Mazumder, R

2017-03-01

Advancements in next-generation sequencing (NGS) technologies are generating a vast amount of data. This exacerbates the current challenge of translating NGS data into actionable clinical interpretations. We have comprehensively combined germline and somatic nonsynonymous single-nucleotide variations (nsSNVs) that affect drug binding sites in order to investigate their prevalence. The integrated data thus generated in conjunction with exome or whole-genome sequencing can be used to identify patients who may not respond to a specific drug because of alterations in drug binding efficacy due to nsSNVs in the target protein's gene. To identify the nsSNVs that may affect drug binding, protein-drug complex structures were retrieved from Protein Data Bank (PDB) followed by identification of amino acids in the protein-drug binding sites using an occluded surface method. Then, the germline and somatic mutations were mapped to these amino acids to identify which of these alter protein-drug binding sites. Using this method we identified 12 993 amino acid-drug binding sites across 253 unique proteins bound to 235 unique drugs. The integration of amino acid-drug binding sites data with both germline and somatic nsSNVs data sets revealed 3133 nsSNVs affecting amino acid-drug binding sites. In addition, a comprehensive drug target discovery was conducted based on protein structure similarity and conservation of amino acid-drug binding sites. Using this method, 81 paralogs were identified that could serve as alternative drug targets. In addition, non-human mammalian proteins bound to drugs were used to identify 142 homologs in humans that can potentially bind to drugs. In the current protein-drug pairs that contain somatic mutations within their binding site, we identified 85 proteins with significant differential gene expression changes associated with specific cancer types. Information on protein-drug binding predicted drug target proteins and prevalence of both somatic and germline nsSNVs that disrupt these binding sites can provide valuable knowledge for personalized medicine treatment. A web portal is available where nsSNVs from individual patient can be checked by scanning against DrugVar to determine whether any of the SNVs affect the binding of any drug in the database.

How single mutations affect viral escape from broad and narrow antibodies to H1 influenza hemagglutinin.

PubMed

Doud, Michael B; Lee, Juhye M; Bloom, Jesse D

2018-04-11

Influenza virus can escape most antibodies with single mutations. However, rare antibodies broadly neutralize many viral strains. It is unclear how easily influenza virus might escape such antibodies if there was strong pressure to do so. Here, we map all single amino-acid mutations that increase resistance to broad antibodies to H1 hemagglutinin. Our approach not only identifies antigenic mutations but also quantifies their effect sizes. All antibodies select mutations, but the effect sizes vary widely. The virus can escape a broad antibody to hemagglutinin's receptor-binding site the same way it escapes narrow strain-specific antibodies: via single mutations with huge effects. In contrast, broad antibodies to hemagglutinin's stalk only select mutations with small effects. Therefore, among the antibodies we examine, breadth is an imperfect indicator of the potential for viral escape via single mutations. Antibodies targeting the H1 hemagglutinin stalk are quantifiably harder to escape than the other antibodies tested here.

Enhanced vulnerability of human proteins towards disease-associated inactivation through divergent evolution.

PubMed

Medina-Carmona, Encarnación; Fuchs, Julian E; Gavira, Jose A; Mesa-Torres, Noel; Neira, Jose L; Salido, Eduardo; Palomino-Morales, Rogelio; Burgos, Miguel; Timson, David J; Pey, Angel L

2017-09-15

Human proteins are vulnerable towards disease-associated single amino acid replacements affecting protein stability and function. Interestingly, a few studies have shown that consensus amino acids from mammals or vertebrates can enhance protein stability when incorporated into human proteins. Here, we investigate yet unexplored relationships between the high vulnerability of human proteins towards disease-associated inactivation and recent evolutionary site-specific divergence of stabilizing amino acids. Using phylogenetic, structural and experimental analyses, we show that divergence from the consensus amino acids at several sites during mammalian evolution has caused local protein destabilization in two human proteins linked to disease: cancer-associated NQO1 and alanine:glyoxylate aminotransferase, mutated in primary hyperoxaluria type I. We demonstrate that a single consensus mutation (H80R) acts as a disease suppressor on the most common cancer-associated polymorphism in NQO1 (P187S). The H80R mutation reactivates P187S by enhancing FAD binding affinity through local and dynamic stabilization of its binding site. Furthermore, we show how a second suppressor mutation (E247Q) cooperates with H80R in protecting the P187S polymorphism towards inactivation through long-range allosteric communication within the structural ensemble of the protein. Our results support that recent divergence of consensus amino acids may have occurred with neutral effects on many functional and regulatory traits of wild-type human proteins. However, divergence at certain sites may have increased the propensity of some human proteins towards inactivation due to disease-associated mutations and polymorphisms. Consensus mutations also emerge as a potential strategy to identify structural hot-spots in proteins as targets for pharmacological rescue in loss-of-function genetic diseases. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Homozygous mutation of VPS16 gene is responsible for an autosomal recessive adolescent-onset primary dystonia.

PubMed

Cai, Xiaodong; Chen, Xin; Wu, Song; Liu, Wenlan; Zhang, Xiejun; Zhang, Doudou; He, Sijie; Wang, Bo; Zhang, Mali; Zhang, Yuan; Li, Zongyang; Luo, Kun; Cai, Zhiming; Li, Weiping

2016-05-12

Dystonia is a neurological movement disorder that is clinically and genetically heterogeneous. Herein, we report the identification a novel homozygous missense mutation, c.156 C > A in VPS16, co-segregating with disease status in a Chinese consanguineous family with adolescent-onset primary dystonia by whole exome sequencing and homozygosity mapping. To assess the biological role of c.156 C > A homozygous mutation of VPS16, we generated mice with targeted mutation site of Vps16 through CRISPR-Cas9 genome-editing approach. Vps16 c.156 C > A homozygous mutant mice exhibited significantly impaired motor function, suggesting that VPS16 is a new causative gene for adolescent-onset primary dystonia.

Genotype imputation in a coalescent model with infinitely-many-sites mutation

PubMed Central

Huang, Lucy; Buzbas, Erkan O.; Rosenberg, Noah A.

2012-01-01

Empirical studies have identified population-genetic factors as important determinants of the properties of genotype-imputation accuracy in imputation-based disease association studies. Here, we develop a simple coalescent model of three sequences that we use to explore the theoretical basis for the influence of these factors on genotype-imputation accuracy, under the assumption of infinitely-many-sites mutation. Employing a demographic model in which two populations diverged at a given time in the past, we derive the approximate expectation and variance of imputation accuracy in a study sequence sampled from one of the two populations, choosing between two reference sequences, one sampled from the same population as the study sequence and the other sampled from the other population. We show that under this model, imputation accuracy—as measured by the proportion of polymorphic sites that are imputed correctly in the study sequence—increases in expectation with the mutation rate, the proportion of the markers in a chromosomal region that are genotyped, and the time to divergence between the study and reference populations. Each of these effects derives largely from an increase in information available for determining the reference sequence that is genetically most similar to the sequence targeted for imputation. We analyze as a function of divergence time the expected gain in imputation accuracy in the target using a reference sequence from the same population as the target rather than from the other population. Together with a growing body of empirical investigations of genotype imputation in diverse human populations, our modeling framework lays a foundation for extending imputation techniques to novel populations that have not yet been extensively examined. PMID:23079542

Engineered Cpf1 variants with altered PAM specificities.

PubMed

Gao, Linyi; Cox, David B T; Yan, Winston X; Manteiga, John C; Schneider, Martin W; Yamano, Takashi; Nishimasu, Hiroshi; Nureki, Osamu; Crosetto, Nicola; Zhang, Feng

2017-08-01

The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells. However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, we performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. We engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity using BLISS indicated that these variants retain high DNA-targeting specificity, which we further improved by introducing an additional non-PAM-interacting mutation. Introducing the identified PAM-interacting mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. Together, these variants increase the targeting range of Cpf1 by approximately threefold in human coding sequences to one cleavage site per ∼11 bp.

Point mutations in acetylcholinesterase 1 associated with chlorpyrifos resistance in the brown planthopper, Nilaparvata lugens Stål.

PubMed

Zhang, Y; Yang, B; Li, J; Liu, M; Liu, Z

2017-08-01

Insecticide resistance frequently results from target-site insensitivity, such as point mutations in acetylcholinesterases (AChEs) for resistance to organophosphates and carbamates. From a field-originated population of Nilaparvata lugens, a major rice pest, a resistant population (R9) was obtained by nine-generation continuous selection with chlorpyrifos. From the same field population, a relatively susceptible population (S9) was also constructed through rearing without any insecticides. Compared to the susceptible strain, Sus [medium lethal dose (LC 50 ) = 0.012 mg/l], R9 had a resistance ratio (RR) of 253.08-fold, whereas the RR of S9 was only 2.25-fold. Piperonyl butoxide and triphenyl phosphate synergized chlorpyrifos in R9 less than three-fold, indicating other important mechanisms for high resistance. The target-site insensitivity was supported by the key property differences of crude AChEs between R9 and S9. Compared to S9, three mutations (G119S, F331C and I332L) were detected in NlAChE1 from individuals of the R9 and field populations, but no mutation was detected in NlAChE2. G119S and F331C could decreased insecticide sensitivities in recombinant NlAChE1, whereas I332L took effect through increasing the influence of F331C on target insensitivity. F331C might be deleterious because of its influence on the catalytic efficiency of NlAChE1, whereas I332L would decrease these adverse effects and maintain the normal functions of AChEs. © 2017 The Royal Entomological Society.

Characterisation of ALS genes in the polyploid species Schoenoplectus mucronatus and implications for resistance management.

PubMed

Scarabel, Laura; Locascio, Antonella; Furini, Antonella; Sattin, Maurizio; Varotto, Serena

2010-03-01

The polyploid weed Schoenoplectus mucronatus (L.) Palla has evolved target-site resistance to ALS-inhibiting herbicides in Italian rice crops. Molecular and genetic characterisation of the resistance mechanism is relevant to the evolution and management of herbicide resistance. The authors aimed (a) to study the organisation of the target-site loci in two field-selected S. mucronatus populations with different cross-resistance patterns, (b) to identify the mutations endowing resistance to ALS inhibitors and determine the role of these mutations by using transgenesis and (c) to analyse the implications for the management of the S. mucronatus populations. Two complete ALS genes (ALS1 and ALS2) having an intron and a third partial intronless ALS gene (ALS3) were identified. The presence of multiple ALS genes was confirmed by Southern blot analyses, and ALS loci were characterised by examining cytosine methylation. In S. mucronatus leaves, the transcripts of ALS1, ALS2 and ALS3 were detected. Two mutations endowing resistance (Pro(197) to His and Trp(574) to Leu) were found in both resistant populations, but at different frequencies. Tobacco plants transformed with the two resistant alleles indicated that the Pro(197)-to-His substitution conferred resistance to SU and TP herbicides, while the allele with the Trp(574)-to-Leu substitution conferred cross-resistance to SU, TP, IMI and PTB herbicides. Schoenoplectus mucronatus has multiple ALS genes characterised by methylated sites that can influence the expression profile. The two mutated alleles proved to be responsible for ALS resistance. At population level, the resistance pattern depends on the frequency of various resistant genotypes, and this influences the efficacy of various ALS-inhibiting herbicides.

Species Identification and Resistance Status of Anopheles gambiae s.l. (Diptera: Culicidae) Mosquitoes in Guinea.

PubMed

Keita, K; Camara, D; Barry, Y; Ossè, R; Wang, L; Sylla, M; Miller, D; Leite, L; Schopp, P; Lawrence, G G; Akogbéto, M; Dotson, E M; Guilavogui, T; Keita, M; Irish, S R

2017-05-01

Insecticide resistance is one of the primary threats to the recent gains in malaria control. This is especially true in Guinea, where long-lasting insecticidal nets are currently the primary vector control intervention. To better inform the national malaria control program on the current status of insecticide resistance in Guinea, resistance bioassays were conducted, using Anopheles gambiae s.l. Giles, in three sites. Molecular analyses were also done on An. gambiae s.l. to determine the species and find whether the target-site mutations kdr and Ace1R were present. Susceptibility tests revealed resistance to DDT and pyrethroids, although mosquitoes were susceptible to deltamethrin in two of the three sites tested. Mosquitoes were susceptible to bendiocarb, except in Kissidougou, Guinea. The kdr-west mutation was widespread and the frequency was 60% or more in all sites. However, the Ace1R mutation was present in low levels. Insecticide susceptibility should continue to be monitored in Guinea to ensure insecticide-based vector control methods remain effective. Published by Oxford University Press on behalf of Entomological Society of America 2017. This work is written by US Government employees and is in the public domain in the US.

Mutagenesis and Functional Studies with Succinate Dehydrogenase Inhibitors in the Wheat Pathogen Mycosphaerella graminicola

PubMed Central

Scalliet, Gabriel; Bowler, Judith; Luksch, Torsten; Kirchhofer-Allan, Lucy; Steinhauer, Diana; Ward, Keith; Niklaus, Michael; Verras, Andreas; Csukai, Michael; Daina, Antoine; Fonné-Pfister, Raymonde

2012-01-01

A range of novel carboxamide fungicides, inhibitors of the succinate dehydrogenase enzyme (SDH, EC 1.3.5.1) is currently being introduced to the crop protection market. The aim of this study was to explore the impact of structurally distinct carboxamides on target site resistance development and to assess possible impact on fitness. We used a UV mutagenesis approach in Mycosphaerella graminicola, a key pathogen of wheat to compare the nature, frequencies and impact of target mutations towards five subclasses of carboxamides. From this screen we identified 27 amino acid substitutions occurring at 18 different positions on the 3 subunits constituting the ubiquinone binding (Qp) site of the enzyme. The nature of substitutions and cross resistance profiles indicated significant differences in the binding interaction to the enzyme across the different inhibitors. Pharmacophore elucidation followed by docking studies in a tridimensional SDH model allowed us to propose rational hypotheses explaining some of the differential behaviors for the first time. Interestingly all the characterized substitutions had a negative impact on enzyme efficiency, however very low levels of enzyme activity appeared to be sufficient for cell survival. In order to explore the impact of mutations on pathogen fitness in vivo and in planta, homologous recombinants were generated for a selection of mutation types. In vivo, in contrast to previous studies performed in yeast and other organisms, SDH mutations did not result in a major increase of reactive oxygen species levels and did not display any significant fitness penalty. However, a number of Qp site mutations affecting enzyme efficiency were shown to have a biological impact in planta. Using the combined approaches described here, we have significantly improved our understanding of possible resistance mechanisms to carboxamides and performed preliminary fitness penalty assessment in an economically important plant pathogen years ahead of possible resistance development in the field. PMID:22536383

Targeting Renal Cell Carcinoma with a HIF-2 antagonist

PubMed Central

Chen, Wenfang; Hill, Haley; Christie, Alana; Kim, Min Soo; Holloman, Eboni; Pavia-Jimenez, Andrea; Homayoun, Farrah; Ma, Yuanqing; Patel, Nirav; Yell, Paul; Hao, Guiyang; Yousuf, Qurratulain; Joyce, Allison; Pedrosa, Ivan; Geiger, Heather; Zhang, He; Chang, Jenny; Gardner, Kevin H.; Bruick, Richard K.; Reeves, Catherine; Hwang, Tae Hyun; Courtney, Kevin; Frenkel, Eugene; Sun, Xiankai; Zojwalla, Naseem; Wong, Tai; Rizzi, James P.; Wallace, Eli M.; Josey, John A.; Xie, Yang; Xie, Xian-Jin; Kapur, Payal; McKay, Renée M.; Brugarolas, James

2016-01-01

Clear cell Renal Cell Carcinoma (ccRCC) is characterized by VHL inactivation1,2. Because no other gene is mutated as frequently, and VHL mutations are truncal3, VHL inactivation is regarded as the governing event4. VHL loss activates HIF-2, and constitutive HIF-2 restores tumorigenesis in VHL-reconstituted ccRCC cells5. HIF-2 is implicated in angiogenesis and multiple other processes6–9, but angiogenesis is the main target of drugs like sunitinib10. HIF-2, a transcription factor, has been regarded as undruggable11. A structure-based design approach identified a selective HIF-2 antagonist (PT2399) that we evaluate using a tumorgraft (TG)/PDX platform12,13. PT2399 dissociated HIF-2 (an obligatory heterodimer [HIF-2α/HIF-1β])14 in human ccRCC suppressing tumorigenesis in 56% (10/18) lines. PT2399 had greater activity than sunitinib, was active in sunitinib-progressing tumors, and was better tolerated. Unexpectedly, some VHL-mutant ccRCCs were resistant. Resistance occurred despite HIF-2 dissociation in tumors and evidence of Hif-2 inhibition in the mouse as determined by suppression of circulating erythropoietin, a HIF-2 target15 and possible pharmacodynamic marker. We identified a HIF-2-dependent gene signature in sensitive tumors. Illustrating drug specificity, gene expression was largely unaffected by PT2399 in resistant tumors. Sensitive tumors exhibited a distinguishing gene expression signature, and generally higher HIF-2α levels. Prolonged PT2399 treatment led to resistance. We identified a binding site and second site suppressor mutation in HIF-2α and HIF-1β respectively. Both mutations preserved HIF-2 dimers despite treatment with PT2399. Finally, an extensively pretreated patient with a sensitive TG had disease control for >11 months with the close analogue PT2385. We validate HIF-2 as a target in ccRCC, show that some ccRCC are, unexpectedly, HIF-2 independent, and set the stage for biomarker-driven clinical trials. PMID:27595394

Molecular characteristics of endometrial cancer coexisting with peritoneal malignant mesothelioma in Li-Fraumeni-like syndrome.

PubMed

Chao, Angel; Lai, Chyong-Huey; Lee, Yun-Shien; Ueng, Shir-Hwa; Lin, Chiao-Yun; Wang, Tzu-Hao

2015-01-15

Endometrial cancer that occurs concurrently with peritoneal malignant mesothelioma (PMM) is difficult to diagnose preoperatively. A postmenopausal woman had endometrial cancer extending to the cervix, vagina and pelvic lymph nodes, and PMM in bilateral ovaries, cul-de-sac, and multiple peritoneal sites. Adjuvant therapies included chemotherapy and radiotherapy. Targeted, massively parallel DNA sequencing and molecular inversion probe microarray analysis revealed a germline TP53 mutation compatible with Li-Fraumeni-like syndrome, somatic mutations of PIK3CA in the endometrial cancer, and a somatic mutation of GNA11 and JAK3 in the PMM. Large-scale genomic amplifications and some deletions were found in the endometrial cancer. The patient has been stable for 24 months after therapy. One of her four children was also found to carry the germline TP53 mutation. Molecular characterization of the coexistent tumors not only helps us make the definite diagnosis, but also provides information to select targeted therapies if needed in the future. Identification of germline TP53 mutation further urged us to monitor future development of malignancies in this patient and encourage cancer screening in her family.

Spectrum of mutations in leiomyosarcomas identified by clinical targeted next-generation sequencing.

PubMed

Lee, Paul J; Yoo, Naomi S; Hagemann, Ian S; Pfeifer, John D; Cottrell, Catherine E; Abel, Haley J; Duncavage, Eric J

2017-02-01

Recurrent genomic mutations in uterine and non-uterine leiomyosarcomas have not been well established. Using a next generation sequencing (NGS) panel of common cancer-associated genes, 25 leiomyosarcomas arising from multiple sites were examined to explore genetic alterations, including single nucleotide variants (SNV), small insertions/deletions (indels), and copy number alterations (CNA). Sequencing showed 86 non-synonymous, coding region somatic variants within 151 gene targets in 21 cases, with a mean of 4.1 variants per case; 4 cases had no putative mutations in the panel of genes assayed. The most frequently altered genes were TP53 (36%), ATM and ATRX (16%), and EGFR and RB1 (12%). CNA were identified in 85% of cases, with the most frequent copy number losses observed in chromosomes 10 and 13 including PTEN and RB1; the most frequent gains were seen in chromosomes 7 and 17. Our data show that deletions in canonical cancer-related genes are common in leiomyosarcomas. Further, the spectrum of gene mutations observed shows that defects in DNA repair and chromosomal maintenance are central to the biology of leiomyosarcomas, and that activating mutations observed in other common cancer types are rare in leiomyosarcomas. Copyright © 2017 Elsevier Inc. All rights reserved.

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

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

Jusakul, Apinya; Cutcutache, Ioana; Yong, Chern Han

Cholangiocarcinoma (CCA) is a hepatobiliary malignancy exhibiting high incidence in countries with endemic liver-fluke infection. We analysed 489 CCAs from 10 countries, combining whole-genome (71 cases), targeted/exome, copy-number, gene expression, and DNA methylation information. Integrative clustering defined four CCA clusters - Fluke- Positive CCAs (Clusters 1/2) are enriched in ERBB2 amplifications and TP53 mutations, conversely Fluke-Negative CCAs (Clusters 3/4) exhibit high copy-number alterations and PD-1/PD-L2 expression, or epigenetic mutations (IDH1/2, BAP1) and FGFR/PRKA-related gene rearrangements. Whole-genome analysis highlighted FGFR2 3’UTR deletion as a mechanism of FGFR2 upregulation. Integration of non-coding promoter mutations with protein-DNA binding profiles demonstrates pervasive modulation ofmore » H3K27me3-associated sites in CCA. Clusters 1 and 4 exhibit distinct DNA hypermethylation patterns targeting either CpG islands or shores - mutation signature and subclonality analysis suggests that these reflect different mutational pathways. Lastly, our results exemplify how genetics, epigenetics and environmental carcinogens can interplay across different geographies to generate distinct molecular subtypes of cancer.« less

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

DOE PAGES

Jusakul, Apinya; Cutcutache, Ioana; Yong, Chern Han; ...

2017-06-30

Cholangiocarcinoma (CCA) is a hepatobiliary malignancy exhibiting high incidence in countries with endemic liver-fluke infection. We analysed 489 CCAs from 10 countries, combining whole-genome (71 cases), targeted/exome, copy-number, gene expression, and DNA methylation information. Integrative clustering defined four CCA clusters - Fluke- Positive CCAs (Clusters 1/2) are enriched in ERBB2 amplifications and TP53 mutations, conversely Fluke-Negative CCAs (Clusters 3/4) exhibit high copy-number alterations and PD-1/PD-L2 expression, or epigenetic mutations (IDH1/2, BAP1) and FGFR/PRKA-related gene rearrangements. Whole-genome analysis highlighted FGFR2 3’UTR deletion as a mechanism of FGFR2 upregulation. Integration of non-coding promoter mutations with protein-DNA binding profiles demonstrates pervasive modulation ofmore » H3K27me3-associated sites in CCA. Clusters 1 and 4 exhibit distinct DNA hypermethylation patterns targeting either CpG islands or shores - mutation signature and subclonality analysis suggests that these reflect different mutational pathways. Lastly, our results exemplify how genetics, epigenetics and environmental carcinogens can interplay across different geographies to generate distinct molecular subtypes of cancer.« less

Compound heterozygous MYO7A mutations segregating Usher syndrome type 2 in a Han family.

PubMed

Zong, Ling; Chen, Kaitian; Wu, Xuan; Liu, Min; Jiang, Hongyan

2016-11-01

Identification of rare deafness genes for inherited congenital sensorineural hearing impairment remains difficult, because a large variety of genes are implicated. In this study we applied targeted capture and next-generation sequencing to uncover the underlying gene in a three-generation Han family segregating recessive inherited hearing loss and retinitis pigmentosa. After excluding mutations in common deafness genes GJB2, SLC26A4 and the mitochondrial gene, genomic DNA of the proband of a Han family was subjected to targeted next-generation sequencing. The candidate mutations were confirmed by Sanger sequencing and subsequently analyzed with in silico tools. An unreported splice site mutation c.3924+1G > C compound with c.6028G > A in the MYO7A gene were detected to cosegregate with the phenotype in this pedigree. Both mutations, located in the evolutionarily conserved FERM domain in myosin VIIA, were predicted to be pathogenic. In this family, profound sensorineural hearing impairment and retinitis pigmentosa without vestibular disorder, constituted the typical Usher syndrome type 2. Identification of novel mutation in compound heterozygosity in MYO7A gene revealed the genetic origin of Usher syndrome type 2 in this Han family. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

High-sensitive electrochemical detection of point mutation based on polymerization-induced enzymatic amplification.

PubMed

Feng, Kejun; Zhao, Jingjin; Wu, Zai-Sheng; Jiang, Jianhui; Shen, Guoli; Yu, Ruqin

2011-03-15

Here a highly sensitive electrochemical method is described for the detection of point mutation in DNA. Polymerization extension reaction is applied to specifically initiate enzymatic electrochemical amplification to improve the sensitivity and enhance the performance of point mutation detection. In this work, 5'-thiolated DNA probe sequences complementary to the wild target DNA are assembled on the gold electrode. In the presence of wild target DNA, the probe is extended by DNA polymerase over the free segment of target as the template. After washing with NaOH solution, the target DNA is removed while the elongated probe sequence remains on the sensing surface. Via hybridizing to the designed biotin-labeled detection probe, the extended sequence is capable of capturing detection probe. After introducing streptavidin-conjugated alkaline phosphatase (SA-ALP), the specific binding between streptavidin and biotin mediates a catalytic reaction of ascorbic acid 2-phosphate (AA-P) substrate to produce a reducing agent ascorbic acid (AA). Then the silver ions in solution are reduced by AA, leading to the deposition of silver metal onto the electrode surface. The amount of deposited silver which is determined by the amount of wild target can be quantified by the linear sweep voltammetry (LSV). The present approach proved to be capable of detecting the wild target DNA down to a detection limit of 1.0×10(-14) M in a wide target concentration range and identifying -28 site (A to G) of the β-thalassemia gene, demonstrating that this scheme offers a highly sensitive and specific approach for point mutation detection. Copyright © 2010 Elsevier B.V. All rights reserved.

Thermus Thermophilus as a Model System for the Study of Ribosomal Antibiotic Resistance

NASA Astrophysics Data System (ADS)

Gregory, Steven T.

2018-03-01

Ribosomes are the intracellular ribonucleoprotein machines responsible for the translation of mRNA sequence into protein sequence. As an essential cell component, the ribosome is the target of numerous antibiotics that bind to critical functional sites to impair protein synthesis. Mutations causing resistance to antibiotics arise in antibiotic binding sites, and an understanding of the basis of resistance will be an essential component of efforts to develop new antibiotics by rational drug design. We have identified a number of antibiotic-resistance mutations in ribosomal genes of the thermophilic bacterium Thermus thermophilus. This species offers two primary advantages for examining the structural basis of antibiotic-resistance, in particular, its potential for genetic manipulation and the suitability of its ribosomes for analysis by X-ray crystallography. Mutations we have identified in this organism are in many instances identical to those found in other bacterial species, including important pathogens, a result of the extreme conservation of ribosome functional sites. Here I summarize the advantages of this organism as a model system to study antibiotic-resistance mechanisms at the molecular level.

Toward a generalized computational workflow for exploiting transient pockets as new targets for small molecule stabilizers: Application to the homogentisate 1,2-dioxygenase mutants at the base of rare disease Alkaptonuria.

PubMed

Bernini, Andrea; Galderisi, Silvia; Spiga, Ottavia; Bernardini, Giulia; Niccolai, Neri; Manetti, Fabrizio; Santucci, Annalisa

2017-10-01

Alkaptonuria (AKU) is an inborn error of metabolism where mutation of homogentisate 1,2-dioxygenase (HGD) gene leads to a deleterious or misfolded product with subsequent loss of enzymatic degradation of homogentisic acid (HGA) whose accumulation in tissues causes ochronosis and degeneration. There is no licensed therapy for AKU. Many missense mutations have been individuated as responsible for quaternary structure disruption of the native hexameric HGD. A new approach to the treatment of AKU is here proposed aiming to totally or partially rescue enzyme activity by targeting of HGD with pharmacological chaperones, i.e. small molecules helping structural stability. Co-factor pockets from oligomeric proteins have already been successfully exploited as targets for such a strategy, but no similar sites are present at HGD surface; hence, transient pockets are here proposed as a target for pharmacological chaperones. Transient pockets are detected along the molecular dynamics trajectory of the protein and filtered down to a set of suitable sites for structural stabilization by mean of biochemical and pharmacological criteria. The result is a computational workflow relevant to other inborn errors of metabolism requiring rescue of oligomeric, misfolded enzymes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Gene replacements and insertions in rice by intron targeting using CRISPR-Cas9.

PubMed

Li, Jun; Meng, Xiangbing; Zong, Yuan; Chen, Kunling; Zhang, Huawei; Liu, Jinxing; Li, Jiayang; Gao, Caixia

2016-09-12

Sequence-specific nucleases have been exploited to create targeted gene knockouts in various plants(1), but replacing a fragment and even obtaining gene insertions at specific loci in plant genomes remain a serious challenge. Here, we report efficient intron-mediated site-specific gene replacement and insertion approaches that generate mutations using the non-homologous end joining (NHEJ) pathway using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system. Using a pair of single guide RNAs (sgRNAs) targeting adjacent introns and a donor DNA template including the same pair of sgRNA sites, we achieved gene replacements in the rice endogenous gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) at a frequency of 2.0%. We also obtained targeted gene insertions at a frequency of 2.2% using a sgRNA targeting one intron and a donor DNA template including the same sgRNA site. Rice plants harbouring the OsEPSPS gene with the intended substitutions were glyphosate-resistant. Furthermore, the site-specific gene replacements and insertions were faithfully transmitted to the next generation. These newly developed approaches can be generally used to replace targeted gene fragments and to insert exogenous DNA sequences into specific genomic sites in rice and other plants.

Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration* ♦

PubMed Central

Koenig, Patrick; Lee, Chingwei V.; Sanowar, Sarah; Wu, Ping; Stinson, Jeremy; Harris, Seth F.; Fuh, Germaine

2015-01-01

The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies. PMID:26088137

Muscle RAS oncogene homolog (MRAS) recurrent mutation in Borrmann type IV gastric cancer.

PubMed

Yasumoto, Makiko; Sakamoto, Etsuko; Ogasawara, Sachiko; Isobe, Taro; Kizaki, Junya; Sumi, Akiko; Kusano, Hironori; Akiba, Jun; Torimura, Takuji; Akagi, Yoshito; Itadani, Hiraku; Kobayashi, Tsutomu; Hasako, Shinichi; Kumazaki, Masafumi; Mizuarai, Shinji; Oie, Shinji; Yano, Hirohisa

2017-01-01

The prognosis of patients with Borrmann type IV gastric cancer (Type IV) is extremely poor. Thus, there is an urgent need to elucidate the molecular mechanisms underlying the oncogenesis of Type IV and to identify new therapeutic targets. Although previous studies using whole-exome and whole-genome sequencing have elucidated genomic alterations in gastric cancer, none has focused on comprehensive genetic analysis of Type IV. To discover cancer-relevant genes in Type IV, we performed whole-exome sequencing and genome-wide copy number analysis on 13 patients with Type IV. Exome sequencing identified 178 somatic mutations in protein-coding sequences or at splice sites. Among the mutations, we found a mutation in muscle RAS oncogene homolog (MRAS), which is predicted to cause molecular dysfunction. MRAS belongs to the Ras subgroup of small G proteins, which includes the prototypic RAS oncogenes. We analyzed an additional 46 Type IV samples to investigate the frequency of MRAS mutation. There were eight nonsynonymous mutations (mutation frequency, 17%), showing that MRAS is recurrently mutated in Type IV. Copy number analysis identified six focal amplifications and one homozygous deletion, including insulin-like growth factor 1 receptor (IGF1R) amplification. The samples with IGF1R amplification had remarkably higher IGF1R mRNA and protein expression levels compared with the other samples. This is the first report of MRAS recurrent mutation in human tumor samples. Our results suggest that MRAS mutation and IGF1R amplification could drive tumorigenesis of Type IV and could be new therapeutic targets. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

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

PubMed

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.

Gene duplication in the major insecticide target site, Rdl, in Drosophila melanogaster

PubMed Central

Remnant, Emily J.; Good, Robert T.; Schmidt, Joshua M.; Lumb, Christopher; Robin, Charles; Daborn, Phillip J.; Batterham, Philip

2013-01-01

The Resistance to Dieldrin gene, Rdl, encodes a GABA-gated chloride channel subunit that is targeted by cyclodiene and phenylpyrazole insecticides. The gene was first characterized in Drosophila melanogaster by genetic mapping of resistance to the cyclodiene dieldrin. The 4,000-fold resistance observed was due to a single amino acid replacement, Ala301 to Ser. The equivalent change was subsequently identified in Rdl orthologs of a large range of resistant insect species. Here, we report identification of a duplication at the Rdl locus in D. melanogaster. The 113-kb duplication contains one WT copy of Rdl and a second copy with two point mutations: an Ala301 to Ser resistance mutation and Met360 to Ile replacement. Individuals with this duplication exhibit intermediate dieldrin resistance compared with single copy Ser301 homozygotes, reduced temperature sensitivity, and altered RNA editing associated with the resistant allele. Ectopic recombination between Roo transposable elements is involved in generating this genomic rearrangement. The duplication phenotypes were confirmed by construction of a transgenic, artificial duplication integrating the 55.7-kb Rdl locus with a Ser301 change into an Ala301 background. Gene duplications can contribute significantly to the evolution of insecticide resistance, most commonly by increasing the amount of gene product produced. Here however, duplication of the Rdl target site creates permanent heterozygosity, providing unique potential for adaptive mutations to accrue in one copy, without abolishing the endogenous role of an essential gene. PMID:23959864

Secondary structure prediction and structure-specific sequence analysis of single-stranded DNA.

PubMed

Dong, F; Allawi, H T; Anderson, T; Neri, B P; Lyamichev, V I

2001-08-01

DNA sequence analysis by oligonucleotide binding is often affected by interference with the secondary structure of the target DNA. Here we describe an approach that improves DNA secondary structure prediction by combining enzymatic probing of DNA by structure-specific 5'-nucleases with an energy minimization algorithm that utilizes the 5'-nuclease cleavage sites as constraints. The method can identify structural differences between two DNA molecules caused by minor sequence variations such as a single nucleotide mutation. It also demonstrates the existence of long-range interactions between DNA regions separated by >300 nt and the formation of multiple alternative structures by a 244 nt DNA molecule. The differences in the secondary structure of DNA molecules revealed by 5'-nuclease probing were used to design structure-specific probes for mutation discrimination that target the regions of structural, rather than sequence, differences. We also demonstrate the performance of structure-specific 'bridge' probes complementary to non-contiguous regions of the target molecule. The structure-specific probes do not require the high stringency binding conditions necessary for methods based on mismatch formation and permit mutation detection at temperatures from 4 to 37 degrees C. Structure-specific sequence analysis is applied for mutation detection in the Mycobacterium tuberculosis katG gene and for genotyping of the hepatitis C virus.

TALEN-mediated targeted mutagenesis of fatty acid desaturase 2 (FAD2) in peanut (Arachis hypogaea L.) promotes the accumulation of oleic acid.

PubMed

Wen, Shijie; Liu, Hao; Li, Xingyu; Chen, Xiaoping; Hong, Yanbin; Li, Haifen; Lu, Qing; Liang, Xuanqiang

2018-05-01

A first creation of high oleic acid peanut varieties by using transcription activator-like effecter nucleases (TALENs) mediated targeted mutagenesis of Fatty Acid Desaturase 2 (FAD2). Transcription activator like effector nucleases (TALENs), which allow the precise editing of DNA, have already been developed and applied for genome engineering in diverse organisms. However, they are scarcely used in higher plant study and crop improvement, especially in allopolyploid plants. In the present study, we aimed to create targeted mutagenesis by TALENs in peanut. Targeted mutations in the conserved coding sequence of Arachis hypogaea fatty acid desaturase 2 (AhFAD2) were created by TALENs. Genetic stability of AhFAD2 mutations was identified by DNA sequencing in up to 9.52 and 4.11% of the regeneration plants at two different targeted sites, respectively. Mutation frequencies among AhFAD2 mutant lines were significantly correlated to oleic acid accumulation. Genetically, stable individuals of positive mutant lines displayed a 0.5-2 fold increase in the oleic acid content compared with non-transgenic controls. This finding suggested that TALEN-mediated targeted mutagenesis could increase the oleic acid content in edible peanut oil. Furthermore, this was the first report on peanut genome editing event, and the obtained high oleic mutants could serve for peanut breeding project.

Hyper-activation of HUSH complex function by Charcot-Marie-Tooth disease mutation in MORC2

PubMed Central

Douse, Christopher H.; Roberts, Rhys C.; Dougan, Gordon; Kingston, Robert E.; Modis, Yorgo; Lehner, Paul J.

2017-01-01

Dominant mutations in the MORC2 gene have recently been shown to cause axonal Charcot-Marie-Tooth (CMT) disease, but the cellular function of MORC2 is poorly understood. Here, through a genome-wide CRISPR/Cas9-mediated forward genetic screen, we identify MORC2 as an essential gene required for epigenetic silencing by the HUSH complex. HUSH recruits MORC2 to target sites in heterochromatin. We exploit a new method – Differential Viral Accessibility (DIVA) – to show that loss of MORC2 results in chromatin decompaction at these target loci, which is concomitant with a loss of H3K9me3 deposition and transcriptional derepression. The ATPase activity of MORC2 is critical for HUSH-mediated silencing, and the most common mutation affecting the ATPase domain found in CMT patients (R252W) hyper-activates HUSH-mediated repression in neuronal cells. These data define a critical role for MORC2 in epigenetic silencing by the HUSH complex and provide a mechanistic basis underpinning the role of MORC2 mutations in CMT disease. PMID:28581500

Targeted mutagenesis of aryl hydrocarbon receptor 2a and 2b genes in Atlantic killifish (Fundulus heteroclitus)

PubMed Central

Aluru, Neelakanteswar; Karchner, Sibel I.; Franks, Diana G.; Nacci, Diane; Champlin, Denise; Hahn, Mark E.

2014-01-01

Understanding molecular mechanisms of toxicity is facilitated by experimental manipulations, such as disruption of function by gene targeting, that are especially challenging in non-standard model species with limited genomic resources. While loss-of-function approaches have included gene knock-down using morpholino-modified oligonucleotides and random mutagenesis using mutagens or retroviruses, more recent approaches include targeted mutagenesis using zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology. These latter methods provide more accessible opportunities to explore gene function in non-traditional model species. To facilitate evaluations of toxic mechanisms for important categories of aryl hydrocarbon pollutants, whose actions are known to be receptor mediated, we used ZFN and CRISPR-Cas9 approaches to generate aryl hydrocarbon receptor 2a (AHR2a) and AHR2b gene mutations in Atlantic killifish (Fundulus heteroclitus) embryos. This killifish is a particularly valuble non-traditional model for this study, with multiple paralogs of AHR whose functions are not well characterized. In addition, some populations of this species have evolved resistance to toxicants such as halogenated aromatic hydrocarbons. AHR-null killifish will be valuable for characterizing the role of the individual AHR paralogs in evolved resistance, as well as in normal development. We first used five-finger ZFNs targeting exons 1 and 3 of AHR2a. Subsequently, CRISPR-Cas9 guide RNAs were designed to target regions in exon 2 and 3 of AHR2a and AHR2b. We successfully induced frameshift mutations in AHR2a exon 3 with ZFN and CRISPR-Cas9 guide RNAs, with mutation frequencies of 10% and 16%, respectively. In AHR2b, mutations were induced using CRISPR-Cas9 guide RNAs targeting sites in both exon 2 (17%) and exon 3 (63%). We screened AHR2b exon 2 CRISPR-Cas9-injected embryos for off-target effects in AHR paralogs. No mutations were observed in closely related AHR genes (AHR1a, AHR1b, AHR2a, AHRR) in the CRISPR-Cas9-injected embryos. Overall, our results demonstrate that targeted genome-editing methods are efficient in inducing mutations at specific loci in embryos of a non-traditional model species, without detectable off-target effects in paralogous genes. PMID:25481785

Site-specific genome editing for correction of induced pluripotent stem cells derived from dominant dystrophic epidermolysis bullosa.

PubMed

Shinkuma, Satoru; Guo, Zongyou; Christiano, Angela M

2016-05-17

Genome editing with engineered site-specific endonucleases involves nonhomologous end-joining, leading to reading frame disruption. The approach is applicable to dominant negative disorders, which can be treated simply by knocking out the mutant allele, while leaving the normal allele intact. We applied this strategy to dominant dystrophic epidermolysis bullosa (DDEB), which is caused by a dominant negative mutation in the COL7A1 gene encoding type VII collagen (COL7). We performed genome editing with TALENs and CRISPR/Cas9 targeting the mutation, c.8068_8084delinsGA. We then cotransfected Cas9 and guide RNA expression vectors expressed with GFP and DsRed, respectively, into induced pluripotent stem cells (iPSCs) generated from DDEB fibroblasts. After sorting, 90% of the iPSCs were edited, and we selected four gene-edited iPSC lines for further study. These iPSCs were differentiated into keratinocytes and fibroblasts secreting COL7. RT-PCR and Western blot analyses revealed gene-edited COL7 with frameshift mutations degraded at the protein level. In addition, we confirmed that the gene-edited truncated COL7 could neither associate with normal COL7 nor undergo triple helix formation. Our data establish the feasibility of mutation site-specific genome editing in dominant negative disorders.

Determinants for DNA target structure selectivity of the human LINE-1 retrotransposon endonuclease.

PubMed

Repanas, Kostas; Zingler, Nora; Layer, Liliana E; Schumann, Gerald G; Perrakis, Anastassis; Weichenrieder, Oliver

2007-01-01

The human LINE-1 endonuclease (L1-EN) is the targeting endonuclease encoded by the human LINE-1 (L1) retrotransposon. L1-EN guides the genomic integration of new L1 and Alu elements that presently account for approximately 28% of the human genome. L1-EN bears considerable technological interest, because its target selectivity may ultimately be engineered to allow the site-specific integration of DNA into defined genomic locations. Based on the crystal structure, we generated L1-EN mutants to analyze and manipulate DNA target site recognition. Crystal structures and their dynamic and functional analysis show entire loop grafts to be feasible, resulting in altered specificity, while individual point mutations do not change the nicking pattern of L1-EN. Structural parameters of the DNA target seem more important for recognition than the nucleotide sequence, and nicking profiles on DNA oligonucleotides in vitro are less well defined than the respective integration site consensus in vivo. This suggests that additional factors other than the DNA nicking specificity of L1-EN contribute to the targeted integration of non-LTR retrotransposons.

MODEST: a web-based design tool for oligonucleotide-mediated genome engineering and recombineering

PubMed Central

Bonde, Mads T.; Klausen, Michael S.; Anderson, Mads V.; Wallin, Annika I.N.; Wang, Harris H.; Sommer, Morten O.A.

2014-01-01

Recombineering and multiplex automated genome engineering (MAGE) offer the possibility to rapidly modify multiple genomic or plasmid sites at high efficiencies. This enables efficient creation of genetic variants including both single mutants with specifically targeted modifications as well as combinatorial cell libraries. Manual design of oligonucleotides for these approaches can be tedious, time-consuming, and may not be practical for larger projects targeting many genomic sites. At present, the change from a desired phenotype (e.g. altered expression of a specific protein) to a designed MAGE oligo, which confers the corresponding genetic change, is performed manually. To address these challenges, we have developed the MAGE Oligo Design Tool (MODEST). This web-based tool allows designing of MAGE oligos for (i) tuning translation rates by modifying the ribosomal binding site, (ii) generating translational gene knockouts and (iii) introducing other coding or non-coding mutations, including amino acid substitutions, insertions, deletions and point mutations. The tool automatically designs oligos based on desired genotypic or phenotypic changes defined by the user, which can be used for high efficiency recombineering and MAGE. MODEST is available for free and is open to all users at http://modest.biosustain.dtu.dk. PMID:24838561

The frequency and accuracy of replication past a thymine-thymine cyclobutane dimer are very different in Saccharomyces cerevisiae and Escherichia coli.

PubMed

Gibbs, P E; Kilbey, B J; Banerjee, S K; Lawrence, C W

1993-05-01

We have compared the mutagenic properties of a T-T cyclobutane dimer in baker's yeast, Saccharomyces cerevisiae, with those in Escherichia coli by transforming each of these species with the same single-stranded shuttle vector carrying either the cis-syn or the trans-syn isomer of this UV photoproduct at a unique site. The mutagenic properties investigated were the frequency of replicational bypass of the photoproduct, the error rate of bypass, and the mutation spectrum. In SOS-induced E. coli, the cis-syn dimer was bypassed in approximately 16% of the vector molecules, and 7.6% of the bypass products had targeted mutations. In S. cerevisiae, however, bypass occurred in about 80% of these molecules, and the bypass was at least 19-fold more accurate (approximately 0.4% targeted mutations). Each of these yeast mutations was a single unique event, and none were like those in E. coli, suggesting that in fact the difference in error rate is much greater. Bypass of the trans-syn dimer occurred in about 17% of the vector molecules in both species, but with this isomer the error rate was higher in S. cerevisiae (21 to 36% targeted mutations) than in E. coli (13%). However, the spectra of mutations induced by the latter photoproduct were virtually identical in the two organisms. We conclude that bypass and error frequencies are determined both by the structure of the photoproduct-containing template and by the particular replication proteins concerned but that the types of mutations induced depend predominantly on the structure of the template. Unlike E. coli, bypass in S. cerevisiae did not require UV-induced functions.

Structural Basis of Glyphosate Resistance Resulting from the Double Mutation Thr97 → Ile and Pro101 → Ser in 5-Enolpyruvylshikimate-3-phosphate Synthase from Escherichia coli*S⃞

PubMed Central

Funke, Todd; Yang, Yan; Han, Huijong; Healy-Fried, Martha; Olesen, Sanne; Becker, Andreas; Schönbrunn, Ernst

2009-01-01

The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the target of the broad spectrum herbicide glyphosate. The genetic engineering of EPSPS led to the introduction of glyphosate-resistant crops worldwide. The genetically engineered corn lines NK603 and GA21 carry distinct EPSPS enzymes. CP4 EPSPS, expressed in NK603 corn and transgenic soybean, cotton, and canola, belongs to class II EPSPS, glyphosate-insensitive variants of this enzyme isolated from certain Gram-positive bacteria. GA21 corn, on the other hand, was created by point mutations of class I EPSPS, such as the enzymes from Zea mays or Escherichia coli, which are sensitive to low glyphosate concentrations. The structural basis of the glyphosate resistance resulting from these point mutations has remained obscure. We studied the kinetic and structural effects of the T97I/P101S double mutation, the molecular basis for GA21 corn, using EPSPS from E. coli. The T97I/P101S enzyme is essentially insensitive to glyphosate (Ki = 2.4 mm) but maintains high affinity for the substrate phosphoenolpyruvate (PEP) (Km = 0.1 mm). The crystal structure at 1.7-Å resolution revealed that the dual mutation causes a shift of residue Gly96 toward the glyphosate binding site, impairing efficient binding of glyphosate, while the side chain of Ile97 points away from the substrate binding site, facilitating PEP utilization. The single site T97I mutation renders the enzyme sensitive to glyphosate and causes a substantial decrease in the affinity for PEP. Thus, only the concomitant mutations of Thr97 and Pro101 induce the conformational changes necessary to produce catalytically efficient, glyphosate-resistant class I EPSPS. PMID:19211556

Structural basis of glyphosate resistance resulting from the double mutation Thr97 -> Ile and Pro101 -> Ser in 5-enolpyruvylshikimate-3-phosphate synthase from Escherichia coli.

PubMed

Funke, Todd; Yang, Yan; Han, Huijong; Healy-Fried, Martha; Olesen, Sanne; Becker, Andreas; Schönbrunn, Ernst

2009-04-10

The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the target of the broad spectrum herbicide glyphosate. The genetic engineering of EPSPS led to the introduction of glyphosate-resistant crops worldwide. The genetically engineered corn lines NK603 and GA21 carry distinct EPSPS enzymes. CP4 EPSPS, expressed in NK603 corn and transgenic soybean, cotton, and canola, belongs to class II EPSPS, glyphosate-insensitive variants of this enzyme isolated from certain Gram-positive bacteria. GA21 corn, on the other hand, was created by point mutations of class I EPSPS, such as the enzymes from Zea mays or Escherichia coli, which are sensitive to low glyphosate concentrations. The structural basis of the glyphosate resistance resulting from these point mutations has remained obscure. We studied the kinetic and structural effects of the T97I/P101S double mutation, the molecular basis for GA21 corn, using EPSPS from E. coli. The T97I/P101S enzyme is essentially insensitive to glyphosate (K(i) = 2.4 mm) but maintains high affinity for the substrate phosphoenolpyruvate (PEP) (K(m) = 0.1 mm). The crystal structure at 1.7-A resolution revealed that the dual mutation causes a shift of residue Gly(96) toward the glyphosate binding site, impairing efficient binding of glyphosate, while the side chain of Ile(97) points away from the substrate binding site, facilitating PEP utilization. The single site T97I mutation renders the enzyme sensitive to glyphosate and causes a substantial decrease in the affinity for PEP. Thus, only the concomitant mutations of Thr(97) and Pro(101) induce the conformational changes necessary to produce catalytically efficient, glyphosate-resistant class I EPSPS.

Dissecting the mechanisms responsible for the multiple insecticide resistance phenotype in Anopheles gambiae s.s., M form, from Vallée du Kou, Burkina Faso

PubMed Central

Kwiatkowska, Rachel M.; Platt, Naomi; Poupardin, Rodolphe; Irving, Helen; Dabire, Roch K.; Mitchell, Sara; Jones, Christopher M.; Diabaté, Abdoulaye; Ranson, Hilary; Wondji, Charles S.

2013-01-01

With the exception of target site mutations, insecticide resistance mechanisms in the principle malaria vector Anopheles gambiae, remains largely uncharacterized in Burkina Faso. Here we detected high prevalence of resistance in Vallée du Kou (VK) to pyrethroids, DDT and dieldrin, moderate level for carbamates and full susceptibility to organophosphates. High frequencies of L1014F kdr (75%) and Rdl (87%) mutations were observed showing strong correlation with pyrethroids/DDT and dieldrin resistance. The frequency of ace1R mutation was low even in carbamate resistant mosquitoes. Microarray analysis identified genes significantly over-transcribed in VK. These include the cytochrome P450 genes, CYP6P3 and CYP6Z2, previously associated with pyrethroid resistance. Gene Ontology (GO) enrichment analysis suggested that elevated neurotransmitter activity is associated with resistance, with the over-transcription of target site resistance genes such as acetylcholinesterase and the GABA receptor. A rhodopsin receptor gene previously associated with pyrethroid resistance in Culex pipiens pallens was also over-transcribed in VK. This study highlights the complex network of mechanisms conferring multiple resistance in malaria vectors and such information should be taken into account when designing and implementing resistance control strategies. PMID:23380570

"Reverse fleximers": introduction of a series of 5-substituted carbocyclic uridine analogues.

PubMed

Sadler, Joshua M; Ojewoye, Olubukola; Seley-Radtke, Katherine L

2008-01-01

Nucleosides are ubiquitous in biological systems and as such, have been a focus of medicinal chemistry research in the search for new and potent therapeutic compounds. There are a number of modified nucleosides on the market, however increasing reports of resistance by mutation of either the enzyme binding site or the pathway that they are designed to interrupt are surfacing. As shown in recent reports, a candidate that can change conformation and still maintain recognition by the target enzyme would be highly desirable, and it is for this reason that flexible substrates have recently been sought as potential therapeutics. With this goal in mind, we have begun investigation into novel flexible scaffolds capable of overcoming viral resistance mechanisms resulting from binding site mutations.

Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

PubMed Central

Choi, Mal-Gi; Kurnov, Vladlen; Kersting, Michael C.; Sreenivas, Avula; Carman, George M.

2005-01-01

The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work, we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent, and dependent on the concentrations of choline kinase (Km = 27 μg/ml) and ATP (Km = 15 μM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSS25QRRHS (Vmax/Km = 17.5 mM-1 μmol min-1 mg-1) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo, the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Whereas the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHS30LTRQ) containing Ser30 was a substrate (Vmax/Km = 3.0 mM−1 μmol min−1 mg−1) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C. PMID:15919656

Recurrent mutations within the amino-terminal region of β-catenin are probable key molecular driver events in sinonasal hemangiopericytoma.

PubMed

Haller, Florian; Bieg, Matthias; Moskalev, Evgeny A; Barthelmeß, Sarah; Geddert, Helene; Boltze, Carsten; Diessl, Nicolle; Braumandl, Karin; Brors, Benedikt; Iro, Heinrich; Hartmann, Arndt; Wiemann, Stefan; Agaimy, Abbas

2015-02-01

Sinonasal hemangiopericytoma (SN-HPC) is an uncommon, site-specific, low-grade mesenchymal neoplasm of probable perivascular myoid cell origin. In contrast to solitary fibrous tumors of soft tissue and sinonasal tract origin, SN-HPCs were recently shown to lack recurrent NAB2-STAT6 fusion variants. Other molecular alterations known to occur in some of soft tissue perivascular myoid cell neoplasms were also absent in SN-HPC; thus, the molecular pathogenesis of SN-HPCs remained unknown. Guided by whole-genome sequencing combined with RNA sequencing of an index case, we analyzed a total of six SN-HPCs for mutations within the amino-terminal region of the gene CTNNB1 (cadherin-associated protein), β 1, 88 kDa, encoding β-catenin. All six cases showed missense mutations, with amino acid substitutions clustering at positions 33 to 45, corresponding to the recognition site of the β-catenin destruction complex. Similar CTNNB1 mutations have been described in a variety of epithelial and mesenchymal neoplasms. These mutations prevent β-catenin phosphorylation and proteasomal degradation but promote its nuclear accumulation and subsequent increased transcription of Wingless-related integration site target genes. Consistent with these molecular findings, β-catenin IHC showed consistent diffuse and strong nuclear staining of the tumor cells in all six SN-HPCs. Our results highlight, for the first time, CTNNB1 mutations as the likely initiating molecular events driving SN-HPC tumorigenesis, which places SN-HPC among the growing family of β-catenin-driven mesenchymal neoplasms. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

[Genetic analysis of two children patients affected with CHARGE syndrome].

PubMed

Li, Guoqiang; Li, Niu; Xu, Yufei; Li, Juan; Ding, Yu; Shen, Yiping; Wang, Xiumin; Wang, Jian

2018-04-10

To analyze two Chinese pediatric patients with multiple malformations and growth and development delay. Both patients were subjected to targeted gene sequencing, and the results were analyzed with Ingenuity Variant Analysis software. Suspected pathogenic variations were verified by Sanger sequencing. High-throughput sequencing showed that both patients have carried heterozygous variants of the CHD7 gene. Patient 1 carried a nonsense mutation in exon 36 (c.7957C>T, p.Arg2653*), while patient 2 carried a nonsense mutation of exon 2 (c.718C>T, p.Gln240*). Sanger sequencing confirmed the above mutations in both patients, while their parents were of wild-type for the corresponding sites, indicating that the two mutations have happened de novo. Two patients were diagnosed with CHARGE syndrome by high-throughput sequencing.

Specific UV-induced mutation spectrum in the p53 gene of skin tumors from DNA-repair-deficient xeroderma pigmentosum patients

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

Dumaz, N.; Drougard, C.; Sarasin, A.

1993-11-15

The UV component of sunlight is the major carcinogen involved in the etiology of skin cancers. The authors have studied the rare, hereditary syndrome xeroderma pigmentosum (XP), which is characterized by a very high incidence of cutaneous tumors on exposed skin at an early age, probably due to a deficiency in excision repair of UV-induced lesions. It is interesting to determine the UV mutation spectrum in XP skin tumors in order to correlate the absence of repair of specific DNA lesions and the initiation of skin tumors. The p53 gene is frequently mutated in human cancers and represents a goodmore » target for studying mutation spectra since there are >100 potential sites for phenotypic mutations. Using reverse transcription-PCR and single-strand conformation polymorphism to analyze >40 XP skin tumors (mainly basal and squamous cell carcinomas), the authors have found that 40% (17 out of 43) contained at least one point mutation on the p53 gene. All the mutations were located at dipyrimidine sites, essentially at CC sequences, which are hot spots for UV-induced DNA lesions. Sixty-one percent of these mutations were tandem CC [yields] TT mutations considered to be unique to UV-induced lesions; these mutations are not observed in internal human tumors. All the mutations, except two, must be due to translesion synthesis of unrepaired dipyrimidine lesions left on the nontranscribed strand. These results show the existence of preferential repair of UV lesions [either pyrimidine dimers or pyrimidine-pyrimidone (6-4) photoproducts] on the transcribed strand in human tissues.« less

Genome Engineering in Bacillus anthracis Using Cre Recombinase

PubMed Central

Pomerantsev, Andrei P.; Sitaraman, Ramakrishnan; Galloway, Craig R.; Kivovich, Violetta; Leppla, Stephen H.

2006-01-01

Genome engineering is a powerful method for the study of bacterial virulence. With the availability of the complete genomic sequence of Bacillus anthracis, it is now possible to inactivate or delete selected genes of interest. However, many current methods for disrupting or deleting more than one gene require use of multiple antibiotic resistance determinants. In this report we used an approach that temporarily inserts an antibiotic resistance marker into a selected region of the genome and subsequently removes it, leaving the target region (a single gene or a larger genomic segment) permanently mutated. For this purpose, a spectinomycin resistance cassette flanked by bacteriophage P1 loxP sites oriented as direct repeats was inserted within a selected gene. After identification of strains having the spectinomycin cassette inserted by a double-crossover event, a thermo-sensitive plasmid expressing Cre recombinase was introduced at the permissive temperature. Cre recombinase action at the loxP sites excised the spectinomycin marker, leaving a single loxP site within the targeted gene or genomic segment. The Cre-expressing plasmid was then removed by growth at the restrictive temperature. The procedure could then be repeated to mutate additional genes. In this way, we sequentially mutated two pairs of genes: pepM and spo0A, and mcrB and mrr. Furthermore, loxP sites introduced at distant genes could be recombined by Cre recombinase to cause deletion of large intervening regions. In this way, we deleted the capBCAD region of the pXO2 plasmid and the entire 30 kb of chromosomal DNA between the mcrB and mrr genes, and in the latter case we found that the 32 intervening open reading frames were not essential to growth. PMID:16369025

OSPREY Predicts Resistance Mutations Using Positive and Negative Computational Protein Design.

PubMed

Ojewole, Adegoke; Lowegard, Anna; Gainza, Pablo; Reeve, Stephanie M; Georgiev, Ivelin; Anderson, Amy C; Donald, Bruce R

2017-01-01

Drug resistance in protein targets is an increasingly common phenomenon that reduces the efficacy of both existing and new antibiotics. However, knowledge of future resistance mutations during pre-clinical phases of drug development would enable the design of novel antibiotics that are robust against not only known resistant mutants, but also against those that have not yet been clinically observed. Computational structure-based protein design (CSPD) is a transformative field that enables the prediction of protein sequences with desired biochemical properties such as binding affinity and specificity to a target. The use of CSPD to predict previously unseen resistance mutations represents one of the frontiers of computational protein design. In a recent study (Reeve et al. Proc Natl Acad Sci U S A 112(3):749-754, 2015), we used our OSPREY (Open Source Protein REdesign for You) suite of CSPD algorithms to prospectively predict resistance mutations that arise in the active site of the dihydrofolate reductase enzyme from methicillin-resistant Staphylococcus aureus (SaDHFR) in response to selective pressure from an experimental competitive inhibitor. We demonstrated that our top predicted candidates are indeed viable resistant mutants. Since that study, we have significantly enhanced the capabilities of OSPREY with not only improved modeling of backbone flexibility, but also efficient multi-state design, fast sparse approximations, partitioned continuous rotamers for more accurate energy bounds, and a computationally efficient representation of molecular-mechanics and quantum-mechanical energy functions. Here, using SaDHFR as an example, we present a protocol for resistance prediction using the latest version of OSPREY. Specifically, we show how to use a combination of positive and negative design to predict active site escape mutations that maintain the enzyme's catalytic function but selectively ablate binding of an inhibitor.

Preparation of sumoylated substrates for biochemical analysis.

PubMed

Knipscheer, Puck; Klug, Helene; Sixma, Titia K; Pichler, Andrea

2009-01-01

Covalent modification of proteins with SUMO (small ubiquitin related modifier) affects many cellular processes like transcription, nuclear transport, DNA repair and cell cycle progression. Although hundreds of SUMO targets have been identified, for several of them the function remains obscure. In the majority of cases sumoylation is investigated via "loss of modification" analysis by mutating the relevant target lysine. However, in other cases this approach is not successful since mapping of the modification site is problematic or mutation does not cause an obvious phenotype. These latter cases ask for different approaches to investigate the target modification. One possibility is to choose the opposite approach, a "gain in modification" analysis by producing both SUMO modified and unmodified protein in vitro and comparing them in functional assays. Here, we describe the purification of the ubiquitin conjugating enzyme E2-25K, its in vitro sumoylation with recombinant enzymes and the subsequent separation and purification of the modified and the unmodified forms.

Bactobolin resistance is conferred by mutations in the L2 ribosomal protein.

PubMed

Chandler, Josephine R; Truong, Thao T; Silva, Patricia M; Seyedsayamdost, Mohammad R; Carr, Gavin; Radey, Matthew; Jacobs, Michael A; Sims, Elizabeth H; Clardy, Jon; Greenberg, E Peter

2012-12-18

Burkholderia thailandensis produces a family of polyketide-peptide molecules called bactobolins, some of which are potent antibiotics. We found that growth of B. thailandensis at 30°C versus that at 37°C resulted in increased production of bactobolins. We purified the three most abundant bactobolins and determined their activities against a battery of bacteria and mouse fibroblasts. Two of the three compounds showed strong activities against both bacteria and fibroblasts. The third analog was much less potent in both assays. These results suggested that the target of bactobolins might be conserved across bacteria and mammalian cells. To learn about the mechanism of bactobolin activity, we isolated four spontaneous bactobolin-resistant Bacillus subtilis mutants. We used genomic sequencing technology to show that each of the four resistant variants had mutations in rplB, which codes for the 50S ribosome-associated L2 protein. Ectopic expression of a mutant rplB gene in wild-type B. subtilis conferred bactobolin resistance. Finally, the L2 mutations did not confer resistance to other antibiotics known to interfere with ribosome function. Our data indicate that bactobolins target the L2 protein or a nearby site and that this is not the target of other antibiotics. We presume that the mammalian target of bactobolins involves the eukaryotic homolog of L2 (L8e). Currently available antibiotics target surprisingly few cellular functions, and there is a need to identify novel antibiotic targets. We have been interested in the Burkholderia thailandensis bactobolins, and we sought to learn about the target of bactobolin activity by mapping spontaneous resistance mutations in the bactobolin-sensitive Bacillus subtilis. Our results indicate that the bactobolin target is the 50S ribosome-associated L2 protein or a region of the ribosome affected by L2. Bactobolin-resistant mutants are not resistant to other known ribosome inhibitors. Our evidence indicates that bactobolins interact with a novel antibiotic target.

Target and Non-target Site Mechanisms Developed by Glyphosate-Resistant Hairy beggarticks (Bidens pilosa L.) Populations from Mexico

PubMed Central

Alcántara-de la Cruz, Ricardo; Fernández-Moreno, Pablo T.; Ozuna, Carmen V.; Rojano-Delgado, Antonia M.; Cruz-Hipolito, Hugo E.; Domínguez-Valenzuela, José A.; Barro, Francisco; De Prado, Rafael

2016-01-01

In 2014 hairy beggarticks (Bidens pilosa L.) has been identified as being glyphosate-resistant in citrus orchards from Mexico. The target and non-target site mechanisms involved in the response to glyphosate of two resistant populations (R1 and R2) and one susceptible (S) were studied. Experiments of dose-response, shikimic acid accumulation, uptake-translocation, enzyme activity and 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) gene sequencing were carried out in each population. The R1 and R2 populations were 20.4 and 2.8-fold less glyphosate sensitive, respectively, than the S population. The resistant populations showed a lesser shikimic acid accumulation than the S population. In the latter one, 24.9% of 14C-glyphosate was translocated to the roots at 96 h after treatment; in the R1 and R2 populations only 12.9 and 15.5%, respectively, was translocated. Qualitative results confirmed the reduced 14C-glyphosate translocation in the resistant populations. The EPSPS enzyme activity of the S population was 128.4 and 8.5-fold higher than the R1 and R2 populations of glyphosate-treated plants, respectively. A single (Pro-106-Ser), and a double (Thr-102-Ile followed by Pro-106-Ser) mutations were identified in the EPSPS2 gene conferred high resistance in R1 population. Target-site mutations associated with a reduced translocation were responsible for the higher glyphosate resistance in the R1 population. The low-intermediate resistance of the R2 population was mediated by reduced translocation. This is the first glyphosate resistance case confirmed in hairy beggarticks in the world. PMID:27752259

CRISPR/Cas9-mediated targeted mutagenesis of GmFT2a delays flowering time in soya bean.

PubMed

Cai, Yupeng; Chen, Li; Liu, Xiujie; Guo, Chen; Sun, Shi; Wu, Cunxiang; Jiang, Bingjun; Han, Tianfu; Hou, Wensheng

2018-01-01

Flowering is an indication of the transition from vegetative growth to reproductive growth and has considerable effects on the life cycle of soya bean (Glycine max). In this study, we employed the CRISPR/Cas9 system to specifically induce targeted mutagenesis of GmFT2a, an integrator in the photoperiod flowering pathway in soya bean. The soya bean cultivar Jack was transformed with three sgRNA/Cas9 vectors targeting different sites of endogenous GmFT2a via Agrobacterium tumefaciens-mediated transformation. Site-directed mutations were observed at all targeted sites by DNA sequencing analysis. T1-generation soya bean plants homozygous for null alleles of GmFT2a frameshift mutated by a 1-bp insertion or short deletion exhibited late flowering under natural conditions (summer) in Beijing, China (N39°58', E116°20'). We also found that the targeted mutagenesis was stably heritable in the following T2 generation, and the homozygous GmFT2a mutants exhibited late flowering under both long-day and short-day conditions. We identified some 'transgene-clean' soya bean plants that were homozygous for null alleles of endogenous GmFT2a and without any transgenic element from the T1 and T2 generations. These 'transgene-clean' mutants of GmFT2a may provide materials for more in-depth research of GmFT2a functions and the molecular mechanism of photoperiod responses in soya bean. They will also contribute to soya bean breeding and regional introduction. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Application of Mutated miR-206 Target Sites Enables Skeletal Muscle-specific Silencing of Transgene Expression of Cardiotropic AAV9 Vectors

PubMed Central

Geisler, Anja; Schön, Christian; Größl, Tobias; Pinkert, Sandra; Stein, Elisabeth A; Kurreck, Jens; Vetter, Roland; Fechner, Henry

2013-01-01

Insertion of completely complementary microRNA (miR) target sites (miRTS) into a transgene has been shown to be a valuable approach to specifically repress transgene expression in non-targeted tissues. miR-122TS have been successfully used to silence transgene expression in the liver following systemic application of cardiotropic adeno-associated virus (AAV) 9 vectors. For miR-206–mediated skeletal muscle-specific silencing of miR-206TS–bearing AAV9 vectors, however, we found this approach failed due to the expression of another member (miR-1) of the same miR family in heart tissue, the intended target. We introduced single-nucleotide substitutions into the miR-206TS and searched for those which prevented miR-1–mediated cardiac repression. Several mutated miR-206TS (m206TS), in particular m206TS-3G, were resistant to miR-1, but remained fully sensitive to miR-206. All these variants had mismatches in the seed region of the miR/m206TS duplex in common. Furthermore, we found that some m206TS, containing mismatches within the seed region or within the 3′ portion of the miR-206, even enhanced the miR-206– mediated transgene repression. In vivo expression of m206TS-3G– and miR-122TS–containing transgene of systemically applied AAV9 vectors was strongly repressed in both skeletal muscle and the liver but remained high in the heart. Thus, site-directed mutagenesis of miRTS provides a new strategy to differentiate transgene de-targeting of related miRs. PMID:23439498

Calcium binding to calmodulin mutants monitored by domain-specific intrinsic phenylalanine and tyrosine fluorescence.

PubMed Central

VanScyoc, Wendy S; Sorensen, Brenda R; Rusinova, Elena; Laws, William R; Ross, J B Alexander; Shea, Madeline A

2002-01-01

Cooperative calcium binding to the two homologous domains of calmodulin (CaM) induces conformational changes that regulate its association with and activation of numerous cellular target proteins. Calcium binding to the pair of high-affinity sites (III and IV in the C-domain) can be monitored by observing calcium-dependent changes in intrinsic tyrosine fluorescence intensity (lambda(ex)/lambda(em) of 277/320 nm). However, calcium binding to the low-affinity sites (I and II in the N-domain) is more difficult to measure with optical spectroscopy because that domain of CaM does not contain tryptophan or tyrosine. We recently demonstrated that calcium-dependent changes in intrinsic phenylalanine fluorescence (lambda(ex)/lambda(em) of 250/280 nm) of an N-domain fragment of CaM reflect occupancy of sites I and II (VanScyoc, W. S., and M. A. Shea, 2001, Protein Sci. 10:1758-1768). Using steady-state and time-resolved fluorescence methods, we now show that these excitation and emission wavelength pairs for phenylalanine and tyrosine fluorescence can be used to monitor equilibrium calcium titrations of the individual domains in full-length CaM. Calcium-dependent changes in phenylalanine fluorescence specifically indicate ion occupancy of sites I and II in the N-domain because phenylalanine residues in the C-domain are nonemissive. Tyrosine emission from the C-domain does not interfere with phenylalanine fluorescence signals from the N-domain. This is the first demonstration that intrinsic fluorescence may be used to monitor calcium binding to each domain of CaM. In this way, we also evaluated how mutations of two residues (Arg74 and Arg90) located between sites II and III can alter the calcium-binding properties of each of the domains. The mutation R74A caused an increase in the calcium affinity of sites I and II in the N-domain. The mutation R90A caused an increase in calcium affinity of sites III and IV in the C-domain whereas R90G caused an increase in calcium affinity of sites in both domains. This approach holds promise for exploring the linked energetics of calcium binding and target recognition. PMID:12414709

Structural insights into substrate and inhibitor binding sites in human indoleamine 2,3-dioxygenase 1

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

Lewis-Ballester, Ariel; Pham, Khoa N.; Batabyal, Dipanwita

Human indoleamine 2,3-dioxygenase 1 (hIDO1) is an attractive cancer immunotherapeutic target owing to its role in promoting tumoral immune escape. However, drug development has been hindered by limited structural information. Here, we report the crystal structures of hIDO1 in complex with its substrate, Trp, an inhibitor, epacadostat, and/or an effector, indole ethanol (IDE). The data reveal structural features of the active site (Sa) critical for substrate activation; in addition, they disclose a new inhibitor-binding mode and a distinct small molecule binding site (Si). Structure-guided mutation of a critical residue, F270, to glycine perturbs the Si site, allowing structural determination ofmore » an inhibitory complex, where both the Sa and Si sites are occupied by Trp. The Si site offers a novel target site for allosteric inhibitors and a molecular explanation for the previously baffling substrate-inhibition behavior of the enzyme. Taken together, the data open exciting new avenues for structure-based drug design.« less

Characterization of sulfonylurea-resistant Schoenoplectus juncoides having a target-site Asp(376)Glu mutation in the acetolactate synthase.

PubMed

Sada, Yoshinao; Ikeda, Hajime; Yamato, Seiji; Kizawa, Satoru

2013-09-01

Schoenoplectus juncoides, a noxious weed for paddy rice, is known to become resistant to sulfonylurea (SU) herbicides by a target-site mutation in either of the two acetolactate synthase (ALS) genes (ALS1 and ALS2). SU-resistant S. juncoides plants having an Asp376Glu mutation in ALS2 were found from a paddy rice field in Japan, but their resistance profile has not been quantitatively investigated. In this study, dose-response of the SU-resistant accession was compared with that of a SU-susceptible accession at in vivo whole-plant level as well as at in vitro enzymatic level. In whole-plant tests, resistance factors (RFs) based on 50% growth reduction (GR50) for imazosulfuron (ISF), bensulfuron-methyl (BSM), metsulfuron-methyl (MSM), bispyribac-sodium (BPS), and imazaquin (IMQ) were 176, 40, 14, 5.2 and 1.5, respectively. Thus, the accession having an Asp376Glu mutation in ALS2 was highly resistant to the three SU herbicides and moderately resistant to BPS, but was not substantially resistant to IMQ. This is slightly different from the earlier results reported from other weeds with an Asp376Glu mutation, in which the mutation confers resistance to broadly all the chemical classes of ALS-inhibiting herbicides. In enzymatic tests, ALS2 of S. juncoides was expressed in E. coli; the resultant ALS2 was subjected to an in vitro assay. RFs of the mutated ALS2 based on 50% enzymatic inhibition (I50) for ISF, BSM, MSM, BPS, and IMQ were 3699, 2438, 322, 80, and 4.8, respectively. The RFs of ALS2 were highly correlated with those of the whole-plant; this suggests that the Asp376Glu mutation in ALS2 is a molecular basis for the whole-plant resistance. The presence of two ALS genes in S. juncoides can at least partially explain why the whole-plant RFs were less than those of the expressed ALS2 enzymes. Copyright © 2013 Elsevier Inc. All rights reserved.

Genomic profiles of lung cancer associated with idiopathic pulmonary fibrosis.

PubMed

Hwang, Ji An; Kim, Deokhoon; Chun, Sung-Min; Bae, SooHyun; Song, Joon Seon; Kim, Mi Young; Koo, Hyun Jung; Song, Jin Woo; Kim, Woo Sung; Lee, Jae Cheol; Kim, Hyeong Ryul; Choi, Chang-Min; Jang, Se Jin

2018-01-01

Little is known about the pathogenesis or molecular profiles of idiopathic pulmonary fibrosis-associated lung cancer (IPF-LC). This study was performed to investigate the genomic profiles of IPF-LC and to explore the possibility of defining potential therapeutic targets in IPF-LC. We assessed genomic profiles of IPF-LC by using targeted exome sequencing (OncoPanel version 2) in 35 matched tumour/normal pairs surgically resected between 2004 and 2014. Germline and somatic variant calling was performed with GATK HaplotypeCaller and MuTect with GATK SomaticIndelocator, respectively. Copy number analysis was conducted with CNVkit, with focal events determined by Genomic Identification of Significant Targets in Cancer 2.0, and pathway analysis (KEGG) with DAVID. Germline mutations in TERT (rs2736100, n = 33) and CDKN1A (rs2395655, n = 27) associated with idiopathic pulmonary fibrosis risk were detected in most samples. A total of 410 somatic mutations were identified, with an average of 11.7 per tumour, including 69 synonymous, 177 missense, 17 nonsense, 1 nonstop and 11 splice-site mutations, and 135 small coding indels. Spectra of the somatic mutations revealed predominant C > T transitions despite an extensive smoking history in most patients, suggesting a potential association between APOBEC-related mutagenesis and the development of IPF-LC. TP53 (22/35, 62.9%) and BRAF (6/35, 17.1%) were found to be significantly mutated in IPF-LC. Recurrent focal amplifications in three chromosomal loci (3q26.33, 7q31.2, and 12q14.3) and 9p21.3 deletion were identified, and genes associated with the JAK-STAT signalling pathway were significantly amplified in IPF-LC (P = 0.012). This study demonstrates that IPF-LC is genetically characterized by the presence of somatic mutations reflecting a variety of environmental exposures on the background of specific germline mutations, and is associated with potentially targetable alterations such as BRAF mutations. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A novel splicing site IRP1 somatic mutation in a patient with pheochromocytoma and JAK2V617F positive polycythemia vera: a case report.

PubMed

Pang, Ying; Gupta, Garima; Yang, Chunzhang; Wang, Herui; Huynh, Thanh-Truc; Abdullaev, Ziedulla; Pack, Svetlana D; Percy, Melanie J; Lappin, Terence R J; Zhuang, Zhengping; Pacak, Karel

2018-03-13

The role of the hypoxia signaling pathway in the pathogenesis of pheochromocytoma/paraganglioma (PPGL)-polycythemia syndrome has been elucidated. Novel somatic mutations in hypoxia-inducible factor type 2A (HIF2A) and germline mutations in prolyl hydroxylase type 1 and type 2 (PHD1 and PHD2) have been identified to cause upregulation of the hypoxia signaling pathway and its target genes including erythropoietin (EPO) and its receptor (EPOR). However, in a minority of patients presenting with this syndrome, the genetics and molecular pathogenesis remain unexplained. The aim of the present study was to uncover novel genetic causes of PPGL-polycythemia syndrome. A female presented with a history of JAK2 V617F positive PV, diagnosed in 2007, and right adrenal pheochromocytoma diagnosed and resected in 2011. Her polycythemia symptoms and hematocrit levels continued to worsen from 2007 to 2011, with an increased frequency of phlebotomies. Postoperatively, until early 2013, her hematocrit levels remained normalized. Following this, the hematocrit levels ranged between 46.4 and 48.9% [35-45%]. Tumor tissue from the patient was further tested for mutations in genes related to upregulation of the hypoxia signaling pathway including iron regulatory protein 1 (IRP1), which is a known regulator of HIF-2α mRNA translation. Functional studies were performed to investigate the consequences of these mutations, especially their effect on the HIF signaling pathway and EPO. Indel mutations (c.267-1_267delGGinsTA) were discovered at the exon 3 splicing site of IRP1. Minigene construct and splicing site analysis showed that the mutation led to a new splicing site and a frameshift mutation of IRP1, which caused a truncated protein. Fluorescence in situ hybridization analysis demonstrated heterozygous IRP1 deletions in tumor cells. Immunohistochemistry results confirmed the truncated IRP1 and overexpressed HIF-2α, EPO and EPOR in tumor cells. This is the first report which provides direct molecular genetic evidence of association between a somatic IRP1 loss-of-function mutation and PHEO and secondary polycythemia. In patients diagnosed with PHEO/PGL and polycythemia with negative genetic testing for mutations in HIF2A, PHD1/2, and VHL, IRP1 should be considered as a candidate gene.

All known patient mutations in the ASH-RhoGAP domains of OCRL affect targeting and APPL1 binding

PubMed Central

McCrea, Heather J.; Paradise, Summer; Tomasini, Livia; Addis, Maria; Melis, Maria Antonietta; De Matteis, Maria Antonietta; De Camilli, Pietro

2008-01-01

Mutations in the inositol 5-phosphatase OCRL are responsible for Lowe syndrome, an X-linked disorder characterized by bilateral cataracts, mental retardation, neonatal hypotonia, and renal Fanconi syndrome, and for Dent disease, another X-linked condition characterized by kidney reabsorption defects. We have previously described an interaction of OCRL with the endocytic adaptor APPL1 that links OCRL to protein networks involved in the disease phenotype. Here we provide new evidence showing that among the interactions which target OCRL to membranes of the endocytic pathway, binding to APPL1 is the only one abolished by all known disease-causing missense mutations in the ASH-RhoGAP domains of the protein. Furthermore, we demonstrate that APPL1 and rab5 independently contribute to recruit OCRL to enlarged endosomes induced by the expression of constitutively active Rab5. Thus, binding to APPL1 helps localize OCRL at specific cellular sites, and disruption of this interaction may play a role in disease. PMID:18307981

Glyphosate resistance: state of knowledge

PubMed Central

Sammons, Robert Douglas; Gaines, Todd A

2014-01-01

Studies of mechanisms of resistance to glyphosate have increased current understanding of herbicide resistance mechanisms. Thus far, single-codon non-synonymous mutations of EPSPS (5-enolypyruvylshikimate-3-phosphate synthase) have been rare and, relative to other herbicide mode of action target-site mutations, unconventionally weak in magnitude for resistance to glyphosate. However, it is possible that weeds will emerge with non-synonymous mutations of two codons of EPSPS to produce an enzyme endowing greater resistance to glyphosate. Today, target-gene duplication is a common glyphosate resistance mechanism and could become a fundamental process for developing any resistance trait. Based on competition and substrate selectivity studies in several species, rapid vacuole sequestration of glyphosate occurs via a transporter mechanism. Conversely, as the chloroplast requires transporters for uptake of important metabolites, transporters associated with the two plastid membranes may separately, or together, successfully block glyphosate delivery. A model based on finite glyphosate dose and limiting time required for chloroplast loading sets the stage for understanding how uniquely different mechanisms can contribute to overall glyphosate resistance. PMID:25180399

Emergence of Ebola Virus Escape Variants in Infected Nonhuman Primates Treated with the MB-003 Antibody Cocktail.

PubMed

Kugelman, Jeffrey R; Kugelman-Tonos, Johanny; Ladner, Jason T; Pettit, James; Keeton, Carolyn M; Nagle, Elyse R; Garcia, Karla Y; Froude, Jeffrey W; Kuehne, Ana I; Kuhn, Jens H; Bavari, Sina; Zeitlin, Larry; Dye, John M; Olinger, Gene G; Sanchez-Lockhart, Mariano; Palacios, Gustavo F

2015-09-29

MB-003, a plant-derived monoclonal antibody cocktail used effectively in treatment of Ebola virus infection in non-human primates, was unable to protect two of six animals when initiated 1 or 2 days post-infection. We characterized a mechanism of viral escape in one of the animals, after observation of two clusters of genomic mutations that resulted in five nonsynonymous mutations in the monoclonal antibody target sites. These mutations were linked to a reduction in antibody binding and later confirmed to be present in a viral isolate that was not neutralized in vitro. Retrospective evaluation of a second independent study allowed the identification of a similar case. Four SNPs in previously identified positions were found in this second fatality, suggesting that genetic drift could be a potential cause for treatment failure. These findings highlight the importance selecting different target domains for each component of the cocktail to minimize the potential for viral escape. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Regulation of the DNA damage response by DNA-PKcs inhibitory phosphorylation of ATM

PubMed Central

Zhou, Yi; Lee, Ji-Hoon; Jiang, Wenxia; Crowe, Jennie L; Zha, Shan; Paull, Tanya T.

2017-01-01

SUMMARY Ataxia-Telangiectasia Mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice. PMID:27939942

Regulation of the DNA Damage Response by DNA-PKcs Inhibitory Phosphorylation of ATM.

PubMed

Zhou, Yi; Lee, Ji-Hoon; Jiang, Wenxia; Crowe, Jennie L; Zha, Shan; Paull, Tanya T

2017-01-05

Ataxia-telangiectasia mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice. Copyright © 2017 Elsevier Inc. All rights reserved.

TERT promoter mutations in bladder cancer affect patient survival and disease recurrence through modification by a common polymorphism.

PubMed

Rachakonda, P Sivaramakrishna; Hosen, Ismail; de Verdier, Petra J; Fallah, Mahdi; Heidenreich, Barbara; Ryk, Charlotta; Wiklund, N Peter; Steineck, Gunnar; Schadendorf, Dirk; Hemminki, Kari; Kumar, Rajiv

2013-10-22

The telomerase reverse transcriptase (TERT) promoter, an important element of telomerase expression, has emerged as a target of cancer-specific mutations. Originally described in melanoma, the mutations in TERT promoter have been shown to be common in certain other tumor types that include glioblastoma, hepatocellular carcinoma, and bladder cancer. To fully define the occurrence and effect of the TERT promoter mutations, we investigated tumors from a well-characterized series of 327 patients with urothelial cell carcinoma of bladder. The somatic mutations, mainly at positions -124 and -146 bp from ATG start site that create binding motifs for E-twenty six/ternary complex factors (Ets/TCF), affected 65.4% of the tumors, with even distribution across different stages and grades. Our data showed that a common polymorphism rs2853669, within a preexisting Ets2 binding site in the TERT promoter, acts as a modifier of the effect of the mutations on survival and tumor recurrence. The patients with the mutations showed poor survival in the absence [hazard ratio (HR) 2.19, 95% confidence interval (CI) 1.02-4.70] but not in the presence (HR 0.42, 95% CI 0.18-1.01) of the variant allele of the polymorphism. The mutations in the absence of the variant allele were highly associated with the disease recurrence in patients with Tis, Ta, and T1 tumors (HR 1.85, 95% CI 1.11-3.08). The TERT promoter mutations are the most common somatic lesions in bladder cancer with clinical implications. The association of the mutations with patient survival and disease recurrence, subject to modification by a common polymorphism, can be a unique putative marker with individualized prognostic potential.

Molecular dynamics simulations show how the FMRP Ile304Asn mutation destabilizes the KH2 domain structure and affects its function.

PubMed

Di Marino, Daniele; Achsel, Tilmann; Lacoux, Caroline; Falconi, Mattia; Bagni, Claudia

2014-01-01

Mutations or deletions of FMRP, involved in the regulation of mRNA metabolism in brain, lead to the Fragile X syndrome (FXS), the most frequent form of inherited intellectual disability. A severe manifestation of the disease has been associated with the Ile304Asn mutation, located on the KH2 domain of the protein. Several hypotheses have been proposed to explain the possible molecular mechanism responsible for the drastic effect of this mutation in humans. Here, we performed a molecular dynamics simulation and show that the Ile304Asn mutation destabilizes the hydrophobic core producing a partial unfolding of two α-helices and a displacement of a third one. The affected regions show increased residue flexibility and motion. Molecular docking analysis revealed strongly reduced binding to a model single-stranded nucleic acid in agreement with known data that the two partially unfolded helices form the RNA-binding surface. The third helix, which we show here to be also affected, is involved in the PAK1 protein interaction. These two functional binding sites on the KH2 domain do not overlap spatially, and therefore, they can simultaneously bind their targets. Since the Ile304Asn mutation affects both binding sites, this may justify the severe clinical manifestation observed in the patient in which both mRNA metabolism activity and cytoskeleton remodeling would be affected.

Determinants of RNA binding and translational repression by the Bicaudal-C regulatory protein.

PubMed

Zhang, Yan; Park, Sookhee; Blaser, Susanne; Sheets, Michael D

2014-03-14

Bicaudal-C (Bic-C) RNA binding proteins function as important translational repressors in multiple biological contexts within metazoans. However, their RNA binding sites are unknown. We recently demonstrated that Bic-C functions in spatially regulated translational repression of the xCR1 mRNA during Xenopus development. This repression contributes to normal development by confining the xCR1 protein, a regulator of key signaling pathways, to specific cells of the embryo. In this report, we combined biochemical approaches with in vivo mRNA reporter assays to define the minimal Bic-C target site within the xCR1 mRNA. This 32-nucleotide Bic-C target site is predicted to fold into a stem-loop secondary structure. Mutational analyses provided evidence that this stem-loop structure is important for Bic-C binding. The Bic-C target site was sufficient for Bic-C mediated repression in vivo. Thus, we describe the first RNA binding site for a Bic-C protein. This identification provides an important step toward understanding the mechanisms by which evolutionarily conserved Bic-C proteins control cellular function in metazoans.

Structure-Based Analysis Reveals Cancer Missense Mutations Target Protein Interaction Interfaces.

PubMed

Engin, H Billur; Kreisberg, Jason F; Carter, Hannah

2016-01-01

Recently it has been shown that cancer mutations selectively target protein-protein interactions. We hypothesized that mutations affecting distinct protein interactions involving established cancer genes could contribute to tumor heterogeneity, and that novel mechanistic insights might be gained into tumorigenesis by investigating protein interactions under positive selection in cancer. To identify protein interactions under positive selection in cancer, we mapped over 1.2 million nonsynonymous somatic cancer mutations onto 4,896 experimentally determined protein structures and analyzed their spatial distribution. In total, 20% of mutations on the surface of known cancer genes perturbed protein-protein interactions (PPIs), and this enrichment for PPI interfaces was observed for both tumor suppressors (Odds Ratio 1.28, P-value < 10(-4)) and oncogenes (Odds Ratio 1.17, P-value < 10(-3)). To study this further, we constructed a bipartite network representing structurally resolved PPIs from all available human complexes in the Protein Data Bank (2,864 proteins, 3,072 PPIs). Analysis of frequently mutated cancer genes within this network revealed that tumor-suppressors, but not oncogenes, are significantly enriched with functional mutations in homo-oligomerization regions (Odds Ratio 3.68, P-Value < 10(-8)). We present two important examples, TP53 and beta-2-microglobulin, for which the patterns of somatic mutations at interfaces provide insights into specifically perturbed biological circuits. In patients with TP53 mutations, patient survival correlated with the specific interactions that were perturbed. Moreover, we investigated mutations at the interface of protein-nucleotide interactions and observed an unexpected number of missense mutations but not silent mutations occurring within DNA and RNA binding sites. Finally, we provide a resource of 3,072 PPI interfaces ranked according to their mutation rates. Analysis of this list highlights 282 novel candidate cancer genes that encode proteins participating in interactions that are perturbed recurrently across tumors. In summary, mutation of specific protein interactions is an important contributor to tumor heterogeneity and may have important implications for clinical outcomes.

Deep Sequencing-guided Design of a High Affinity Dual Specificity Antibody to Target Two Angiogenic Factors in Neovascular Age-related Macular Degeneration.

PubMed

Koenig, Patrick; Lee, Chingwei V; Sanowar, Sarah; Wu, Ping; Stinson, Jeremy; Harris, Seth F; Fuh, Germaine

2015-09-04

The development of dual targeting antibodies promises therapies with improved efficacy over mono-specific antibodies. Here, we engineered a Two-in-One VEGF/angiopoietin 2 antibody with dual action Fab (DAF) as a potential therapeutic for neovascular age-related macular degeneration. Crystal structures of the VEGF/angiopoietin 2 DAF in complex with its two antigens showed highly overlapping binding sites. To achieve sufficient affinity of the DAF to block both angiogenic factors, we turned to deep mutational scanning in the complementarity determining regions (CDRs). By mutating all three CDRs of each antibody chain simultaneously, we were able not only to identify affinity improving single mutations but also mutation pairs from different CDRs that synergistically improve both binding functions. Furthermore, insights into the cooperativity between mutations allowed us to identify fold-stabilizing mutations in the CDRs. The data obtained from deep mutational scanning reveal that the majority of the 52 CDR residues are utilized differently for the two antigen binding function and permit, for the first time, the engineering of several DAF variants with sub-nanomolar affinity against two structurally unrelated antigens. The improved variants show similar blocking activity of receptor binding as the high affinity mono-specific antibodies against these two proteins, demonstrating the feasibility of generating a dual specificity binding surface with comparable properties to individual high affinity mono-specific antibodies. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Genetic Testing Confirmed the Early Diagnosis of X-Linked Hypophosphatemic Rickets in a 7-Month-Old Infant

PubMed Central

Poon, Kok Siong; Sng, Andrew Anjian; Ho, Cindy Weili; Koay, Evelyn Siew-Chuan

2015-01-01

Loss-of-function mutations in the phosphate regulating gene with homologies to endopeptidases on the X-chromosome (PHEX) have been causally associated with X-linked hypophosphatemic rickets (XLHR). The early diagnosis of XLHR in infants is challenging when it is based solely on clinical features and biochemical findings. We report a 7-month-old boy with a family history of hypophosphatemic rickets., who demonstrated early clinical evidence of rickets, although serial biochemical findings could not definitively confirm rickets. A sequencing assay targeting the PHEX gene was first performed on the mother’s DNA to screen for mutations in the 5′UTR, 22 coding exons, and the exon-intron junctions. Targeted mutation analysis and mRNA studies were subsequently performed on the boys’ DNA to investigate the pathogenicity of the identified mutation. Genetic screening of the PHEX gene revealed a novel mutation, c.1080-2A>C, at the splice acceptor site in intron 9. The detection of an aberrant mRNA transcript with skipped (loss of) exon 10 establishes its pathogenicity and confirms the diagnosis of XLHR in this infant. Genetic testing of the PHEX gene resulted in early diagnosis of XLHR, thus enabling initiation of therapy and prevention of progressive rachitic changes in the infant. PMID:26904698

Molecular dynamics simulations reveal the allosteric effect of F1174C resistance mutation to ceritinib in ALK-associated lung cancer.

PubMed

Ni, Zhong; Wang, Xiting; Zhang, Tianchen; Jin, Rong Zhong

2016-12-01

Anaplastic lymphoma kinase (ALK) has become as an important target for the treatment of various human cancers, especially non-small-cell lung cancer. A mutation, F1174C, suited in the C-terminal helix αC of ALK and distal from the small-molecule inhibitor ceritinib bound to the ATP-binding site, causes the emergence of drug resistance to ceritinib. However, the detailed mechanism for the allosteric effect of F1174C resistance mutation to ceritinib remains unclear. Here, molecular dynamics (MD) simulations and binding free energy calculations [Molecular Mechanics/Generalized Born Surface Area (MM/GBSA)] were carried out to explore the advent of drug resistance mutation in ALK. MD simulations observed that the exquisite aromatic-aromatic network formed by residues F1098, F1174, F1245, and F1271 in the wild-type ALK-ceritinib complex was disrupted by the F1174C mutation. The resulting mutation allosterically affected the conformational dynamic of P-loop and caused the upward movement of the P-loop from the ATP-binding site, thereby weakening the interaction between ceritinib and the P-loop. The subsequent MM/GBSA binding free energy calculations and decomposition analysis of binding free energy validated this prediction. This study provides mechanistic insight into the allosteric effect of F1174C resistance mutation to ceritinib in ALK and is expected to contribute to design the next-generation of ALK inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

CRISPR/Cas9-Based Multiplex Genome Editing in Monocot and Dicot Plants.

PubMed

Ma, Xingliang; Liu, Yao-Guang

2016-07-01

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome targeting system has been applied to a variety of organisms, including plants. Compared to other genome-targeting technologies such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), the CRISPR/Cas9 system is easier to use and has much higher editing efficiency. In addition, multiple "single guide RNAs" (sgRNAs) with different target sequences can be designed to direct the Cas9 protein to multiple genomic sites for simultaneous multiplex editing. Here, we present a procedure for highly efficient multiplex genome targeting in monocot and dicot plants using a versatile and robust CRISPR/Cas9 vector system, emphasizing the construction of binary constructs with multiple sgRNA expression cassettes in one round of cloning using Golden Gate ligation. We also describe the genotyping of targeted mutations in transgenic plants by direct Sanger sequencing followed by decoding of superimposed sequencing chromatograms containing biallelic or heterozygous mutations using the Web-based tool DSDecode. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Highly efficient targeted mutagenesis in one-cell mouse embryos mediated by the TALEN and CRISPR/Cas systems.

PubMed

Yasue, Akihiro; Mitsui, Silvia Naomi; Watanabe, Takahito; Sakuma, Tetsushi; Oyadomari, Seiichi; Yamamoto, Takashi; Noji, Sumihare; Mito, Taro; Tanaka, Eiji

2014-07-16

Since the establishment of embryonic stem (ES) cell lines, the combined use of gene targeting with homologous recombination has aided in elucidating the functions of various genes. However, the ES cell technique is inefficient and time-consuming. Recently, two new gene-targeting technologies have been developed: the transcription activator-like effector nuclease (TALEN) system, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system. In addition to aiding researchers in solving conventional problems, these technologies can be used to induce site-specific mutations in various species for which ES cells have not been established. Here, by targeting the Fgf10 gene through RNA microinjection in one-cell mouse embryos with the TALEN and CRISPR/Cas systems, we produced the known limb-defect phenotypes of Fgf10-deficient embryos at the F0 generation. Compared to the TALEN system, the CRISPR/Cas system induced the limb-defect phenotypes with a strikingly higher efficiency. Our results demonstrate that although both gene-targeting technologies are useful, the CRISPR/Cas system more effectively elicits single-step biallelic mutations in mice.

Both point mutations and low expression levels of the nicotinic acetylcholine receptor β1 subunit are associated with imidacloprid resistance in an Aphis gossypii (Glover) population from a Bt cotton field in China.

PubMed

Chen, Xuewei; Li, Fen; Chen, Anqi; Ma, Kangsheng; Liang, Pingzhuo; Liu, Ying; Song, Dunlun; Gao, Xiwu

2017-09-01

Aphis gossypii Glover is a destructive pest of numerous crops throughout the world. Although the expansion of Bt cotton cultivation has helped to control some insect pests, the damage from cotton aphids has not been mitigated. The evolution of aphid resistance to imidacloprid has made its chemical control more difficult since its introduction in 1991. Field populations of A. gossypii that were collected from different transgenic (Bt) cotton planting areas of China in 2014 developed different levels of resistance to imidacloprid. The IMI_R strain has developed high resistance to imidacloprid with the resistance ratio >1200-fold. Compared with the susceptible IMI_S strain, the IMI_R strain also developed a high level cross resistance to sulfoxaflor and acetamiprid. The limited synergism with either PBO or DEF suggests that resistance may be due to the site mutation of molecular target rather than to enhanced detoxification. Three target-site mutations within the nicotinic acetylcholine receptor (nAChR) β1 subunit were detected in the IMI_R strain. The R81T mutation has been reported to be responsible for imidacloprid resistance in A. gossypii and M. persicae. Both V62I and K264E were first detected in A. gossypii. These point mutations are also present in field populations, suggesting that they play a role in the resistance to imidacloprid. Furthermore, the expression level of transcripts encoding β1 subunit was decreased significantly in the IMI_R strain compared with the IMI_S strain, suggesting that both point mutations and the down-regulation of nAChR β1 subunit expression may be involved in the resistance mechanism for imidacloprid in A. gossypii. These results should be useful for the management of imidacloprid-resistant cotton aphids in Bt cotton fields in China. Copyright © 2016 Elsevier B.V. All rights reserved.

Effective immuno-targeting of the IDH1 mutation R132H in a murine model of intracranial glioma.

PubMed

Pellegatta, Serena; Valletta, Lorella; Corbetta, Cristina; Patanè, Monica; Zucca, Ileana; Riccardi Sirtori, Federico; Bruzzone, Maria Grazia; Fogliatto, Gianpaolo; Isacchi, Antonella; Pollo, Bianca; Finocchiaro, Gaetano

2015-01-21

The R132H mutation of cytosolic isocitrate dehydrogenase (IDH1) is present in the majority of low grade gliomas.Immunotherapy in these tumors has an interesting, still unexploited, therapeutic potential, as they are less immunosuppressive than glioblastomas. Using site-directed mutagenesis we introduced the R132H mutation into the murine glioma cell line GL261,creating mIDH1-GL261. Presence of the mutation was confirmed by immunoblotting and production of the oncometabolite 2-hydroxyglutarate (2HG), demonstrated by mass spectrometry (LC-MS/MS) performed on cell supernatant. In vitro mIDH1-GL261 had different morphology but similar growth rate than parental GL261 (p-GL261). After intracranial injection, MRI suggested that the initial growth rate was slower in mIDH1-GL261 than p-GL261 gliomas but overall survival was similar. mIDH1-GL261 gliomas showed evidence of R132H expression and of intratumoral 2HG production (evaluated by MRS and LC-MS/MS). Immunizations were performed nine days after intracranial implantation of mIDH1- or p-GL261 cells by three subcutaneous injections of five different peptides encompassing the IDH1 mutation site, all emulsified with Montanide ISA-51, in association with GM-CSF. Control mice were injected with four ovalbumin peptides or vehicle. Mice with mIDH1-GL261 but not p-GL261 gliomas treated with mIDH1 peptides survived longer than controls; 25% of them were cured. Immunized mice showed higher amounts of peripheral CD8+ T cells, higher production of IFN-γ, and evidence of anti-mIDH1 antibodies.Immunizations led to intratumoral up-regulation of IFN-γ, granzyme-b and perforin-1 and down-regulation of TGF-β2 and IL-10. These results support the translational potential of immunotherapeutic targeting of gliomas carrying IDH1 mutations.

The prototype HIV-1 maturation inhibitor, bevirimat, binds to the CA-SP1 cleavage site in immature Gag particles.

PubMed

Nguyen, Albert T; Feasley, Christa L; Jackson, Ken W; Nitz, Theodore J; Salzwedel, Karl; Air, Gillian M; Sakalian, Michael

2011-12-07

Bevirimat, the prototype Human Immunodeficiency Virus type 1 (HIV-1) maturation inhibitor, is highly potent in cell culture and efficacious in HIV-1 infected patients. In contrast to inhibitors that target the active site of the viral protease, bevirimat specifically inhibits a single cleavage event, the final processing step for the Gag precursor where p25 (CA-SP1) is cleaved to p24 (CA) and SP1. In this study, photoaffinity analogs of bevirimat and mass spectrometry were employed to map the binding site of bevirimat to Gag within immature virus-like particles. Bevirimat analogs were found to crosslink to sequences overlapping, or proximal to, the CA-SP1 cleavage site, consistent with previous biochemical data on the effect of bevirimat on Gag processing and with genetic data from resistance mutations, in a region predicted by NMR and mutational studies to have α-helical character. Unexpectedly, a second region of interaction was found within the Major Homology Region (MHR). Extensive prior genetic evidence suggests that the MHR is critical for virus assembly. This is the first demonstration of a direct interaction between the maturation inhibitor, bevirimat, and its target, Gag. Information gained from this study sheds light on the mechanisms by which the virus develops resistance to this class of drug and may aid in the design of next-generation maturation inhibitors.

The DEAD box helicase RDE-12 promotes amplification of RNAi in cytoplasmic foci in C. elegans

PubMed Central

Yang, Huan; Vallandingham, Jim; Shiu, Philip; Li, Hua; Hunter, Craig P.; Mak, Ho Yi

2014-01-01

Summary RNA interference (RNAi) is a potent mechanism for down-regulating gene expression. Conserved RNAi pathway components are found in animals, plants, fungi and other eukaryotes [1–3]. In C. elegans, the RNAi response is greatly amplified by the synthesis of abundant secondary siRNAs [4–6]. Exogenous double stranded RNA is processed by Dicer and RDE-1/Argonaute into primary siRNA that guides target mRNA recognition. The RDE-10/RDE-11 complex and the RNA dependent RNA polymerase RRF-1 then engage the target mRNA for secondary siRNA synthesis [7, 8]. However, the molecular link between primary siRNA production and secondary siRNA synthesis remains largely unknown. Furthermore, it is unclear if the sub-cellular sites for target mRNA recognition and degradation coincide with sites where siRNA synthesis and amplification occur. In the C. elegans germline, cytoplasmic P granules at the nuclear pores and perinuclear Mutator foci contribute to target mRNA surveillance and siRNA amplification, respectively [9–11]. We report that RDE-12, a conserved FG domain containing DEAD-box helicase, localizes in P-granules and cytoplasmic foci that are enriched in RSD-6 but are excluded from the Mutator foci. Our results suggest that RDE-12 promotes secondary siRNA synthesis by orchestrating the recruitment of RDE-10 and RRF-1 to primary siRNA targeted mRNA in distinct cytoplasmic compartments. PMID:24684930

The DEAD box helicase RDE-12 promotes amplification of RNAi in cytoplasmic foci in C. elegans.

PubMed

Yang, Huan; Vallandingham, Jim; Shiu, Philip; Li, Hua; Hunter, Craig P; Mak, Ho Yi

2014-04-14

RNAi is a potent mechanism for downregulating gene expression. Conserved RNAi pathway components are found in animals, plants, fungi, and other eukaryotes. In C. elegans, the RNAi response is greatly amplified by the synthesis of abundant secondary small interfering RNAs (siRNAs). Exogenous double-stranded RNA is processed by Dicer and RDE-1/Argonaute into primary siRNA that guides target mRNA recognition. The RDE-10/RDE-11 complex and the RNA-dependent RNA polymerase RRF-1 then engage the target mRNA for secondary siRNA synthesis. However, the molecular link between primary siRNA production and secondary siRNA synthesis remains largely unknown. Furthermore, it is unclear whether the subcellular sites for target mRNA recognition and degradation coincide with sites where siRNA synthesis and amplification occur. In the C. elegans germline, cytoplasmic P granules at the nuclear pores and perinuclear Mutator foci contribute to target mRNA surveillance and siRNA amplification, respectively. We report that RDE-12, a conserved phenylalanine-glycine (FG) domain-containing DEAD box helicase, localizes in P granules and cytoplasmic foci that are enriched in RSD-6 but are excluded from the Mutator foci. Our results suggest that RDE-12 promotes secondary siRNA synthesis by orchestrating the recruitment of RDE-10 and RRF-1 to primary siRNA-targeted mRNA in distinct cytoplasmic compartments. Copyright © 2014 Elsevier Ltd. All rights reserved.

Characterization of the voltage-gated sodium channel of the Asian citrus psyllid, Diaphorina citri.

PubMed

Liu, Bin; Coy, Monique R; Wang, Jin-Jun; Stelinski, Lukasz L

2017-02-01

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is an important insect pest of citrus. It is the vector of 'Candidatus' Liberibacter asiaticus, a phloem-limited bacterium that infects citrus, resulting in the disease Huanglongbing (HLB). Disease management relies heavily on suppression of D. citri populations with insecticides, including pyrethroids. In recent annual surveys to monitor insecticide resistance, reduced susceptibility to fenpropathrin was identified in several field populations of D. citri. The primary target of pyrethroids is the voltage-gated sodium channel (VGSC). The VGSC is prone to target-site insensitivity because of mutations that either reduce pyrethroid binding and/or alter gating kinetics. These mutations, known as knockdown resistance or kdr, have been reported in a wide diversity of arthropod species. Alternative splicing, in combination with kdr mutations, has been also associated with reduced pyrethroid efficacy. Here we report the molecular characterization of the VGSC in D. citri along with a survey of alternative splicing across developmental stages of this species. Previous studies demonstrated that D. citri has an exquisite enzymatic arsenal to detoxify insecticides resulting in reduced efficacy. The results from the current investigation demonstrate that target-site insensitivity is also a potential basis for insecticide resistance to pyrethroids in D. citri. The VGSC sequence and its molecular characterization should facilitate early elucidation of the underlying cause of an established case of resistance to pyrethroids. This is the first characterization of a VGSC from a hemipteran to this level of detail, with the majority of the previous studies on dipterans and lepidopterans. © 2015 Institute of Zoology, Chinese Academy of Sciences.

Allele-Specific Chromatin Recruitment and Therapeutic Vulnerabilities of ESR1 Activating Mutations.

PubMed

Jeselsohn, Rinath; Bergholz, Johann S; Pun, Matthew; Cornwell, MacIntosh; Liu, Weihan; Nardone, Agostina; Xiao, Tengfei; Li, Wei; Qiu, Xintao; Buchwalter, Gilles; Feiglin, Ariel; Abell-Hart, Kayley; Fei, Teng; Rao, Prakash; Long, Henry; Kwiatkowski, Nicholas; Zhang, Tinghu; Gray, Nathanael; Melchers, Diane; Houtman, Rene; Liu, X Shirley; Cohen, Ofir; Wagle, Nikhil; Winer, Eric P; Zhao, Jean; Brown, Myles

2018-02-12

Estrogen receptor α (ER) ligand-binding domain (LBD) mutations are found in a substantial number of endocrine treatment-resistant metastatic ER-positive (ER + ) breast cancers. We investigated the chromatin recruitment, transcriptional network, and genetic vulnerabilities in breast cancer models harboring the clinically relevant ER mutations. These mutants exhibit both ligand-independent functions that mimic estradiol-bound wild-type ER as well as allele-specific neomorphic properties that promote a pro-metastatic phenotype. Analysis of the genome-wide ER binding sites identified mutant ER unique recruitment mediating the allele-specific transcriptional program. Genetic screens identified genes that are essential for the ligand-independent growth driven by the mutants. These studies provide insights into the mechanism of endocrine therapy resistance engendered by ER mutations and potential therapeutic targets. Copyright © 2018 Elsevier Inc. All rights reserved.

“Reverse Fleximers”: Introduction of a series of 5-substituted carbocyclic uridine analogues

PubMed Central

Sadler, Joshua M.; Ojewoye, Olubukola; Seley-Radtke, Katherine L.

2009-01-01

Nucleosides are ubiquitous in biological systems and as such, have been a focus of medicinal chemistry research in the search for new and potent therapeutic compounds. There are a number of modified nucleosides on the market, however increasing reports of resistance by mutation of either the enzyme binding site or the pathway that they are designed to interrupt are surfacing. As shown in recent reports, a candidate that can change conformation and still maintain recognition by the target enzyme would be highly desirable, and it is for this reason that flexible substrates have recently been sought as potential therapeutics. With this goal in mind, we have begun investigation into novel flexible scaffolds capable of overcoming viral resistance mechanisms resulting from binding site mutations. PMID:18776508

Recovery of phenotypes obtained by adaptive evolution through inverse metabolic engineering.

PubMed

Hong, Kuk-Ki; Nielsen, Jens

2012-11-01

In a previous study, system level analysis of adaptively evolved yeast mutants showing improved galactose utilization revealed relevant mutations. The governing mutations were suggested to be in the Ras/PKA signaling pathway and ergosterol metabolism. Here, site-directed mutants having one of the mutations RAS2(Lys77), RAS2(Tyr112), and ERG5(Pro370) were constructed and evaluated. The mutants were also combined with overexpression of PGM2, earlier proved as a beneficial target for galactose utilization. The constructed strains were analyzed for their gross phenotype, transcriptome and targeted metabolites, and the results were compared to those obtained from reference strains and the evolved strains. The RAS2(Lys77) mutation resulted in the highest specific galactose uptake rate among all of the strains with an increased maximum specific growth rate on galactose. The RAS2(Tyr112) mutation also improved the specific galactose uptake rate and also resulted in many transcriptional changes, including ergosterol metabolism. The ERG5(Pro370) mutation only showed a small improvement, but when it was combined with PGM2 overexpression, the phenotype was almost the same as that of the evolved mutants. Combination of the RAS2 mutations with PGM2 overexpression also led to a complete recovery of the adaptive phenotype in galactose utilization. Recovery of the gross phenotype by the reconstructed mutants was achieved with much fewer changes in the genome and transcriptome than for the evolved mutants. Our study demonstrates how the identification of specific mutations by systems biology can direct new metabolic engineering strategies for improving galactose utilization by yeast.

Non-neural androgen receptors affect sexual differentiation of brain and behaviour.

PubMed

Monks, D A; Swift-Gallant, A

2018-02-01

Although gonadal testosterone is the principal endocrine factor that promotes masculine traits in mammals, the development of a male phenotype requires local production of both androgenic and oestrogenic signals within target tissues. Much of our knowledge concerning androgenic components of testosterone signalling in sexual differentiation comes from studies of androgen receptor (Ar) loss of function mutants. Here, we review these studies of loss of Ar function and of AR overexpression either globally or selectively in the nervous system of mice. Global and neural mutations affect socio-sexual behaviour and the neuroanatomy of these mice in a sexually differentiated manner. Some masculine traits are affected by both global and neural mutation, indicative of neural mediation, whereas other masculine traits are affected only by global mutation, indicative of an obligatory non-neural androgen target. These results support a model in which multiple sites of androgen action coordinate to produce masculine phenotypes. Furthermore, AR overexpression does not always have a phenotype opposite to that of loss of Ar function mutants, indicative of a nonlinear relationship between androgen dose and masculine phenotype in some cases. Potential mechanisms of Ar gene function in non-neural targets in producing masculine phenotypes are discussed. © 2017 British Society for Neuroendocrinology.

Trade-offs with stability modulate innate and mutationally acquired drug-resistance in bacterial dihydrofolate reductase enzymes.

PubMed

Matange, Nishad; Bodkhe, Swapnil; Patel, Maitri; Shah, Pooja

2018-06-05

Structural stability is a major constraint on the evolution of protein sequences. However, under strong directional selection, mutations that confer novel phenotypes but compromise structural stability of proteins may be permissible. During the evolution of antibiotic resistance, mutations that confer drug resistance often have pleiotropic effects on the structure and function of antibiotic-target proteins, usually essential metabolic enzymes. In this study, we show that trimethoprim-resistant alleles of dihydrofolate reductase from Escherichia coli (EcDHFR) harbouring the Trp30Gly, Trp30Arg or Trp30Cys mutations are significantly less stable than the wild type making them prone to aggregation and proteolysis. This destabilization is associated with lower expression level resulting in a fitness cost and negative epistasis with other TMP-resistant mutations in EcDHFR. Using structure-based mutational analysis we show that perturbation of critical stabilizing hydrophobic interactions in wild type EcDHFR enzyme explains the phenotypes of Trp30 mutants. Surprisingly, though crucial for the stability of EcDHFR, significant sequence variation is found at this site among bacterial DHFRs. Mutational and computational analyses in EcDHFR as well as in DHFR enzymes from Staphylococcus aureus and Mycobacterium tuberculosis demonstrate that natural variation at this site and its interacting hydrophobic residues, modulates TMP-resistance in other bacterial DHFRs as well, and may explain the different susceptibilities of bacterial pathogens to trimethoprim. Our study demonstrates that trade-offs between structural stability and function can influence innate drug resistance as well as the potential for mutationally acquired drug resistance of an enzyme. ©2018 The Author(s).

Biological and functional relevance of CASP predictions

PubMed Central

Liu, Tianyun; Ish‐Shalom, Shirbi; Torng, Wen; Lafita, Aleix; Bock, Christian; Mort, Matthew; Cooper, David N; Bliven, Spencer; Capitani, Guido; Mooney, Sean D.

2017-01-01

Abstract Our goal is to answer the question: compared with experimental structures, how useful are predicted models for functional annotation? We assessed the functional utility of predicted models by comparing the performances of a suite of methods for functional characterization on the predictions and the experimental structures. We identified 28 sites in 25 protein targets to perform functional assessment. These 28 sites included nine sites with known ligand binding (holo‐sites), nine sites that are expected or suggested by experimental authors for small molecule binding (apo‐sites), and Ten sites containing important motifs, loops, or key residues with important disease‐associated mutations. We evaluated the utility of the predictions by comparing their microenvironments to the experimental structures. Overall structural quality correlates with functional utility. However, the best‐ranked predictions (global) may not have the best functional quality (local). Our assessment provides an ability to discriminate between predictions with high structural quality. When assessing ligand‐binding sites, most prediction methods have higher performance on apo‐sites than holo‐sites. Some servers show consistently high performance for certain types of functional sites. Finally, many functional sites are associated with protein‐protein interaction. We also analyzed biologically relevant features from the protein assemblies of two targets where the active site spanned the protein‐protein interface. For the assembly targets, we find that the features in the models are mainly determined by the choice of template. PMID:28975675

Functional analysis of dengue virus (DENV) type 2 envelope protein domain 3 type-specific and DENV complex-reactive critical epitope residues.

PubMed

Pitcher, Trevor J; Sarathy, Vanessa V; Matsui, Kiyohiko; Gromowski, Gregory D; Huang, Claire Y-H; Barrett, Alan D T

2015-02-01

The dengue virus (DENV) envelope protein domain 3 (ED3) is the target of potent virus neutralizing antibodies. The DENV-2 ED3 contains adjacent type-specific and DENV complex-reactive antigenic sites that are composed of a small number of residues that were previously demonstrated to be critical for antibody binding. Site-directed mutagenesis of a DENV-2 16681 infectious clone was used to mutate critical residues in the DENV-2 type-specific (K305A and P384A) and DENV complex-reactive (K310A) antigenic sites. The K305A mutant virus multiplied like the parent virus in mosquito and mammalian cells, as did the P384A mutant virus, which required a compensatory mutation (G330D) for viability. However, the K310A mutant virus could not be recovered. The DENV-2 type-specific critical residue mutations K305A and P384A+G330D reduced the ability of DENV-2 type-specific, but not DENV complex-reactive, mAbs to neutralize virus infectivity and this was directly correlated with mAb binding affinity to the rED3 mutants. © 2015 The Authors.

Non-RVD mutations that enhance the dynamics of the TAL repeat array along the superhelical axis improve TALEN genome editing efficacy

PubMed Central

Tochio, Naoya; Umehara, Kohei; Uewaki, Jun-ichi; Flechsig, Holger; Kondo, Masaharu; Dewa, Takehisa; Sakuma, Tetsushi; Yamamoto, Takashi; Saitoh, Takashi; Togashi, Yuichi; Tate, Shin-ichi

2016-01-01

Transcription activator-like effector (TALE) nuclease (TALEN) is widely used as a tool in genome editing. The DNA binding part of TALEN consists of a tandem array of TAL-repeats that form a right-handed superhelix. Each TAL-repeat recognises a specific base by the repeat variable diresidue (RVD) at positions 12 and 13. TALEN comprising the TAL-repeats with periodic mutations to residues at positions 4 and 32 (non-RVD sites) in each repeat (VT-TALE) exhibits increased efficacy in genome editing compared with a counterpart without the mutations (CT-TALE). The molecular basis for the elevated efficacy is unknown. In this report, comparison of the physicochemical properties between CT- and VT-TALEs revealed that VT-TALE has a larger amplitude motion along the superhelical axis (superhelical motion) compared with CT-TALE. The greater superhelical motion in VT-TALE enabled more TAL-repeats to engage in the target sequence recognition compared with CT-TALE. The extended sequence recognition by the TAL-repeats improves site specificity with limiting the spatial distribution of FokI domains to facilitate their dimerization at the desired site. Molecular dynamics simulations revealed that the non-RVD mutations alter inter-repeat hydrogen bonding to amplify the superhelical motion of VT-TALE. The TALEN activity is associated with the inter-repeat hydrogen bonding among the TAL repeats. PMID:27883072

Crystal Structures of Penicillin-Binding Protein 2 From Penicillin-Susceptible And -Resistant Strains of Neisseria Gonorrhoeae Reveal An Unexpectedly Subtle Mechanism for Antibiotic Resistance

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

Powell, A.J.; Tomberg, J.; Deacon, A.M.

Penicillin-binding protein 2 (PBP2) from N. gonorrhoeae is the major molecular target for {beta}-lactam antibiotics used to treat gonococcal infections. PBP2 from penicillin-resistant strains of N. gonorrhoeae harbors an aspartate insertion after position 345 (Asp-345a) and 4-8 additional mutations, but how these alter the architecture of the protein is unknown. We have determined the crystal structure of PBP2 derived from the penicillin-susceptible strain FA19, which shows that the likely effect of Asp-345a is to alter a hydrogen-bonding network involving Asp-346 and the SXN triad at the active site. We have also solved the crystal structure of PBP2 derived from themore » penicillin-resistant strain FA6140 that contains four mutations near the C terminus of the protein. Although these mutations lower the second order rate of acylation for penicillin by 5-fold relative to wild type, comparison of the two structures shows only minor structural differences, with the positions of the conserved residues in the active site essentially the same in both. Kinetic analyses indicate that two mutations, P551S and F504L, are mainly responsible for the decrease in acylation rate. Melting curves show that the four mutations lower the thermal stability of the enzyme. Overall, these data suggest that the molecular mechanism underlying antibiotic resistance contributed by the four mutations is subtle and involves a small but measurable disordering of residues in the active site region that either restricts the binding of antibiotic or impedes conformational changes that are required for acylation by {beta}-lactam antibiotics.« less

Molecular modeling and residue interaction network studies on the mechanism of binding and resistance of the HCV NS5B polymerase mutants to VX-222 and ANA598.

PubMed

Xue, Weiwei; Jiao, Pingzu; Liu, Huanxiang; Yao, Xiaojun

2014-04-01

Hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase (RdRp) with essential functions in viral genome replication and represents a promising therapeutic target to develop direct-acting antivirals (DAAs). Multiple nonnucleoside inhibitors (NNIs) binding sites have been identified within the polymerase. VX-222 and ANA598 are two NNIs targeting thumb II site and palm I site of HCV NS5B polymerase, respectively. These two molecules have been shown to be very effective in phase II clinical trials. However, the emergence of resistant HCV replicon variants (L419M, M423T, I482L mutants to VX-222 and M414T, M414L, G554D mutants to ANA598) has significantly decreased their efficacy. To elucidate the molecular mechanism about how these mutations influenced the drug binding mode and decreased drug efficacy, we studied the binding modes of VX-222 and ANA598 to wild-type and mutant polymerase by molecular modeling approach. Molecular dynamics (MD) simulations results combined with binding free energy calculations indicated that the mutations significantly altered the binding free energy and the interaction for the drugs to polymerase. The further per-residue binding free energy decomposition analysis revealed that the mutations decreased the interactions with several key residues, such as L419, M423, L474, S476, I482, L497, for VX-222 and L384, N411, M414, Y415, Q446, S556, G557 for ANA598. These were the major origins for the resistance to these two drugs. In addition, by analyzing the residue interaction network (RIN) of the complexes between the drugs with wild-type and the mutant polymerase, we found that the mutation residues in the networks involved in the drug resistance possessed a relatively lower size of topology centralities. The shift of betweenness and closeness values of binding site residues in the mutant polymerase is relevant to the mechanism of drug resistance of VX-222 and ANA598. These results can provide an atomic-level understanding about the mechanisms of drug resistance conferred by the studied mutations and will be helpful to design more potent inhibitors which could effectively overcome drug resistance of antivirus agents. Copyright © 2014 Elsevier B.V. All rights reserved.

Targeted next-generation sequencing reveals novel USH2A mutations associated with diverse disease phenotypes: implications for clinical and molecular diagnosis.

PubMed

Chen, Xue; Sheng, Xunlun; Liu, Xiaoxing; Li, Huiping; Liu, Yani; Rong, Weining; Ha, Shaoping; Liu, Wenzhou; Kang, Xiaoli; Zhao, Kanxing; Zhao, Chen

2014-01-01

USH2A mutations have been implicated in the disease etiology of several inherited diseases, including Usher syndrome type 2 (USH2), nonsyndromic retinitis pigmentosa (RP), and nonsyndromic deafness. The complex genetic and phenotypic spectrums relevant to USH2A defects make it difficult to manage patients with such mutations. In the present study, we aim to determine the genetic etiology and to characterize the correlated clinical phenotypes for three Chinese pedigrees with nonsyndromic RP, one with RP sine pigmento (RPSP), and one with USH2. Family histories and clinical details for all included patients were reviewed. Ophthalmic examinations included best corrected visual acuities, visual field measurements, funduscopy, and electroretinography. Targeted next-generation sequencing (NGS) was applied using two sequence capture arrays to reveal the disease causative mutations for each family. Genotype-phenotype correlations were also annotated. Seven USH2A mutations, including four missense substitutions (p.P2762A, p.G3320C, p.R3719H, and p.G4763R), two splice site variants (c.8223+1G>A and c.8559-2T>C), and a nonsense mutation (p.Y3745*), were identified as disease causative in the five investigated families, of which three reported to have consanguineous marriage. Among all seven mutations, six were novel, and one was recurrent. Two homozygous missense mutations (p.P2762A and p.G3320C) were found in one individual family suggesting a potential double hit effect. Significant phenotypic divergences were revealed among the five families. Three families of the five families were affected with early, moderated, or late onset RP, one with RPSP, and the other one with USH2. Our study expands the genotypic and phenotypic variability relevant to USH2A mutations, which would help with a clear insight into the complex genetic and phenotypic spectrums relevant to USH2A defects, and is complementary for a better management of patients with such mutations. We have also demonstrated that a targeted NGS approach is a valuable tool for the genetic diagnosis of USH2 and RP.

Targeted Next-Generation Sequencing Reveals Novel USH2A Mutations Associated with Diverse Disease Phenotypes: Implications for Clinical and Molecular Diagnosis

PubMed Central

Li, Huiping; Liu, Yani; Rong, Weining; Ha, Shaoping; Liu, Wenzhou; Kang, Xiaoli; Zhao, Kanxing; Zhao, Chen

2014-01-01

USH2A mutations have been implicated in the disease etiology of several inherited diseases, including Usher syndrome type 2 (USH2), nonsyndromic retinitis pigmentosa (RP), and nonsyndromic deafness. The complex genetic and phenotypic spectrums relevant to USH2A defects make it difficult to manage patients with such mutations. In the present study, we aim to determine the genetic etiology and to characterize the correlated clinical phenotypes for three Chinese pedigrees with nonsyndromic RP, one with RP sine pigmento (RPSP), and one with USH2. Family histories and clinical details for all included patients were reviewed. Ophthalmic examinations included best corrected visual acuities, visual field measurements, funduscopy, and electroretinography. Targeted next-generation sequencing (NGS) was applied using two sequence capture arrays to reveal the disease causative mutations for each family. Genotype-phenotype correlations were also annotated. Seven USH2A mutations, including four missense substitutions (p.P2762A, p.G3320C, p.R3719H, and p.G4763R), two splice site variants (c.8223+1G>A and c.8559-2T>C), and a nonsense mutation (p.Y3745*), were identified as disease causative in the five investigated families, of which three reported to have consanguineous marriage. Among all seven mutations, six were novel, and one was recurrent. Two homozygous missense mutations (p.P2762A and p.G3320C) were found in one individual family suggesting a potential double hit effect. Significant phenotypic divergences were revealed among the five families. Three families of the five families were affected with early, moderated, or late onset RP, one with RPSP, and the other one with USH2. Our study expands the genotypic and phenotypic variability relevant to USH2A mutations, which would help with a clear insight into the complex genetic and phenotypic spectrums relevant to USH2A defects, and is complementary for a better management of patients with such mutations. We have also demonstrated that a targeted NGS approach is a valuable tool for the genetic diagnosis of USH2 and RP. PMID:25133613

Metabolic and Target-Site Mechanisms Combine to Confer Strong DDT Resistance in Anopheles gambiae

PubMed Central

Mitchell, Sara N.; Rigden, Daniel J.; Dowd, Andrew J.; Lu, Fang; Wilding, Craig S.; Weetman, David; Dadzie, Samuel; Jenkins, Adam M.; Regna, Kimberly; Boko, Pelagie; Djogbenou, Luc; Muskavitch, Marc A. T.; Ranson, Hilary; Paine, Mark J. I.; Mayans, Olga; Donnelly, Martin J.

2014-01-01

The development of resistance to insecticides has become a classic exemplar of evolution occurring within human time scales. In this study we demonstrate how resistance to DDT in the major African malaria vector Anopheles gambiae is a result of both target-site resistance mechanisms that have introgressed between incipient species (the M- and S-molecular forms) and allelic variants in a DDT-detoxifying enzyme. Sequencing of the detoxification enzyme, Gste2, from DDT resistant and susceptible strains of An. gambiae, revealed a non-synonymous polymorphism (I114T), proximal to the DDT binding domain, which segregated with strain phenotype. Recombinant protein expression and DDT metabolism analysis revealed that the proteins from the susceptible strain lost activity at higher DDT concentrations, characteristic of substrate inhibition. The effect of I114T on GSTE2 protein structure was explored through X-ray crystallography. The amino acid exchange in the DDT-resistant strain introduced a hydroxyl group nearby the hydrophobic DDT-binding region. The exchange does not result in structural alterations but is predicted to facilitate local dynamics and enzyme activity. Expression of both wild-type and 114T alleles the allele in Drosophila conferred an increase in DDT tolerance. The 114T mutation was significantly associated with DDT resistance in wild caught M-form populations and acts in concert with target-site mutations in the voltage gated sodium channel (Vgsc-1575Y and Vgsc-1014F) to confer extreme levels of DDT resistance in wild caught An. gambiae. PMID:24675797

Low incidence of SNVs and indels in trio genomes of Cas9-mediated multiplex edited sheep.

PubMed

Wang, Xiaolong; Liu, Jing; Niu, Yiyuan; Li, Yan; Zhou, Shiwei; Li, Chao; Ma, Baohua; Kou, Qifang; Petersen, Bjoern; Sonstegard, Tad; Huang, Xingxu; Jiang, Yu; Chen, Yulin

2018-05-25

The simplicity of the CRISPR/Cas9 system has enabled its widespread applications in generating animal models, functional genomic screening and in treating genetic and infectious diseases. However, unintended mutations produced by off-target CRISPR/Cas9 nuclease activity may lead to negative consequences. Especially, a very recent study found that gene editing can introduce hundreds of unintended mutations into the genome, and have attracted wide attention. To address the off-target concerns, urgent characterization of the CRISPR/Cas9-mediated off-target mutagenesis is highly anticipated. Here we took advantage of our previously generated gene-edited sheep and performed family trio-based whole genome sequencing which is capable of discriminating variants in the edited progenies that are inherited, naturally generated, or induced by genetic modification. Three family trios were re-sequenced at a high average depth of genomic coverage (~ 25.8×). After developing a pipeline to comprehensively analyze the sequence data for de novo single nucleotide variants, indels and structural variations from the genome; we only found a single unintended event in the form of a 2.4 kb inversion induced by site-specific double-strand breaks between two sgRNA targeting sites at the MSTN locus with a low incidence. We provide the first report on the fidelity of CRISPR-based modification for sheep genomes targeted simultaneously for gene breaks at three coding sequence locations. The trio-based sequencing approach revealed almost negligible off-target modifications, providing timely evidences of the safe application of genome editing in vivo with CRISPR/Cas9.

Reasons of carcinogenesis indicate a big-bang inside: a hypothesis for the aberration of DNA methylation.

PubMed

Roy, A; Roy Chattopadhyay, N

2013-07-01

Cancer involves various sets of altered gene functions which embrace all the three basic mechanisms of regulation of gene expression. However, no common mechanism is inferred till date for this versatile disease and thus no full proof remedy can be offered. Here we show that the basic mechanisms are interlinked and indicate towards one of those mechanisms as being the superior one; the methylation of cytosines in specific DNA sequences, for the initiation and maintenance of carcinogenesis. The analyses of the previous reports and the nucleotide sequences of the DNA methyltransferases strongly support the assumption that the mutation(s) in the DNA-binding site(s) of DNA-methyltransferases acts as a master regulator; though it continues the cycle from mutation to repair to methylation. We anticipate that our hypothesis will start a line of study for the proposal of a treatment regime for cancers by introducing wild type methyltransferases in the diseased cells and/or germ cells, and/or by targeting ligands to the altered binding domain(s) where a mutation in the concerned enzyme(s) is seen. Copyright © 2013. Published by Elsevier Ltd.

Mutations of the phage lambda nutL region that prevent the action of Nun, a site-specific transcription termination factor.

PubMed Central

Baron, J; Weisberg, R A

1992-01-01

Phage HK022 encodes a protein, Nun, that promotes transcription termination within the pL and pR operons of its relative, phage lambda. The lambda sequences required for termination had previously been shown to overlap the nut sites, which are essential for transcription antitermination during normal lambda growth. To further specify the Nun target and to determine its relation to the nut sites, we constructed deletion and base substitution mutations of the lambda nutL region and measured Nun-dependent reduction of the expression of a downstream reporter gene. The shortest construct that retained full Nun responsiveness was a 42-bp segment that included both boxA and boxB, sequences that have been implicated in lambda antitermination. Deletion of boxA reduced Nun termination, and deletion of both sequences eliminated Nun termination. Base substitutions in boxA and the proximal portion of boxB impaired Nun termination, while base substitutions between boxA and boxB, in the distal portion of boxB, and immediately downstream from boxB had no appreciable effect. The termination defect of all of the base substitution mutations was relieved by increasing the level of Nun protein; in contrast, the deletions and a multiple-base substitution did not regain full Nun responsiveness at elevated Nun concentrations. We also asked if these mutant nut regions retained their ability to interact with N, the lambda-encoded antitermination protein. A qualitative assay showed that mutations within boxA or boxB reduced interaction, while mutations outside boxA and boxB did not. These data show that (i) the recognition sites for N and Nun overlap to a very considerable extent but are probably not identical and (ii) a high concentration of Nun promotes its interaction with mutant nut sites, a behavior also reported to be characteristic of N. PMID:1532174

The methylenetetrahydrofolate reductase C677T mutation induces cell-specific changes in genomic DNA methylation and uracil misincorporation: A possible molecular basis for the site-specific cancer risk modification

PubMed Central

Sohn, Kyoung-Jin; Jang, Hyeran; Campan, Mihaela; Weisenberger, Daniel J.; Dickhout, Jeffrey; Wang, Yi-Cheng; Cho, Robert C.; Yates, Zoe; Lucock, Mark; Chiang, En-Pei; Austin, Richard C.; Choi, Sang-Woon; Laird, Peter W.; Kim, Young-In

2009-01-01

The C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene is associated with a decreased risk of colon cancer while it may increase the risk of breast cancer. This polymorphism is associated with changes in intracellular folate cofactors, which may affect DNA methylation and synthesis via altered one-carbon transfer reactions. We investigated the effect of this mutation on DNA methylation and uracil misincorporation and its interaction with exogenous folate in further modulating these biomarkers of one-carbon transfer reactions in an in vitro model of the MTHFR 677T mutation in HCT116 colon and MDA-MB-435 breast adenocarcinoma cells. In HCT116 cells, the MTHFR 677T mutation was associated with significantly increased genomic DNA methylation when folate supply was adequate or high; however, in the setting of folate insufficiency, this mutation was associated with significantly decreased genomic DNA methylation. In contrast, in MDA-MB-435 cells, the MTHFR 677T mutation was associated with significantly decreased genomic DNA methylation when folate supply was adequate or high and with no effect when folate supply was low. The MTHFR 677T mutation was associated with a nonsignificant trend toward decreased and increased uracil misincorporation in HCT116 and MDA-MB-435 cells, respectively. Our data demonstrate for the first time a functional consequence of changes in intracellular folate cofactors resulting from the MTHFR 677T mutation in cells derived from the target organs of interest, thus providing a plausible cellular mechanism that may partly explain the site-specific modification of colon and breast cancer risks associated with the MTHFR C677T mutation. PMID:19123462

Changes in RNA polymerase II progression influence somatic hypermutation of Ig-related genes by AID

PubMed Central

Kodgire, Prashant; Mukkawar, Priyanka; Ratnam, Sarayu; Martin, Terence E.

2013-01-01

Somatic hypermutation (SHM) of Ig genes is initiated by the activation-induced cytidine deaminase (AID), and requires target gene transcription. We previously proposed that AID may associate with the RNA polymerase II (Pol). Here, to determine aspects of the transcription process required for SHM, we knocked-in a transcription terminator into an Ig gene variable region in DT40 chicken B cell line. We found that the human β-globin terminator was an efficient inhibitor of downstream transcription in these cells. The terminator reduced mutations downstream of the poly(A) signal, suggesting that the process of transcription is essential for efficient SHM and that AID has better access to its target when Pol is in the elongating rather than terminating mode. Mutations upstream of the poly(A) site were almost doubled in the active terminator clones compared with an inactivated terminator, and this region showed more single-stranded DNA, indicating that Pol pausing assists SHM. Moreover, the nontranscribed DNA strand was the preferred SHM target upstream of the active terminator. Pol pausing during poly(A) site recognition may facilitate persistence of negative supercoils, exposing the coding single strand and possibly allowing the nascent RNA intermittent reannealing with the template strand, for prolonged access of AID. PMID:23752228

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

Hodge, Curtis D.; Ismail, Ismail H.; Edwards, Ross A.

DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63-linked ubiquitin chains in DSB signaling. In this paper, we defined the activated RNF8-Ubc13~ubiquitin complex by x-ray crystallography and its functional solution conformations by x-ray scattering, as tested by separation-of-function mutations imaged in cells by immunofluorescence. The collective results show that the RING E3 RNF8 targets E2 Ubc13 to DSB sites and plays a critical role in damage signaling by stimulating polyubiquitination through modulating conformations of ubiquitin covalently linked to the Ubc13more » active site. Structure-guided separation-of-function mutations show that the RNF8 E2 stimulating activity is essential for DSB signaling in mammalian cells and is necessary for downstream recruitment of 53BP1 and BRCA1. Chromatin-targeted RNF168 rescues 53BP1 recruitment involved in non-homologous end joining but not BRCA1 recruitment for homologous recombination. Finally, these findings suggest an allosteric approach to targeting the ubiquitin-docking cleft at the E2-E3 interface for possible interventions in cancer and chronic inflammation, and moreover, they establish an independent RNF8 role in BRCA1 recruitment.« less

Correction of Dystrophin Expression in Cells From Duchenne Muscular Dystrophy Patients Through Genomic Excision of Exon 51 by Zinc Finger Nucleases

PubMed Central

Ousterout, David G; Kabadi, Ami M; Thakore, Pratiksha I; Perez-Pinera, Pablo; Brown, Matthew T; Majoros, William H; Reddy, Timothy E; Gersbach, Charles A

2015-01-01

Duchenne muscular dystrophy (DMD) is caused by genetic mutations that result in the absence of dystrophin protein expression. Oligonucleotide-induced exon skipping can restore the dystrophin reading frame and protein production. However, this requires continuous drug administration and may not generate complete skipping of the targeted exon. In this study, we apply genome editing with zinc finger nucleases (ZFNs) to permanently remove essential splicing sequences in exon 51 of the dystrophin gene and thereby exclude exon 51 from the resulting dystrophin transcript. This approach can restore the dystrophin reading frame in ~13% of DMD patient mutations. Transfection of two ZFNs targeted to sites flanking the exon 51 splice acceptor into DMD patient myoblasts led to deletion of this genomic sequence. A clonal population was isolated with this deletion and following differentiation we confirmed loss of exon 51 from the dystrophin mRNA transcript and restoration of dystrophin protein expression. Furthermore, transplantation of corrected cells into immunodeficient mice resulted in human dystrophin expression localized to the sarcolemmal membrane. Finally, we quantified ZFN toxicity in human cells and mutagenesis at predicted off-target sites. This study demonstrates a powerful method to restore the dystrophin reading frame and protein expression by permanently deleting exons. PMID:25492562

Targeting renal cell carcinoma with a HIF-2 antagonist.

PubMed

Chen, Wenfang; Hill, Haley; Christie, Alana; Kim, Min Soo; Holloman, Eboni; Pavia-Jimenez, Andrea; Homayoun, Farrah; Ma, Yuanqing; Patel, Nirav; Yell, Paul; Hao, Guiyang; Yousuf, Qurratulain; Joyce, Allison; Pedrosa, Ivan; Geiger, Heather; Zhang, He; Chang, Jenny; Gardner, Kevin H; Bruick, Richard K; Reeves, Catherine; Hwang, Tae Hyun; Courtney, Kevin; Frenkel, Eugene; Sun, Xiankai; Zojwalla, Naseem; Wong, Tai; Rizzi, James P; Wallace, Eli M; Josey, John A; Xie, Yang; Xie, Xian-Jin; Kapur, Payal; McKay, Renée M; Brugarolas, James

2016-11-03

Clear cell renal cell carcinoma (ccRCC) is characterized by inactivation of the von Hippel-Lindau tumour suppressor gene (VHL). Because no other gene is mutated as frequently in ccRCC and VHL mutations are truncal, VHL inactivation is regarded as the governing event. VHL loss activates the HIF-2 transcription factor, and constitutive HIF-2 activity restores tumorigenesis in VHL-reconstituted ccRCC cells. HIF-2 has been implicated in angiogenesis and multiple other processes, but angiogenesis is the main target of drugs such as the tyrosine kinase inhibitor sunitinib. HIF-2 has been regarded as undruggable. Here we use a tumourgraft/patient-derived xenograft platform to evaluate PT2399, a selective HIF-2 antagonist that was identified using a structure-based design approach. PT2399 dissociated HIF-2 (an obligatory heterodimer of HIF-2α-HIF-1β) in human ccRCC cells and suppressed tumorigenesis in 56% (10 out of 18) of such lines. PT2399 had greater activity than sunitinib, was active in sunitinib-progressing tumours, and was better tolerated. Unexpectedly, some VHL-mutant ccRCCs were resistant to PT2399. Resistance occurred despite HIF-2 dissociation in tumours and evidence of Hif-2 inhibition in the mouse, as determined by suppression of circulating erythropoietin, a HIF-2 target and possible pharmacodynamic marker. We identified a HIF-2-dependent gene signature in sensitive tumours. Gene expression was largely unaffected by PT2399 in resistant tumours, illustrating the specificity of the drug. Sensitive tumours exhibited a distinguishing gene expression signature and generally higher levels of HIF-2α. Prolonged PT2399 treatment led to resistance. We identified binding site and second site suppressor mutations in HIF-2α and HIF-1β, respectively. Both mutations preserved HIF-2 dimers despite treatment with PT2399. Finally, an extensively pretreated patient whose tumour had given rise to a sensitive tumourgraft showed disease control for more than 11 months when treated with a close analogue of PT2399, PT2385. We validate HIF-2 as a target in ccRCC, show that some ccRCCs are HIF-2 independent, and set the stage for biomarker-driven clinical trials.

Cross-resistance patterns to acetolactate synthase (ALS)-inhibiting herbicides of flixweed (Descurainia sophia L.) conferred by different combinations of ALS isozymes with a Pro-197-Thr mutation or a novel Trp-574-Leu mutation.

PubMed

Deng, Wei; Yang, Qian; Zhang, Yongzhi; Jiao, Hongtao; Mei, Yu; Li, Xuefeng; Zheng, Mingqi

2017-03-01

Acetolactate synthase (ALS) is the common target of ALS-inhibiting herbicides, and target-site ALS mutations are the main mechanism of resistance to ALS-inhibiting herbicides. In this study, ALS1 and ALS2 genes with full lengths of 2004bp and 1998bp respectively were cloned in individual plants of susceptible (S) or resistant (R) flixweed (Descurainia sophia L.) populations. Two ALS mutations of Pro-197-Thr and/or Trp-574-Leu were identified in plants of three R biotypes (HB24, HB30 and HB42). In order to investigate the function of ALS isozymes in ALS-inhibiting herbicide resistance, pHB24 (a Pro-197-Thr mutation in ALS1 and a wild type ALS2), pHB42 (a Trp-574-Leu mutation in ALS1 and a wild type ALS2) and pHB30 (a Trp-574-Leu mutation in ALS1 and a Pro-197-Thr mutation in ALS2) subpopulations individually homozygous for different ALS mutations were generated. Individuals of pHB30 had mutations in each isozyme of ALS and had higher resistance than pHB24 and pHB42 populations containing mutations in only one ALS isozyme. Moreover, the pHB24 had resistance to SU, TP and SCT herbicides, whereas pHB24 and pHB42 had resistance to these classes of herbicides as well as IMI and PTB herbicides. The sensitivity of isolated ALS enzyme to inhibition by herbicides in these populations correlated with whole plant resistance levels. Therefore, reduced ALS sensitivity resulting from the mutations in ALS was responsible for resistance to ALS-inhibiting herbicides in flixweed. Copyright © 2016 Elsevier Inc. All rights reserved.

Detailed analysis of targeted gene mutations caused by the Platinum-Fungal TALENs in Aspergillus oryzae RIB40 strain and a ligD disruptant.

PubMed

Mizutani, Osamu; Arazoe, Takayuki; Toshida, Kenji; Hayashi, Risa; Ohsato, Shuichi; Sakuma, Tetsushi; Yamamoto, Takashi; Kuwata, Shigeru; Yamada, Osamu

2017-03-01

Transcription activator-like effector nucleases (TALENs), which can generate DNA double-strand breaks at specific sites in the desired genome locus, have been used in many organisms as a tool for genome editing. In Aspergilli, including Aspergillus oryzae, however, the use of TALENs has not been validated. In this study, we performed genome editing of A. oryzae wild-type strain via error of nonhomologous end-joining (NHEJ) repair by transient expression of high-efficiency Platinum-Fungal TALENs (PtFg TALENs). Targeted mutations were observed as various mutation patterns. In particular, approximately half of the PtFg TALEN-mediated deletion mutants had deletions larger than 1 kb in the TALEN-targeting region. We also conducted PtFg TALEN-based genome editing in A. oryzae ligD disruptant (ΔligD) lacking the ligD gene involved in the final step of the NHEJ repair and found that mutations were still obtained as well as wild-type. In this case, the ratio of the large deletions reduced compared to PtFg TALEN-based genome editing in the wild-type. In conclusion, we demonstrate that PtFg TALENs are sufficiently functional to cause genome editing via error of NHEJ in A. oryzae. In addition, we reveal that genome editing using TALENs in A. oryzae tends to cause large deletions at the target region, which were partly suppressed by deletion of ligD. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Cas9-Guide RNA Directed Genome Editing in Soybean[OPEN

PubMed Central

Li, Zhongsen; Liu, Zhan-Bin; Xing, Aiqiu; Moon, Bryan P.; Koellhoffer, Jessica P.; Huang, Lingxia; Ward, R. Timothy; Clifton, Elizabeth; Falco, S. Carl; Cigan, A. Mark

2015-01-01

Recently discovered bacteria and archaea adaptive immune system consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) endonuclease has been explored in targeted genome editing in different species. Streptococcus pyogenes Cas9-guide RNA (gRNA) was successfully applied to generate targeted mutagenesis, gene integration, and gene editing in soybean (Glycine max). Two genomic sites, DD20 and DD43 on chromosome 4, were mutagenized with frequencies of 59% and 76%, respectively. Sequencing randomly selected transgenic events confirmed that the genome modifications were specific to the Cas9-gRNA cleavage sites and consisted of small deletions or insertions. Targeted gene integrations through homology-directed recombination were detected by border-specific polymerase chain reaction analysis for both sites at callus stage, and one DD43 homology-directed recombination event was transmitted to T1 generation. T1 progenies of the integration event segregated according to Mendelian laws and clean homozygous T1 plants with the donor gene precisely inserted at the DD43 target site were obtained. The Cas9-gRNA system was also successfully applied to make a directed P178S mutation of acetolactate synthase1 gene through in planta gene editing. PMID:26294043

Dysferlin rescue by spliceosome-mediated pre-mRNA trans-splicing targeting introns harbouring weakly defined 3' splice sites.

PubMed

Philippi, Susanne; Lorain, Stéphanie; Beley, Cyriaque; Peccate, Cécile; Précigout, Guillaume; Spuler, Simone; Garcia, Luis

2015-07-15

The modification of the pre-mRNA cis-splicing process employing a pre-mRNA trans-splicing molecule (PTM) is an attractive strategy for the in situ correction of genes whose careful transcription regulation and full-length expression is determinative for protein function, as it is the case for the dysferlin (DYSF, Dysf) gene. Loss-of-function mutations of DYSF result in different types of muscular dystrophy mainly manifesting as limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi muscular dystrophy 1 (MMD1). We established a 3' replacement strategy for mutated DYSF pre-mRNAs induced by spliceosome-mediated pre-mRNA trans-splicing (SmaRT) by the use of a PTM. In contrast to previously established SmaRT strategies, we particularly focused on the identification of a suitable pre-mRNA target intron other than the optimization of the PTM design. By targeting DYSF pre-mRNA introns harbouring differentially defined 3' splice sites (3' SS), we found that target introns encoding weakly defined 3' SSs were trans-spliced successfully in vitro in human LGMD2B myoblasts as well as in vivo in skeletal muscle of wild-type and Dysf(-/-) mice. For the first time, we demonstrate rescue of Dysf protein by SmaRT in vivo. Moreover, we identified concordant qualities among the successfully targeted Dysf introns and targeted endogenous introns in previously reported SmaRT approaches that might facilitate a selective choice of target introns in future SmaRT strategies. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Identification of a Second Substrate-binding Site in Solute-Sodium Symporters*

PubMed Central

Li, Zheng; Lee, Ashley S. E.; Bracher, Susanne; Jung, Heinrich; Paz, Aviv; Kumar, Jay P.; Abramson, Jeff; Quick, Matthias; Shi, Lei

2015-01-01

The structure of the sodium/galactose transporter (vSGLT), a solute-sodium symporter (SSS) from Vibrio parahaemolyticus, shares a common structural fold with LeuT of the neurotransmitter-sodium symporter family. Structural alignments between LeuT and vSGLT reveal that the crystallographically identified galactose-binding site in vSGLT is located in a more extracellular location relative to the central substrate-binding site (S1) in LeuT. Our computational analyses suggest the existence of an additional galactose-binding site in vSGLT that aligns to the S1 site of LeuT. Radiolabeled galactose saturation binding experiments indicate that, like LeuT, vSGLT can simultaneously bind two substrate molecules under equilibrium conditions. Mutating key residues in the individual substrate-binding sites reduced the molar substrate-to-protein binding stoichiometry to ∼1. In addition, the related and more experimentally tractable SSS member PutP (the Na+/proline transporter) also exhibits a binding stoichiometry of 2. Targeting residues in the proposed sites with mutations results in the reduction of the binding stoichiometry and is accompanied by severely impaired translocation of proline. Our data suggest that substrate transport by SSS members requires both substrate-binding sites, thereby implying that SSSs and neurotransmitter-sodium symporters share common mechanistic elements in substrate transport. PMID:25398883

HIV protease drug resistance and its impact on inhibitor design.

PubMed

Ala, P J; Rodgers, J D; Chang, C H

1999-07-01

The primary cause of resistance to the currently available HIV protease inhibitors is the accumulation of multiple mutations in the viral protease. So far more than 20 substitutions have been observed in the active site, dimer interface, surface loops and flaps of the homodimer. While many mutations reduce the protease's affinity for inhibitors, others appear to enhance its catalytic efficiency. This high degree of genetic flexibility has made the protease an elusive drug target. The design of the next generation of HIV protease inhibitors will be discussed in light of the current structural information.

The Chromodomain of Tf1 Integrase Promotes Binding to cDNA and Mediates Target Site Selection▿ †

PubMed Central

Chatterjee, Atreyi Ghatak; Leem, Young Eun; Kelly, Felice D.; Levin, Henry L.

2009-01-01

The long terminal repeat (LTR) retrotransposon Tf1 of Schizosaccharomyces pombe integrates specifically into the promoters of pol II-transcribed genes. Its integrase (IN) contains a C-terminal chromodomain related to the chromodomains that bind to the N-terminal tail of histone H3. Although we have been unable to detect an interaction between histone tails and the chromodomain of Tf1 IN, it is possible that the chromodomain plays a role in directing IN to its target sites. To test this idea, we generated transposons with single amino acid substitutions in highly conserved residues of the chromodomain and created a chromodomain-deleted mutant. The mutations, V1290A, Y1292A, W1305A, and CHDΔ, substantially reduced transposition activity in vivo. Blotting assays showed that there was little or no reduction in the levels of IN or cDNA. By measuring the homologous recombination between cDNA and the plasmid copy of Tf1, we found that two of the mutations did not reduce the import of cDNA into the nucleus, while another caused a 33% reduction. Chromatin immunoprecipitation assays revealed that CHDΔ caused an approximately threefold reduction in the binding of IN to the downstream LTR of the cDNA. These data indicate that the chromodomain contributed directly to integration. We therefore tested whether the chromodomain contributed to selecting insertion sites. Results of a target plasmid assay showed that the deletion of the chromodomain resulted in a drastic reduction in the preference for pol II promoters. Collectively, these data indicate that the chromodomain promotes binding of cDNA and plays a key role in efficient targeting. PMID:19109383

The chromodomain of Tf1 integrase promotes binding to cDNA and mediates target site selection.

PubMed

Chatterjee, Atreyi Ghatak; Leem, Young Eun; Kelly, Felice D; Levin, Henry L

2009-03-01

The long terminal repeat (LTR) retrotransposon Tf1 of Schizosaccharomyces pombe integrates specifically into the promoters of pol II-transcribed genes. Its integrase (IN) contains a C-terminal chromodomain related to the chromodomains that bind to the N-terminal tail of histone H3. Although we have been unable to detect an interaction between histone tails and the chromodomain of Tf1 IN, it is possible that the chromodomain plays a role in directing IN to its target sites. To test this idea, we generated transposons with single amino acid substitutions in highly conserved residues of the chromodomain and created a chromodomain-deleted mutant. The mutations, V1290A, Y1292A, W1305A, and CHDDelta, substantially reduced transposition activity in vivo. Blotting assays showed that there was little or no reduction in the levels of IN or cDNA. By measuring the homologous recombination between cDNA and the plasmid copy of Tf1, we found that two of the mutations did not reduce the import of cDNA into the nucleus, while another caused a 33% reduction. Chromatin immunoprecipitation assays revealed that CHDDelta caused an approximately threefold reduction in the binding of IN to the downstream LTR of the cDNA. These data indicate that the chromodomain contributed directly to integration. We therefore tested whether the chromodomain contributed to selecting insertion sites. Results of a target plasmid assay showed that the deletion of the chromodomain resulted in a drastic reduction in the preference for pol II promoters. Collectively, these data indicate that the chromodomain promotes binding of cDNA and plays a key role in efficient targeting.

Amino-Acid Network Clique Analysis of Protein Mutation Non-Additive Effects: A Case Study of Lysozme.

PubMed

Ming, Dengming; Chen, Rui; Huang, He

2018-05-10

Optimizing amino-acid mutations in enzyme design has been a very challenging task in modern bio-industrial applications. It is well known that many successful designs often hinge on extensive correlations among mutations at different sites within the enzyme, however, the underpinning mechanism for these correlations is far from clear. Here, we present a topology-based model to quantitively characterize non-additive effects between mutations. The method is based on the molecular dynamic simulations and the amino-acid network clique analysis. It examines if the two mutation sites of a double-site mutation fall into to a 3-clique structure, and associates such topological property of mutational site spatial distribution with mutation additivity features. We analyzed 13 dual mutations of T4 phage lysozyme and found that the clique-based model successfully distinguishes highly correlated or non-additive double-site mutations from those additive ones whose component mutations have less correlation. We also applied the model to protein Eglin c whose structural topology is significantly different from that of T4 phage lysozyme, and found that the model can, to some extension, still identify non-additive mutations from additive ones. Our calculations showed that mutation non-additive effects may heavily depend on a structural topology relationship between mutation sites, which can be quantitatively determined using amino-acid network k -cliques. We also showed that double-site mutation correlations can be significantly altered by exerting a third mutation, indicating that more detailed physicochemical interactions should be considered along with the network clique-based model for better understanding of this elusive mutation-correlation principle.

Generation of TALE nickase-mediated gene-targeted cows expressing human serum albumin in mammary glands.

PubMed

Luo, Yan; Wang, Yongsheng; Liu, Jun; Cui, Chenchen; Wu, Yongyan; Lan, Hui; Chen, Qi; Liu, Xu; Quan, Fusheng; Guo, Zekun; Zhang, Yong

2016-02-08

Targeting exogenous genes at milk protein loci via gene-targeting technology is an ideal strategy for producing large quantities of pharmaceutical proteins. Transcription-activator-like effector (TALE) nucleases (TALENs) are an efficient genome-editing tool. However, the off-target effects may lead to unintended gene mutations. In this study, we constructed TALENs and TALE nickases directed against exon 2 of the bovine β-lactoglobulin (BLG) locus. The nickases can induce a site-specific DNA single-strand break, without inducing double-strand break and nonhomologous end joining mediated gene mutation, and lower cell apoptosis rate than TALENs. After co-transfecting the bovine fetal fibroblasts with human serum albumin (HSA) gene-targeting vector and TALE nickase expression vectors, approximately 4.8% (40/835) of the cell clones contained HSA at BLG locus. Unexpectedly, one homozygous gene-targeted cell clone (1/835, 0.1%) was obtained by targeting both alleles of BLG in a single round of transfection. The recombinant protein mimicking the endogenous BLG was highly expressed and correctly folded in the mammary glands of the targeted cows, and the expression level of HSA was significantly increased in the homozygous targeted cows. Results suggested that the combination of TALE nickase-mediated gene targeting and somatic cell nuclear transfer is a feasible and safe approach in producing gene-targeted livestock.

Functional analysis of the EspR binding sites upstream of espR in Mycobacterium tuberculosis.

PubMed

Cao, Guangxiang; Howard, Susan T; Zhang, Peipei; Hou, Guihua; Pang, Xiuhua

2013-11-01

The ESX-1 secretion system exports substrate proteins into host cells and is crucial for the pathogenesis of Mycobacterium tuberculosis. EspR is one of the characterized transcriptional regulators that modulates the ESX-1 system by binding the conserved EspR binding sites in the promoter of espA, the encoding gene of EspA, which is also a substrate protein of the ESX-1 system and is required for the ESX-1 activity. EspR is autoregulatory and conserved EspR binding sites are present upstream of espR. In this study, we showed that these EspR sites had varying affinities for EspR, with site B being the strongest one. Point mutations of the DNA sequence at site B abolished binding of EspR to oligonucleotides containing site B alone or with other sites, further suggesting that site B is a major binding site for EspR. Complementation studies showed that constructs containing espR, and the upstream intergenic region fully restored espR expression in a ΔespR mutant strain. Although recombinant strains with mutations at more than one EspR site showed minimal differences in espR expression, reduced expression of other EspR target genes was observed, suggesting that slight changes in EspR levels can have downstream regulatory effects. These findings contribute to our understanding of the regulation of the ESX-1 system.

Calcium Sensing Receptor Mutations Implicated in Pancreatitis and Idiopathic Epilepsy Syndrome Disrupt an Arginine-rich Retention Motif

PubMed Central

Stepanchick, Ann; McKenna, Jennifer; McGovern, Olivia; Huang, Ying; Breitwieser, Gerda E.

2010-01-01

Calcium sensing receptor (CaSR) mutations implicated in familial hypocalciuric hypercalcemia, pancreatitis and idiopathic epilepsy syndrome map to an extended arginine-rich region in the proximal carboxyl terminus. Arginine-rich motifs mediate endoplasmic reticulum retention and/or retrieval of multisubunit proteins so we asked whether these mutations, R886P, R896H or R898Q, altered CaSR targeting to the plasma membrane. Targeting was enhanced by all three mutations, and Ca2+-stimulated ERK1/2 phosphorylation was increased for R896H and R898Q. To define the role of the extended arginine-rich region in CaSR trafficking, we independently determined the contributions of R890/R891 and/or R896/K897/R898 motifs by mutation to alanine. Disruption of the motif(s) significantly increased surface expression and function relative to wt CaSR. The arginine-rich region is flanked by phosphorylation sites at S892 (protein kinase C) and S899 (protein kinase A). The phosphorylation state of S899 regulated recognition of the arginine-rich region; S899D showed increased surface localization. CaSR assembles in the endoplasmic reticulum as a covalent disulfide-linked dimer and we determined whether retention requires the presence of arginine-rich regions in both subunits. A single arginine-rich region within the dimer was sufficient to confer intracellular retention comparable to wt CaSR. We have identified an extended arginine-rich region in the proximal carboxyl terminus of CaSR (residues R890 - R898) which fosters intracellular retention of CaSR and is regulated by phosphorylation. Mutation(s) identified in chronic pancreatitis and idiopathic epilepsy syndrome therefore increase plasma membrane targeting of CaSR, likely contributing to the altered Ca2+ signaling characteristic of these diseases. PMID:20798521

Activation of tyrosine kinases by mutation of the gatekeeper threonine

PubMed Central

Azam, Mohammad; Seeliger, Markus A; Gray, Nathanael S; Kuriyan, John; Daley, George Q

2008-01-01

Protein kinases targeted by small-molecule inhibitors develop resistance through mutation of the ‘gatekeeper’ threonine residue of the active site. Here we show that the gatekeeper mutation in the cellular forms of c-ABL, c-SRC, platelet-derived growth factor receptor-α and -β, and epidermal growth factor receptor activates the kinase and promotes malignant transformation of BaF3 cells. Structural analysis reveals that a network of hydrophobic interactions—the hydrophobic spine—characteristic of the active kinase conformation is stabilized by the gatekeeper substitution. Substitution of glycine for the residues constituting the spine disrupts the hydrophobic connectivity and inactivates the kinase. Furthermore, a small-molecule inhibitor that maximizes complementarity with the dismantled spine (compound 14) inhibits the gatekeeper mutation of BCR-ABL-T315I. These results demonstrate that mutation of the gatekeeper threonine is a common mechanism of activation for tyrosine kinases and provide structural insights to guide the development of next-generation inhibitors. PMID:18794843

Study of hepatitis B virus gene mutations with enzymatic colorimetry-based DNA microarray.

PubMed

Mao, Hailei; Wang, Huimin; Zhang, Donglei; Mao, Hongju; Zhao, Jianlong; Shi, Jian; Cui, Zhichu

2006-01-01

To establish a modified microarray method for detecting HBV gene mutations in the clinic. Site-specific oligonucleotide probes were immobilized to microarray slides and hybridized to biotin-labeled HBV gene fragments amplified from two-step PCR. Hybridized targets were transferred to nitrocellulose membranes, followed by intensity measurement using BCIP/NBT colorimetry. HBV genes from 99 Hepatitis B patients and 40 healthy blood donors were analyzed. Mutation frequencies of HBV pre-core/core and basic core promoter (BCP) regions were found to be significantly higher in the patient group (42%, 40% versus 2.5%, 5%, P < 0.01). Compared with a traditional fluorescence method, the colorimetry method exhibited the same level of sensitivity and reproducibility. An enzymatic colorimetry-based DNA microarray assay was successfully established to monitor HBV mutations. Pre-core/core and BCP mutations of HBV genes could be major causes of HBV infection in HBeAg-negative patients and could also be relevant to chronicity and aggravation of hepatitis B.

L1014F-kdr Mutation in Indian Anopheles subpictus (Diptera: Culicidae) Arising From Two Alternative Transversions in the Voltage-Gated Sodium Channel and a Single PIRA-PCR for Their Detection

PubMed Central

Singh, O. P.; Dykes, C. L.; Sharma, G.; Das, M. K.

2015-01-01

Leucine-to-phenylalanine substitution at residue L1014 in the voltage-gated sodium channel, target site of action for dichlorodiphenyltrichloroethane (DDT) and pyrethroids, is the most common knockdown resistance (kdr) mutation reported in several insects conferring resistance against DDT and pyrethroids. Here, we report presence of two coexisting alternative transversions, A>T and A>C, on the third codon position of L1014 residue in malaria vector Anopheles subpictus Grassi (species A) from Jamshedpur (India), both leading to the same amino acid substitution of Leu-to-Phe with allelic frequencies of 19 and 67%, respectively. A single primer-introduced restriction analysis–polymerase chain reaction (PIRA-PCR) was devised for the identification of L1014F-kdr mutation in An. subpictus resulting from either type of point mutation. Genotyping of samples with PIRA-PCR revealed high frequency (82%) of L1014F-kdr mutation in the study area. PMID:26336276

Production of Mutated Porcine Embryos Using Zinc Finger Nucleases and a Reporter-based Cell Enrichment System.

PubMed

Koo, Ok Jae; Park, Sol Ji; Lee, Choongil; Kang, Jung Taek; Kim, Sujin; Moon, Joon Ho; Choi, Ji Yei; Kim, Hyojin; Jang, Goo; Kim, Jin-Soo; Kim, Seokjoong; Lee, Byeong-Chun

2014-03-01

To facilitate the construction of genetically-modified pigs, we produced cloned embryos derived from porcine fibroblasts transfected with a pair of engineered zinc finger nuclease (ZFN) plasmids to create targeted mutations and enriched using a reporter plasmid system. The reporter expresses RFP and eGFP simultaneously when ZFN-mediated site-specific mutations occur. Thus, double positive cells (RFP(+)/eGFP(+)) were selected and used for somatic cell nuclear transfer. Two types of reporter based enrichment systems were used in this study; the cloned embryos derived from cells enriched using a magnetic sorting-based system showed better developmental competence than did those derived from cells enriched by flow cytometry. Mutated sequences, such as insertions, deletions, or substitutions, together with the wild-type sequence, were found in the cloned porcine blastocysts. Therefore, genetic mutations can be achieved in cloned porcine embryos reconstructed with ZFN-treated cells that were enriched by a reporter-based system.

Genetic Correction and Hepatic Differentiation of Hemophilia B-specific Human Induced Pluripotent Stem Cells.

PubMed

He, Qiong; Wang, Hui-Hui; Cheng, Tao; Yuan, Wei-Ping; Ma, Yu-Po; Jiang, Yong-Ping; Ren, Zhi-Hua

2017-09-27

Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells (iPSCs) derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by sequencing the encoding area of human coagulation factor IX (F IX) gene. Genomic DNA was extracted from the iPSCs, and the primers were designed to amplify the eight exons of F IX. Next, the point mutation in those iPSCs was genetically corrected using CRISPR/Cas9 technology in the presence of a 129-nucleotide homologous repair template that contained two synonymous mutations. Then, top 8 potential off-target sites were subsequently analyzed using Sanger sequencing. Finally, the corrected clones were differentiated into hepatocyte-like cells, and the secretion of F IX was validated by immunocytochemistry and ELISA assay. Results The cell line bore a missense mutation in the 6 th coding exon (c.676 C>T) of F IX gene. Correction of the point mutation was achieved via CRISPR/Cas9 technology in situ with a high efficacy at about 22% (10/45) and no off-target effects detected in the corrected iPSC clones. F IX secretion, which was further visualized by immunocytochemistry and quantified by ELISA in vitro, reached about 6 ng/ml on day 21 of differentiation procedure. Conclusions Mutations in human disease-specific iPSCs could be precisely corrected by CRISPR/Cas9 technology, and corrected cells still maintained hepatic differentiation capability. Our findings might throw a light on iPSC-based personalized therapies in the clinical application, especially for hemophilia B.

Mutation Analysis of SLC26A4 for Pendred Syndrome and Nonsyndromic Hearing Loss by High-Resolution Melting

PubMed Central

Chen, Neng; Tranebjærg, Lisbeth; Rendtorff, Nanna Dahl; Schrijver, Iris

2011-01-01

Pendred syndrome and DFNB4 (autosomal recessive nonsyndromic congenital deafness, locus 4) are associated with autosomal recessive congenital sensorineural hearing loss and mutations in the SLC26A4 gene. Extensive allelic heterogeneity, however, necessitates analysis of all exons and splice sites to identify mutations for individual patients. Although Sanger sequencing is the gold standard for mutation detection, screening methods supplemented with targeted sequencing can provide a cost-effective alternative. One such method, denaturing high-performance liquid chromatography, was developed for clinical mutation detection in SLC26A4. However, this method inherently cannot distinguish homozygous changes from wild-type sequences. High-resolution melting (HRM), on the other hand, can detect heterozygous and homozygous changes cost-effectively, without any post-PCR modifications. We developed a closed-tube HRM mutation detection method specific for SLC26A4 that can be used in the clinical diagnostic setting. Twenty-eight primer pairs were designed to cover all 21 SLC26A4 exons and splice junction sequences. Using the resulting amplicons, initial HRM analysis detected all 45 variants previously identified by sequencing. Subsequently, a 384-well plate format was designed for up to three patient samples per run. Blinded HRM testing on these plates of patient samples collected over 1 year in a clinical diagnostic laboratory accurately detected all variants identified by sequencing. In conclusion, HRM with targeted sequencing is a reliable, simple, and cost-effective method for SLC26A4 mutation screening and detection. PMID:21704276

Comparative sequencing analysis reveals high genomic concordance between matched primary and metastatic colorectal cancer lesions.

PubMed

Brannon, A Rose; Vakiani, Efsevia; Sylvester, Brooke E; Scott, Sasinya N; McDermott, Gregory; Shah, Ronak H; Kania, Krishan; Viale, Agnes; Oschwald, Dayna M; Vacic, Vladimir; Emde, Anne-Katrin; Cercek, Andrea; Yaeger, Rona; Kemeny, Nancy E; Saltz, Leonard B; Shia, Jinru; D'Angelica, Michael I; Weiser, Martin R; Solit, David B; Berger, Michael F

2014-08-28

Colorectal cancer is the second leading cause of cancer death in the United States, with over 50,000 deaths estimated in 2014. Molecular profiling for somatic mutations that predict absence of response to anti-EGFR therapy has become standard practice in the treatment of metastatic colorectal cancer; however, the quantity and type of tissue available for testing is frequently limited. Further, the degree to which the primary tumor is a faithful representation of metastatic disease has been questioned. As next-generation sequencing technology becomes more widely available for clinical use and additional molecularly targeted agents are considered as treatment options in colorectal cancer, it is important to characterize the extent of tumor heterogeneity between primary and metastatic tumors. We performed deep coverage, targeted next-generation sequencing of 230 key cancer-associated genes for 69 matched primary and metastatic tumors and normal tissue. Mutation profiles were 100% concordant for KRAS, NRAS, and BRAF, and were highly concordant for recurrent alterations in colorectal cancer. Additionally, whole genome sequencing of four patient trios did not reveal any additional site-specific targetable alterations. Colorectal cancer primary tumors and metastases exhibit high genomic concordance. As current clinical practices in colorectal cancer revolve around KRAS, NRAS, and BRAF mutation status, diagnostic sequencing of either primary or metastatic tissue as available is acceptable for most patients. Additionally, consistency between targeted sequencing and whole genome sequencing results suggests that targeted sequencing may be a suitable strategy for clinical diagnostic applications.

Mapping structural landmarks, ligand binding sites and missense mutations to the collagen IV heterotrimers predicts major functional domains, novel interactions and variation in phenotypes in inherited diseases affecting basement membranes

PubMed Central

Des Parkin, J.; San Antonio, James D.; Pedchenko, Vadim; Hudson, Billy; Jensen, Shane T.; Savige, Judy

2016-01-01

Collagen IV is the major protein found in basement membranes. It comprises 3 heterotrimers (α1α1α2, α3α4α5, and α5α5α6) that form distinct networks, and are responsible for membrane strength and integrity. We constructed linear maps of the collagen IV heterotrimers (‘interactomes’) that indicated major structural landmarks, known and predicted ligand-binding sites, and missense mutations, in order to identify functional and disease-associated domains, potential interactions between ligands, and genotype-phenotype relationships. The maps documented more than 30 known ligand-binding sites as well as motifs for integrins, heparin, von Willebrand factor (VWF), decorin and bone morphogenetic protein (BMP). They predicted functional domains for angiogenesis and haemostasis, and disease domains for autoimmunity, tumor growth and inhibition, infection and glycation. Cooperative ligand interactions were indicated by binding site proximity, for example, between integrins, matrix metalloproteinases and heparin. The maps indicated that mutations affecting major ligand-binding sites, for example for Von Hippel Lindau (VHL) protein in the α1 chain or integrins in the α5 chain, resulted in distinctive phenotypes (Hereditary Angiopathy, Nephropathy, Aneurysms and muscle Cramps (HANAC) syndrome, and early onset Alport syndrome respectively). These maps further our understanding of basement membrane biology and disease, and suggest novel membrane interactions, functions, and therapeutic targets. PMID:21280145

FR171456 is a specific inhibitor of mammalian NSDHL and yeast Erg26p

PubMed Central

Helliwell, Stephen B.; Karkare, Shantanu; Bergdoll, Marc; Rahier, Alain; Leighton-Davis, Juliet R.; Fioretto, Celine; Aust, Thomas; Filipuzzi, Ireos; Frederiksen, Mathias; Gounarides, John; Hoepfner, Dominic; Hofmann, Andreas; Imbert, Pierre-Eloi; Jeker, Rolf; Knochenmuss, Richard; Krastel, Philipp; Margerit, Anais; Memmert, Klaus; Miault, Charlotte V.; Rao Movva, N.; Muller, Alban; Naegeli, Hans-Ulrich; Oberer, Lukas; Prindle, Vivian; Riedl, Ralph; Schuierer, Sven; Sexton, Jessica A.; Tao, Jianshi; Wagner, Trixie; Yin, Hong; Zhang, Juan; Roggo, Silvio; Reinker, Stefan; Parker, Christian N.

2015-01-01

FR171456 is a natural product with cholesterol-lowering properties in animal models, but its molecular target is unknown, which hinders further drug development. Here we show that FR171456 specifically targets the sterol-4-alpha-carboxylate-3-dehydrogenase (Saccharomyces cerevisiae—Erg26p, Homo sapiens—NSDHL (NAD(P) dependent steroid dehydrogenase-like)), an essential enzyme in the ergosterol/cholesterol biosynthesis pathway. FR171456 significantly alters the levels of cholesterol pathway intermediates in human and yeast cells. Genome-wide yeast haploinsufficiency profiling experiments highlight the erg26/ERG26 strain, and multiple mutations in ERG26 confer resistance to FR171456 in growth and enzyme assays. Some of these ERG26 mutations likely alter Erg26 binding to FR171456, based on a model of Erg26. Finally, we show that FR171456 inhibits an artificial Hepatitis C viral replicon, and has broad antifungal activity, suggesting potential additional utility as an anti-infective. The discovery of the target and binding site of FR171456 within the target will aid further development of this compound. PMID:26456460

Genomic characterization of pediatric T-cell acute lymphoblastic leukemia reveals novel recurrent driver mutations

PubMed Central

Spinella, Jean-François; Cassart, Pauline; Richer, Chantal; Saillour, Virginie; Ouimet, Manon; Langlois, Sylvie; St-Onge, Pascal; Sontag, Thomas; Healy, Jasmine; Minden, Mark D.; Sinnett, Daniel

2016-01-01

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with variable prognosis. It represents 15% of diagnosed pediatric ALL cases and has a threefold higher incidence among males. Many recurrent alterations have been identified and help define molecular subgroups of T-ALL, however the full range of events involved in driving transformation remain to be defined. Using an integrative approach combining genomic and transcriptomic data, we molecularly characterized 30 pediatric T-ALLs and identified common recurrent T-ALL targets such as FBXW7, JAK1, JAK3, PHF6, KDM6A and NOTCH1 as well as novel candidate T-ALL driver mutations including the p.R35L missense mutation in splicesome factor U2AF1 found in 3 patients and loss of function mutations in the X-linked tumor suppressor genes MED12 (frameshit mutation p.V167fs, splice site mutation g.chrX:70339329T>C, missense mutation p.R1989H) and USP9X (nonsense mutation p.Q117*). In vitro functional studies further supported the putative role of these novel T-ALL genes in driving transformation. U2AF1 p.R35L was shown to induce aberrant splicing of downstream target genes, and shRNA knockdown of MED12 and USP9X was shown to confer resistance to apoptosis following T-ALL relevant chemotherapy drug treatment in Jurkat leukemia cells. Interestingly, nearly 60% of novel candidate driver events were identified among immature T-ALL cases, highlighting the underlying genomic complexity of pediatric T-ALL, and the need for larger integrative studies to decipher the mechanisms that contribute to its various subtypes and provide opportunities to refine patient stratification and treatment. PMID:27602765

Improvement of the optimum pH of Aspergillus niger xylanase towards an alkaline pH by site-directed mutagenesis.

PubMed

Li, Fei; Xie, Jingcong; Zhang, Xuesong; Zhao, Linguo

2015-01-01

In an attempt to shift the optimal pH of the xylanase B (XynB) from Aspergillus niger towards alkalinity, target mutation sites were selected by alignment between Aspergillus niger xylanase B and other xylanases that have alkalophilic pH optima that highlight charged residues in the eight-residues-longer loop in the alkalophilic xylanase. Multiple engineered XynB mutants were created by site-directed mutagenesis with substitutions Q164K and Q164K+D117N. The variant XynB-117 had the highest optimum pH (at 5.5), which corresponded to a basic 0.5 pH unit shift when compared with the wild-type enzyme. However, the optimal pH of the XynB- 164 mutation was not changed, similar to the wild type. These results suggest that the residues at positions 164 and 117 in the eight-residues-longer loop and the cleft's edge are important in determining the pH optima of XynB from Aspergillus niger.

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

PubMed Central

2012-01-01

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

An interaction map of small-molecule kinase inhibitors with anaplastic lymphoma kinase (ALK) mutants in ALK-positive non-small cell lung cancer.

PubMed

Ai, Xinghao; Shen, Shengping; Shen, Lan; Lu, Shun

2015-05-01

Human anaplastic lymphoma kinase (ALK) has become a well-established target for the treatment of ALK-positive non-small cell lung cancer (NSCLC). Here, we have profiled seven small-molecule inhibitors, including 2 that are approved drugs, against a panel of clinically relevant mutations in ALK tyrosine kinase (TK) domain, aiming at a comprehensive understanding of molecular mechanism and biological implication underlying inhibitor response to ALK TK mutation. We find that (i) the gatekeeper mutation L1196M causes crizotinib resistance by simultaneously increasing and decreasing the binding affinities of, respectively, ATP and inhibitor to ALK, whereas the secondary mutation C1156Y, which is located far away from the ATP-binding site of ALK TK domain, causes the resistance by inducing marked allosteric effect on the site, (ii) the 2nd and 3rd generation kinase inhibitors exhibit relatively high sensitivity towards ALK mutants as compared to 1st generation inhibitors, (iii) the pan-kinase inhibitor staurosporine is insensitive for most mutations due to its high structural compatibility, and (iv) ATP affinity to ALK is generally reduced upon most clinically relevant mutations. Furthermore, we also identify six novel mutation-inhibitor pairs that are potentially associated with drug resistance. In addition, the G1202R and C1156Y mutations are expected to generally cause resistance for many existing inhibitors, since they can address significant effect on the geometric shape and physicochemical property of ALK active pocket. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Multiscale mutation clustering algorithm identifies pan-cancer mutational clusters associated with pathway-level changes in gene expression

PubMed Central

Poole, William; Leinonen, Kalle; Shmulevich, Ilya

2017-01-01

Cancer researchers have long recognized that somatic mutations are not uniformly distributed within genes. However, most approaches for identifying cancer mutations focus on either the entire-gene or single amino-acid level. We have bridged these two methodologies with a multiscale mutation clustering algorithm that identifies variable length mutation clusters in cancer genes. We ran our algorithm on 539 genes using the combined mutation data in 23 cancer types from The Cancer Genome Atlas (TCGA) and identified 1295 mutation clusters. The resulting mutation clusters cover a wide range of scales and often overlap with many kinds of protein features including structured domains, phosphorylation sites, and known single nucleotide variants. We statistically associated these multiscale clusters with gene expression and drug response data to illuminate the functional and clinical consequences of mutations in our clusters. Interestingly, we find multiple clusters within individual genes that have differential functional associations: these include PTEN, FUBP1, and CDH1. This methodology has potential implications in identifying protein regions for drug targets, understanding the biological underpinnings of cancer, and personalizing cancer treatments. Toward this end, we have made the mutation clusters and the clustering algorithm available to the public. Clusters and pathway associations can be interactively browsed at m2c.systemsbiology.net. The multiscale mutation clustering algorithm is available at https://github.com/IlyaLab/M2C. PMID:28170390

Multiscale mutation clustering algorithm identifies pan-cancer mutational clusters associated with pathway-level changes in gene expression.

PubMed

Poole, William; Leinonen, Kalle; Shmulevich, Ilya; Knijnenburg, Theo A; Bernard, Brady

2017-02-01

Cancer researchers have long recognized that somatic mutations are not uniformly distributed within genes. However, most approaches for identifying cancer mutations focus on either the entire-gene or single amino-acid level. We have bridged these two methodologies with a multiscale mutation clustering algorithm that identifies variable length mutation clusters in cancer genes. We ran our algorithm on 539 genes using the combined mutation data in 23 cancer types from The Cancer Genome Atlas (TCGA) and identified 1295 mutation clusters. The resulting mutation clusters cover a wide range of scales and often overlap with many kinds of protein features including structured domains, phosphorylation sites, and known single nucleotide variants. We statistically associated these multiscale clusters with gene expression and drug response data to illuminate the functional and clinical consequences of mutations in our clusters. Interestingly, we find multiple clusters within individual genes that have differential functional associations: these include PTEN, FUBP1, and CDH1. This methodology has potential implications in identifying protein regions for drug targets, understanding the biological underpinnings of cancer, and personalizing cancer treatments. Toward this end, we have made the mutation clusters and the clustering algorithm available to the public. Clusters and pathway associations can be interactively browsed at m2c.systemsbiology.net. The multiscale mutation clustering algorithm is available at https://github.com/IlyaLab/M2C.

Biological and functional relevance of CASP predictions.

PubMed

Liu, Tianyun; Ish-Shalom, Shirbi; Torng, Wen; Lafita, Aleix; Bock, Christian; Mort, Matthew; Cooper, David N; Bliven, Spencer; Capitani, Guido; Mooney, Sean D; Altman, Russ B

2018-03-01

Our goal is to answer the question: compared with experimental structures, how useful are predicted models for functional annotation? We assessed the functional utility of predicted models by comparing the performances of a suite of methods for functional characterization on the predictions and the experimental structures. We identified 28 sites in 25 protein targets to perform functional assessment. These 28 sites included nine sites with known ligand binding (holo-sites), nine sites that are expected or suggested by experimental authors for small molecule binding (apo-sites), and Ten sites containing important motifs, loops, or key residues with important disease-associated mutations. We evaluated the utility of the predictions by comparing their microenvironments to the experimental structures. Overall structural quality correlates with functional utility. However, the best-ranked predictions (global) may not have the best functional quality (local). Our assessment provides an ability to discriminate between predictions with high structural quality. When assessing ligand-binding sites, most prediction methods have higher performance on apo-sites than holo-sites. Some servers show consistently high performance for certain types of functional sites. Finally, many functional sites are associated with protein-protein interaction. We also analyzed biologically relevant features from the protein assemblies of two targets where the active site spanned the protein-protein interface. For the assembly targets, we find that the features in the models are mainly determined by the choice of template. © 2017 The Authors Proteins: Structure, Function and Bioinformatics Published by Wiley Periodicals, Inc.

L925I mutation in the Para-type sodium channel is associated with pyrethroid resistance in Triatoma infestans from the Gran Chaco region.

PubMed

Capriotti, Natalia; Mougabure-Cueto, Gastón; Rivera-Pomar, Rolando; Ons, Sheila

2014-01-01

Chagas' disease is an important public health concern in Latin America. Despite intensive vector control efforts using pyrethroid insecticides, the elimination of Triatoma infestans has failed in the Gran Chaco, an ecoregion that extends over Argentina, Paraguay, Bolivia and Brazil. The voltage-gated sodium channel is the target site of pyrethroid insecticides. Point mutations in domain II region of the channel have been implicated in pyrethroid resistance of several insect species. In the present paper, we identify L925I, a new pyrethroid resistance-conferring mutation in T. infestans. This mutation has been found only in hemipterans. In T. infestans, L925I mutation occurs in a resistant population from the Gran Chaco region and is associated with inefficiency in the control campaigns. We also describe a method to detect L925I mutation in individuals from the field. The findings have important implications in the implementation of strategies for resistance management and in the rational design of campaigns for the control of Chagas' disease transmission.

L925I Mutation in the Para-Type Sodium Channel Is Associated with Pyrethroid Resistance in Triatoma infestans from the Gran Chaco Region

PubMed Central

Capriotti, Natalia; Mougabure-Cueto, Gastón; Rivera-Pomar, Rolando; Ons, Sheila

2014-01-01

Background Chagas' disease is an important public health concern in Latin America. Despite intensive vector control efforts using pyrethroid insecticides, the elimination of Triatoma infestans has failed in the Gran Chaco, an ecoregion that extends over Argentina, Paraguay, Bolivia and Brazil. The voltage-gated sodium channel is the target site of pyrethroid insecticides. Point mutations in domain II region of the channel have been implicated in pyrethroid resistance of several insect species. Methods and Findings In the present paper, we identify L925I, a new pyrethroid resistance-conferring mutation in T. infestans. This mutation has been found only in hemipterans. In T. infestans, L925I mutation occurs in a resistant population from the Gran Chaco region and is associated with inefficiency in the control campaigns. We also describe a method to detect L925I mutation in individuals from the field. Conclusions and Significance The findings have important implications in the implementation of strategies for resistance management and in the rational design of campaigns for the control of Chagas' disease transmission. PMID:24466362

Cas9-nickase-mediated genome editing corrects hereditary tyrosinemia in rats.

PubMed

Shao, Yanjiao; Wang, Liren; Guo, Nana; Wang, Shengfei; Yang, Lei; Li, Yajing; Wang, Mingsong; Yin, Shuming; Han, Honghui; Zeng, Li; Zhang, Ludi; Hui, Lijian; Ding, Qiurong; Zhang, Jiqin; Geng, Hongquan; Liu, Mingyao; Li, Dali

2018-05-04

Hereditary tyrosinemia type I (HTI) is a metabolic genetic disorder caused by mutation of fumarylacetoacetate hydrolase (FAH). Because of the accumulation of toxic metabolites, HTI causes severe liver cirrhosis, liver failure, and even hepatocellular carcinoma. HTI is an ideal model for gene therapy, and several strategies have been shown to ameliorate HTI symptoms in animal models. Although CRISPR/Cas9-mediated genome editing is able to correct the Fah mutation in mouse models, WT Cas9 induces numerous undesired mutations that have raised safety concerns for clinical applications. To develop a new method for gene correction with high fidelity, we generated a Fah mutant rat model to investigate whether Cas9 nickase (Cas9n)-mediated genome editing can efficiently correct the Fah First, we confirmed that Cas9n rarely induces indels in both on-target and off-target sites in cell lines. Using WT Cas9 as a positive control, we delivered Cas9n and the repair donor template/single guide (sg)RNA through adenoviral vectors into HTI rats. Analyses of the initial genome editing efficiency indicated that only WT Cas9 but not Cas9n causes indels at the on-target site in the liver tissue. After receiving either Cas9n or WT Cas9-mediated gene correction therapy, HTI rats gained weight steadily and survived. Fah-expressing hepatocytes occupied over 95% of the liver tissue 9 months after the treatment. Moreover, CRISPR/Cas9-mediated gene therapy prevented the progression of liver cirrhosis, a phenotype that could not be recapitulated in the HTI mouse model. These results strongly suggest that Cas9n-mediated genome editing is a valuable and safe gene therapy strategy for this genetic disease. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

A Guide to Computational Tools and Design Strategies for Genome Editing Experiments in Zebrafish Using CRISPR/Cas9.

PubMed

Prykhozhij, Sergey V; Rajan, Vinothkumar; Berman, Jason N

2016-02-01

The development of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology for mainstream biotechnological use based on its discovery as an adaptive immune mechanism in bacteria has dramatically improved the ability of molecular biologists to modify genomes of model organisms. The zebrafish is highly amenable to applications of CRISPR/Cas9 for mutation generation and a variety of DNA insertions. Cas9 protein in complex with a guide RNA molecule recognizes where to cut the homologous DNA based on a short stretch of DNA termed the protospacer-adjacent motif (PAM). Rapid and efficient identification of target sites immediately preceding PAM sites, quantification of genomic occurrences of similar (off target) sites and predictions of cutting efficiency are some of the features where computational tools play critical roles in CRISPR/Cas9 applications. Given the rapid advent and development of this technology, it can be a challenge for researchers to remain up to date with all of the important technological developments in this field. We have contributed to the armamentarium of CRISPR/Cas9 bioinformatics tools and trained other researchers in the use of appropriate computational programs to develop suitable experimental strategies. Here we provide an in-depth guide on how to use CRISPR/Cas9 and other relevant computational tools at each step of a host of genome editing experimental strategies. We also provide detailed conceptual outlines of the steps involved in the design and execution of CRISPR/Cas9-based experimental strategies, such as generation of frameshift mutations, larger chromosomal deletions and inversions, homology-independent insertion of gene cassettes and homology-based knock-in of defined point mutations and larger gene constructs.

Analysis of SOST expression using large minigenes reveals the MEF2C binding site in the evolutionarily conserved region (ECR5) enhancer mediates forskolin, but not 1,25-dihydroxyvitamin D3 or TGFβ1 responsiveness.

PubMed

St John, Hillary C; Hansen, Sydney J; Pike, J Wesley

2016-11-01

Transcribed from the SOST gene, sclerostin is an osteocyte-derived negative regulator of bone formation that inhibits osteoblastogenesis via antagonism of the Wnt pathway. Sclerostin is a promising therapeutic target for low bone mass diseases and neutralizing antibody therapies that target sclerostin are in development. Diverse stimuli regulate SOST including the vitamin D hormone, forskolin (Fsk), bone morphogenic protein 2 (BMP-2), oncostatin M (OSM), dexamethasone (Dex), and transforming growth factor (TGFβ 1 ). To explore the mechanisms by which these compounds regulate SOST expression, we examined their ability to regulate a SOST reporter minigene containing the entire SOST locus including the downstream regionor mutant minigenes containing a deletion of the -1kb to -2kb promoter proximal region (-1kb), ECR2, ECR5, or two point mutations in the MEF2 binding site of ECR5 (ECR5/MEF2). Previous reports suggest that both the PTH and TGFβ 1 effects on SOST are mediated through ECR5 and that the action of PTH is mediated specifically via the MEF2 binding site at ECR5. Consistent with these reports, the suppressive effects of Fsk were abrogated following both ECR5 deletion and ECR5/MEF2 mutation. In contrast, we found that TGFβ 1 negatively regulated SOST and that neither ECR5 nor ECR5/MEF2 was involved. Surprisingly, none of these four deletions/mutations abrogated the suppressive effects of the vitamin D hormone, OSM, Dex, or TGFβ 1 , or the positive effects of BMP-2. These data suggest that we need to move beyond ECR5 to understand SOST regulation. Copyright © 2015 Elsevier Ltd. All rights reserved.

MutScan: fast detection and visualization of target mutations by scanning FASTQ data.

PubMed

Chen, Shifu; Huang, Tanxiao; Wen, Tiexiang; Li, Hong; Xu, Mingyan; Gu, Jia

2018-01-22

Some types of clinical genetic tests, such as cancer testing using circulating tumor DNA (ctDNA), require sensitive detection of known target mutations. However, conventional next-generation sequencing (NGS) data analysis pipelines typically involve different steps of filtering, which may cause miss-detection of key mutations with low frequencies. Variant validation is also indicated for key mutations detected by bioinformatics pipelines. Typically, this process can be executed using alignment visualization tools such as IGV or GenomeBrowse. However, these tools are too heavy and therefore unsuitable for validating mutations in ultra-deep sequencing data. We developed MutScan to address problems of sensitive detection and efficient validation for target mutations. MutScan involves highly optimized string-searching algorithms, which can scan input FASTQ files to grab all reads that support target mutations. The collected supporting reads for each target mutation will be piled up and visualized using web technologies such as HTML and JavaScript. Algorithms such as rolling hash and bloom filter are applied to accelerate scanning and make MutScan applicable to detect or visualize target mutations in a very fast way. MutScan is a tool for the detection and visualization of target mutations by only scanning FASTQ raw data directly. Compared to conventional pipelines, this offers a very high performance, executing about 20 times faster, and offering maximal sensitivity since it can grab mutations with even one single supporting read. MutScan visualizes detected mutations by generating interactive pile-ups using web technologies. These can serve to validate target mutations, thus avoiding false positives. Furthermore, MutScan can visualize all mutation records in a VCF file to HTML pages for cloud-friendly VCF validation. MutScan is an open source tool available at GitHub: https://github.com/OpenGene/MutScan.

Tricyclic GyrB/ParE (TriBE) Inhibitors: A New Class of Broad-Spectrum Dual-Targeting Antibacterial Agents

PubMed Central

Tari, Leslie W.; Li, Xiaoming; Trzoss, Michael; Bensen, Daniel C.; Chen, Zhiyong; Lam, Thanh; Zhang, Junhu; Lee, Suk Joong; Hough, Grayson; Phillipson, Doug; Akers-Rodriguez, Suzanne; Cunningham, Mark L.; Kwan, Bryan P.; Nelson, Kirk J.; Castellano, Amanda; Locke, Jeff B.; Brown-Driver, Vickie; Murphy, Timothy M.; Ong, Voon S.; Pillar, Chris M.; Shinabarger, Dean L.; Nix, Jay; Lightstone, Felice C.; Wong, Sergio E.; Nguyen, Toan B.; Shaw, Karen J.; Finn, John

2013-01-01

Increasing resistance to every major class of antibiotics and a dearth of novel classes of antibacterial agents in development pipelines has created a dwindling reservoir of treatment options for serious bacterial infections. The bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV, are validated antibacterial drug targets with multiple prospective drug binding sites, including the catalytic site targeted by the fluoroquinolone antibiotics. However, growing resistance to fluoroquinolones, frequently mediated by mutations in the drug-binding site, is increasingly limiting the utility of this antibiotic class, prompting the search for other inhibitor classes that target different sites on the topoisomerase complexes. The highly conserved ATP-binding subunits of DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as excellent candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, to date, no natural product or small molecule inhibitors targeting these sites have succeeded in the clinic, and no inhibitors of these enzymes have yet been reported with broad-spectrum antibacterial activity encompassing the majority of Gram-negative pathogens. Using structure-based drug design (SBDD), we have created a novel dual-targeting pyrimidoindole inhibitor series with exquisite potency against GyrB and ParE enzymes from a broad range of clinically important pathogens. Inhibitors from this series demonstrate potent, broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens of clinical importance, including fluoroquinolone resistant and multidrug resistant strains. Lead compounds have been discovered with clinical potential; they are well tolerated in animals, and efficacious in Gram-negative infection models. PMID:24386374

High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients.

PubMed

Kukita, Yoji; Matoba, Ryo; Uchida, Junji; Hamakawa, Takuya; Doki, Yuichiro; Imamura, Fumio; Kato, Kikuya

2015-08-01

Circulating tumour DNA (ctDNA) is an emerging field of cancer research. However, current ctDNA analysis is usually restricted to one or a few mutation sites due to technical limitations. In the case of massively parallel DNA sequencers, the number of false positives caused by a high read error rate is a major problem. In addition, the final sequence reads do not represent the original DNA population due to the global amplification step during the template preparation. We established a high-fidelity target sequencing system of individual molecules identified in plasma cell-free DNA using barcode sequences; this system consists of the following two steps. (i) A novel target sequencing method that adds barcode sequences by adaptor ligation. This method uses linear amplification to eliminate the errors introduced during the early cycles of polymerase chain reaction. (ii) The monitoring and removal of erroneous barcode tags. This process involves the identification of individual molecules that have been sequenced and for which the number of mutations have been absolute quantitated. Using plasma cell-free DNA from patients with gastric or lung cancer, we demonstrated that the system achieved near complete elimination of false positives and enabled de novo detection and absolute quantitation of mutations in plasma cell-free DNA. © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

An integrated molecular analysis of lung adenocarcinomas identifies potential therapeutic targets among TTF1-negative tumors including DNA repair proteins and Nrf2

PubMed Central

Cardnell, Robert J.G.; Behrens, Carmen; Diao, Lixia; Fan, YouHong; Tang, Ximing; Tong, Pan; John D., Minna; Mills, Gordon B.; Heymach, John V.; Wistuba, Ignacio I.; Wang, Jing; Byers., Lauren A.

2015-01-01

Purpose Thyroid transcription factor-1 (TTF1) immunohistochemistry (IHC) is used clinically to differentiate primary lung adenocarcinomas (LUAD) from squamous lung cancers and metastatic adenocarcinomas from other primary sites. However, a subset of LUAD (15-20%) does not express TTF1 and TTF1-negative patients have worse clinical outcomes. As there are no established targeted agents with activity in TTF1-negative LUAD, we performed an integrated molecular analysis to identify potential therapeutic targets. Experimental Design Using two clinical LUAD cohorts (274 tumors), one from our institution (PROSPECT) and the TCGA, we interrogated proteomic profiles (by reverse-phase protein array (RPPA)), gene expression, and mutational data. Drug response data from 74 cell lines were used to validate potential therapeutic agents. Results Strong correlations were observed between TTF1 IHC and TTF1 measurements by RPPA (Rho=0.57, p<0.001) and gene expression (NKX2-1, Rho=0.61, p<0.001). Established driver mutations (e.g. BRAF and EGFR) were associated with high TTF1 expression. In contrast, TTF1-negative LUAD had a higher frequency of inactivating KEAP1 mutations (p=0.001). Proteomic profiling identified increased expression of DNA repair proteins (e.g., Chk1 and the DNA repair score) and suppressed PI3K/MAPK signaling among TTF1-negative tumors, with differences in total proteins confirmed at the mRNA level. Cell line analysis showed drugs targeting DNA repair to be more active in TTF1-low cell lines. Conclusions Combined genomic and proteomic analyses demonstrated infrequent alteration of validated lung cancer targets (including the absence of BRAF mutations in TTF1-negative LUAD), but identified novel potential targets for TTF1-negative LUAD includingKEAP1/Nrf2 and DNA repair pathways. PMID:25878335

A nicotinic acetylcholine receptor mutation conferring target-site resistance to imidacloprid in Nilaparvata lugens (brown planthopper).

PubMed

Liu, Zewen; Williamson, Martin S; Lansdell, Stuart J; Denholm, Ian; Han, Zhaojun; Millar, Neil S

2005-06-14

Neonicotinoids, such as imidacloprid, are nicotinic acetylcholine receptor (nAChR) agonists with potent insecticidal activity. Since its introduction in the early 1990s, imidacloprid has become one of the most extensively used insecticides for both crop protection and animal health applications. As with other classes of insecticides, resistance to neonicotinoids is a significant threat and has been identified in several pest species, including the brown planthopper, Nilaparvata lugens, a major rice pest in many parts of Asia. In this study, radioligand binding experiments have been conducted with whole-body membranes prepared from imidacloprid-susceptible and imidacloprid-resistant strains of N. lugens. The results reveal a much higher level of [3H]imidacloprid-specific binding to the susceptible strain than to the resistant strain (16.7 +/- 1.0 and 0.34 +/- 0.21 fmol/mg of protein, respectively). With the aim of understanding the molecular basis of imidacloprid resistance, five nAChR subunits (Nlalpha1-Nlalpha4 and Nlbeta1) have been cloned from N. lugens.A comparison of nAChR subunit genes from imidacloprid-sensitive and imidacloprid-resistant populations has identified a single point mutation at a conserved position (Y151S) in two nAChR subunits, Nlalpha1 and Nlalpha3. A strong correlation between the frequency of the Y151S point mutation and the level of resistance to imidacloprid has been demonstrated by allele-specific PCR. By expression of hybrid nAChRs containing N. lugens alpha and rat beta2 subunits, evidence was obtained that demonstrates that mutation Y151S is responsible for a substantial reduction in specific [3H]imidacloprid binding. This study provides direct evidence for the occurrence of target-site resistance to a neonicotinoid insecticide.

Uterine metastasis of lung adenocarcinoma under molecular target therapy with epidermal growth factor receptor tyrosine kinase inhibitors: A case report and review of the literature.

PubMed

Shibata, Mayu; Shizu, Masato; Watanabe, Kazuko; Takeda, Akihiro

2018-02-01

A 63-year-old woman presented with abnormal vaginal bleeding. Her disease history was significant, and included advanced lung adenocarcinoma with a deletion mutation in exon 19 of the epidermal growth factor receptor (EGFR) gene, which was managed by concurrent chemoradiotherapy, followed by molecular targeted therapy with tyrosine kinase inhibitors (TKIs) for a two-year period. Contrast-enhanced computed tomography showed the enlargement of a previously suspicious myoma node, with peripheral enhancement. Hemorrhagic necrosis was also observed on magnetic resonance imaging. Transabdominal hysterectomy and bilateral salpingo-oophorectomy showed solitary intramyometrial metastatic lung adenocarcinoma with a second-site T790M gatekeeper mutation in exon 20 of the EGFR gene. In conclusion, uterine metastasis from lung adenocarcinoma can present a diagnostic challenge. The possibility of lung cancer metastasis should be considered when a uterine mass increases in size during treatment. Molecular analysis of the EGFR gene to detect mutations could provide useful information for planning the treatment strategy. © 2017 Japan Society of Obstetrics and Gynecology.

Hyperactivation of HUSH complex function by Charcot-Marie-Tooth disease mutation in MORC2.

PubMed

Tchasovnikarova, Iva A; Timms, Richard T; Douse, Christopher H; Roberts, Rhys C; Dougan, Gordon; Kingston, Robert E; Modis, Yorgo; Lehner, Paul J

2017-07-01

Dominant mutations in the MORC2 gene have recently been shown to cause axonal Charcot-Marie-Tooth (CMT) disease, but the cellular function of MORC2 is poorly understood. Here, through a genome-wide CRISPR-Cas9-mediated forward genetic screen, we identified MORC2 as an essential gene required for epigenetic silencing by the HUSH complex. HUSH recruits MORC2 to target sites in heterochromatin. We exploited a new method, differential viral accessibility (DIVA), to show that loss of MORC2 results in chromatin decompaction at these target loci, which is concomitant with a loss of H3K9me3 deposition and transcriptional derepression. The ATPase activity of MORC2 is critical for HUSH-mediated silencing, and the most common alteration affecting the ATPase domain in CMT patients (p.Arg252Trp) hyperactivates HUSH-mediated repression in neuronal cells. These data define a critical role for MORC2 in epigenetic silencing by the HUSH complex and provide a mechanistic basis underpinning the role of MORC2 mutations in CMT disease.

Parkin targets HIF-1α for ubiquitination and degradation to inhibit breast tumor progression.

PubMed

Liu, Juan; Zhang, Cen; Zhao, Yuhan; Yue, Xuetian; Wu, Hao; Huang, Shan; Chen, James; Tomsky, Kyle; Xie, Haiyang; Khella, Christen A; Gatza, Michael L; Xia, Dajing; Gao, Jimin; White, Eileen; Haffty, Bruce G; Hu, Wenwei; Feng, Zhaohui

2017-11-28

Mutations in E3 ubiquitin ligase Parkin have been linked to familial Parkinson's disease. Accumulating evidence suggests that Parkin is a tumor suppressor, but the underlying mechanism is poorly understood. Here we show that Parkin is an E3 ubiquitin ligase for hypoxia-inducible factor 1α (HIF-1α). Parkin interacts with HIF-1α and promotes HIF-1α degradation through ubiquitination, which in turn inhibits metastasis of breast cancer cells. Parkin downregulation in breast cancer cells promotes metastasis, which can be inhibited by targeting HIF-1α with RNA interference or the small-molecule inhibitor YC-1. We further identify lysine 477 (K477) of HIF-1α as a major ubiquitination site for Parkin. K477R HIF-1α mutation and specific cancer-associated Parkin mutations largely abolish the functions of Parkin to ubiquitinate HIF-1α and inhibit cancer metastasis. Importantly, Parkin expression is inversely correlated with HIF-1α expression and metastasis in breast cancer. Our results reveal an important mechanism for Parkin in tumor suppression and HIF-1α regulation.

CRISPR/Cas9-mediated targeted mutagenesis in grape

PubMed Central

Ban, Yusuke; Azuma, Akifumi; Onoue, Noriyuki; Moriguchi, Takaya; Yamamoto, Toshiya; Toki, Seiichi

2017-01-01

RNA-guided genome editing using the CRISPR/Cas9 CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system has been applied successfully in several plant species. However, to date, there are few reports on the use of any of the current genome editing approaches in grape—an important fruit crop with a large market not only for table grapes but also for wine. Here, we report successful targeted mutagenesis in grape (Vitis vinifera L., cv. Neo Muscat) using the CRISPR/Cas9 system. When a Cas9 expression construct was transformed to embryonic calli along with a synthetic sgRNA expression construct targeting the Vitis vinifera phytoene desaturase (VvPDS) gene, regenerated plants with albino leaves were obtained. DNA sequencing confirmed that the VvPDS gene was mutated at the target site in regenerated grape plants. Interestingly, the ratio of mutated cells was higher in lower, older, leaves compared to that in newly appearing upper leaves. This result might suggest either that the proportion of targeted mutagenized cells is higher in older leaves due to the repeated induction of DNA double strand breaks (DSBs), or that the efficiency of precise DSBs repair in cells of old grape leaves is decreased. PMID:28542349

Point mutation impairs centromeric CENH3 loading and induces haploid plants.

PubMed

Karimi-Ashtiyani, Raheleh; Ishii, Takayoshi; Niessen, Markus; Stein, Nils; Heckmann, Stefan; Gurushidze, Maia; Banaei-Moghaddam, Ali Mohammad; Fuchs, Jörg; Schubert, Veit; Koch, Kerstin; Weiss, Oda; Demidov, Dmitri; Schmidt, Klaus; Kumlehn, Jochen; Houben, Andreas

2015-09-08

The chromosomal position of the centromere-specific histone H3 variant CENH3 (also called "CENP-A") is the assembly site for the kinetochore complex of active centromeres. Any error in transcription, translation, modification, or incorporation can affect the ability to assemble intact CENH3 chromatin and can cause centromere inactivation [Allshire RC, Karpen GH (2008) Nat Rev Genet 9 (12):923-937]. Here we show that a single-point amino acid exchange in the centromere-targeting domain of CENH3 leads to reduced centromere loading of CENH3 in barley, sugar beet, and Arabidopsis thaliana. Haploids were obtained after cenh3 L130F-complemented cenh3-null mutant plants were crossed with wild-type A. thaliana. In contrast, in a noncompeting situation (i.e., centromeres possessing only mutated or only wild-type CENH3), no uniparental chromosome elimination occurs during early embryogenesis. The high degree of evolutionary conservation of the identified mutation site offers promising opportunities for application in a wide range of crop species in which haploid technology is of interest.

Point mutation impairs centromeric CENH3 loading and induces haploid plants

PubMed Central

Karimi-Ashtiyani, Raheleh; Ishii, Takayoshi; Niessen, Markus; Stein, Nils; Heckmann, Stefan; Gurushidze, Maia; Banaei-Moghaddam, Ali Mohammad; Fuchs, Jörg; Schubert, Veit; Koch, Kerstin; Weiss, Oda; Demidov, Dmitri; Schmidt, Klaus; Kumlehn, Jochen; Houben, Andreas

2015-01-01

The chromosomal position of the centromere-specific histone H3 variant CENH3 (also called “CENP-A”) is the assembly site for the kinetochore complex of active centromeres. Any error in transcription, translation, modification, or incorporation can affect the ability to assemble intact CENH3 chromatin and can cause centromere inactivation [Allshire RC, Karpen GH (2008) Nat Rev Genet 9 (12):923–937]. Here we show that a single-point amino acid exchange in the centromere-targeting domain of CENH3 leads to reduced centromere loading of CENH3 in barley, sugar beet, and Arabidopsis thaliana. Haploids were obtained after cenh3 L130F-complemented cenh3-null mutant plants were crossed with wild-type A. thaliana. In contrast, in a noncompeting situation (i.e., centromeres possessing only mutated or only wild-type CENH3), no uniparental chromosome elimination occurs during early embryogenesis. The high degree of evolutionary conservation of the identified mutation site offers promising opportunities for application in a wide range of crop species in which haploid technology is of interest. PMID:26294252

A Noncoding, Regulatory Mutation Implicates HCFC1 in Nonsyndromic Intellectual Disability

PubMed Central

Huang, Lingli; Jolly, Lachlan A.; Willis-Owen, Saffron; Gardner, Alison; Kumar, Raman; Douglas, Evelyn; Shoubridge, Cheryl; Wieczorek, Dagmar; Tzschach, Andreas; Cohen, Monika; Hackett, Anna; Field, Michael; Froyen, Guy; Hu, Hao; Haas, Stefan A.; Ropers, Hans-Hilger; Kalscheuer, Vera M.; Corbett, Mark A.; Gecz, Jozef

2012-01-01

The discovery of mutations causing human disease has so far been biased toward protein-coding regions. Having excluded all annotated coding regions, we performed targeted massively parallel resequencing of the nonrepetitive genomic linkage interval at Xq28 of family MRX3. We identified in the binding site of transcription factor YY1 a regulatory mutation that leads to overexpression of the chromatin-associated transcriptional regulator HCFC1. When tested on embryonic murine neural stem cells and embryonic hippocampal neurons, HCFC1 overexpression led to a significant increase of the production of astrocytes and a considerable reduction in neurite growth. Two other nonsynonymous, potentially deleterious changes have been identified by X-exome sequencing in individuals with intellectual disability, implicating HCFC1 in normal brain function. PMID:23000143

Targeted disruption of sp7 and myostatin with CRISPR-Cas9 results in severe bone defects and more muscular cells in common carp

PubMed Central

Zhong, Zhaomin; Niu, Pengfei; Wang, Mingyong; Huang, Guodong; Xu, Shuhao; Sun, Yi; Xu, Xiaona; Hou, Yi; Sun, Xiaowen; Yan, Yilin; Wang, Han

2016-01-01

The common carp (Cyprinus carpio) as one of the most important aquaculture fishes produces over 3 million metric tones annually, approximately 10% the annual production of the all farmed freshwater fish worldwide. However, the tetraploidy genome and long generation-time of the common carp have made its breeding and genetic studies extremely difficult. Here, TALEN and CRISPR-Cas9, two versatile genome-editing tools, are employed to target common carp bone-related genes sp7, runx2, bmp2a, spp1, opg, and muscle suppressor gene mstn. TALEN were shown to induce mutations in the target coding sites of sp7, runx2, spp1 and mstn. With CRISPR-Cas9, the two common carp sp7 genes, sp7a and sp7b, were mutated individually, all resulting in severe bone defects; while mstnba mutated fish have grown significantly more muscle cells. We also employed CRISPR-Cas9 to generate double mutant fish of sp7a;mstnba with high efficiencies in a single step. These results demonstrate that both TALEN and CRISPR-Cas9 are highly efficient tools for modifying the common carp genome, and open avenues for facilitating common carp genetic studies and breeding. PMID:26976234

Characterization of spontaneous mutants of Magnaporthe grisea expressing stable resistance to the Qo-inhibiting fungicide azoxystrobin.

PubMed

Avila-Adame, Cruz; Köller, Wolfram

2003-03-01

The class of Qo-inhibiting fungicides (QoIs) act as respiration inhibitors by binding to the Qo center of cytochrome b. The longevity of these fungicides has been challenged by the selection of fungal sub-populations resisting high doses of QoI fungicides, with a G143A amino acid exchange in the cytochrome b target site identified as the most common cause of resistance. In contrast, the mechanism of alternative respiration, as another mechanism of fungal QoI resistance, has thus far not been affiliated with practical resistance. In the present study, azoxystrobin-resistant mutants of Magnaporthe grisea were generated and characterized. Emergence of these spontaneous mutants was facilitated when resting melanized mycelia were allowed to escape full inhibition by azoxystrobin. This escape was related to the intactness of alternative respiration, indicating that residual expression of this rescue mechanism was involved in the spontaneous emergence of target-site mutants. The two mutants characterized resisted high doses of the QoI, azoxystrobin, with resistance factors exceeding 1,000. Two different mutations of the cytochrome b gene were identified as exchanges of guanine, leading to a G143A or a G143S amino acid exchange. Resistance of both target-site mutants remained stable during four consecutive disease cycles in the absence of azoxystrobin. Several parameters tested to measure fitness penalties inherent to the mutational changes revealed that the G143A mutant was not compromised. In contrast, the conidia production of the G143S mutant was significantly lower under both saprophytic and pathogenic conditions of reproduction.

Characterising variation in five genetic loci of cytomegalovirus during treatment for congenital infection.

PubMed

Kadambari, Seilesh; Atkinson, Claire; Luck, Suzanne; Macartney, Malcolm; Conibear, Tim; Harrison, Ian; Booth, Clare; Sharland, Mike; Griffiths, Paul D

2017-03-01

Cytomegalovirus (CMV) is the most common congenital infection in humans and a leading cause of sensorineural hearing loss. Ganciclovir (6 mg/kg twice daily for 42 days) has been shown to reduce hearing deterioration and is used in clinical practice. Vaccines and passive administration of antibody are being evaluated in randomized controlled trials in allograft candidates, women of childbearing age, and pregnant women with primary CMV infection. To help define genetic variation in each of the targets of these therapeutic interventions, we amplified and sequenced genes UL97 (site utilised for ganciclovir phosphorylation), UL55 (glycoprotein B (gB) vaccine target) and UL128, UL130, and UL131a (specific monoclonal antibody targets). Serial blood, saliva, and urine samples (total 120) obtained from nine infants with symptomatic congenital CMV treated with 42 days' ganciclovir were analyzed. All samples tested were UL97 wild type at baseline and none developed mutations during treatment, showing no selection of resistance. The prevalences of UL55 genotypes were 28% gB1, 22% gB2, 1% gB3, and mixed in 20% samples. No mutations were noted in UL128-131a. Phylogenetic tree analysis showed that sequences with variations were found in multiple body sites of individual patients, so there was no evidence of body site compartmentalization of particular strains of CMV. The significance of these results for changes in diagnostic practices and therapeutic interventions against CMV are discussed. J. Med. Virol. 89:502-507, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Insights into the binding specificity of wild type and mutated wheat germ agglutinin towards Neu5Acα(2-3)Gal: a study by in silico mutations and molecular dynamics simulations.

PubMed

Parasuraman, Ponnusamy; Murugan, Veeramani; Selvin, Jeyasigamani F A; Gromiha, M Michael; Fukui, Kazuhiko; Veluraja, Kasinadar

2014-08-01

Wheat germ agglutinin (WGA) is a plant lectin, which specifically recognizes the sugars NeuNAc and GlcNAc. Mutated WGA with enhanced binding specificity can be used as biomarkers for cancer. In silico mutations are performed at the active site of WGA to enhance the binding specificity towards sialylglycans, and molecular dynamics simulations of 20 ns are carried out for wild type and mutated WGAs (WGA1, WGA2, and WGA3) in complex with sialylgalactose to examine the change in binding specificity. MD simulations reveal the change in binding specificity of wild type and mutated WGAs towards sialylgalactose and bound conformational flexibility of sialylgalactose. The mutated polar amino acid residues Asn114 (S114N), Lys118 (G118K), and Arg118 (G118R) make direct and water mediated hydrogen bonds and hydrophobic interactions with sialylgalactose. An analysis of possible hydrogen bonds, hydrophobic interactions, total pair wise interaction energy between active site residues and sialylgalactose and MM-PBSA free energy calculation reveals the plausible binding modes and the role of water in stabilizing different binding modes. An interesting observation is that the binding specificity of mutated WGAs (cyborg lectin) towards sialylgalactose is found to be higher in double point mutation (WGA3). One of the substituted residues Arg118 plays a crucial role in sugar binding. Based on the interactions and energy calculations, it is concluded that the order of binding specificity of WGAs towards sialylgalactose is WGA3 > WGA1 > WGA2 > WGA. On comparing with the wild type, double point mutated WGA (WGA3) exhibits increased specificity towards sialylgalactose, and thus, it can be effectively used in targeted drug delivery and as biological cell marker in cancer therapeutics. Copyright © 2014 John Wiley & Sons, Ltd.

Introduction of a point mutation into the mouse genome by homologous recombination in embryonic stem cells using a replacement type vector with a selectable marker.

PubMed

Rubinstein, M; Japón, M A; Low, M J

1993-06-11

The introduction of small mutations instead of null alleles into the mouse genome has broad applications to the study of protein structure-function relationships and the creation of animal models of human genetic diseases. To test a simple mutational strategy we designed a targeting vector for the mouse proopiomelanocortin (POMC) gene containing a single nucleotide insertion that converts the initial tyrosine codon of beta-endorphin 1-31 to a premature translational termination codon and introduces a unique Hpal endonuclease restriction site. The targeting vector also contains a neo cassette immediately 3' to the last POMC exon and a herpes simplex virus thymidine kinase cassette to allow positive and negative selection. Homologous recombination occurred at a frequency of 1/30 clones of electroporated embryonic stem cells selected in G418 and gancyclovir. 10/11 clones identified initially by a polymerase chain reaction (PCR) strategy had the predicted structure without evidence of concatemer formation by Southern blot analysis. We used a combination of Hpa I digestion of PCR amplified fragments and direct nucleotide sequencing to further confirm that the point mutation was retained in 9/10 clones. The POMC gene was transcriptionally silent in embryonic stem cells and the targeted allele was not activated by the downstream phosphoglycerate kinase-1 promoter that transcribed the neo gene. Under the electroporation conditions used, we have demonstrated that a point mutation can be introduced with high efficiency and precision into the POMC gene using a replacement type vector containing a retained selectable marker without affecting expression of the allele in the embryonic stem cells. A similar strategy may be useful for a wide range of genes.

Introduction of a point mutation into the mouse genome by homologous recombination in embryonic stem cells using a replacement type vector with a selectable marker.

PubMed Central

Rubinstein, M; Japón, M A; Low, M J

1993-01-01

The introduction of small mutations instead of null alleles into the mouse genome has broad applications to the study of protein structure-function relationships and the creation of animal models of human genetic diseases. To test a simple mutational strategy we designed a targeting vector for the mouse proopiomelanocortin (POMC) gene containing a single nucleotide insertion that converts the initial tyrosine codon of beta-endorphin 1-31 to a premature translational termination codon and introduces a unique Hpal endonuclease restriction site. The targeting vector also contains a neo cassette immediately 3' to the last POMC exon and a herpes simplex virus thymidine kinase cassette to allow positive and negative selection. Homologous recombination occurred at a frequency of 1/30 clones of electroporated embryonic stem cells selected in G418 and gancyclovir. 10/11 clones identified initially by a polymerase chain reaction (PCR) strategy had the predicted structure without evidence of concatemer formation by Southern blot analysis. We used a combination of Hpa I digestion of PCR amplified fragments and direct nucleotide sequencing to further confirm that the point mutation was retained in 9/10 clones. The POMC gene was transcriptionally silent in embryonic stem cells and the targeted allele was not activated by the downstream phosphoglycerate kinase-1 promoter that transcribed the neo gene. Under the electroporation conditions used, we have demonstrated that a point mutation can be introduced with high efficiency and precision into the POMC gene using a replacement type vector containing a retained selectable marker without affecting expression of the allele in the embryonic stem cells. A similar strategy may be useful for a wide range of genes. Images PMID:8392702

Epitope Dampening Monotypic Measles Virus Hemagglutinin Glycoprotein Results in Resistance to Cocktail of Monoclonal Antibodies

PubMed Central

Lech, Patrycja J.; Tobin, Gregory J.; Bushnell, Ruth; Gutschenritter, Emily; Pham, Linh D.; Nace, Rebecca; Verhoeyen, Els; Cosset, François-Loïc; Muller, Claude P.; Russell, Stephen J.; Nara, Peter L.

2013-01-01

The measles virus (MV) is serologically monotypic. Life-long immunity is conferred by a single attack of measles or following vaccination with the MV vaccine. This is contrary to viruses such as influenza, which readily develop resistance to the immune system and recur. A better understanding of factors that restrain MV to one serotype may allow us to predict if MV will remain monotypic in the future and influence the design of novel MV vaccines and therapeutics. MV hemagglutinin (H) glycoprotein, binds to cellular receptors and subsequently triggers the fusion (F) glycoprotein to fuse the virus into the cell. H is also the major target for neutralizing antibodies. To explore if MV remains monotypic due to a lack of plasticity of the H glycoprotein, we used the technology of Immune Dampening to generate viruses with rationally designed N-linked glycosylation sites and mutations in different epitopes and screened for viruses that escaped monoclonal antibodies (mAbs). We then combined rationally designed mutations with naturally selected mutations to generate a virus resistant to a cocktail of neutralizing mAbs targeting four different epitopes simultaneously. Two epitopes were protected by engineered N-linked glycosylations and two epitopes acquired escape mutations via two consecutive rounds of artificial selection in the presence of mAbs. Three of these epitopes were targeted by mAbs known to interfere with receptor binding. Results demonstrate that, within the epitopes analyzed, H can tolerate mutations in different residues and additional N-linked glycosylations to escape mAbs. Understanding the degree of change that H can tolerate is important as we follow its evolution in a host whose immunity is vaccine induced by genotype A strains instead of multiple genetically distinct wild-type MVs. PMID:23300970

Merkel Cell Carcinoma Therapeutic Update.

PubMed

Cassler, Nicole M; Merrill, Dean; Bichakjian, Christopher K; Brownell, Isaac

2016-07-01

Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine tumor of the skin. Early-stage disease can be cured with surgical resection and radiotherapy (RT). Sentinel lymph node biopsy (SLNB) is an important staging tool, as a microscopic MCC is frequently identified. Adjuvant RT to the primary excision site and regional lymph node bed may improve locoregional control. However, newer studies confirm that patients with biopsy-negative sentinel lymph nodes may not benefit from regional RT. Advanced MCC currently lacks a highly effective treatment as responses to chemotherapy are not durable. Recent work suggests that immunotherapy targeting the programmed cell death receptor 1/programmed cell death ligand 1 (PD-1/PD-L1) checkpoint holds great promise in treating advanced MCC and may provide durable responses in a portion of patients. At the same time, high-throughput sequencing studies have demonstrated significant differences in the mutational profiles of tumors with and without the Merkel cell polyomavirus (MCV). An important secondary endpoint in the ongoing immunotherapy trials for MCC will be determining if there is a response difference between the virus-positive MCC tumors that typically lack a large mutational burden and the virus-negative tumors that have a large number of somatic mutations and predicted tumor neoantigens. Interestingly, sequencing studies have failed to identify a highly recurrent activated driver pathway in the majority of MCC tumors. This may explain why targeted therapies can demonstrate exceptional responses in case reports but fail when treating all comers with MCC. Ultimately, a precision medicine approach may be more appropriate for treating MCC, where identified driver mutations are used to direct targeted therapies. At a minimum, stratifying patients in future clinical trials based on tumor viral status should be considered as virus-negative tumors are more likely to harbor activating driver mutations.

Simultaneous fluorescence light-up and selective multicolor nucleobase recognition based on sequence-dependent strong binding of berberine to DNA abasic site.

PubMed

Wu, Fei; Shao, Yong; Ma, Kun; Cui, Qinghua; Liu, Guiying; Xu, Shujuan

2012-04-28

Label-free DNA nucleobase recognition by fluorescent small molecules has received much attention due to its simplicity in mutation identification and drug screening. However, sequence-dependent fluorescence light-up nucleobase recognition and multicolor emission with individual emission energy for individual nucleobases have been seldom realized. Herein, an abasic site (AP site) in a DNA duplex was employed as a binding field for berberine, one of isoquinoline alkaloids. Unlike weak binding of berberine to the fully matched DNAs without the AP site, strong binding of berberine to the AP site occurs and the berberine's fluorescence light-up behaviors are highly dependent on the target nucleobases opposite the AP site in which the targets thymine and cytosine produce dual emission bands, while the targets guanine and adenine only give a single emission band. Furthermore, more intense emissions are observed for the target pyrimidines than purines. The flanking bases of the AP site also produce some modifications of the berberine's emission behavior. The binding selectivity of berberine at the AP site is also confirmed by measurements of fluorescence resonance energy transfer, excited-state lifetime, DNA melting and fluorescence quenching by ferrocyanide and sodium chloride. It is expected that the target pyrimidines cause berberine to be stacked well within DNA base pairs near the AP site, which results in a strong resonance coupling of the electronic transitions to the particular vibration mode to produce the dual emissions. The fluorescent signal-on and emission energy-modulated sensing for nucleobases based on this fluorophore is substantially advantageous over the previously used fluorophores. We expect that this approach will be developed as a practical device for differentiating pyrimidines from purines by positioning an AP site toward a target that is available for readout by this alkaloid probe. This journal is © The Royal Society of Chemistry 2012

indCAPS: A tool for designing screening primers for CRISPR/Cas9 mutagenesis events.

PubMed

Hodgens, Charles; Nimchuk, Zachary L; Kieber, Joseph J

2017-01-01

Genetic manipulation of organisms using CRISPR/Cas9 technology generally produces small insertions/deletions (indels) that can be difficult to detect. Here, we describe a technique to easily and rapidly identify such indels. Sequence-identified mutations that alter a restriction enzyme recognition site can be readily distinguished from wild-type alleles using a cleaved amplified polymorphic sequence (CAPS) technique. If a restriction site is created or altered by the mutation such that only one allele contains the restriction site, a polymerase chain reaction (PCR) followed by a restriction digest can be used to distinguish the two alleles. However, in the case of most CRISPR-induced alleles, no such restriction sites are present in the target sequences. In this case, a derived CAPS (dCAPS) approach can be used in which mismatches are purposefully introduced in the oligonucleotide primers to create a restriction site in one, but not both, of the amplified templates. Web-based tools exist to aid dCAPS primer design, but when supplied sequences that include indels, the current tools often fail to suggest appropriate primers. Here, we report the development of a Python-based, species-agnostic web tool, called indCAPS, suitable for the design of PCR primers used in dCAPS assays that is compatible with indels. This tool should have wide utility for screening editing events following CRISPR/Cas9 mutagenesis as well as for identifying specific editing events in a pool of CRISPR-mediated mutagenesis events. This tool was field-tested in a CRISPR mutagenesis experiment targeting a cytokinin receptor (AHK3) in Arabidopsis thaliana. The tool suggested primers that successfully distinguished between wild-type and edited alleles of a target locus and facilitated the isolation of two novel ahk3 null alleles. Users can access indCAPS and design PCR primers to employ dCAPS to identify CRISPR/Cas9 alleles at http://indcaps.kieber.cloudapps.unc.edu/.

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

PubMed

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

2016-05-01

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

A Simple Combinatorial Codon Mutagenesis Method for Targeted Protein Engineering.

PubMed

Belsare, Ketaki D; Andorfer, Mary C; Cardenas, Frida S; Chael, Julia R; Park, Hyun June; Lewis, Jared C

2017-03-17

Directed evolution is a powerful tool for optimizing enzymes, and mutagenesis methods that improve enzyme library quality can significantly expedite the evolution process. Here, we report a simple method for targeted combinatorial codon mutagenesis (CCM). To demonstrate the utility of this method for protein engineering, CCM libraries were constructed for cytochrome P450 BM3 , pfu prolyl oligopeptidase, and the flavin-dependent halogenase RebH; 10-26 sites were targeted for codon mutagenesis in each of these enzymes, and libraries with a tunable average of 1-7 codon mutations per gene were generated. Each of these libraries provided improved enzymes for their respective transformations, which highlights the generality, simplicity, and tunability of CCM for targeted protein engineering.

Seamless correction of the sickle cell disease mutation of the HBB gene in human induced pluripotent stem cells using TALENs.

PubMed

Sun, Ning; Zhao, Huimin

2014-05-01

Sickle cell disease (SCD) is the most common human genetic disease which is caused by a single mutation of human β-globin (HBB) gene. The lack of long-term treatment makes the development of reliable cell and gene therapies highly desirable. Disease-specific patient-derived human induced pluripotent stem cells (hiPSCs) have great potential for developing novel cell and gene therapies. With the disease-causing mutations corrected in situ, patient-derived hiPSCs can restore normal cell functions and serve as a renewable autologous cell source for the treatment of genetic disorders. Here we successfully utilized transcription activator-like effector nucleases (TALENs), a recently emerged novel genome editing tool, to correct the SCD mutation in patient-derived hiPSCs. The TALENs we have engineered are highly specific and generate minimal off-target effects. In combination with piggyBac transposon, TALEN-mediated gene targeting leaves no residual ectopic sequences at the site of correction and the corrected hiPSCs retain full pluripotency and a normal karyotype. Our study demonstrates an important first step of using TALENs for the treatment of genetic diseases such as SCD, which represents a significant advance toward hiPSC-based cell and gene therapies. © 2013 Wiley Periodicals, Inc.

Insight into the Meligethes aeneus voltage-sensitive sodium channel structure and an attempt to select the best pyrethroid ligands.

PubMed

Obrępalska-Stęplowska, Aleksandra; Czerwoniec, Anna; Wieczorek, Przemysław; Wrzesińska, Barbara

2016-01-01

The voltage-sensitive sodium channel (VSSC) is a target for the pharmacological action of pyrethroids which are used in controlling pests, including those of agricultural importance. Among these is the pollen beetle (Meligethes aeneus F.) - the most serious pest of Brassica napus. Owing to the heavy use of pyrethroids, a widespread build-up of resistance has occurred. The main cause of pyrethroid insensitivity in M. aeneus is considered to be an increased oxidative metabolism; however, the additional mechanism of resistance associated with mutations in the VSSC might contribute to this phenomenon. We generated a VSSC 3D model to study the docking affinities of pyrethroids to their target site within the channel. Our goal was to identify the pyrethroids for which docking affinity scores were high and not affected by potential mutations in the VSSC. We found that the docking scores of cypermethrin are hardly influenced by the appearance of point mutations. Additionally, tau-fluvalinate, deltamethrin and bifenthrin are VSSC ligands with high affinity scores. Our docking models suggest that point mutations in the VSSC binding pocket might affect the stability of ligand interactions and change the pattern of ligand docking locations, which might have a potential effect on VSSC gating properties. © 2015 Society of Chemical Industry.

Genomic Characterization of Upper Tract Urothelial Carcinoma.

PubMed

Sfakianos, John P; Cha, Eugene K; Iyer, Gopa; Scott, Sasinya N; Zabor, Emily C; Shah, Ronak H; Ren, Qinghu; Bagrodia, Aditya; Kim, Philip H; Hakimi, A Ari; Ostrovnaya, Irina; Ramirez, Ricardo; Hanrahan, Aphrothiti J; Desai, Neil B; Sun, Arony; Pinciroli, Patrizia; Rosenberg, Jonathan E; Dalbagni, Guido; Schultz, Nikolaus; Bajorin, Dean F; Reuter, Victor E; Berger, Michael F; Bochner, Bernard H; Al-Ahmadie, Hikmat A; Solit, David B; Coleman, Jonathan A

2015-12-01

Despite a similar histologic appearance, upper tract urothelial carcinoma (UTUC) and urothelial carcinoma of the bladder (UCB) tumors have distinct epidemiologic and clinicopathologic differences. To investigate whether the differences between UTUC and UCB result from intrinsic biological diversity. Tumor and germline DNA from patients with UTUC (n=83) and UCB (n=102) were analyzed using a custom next-generation sequencing assay to identify somatic mutations and copy number alterations in 300 cancer-associated genes. We described co-mutation patterns and copy number alterations in UTUC. We also compared mutation frequencies in high-grade UTUC (n=59) and high-grade UCB (n=102). Comparison of high-grade UTUC and UCB revealed significant differences in the prevalence of somatic alterations. Genes altered more commonly in high-grade UTUC included FGFR3 (35.6% vs 21.6%; p=0.065), HRAS (13.6% vs 1.0%; p=0.001), and CDKN2B (15.3% vs 3.9%; p=0.016). Genes less frequently mutated in high-grade UTUC included TP53 (25.4% vs 57.8%; p<0.001), RB1 (0.0% vs 18.6%; p<0.001), and ARID1A (13.6% vs 27.5%; p=0.050). Because our assay was restricted to genomic alterations in a targeted panel, rare mutations and epigenetic changes were not analyzed. High-grade UTUC tumors display a spectrum of genetic alterations similar to high-grade UCB. However, there were significant differences in the prevalence of several recurrently mutated genes including HRAS, TP53, and RB1. As relevant targeted inhibitors are being developed and tested, these results may have important implications for the site-specific management of patients with urothelial carcinoma. Comparison of next-generation sequencing of upper tract urothelial carcinoma (UTUC) with urothelial bladder cancer identified that similar mutations were present in both cancer types but at different frequencies, indicating a potential need for unique management strategies. UTUC tumors were found to have a high rate of mutations that could be targeted with novel therapies. Copyright © 2015 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi.

PubMed

Kaulich, Manuel; Lee, Yeon J; Lönn, Peter; Springer, Aaron D; Meade, Bryan R; Dowdy, Steven F

2015-04-20

Gene knockout strategies, RNAi and rescue experiments are all employed to study mammalian gene function. However, the disadvantages of these approaches include: loss of function adaptation, reduced viability and gene overexpression that rarely matches endogenous levels. Here, we developed an endogenous gene knockdown/rescue strategy that combines RNAi selectivity with a highly efficient CRISPR directed recombinant Adeno-Associated Virus (rAAV) mediated gene targeting approach to introduce allele-specific mutations plus an allele-selective siRNA Sensitive (siSN) site that allows for studying gene mutations while maintaining endogenous expression and regulation of the gene of interest. CRISPR/Cas9 plus rAAV targeted gene-replacement and introduction of allele-specific RNAi sensitivity mutations in the CDK2 and CDK1 genes resulted in a >85% site-specific recombination of Neo-resistant clones versus ∼8% for rAAV alone. RNAi knockdown of wild type (WT) Cdk2 with siWT in heterozygotic knockin cells resulted in the mutant Cdk2 phenotype cell cycle arrest, whereas allele specific knockdown of mutant CDK2 with siSN resulted in a wild type phenotype. Together, these observations demonstrate the ability of CRISPR plus rAAV to efficiently recombine a genomic locus and tag it with a selective siRNA sequence that allows for allele-selective phenotypic assays of the gene of interest while it remains expressed and regulated under endogenous control mechanisms. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Neutrophil Elastase, Proteinase 3, and Cathepsin G as Therapeutic Targets in Human Diseases

PubMed Central

Horwitz, Marshall S.; Jenne, Dieter E.; Gauthier, Francis

2010-01-01

Polymorphonuclear neutrophils are the first cells recruited to inflammatory sites and form the earliest line of defense against invading microorganisms. Neutrophil elastase, proteinase 3, and cathepsin G are three hematopoietic serine proteases stored in large quantities in neutrophil cytoplasmic azurophilic granules. They act in combination with reactive oxygen species to help degrade engulfed microorganisms inside phagolysosomes. These proteases are also externalized in an active form during neutrophil activation at inflammatory sites, thus contributing to the regulation of inflammatory and immune responses. As multifunctional proteases, they also play a regulatory role in noninfectious inflammatory diseases. Mutations in the ELA2/ELANE gene, encoding neutrophil elastase, are the cause of human congenital neutropenia. Neutrophil membrane-bound proteinase 3 serves as an autoantigen in Wegener granulomatosis, a systemic autoimmune vasculitis. All three proteases are affected by mutations of the gene (CTSC) encoding dipeptidyl peptidase I, a protease required for activation of their proform before storage in cytoplasmic granules. Mutations of CTSC cause Papillon-Lefèvre syndrome. Because of their roles in host defense and disease, elastase, proteinase 3, and cathepsin G are of interest as potential therapeutic targets. In this review, we describe the physicochemical functions of these proteases, toward a goal of better delineating their role in human diseases and identifying new therapeutic strategies based on the modulation of their bioavailability and activity. We also describe how nonhuman primate experimental models could assist with testing the efficacy of proposed therapeutic strategies. PMID:21079042

Abundant off-target edits from site-directed RNA editing can be reduced by nuclear localization of the editing enzyme.

PubMed

Vallecillo-Viejo, Isabel C; Liscovitch-Brauer, Noa; Montiel-Gonzalez, Maria Fernanda; Eisenberg, Eli; Rosenthal, Joshua J C

2018-01-02

Site-directed RNA editing (SDRE) is a general strategy for making targeted base changes in RNA molecules. Although the approach is relatively new, several groups, including our own, have been working on its development. The basic strategy has been to couple the catalytic domain of an adenosine (A) to inosine (I) RNA editing enzyme to a guide RNA that is used for targeting. Although highly efficient on-target editing has been reported, off-target events have not been rigorously quantified. In this report we target premature termination codons (PTCs) in messages encoding both a fluorescent reporter protein and the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein transiently transfected into human epithelial cells. We demonstrate that while on-target editing is efficient, off-target editing is extensive, both within the targeted message and across the entire transcriptome of the transfected cells. By redirecting the editing enzymes from the cytoplasm to the nucleus, off-target editing is reduced without compromising the on-target editing efficiency. The addition of the E488Q mutation to the editing enzymes, a common strategy for increasing on-target editing efficiency, causes a tremendous increase in off-target editing. These results underscore the need to reduce promiscuity in current approaches to SDRE.

ClpXP protease targets long-lived DNA translocation states of a helicase-like motor to cause restriction alleviation

PubMed Central

Simons, Michelle; Diffin, Fiona M.; Szczelkun, Mark D.

2014-01-01

We investigated how Escherichia coli ClpXP targets the helicase-nuclease (HsdR) subunit of the bacterial Type I restriction–modification enzyme EcoKI during restriction alleviation (RA). RA is a temporary reduction in endonuclease activity that occurs when Type I enzymes bind unmodified recognition sites on the host genome. These conditions arise upon acquisition of a new system by a naïve host, upon generation of new sites by genome rearrangement/mutation or during homologous recombination between hemimethylated DNA. Using recombinant DNA and proteins in vitro, we demonstrate that ClpXP targets EcoKI HsdR during dsDNA translocation on circular DNA but not on linear DNA. Protein roadblocks did not activate HsdR proteolysis. We suggest that DNA translocation lifetime, which is elevated on circular DNA relative to linear DNA, is important to RA. To identify the ClpX degradation tag (degron) in HsdR, we used bioinformatics and biochemical assays to design N- and C-terminal mutations that were analysed in vitro and in vivo. None of the mutants produced a phenotype consistent with loss of the degron, suggesting an as-yet-unidentified recognition pathway. We note that an EcoKI nuclease mutant still produces cell death in a clpx− strain, consistent with DNA damage induced by unregulated motor activity. PMID:25260590

Progress in Genome Editing Technology and Its Application in Plants

PubMed Central

Zhang, Kai; Raboanatahiry, Nadia; Zhu, Bin; Li, Maoteng

2017-01-01

Genome editing technology (GET) is a versatile approach that has progressed rapidly as a mechanism to alter the genotype and phenotype of organisms. However, conventional genome modification using GET cannot satisfy current demand for high-efficiency and site-directed mutagenesis, retrofitting of artificial nucleases has developed into a new avenue within this field. Based on mechanisms to recognize target genes, newly-developed GETs can generally be subdivided into three cleavage systems, protein-dependent DNA cleavage systems (i.e., zinc-finger nucleases, ZFN, and transcription activator-like effector nucleases, TALEN), RNA-dependent DNA cleavage systems (i.e., clustered regularly interspaced short palindromic repeats-CRISPR associated proteins, CRISPR-Cas9, CRISPR-Cpf1, and CRISPR-C2c1), and RNA-dependent RNA cleavage systems (i.e., RNA interference, RNAi, and CRISPR-C2c2). All these techniques can lead to double-stranded (DSB) or single-stranded breaks (SSB), and result in either random mutations via non-homologous end-joining (NHEJ) or targeted mutation via homologous recombination (HR). Thus, site-directed mutagenesis can be induced via targeted gene knock-out, knock-in, or replacement to modify specific characteristics including morphology-modification, resistance-enhancement, and physiological mechanism-improvement along with plant growth and development. In this paper, an non-comprehensive review on the development of different GETs as applied to plants is presented. PMID:28261237

A variable DNA recognition site organization establishes the LiaR-mediated cell envelope stress response of enterococci to daptomycin

DOE PAGES

Davlieva, Milya; Shi, Yiwen; Leonard, Paul G.; ...

2015-04-19

LiaR is a ‘master regulator’ of the cell envelope stress response in enterococci and many other Gram-positive organisms. Mutations to liaR can lead to antibiotic resistance to a variety of antibiotics including the cyclic lipopeptide daptomycin. LiaR is phosphorylated in response to membrane stress to regulate downstream target operons. Using DNA footprinting of the regions upstream of the liaXYZ and liaFSR operons we show that LiaR binds an extended stretch of DNA that extends beyond the proposed canonical consensus sequence suggesting a more complex level of regulatory control of target operons. We go on to determine the biochemical and structuralmore » basis for increased resistance to daptomycin by the adaptive mutation to LiaR (D191N) first identified from the pathogen Enterococcus faecalis S613. LiaR D191N increases oligomerization of LiaR to form a constitutively activated tetramer that has high affinity for DNA even in the absence of phosphorylation leading to increased resistance. The crystal structures of the LiaR DNA binding domain complexed to the putative consensus sequence as well as an adjoining secondary sequence show that upon binding, LiaR induces DNA bending that is consistent with increased recruitment of RNA polymerase to the transcription start site and upregulation of target operons.« less

In silico identification of novel kinase inhibitors targeting wild-type and T315I mutant ABL1 from FDA-approved drugs.

PubMed

Xu, Huai-long; Wang, Zi-jie; Liang, Xiao-meng; Li, Xin; Shi, Zheng; Zhou, Nan; Bao, Jin-ku

2014-06-01

The constitutively active fusion protein BCR-ABL1 is the major cause of chronic myeloid leukemia (CML), and selective inhibition of ABL1 is a promising approach for the treatment of CML. Reported drugs worked well in clinical practice, such as imatinib, dasatinib, nilotinib and bosutinib. However, resistance arises due to ABL1 mutation in patients, especially the T315I gate-keeper mutation. Thus, wide spectrum drugs targeting ABL1 are urgently needed. In order to screen potential drugs targeting wild-type ABL1 and T315I mutant ABL1, 1408 FDA approved small molecule drugs were subjected to molecular docking. With subsequent molecular dynamic (MD) simulation and MM/GBSA binding free energy calculation and energy decomposition, we identified chlorhexidine and sorafenib as potential "new use" drugs targeting wild-type ABL1, while nicergoline and plerixafor targeted T315I ABL1. Meanwhile, we also found that residues located in the ATP-binding site and A-loop motif played key roles in drug discovery towards ABL1. These findings may not only serve as a paradigm for the repositioning of existing approved drugs, but also instill new vitality to ABL1-targeted anti-CML therapeutics.

The role of small molecule Kit protein-tyrosine kinase inhibitors in the treatment of neoplastic disorders.

PubMed

Roskoski, Robert

2018-04-25

The Kit proto-oncogene was found as a consequence of the discovery of the feline v-kit sarcoma oncogene. Stem cell factor (SCF) is the Kit ligand and it mediates Kit dimerization and activation. The Kit receptor contains an extracellular segment that is made up of five immunoglobulin-like domains (D1/2/3/4/5), a transmembrane segment, a juxtamembrane segment, a protein-tyrosine kinase domain that contains an insert of 77 amino acid residues, and a carboxyterminal tail. Activating somatic mutations in Kit have been documented in various neoplasms including gastrointestinal stromal tumors (GIST), mast cell overexpression (systemic mastocytosis), core-binding factor acute myeloid leukemias (AML), melanomas, and seminomas. In the case of gastrointestinal stromal tumors, most activating mutations occur in the juxtamembrane segment and these mutants are initially sensitive to imatinib. As with many targeted anticancer drugs, resistance to Kit antagonists occurs in about two years and is the result of secondary KIT mutations. An activation segment exon 17 D816V mutation is one of the more common resistance mutations in Kit and this mutant is resistant to imatinib and sorafenib. Type I protein kinase inhibitors interact with the active enzyme form with DFG-D of the proximal activation segment directed inward toward the active site (DFG-D in ). In contrast, type II inhibitors bind to their target with the DFG-D pointing away from the active site (DFG-D out ). Based upon the X-ray crystallographic structures, imatinib, sunitinib, and ponatinib are Type II Kit inhibitors. We used the Schrödinger induced fit docking protocol to model the interaction of midostaurin with Kit and the result indicates that it binds to the DFG-D in conformation of the receptor and is thus classified as type I inhibitor. This medication inhibits the notoriously resistant Kit D816V mutant and is approved for the treatment of systemic mastocytosis and is effective against tumors bearing the D816V activation/resistance mutation. Copyright © 2018 Elsevier Ltd. All rights reserved.

Pitfalls in the molecular genetic diagnosis of Leber hereditary optic neuropathy (LHON)

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

Johns, D.R.; Neufeld, M.J.

1993-10-01

Pathogenetic mutations in mtDNA are found in the majority of patients with Leber hereditary optic neuropathy (LHON), and molecular genetic techniques to detect them are important for diagnosis. A false-positive molecular genetic error has adverse consequences for the diagnosis of this maternally inherited disease. The authors found a number of mtDNA polymorphisms that occur adjacent to known LHON-associated mutations and that confound their molecular genetic detection. These transition mutations occur at mtDNA nt 11779 (SfaNI site loss, 11778 mutation), nt 3459 (BsaHI site loss, 3460 mutation), nt 15258 (AccI site loss, 15257 mutation), nt 14485 (mismatch primer Sau3AI site loss,more » 14484 mutation), and nt 13707 (BstNI site loss, 13708 mutation). Molecular genetic detection of the most common pathogenetic mtDNA mutations in LHON, using a single restriction enzyme, may be confounded by adjacent polymorphisms that occur with a false-positive rate of 2%-7%. 19 refs.« less

Genomic signatures of selection at linked sites: unifying the disparity among species

PubMed Central

Cutter, Asher D.; Payseur, Bret A.

2014-01-01

Population genetics theory supplies powerful predictions about how natural selection interacts with genetic linkage to sculpt the genomic landscape of nucleotide polymorphism. Both the spread of beneficial mutations and removal of deleterious mutations act to depress polymorphism levels, especially in low-recombination regions. However, empiricists have documented extreme disparities among species. Here we characterize the dominant features that could drive variation in linked selection among species, including roles for selective sweeps being ‘hard’ or ‘soft’, and concealing by demography and genomic confounds. We advocate targeted studies of close relatives to unify our understanding of how selection and linkage interact to shape genome evolution. PMID:23478346

Mutants of Cre recombinase with improved accuracy

PubMed Central

Eroshenko, Nikolai; Church, George M.

2013-01-01

Despite rapid advances in genome engineering technologies, inserting genes into precise locations in the human genome remains an outstanding problem. It has been suggested that site-specific recombinases can be adapted towards use as transgene delivery vectors. The specificity of recombinases can be altered either with directed evolution or via fusions to modular DNA-binding domains. Unfortunately, both wildtype and altered variants often have detectable activities at off-target sites. Here we use bacterial selections to identify mutations in the dimerization surface of Cre recombinase (R32V, R32M, and 303GVSdup) that improve the accuracy of recombination. The mutants are functional in bacteria, in human cells, and in vitro (except for 303GVSdup, which we did not purify), and have improved selectivity against both model off-target sites and the entire E. coli genome. We propose that destabilizing binding cooperativity may be a general strategy for improving the accuracy of dimeric DNA-binding proteins. PMID:24056590

Multifocal clonal evolution characterized using circulating tumour DNA in a case of metastatic breast cancer

PubMed Central

Murtaza, Muhammed; Dawson, Sarah-Jane; Pogrebniak, Katherine; Rueda, Oscar M.; Provenzano, Elena; Grant, John; Chin, Suet-Feung; Tsui, Dana W. Y.; Marass, Francesco; Gale, Davina; Ali, H. Raza; Shah, Pankti; Contente-Cuomo, Tania; Farahani, Hossein; Shumansky, Karey; Kingsbury, Zoya; Humphray, Sean; Bentley, David; Shah, Sohrab P.; Wallis, Matthew; Rosenfeld, Nitzan; Caldas, Carlos

2015-01-01

Circulating tumour DNA analysis can be used to track tumour burden and analyse cancer genomes non-invasively but the extent to which it represents metastatic heterogeneity is unknown. Here we follow a patient with metastatic ER-positive and HER2-positive breast cancer receiving two lines of targeted therapy over 3 years. We characterize genomic architecture and infer clonal evolution in eight tumour biopsies and nine plasma samples collected over 1,193 days of clinical follow-up using exome and targeted amplicon sequencing. Mutation levels in the plasma samples reflect the clonal hierarchy inferred from sequencing of tumour biopsies. Serial changes in circulating levels of sub-clonal private mutations correlate with different treatment responses between metastatic sites. This comparison of biopsy and plasma samples in a single patient with metastatic breast cancer shows that circulating tumour DNA can allow real-time sampling of multifocal clonal evolution. PMID:26530965

Multifocal clonal evolution characterized using circulating tumour DNA in a case of metastatic breast cancer.

PubMed

Murtaza, Muhammed; Dawson, Sarah-Jane; Pogrebniak, Katherine; Rueda, Oscar M; Provenzano, Elena; Grant, John; Chin, Suet-Feung; Tsui, Dana W Y; Marass, Francesco; Gale, Davina; Ali, H Raza; Shah, Pankti; Contente-Cuomo, Tania; Farahani, Hossein; Shumansky, Karey; Kingsbury, Zoya; Humphray, Sean; Bentley, David; Shah, Sohrab P; Wallis, Matthew; Rosenfeld, Nitzan; Caldas, Carlos

2015-11-04

Circulating tumour DNA analysis can be used to track tumour burden and analyse cancer genomes non-invasively but the extent to which it represents metastatic heterogeneity is unknown. Here we follow a patient with metastatic ER-positive and HER2-positive breast cancer receiving two lines of targeted therapy over 3 years. We characterize genomic architecture and infer clonal evolution in eight tumour biopsies and nine plasma samples collected over 1,193 days of clinical follow-up using exome and targeted amplicon sequencing. Mutation levels in the plasma samples reflect the clonal hierarchy inferred from sequencing of tumour biopsies. Serial changes in circulating levels of sub-clonal private mutations correlate with different treatment responses between metastatic sites. This comparison of biopsy and plasma samples in a single patient with metastatic breast cancer shows that circulating tumour DNA can allow real-time sampling of multifocal clonal evolution.

Whole organism lineage tracing by combinatorial and cumulative genome editing

PubMed Central

McKenna, Aaron; Findlay, Gregory M.; Gagnon, James A.; Horwitz, Marshall S.; Schier, Alexander F.; Shendure, Jay

2016-01-01

Multicellular systems develop from single cells through distinct lineages. However, current lineage tracing approaches scale poorly to whole, complex organisms. Here we use genome editing to progressively introduce and accumulate diverse mutations in a DNA barcode over multiple rounds of cell division. The barcode, an array of CRISPR/Cas9 target sites, marks cells and enables the elucidation of lineage relationships via the patterns of mutations shared between cells. In cell culture and zebrafish, we show that rates and patterns of editing are tunable, and that thousands of lineage-informative barcode alleles can be generated. By sampling hundreds of thousands of cells from individual zebrafish, we find that most cells in adult organs derive from relatively few embryonic progenitors. In future analyses, genome editing of synthetic target arrays for lineage tracing (GESTALT) can be used to generate large-scale maps of cell lineage in multicellular systems for normal development and disease. PMID:27229144

The loss-of-allele assay for ES cell screening and mouse genotyping.

PubMed

Frendewey, David; Chernomorsky, Rostislav; Esau, Lakeisha; Om, Jinsop; Xue, Yingzi; Murphy, Andrew J; Yancopoulos, George D; Valenzuela, David M

2010-01-01

Targeting vectors used to create directed mutations in mouse embryonic stem (ES) cells consist, in their simplest form, of a gene for drug selection flanked by mouse genomic sequences, the so-called homology arms that promote site-directed homologous recombination between the vector and the target gene. The VelociGene method for the creation of targeted mutations in ES cells employs targeting vectors, called BACVecs, that are based on bacterial artificial chromosomes. Compared with conventional short targeting vectors, BacVecs provide two major advantages: (1) their much larger homology arms promote high targeting efficiencies without the need for isogenicity or negative selection strategies; and (2) they enable deletions and insertions of up to 100kb in a single targeting event, making possible gene-ablating definitive null alleles and other large-scale genomic modifications. Because of their large arm sizes, however, BACVecs do not permit screening by conventional assays, such as long-range PCR or Southern blotting, that link the inserted targeting vector to the targeted locus. To exploit the advantages of BACVecs for gene targeting, we inverted the conventional screening logic in developing the loss-of-allele (LOA) assay, which quantifies the number of copies of the native locus to which the mutation was directed. In a correctly targeted ES cell clone, the LOA assay detects one of the two native alleles (for genes not on the X or Y chromosome), the other allele being disrupted by the targeted modification. We apply the same principle in reverse as a gain-of-allele assay to quantify the copy number of the inserted targeting vector. The LOA assay reveals a correctly targeted clone as having lost one copy of the native target gene and gained one copy of the drug resistance gene or other inserted marker. The combination of these quantitative assays makes LOA genotyping unequivocal and amenable to automated scoring. We use the quantitative polymerase chain reaction (qPCR) as our method of allele quantification, but any method that can reliably distinguish the difference between one and two copies of the target gene can be used to develop an LOA assay. We have designed qPCR LOA assays for deletions, insertions, point mutations, domain swaps, conditional, and humanized alleles and have used the insert assays to quantify the copy number of random insertion BAC transgenics. Because of its quantitative precision, specificity, and compatibility with high throughput robotic operations, the LOA assay eliminates bottlenecks in ES cell screening and mouse genotyping and facilitates maximal speed and throughput for knockout mouse production. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Amicoumacin A inhibits translation by stabilizing mRNA interaction with the ribosome

PubMed Central

Polikanov, Yury S.; Osterman, Ilya A.; Szal, Teresa; Tashlitsky, Vadim N.; Serebryakova, Marina V.; Kusochek, Pavel; Bulkley, David; Malanicheva, Irina A.; Efimenko, Tatyana A.; Efremenkova, Olga V.; Konevega, Andrey L.; Shaw, Karen J.; Bogdanov, Alexey A.; Rodnina, Marina V.; Dontsova, Olga A.; Mankin, Alexander S.; Steitz, Thomas A.; Sergiev, Petr V.

2014-01-01

SUMMARY We demonstrate that the antibiotic amicoumacin A (AMI) whose cellular target was unknown, is a potent inhibitor of protein synthesis. Resistance mutations in helix 24 of the 16S rRNA mapped the AMI binding site to the small ribosomal subunit. The crystal structure of bacterial ribosome in complex with AMI solved at 2.4 Å resolution revealed that the antibiotic makes contacts with universally conserved nucleotides of 16S rRNA in the E site and the mRNA backbone. Simultaneous interactions of AMI with 16S rRNA and mRNA and the in vivo experimental evidence suggest that it may inhibit the progression of the ribosome along mRNA. Consistent with this proposal, binding of AMI interferes with translocation in vitro. The inhibitory action of AMI can be partly compensated by mutations in the translation elongation factor G. PMID:25306919

DNA abasic site-directed formation of fluorescent silver nanoclusters for selective nucleobase recognition

NASA Astrophysics Data System (ADS)

Ma, Kun; Cui, Qinghua; Liu, Guiying; Wu, Fei; Xu, Shujuan; Shao, Yong

2011-07-01

DNA single-nucleotide polymorphism (SNP) detection has attracted much attention due to mutation related diseases. Various methods for SNP detection have been proposed and many are already in use. Here, we find that the abasic site (AP site) in the DNA duplex can be developed as a capping scaffold for the generation of fluorescent silver nanoclusters (Ag NCs). As a proof of concept, the DNA sequences from fragments near codon 177 of cancer supression gene p53 were used as a model for SNP detection by in situ formed Ag NCs. The formation of fluorescent Ag NCs in the AP site-containing DNA duplex is highly selective for cytosine facing the AP site and guanines flanking the site and can be employed in situ as readout for SNP detection. The fluorescent signal-on sensing for SNP based on this inorganic fluorophore is substantially advantageous over the previously reported signal-off responses using low-molecular-weight organic ligands. The strong dependence of fluorescent Ag NC formation on the sequences surrounding the AP site was successfully used to identify mutations in codon 177 of cancer supression gene p53. We anticipate that this approach will be employed to develop a practical SNP detection method by locating an AP site toward the midway cytosine in a target strand containing more than three consecutive cytosines.

Enhanced thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 by rational engineering of a glycine to proline mutation.

PubMed

Tian, Jian; Wang, Ping; Gao, Shan; Chu, Xiaoyu; Wu, Ningfeng; Fan, Yunliu

2010-12-01

Protein thermostability can be increased by some glycine to proline mutations in a target protein. However, not all glycine to proline mutations can improve protein thermostability, and this method is suitable only at carefully selected mutation sites that can accommodate structural stabilization. In this study, homology modeling and molecular dynamics simulations were used to select appropriate glycine to proline mutations to improve protein thermostability, and the effect of the selected mutations was proved by the experiments. The structure of methyl parathion hydrolase (MPH) from Ochrobactrum sp. M231 (Ochr-MPH) was constructed by homology modeling, and molecular dynamics simulations were performed on the modeled structure. A profile of the root mean square fluctuations of Ochr-MPH was calculated at the nanosecond timescale, and an eight-amino acid loop region (residues 186-193) was identified as having high conformational fluctuation. The two glycines nearest to this region were selected as mutation targets that might affect protein flexibility in the vicinity. The structures and conformational fluctuations of two single mutants (G194P and G198P) and one double mutant (G194P/G198P) were modeled and analyzed using molecular dynamics simulations. The results predicted that the mutant G194P had the decreased conformational fluctuation in the loop region and might increase the thermostability of Ochr-MPH. The thermostability and kinetic behavior of the wild-type and three mutant enzymes were measured. The results were consistent with the computational predictions, and the mutant G194P was found to have higher thermostability than the wild-type enzyme. © 2010 The Authors Journal compilation © 2010 FEBS.

Convergent pathogenic pathways in Alzheimer’s and Huntington disease: Shared targets for drug development

PubMed Central

Ehrnhoefer, Dagmar E.; Wong, Bibiana K.Y.; Hayden, Michael R.

2011-01-01

Neurodegenerative diseases exemplified by Alzheimer’s and Huntington disease are characterized by the progressive neuropsychiatric dysfunction and loss of specific neuronal subtypes. Even though there are differences in the exact sites of pathology and clinical profiles only partially overlap, considerable similarities in disease mechanisms and pathogenic pathways can be observed. These shared mechanisms raise the possibility of common therapeutic targets for drug development. Huntington disease with a monogenic cause and the possibility to accurately identify pre-manifest mutation carriers could be exploited as a ‘model’ for Alzheimer’s disease to test the efficacy of therapeutic interventions targeting shared pathogenic pathways. PMID:22015920

Mutation of the C/EBP binding sites in the Rous sarcoma virus long terminal repeat and gag enhancers.

PubMed Central

Ryden, T A; de Mars, M; Beemon, K

1993-01-01

Several C/EBP binding sites within the Rous sarcoma virus (RSV) long terminal repeat (LTR) and gag enhancers were mutated, and the effect of these mutations on viral gene expression was assessed. Minimal site-specific mutations in each of three adjacent C/EBP binding sites in the LTR reduced steady-state viral RNA levels. Double mutation of the two 5' proximal LTR binding sites resulted in production of 30% of wild-type levels of virus. DNase I footprinting analysis of mutant DNAs indicated that the mutations blocked C/EBP binding at the affected sites. Additional C/EBP binding sites were identified upstream of the 3' LTR and within the 5' end of the LTRs. Point mutations in the RSV gag intragenic enhancer region, which blocked binding of C/EBP at two of three adjacent C/EBP sites, also reduced virus production significantly. Nuclear extracts prepared from both chicken embryo fibroblasts (CEFs) and chicken muscle contained proteins binding to the same RSV DNA sites as did C/EBP, and mutations that prevented C/EBP binding also blocked binding of these chicken proteins. It appears that CEFs and chicken muscle contain distinct proteins binding to these RSV DNA sites; the CEF binding protein was heat stable, as is C/EBP, while the chicken muscle protein was heat sensitive. Images PMID:8386280

Co-evolution of somatic variation in primary and metastatic colorectal cancer may expand biopsy indications in the molecular era.

PubMed

Kim, Richard; Schell, Michael J; Teer, Jamie K; Greenawalt, Danielle M; Yang, Mingli; Yeatman, Timothy J

2015-01-01

Metastasis is thought to be a clonal event whereby a single cell initiates the development of a new tumor at a distant site. However the degree to which primary and metastatic tumors differ on a molecular level remains unclear. To further evaluate these concepts, we used next generation sequencing (NGS) to assess the molecular composition of paired primary and metastatic colorectal cancer tissue specimens. 468 colorectal tumor samples from a large personalized medicine initiative were assessed by targeted gene sequencing of 1,321 individual genes. Eighteen patients produced genomic profiles for 17 paired primary:metastatic (and 2 metastatic:metastatic) specimens. An average of 33.3 mutations/tumor were concordant (shared) between matched samples, including common well-known genes (APC, KRAS, TP53). An average of 2.3 mutations/tumor were discordant (unshared) among paired sites. KRAS mutational status was always concordant. The overall concordance rate for mutations was 93.5%; however, nearly all (18/19 (94.7%)) paired tumors showed at least one mutational discordance. Mutations were seen in: TTN, the largest gene (5 discordant pairs), ADAMTS20, APC, MACF1, RASA1, TP53, and WNT2 (2 discordant pairs), SMAD2, SMAD3, SMAD4, FBXW7, and 66 others (1 discordant pair). Whereas primary and metastatic tumors displayed little variance overall, co-evolution produced incremental mutations in both. These results suggest that while biopsy of the primary tumor alone is likely sufficient in the chemotherapy-naïve patient, additional biopsies of primary or metastatic disease may be necessary to precisely tailor therapy following chemotherapy resistance or insensitivity in order to adequately account for tumor evolution.

Co-Evolution of Somatic Variation in Primary and Metastatic Colorectal Cancer May Expand Biopsy Indications in the Molecular Era

PubMed Central

Kim, Richard; Schell, Michael J.; Teer, Jamie K.; Greenawalt, Danielle M.; Yang, Mingli; Yeatman, Timothy J.

2015-01-01

Introduction Metastasis is thought to be a clonal event whereby a single cell initiates the development of a new tumor at a distant site. However the degree to which primary and metastatic tumors differ on a molecular level remains unclear. To further evaluate these concepts, we used next generation sequencing (NGS) to assess the molecular composition of paired primary and metastatic colorectal cancer tissue specimens. Methods 468 colorectal tumor samples from a large personalized medicine initiative were assessed by targeted gene sequencing of 1,321 individual genes. Eighteen patients produced genomic profiles for 17 paired primary:metastatic (and 2 metastatic:metastatic) specimens. Results An average of 33.3 mutations/tumor were concordant (shared) between matched samples, including common well-known genes (APC, KRAS, TP53). An average of 2.3 mutations/tumor were discordant (unshared) among paired sites. KRAS mutational status was always concordant. The overall concordance rate for mutations was 93.5%; however, nearly all (18/19 (94.7%)) paired tumors showed at least one mutational discordance. Mutations were seen in: TTN, the largest gene (5 discordant pairs), ADAMTS20, APC, MACF1, RASA1, TP53, and WNT2 (2 discordant pairs), SMAD2, SMAD3, SMAD4, FBXW7, and 66 others (1 discordant pair). Conclusions Whereas primary and metastatic tumors displayed little variance overall, co-evolution produced incremental mutations in both. These results suggest that while biopsy of the primary tumor alone is likely sufficient in the chemotherapy-naïve patient, additional biopsies of primary or metastatic disease may be necessary to precisely tailor therapy following chemotherapy resistance or insensitivity in order to adequately account for tumor evolution. PMID:25974029

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

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

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

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

Alanine to valine substitutions in the pore helix IIIP1 and linker-helix IIIL45 confer cockroach sodium channel resistance to DDT and pyrethroids.

PubMed

Chen, Mengli; Du, Yuzhe; Nomura, Yoshiko; Zhu, Guonian; Zhorov, Boris S; Dong, Ke

2017-05-01

Pyrethroid insecticides exert toxic effects by prolonging the opening of voltage-gated sodium channels. More than 20 sodium channel mutations from arthropod pests and disease vectors have been confirmed to confer pyrethroid resistance. These mutations have been valuable in elucidating the molecular interaction between pyrethroids and sodium channels, including identification of two pyrethroid receptor sites. Previously, two alanine to valine substitutions, one in the pore helix IIIP1 and the other in the linker-helix connecting S4 and S5 in domain III (IIIL45), were found in Drosophila melanogaster mutants that are resistant to DDT and deltamethrin (a type II pyrethroid with an α-cyano group at the phenylbenzyl alcohol position, which is lacking in type I pyrethroids), but their role in target-site-mediated insecticide resistance has not been functionally confirmed. In this study, we functionally examined the two mutations in cockroach sodium channels expressed in Xenopus laevis oocytes. Both mutations caused depolarizing shifts in the voltage dependence of activation, conferred DDT resistance and also resistance to two Type I pyrethroids by almost abolishing the tail currents induced by Type I pyrethroids. In contrast, neither mutation reduced the amplitude of tail currents induced by the Type II pyrethroids, deltamethrin or cypermethrin. However, both mutations accelerated the decay of Type II pyrethroid-induced tail currents, which normally decay extremely slowly. These results provided new insight into the molecular basis of different actions of Type I and Type II pyrethroids on sodium channels. Computer modeling predicts that both mutations may allosterically affect pyrethroid binding. Copyright © 2016 Elsevier B.V. All rights reserved.

CRISPR/Cas9/sgRNA-mediated targeted gene modification confirms the cause-effect relationship between gyrA mutation and quinolone resistance in Escherichia coli.

PubMed

Qiu, Haixiang; Gong, Jiansen; Butaye, Patrick; Lu, Guangwu; Huang, Ke; Zhu, Guoqiang; Zhang, Jilei; Hathcock, Terri; Cheng, Darong; Wang, Chengming

2018-05-14

Quinolones are broad-spectrum antibiotics that have been used for decades in treating bacterial infections in humans and animals, and subsequently bacterial resistance to these agents has increased. While studies indicated the relationship between gyrA mutations and bacterial resistance to quinolones, CRISPR/Cas9 was used in this study to investigate causal role of gyrA mutation in the quinolone resistance. In this study, 818 clinical Escherichia coli isolates were analyzed for gyrA mutations and their resistance to quinolones. The CRISPR/Cas9 system was used to generate gyrA mutations in quinolone-susceptible E. coli ATCC 25922, and quinolone-resistant clinical E. coli. The antimicrobial resistance prevalence rate in E. coli against nalidixic acid, ciprofloxacin and enrofloxacin was 77.1% (631/818), 51.1% (418/818) and 49.8% (407/818), respectively. The gyrA mutations were identified in nucleotide positions 248, 255, 259, 260, 261, 273 and 300, and mutations at positions 248 and 259 resulting in amino acid changes at positions 83 and 87 were associated with quinolone resistance. Double-site amino acid mutations increase resistance to quinolones. The gyrA mutations causing changes at amino acids 83 and 87 reversed the features of quinolone resistance in ATCC and clinical strains, verifying the causal role of gyrA mutation in the quinolone resistance of E. coli.

Recent Advances in CRISPR-Cas9 Genome Editing Technology for Biological and Biomedical Investigations.

PubMed

Singh, Vijai; Gohil, Nisarg; Ramírez García, Robert; Braddick, Darren; Fofié, Christian Kuete

2018-01-01

The Type II CRISPR-Cas9 system is a simple, efficient, and versatile tool for targeted genome editing in a wide range of organisms and cell types. It continues to gain more scientific interest and has established itself as an extremely powerful technology within our synthetic biology toolkit. It works upon a targeted site and generates a double strand breaks that become repaired by either the NHEJ or the HDR pathway, modifying or permanently replacing the genomic target sequences of interest. These can include viral targets, single-mutation genetic diseases, and multiple-site corrections for wide scale disease states, offering the potential to manage and cure some of mankind's most persistent biomedical menaces. Here, we present the developing progress and future potential of CRISPR-Cas9 in biological and biomedical investigations, toward numerous therapeutic, biomedical, and biotechnological applications, as well as some of the challenges within. J. Cell. Biochem. 119: 81-94, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Pyrethroid resistance in Sitophilus zeamais is associated with a mutation (T929I) in the voltage-gated sodium channel.

PubMed

Araújo, Rúbia A; Williamson, Martin S; Bass, Christopher; Field, Linda M; Duce, Ian R

2011-08-01

The maize weevil, Sitophilus zeamais, is the most important pest affecting stored grain in Brazil and its control relies heavily on the use of insecticides. The intensive use of compounds such as the pyrethroids has led to the emergence of resistance, and previous studies have suggested that resistance to both pyrethroids and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) may result from reduced sensitivity of the insecticide target, the voltage-gated sodium channel. To identify the molecular mechanisms underlying pyrethroid resistance in S. zeamais, the domain II region of the voltage-gated sodium channel (para-orthologue) gene was amplified by PCR and sequenced from susceptible and resistant laboratory S. zeamais strains that were selected with a discriminating dose of DDT. A single point mutation, T929I, was found in the para gene of the resistant S. zeamais populations and its presence in individual weevils was strongly associated with survival after DDT exposure. This is the first identification of a target-site resistance mutation in S. zeamais and unusually it is a super-kdr type mutation occurring in the absence of the more common kdr (L1014F) substitution. A high-throughput assay based on TaqMan single nucleotide polymorphism genotyping was developed for sensitive detection of the mutation and used to screen field-collected strains of S. zeamais. This showed that the mutation is present at low frequency in field populations and is a useful tool for informing control strategies. © 2011 The Authors. Insect Molecular Biology © 2011 The Royal Entomological Society.

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

PubMed

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

2017-10-01

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

Correlation of somatic mutations and clinical outcome in melanoma patients treated with carboplatin, paclitaxel, and sorafenib

PubMed Central

Wilson, Melissa A.; Zhao, Fengmin; Letrero, Richard; D’Andrea, Kurt; Rimm, David L.; Kirkwood, John M.; Kluger, Harriet M.; Lee, Sandra J.; Schuchter, Lynn M.; Flaherty, Keith T.; Nathanson, Katherine L.

2014-01-01

Purpose Sorafenib is an inhibitor of VEGFR, PDGFR, and RAF kinases, amongst others. We assessed the association of somatic mutations with clinicopathologic features and clinical outcomes in patients with metastatic melanoma treated on E2603, comparing treatment with carboplatin, paclitaxel +/− sorafenib (CP vs. CPS). Experimental Design Pre-treatment tumor samples from 179 unique individuals enrolled on E2603 were analyzed. Genotyping was performed using a custom iPlex panel interrogating 74 mutations in 13 genes. Statistical analysis was performed using Fisher’s exact test, logistic regression, and Cox’s proportional-hazards models. Progression free survival and overall survival were estimated using Kaplan-Meier methods. Results BRAF and NRAS mutations were found at frequencies consistent with other metastatic melanoma cohorts. BRAF-mutant melanoma was associated with worse performance status, increased number of disease sites, and younger age at diagnosis; NRAS-mutant melanoma was associated with better performance status, fewer sites of disease, and female gender. BRAF and NRAS mutations were not significantly predictive of response or survival when treated with CPS vs. CP. However, patients with NRAS-mutant melanoma trended towards a worse response and PFS on CP than those with BRAF-mutant or WT/WT melanoma, an association that was reversed for this group on the CPS arm. Conclusions This study of somatic mutations in melanoma is the last prospectively collected phase III clinical trial population prior to the era of BRAF targeted therapy. A trend towards improved clinical response in patients with NRAS-mutant melanoma treated with CPS was observed, possibly due to sorafenib’s effect on CRAF. PMID:24714776

Mutation Induced Conformational Change In CaMKII Peptide Alters Binding Affinity to CaM Through Alternate Binding Site

NASA Astrophysics Data System (ADS)

Ezerski, Jacob; Cheung, Margaret

CaM forms distinct conformation states through modifications in its charge distribution upon binding to Ca2+ ions. The occurrence of protein structural change resulting from an altered charge distribution is paramount in the scheme of cellular signaling. Not only is charge induced structural change observed in CaM, it is also seen in an essential binding target: calmodulin-depended protein kinase II (CaMKII). In order to investigate the mechanism of selectivity in relation to changes in secondary structure, the CaM binding domain of CaMKII is isolated. Experimentally, charged residues of the CaMKII peptide are systematically mutated to alanine, resulting in altered binding kinetics between the peptide and the Ca2+ saturated state of CaM. We perform an all atom simulation of the wildtype (RRK) and mutated (AAA) CaMKII peptides and generate structures from the trajectory. We analyze RRK and AAA using DSSP and find significant structural differences due to the mutation. Structures from the RRK and AAA ensembles are then selected and docked onto the crystal structure of Ca2+ saturated CaM. We observe that RRK binds to CaM at the C-terminus, whereas the 3-residue mutation, AAA, shows increased patterns of binding to the N-terminus and linker regions of CaM. Due to the conformational change of the peptide ensemble from charged residue mutation, a distinct change in the binding site can be seen, which offers an explanation to experimentally observed changes in kinetic binding rates

Cell-cycle reprogramming for PI3K inhibition overrides a relapse-specific C481S BTK mutation revealed by longitudinal functional genomics in mantle cell lymphoma.

PubMed

Chiron, David; Di Liberto, Maurizio; Martin, Peter; Huang, Xiangao; Sharman, Jeff; Blecua, Pedro; Mathew, Susan; Vijay, Priyanka; Eng, Ken; Ali, Siraj; Johnson, Amy; Chang, Betty; Ely, Scott; Elemento, Olivier; Mason, Christopher E; Leonard, John P; Chen-Kiang, Selina

2014-09-01

Despite the unprecedented clinical activity of the Bruton tyrosine kinase (BTK) inhibitor ibrutinib in mantle cell lymphoma (MCL), acquired resistance is common. By longitudinal integrative whole-exome and whole-transcriptome sequencing and targeted sequencing, we identified the first relapse-specific C481S mutation at the ibrutinib binding site of BTK in MCL cells at progression following a durable response. This mutation enhanced BTK and AKT activation and tissue-specific proliferation of resistant MCL cells driven by CDK4 activation. It was absent, however, in patients with primary resistance or progression following transient response to ibrutinib, suggesting alternative mechanisms of resistance. Through synergistic induction of PIK3IP1 and inhibition of PI3K-AKT activation, prolonged early G1 arrest induced by PD 0332991 (palbociclib) inhibition of CDK4 sensitized resistant lymphoma cells to ibrutinib killing when BTK was unmutated, and to PI3K inhibitors independent of C481S mutation. These data identify a genomic basis for acquired ibrutinib resistance in MCL and suggest a strategy to override both primary and acquired ibrutinib resistance. We have discovered the first relapse-specific BTK mutation in patients with MCL with acquired resistance, but not primary resistance, to ibrutinib, and demonstrated a rationale for targeting the proliferative resistant MCL cells by inhibiting CDK4 and the cell cycle in combination with ibrutinib in the presence of BTK(WT) or a PI3K inhibitor independent of BTK mutation. As drug resistance remains a major challenge and CDK4 and PI3K are dysregulated at a high frequency in human cancers, targeting CDK4 in genome-based combination therapy represents a novel approach to lymphoma and cancer therapy. Cancer Discov; 4(9); 1022-35. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 973. ©2014 American Association for Cancer Research.

Drug Resistance Mechanism of L10F, L10F/N88S and L90M mutations in CRF01_AE HIV-1 protease: Molecular dynamics simulations and binding free energy calculations.

PubMed

Vasavi, C S; Tamizhselvi, Ramasamy; Munusami, Punnagai

2017-08-01

HIV-1 protease plays a crucial role in viral replication and maturation, which makes it one of the most attractive targets for anti-retroviral therapy. The majority of HIV infections in developing countries are due to non-B subtype. Subtype AE is spreading rapidly and infecting huge population worldwide. The mutations in the active site of subtype AE directly impair the interactions with the inhibitor. The non-active site mutations influence the binding of the inhibitor indirectly and their resistance mechanism is not well understood. It is important to design new effective inhibitors that combat drug resistance in subtype AE protease. In this work, we examined the effect of non active site mutations L10F, L10F/N88S and L90M with nelfinavir using molecular dynamics simulation and binding free energy calculations. The simulations suggested that the L10F and L10F/N88S mutants decrease the binding affinity of nelfinavir, whereas the L90M mutant increases the binding affinity. The formation of hydrogen bonds between nelfinavir and Asp30 is crucial for effective binding. The benzamide moiety of nelfinavir shows large positional deviation in L10F and L10F/N88S complexes and the L10F/N88S mutation changes the hydrogen bond between the side chain atoms of 30th residue and the 88th residue. Consequently the hydrogen bond interaction between Asp30 and nelfinavir are destroyed leading to drug resistance. Our present study shed light on the resistance mechanism of the strongly linked mutation L10F/N88S observed experimentally in AE subtype. Copyright © 2017 Elsevier Inc. All rights reserved.

ACE: an efficient and sensitive tool to detect insecticide resistance-associated mutations in insect acetylcholinesterase from RNA-Seq data.

PubMed

Guo, Dianhao; Luo, Jiapeng; Zhou, Yuenan; Xiao, Huamei; He, Kang; Yin, Chuanlin; Xu, Jianhua; Li, Fei

2017-07-10

Insecticide resistance is a substantial problem in controlling agricultural and medical pests. Detecting target site mutations is crucial to manage insecticide resistance. Though PCR-based methods have been widely used in this field, they are time-consuming and inefficient, and typically have a high false positive rate. Acetylcholinesterases (Ace) is the neural target of the widely used organophosphate (OP) and carbamate insecticides. However, there is not any software available to detect insecticide resistance associated mutations in RNA-Seq data at present. A computational pipeline ACE was developed to detect resistance mutations of ace in insect RNA-Seq data. Known ace resistance mutations were collected and used as a reference. We constructed a Web server for ACE, and the standalone software in both Linux and Windows versions is available for download. ACE was used to analyse 971 RNA-Seq data from 136 studies in 7 insect pests. The mutation frequency of each RNA-Seq dataset was calculated. The results indicated that the resistance frequency was 30%-44% in an eastern Ugandan Anopheles population, thus suggesting this resistance-conferring mutation has reached high frequency in these mosquitoes in Uganda. Analyses of RNA-Seq data from the diamondback moth Plutella xylostella indicated that the G227A mutation was positively related with resistance levels to organophosphate or carbamate insecticides. The wasp Nasonia vitripennis had a low frequency of resistant reads (<5%), but the agricultural pests Chilo suppressalis and Bemisia tabaci had a high resistance frequency. All ace reads in the 30 B. tabaci RNA-Seq data were resistant reads, suggesting that insecticide resistance has spread to very high frequency in B. tabaci. To the best of our knowledge, the ACE pipeline is the first tool to detect resistance mutations from RNA-Seq data, and it facilitates the full utilization of large-scale genetic data obtained by using next-generation sequencing.

Genetic Pathway of HIV-1 Resistance to Novel Fusion Inhibitors Targeting the Gp41 Pocket

PubMed Central

Su, Yang; Chong, Huihiui; Xiong, Shengwen; Qiao, Yuanyuan; Qiu, Zonglin

2015-01-01

ABSTRACT The peptide drug enfuvirtide (T20) is the only HIV-1 fusion inhibitor in clinical use, but it easily induces drug resistance, calling for new strategies for developing effective drugs. On the basis of the M-T hook structure, we recently developed highly potent short-peptide HIV-1 fusion inhibitors (MTSC22 and HP23), which mainly target the conserved gp41 pocket and possess high genetic barriers to resistance. Here, we focused on the selection and characterization of HIV-1 escape mutants of MTSC22, which revealed new resistance pathways and mechanisms. Two mutations (E49K and L57R) located at the inhibitor-binding site and two mutations (N126K and E136G) located at the C-terminal heptad repeat region of gp41 were identified as conferring high resistance either singly or in combination. While E49K reduced the C-terminal binding of inhibitors via an electrostatic repulsion, L57R dramatically disrupted the N-terminal binding of M-T hook structure and pocket-binding domain. Unlike E49K and N126K, which enhanced the stability of the endogenous viral six-helical bundle core (6-HB), L57R and E136G conversely destabilized the 6-HB structure. We also demonstrated that both primary and secondary mutations caused the structural changes in 6-HB and severely impaired the capability for HIV-1 entry. Collectively, our data provide novel insights into the mechanisms of short-peptide fusion inhibitors targeting the gp41 pocket site and help increase our understanding of the structure and function of gp41 and HIV-1 evolution. IMPORTANCE The deep pocket on the N-trimer of HIV-1 gp41 has been considered an ideal drug target because of its high degree of conservation and essential role in viral entry. Short-peptide fusion inhibitors, which contain an M-T hook structure and mainly target the pocket site, show extremely high binding and inhibitory activities as well as high genetic barriers to resistance. In this study, the HIV-1 mutants resistant to MTSC22 were selected and characterized, which revealed that the E49K and L57R substitutions at the inhibitor-binding site and the N126K and E136G substitutions at the C-terminal heptad repeat region of gp41 critically determine the resistance phenotype. The data provide novel insights into the mechanisms of action of the M-T hook structure-based fusion inhibitors which will help further our understanding of the structure-function relationship of gp41 and molecular pathways of HIV-1 evolution and eventually facilitate the development of new anti-HIV drugs. PMID:26446597

A fully humanized transgenic mouse model of Huntington disease

PubMed Central

Southwell, Amber L.; Warby, Simon C.; Carroll, Jeffrey B.; Doty, Crystal N.; Skotte, Niels H.; Zhang, Weining; Villanueva, Erika B.; Kovalik, Vlad; Xie, Yuanyun; Pouladi, Mahmoud A.; Collins, Jennifer A.; Yang, X. William; Franciosi, Sonia; Hayden, Michael R.

2013-01-01

Silencing the mutant huntingtin gene (muHTT) is a direct and simple therapeutic strategy for the treatment of Huntington disease (HD) in principle. However, targeting the HD mutation presents challenges because it is an expansion of a common genetic element (a CAG tract) that is found throughout the genome. Moreover, the HTT protein is important for neuronal health throughout life, and silencing strategies that also reduce the wild-type HTT allele may not be well tolerated during the long-term treatment of HD. Several HTT silencing strategies are in development that target genetic sites in HTT that are outside of the CAG expansion, including HD mutation-linked single-nucleotide polymorphisms and the HTT promoter. Preclinical testing of these genetic therapies has required the development of a new mouse model of HD that carries these human-specific genetic targets. To generate a fully humanized mouse model of HD, we have cross-bred BACHD and YAC18 on the Hdh−/− background. The resulting line, Hu97/18, is the first murine model of HD that fully genetically recapitulates human HD having two human HTT genes, no mouse Hdh genes and heterozygosity of the HD mutation. We find that Hu97/18 mice display many of the behavioral changes associated with HD including motor, psychiatric and cognitive deficits, as well as canonical neuropathological abnormalities. This mouse line will be useful for gaining additional insights into the disease mechanisms of HD as well as for testing genetic therapies targeting human HTT. PMID:23001568

Mutagenesis Studies of the H5 Influenza Hemagglutinin Stem Loop Region*

PubMed Central

Antanasijevic, Aleksandar; Basu, Arnab; Bowlin, Terry L.; Mishra, Rama K.; Rong, Lijun; Caffrey, Michael

2014-01-01

Influenza outbreaks, particularly the pandemic 1918 H1 and avian H5 strains, are of high concern to public health. The hemagglutinin envelope protein of influenza plays a critical role in viral entry and thus is an attractive target for inhibition of virus entry. The highly conserved stem loop region of hemagglutinin has been shown to undergo critically important conformational changes during the entry process and, moreover, to be a site for inhibition of virus entry by antibodies, small proteins, and small drug-like molecules. In this work we probe the structure-function properties of the H5 hemagglutinin stem loop region by site-directed mutagenesis. We find that most mutations do not disrupt expression, proteolytic processing, incorporation into virus, or receptor binding; however, many of the mutations disrupt the entry process. We further assess the effects of mutations on inhibition of entry by a neutralizing monoclonal antibody (C179) and find examples of increased and decreased sensitivity to the antibody, consistent with the antibody binding site observed by x-ray crystallography. In addition, we tested the sensitivity of the mutants to MBX2329, a small molecule inhibitor of influenza entry. Interestingly, the mutants exhibit increased and decreased sensitivities to MBX2329, which gives further insight into the binding site of the compound on HA and potential mechanisms of escape. Finally, we have modeled the binding site of MBX2329 using molecular dynamics and find that the resulting structure is in good agreement with the mutagenesis results. Together these studies underscore the importance of the stem loop region to HA function and suggest potential sites for therapeutic intervention of influenza entry. PMID:24947513

Mutagenesis studies of the H5 influenza hemagglutinin stem loop region.

PubMed

Antanasijevic, Aleksandar; Basu, Arnab; Bowlin, Terry L; Mishra, Rama K; Rong, Lijun; Caffrey, Michael

2014-08-08

Influenza outbreaks, particularly the pandemic 1918 H1 and avian H5 strains, are of high concern to public health. The hemagglutinin envelope protein of influenza plays a critical role in viral entry and thus is an attractive target for inhibition of virus entry. The highly conserved stem loop region of hemagglutinin has been shown to undergo critically important conformational changes during the entry process and, moreover, to be a site for inhibition of virus entry by antibodies, small proteins, and small drug-like molecules. In this work we probe the structure-function properties of the H5 hemagglutinin stem loop region by site-directed mutagenesis. We find that most mutations do not disrupt expression, proteolytic processing, incorporation into virus, or receptor binding; however, many of the mutations disrupt the entry process. We further assess the effects of mutations on inhibition of entry by a neutralizing monoclonal antibody (C179) and find examples of increased and decreased sensitivity to the antibody, consistent with the antibody binding site observed by x-ray crystallography. In addition, we tested the sensitivity of the mutants to MBX2329, a small molecule inhibitor of influenza entry. Interestingly, the mutants exhibit increased and decreased sensitivities to MBX2329, which gives further insight into the binding site of the compound on HA and potential mechanisms of escape. Finally, we have modeled the binding site of MBX2329 using molecular dynamics and find that the resulting structure is in good agreement with the mutagenesis results. Together these studies underscore the importance of the stem loop region to HA function and suggest potential sites for therapeutic intervention of influenza entry. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Tricyclic GyrB/ParE (TriBE) Inhibitors. A new class of broad-spectrum dual-targeting antibacterial agents

DOE PAGES

Tari, Leslie W.; Li, Xiaoming; Trzoss, Michael; ...

2013-12-26

Increasing resistance to every major class of antibiotics and a dearth of novel classes of antibacterial agents in development pipelines has created a dwindling reservoir of treatment options for serious bacterial infections. The bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV, are validated antibacterial drug targets with multiple prospective drug binding sites, including the catalytic site targeted by the fluoroquinolone antibiotics. Growing resistance to fluoroquinolones, frequently mediated by mutations in the drug-binding site, is increasingly limiting the utility of this antibiotic class, prompting the search for other inhibitor classes that target different sites on the topoisomerase complexes. The highlymore » conserved ATP-binding subunits of DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as excellent candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, to date, no natural product or small molecule inhibitors targeting these sites have succeeded in the clinic, and no inhibitors of these enzymes have yet been reported with broad-spectrum antibacterial activity encompassing the majority of Gram-negative pathogens. Using structure-based drug design (SBDD), we have created a novel dual-targeting pyrimidoindole inhibitor series with exquisite potency against GyrB and ParE enzymes from a broad range of clinically important pathogens. Inhibitors from this series demonstrate potent, broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens of clinical importance, including fluoroquinolone resistant and multidrug resistant strains. Moreover, lead compounds have been discovered with clinical potential; they are well tolerated in animals, and efficacious in Gram-negative infection models.« less

Tricyclic GyrB/ParE (TriBE) Inhibitors. A new class of broad-spectrum dual-targeting antibacterial agents

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

Tari, Leslie W.; Li, Xiaoming; Trzoss, Michael

Increasing resistance to every major class of antibiotics and a dearth of novel classes of antibacterial agents in development pipelines has created a dwindling reservoir of treatment options for serious bacterial infections. The bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV, are validated antibacterial drug targets with multiple prospective drug binding sites, including the catalytic site targeted by the fluoroquinolone antibiotics. Growing resistance to fluoroquinolones, frequently mediated by mutations in the drug-binding site, is increasingly limiting the utility of this antibiotic class, prompting the search for other inhibitor classes that target different sites on the topoisomerase complexes. The highlymore » conserved ATP-binding subunits of DNA gyrase (GyrB) and topoisomerase IV (ParE) have long been recognized as excellent candidates for the development of dual-targeting antibacterial agents with broad-spectrum potential. However, to date, no natural product or small molecule inhibitors targeting these sites have succeeded in the clinic, and no inhibitors of these enzymes have yet been reported with broad-spectrum antibacterial activity encompassing the majority of Gram-negative pathogens. Using structure-based drug design (SBDD), we have created a novel dual-targeting pyrimidoindole inhibitor series with exquisite potency against GyrB and ParE enzymes from a broad range of clinically important pathogens. Inhibitors from this series demonstrate potent, broad-spectrum antibacterial activity against Gram-positive and Gram-negative pathogens of clinical importance, including fluoroquinolone resistant and multidrug resistant strains. Moreover, lead compounds have been discovered with clinical potential; they are well tolerated in animals, and efficacious in Gram-negative infection models.« less

PoPMuSiC 2.1: a web server for the estimation of protein stability changes upon mutation and sequence optimality.

PubMed

Dehouck, Yves; Kwasigroch, Jean Marc; Gilis, Dimitri; Rooman, Marianne

2011-05-13

The rational design of modified proteins with controlled stability is of extreme importance in a whole range of applications, notably in the biotechnological and environmental areas, where proteins are used for their catalytic or other functional activities. Future breakthroughs in medical research may also be expected from an improved understanding of the effect of naturally occurring disease-causing mutations on the molecular level. PoPMuSiC-2.1 is a web server that predicts the thermodynamic stability changes caused by single site mutations in proteins, using a linear combination of statistical potentials whose coefficients depend on the solvent accessibility of the mutated residue. PoPMuSiC presents good prediction performances (correlation coefficient of 0.8 between predicted and measured stability changes, in cross validation, after exclusion of 10% outliers). It is moreover very fast, allowing the prediction of the stability changes resulting from all possible mutations in a medium size protein in less than a minute. This unique functionality is user-friendly implemented in PoPMuSiC and is particularly easy to exploit. Another new functionality of our server concerns the estimation of the optimality of each amino acid in the sequence, with respect to the stability of the structure. It may be used to detect structural weaknesses, i.e. clusters of non-optimal residues, which represent particularly interesting sites for introducing targeted mutations. This sequence optimality data is also expected to have significant implications in the prediction and the analysis of particular structural or functional protein regions. To illustrate the interest of this new functionality, we apply it to a dataset of known catalytic sites, and show that a much larger than average concentration of structural weaknesses is detected, quantifying how these sites have been optimized for function rather than stability. The freely available PoPMuSiC-2.1 web server is highly useful for identifying very rapidly a list of possibly relevant mutations with the desired stability properties, on which subsequent experimental studies can be focused. It can also be used to detect sequence regions corresponding to structural weaknesses, which could be functionally important or structurally delicate regions, with obvious applications in rational protein design.

Recurrent SETBP1 mutations in atypical chronic myeloid leukemia

PubMed Central

Piazza, Rocco; Valletta, Simona; Winkelmann, Nils; Redaelli, Sara; Spinelli, Roberta; Pirola, Alessandra; Antolini, Laura; Mologni, Luca; Donadoni, Carla; Papaemmanuil, Elli; Schnittger, Susanne; Kim, Dong-Wook; Boultwood, Jacqueline; Rossi, Fabio; Gaipa, Giuseppe; De Martini, Greta P; di Celle, Paola Francia; Jang, Hyun Gyung; Fantin, Valeria; Bignell, Graham R; Magistroni, Vera; Haferlach, Torsten; Pogliani, Enrico Maria; Campbell, Peter J; Chase, Andrew J; Tapper, William J; Cross, Nicholas C P; Gambacorti-Passerini, Carlo

2013-01-01

Atypical chronic myeloid leukemia (aCML) shares clinical and laboratory features with CML, but it lacks the BCR-ABL1 fusion. We performed exome sequencing of eight aCMLs and identified somatic alterations of SETBP1 (encoding a p.Gly870Ser alteration) in two cases. Targeted resequencing of 70 aCMLs, 574 diverse hematological malignancies and 344 cancer cell lines identified SETBP1 mutations in 24 cases, including 17 of 70 aCMLs (24.3%; 95% confidence interval (CI) = 16–35%). Most mutations (92%) were located between codons 858 and 871 and were identical to changes seen in individuals with Schinzel-Giedion syndrome. Individuals with mutations had higher white blood cell counts (P = 0.008) and worse prognosis (P = 0.01). The p.Gly870Ser alteration abrogated a site for ubiquitination, and cells exogenously expressing this mutant exhibited higher amounts of SETBP1 and SET protein, lower PP2A activity and higher proliferation rates relative to those expressing the wild-type protein. In summary, mutated SETBP1 represents a newly discovered oncogene present in aCML and closely related diseases. PMID:23222956

Recurrent SETBP1 mutations in atypical chronic myeloid leukemia.

PubMed

Piazza, Rocco; Valletta, Simona; Winkelmann, Nils; Redaelli, Sara; Spinelli, Roberta; Pirola, Alessandra; Antolini, Laura; Mologni, Luca; Donadoni, Carla; Papaemmanuil, Elli; Schnittger, Susanne; Kim, Dong-Wook; Boultwood, Jacqueline; Rossi, Fabio; Gaipa, Giuseppe; De Martini, Greta P; di Celle, Paola Francia; Jang, Hyun Gyung; Fantin, Valeria; Bignell, Graham R; Magistroni, Vera; Haferlach, Torsten; Pogliani, Enrico Maria; Campbell, Peter J; Chase, Andrew J; Tapper, William J; Cross, Nicholas C P; Gambacorti-Passerini, Carlo

2013-01-01

Atypical chronic myeloid leukemia (aCML) shares clinical and laboratory features with CML, but it lacks the BCR-ABL1 fusion. We performed exome sequencing of eight aCMLs and identified somatic alterations of SETBP1 (encoding a p.Gly870Ser alteration) in two cases. Targeted resequencing of 70 aCMLs, 574 diverse hematological malignancies and 344 cancer cell lines identified SETBP1 mutations in 24 cases, including 17 of 70 aCMLs (24.3%; 95% confidence interval (CI) = 16-35%). Most mutations (92%) were located between codons 858 and 871 and were identical to changes seen in individuals with Schinzel-Giedion syndrome. Individuals with mutations had higher white blood cell counts (P = 0.008) and worse prognosis (P = 0.01). The p.Gly870Ser alteration abrogated a site for ubiquitination, and cells exogenously expressing this mutant exhibited higher amounts of SETBP1 and SET protein, lower PP2A activity and higher proliferation rates relative to those expressing the wild-type protein. In summary, mutated SETBP1 represents a newly discovered oncogene present in aCML and closely related diseases.

High-Throughput Analysis of Promoter Occupancy Reveals Direct Neural Targets of FOXP2, a Gene Mutated in Speech and Language Disorders

PubMed Central

Vernes, Sonja C. ; Spiteri, Elizabeth ; Nicod, Jérôme ; Groszer, Matthias ; Taylor, Jennifer M. ; Davies, Kay E. ; Geschwind, Daniel H. ; Fisher, Simon E. 

2007-01-01

We previously discovered that mutations of the human FOXP2 gene cause a monogenic communication disorder, primarily characterized by difficulties in learning to make coordinated sequences of articulatory gestures that underlie speech. Affected people have deficits in expressive and receptive linguistic processing and display structural and/or functional abnormalities in cortical and subcortical brain regions. FOXP2 provides a unique window into neural processes involved in speech and language. In particular, its role as a transcription factor gene offers powerful functional genomic routes for dissecting critical neurogenetic mechanisms. Here, we employ chromatin immunoprecipitation coupled with promoter microarrays (ChIP-chip) to successfully identify genomic sites that are directly bound by FOXP2 protein in native chromatin of human neuron-like cells. We focus on a subset of downstream targets identified by this approach, showing that altered FOXP2 levels yield significant changes in expression in our cell-based models and that FOXP2 binds in a specific manner to consensus sites within the relevant promoters. Moreover, we demonstrate significant quantitative differences in target expression in embryonic brains of mutant mice, mediated by specific in vivo Foxp2-chromatin interactions. This work represents the first identification and in vivo verification of neural targets regulated by FOXP2. Our data indicate that FOXP2 has dual functionality, acting to either repress or activate gene expression at occupied promoters. The identified targets suggest roles in modulating synaptic plasticity, neurodevelopment, neurotransmission, and axon guidance and represent novel entry points into in vivo pathways that may be disturbed in speech and language disorders. PMID:17999362

A prohormone convertase cleavage site within a predicted alpha-helix mediates sorting of the neuronal and endocrine polypeptide VGF into the regulated secretory pathway.

PubMed

Garcia, Angelo L; Han, Shan-Kuo; Janssen, William G; Khaing, Zin Z; Ito, Timothy; Glucksman, Marc J; Benson, Deanna L; Salton, Stephen R J

2005-12-16

Distinct intracellular pathways are involved in regulated and constitutive protein secretion from neuronal and endocrine cells, yet the peptide signals and molecular mechanisms responsible for targeting and retention of soluble proteins in secretory granules are incompletely understood. By using confocal microscopy and subcellular fractionation, we examined trafficking of the neuronal and endocrine peptide precursor VGF that is stored in large dense core vesicles and undergoes regulated secretion. VGF cofractionated with secretory vesicle membranes but was not detected in detergent-resistant lipid rafts. Deletional analysis using epitope-tagged VGF suggested that the C-terminal 73-amino acid fragment of VGF, containing two predicted alpha-helical loops and four potential prohormone convertase (PC) cleavage sites, was necessary and sufficient with an N-terminal signal peptide-containing domain, for large dense core vesicle sorting and regulated secretion from PC12 and INS-1 cells. Further transfection analysis identified the sorting sequence as a compact C-terminal alpha-helix and embedded 564RRR566 PC cleavage site; mutation of the 564RRR566 PC site in VGF-(1-65): GFP:VGF-(545-617) blocked regulated secretion, whereas disruption of the alpha-helix had no effect. Mutation of the adjacent 567HFHH570 motif, a charged region that might enhance PC cleavage in acidic environments, also blocked regulated release. Finally, inhibition of PC cleavage in PC12 cells using the membrane-permeable synthetic peptide chloromethyl ketone (decanoyl-RVKR-CMK) blocked regulated secretion of VGF. Our studies define a critical RRR-containing C-terminal domain that targets VGF into the regulated pathway in neuronal PC12 and endocrine INS-1 cells, providing additional support for the proposed role that PCs and their cleavage sites play in regulated peptide secretion.

Reconstitutional Mutagenesis of the Maize P Gene by Short-Range Ac Transpositions

PubMed Central

Moreno, M. A.; Chen, J.; Greenblatt, I.; Dellaporta, S. L.

1992-01-01

The tendency for Ac to transpose over short intervals has been utilized to develop insertional mutagenesis and fine structure genetic mapping strategies in maize. We recovered excisions of Ac from the P gene and insertions into nearby chromosomal sites. These closely linked Ac elements reinserted into the P gene, reconstituting over 250 unstable variegated alleles. Reconstituted alleles condition a variety of variegation patterns that reflect the position and orientation of Ac within the P gene. Molecular mapping and DNA sequence analyses have shown that reinsertion sites are dispersed throughout a 12.3-kb chromosomal region in the promoter, exons and introns of the P gene, but in some regions insertions sites were clustered in a nonrandom fashion. Transposition profiles and target site sequence data obtained from these studies have revealed several features of Ac transposition including its preference for certain target sites. These results clearly demonstrate the tendency of Ac to transpose to nearby sites in both proximal and distal directions from the donor site. With minor modifications, reconstitutional mutagenesis should be applicable to many Ac-induced mutations in maize and in other plant species and can possibly be extended to other eukaryotic transposon systems as well. PMID:1325389

Identifying three-dimensional structures of autophosphorylation complexes in crystals of protein kinases

PubMed Central

Xu, Qifang; Malecka, Kimberly L.; Fink, Lauren; Jordan, E. Joseph; Duffy, Erin; Kolander, Samuel; Peterson, Jeffrey; Dunbrack, Roland L.

2016-01-01

Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Crystal structures of several homomeric protein kinase complexes have a serine, threonine, or tyrosine autophosphorylation site of one kinase monomer located in the active site of another monomer, a structural complex that we call an “autophosphorylation complex.” We developed and applied a structural bioinformatics method to identify all such autophosphorylation kinase complexes in X-ray crystallographic structures in the Protein Data Bank (PDB). We identified 15 autophosphorylation complexes in the PDB, of which 5 complexes had not previously been described in the publications describing the crystal structures. These 5 consist of tyrosine residues in the N-terminal juxtamembrane regions of colony stimulating factor 1 receptor (CSF1R, Tyr561) and EPH receptor A2 (EPHA2, Tyr594), tyrosine residues in the activation loops of the SRC kinase family member LCK (Tyr394) and insulin-like growth factor 1 receptor (IGF1R, Tyr1166), and a serine in a nuclear localization signal region of CDC-like kinase 2 (CLK2, Ser142). Mutations in the complex interface may alter autophosphorylation activity and contribute to disease; therefore we mutated residues in the autophosphorylation complex interface of LCK and found that two mutations impaired autophosphorylation (T445V and N446A) and mutation of Pro447 to Ala, Gly, or Leu increased autophosphorylation. The identified autophosphorylation sites are conserved in many kinases, suggesting that, by homology, these complexes may provide insight into autophosphorylation complex interfaces of kinases that are relevant drug targets. PMID:26628682

Importance of the Extracellular Loop 4 in the Human Serotonin Transporter for Inhibitor Binding and Substrate Translocation*

PubMed Central

Rannversson, Hafsteinn; Wilson, Pamela; Kristensen, Kristina Birch; Sinning, Steffen; Kristensen, Anders Skov; Strømgaard, Kristian; Andersen, Jacob

2015-01-01

The serotonin transporter (SERT) terminates serotonergic neurotransmission by performing reuptake of released serotonin, and SERT is the primary target for antidepressants. SERT mediates the reuptake of serotonin through an alternating access mechanism, implying that a central substrate site is connected to both sides of the membrane by permeation pathways, of which only one is accessible at a time. The coordinated conformational changes in SERT associated with substrate translocation are not fully understood. Here, we have identified a Leu to Glu mutation at position 406 (L406E) in the extracellular loop 4 (EL4) of human SERT, which induced a remarkable gain-of-potency (up to >40-fold) for a range of SERT inhibitors. The effects were highly specific for L406E relative to six other mutations in the same position, including the closely related L406D mutation, showing that the effects induced by L406E are not simply charge-related effects. Leu406 is located >10 Å from the central inhibitor binding site indicating that the mutation affects inhibitor binding in an indirect manner. We found that L406E decreased accessibility to a residue in the cytoplasmic pathway. The shift in equilibrium to favor a more outward-facing conformation of SERT can explain the reduced turnover rate and increased association rate of inhibitor binding we found for L406E. Together, our findings show that EL4 allosterically can modulate inhibitor binding within the central binding site, and substantiates that EL4 has an important role in controlling the conformational equilibrium of human SERT. PMID:25903124

Integrative genome-wide analysis reveals HLP1, a novel RNA-binding protein, regulates plant flowering by targeting alternative polyadenylation

PubMed Central

Zhang, Yong; Gu, Lianfeng; Hou, Yifeng; Wang, Lulu; Deng, Xian; Hang, Runlai; Chen, Dong; Zhang, Xiansheng; Zhang, Yi; Liu, Chunyan; Cao, Xiaofeng

2015-01-01

Alternative polyadenylation (APA) is a widespread mechanism for gene regulation and has been implicated in flowering, but the molecular basis governing the choice of a specific poly(A) site during the vegetative-to-reproductive growth transition remains unclear. Here we characterize HLP1, an hnRNP A/B protein as a novel regulator for pre-mRNA 3′-end processing in Arabidopsis. Genetic analysis reveals that HLP1 suppresses Flowering Locus C (FLC), a key repressor of flowering in Arabidopsis. Genome-wide mapping of HLP1-RNA interactions indicates that HLP1 binds preferentially to A-rich and U-rich elements around cleavage and polyadenylation sites, implicating its role in 3′-end formation. We show HLP1 is significantly enriched at transcripts involved in RNA metabolism and flowering. Comprehensive profiling of the poly(A) site usage reveals that HLP1 mutations cause thousands of poly(A) site shifts. A distal-to-proximal poly(A) site shift in the flowering regulator FCA, a direct target of HLP1, leads to upregulation of FLC and delayed flowering. Our results elucidate that HLP1 is a novel factor involved in 3′-end processing and controls reproductive timing via targeting APA. PMID:26099751

Polymorphisms in Gag spacer peptide 1 confer varying levels of resistance to the HIV- 1 maturation inhibitor bevirimat.

PubMed

Adamson, Catherine S; Sakalian, Michael; Salzwedel, Karl; Freed, Eric O

2010-04-20

The maturation inhibitor bevirimat (BVM) potently inhibits human immunodeficiency virus type 1 (HIV-1) replication by blocking capsid-spacer peptide 1 (CA-SP1) cleavage. Recent clinical trials demonstrated that a significant proportion of HIV-1-infected patients do not respond to BVM. A patient's failure to respond correlated with baseline polymorphisms at SP1 residues 6-8. In this study, we demonstrate that varying levels of BVM resistance are associated with point mutations at these residues. BVM susceptibility was maintained by SP1-Q6A, -Q6H and -T8A mutations. However, an SP1-V7A mutation conferred high-level BVM resistance, and SP1-V7M and T8Delta mutations conferred intermediate levels of BVM resistance. Future exploitation of the CA-SP1 cleavage site as an antiretroviral drug target will need to overcome the baseline variability in the SP1 region of Gag.

Conservative Secondary Shell Substitution In Cyclooxygenase-2 Reduces Inhibition by Indomethacin Amides and Esters via Altered Enzyme Dynamics

PubMed Central

2015-01-01

The cyclooxygenase enzymes (COX-1 and COX-2) are the therapeutic targets of nonsteroidal anti-inflammatory drugs (NSAIDs). Neutralization of the carboxylic acid moiety of the NSAID indomethacin to an ester or amide functionality confers COX-2 selectivity, but the molecular basis for this selectivity has not been completely revealed through mutagenesis studies and/or X-ray crystallographic attempts. We expressed and assayed a number of divergent secondary shell COX-2 active site mutants and found that a COX-2 to COX-1 change at position 472 (Leu in COX-2, Met in COX-1) reduced the potency of enzyme inhibition by a series of COX-2-selective indomethacin amides and esters. In contrast, the potencies of indomethacin, arylacetic acid, propionic acid, and COX-2-selective diarylheterocycle inhibitors were either unaffected or only mildly affected by this mutation. Molecular dynamics simulations revealed identical equilibrium enzyme structures around residue 472; however, calculations indicated that the L472M mutation impacted local low-frequency dynamical COX constriction site motions by stabilizing the active site entrance and slowing constriction site dynamics. Kinetic analysis of inhibitor binding is consistent with the computational findings. PMID:26704937

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

NASA Astrophysics Data System (ADS)

Qian, Long; Kussell, Edo

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

Deletion of transcription factor binding motifs using the CRISPR/spCas9 system in the β-globin LCR.

PubMed

Kim, Yea Woon; Kim, AeRi

2017-07-20

Transcription factors play roles in gene transcription through direct binding to their motifs in genome, and inhibiting this binding provides an effective strategy for studying their roles. Here we applied the CRISPR/spCas9 system to mutate the binding motifs of transcription factors. Binding motifs for erythroid specific transcription factors were mutated in the locus control region hypersensitive sites of the human β-globin locus. Guide RNAs targeting binding motifs were cloned into lentiviral CRISPR vector containing the spCas9 gene, and transduced into MEL/ch11 cells carrying a human chromosome 11. DNA mutations in clonal cells were initially screened by quantitative PCR in genomic DNA and then clarified by sequencing. Mutations in binding motifs reduced occupancy by transcription factors in a chromatin environment. Characterization of mutations revealed that the CRISPR/spCas9 system mainly induced deletions in short regions of <20 bp and preferentially deleted nucleotides around the fifth nucleotide upstream of Protospacer adjacent motifs. These results indicate that the CRISPR/Cas9 system is suitable for mutating the binding motifs of transcription factors, and, consequently, would contribute to elucidate the direct roles of transcription factors. ©2017 The Author(s).

Resistance to herbicides caused by single amino acid mutations in acetyl-CoA carboxylase in resistant populations of grassy weeds.

PubMed

Jang, SoRi; Marjanovic, Jasmina; Gornicki, Piotr

2013-03-01

Eleven spontaneous mutations of acetyl-CoA carboxylase have been identified in many herbicide-resistant populations of 42 species of grassy weeds, hampering application of aryloxyphenoxypropionate, cyclohexadione and phenylpyrazoline herbicides in agriculture. IC(50) shifts (resistance indices) caused by herbicide-resistant mutations were determined using a recombinant yeast system that allows comparison of the effects of single amino acid mutations in the same biochemical background, avoiding the complexity inherent in the in planta experiments. The effect of six mutations on the sensitivity of acetyl-CoA carboxylase to nine herbicides representing the three chemical classes was studied. A combination of partially overlapping binding sites of the three classes of herbicides and the structure of their variable parts explains cross-resistance among and between the three classes of inhibitors, as well as differences in their specificity. Some degree of resistance was detected for 51 of 54 herbicide/mutation combinations. Introduction of new herbicides targeting acetyl-CoA carboxylase will depend on their ability to overcome the high degree of cross-resistance already existing in weed populations. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

Targeted mutagenesis in sea urchin embryos using TALENs.

PubMed

Hosoi, Sayaka; Sakuma, Tetsushi; Sakamoto, Naoaki; Yamamoto, Takashi

2014-01-01

Genome editing with engineered nucleases such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) has been reported in various animals. We previously described ZFN-mediated targeted mutagenesis and insertion of reporter genes in sea urchin embryos. In this study, we demonstrate that TALENs can induce mutagenesis at specific genomic loci of sea urchin embryos. Injection of TALEN mRNAs targeting the HpEts transcription factor into fertilized eggs resulted in the impairment of skeletogenesis. Sequence analyses of the mutations showed that deletions and/or insertions occurred at the HpEts target site in the TALEN mRNAs-injected embryos. The results suggest that targeted gene disruption using TALENs is feasible in sea urchin embryos. © 2013 The Authors Development, Growth & Differentiation © 2013 Japanese Society of Developmental Biologists.

Uveal melanoma hepatic metastases mutation spectrum analysis using targeted next-generation sequencing of 400 cancer genes.

PubMed

Luscan, A; Just, P A; Briand, A; Burin des Roziers, C; Goussard, P; Nitschké, P; Vidaud, M; Avril, M F; Terris, B; Pasmant, E

2015-04-01

Uveal melanoma (UM) is the most common malignant tumour of the eye. Diagnosis often occurs late in the course of disease, and prognosis is generally poor. Recently, recurrent somatic mutations were described, unravelling additional specific altered pathways in UM. Targeted next-generation sequencing (NGS) can now be applied to an accurate and fast identification of somatic mutations in cancer. The aim of the present study was to characterise the mutation pattern of five UM hepatic metastases with well-defined clinical and pathological features. We analysed the UM mutation spectrum using targeted NGS on 409 cancer genes. Four previous reported genes were found to be recurrently mutated. All tumours presented mutually exclusive GNA11 or GNAQ missense mutations. BAP1 loss-of-function mutations were found in three UMs. SF3B1 missense mutations were found in the two UMs with no BAP1 mutations. We then searched for additional mutation targets. We identified the Arg505Cys mutation in the tumour suppressor FBXW7. The same mutation was previously described in different cancer types, and FBXW7 was recently reported to be mutated in UM exomes. Further studies are required to confirm FBXW7 implication in UM tumorigenesis. Elucidating the molecular mechanisms underlying UM tumorigenesis holds the promise for novel and effective targeted UM therapies. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Site-to-site interdomain communication may mediate different loss-of-function mechanisms in a cancer-associated NQO1 polymorphism

NASA Astrophysics Data System (ADS)

Medina-Carmona, Encarnación; Neira, Jose L.; Salido, Eduardo; Fuchs, Julian E.; Palomino-Morales, Rogelio; Timson, David J.; Pey, Angel L.

2017-03-01

Disease associated genetic variations often cause intracellular enzyme inactivation, dysregulation and instability. However, allosteric communication of mutational effects to distant functional sites leading to loss-of-function remains poorly understood. We characterize here interdomain site-to-site communication by which a common cancer-associated single nucleotide polymorphism (c.C609T/p.P187S) reduces the activity and stability in vivo of NAD(P)H:quinone oxidoreductase 1 (NQO1). NQO1 is a FAD-dependent, two-domain multifunctional stress protein acting as a Phase II enzyme, activating cancer pro-drugs and stabilizing p53 and p73α oncosuppressors. We show that p.P187S causes structural and dynamic changes communicated to functional sites far from the mutated site, affecting the FAD binding site located at the N-terminal domain (NTD) and accelerating proteasomal degradation through dynamic effects on the C-terminal domain (CTD). Structural protein:protein interaction studies reveal that the cancer-associated polymorphism does not abolish the interaction with p73α, indicating that oncosuppressor destabilization largely mirrors the low intracellular stability of p.P187S. In conclusion, we show how a single disease associated amino acid change may allosterically perturb several functional sites in an oligomeric and multidomain protein. These results have important implications for the understanding of loss-of-function genetic diseases and the identification of novel structural hot spots as targets for pharmacological intervention.

The molecular genetics of insecticide resistance.

PubMed

Ffrench-Constant, Richard H

2013-08-01

The past 60 years have seen a revolution in our understanding of the molecular genetics of insecticide resistance. While at first the field was split by arguments about the relative importance of mono- vs. polygenic resistance and field- vs. laboratory-based selection, the application of molecular cloning to insecticide targets and to the metabolic enzymes that degrade insecticides before they reach those targets has brought out an exponential growth in our understanding of the mutations involved. Molecular analysis has confirmed the relative importance of single major genes in target-site resistance and has also revealed some interesting surprises about the multi-gene families, such as cytochrome P450s, involved in metabolic resistance. Identification of the mutations involved in resistance has also led to parallel advances in our understanding of the enzymes and receptors involved, often with implications for the role of these receptors in humans. This Review seeks to provide an historical perspective on the impact of molecular biology on our understanding of resistance and to begin to look forward to the likely impact of rapid advances in both sequencing and genome-wide association analysis.

Selection of drug-resistant feline immunodeficiency virus (FIV) encoding FIV/HIV chimeric protease in the presence of HIV-specific protease inhibitors.

PubMed

Lin, Ying-Chuan; Happer, Meaghan; Elder, John H

2013-08-01

An infectious chimeric feline immunodeficiency virus (FIV)/HIV strain carrying six HIV-like protease (PR) mutations (I37V/N55M/V59I/I98S/Q99V/P100N) was subjected to selection in culture against the PR inhibitor lopinavir (LPV), darunavir (DRV), or TL-3. LPV selection resulted in the sequential emergence of V99A (strain S-1X), I59V (strain S-2X), and I108V (strain S-3X) mutations, followed by V37I (strain S-4X). Mutant PRs were analyzed in vitro, and an isogenic virus producing each mutant PR was analyzed in culture for LPV sensitivity, yielding results consistent with the original selection. The 50% inhibitory concentrations (IC50s) for S-1X, S-2X, S-3X, and S-4X were 95, 643, 627, and 1,543 nM, respectively. The primary resistance mutations, V99(82)A, I59(50)V, and V37(32)I, are consistent with the resistance pattern developed by HIV-1 under similar selection conditions. While resistance to LPV emerged readily, similar PR mutations causing resistance to either DRV or TL-3 failed to emerge after passage for more than a year. However, a G37D mutation in the nucleocapsid (NC) was observed in both selections and an isogenic G37D mutant replicated in the presence of 100 nM DRV or TL-3, whereas parental chimeric FIV could not. An additional mutation, L92V, near the PR active site in the folded structure recently emerged during TL-3 selection. The L92V mutant PR exhibited an IC50 of 50 nM, compared to 35 nM for 6s-98S PR, and processed the NC-p2 junction more efficiently, consistent with increased viral fitness. These findings emphasize the role of mutations outside the active site of PR in increasing viral resistance to active-site inhibitors and suggest additional targets for inhibitor development.

Selection of Drug-Resistant Feline Immunodeficiency Virus (FIV) Encoding FIV/HIV Chimeric Protease in the Presence of HIV-Specific Protease Inhibitors

PubMed Central

Lin, Ying-Chuan; Happer, Meaghan

2013-01-01

An infectious chimeric feline immunodeficiency virus (FIV)/HIV strain carrying six HIV-like protease (PR) mutations (I37V/N55M/V59I/I98S/Q99V/P100N) was subjected to selection in culture against the PR inhibitor lopinavir (LPV), darunavir (DRV), or TL-3. LPV selection resulted in the sequential emergence of V99A (strain S-1X), I59V (strain S-2X), and I108V (strain S-3X) mutations, followed by V37I (strain S-4X). Mutant PRs were analyzed in vitro, and an isogenic virus producing each mutant PR was analyzed in culture for LPV sensitivity, yielding results consistent with the original selection. The 50% inhibitory concentrations (IC50s) for S-1X, S-2X, S-3X, and S-4X were 95, 643, 627, and 1,543 nM, respectively. The primary resistance mutations, V9982A, I5950V, and V3732I, are consistent with the resistance pattern developed by HIV-1 under similar selection conditions. While resistance to LPV emerged readily, similar PR mutations causing resistance to either DRV or TL-3 failed to emerge after passage for more than a year. However, a G37D mutation in the nucleocapsid (NC) was observed in both selections and an isogenic G37D mutant replicated in the presence of 100 nM DRV or TL-3, whereas parental chimeric FIV could not. An additional mutation, L92V, near the PR active site in the folded structure recently emerged during TL-3 selection. The L92V mutant PR exhibited an IC50 of 50 nM, compared to 35 nM for 6s-98S PR, and processed the NC-p2 junction more efficiently, consistent with increased viral fitness. These findings emphasize the role of mutations outside the active site of PR in increasing viral resistance to active-site inhibitors and suggest additional targets for inhibitor development. PMID:23720716

HER2 activating mutations are targets for colorectal cancer treatment.

PubMed

Kavuri, Shyam M; Jain, Naveen; Galimi, Francesco; Cottino, Francesca; Leto, Simonetta M; Migliardi, Giorgia; Searleman, Adam C; Shen, Wei; Monsey, John; Trusolino, Livio; Jacobs, Samuel A; Bertotti, Andrea; Bose, Ron

2015-08-01

The Cancer Genome Atlas project identified HER2 somatic mutations and gene amplification in 7% of patients with colorectal cancer. Introduction of the HER2 mutations S310F, L755S, V777L, V842I, and L866M into colon epithelial cells increased signaling pathways and anchorage-independent cell growth, indicating that they are activating mutations. Introduction of these HER2 activating mutations into colorectal cancer cell lines produced resistance to cetuximab and panitumumab by sustaining MAPK phosphorylation. HER2 mutants are potently inhibited by low nanomolar doses of the irreversible tyrosine kinase inhibitors neratinib and afatinib. HER2 gene sequencing of 48 cetuximab-resistant, quadruple (KRAS, NRAS, BRAF, and PIK3CA) wild-type (WT) colorectal cancer patient-derived xenografts (PDX) identified 4 PDXs with HER2 mutations. HER2-targeted therapies were tested on two PDXs. Treatment with a single HER2-targeted drug (trastuzumab, neratinib, or lapatinib) delayed tumor growth, but dual HER2-targeted therapy with trastuzumab plus tyrosine kinase inhibitors produced regression of these HER2-mutated PDXs. HER2 activating mutations cause EGFR antibody resistance in colorectal cell lines, and PDXs with HER2 mutations show durable tumor regression when treated with dual HER2-targeted therapy. These data provide a strong preclinical rationale for clinical trials targeting HER2 activating mutations in metastatic colorectal cancer. ©2015 American Association for Cancer Research.

First report of HGD mutations in a Chinese with alkaptonuria.

PubMed

Yang, Yong-jia; Guo, Ji-hong; Chen, Wei-jian; Zhao, Rui; Tang, Jin-song; Meng, Xiao-hua; Zhao, Liu; Tu, Ming; He, Xin-yu; Wu, Ling-qian; Zhu, Yi-min

2013-04-15

Alkaptonuria (AKU) is one of the first prototypic inborn errors in metabolism and the first human disease found to be transmitted via Mendelian autosomal recessive inheritance. It is caused by HGD mutations, which leads to a deficiency in homogentisate 1,2-dioxygenase (HGD) activity. To date, several HGD mutations have been identified as the cause of the prototypic disease across different ethnic populations worldwide. However, in Asia, the HGD mutation is very rarely reported. For the Chinese population, no literature on HGD mutation screening is available to date. In this paper, we describe two novel HGD mutations in a Chinese AKU family, the splicing mutation of IVS7+1G>C, a donor splice site of exon 7, and a missense mutation of F329C in exon 12. The predicted new splicing site of the mutated exon 7 sequence demonstrated a 303bp extension after the mutation site. The F329C mutation most probably disturbed the stability of the conformation of the two loops critical to the Fe(2+) active site of the HGD enzyme. Copyright © 2013 Elsevier B.V. All rights reserved.

Molecular basis for resistance to ACCase-inhibiting fluazifop in Eleusine indica from Malaysia.

PubMed

Cha, Thye San; Najihah, Mohamed Ghazani; Sahid, Ismail Bin; Chuah, Tse Seng

2014-05-01

Eleusine indica (goosegrass) populations resistant to fluazifop, an acetyl-CoA carboxylase (ACCase: EC6.4.1.2)-inhibiting herbicide, were found in several states in Malaysia. Dose-response assay indicated a resistance factor of 87.5, 62.5 and 150 for biotypes P2, P3 and P4, respectively. DNA sequencing and allele-specific PCR revealed that both biotypes P2 and P3 exhibit a single non-synonymous point mutation from TGG to TGC that leads to a well known Trp-2027-Cys mutation. Interestingly, the highly resistant biotype, P4, did not contain any of the known mutation except the newly discovered target point Asn-2097-Asp, which resulted from a nucleotide change in the codon AAT to GAT. ACCase gene expression was found differentially regulated in the susceptible biotype (P1) and highly resistant biotype P4 from 24 to 72h after treatment (HAT) when being treated with the recommended field rate (198gha(-1)) of fluazifop. However, the small and erratic differences of ACCase gene expression between biotype P1 and P4 does not support the 150-fold resistance in biotype P4. Therefore, the involvement of the target point Asn-2097-Asp and other non-target-site-based resistance mechanisms in the biotype P4 could not be ruled out. Copyright © 2014 Elsevier Inc. All rights reserved.

Zinc-finger Nuclease-induced Gene Repair With Oligodeoxynucleotides: Wanted and Unwanted Target Locus Modifications

PubMed Central

Radecke, Sarah; Radecke, Frank; Cathomen, Toni; Schwarz, Klaus

2010-01-01

Correcting a mutated gene directly at its endogenous locus represents an alternative to gene therapy protocols based on viral vectors with their risk of insertional mutagenesis. When solely a single-stranded oligodeoxynucleotide (ssODN) is used as a repair matrix, the efficiency of the targeted gene correction is low. However, as shown with the homing endonuclease I-SceI, ssODN-mediated gene correction can be enhanced by concomitantly inducing a DNA double-strand break (DSB) close to the mutation. Because I-SceI is hardly adjustable to cut at any desired position in the human genome, here, customizable zinc-finger nucleases (ZFNs) were used to stimulate ssODN-mediated repair of a mutated single-copy reporter locus stably integrated into human embryonic kidney-293 cells. The ZFNs induced faithful gene repair at a frequency of 0.16%. Six times more often, ZFN-induced DSBs were found to be modified by unfaithful addition of ssODN between the termini and about 60 times more often by nonhomologous end joining-related deletions and insertions. Additionally, ZFN off-target activity based on binding mismatch sites at the locus of interest was detected in in vitro cleavage assays and also in chromosomal DNA isolated from treated cells. Therefore, the specificity of ZFN-induced ssODN-mediated gene repair needs to be improved, especially regarding clinical applications. PMID:20068556

Generation and Inheritance of Targeted Mutations in Potato (Solanum tuberosum L.) Using the CRISPR/Cas System

PubMed Central

Butler, Nathaniel M.; Atkins, Paul A.; Voytas, Daniel F.; Douches, David S.

2015-01-01

Genome editing using sequence-specific nucleases (SSNs) offers an alternative approach to conventional genetic engineering and an opportunity to extend the benefits of genetic engineering in agriculture. Currently available SSN platforms, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems (Cas)) have been used in a range of plant species for targeted mutagenesis via non-homologous end joining (NHEJ) are just beginning to be explored in crops such as potato (Solanum tuberosum Group Tuberosum L.). In this study, CRISPR/Cas reagents expressing one of two single-guide RNA (sgRNA) targeting the potato ACETOLACTATE SYNTHASE1 (StALS1) gene were tested for inducing targeted mutations in callus and stable events of diploid and tetraploid potato using Agrobacterium-mediated transformation with either a conventional T-DNA or a modified geminivirus T-DNA. The percentage of primary events with targeted mutations ranged from 3–60% per transformation and from 0–29% above an expected threshold based on the number of ALS alleles. Primary events with targeted mutation frequencies above the expected threshold were used for mutation cloning and inheritance studies using clonal propagation and crosses or selfing. Four of the nine primary events used for mutation cloning had more than one mutation type, and eight primary events contained targeted mutations that were maintained across clonal generations. Somatic mutations were most evident in the diploid background with three of the four primary events having more than two mutation types at a single ALS locus. Conversely, in the tetraploid background, four of the five candidates carried only one mutation type. Single targeted mutations were inherited through the germline of both diploid and tetraploid primary events with transmission percentages ranging from 87–100%. This demonstration of CRISPR/Cas in potato extends the range of plant species modified using CRISPR/Cas and provides a framework for future studies. PMID:26657719

Generation and Inheritance of Targeted Mutations in Potato (Solanum tuberosum L.) Using the CRISPR/Cas System.

PubMed

Butler, Nathaniel M; Atkins, Paul A; Voytas, Daniel F; Douches, David S

2015-01-01

Genome editing using sequence-specific nucleases (SSNs) offers an alternative approach to conventional genetic engineering and an opportunity to extend the benefits of genetic engineering in agriculture. Currently available SSN platforms, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems (Cas)) have been used in a range of plant species for targeted mutagenesis via non-homologous end joining (NHEJ) are just beginning to be explored in crops such as potato (Solanum tuberosum Group Tuberosum L.). In this study, CRISPR/Cas reagents expressing one of two single-guide RNA (sgRNA) targeting the potato ACETOLACTATE SYNTHASE1 (StALS1) gene were tested for inducing targeted mutations in callus and stable events of diploid and tetraploid potato using Agrobacterium-mediated transformation with either a conventional T-DNA or a modified geminivirus T-DNA. The percentage of primary events with targeted mutations ranged from 3-60% per transformation and from 0-29% above an expected threshold based on the number of ALS alleles. Primary events with targeted mutation frequencies above the expected threshold were used for mutation cloning and inheritance studies using clonal propagation and crosses or selfing. Four of the nine primary events used for mutation cloning had more than one mutation type, and eight primary events contained targeted mutations that were maintained across clonal generations. Somatic mutations were most evident in the diploid background with three of the four primary events having more than two mutation types at a single ALS locus. Conversely, in the tetraploid background, four of the five candidates carried only one mutation type. Single targeted mutations were inherited through the germline of both diploid and tetraploid primary events with transmission percentages ranging from 87-100%. This demonstration of CRISPR/Cas in potato extends the range of plant species modified using CRISPR/Cas and provides a framework for future studies.

Rules of co-occurring mutations characterize the antigenic evolution of human influenza A/H3N2, A/H1N1 and B viruses.

PubMed

Chen, Haifen; Zhou, Xinrui; Zheng, Jie; Kwoh, Chee-Keong

2016-12-05

The human influenza viruses undergo rapid evolution (especially in hemagglutinin (HA), a glycoprotein on the surface of the virus), which enables the virus population to constantly evade the human immune system. Therefore, the vaccine has to be updated every year to stay effective. There is a need to characterize the evolution of influenza viruses for better selection of vaccine candidates and the prediction of pandemic strains. Studies have shown that the influenza hemagglutinin evolution is driven by the simultaneous mutations at antigenic sites. Here, we analyze simultaneous or co-occurring mutations in the HA protein of human influenza A/H3N2, A/H1N1 and B viruses to predict potential mutations, characterizing the antigenic evolution. We obtain the rules of mutation co-occurrence using association rule mining after extracting HA1 sequences and detect co-mutation sites under strong selective pressure. Then we predict the potential drifts with specific mutations of the viruses based on the rules and compare the results with the "observed" mutations in different years. The sites under frequent mutations are in antigenic regions (epitopes) or receptor binding sites. Our study demonstrates the co-occurring site mutations obtained by rule mining can capture the evolution of influenza viruses, and confirms that cooperative interactions among sites of HA1 protein drive the influenza antigenic evolution.

Translational read-through of a nonsense mutation causing Bartter syndrome.

PubMed

Cho, Hee Yeon; Lee, Beom Hee; Cheong, Hae Il

2013-06-01

Bartter syndrome (BS) is classified into 5 genotypes according to underlying mutant genes and BS III is caused by loss-of-function mutations in the CLCNKB gene encoding for basolateral ClC-Kb. BS III is the most common genotype in Korean patients with BS and W610X is the most common CLCNKB mutation in Korean BS III. In this study, we tested the hypothesis that the CLCNKB W610X mutation can be rescued in vitro using aminoglycoside antibiotics, which are known to induce translational read-through of a nonsense mutation. The CLCNKB cDNA was cloned into a eukaryotic expression vector and the W610X nonsense mutation was generated by site-directed mutagenesis. Cultured polarized MDCK cells were transfected with the vectors, and the read-through was induced using an aminoglycoside derivative, G418. Cellular expression of the target protein was monitored via immunohistochemistry. While cells transfected with the mutant CLCNKB failed to express ClC-Kb, G418 treatment of the cells induced the full-length protein expression, which was localized to the basolateral plasma membranes. It is demonstrated that the W610X mutation in CLCNKB can be a good candidate for trial of translational read-through induction as a therapeutic modality.

A mutation (L1014F) in the voltage-gated sodium channel of the grain aphid, Sitobion avenae, is associated with resistance to pyrethroid insecticides.

PubMed

Foster, Stephen P; Paul, Verity L; Slater, Russell; Warren, Anne; Denholm, Ian; Field, Linda M; Williamson, Martin S

2014-08-01

The grain aphid, Sitobion avenae Fabricius (Hemiptera: Aphididae), is an important pest of cereal crops. Pesticides are the main method for control but carry the risk of selecting for resistance. In response to reports of reduced efficacy of pyrethroid sprays applied to S. avenae, field samples were collected and screened for mutations in the voltage-gated sodium channel, the primary target site for pyrethroids. Aphid mobility and mortality to lambda-cyhalothrin were measured in coated glass vial bioassays. A single amino acid substitution (L1014F) was identified in the domain IIS6 segment of the sodium channel from the S. avenae samples exhibiting reduced pyrethroid efficacy. Bioassays on aphids heterozygous for the kdr mutation (SR) or homozygous for the wild-type allele (SS) showed that those carrying the mutation had significantly lower susceptibility to lambda-cyhalothrin. The L1014F (kdr) mutation, known to confer pyrethroid resistance in many insect pests, has been identified for the first time in S. avenae. Clonal lines heterozygous for the mutation showed 35-40-fold resistance to lambda-cyhalothrin in laboratory bioassays, consistent with the reported effect of this mutation on pyrethroid sensitivity in other aphid species. © 2013 Society of Chemical Industry.

Mechanism-based corrector combination restores ΔF508-CFTR folding and function

PubMed Central

Okiyoneda, Tsukasa; Veit, Guido; Dekkers, Johanna F.; Bagdany, Miklos; Soya, Naoto; Xu, Haijin; Roldan, Ariel; Verkman, Alan S.; Kurth, Mark; Simon, Agnes; Hegedus, Tamas; Beekman, Jeffrey M.; Lukacs, Gergely L.

2013-01-01

The most common cystic fibrosis (CF) mutation, ΔF508 in the nucleotide binding domain-1 (NBD1), impairs CFTR coupled-domain folding, plasma membrane (PM) expression, function and stability. VX-809, a promising investigational corrector of ΔF508-CFTR misprocessing, has limited clinical benefit and incompletely understood mechanism, hampering drug development. Based on the effect of second site suppressor mutations, robust ΔF508-CFTR correction likely requires stabilization of NBD1 and the membrane spanning domains (MSDs)-NBD1 interface, both established primary conformational defects. Here, we elucidated the molecular targets of available correctors; class-I stabilizes the NBD1-MSD1/2 interface, class-II targets NBD2, and only chemical chaperones, surrogates of class-III correctors, stabilize the human ΔF508-NBD1. While VX-809 can correct missense mutations primarily destabilizing the NBD1-MSD1/2 interface, functional PM expression of ΔF508-CFTR also requires compounds that counteract the NBD1 and NBD2 stability defects in CF bronchial epithelial cells and intestinal organoids. Thus, structure-guided corrector combination represents an effective approach for CF therapy. PMID:23666117

Mutations in the kinesin-like protein Eg5 disrupting localization to the mitotic spindle.

PubMed Central

Sawin, K E; Mitchison, T J

1995-01-01

Eg5, a member of the bimC subfamily of kinesin-like microtubule motor proteins, localizes to spindle microtubules in mitosis but not to interphase microtubules. We investigated the molecular basis for spindle localization by transient transfection of Xenopus A6 cells with myc-tagged derivatives of Eg5. Expressed at constitutively high levels from a cytomegalovirus promoter, mycEg5 protein is cytoplasmic throughout interphase, begins to bind microtubules in early prophase, and remains localized to spindle and/or midbody microtubules through mitosis to the end of telophase. Both N- and C-terminal regions of Eg5 are required for this cell-cycle-regulated targeting. Eg5 also contains within its C-terminal domain a sequence conserved among bimC subfamily proteins that includes a potential p34cdc2 phosphorylation site. We show that mutation of a single threonine (T937) within this site to nonphosphorylatable alanine abolishes localization of the mutant protein to the spindle, whereas mutation of T937 to serine preserves spindle localization. We hypothesize that phosphorylation of Eg5 may regulate its localization to the spindle in the cell cycle. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7753799

Drosophila as a model for intractable epilepsy: gilgamesh suppresses seizures in para(bss1) heterozygote flies.

PubMed

Howlett, Iris C; Rusan, Zeid M; Parker, Louise; Tanouye, Mark A

2013-08-07

Intractable epilepsies, that is, seizure disorders that do not respond to currently available therapies, are difficult, often tragic, neurological disorders. Na(+) channelopathies have been implicated in some intractable epilepsies, including Dravet syndrome (Dravet 1978), but little progress has been forthcoming in therapeutics. Here we examine a Drosophila model for intractable epilepsy, the Na(+) channel gain-of-function mutant para(bss1) that resembles Dravet syndrome in some aspects (parker et al. 2011a). In particular, we identify second-site mutations that interact with para(bss1), seizure enhancers, and seizure suppressors. We describe one seizure-enhancer mutation named charlatan (chn). The chn gene normally encodes an Neuron-Restrictive Silencer Factor/RE1-Silencing Transcription factor transcriptional repressor of neuronal-specific genes. We identify a second-site seizure-suppressor mutation, gilgamesh (gish), that reduces the severity of several seizure-like phenotypes of para(bss1)/+ heterozygotes. The gish gene normally encodes the Drosophila ortholog of casein kinase CK1g3, a member of the CK1 family of serine-threonine kinases. We suggest that CK1g3 is an unexpected but promising new target for seizure therapeutics.

UVB-induced mutagenesis in hairless {lambda}lacZ-transgenic mice

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

Frijhoff, A.F.W.; Rebel, H.; Mientjes, E.J.

UVB-induced mutagenesis was studied in hairless 40.6 transgenic mice (Muta{trademark}Mouse), which contain the {lambda}gt1OlacZ shuttle vector as a target for mutagenesis. Mice were exposed at the dorsal side to either single doses of 200, 500, 800, or 1000 J/m{sup 2} UVB or to two successive irradiations of either 200 and 800 J/m{sup 2} UVB, with intervals of 1,3, or 5 days, or to 800 and 200 J/m{sup 2} UVB with a 5-day interval. At 23 days after the last exposure, lacZ mutant frequencies (MF) were determined in the epidermis. The lacZ MF increased linearly with increasing dose of UVB. Themore » mutagenic effect of two successive irradiations appeared to be additive. The UV-induced mutation spectrum was dominated by G:C{r_arrow}A:T transitions at dipyrimidine sites. DNA-sequence analysis of spontaneously mutated phages showed a diverse spectrum consisting of insertions, deletions and G:C {r_arrow} A:T transitions at CpG sites. the results indicate that the hairless {lambda}lacZ-transgenic mouse is a suitable in vivo model for studying UVB-induced mutations. 29 refs., 5 tabs.« less

Role of Host-Driven Mutagenesis in Determining Genome Evolution of Sigma Virus (DMelSV; Rhabdoviridae) in Drosophila melanogaster.

PubMed

Piontkivska, Helen; Matos, Luis F; Paul, Sinu; Scharfenberg, Brian; Farmerie, William G; Miyamoto, Michael M; Wayne, Marta L

2016-10-05

Sigma virus (DMelSV) is ubiquitous in natural populations of Drosophila melanogaster. Host-mediated, selective RNA editing of adenosines to inosines (ADAR) may contribute to control of viral infection by preventing transcripts from being transported into the cytoplasm or being translated accurately; or by increasing the viral genomic mutation rate. Previous PCR-based studies showed that ADAR mutations occur in DMelSV at low frequency. Here we use SOLiD TM deep sequencing of flies from a single host population from Athens, GA, USA to comprehensively evaluate patterns of sequence variation in DMelSV with respect to ADAR. GA dinucleotides, which are weak targets of ADAR, are strongly overrepresented in the positive strand of the virus, consistent with selection to generate ADAR resistance on this complement of the transient, double-stranded RNA intermediate in replication and transcription. Potential ADAR sites in a worldwide sample of viruses are more likely to be "resistant" if the sites do not vary among samples. Either variable sites are less constrained and hence are subject to weaker selection than conserved sites, or the variation is driven by ADAR. We also find evidence of mutations segregating within hosts, hereafter referred to as hypervariable sites. Some of these sites were variable only in one or two flies (i.e., rare); others were shared by four or even all five of the flies (i.e., common). Rare and common hypervariable sites were indistinguishable with respect to susceptibility to ADAR; however, polymorphism in rare sites were more likely to be consistent with the action of ADAR than in common ones, again suggesting that ADAR is deleterious to the virus. Thus, in DMelSV, host mutagenesis is constraining viral evolution both within and between hosts. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells.

PubMed

Byrne, Susan M; Ortiz, Luis; Mali, Prashant; Aach, John; Church, George M

2015-02-18

Sequence-specific nucleases such as TALEN and the CRISPR/Cas9 system have so far been used to disrupt, correct or insert transgenes at precise locations in mammalian genomes. We demonstrate efficient 'knock-in' targeted replacement of multi-kilobase genes in human induced pluripotent stem cells (iPSC). Using a model system replacing endogenous human genes with their mouse counterpart, we performed a comprehensive study of targeting vector design parameters for homologous recombination. A 2.7 kilobase (kb) homozygous gene replacement was achieved in up to 11% of iPSC without selection. The optimal homology arm length was around 2 kb, with homology length being especially critical on the arm not adjacent to the cut site. Homologous sequence inside the cut sites was detrimental to targeting efficiency, consistent with a synthesis-dependent strand annealing (SDSA) mechanism. Using two nuclease sites, we observed a high degree of gene excisions and inversions, which sometimes occurred more frequently than indel mutations. While homozygous deletions of 86 kb were achieved with up to 8% frequency, deletion frequencies were not solely a function of nuclease activity and deletion size. Our results analyzing the optimal parameters for targeting vector design will inform future gene targeting efforts involving multi-kilobase gene segments, particularly in human iPSC. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

A novel mutation of the EYA4 gene associated with post-lingual hearing loss in a proband is co-segregating with a novel PAX3 mutation in two congenitally deaf family members.

PubMed

Cesca, Federica; Bettella, Elisa; Polli, Roberta; Cama, Elona; Scimemi, Pietro; Santarelli, Rosamaria; Murgia, Alessandra

2018-01-01

This work was aimed at establishing the molecular etiology of hearing loss in a 9-year old girl with post-lingual non-syndromic mild sensorineural hearing loss with a complex family history of clinically heterogeneous deafness. The proband's DNA was subjected to NGS analysis of a 59-targeted gene panel, with the use of the Ion Torrent PGM platform. Conventional Sanger sequencing was used for segregation analysis in all the affected relatives. The proband and all the other hearing impaired members of the family underwent a thorough clinical and audiological evaluation. A new likely pathogenic mutation in the EYA4 gene (c.1154C > T; p.Ser385Leu) was identified in the proband and in her 42-year-old father with post-lingual non-syndromic profound sensorineural hearing loss. The EYA4 mutation was also found in the proband's grandfather and uncle, both showing clinical features of Waardenburg syndrome type 1. A novel pathogenic splice-site mutation (c.321+1G > A) of the PAX3 gene was found to co-segregate with the EYA4 mutation in these two subjects. The identified novel EYA4 mutation can be considered responsible of the hearing loss observed in the proband and her father, while a dual molecular diagnosis was reached in the relatives co-segregating the EYA4 and the PAX3 mutations. In these two subjects the DFNA10 phenotype was masked by Waardenburg syndrome. The use of NGS targeted gene-panel, in combination with an extensive clinical and audiological examination led us to identify the genetic cause of the hearing loss in members of a family in which different forms of autosomal dominant deafness segregate. These results provide precise and especially important prognostic and follow-up information for the future audiologic management in the youngest affected member. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantification of mutant SPOP proteins in prostate cancer using mass spectrometry-based targeted proteomics

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

Wang, Hui; Barbieri, Christopher E.; He, Jintang

Speckle-type POZ protein (SPOP) is an E3 ubiquitin ligase adaptor protein that functions as a potential tumor suppressor, and SPOP mutations have been identified in ~10% of human prostate cancers. However, it remains unclear if mutant SPOP proteins can be utilized as biomarkers for early detection, diagnosis, prognosis or targeted therapy of prostate cancer. Moreover, the SPOP mutation sites are distributed in a relatively short region where multiple lysine residues, posing significant challenges for bottom-up proteomics analysis of the SPOP mutations. To address this issue, PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing)-SRM (selected reaction monitoring) mass spectrometrymore » assays have been developed for quantifying wild-type SPOP protein and 11 prostate cancer-derived SPOP mutations. Despite inherent limitations due to amino acid sequence constraints, all the PRISM-SRM assays developed using Arg-C digestion showed a linear dynamic range of at least two orders of magnitude, with limits of quantification range from 0.1 to 1 fmol/μg of total protein in the cell lysate. Applying these SRM assays to analyze HEK293T cells with and without expression of the three most frequent SPOP mutations in prostate cancer (Y87N, F102C or F133V) led to confident detection of all three SPOP mutations in corresponding positive cell lines but not in the negative cell lines. Expression of the F133V mutation and wild-type SPOP was at much lower levels compared to that of F102C and Y87N mutations; however, at present it is unknown if this also affects the activity of the SPOP protein. In summary, PRISM-SRM enables multiplexed, isoform-specific detection of mutant SPOP proteins in cell lysates, which holds great potential in biomarker development for prostate cancer.« less

Sequences in Glycoprotein gp41, the CD4 Binding Site, and the V2 Domain Regulate Sensitivity and Resistance of HIV-1 to Broadly Neutralizing Antibodies

PubMed Central

O'Rourke, Sara M.; Schweighardt, Becky; Phung, Pham; Mesa, Kathryn A.; Vollrath, Aaron L.; Tatsuno, Gwen P.; To, Briana; Sinangil, Faruk; Limoli, Kay; Wrin, Terri

2012-01-01

The swarm of quasispecies that evolves in each HIV-1-infected individual represents a source of closely related Env protein variants that can be used to explore various aspects of HIV-1 biology. In this study, we made use of these variants to identify mutations that confer sensitivity and resistance to the broadly neutralizing antibodies found in the sera of selected HIV-1-infected individuals. For these studies, libraries of Env proteins were cloned from infected subjects and screened for infectivity and neutralization sensitivity. The nucleotide sequences of the Env proteins were then compared for pairs of neutralization-sensitive and -resistant viruses. In vitro mutagenesis was used to identify the specific amino acids responsible for the neutralization phenotype. All of the mutations altering neutralization sensitivity/resistance appeared to induce conformational changes that simultaneously enhanced the exposure of two or more epitopes located in different regions of gp160. These mutations appeared to occur at unique positions required to maintain the quaternary structure of the gp160 trimer, as well as conformational masking of epitopes targeted by neutralizing antibodies. Our results show that sequences in gp41, the CD4 binding site, and the V2 domain all have the ability to act as global regulators of neutralization sensitivity. Our results also suggest that neutralization assays designed to support the development of vaccines and therapeutics targeting the HIV-1 Env protein should consider virus variation within individuals as well as virus variation between individuals. PMID:22933284

Molecular principles behind pyrazinamide resistance due to mutations in panD gene in Mycobacterium tuberculosis.

PubMed

Pandey, Bharati; Grover, Sonam; Tyagi, Chetna; Goyal, Sukriti; Jamal, Salma; Singh, Aditi; Kaur, Jagdeep; Grover, Abhinav

2016-04-25

The latest resurrection of drug resistance poses serious threat to the treatment and control of the disease. Mutations have been detected in panD gene in the Mycobacterium tuberculosis (Mtb) strains. Mutation of histidine to arginine at residue 21 (H21R) and isoleucine to valine at residue 29 (I49V) in the non-active site of panD gene has led to PZA resistance. This study will help in reconnoitering the mechanism of pyrazinamide (PZA) resistance caused due to double mutation identified in the panD gene of M. tuberculosis clinical isolates. It is known that panD gene encodes aspartate decarboxylase essential for β-alanine synthesis that makes it a potential therapeutic drug target for tuberculosis treatment. The knowledge about the molecular mechanism conferring drug resistance in M. tuberculosis is scarce, which is a significant challenge in designing successful therapeutic drug. In this study, structural and dynamic repercussions of H21R-I49V double mutation in panD complexed with PZA have been corroborated through docking and molecular dynamics based simulation. The double mutant (DM) shows low docking score and thus, low binding affinity for PZA as compared to the native protein. It was observed that the mutant protein exhibits more structural fluctuation at the ligand binding site in comparison to the native type. Furthermore, the flexibility and compactness analyses indicate that the double mutation influence interaction of PZA with the protein. The hydrogen-bond interaction patterns further supported our results. The covariance and PCA analysis elucidated that the double mutation affects the collective motion of residues in phase space. The results have been presented with an explanation for the induced drug resistance conferred by the H21R-I49V double mutation in panD gene and gain valuable insight to facilitate the advent of efficient therapeutics for combating resistance against PZA. Copyright © 2016 Elsevier B.V. All rights reserved.

Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site.

PubMed

Mazhab-Jafari, Mohammad T; Marshall, Christopher B; Smith, Matthew J; Gasmi-Seabrook, Geneviève M C; Stathopulos, Peter B; Inagaki, Fuyuhiko; Kay, Lewis E; Neel, Benjamin G; Ikura, Mitsuhiko

2015-05-26

K-RAS4B (Kirsten rat sarcoma viral oncogene homolog 4B) is a prenylated, membrane-associated GTPase protein that is a critical switch for the propagation of growth factor signaling pathways to diverse effector proteins, including rapidly accelerated fibrosarcoma (RAF) kinases and RAS-related protein guanine nucleotide dissociation stimulator (RALGDS) proteins. Gain-of-function KRAS mutations occur frequently in human cancers and predict poor clinical outcome, whereas germ-line mutations are associated with developmental syndromes. However, it is not known how these mutations affect K-RAS association with biological membranes or whether this impacts signal transduction. Here, we used solution NMR studies of K-RAS4B tethered to nanodiscs to investigate lipid bilayer-anchored K-RAS4B and its interactions with effector protein RAS-binding domains (RBDs). Unexpectedly, we found that the effector-binding region of activated K-RAS4B is occluded by interaction with the membrane in one of the NMR-observable, and thus highly populated, conformational states. Binding of the RAF isoform ARAF and RALGDS RBDs induced marked reorientation of K-RAS4B from the occluded state to RBD-specific effector-bound states. Importantly, we found that two Noonan syndrome-associated mutations, K5N and D153V, which do not affect the GTPase cycle, relieve the occluded orientation by directly altering the electrostatics of two membrane interaction surfaces. Similarly, the most frequent KRAS oncogenic mutation G12D also drives K-RAS4B toward an exposed configuration. Further, the D153V and G12D mutations increase the rate of association of ARAF-RBD with lipid bilayer-tethered K-RAS4B. We revealed a mechanism of K-RAS4B autoinhibition by membrane sequestration of its effector-binding site, which can be disrupted by disease-associated mutations. Stabilizing the autoinhibitory interactions between K-RAS4B and the membrane could be an attractive target for anticancer drug discovery.

Dominant Drop mutants are gain-of-function alleles of the muscle segment homeobox gene (msh) whose overexpression leads to the arrest of eye development.

PubMed

Mozer, B A

2001-05-15

Dominant Drop (Dr) mutations are nearly eyeless and have additional recessive phenotypes including lethality and patterning defects in eye and sensory bristles due to cis-regulatory lesions in the cell cycle regulator string (stg). Genetic analysis demonstrates that the dominant small eye phenotype is the result of separate gain-of-function mutations in the closely linked muscle segment homeobox (msh) gene, encoding a homeodomain transcription factor required for patterning of muscle and nervous system. Reversion of the Dr(Mio) allele was coincident with the generation of lethal loss-of-function mutations in msh in cis, suggesting that the dominant eye phenotype is the result of ectopic expression. Molecular genetic analysis revealed that two dominant Dr alleles contain lesions upstream of the msh transcription start site. In the Dr(Mio) mutant, a 3S18 retrotransposon insertion is the target of second-site mutations (P-element insertions or deletions) which suppress the dominant eye phenotype following reversion. The pattern of 3S18 expression and the absence of msh in eye imaginal discs suggest that transcriptional activation of the msh promoter accounts for ectopic expression. Dr dominant mutations arrest eye development by blocking the progression of the morphogenetic furrow leading to photoreceptor cell loss via apoptosis. Gal4-mediated ubiquitous expression of msh in third-instar larvae was sufficient to arrest the morphogenetic furrow in the eye imaginal disc and resulted in lethality prior to eclosion. Dominant mutations in the human msx2 gene, one of the vertebrate homologs of msh, are associated with craniosynostosis, a disease affecting cranial development. The Dr mutations are the first example of gain-of-function mutations in the msh/msx gene family identified in a genetically tractible model organism and may serve as a useful tool to identify additional genes that regulate this class of homeodomain proteins. Copyright 2001 Academic Press.

First report of the East African kdr mutation in an Anopheles gambiae mosquito in Côte d'Ivoire.

PubMed

Chouaïbou, Mouhamadou; Kouadio, Fodjo Behi; Tia, Emmanuel; Djogbenou, Luc

2017-02-09

Background . The intensive use of insecticides in public health and agriculture has led to the development of insecticide resistances in malaria vectors across sub-Saharan Africa countries in the last two decades. The kdr target site point mutation which is among the best characterised resistance mechanisms seems to be changing its distribution patterns on the African continent. The 1014F  kdr mutation originally described only in West Africa is spreading to East Africa while the 1014S  kdr mutation originally described in East Africa, is spreading to West and Central Africa. However, the East- kdr mutation has not been reported in Côte d'Ivoire so far. Methods . Immature stages of Anopheles gambiae s.l. were collected from breeding sites at the outskirts of Yamoussoukro, Côte d'Ivoire. Emerging 3-5 day old adult female mosquitoes were tested for susceptibility to deltamethrin 0.05%, malathion 5%, bendiocarb 1% and dichlorodiphenyltrichloroethane (DDT) 4% according to WHO standard procedures. A total of 50 An. gambiae s.l. specimens were drawn at random for DNA extraction and identification down to the species level. A subsample of 30 mosquitoes was tested for the East-African kdr mutation using a Taqman assay. Results . The tested mosquito population appeared to be strongly resistant to deltamethrin (1.03% mortality), bendiocarb (38.46% mortality) and DDT (0% mortality) with probable resistance observed for malathion (92.47%). Among the 41 mosquitoes that were successfully characterized, An. coluzzii was predominant (68.3%) followed by An. gambiae   s.s. (19.5%) and a few hybrids (7.3%). Out of 30 specimens genotyped for East- kdr , a single hybrid mosquito appeared to be heterozygous for the mutation. Conclusion . The present study revealed the presence of the East- kdr mutation in Côte d'Ivoire for the first time in An. gambiae and highlights the urgent need to start monitoring the allele and genotype frequencies.

Target-site resistance to pyrethroid insecticides in German populations of the cabbage stem flea beetle, Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae).

PubMed

Zimmer, Christoph T; Müller, Andreas; Heimbach, Udo; Nauen, Ralf

2014-01-01

Cabbage stem flea beetle, Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae) is a major pest of winter oilseed rape in several European countries particularly attacking young emerging plants in autumn. Over the last several decades, pyrethroid insecticides have been foliarly applied to control flea beetle outbreaks. Recent control failures in northern Germany suggested pyrethroid resistance development in cabbage stem flea beetles, which were confirmed by resistance monitoring bioassays using lambda-cyhalothrin in an adult vial test. The purpose of this study was to investigate the presence of polymorphisms in the para-type voltage-gated sodium channel gene of P. chrysocephala known to be involved in knock-down resistance (kdr). By using a degenerate primer approach we PCR amplified part of the para-type sodium channel gene and identified in resistant flea beetles a single nucleotide polymorphism resulting in an L1014F (kdr) mutation within domain IIS6 of the channel protein, known as one of the chief pyrethroid target-site resistance mechanisms in several other pest insects. Twenty populations including four archived museum samples collected between 1945 and 1958 were analyzed using a newly developed pyrosequencing diagnostic assay. The assay revealed a kdr allele frequency of 90-100% in those flea beetle populations expressing high-level cross-resistance in discriminating dose bioassays against different pyrethroids such as lambda-cyhalothrin, tau-fluvalinate, etofenprox and bifenthrin. The presence of target-site resistance to pyrethroids in cabbage stem flea beetle is extremely worrying considering the lack of effective alternative modes of action to control this pest in Germany and other European countries, and is likely to result in major control problems once it expands to other geographies. The striking fact that cabbage stem flea beetle is next to pollen beetle, Meligethes aeneus the second coleopteran pest in European winter oilseed rape resisting pyrethroid treatments by expressing a target-site mutation, underpins the importance of diversity in available chemistry for resistance management tactics based on mode of action rotation in order to guarantee sustainable winter oilseed rape cultivation in Europe. Copyright © 2013 Elsevier Inc. All rights reserved.

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

PubMed

Supek, Fran; Lehner, Ben

2017-07-27

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

A Novel Splice-Site Mutation in Angiotensin I-Converting Enzyme (ACE) Gene, c.3691+1G>A (IVS25+1G>A), Causes a Dramatic Increase in Circulating ACE through Deletion of the Transmembrane Anchor

PubMed Central

Persu, Alexandre; Lambert, Michel; Deinum, Jaap; Cossu, Marta; de Visscher, Nathalie; Irenge, Leonid; Ambroise, Jerôme; Minon, Jean-Marc; Nesterovitch, Andrew B.; Churbanov, Alexander; Popova, Isolda A.; Danilov, Sergei M.; Danser, A. H. Jan; Gala, Jean-Luc

2013-01-01

Background Angiotensin-converting enzyme (ACE) (EC 4.15.1) metabolizes many biologically active peptides and plays a key role in blood pressure regulation and vascular remodeling. Elevated ACE levels are associated with different cardiovascular and respiratory diseases. Methods and Results Two Belgian families with a 8-16-fold increase in blood ACE level were incidentally identified. A novel heterozygous splice site mutation of intron 25 - IVS25+1G>A (c.3691+1G>A) - cosegregating with elevated plasma ACE was identified in both pedigrees. Messenger RNA analysis revealed that the mutation led to the retention of intron 25 and Premature Termination Codon generation. Subjects harboring the mutation were mostly normotensive, had no left ventricular hypertrophy or cardiovascular disease. The levels of renin-angiotensin-aldosterone system components in the mutated cases and wild-type controls were similar, both at baseline and after 50 mg captopril. Compared with non-affected members, quantification of ACE surface expression and shedding using flow cytometry assay of dendritic cells derived from peripheral blood monocytes of affected members, demonstrated a 50% decrease and 3-fold increase, respectively. Together with a dramatic increase in circulating ACE levels, these findings argue in favor of deletion of transmembrane anchor, leading to direct secretion of ACE out of cells. Conclusions We describe a novel mutation of the ACE gene associated with a major familial elevation of circulating ACE, without evidence of activation of the renin-angiotensin system, target organ damage or cardiovascular complications. These data are consistent with the hypothesis that membrane-bound ACE, rather than circulating ACE, is responsible for Angiotensin II generation and its cardiovascular consequences. PMID:23560051

Thermal Stabilization of Dihydrofolate Reductase Using Monte Carlo Unfolding Simulations and Its Functional Consequences

PubMed Central

Whitney, Anna; Shakhnovich, Eugene I.

2015-01-01

Design of proteins with desired thermal properties is important for scientific and biotechnological applications. Here we developed a theoretical approach to predict the effect of mutations on protein stability from non-equilibrium unfolding simulations. We establish a relative measure based on apparent simulated melting temperatures that is independent of simulation length and, under certain assumptions, proportional to equilibrium stability, and we justify this theoretical development with extensive simulations and experimental data. Using our new method based on all-atom Monte-Carlo unfolding simulations, we carried out a saturating mutagenesis of Dihydrofolate Reductase (DHFR), a key target of antibiotics and chemotherapeutic drugs. The method predicted more than 500 stabilizing mutations, several of which were selected for detailed computational and experimental analysis. We find a highly significant correlation of r = 0.65–0.68 between predicted and experimentally determined melting temperatures and unfolding denaturant concentrations for WT DHFR and 42 mutants. The correlation between energy of the native state and experimental denaturation temperature was much weaker, indicating the important role of entropy in protein stability. The most stabilizing point mutation was D27F, which is located in the active site of the protein, rendering it inactive. However for the rest of mutations outside of the active site we observed a weak yet statistically significant positive correlation between thermal stability and catalytic activity indicating the lack of a stability-activity tradeoff for DHFR. By combining stabilizing mutations predicted by our method, we created a highly stable catalytically active E. coli DHFR mutant with measured denaturation temperature 7.2°C higher than WT. Prediction results for DHFR and several other proteins indicate that computational approaches based on unfolding simulations are useful as a general technique to discover stabilizing mutations. PMID:25905910

RNF8 E3 Ubiquitin Ligase Stimulates Ubc13 E2 Conjugating Activity That Is Essential for DNA Double Strand Break Signaling and BRCA1 Tumor Suppressor Recruitment

DOE PAGES

Hodge, Curtis D.; Ismail, Ismail H.; Edwards, Ross A.; ...

2016-02-22

DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63-linked ubiquitin chains in DSB signaling. In this paper, we defined the activated RNF8-Ubc13~ubiquitin complex by x-ray crystallography and its functional solution conformations by x-ray scattering, as tested by separation-of-function mutations imaged in cells by immunofluorescence. The collective results show that the RING E3 RNF8 targets E2 Ubc13 to DSB sites and plays a critical role in damage signaling by stimulating polyubiquitination through modulating conformations of ubiquitin covalently linked to the Ubc13more » active site. Structure-guided separation-of-function mutations show that the RNF8 E2 stimulating activity is essential for DSB signaling in mammalian cells and is necessary for downstream recruitment of 53BP1 and BRCA1. Chromatin-targeted RNF168 rescues 53BP1 recruitment involved in non-homologous end joining but not BRCA1 recruitment for homologous recombination. Finally, these findings suggest an allosteric approach to targeting the ubiquitin-docking cleft at the E2-E3 interface for possible interventions in cancer and chronic inflammation, and moreover, they establish an independent RNF8 role in BRCA1 recruitment.« less

Novel MSH2 splice-site mutation in a young patient with Lynch syndrome

PubMed Central

Liccardo, Raffaella; De Rosa, Marina; Izzo, Paola; Duraturo, Francesca

2018-01-01

Lynch Syndrome (LS) is associated with germline mutations in one of the mismatch repair (MMR) genes, including MutL homolog 1 (MLH1), MutS homolog 2 (MSH2), MSH6, PMS1 homolog 2, mismatch repair system component (PMS2), MLH3 and MSH3. The mutations identified in MMR genes are point mutations or large rearrangements. The point mutations are certainly pathogenetic whether they determine formation of truncated protein. The mutations that arise in splice sites are classified as ‘likely pathogenic’ variants. In the present study, a novel splicing mutation was identified, (named c.212-1g>a), in the MSH2 gene. This novel mutation in the consensus splice site of MSH2 exon 2 leads to the loss of the canonical splice site, without skipping in-frame of exon 2; also with the formation of 2 aberrant transcripts, due to the activation of novel splice sites in exon 2. This mutation was identified in a young patient who developed colon cancer at the age of 26 years and their belongs to family that met the ‘Revised Amsterdam Criteria’. The present study provided insight into the molecular mechanism determining the pathogenicity of this novel MSH2 mutation and it reaffirms the importance of genetic testing in LS. PMID:29568967

Levels of DNA Methylation Vary at CpG Sites across the BRCA1 Promoter, and Differ According to Triple Negative and "BRCA-Like" Status, in Both Blood and Tumour DNA.

PubMed

Daniels, Sarah L; Burghel, George J; Chambers, Philip; Al-Baba, Shadi; Connley, Daniel D; Brock, Ian W; Cramp, Helen E; Dotsenko, Olena; Wilks, Octavia; Wyld, Lynda; Cross, Simon S; Cox, Angela

2016-01-01

Triple negative breast cancer is typically an aggressive and difficult to treat subtype. It is often associated with loss of function of the BRCA1 gene, either through mutation, loss of heterozygosity or methylation. This study aimed to measure methylation of the BRCA1 gene promoter at individual CpG sites in blood, tumour and normal breast tissue, to assess whether levels were correlated between different tissues, and with triple negative receptor status, histopathological scoring for BRCA-like features and BRCA1 protein expression. Blood DNA methylation levels were significantly correlated with tumour methylation at 9 of 11 CpG sites examined (p<0.0007). The levels of tumour DNA methylation were significantly higher in triple negative tumours, and in tumours with high BRCA-like histopathological scores (10 of 11 CpG sites; p<0.01 and p<0.007 respectively). Similar results were observed in blood DNA (6 of 11 CpG sites; p<0.03 and 7 of 11 CpG sites; p<0.02 respectively). This study provides insight into the pattern of CpG methylation across the BRCA1 promoter, and supports previous studies suggesting that tumours with BRCA1 promoter methylation have similar features to those with BRCA1 mutations, and therefore may be suitable for the same targeted therapies.

Opaque-2 is a transcriptional activator that recognizes a specific target site in 22-kD zein genes.

PubMed Central

Schmidt, R J; Ketudat, M; Aukerman, M J; Hoschek, G

1992-01-01

opaque-2 (o2) is a regulatory locus in maize that plays an essential role in controlling the expression of genes encoding the 22-kD zein proteins. Through DNase I footprinting and DNA binding analyses, we have identified the binding site for the O2 protein (O2) in the promoter of 22-kD zein genes. The sequence in the 22-kD zein gene promoter that is recognized by O2 is similar to the target site recognized by other "basic/leucine zipper" (bZIP) proteins in that it contains an ACGT core that is necessary for DNA binding. The site is located in the -300 region relative to the translation start and lies about 20 bp downstream of the highly conserved zein gene sequence motif known as the "prolamin box." Employing gel mobility shift assays, we used O2 antibodies and nuclear extracts from an o2 null mutant to demonstrate that the O2 protein in maize endosperm nuclei recognizes the target site in the zein gene promoter. Mobility shift assays using nuclear proteins from an o2 null mutant indicated that other endosperm proteins in addition to O2 can bind the O2 target site and that O2 may be associated with one of these proteins. We also demonstrated that in yeast cells the O2 protein can activate expression of a lacZ gene containing a multimer of the O2 target sequence as part of its promoter, thus confirming its role as a transcriptional activator. A computer-assisted search indicated that the O2 target site is not present in the promoters of zein genes other than those of the 22-kD class. These data suggest a likely explanation at the molecular level for the differential effect of o2 mutations on expression of certain members of the zein gene family. PMID:1392590

Effect of herbicide resistance endowing Ile-1781-Leu and Asp-2078-Gly ACCase gene mutations on ACCase kinetics and growth traits in Lolium rigidum

PubMed Central

Vila-Aiub, Martin M.; Yu, Qin; Han, Heping; Powles, Stephen B.

2015-01-01

The rate of herbicide resistance evolution in plants depends on fitness traits endowed by alleles in both the presence and absence (resistance cost) of herbicide selection. The effect of two Lolium rigidum spontaneous homozygous target-site resistance-endowing mutations (Ile-1781-Leu, Asp-2078-Gly) on both ACCase activity and various plant growth traits have been investigated here. Relative growth rate (RGR) and components (net assimilation rate, leaf area ratio), resource allocation to different organs, and growth responses in competition with a wheat crop were assessed. Unlike plants carrying the Ile-1781-Leu resistance mutation, plants homozygous for the Asp-2078-Gly mutation exhibited a significantly lower RGR (30%), which translated into lower allocation of biomass to roots, shoots, and leaves, and poor responses to plant competition. Both the negligible and significant growth reductions associated, respectively, with the Ile-1781-Leu and Asp-2078-Gly resistance mutations correlated with their impact on ACCase activity. Whereas the Ile-1781-Leu mutation showed no pleiotropic effects on ACCase kinetics, the Asp-2078-Gly mutation led to a significant reduction in ACCase activity. The impaired growth traits are discussed in the context of resistance costs and the effects of each resistance allele on ACCase activity. Similar effects of these two particular ACCase mutations on the ACCase activity of Alopecurus myosuroides were also confirmed. PMID:26019257

Single 23S rRNA mutations at the ribosomal peptidyl transferase centre confer resistance to valnemulin and other antibiotics in Mycobacterium smegmatis by perturbation of the drug binding pocket.

PubMed

Long, Katherine S; Poehlsgaard, Jacob; Hansen, Lykke H; Hobbie, Sven N; Böttger, Erik C; Vester, Birte

2009-03-01

Tiamulin and valnemulin target the peptidyl transferase centre (PTC) on the bacterial ribosome. They are used in veterinary medicine to treat infections caused by a variety of bacterial pathogens, including the intestinal spirochetes Brachyspira spp. Mutations in ribosomal protein L3 and 23S rRNA have previously been associated with tiamulin resistance in Brachyspira spp. isolates, but as multiple mutations were isolated together, the roles of the individual mutations are unclear. In this work, individual 23S rRNA mutations associated with pleuromutilin resistance at positions 2055, 2447, 2504 and 2572 (Escherichia coli numbering) are introduced into a Mycobacterium smegmatis strain with a single rRNA operon. The single mutations each confer a significant and similar degree of valnemulin resistance and those at 2447 and 2504 also confer cross-resistance to other antibiotics that bind to the PTC in M. smegmatis. Antibiotic footprinting experiments on mutant ribosomes show that the introduced mutations cause structural perturbations at the PTC and reduced binding of pleuromutilin antibiotics. This work underscores the fact that mutations at nucleotides distant from the pleuromutilin binding site can confer the same level of valnemulin resistance as those at nucleotides abutting the bound drug, and suggests that the former function indirectly by altering local structure and flexibility at the drug binding pocket.

HIV-1 reverse transcriptase and antiviral drug resistance. Part 2.

PubMed

Das, Kalyan; Arnold, Eddy

2013-04-01

Structures of RT and its complexes combined with biochemical and clinical data help in illuminating the molecular mechanisms of different drug-resistance mutations. The NRTI drugs that are used in combinations have different primary mutation sites. RT mutations that confer resistance to one drug can be hypersensitive to another RT drug. Structure of an RT-DNA-nevirapine complex revealed how NNRTI binding forbids RT from forming a polymerase competent complex. Collective knowledge about various mechanisms of drug resistance by RT has broader implications for understanding and targeting drug resistance in general. In Part 1, we discussed the role of RT in developing HIV-1 drug resistance, structural and functional states of RT, and the nucleoside/nucleotide analog (NRTI) and non-nucleoside (NNRTI) drugs used in treating HIV-1 infections. In this part, we discuss structural understanding of various mechanisms by which RT confers antiviral drug resistance. Copyright © 2013 Elsevier B.V. All rights reserved.

Exome Sequencing Identified a Splice Site Mutation in FHL1 that Causes Uruguay Syndrome, an X-Linked Disorder With Skeletal Muscle Hypertrophy and Premature Cardiac Death.

PubMed

Xue, Yuan; Schoser, Benedikt; Rao, Aliz R; Quadrelli, Roberto; Vaglio, Alicia; Rupp, Verena; Beichler, Christine; Nelson, Stanley F; Schapacher-Tilp, Gudrun; Windpassinger, Christian; Wilcox, William R

2016-04-01

Previously, we reported a rare X-linked disorder, Uruguay syndrome in a single family. The main features are pugilistic facies, skeletal deformities, and muscular hypertrophy despite a lack of exercise and cardiac ventricular hypertrophy leading to premature death. An ≈19 Mb critical region on X chromosome was identified through identity-by-descent analysis of 3 affected males. Exome sequencing was conducted on one affected male to identify the disease-causing gene and variant. A splice site variant (c.502-2A>G) in the FHL1 gene was highly suspicious among other candidate genes and variants. FHL1A is the predominant isoform of FHL1 in cardiac and skeletal muscle. Sequencing cDNA showed the splice site variant led to skipping of exons 6 of the FHL1A isoform, equivalent to the FHL1C isoform. Targeted analysis showed that this splice site variant cosegregated with disease in the family. Western blot and immunohistochemical analysis of muscle from the proband showed a significant decrease in protein expression of FHL1A. Real-time polymerase chain reaction analysis of different isoforms of FHL1 demonstrated that the FHL1C is markedly increased. Mutations in the FHL1 gene have been reported in disorders with skeletal and cardiac myopathy but none has the skeletal or facial phenotype seen in patients with Uruguay syndrome. Our data suggest that a novel FHL1 splice site variant results in the absence of FHL1A and the abundance of FHL1C, which may contribute to the complex and severe phenotype. Mutation screening of the FHL1 gene should be considered for patients with uncharacterized myopathies and cardiomyopathies. © 2016 American Heart Association, Inc.

Extending the cleavage rules for the hammerhead ribozyme: mutating adenosine15.1 to inosine15.1 changes the cleavage site specificity from N16.2U16.1H17 to N16.2C16.1H17.

PubMed Central

Ludwig, J; Blaschke, M; Sproat, B S

1998-01-01

In this paper, we show that an adenosine to inosine mutation at position 15.1 changes the substrate specificity of the hammerhead ribozyme from N16.2U16.1H17to N16.2C16.1H17(H represents A, C or U). This result extends the hammerhead cleavage triplet definition from N16.2U16.1H17to the more general N16.2Y16.1H17. Comparison of cleavage rates using I15.1ribozymes for NCH triplets and standard A15.1 ribozymes for NUH triplets under single turnover conditions shows similar or slightly enhanced levels of reactivity for the I15. 1-containing structures. The effect of I15.1 substitution was also tested in nuclease-resistant 2'- O -alkyl substituted derivatives (oligozymes), showing a similar level of activity for the NUH and NCH cleaving structures. The availability of NCH triplets that can be targeted without loss of efficiency increases the flexibility of ribozyme targeting strategies. This was demonstrated by an efficient cleavage of an HCV transcript at a previously inaccessible GCA site in codon 2. PMID:9580675

True-breeding targeted gene knock-out in barley using designer TALE-nuclease in haploid cells.

PubMed

Gurushidze, Maia; Hensel, Goetz; Hiekel, Stefan; Schedel, Sindy; Valkov, Vladimir; Kumlehn, Jochen

2014-01-01

Transcription activator-like effector nucleases (TALENs) are customizable fusion proteins able to cleave virtually any genomic DNA sequence of choice, and thereby to generate site-directed genetic modifications in a wide range of cells and organisms. In the present study, we expressed TALENs in pollen-derived, regenerable cells to establish the generation of instantly true-breeding mutant plants. A gfp-specific TALEN pair was expressed via Agrobacterium-mediated transformation in embryogenic pollen of transgenic barley harboring a functional copy of gfp. Thanks to the haploid nature of the target cells, knock-out mutations were readily detected, and homozygous primary mutant plants obtained following genome duplication. In all, 22% of the TALEN transgenics proved knocked out with respect to gfp, and the loss of function could be ascribed to the deletions of between four and 36 nucleotides in length. The altered gfp alleles were transmitted normally through meiosis, and the knock-out phenotype was consistently shown by the offspring of two independent mutants. Thus, here we describe the efficient production of TALEN-mediated gene knock-outs in barley that are instantaneously homozygous and non-chimeric in regard to the site-directed mutations induced. This TALEN approach has broad applicability for both elucidating gene function and tailoring the phenotype of barley and other crop species.

[Study of gene mutation in 62 hemophilia A children].

PubMed

Hu, Q; Liu, A G; Zhang, L Q; Zhang, A; Wang, Y Q; Wang, S M; Lu, Y J; Wang, X

2017-11-02

Objective: To analyze the mutation type of FⅧ gene in children with hemophilia A and to explore the relationship among hemophilia gene mutation spectrum, gene mutation and clinical phenotype. Method: Sixty-two children with hemophilia A from Department of Pediatric Hematology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology between January 2015 and March 2017 were enrolled. All patients were male, aged from 4 months to 7 years and F Ⅷ activity ranged 0.2%-11.0%. Fifty cases had severe, 10 cases had moderate and 2 cases had mild hemophilia A. DNA was isolated from peripheral blood in hemophilia A children and the target gene fragment was amplified by PCR, in combination with the second generation sequencing, 22 and 1 introns were detected. Negative cases were detected by the second generation sequencing and results were compared with those of the international FⅧ gene mutation database. Result: There were 20 cases (32%) of intron 22 inversion, 2 cases (3%) of intron 1 inversion, 18 cases (29%) of missense mutation, 5 cases (8%) of nonsense mutation, 7 cases (11%) of deletion mutation, 1 case(2%)of splice site mutation, 2 cases (3%) of large fragment deletion and 1 case of insertion mutation (2%). No mutation was detected in 2 cases (3%), and 4 cases (7%) failed to amplify. The correlation between phenotype and genotype showed that the most common gene mutation in severe hemophilia A was intron 22 inversion (20 cases), accounting for 40% of severe patients, followed by 11 cases of missense mutation (22%). The most common mutation in moderate hemophilia A was missense mutation (6 cases), accounting for 60% of moderate patients. Conclusion: The most frequent mutation type in hemophilia A was intron 22 inversion, followed by missense mutation, again for missing mutation. The relationship between phenotype and genotype: the most frequent gene mutation in severe hemophilia A is intron 22 inversion, followed by missense mutation; the most frequent gene mutation in medium hemophilia A is missense mutation.

A novel frameshift GRN mutation results in frontotemporal lobar degeneration with a distinct clinical phenotype in two siblings: case report and literature review.

PubMed

Hosaka, Takashi; Ishii, Kazuhiro; Miura, Takeshi; Mezaki, Naomi; Kasuga, Kensaku; Ikeuchi, Takeshi; Tamaoka, Akira

2017-09-15

Progranulin gene (GRN) mutations are major causes of frontotemporal lobar degeneration. To date, 68 pathogenic GRN mutations have been identified. However, very few of these mutations have been reported in Asians. Moreover, some GRN mutations manifest with familial phenotypic heterogeneity. Here, we present a novel GRN mutation resulting in frontotemporal lobar degeneration with a distinct clinical phenotype, and we review reports of GRN mutations associated with familial phenotypic heterogeneity. We describe the case of a 74-year-old woman with left frontotemporal lobe atrophy who presented with progressive anarthria and non-fluent aphasia. Her brother had been diagnosed with corticobasal syndrome (CBS) with right-hand limb-kinetic apraxia, aphasia, and a similar pattern of brain atrophy. Laboratory blood examinations did not reveal abnormalities that could have caused cognitive dysfunction. In the cerebrospinal fluid, cell counts and protein concentrations were within normal ranges, and concentrations of tau protein and phosphorylated tau protein were also normal. Since similar familial cases due to mutation of GRN and microtubule-associated protein tau gene (MAPT) were reported, we performed genetic analysis. No pathological mutations of MAPT were identified, but we identified a novel GRN frameshift mutation (c.1118_1119delCCinsG: p.Pro373ArgX37) that resulted in progranulin haploinsufficiency. This is the first report of a GRN mutation associated with familial phenotypic heterogeneity in Japan. Literature review of GRN mutations associated with familial phenotypic heterogeneity revealed no tendency of mutation sites. The role of progranulin has been reported in this and other neurodegenerative diseases, and the analysis of GRN mutations may lead to the discovery of a new therapeutic target.

Identifying RNA splicing factors using IFT genes in Chlamydomonas reinhardtii.

PubMed

Lin, Huawen; Zhang, Zhengyan; Iomini, Carlo; Dutcher, Susan K

2018-03-01

Intraflagellar transport moves proteins in and out of flagella/cilia and it is essential for the assembly of these organelles. Using whole-genome sequencing, we identified splice site mutations in two IFT genes, IFT81 ( fla9 ) and IFT121 ( ift121-2 ), which lead to flagellar assembly defects in the unicellular green alga Chlamydomonas reinhardtii The splicing defects in these ift mutants are partially corrected by mutations in two conserved spliceosome proteins, DGR14 and FRA10. We identified a dgr14 deletion mutant, which suppresses the 3' splice site mutation in IFT81 , and a frameshift mutant of FRA10 , which suppresses the 5' splice site mutation in IFT121 Surprisingly, we found dgr14-1 and fra10 mutations suppress both splice site mutations. We suggest these two proteins are involved in facilitating splice site recognition/interaction; in their absence some splice site mutations are tolerated. Nonsense mutations in SMG1 , which is involved in nonsense-mediated decay, lead to accumulation of aberrant transcripts and partial restoration of flagellar assembly in the ift mutants. The high density of introns and the conservation of noncore splicing factors, together with the ease of scoring the ift mutant phenotype, make Chlamydomonas an attractive organism to identify new proteins involved in splicing through suppressor screening. © 2018 The Authors.

Mapping mutational effects along the evolutionary landscape of HIV envelope

PubMed Central

Hilton, Sarah K; Overbaugh, Julie

2018-01-01

The immediate evolutionary space accessible to HIV is largely determined by how single amino acid mutations affect fitness. These mutational effects can shift as the virus evolves. However, the prevalence of such shifts in mutational effects remains unclear. Here, we quantify the effects on viral growth of all amino acid mutations to two HIV envelope (Env) proteins that differ at >100 residues. Most mutations similarly affect both Envs, but the amino acid preferences of a minority of sites have clearly shifted. These shifted sites usually prefer a specific amino acid in one Env, but tolerate many amino acids in the other. Surprisingly, shifts are only slightly enriched at sites that have substituted between the Envs—and many occur at residues that do not even contact substitutions. Therefore, long-range epistasis can unpredictably shift Env’s mutational tolerance during HIV evolution, although the amino acid preferences of most sites are conserved between moderately diverged viral strains. PMID:29590010

Loss of the BMP Antagonist, SMOC-1, Causes Ophthalmo-Acromelic (Waardenburg Anophthalmia) Syndrome in Humans and Mice

PubMed Central

Rainger, Joe; van Beusekom, Ellen; Ramsay, Jacqueline K.; McKie, Lisa; Al-Gazali, Lihadh; Pallotta, Rosanna; Saponari, Anita; Branney, Peter; Fisher, Malcolm; Morrison, Harris; Bicknell, Louise; Gautier, Philippe; Perry, Paul; Sokhi, Kishan; Sexton, David; Bardakjian, Tanya M.; Schneider, Adele S.; Elcioglu, Nursel; Ozkinay, Ferda; Koenig, Rainer; Mégarbané, Andre; Semerci, C. Nur; Khan, Ayesha; Zafar, Saemah; Hennekam, Raoul; Sousa, Sérgio B.; Ramos, Lina; Garavelli, Livia; Furga, Andrea Superti; Wischmeijer, Anita; Jackson, Ian J.; Gillessen-Kaesbach, Gabriele; Brunner, Han G.; Wieczorek, Dagmar; van Bokhoven, Hans; FitzPatrick, David R.

2011-01-01

Ophthalmo-acromelic syndrome (OAS), also known as Waardenburg Anophthalmia syndrome, is defined by the combination of eye malformations, most commonly bilateral anophthalmia, with post-axial oligosyndactyly. Homozygosity mapping and subsequent targeted mutation analysis of a locus on 14q24.2 identified homozygous mutations in SMOC1 (SPARC-related modular calcium binding 1) in eight unrelated families. Four of these mutations are nonsense, two frame-shift, and two missense. The missense mutations are both in the second Thyroglobulin Type-1 (Tg1) domain of the protein. The orthologous gene in the mouse, Smoc1, shows site- and stage-specific expression during eye, limb, craniofacial, and somite development. We also report a targeted pre-conditional gene-trap mutation of Smoc1 (Smoc1tm1a) that reduces mRNA to ∼10% of wild-type levels. This gene-trap results in highly penetrant hindlimb post-axial oligosyndactyly in homozygous mutant animals (Smoc1tm1a/tm1a). Eye malformations, most commonly coloboma, and cleft palate occur in a significant proportion of Smoc1tm1a/tm1a embryos and pups. Thus partial loss of Smoc-1 results in a convincing phenocopy of the human disease. SMOC-1 is one of the two mammalian paralogs of Drosophila Pentagone, an inhibitor of decapentaplegic. The orthologous gene in Xenopus laevis, Smoc-1, also functions as a Bone Morphogenic Protein (BMP) antagonist in early embryogenesis. Loss of BMP antagonism during mammalian development provides a plausible explanation for both the limb and eye phenotype in humans and mice. PMID:21750680

Loss of the BMP antagonist, SMOC-1, causes Ophthalmo-acromelic (Waardenburg Anophthalmia) syndrome in humans and mice.

PubMed

Rainger, Joe; van Beusekom, Ellen; Ramsay, Jacqueline K; McKie, Lisa; Al-Gazali, Lihadh; Pallotta, Rosanna; Saponari, Anita; Branney, Peter; Fisher, Malcolm; Morrison, Harris; Bicknell, Louise; Gautier, Philippe; Perry, Paul; Sokhi, Kishan; Sexton, David; Bardakjian, Tanya M; Schneider, Adele S; Elcioglu, Nursel; Ozkinay, Ferda; Koenig, Rainer; Mégarbané, Andre; Semerci, C Nur; Khan, Ayesha; Zafar, Saemah; Hennekam, Raoul; Sousa, Sérgio B; Ramos, Lina; Garavelli, Livia; Furga, Andrea Superti; Wischmeijer, Anita; Jackson, Ian J; Gillessen-Kaesbach, Gabriele; Brunner, Han G; Wieczorek, Dagmar; van Bokhoven, Hans; Fitzpatrick, David R

2011-07-01

Ophthalmo-acromelic syndrome (OAS), also known as Waardenburg Anophthalmia syndrome, is defined by the combination of eye malformations, most commonly bilateral anophthalmia, with post-axial oligosyndactyly. Homozygosity mapping and subsequent targeted mutation analysis of a locus on 14q24.2 identified homozygous mutations in SMOC1 (SPARC-related modular calcium binding 1) in eight unrelated families. Four of these mutations are nonsense, two frame-shift, and two missense. The missense mutations are both in the second Thyroglobulin Type-1 (Tg1) domain of the protein. The orthologous gene in the mouse, Smoc1, shows site- and stage-specific expression during eye, limb, craniofacial, and somite development. We also report a targeted pre-conditional gene-trap mutation of Smoc1 (Smoc1(tm1a)) that reduces mRNA to ∼10% of wild-type levels. This gene-trap results in highly penetrant hindlimb post-axial oligosyndactyly in homozygous mutant animals (Smoc1(tm1a/tm1a)). Eye malformations, most commonly coloboma, and cleft palate occur in a significant proportion of Smoc1(tm1a/tm1a) embryos and pups. Thus partial loss of Smoc-1 results in a convincing phenocopy of the human disease. SMOC-1 is one of the two mammalian paralogs of Drosophila Pentagone, an inhibitor of decapentaplegic. The orthologous gene in Xenopus laevis, Smoc-1, also functions as a Bone Morphogenic Protein (BMP) antagonist in early embryogenesis. Loss of BMP antagonism during mammalian development provides a plausible explanation for both the limb and eye phenotype in humans and mice.

In-silico evidences for binding of Glucokinase activators to EGFR C797S to overcome EGFR resistance obstacle with mutant-selective allosteric inhibition.

PubMed

Patel, Harun; Pawara, Rahul; Surana, Sanjay

2018-03-29

The tyrosine kinase inhibitors (TKI) against epidermal growth factor receptor (EGFR) are generally utilized as a part of patients with non-small cell lung carcinoma (NSCLC). However, EGFR T790M mutation results in resistance to most clinically available EGFR TKIs. Third-generation EGFR TKIs against the T790M mutation has been in active clinical development to triumph the resistance problem; they covalently bind with conserved Cys797 inside the EGFR active site, offering both potency and kinase-selectivity. Third generation drugs target C797, which makes the C797S resistance mutation more subtle. EGFR C797S mutation was accounted to be a main mechanism of resistance to the third-generation inhibitors. The C797S mutation gives off an impression of being an ideal target for conquering the acquired resistance to the third generation inhibitors. We have performed structure based-virtual screening strategies for binding of glucokinase activator to EGFR C797S, which can overcome EGFR resistance impediment with mutant-selective allosteric inhibition towards all kinds of mutant EGFR (T790M, L858R, TMLR) and WT EGFR. The final filter of Lipinski's Rule of Five, Jargan's Rule of Three and in silico ADME predictions gave 23 hits, which conform to Lipinski's rule and Jorgensen's rule and all their pharmacokinetic parameters are inside the appropriate range characterized for human use, in this manner demonstrating their potential as a drug-like molecule. Copyright © 2018 Elsevier Ltd. All rights reserved.

Molecular networks implicated in speech-related disorders: FOXP2 regulates the SRPX2/uPAR complex.

PubMed

Roll, Patrice; Vernes, Sonja C; Bruneau, Nadine; Cillario, Jennifer; Ponsole-Lenfant, Magali; Massacrier, Annick; Rudolf, Gabrielle; Khalife, Manal; Hirsch, Edouard; Fisher, Simon E; Szepetowski, Pierre

2010-12-15

It is a challenge to identify the molecular networks contributing to the neural basis of human speech. Mutations in transcription factor FOXP2 cause difficulties mastering fluent speech (developmental verbal dyspraxia, DVD), whereas mutations of sushi-repeat protein SRPX2 lead to epilepsy of the rolandic (sylvian) speech areas, with DVD or with bilateral perisylvian polymicrogyria. Pathophysiological mechanisms driven by SRPX2 involve modified interaction with the plasminogen activator receptor (uPAR). Independent chromatin-immunoprecipitation microarray screening has identified the uPAR gene promoter as a potential target site bound by FOXP2. Here, we directly tested for the existence of a transcriptional regulatory network between human FOXP2 and the SRPX2/uPAR complex. In silico searches followed by gel retardation assays identified specific efficient FOXP2-binding sites in each of the promoter regions of SRPX2 and uPAR. In FOXP2-transfected cells, significant decreases were observed in the amounts of both SRPX2 (43.6%) and uPAR (38.6%) native transcripts. Luciferase reporter assays demonstrated that FOXP2 expression yielded a marked inhibition of SRPX2 (80.2%) and uPAR (77.5%) promoter activity. A mutant FOXP2 that causes DVD (p.R553H) failed to bind to SRPX2 and uPAR target sites and showed impaired down-regulation of SRPX2 and uPAR promoter activity. In a patient with polymicrogyria of the left rolandic operculum, a novel FOXP2 mutation (p.M406T) was found in the leucine-zipper (dimerization) domain. p.M406T partially impaired the FOXP2 regulation of SRPX2 promoter activity, whereas that of the uPAR promoter remained unchanged. Together with recently described FOXP2-CNTNAP2 and SRPX2/uPAR links, the FOXP2-SRPX2/uPAR network provides exciting insights into molecular pathways underlying speech-related disorders.

Amyloid protein-mediated differential DNA methylation status regulates gene expression in Alzheimer's disease model cell line

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

Sung, Hye Youn; Choi, Eun Nam; Ahn Jo, Sangmee

2011-11-04

Highlights: Black-Right-Pointing-Pointer Genome-wide DNA methylation pattern in Alzheimer's disease model cell line. Black-Right-Pointing-Pointer Integrated analysis of CpG methylation and mRNA expression profiles. Black-Right-Pointing-Pointer Identify three Swedish mutant target genes; CTIF, NXT2 and DDR2 gene. Black-Right-Pointing-Pointer The effect of Swedish mutation on alteration of DNA methylation and gene expression. -- Abstract: The Swedish mutation of amyloid precursor protein (APP-sw) has been reported to dramatically increase beta amyloid production through aberrant cleavage at the beta secretase site, causing early-onset Alzheimer's disease (AD). DNA methylation has been reported to be associated with AD pathogenesis, but the underlying molecular mechanism of APP-sw-mediated epigenetic alterationsmore » in AD pathogenesis remains largely unknown. We analyzed genome-wide interplay between promoter CpG DNA methylation and gene expression in an APP-sw-expressing AD model cell line. To identify genes whose expression was regulated by DNA methylation status, we performed integrated analysis of CpG methylation and mRNA expression profiles, and identified three target genes of the APP-sw mutant; hypomethylated CTIF (CBP80/CBP20-dependent translation initiation factor) and NXT2 (nuclear exporting factor 2), and hypermethylated DDR2 (discoidin domain receptor 2). Treatment with the demethylating agent 5-aza-2 Prime -deoxycytidine restored mRNA expression of these three genes, implying methylation-dependent transcriptional regulation. The profound alteration in the methylation status was detected at the -435, -295, and -271 CpG sites of CTIF, and at the -505 to -341 region in the promoter of DDR2. In the promoter region of NXT2, only one CpG site located at -432 was differentially unmethylated in APP-sw cells. Thus, we demonstrated the effect of the APP-sw mutation on alteration of DNA methylation and subsequent gene expression. This epigenetic regulatory mechanism may contribute to the pathogenesis of AD.« less

Multiplexed Elimination of Wild-Type DNA and High-Resolution Melting Prior to Targeted Resequencing of Liquid Biopsies.

PubMed

Ladas, Ioannis; Fitarelli-Kiehl, Mariana; Song, Chen; Adalsteinsson, Viktor A; Parsons, Heather A; Lin, Nancy U; Wagle, Nikhil; Makrigiorgos, G Mike

2017-10-01

The use of clinical samples and circulating cell-free DNA (cfDNA) collected from liquid biopsies for diagnostic and prognostic applications in cancer is burgeoning, and improved methods that reduce the influence of excess wild-type (WT) portion of the sample are desirable. Here we present enrichment of mutation-containing sequences using enzymatic degradation of WT DNA. Mutation enrichment is combined with high-resolution melting (HRM) performed in multiplexed closed-tube reactions as a rapid, cost-effective screening tool before targeted resequencing. We developed a homogeneous, closed-tube approach to use a double-stranded DNA-specific nuclease for degradation of WT DNA at multiple targets simultaneously. The No Denaturation Nuclease-assisted Minor Allele Enrichment with Probe Overlap (ND-NaME-PrO) uses WT oligonucleotides overlapping both strands on putative DNA targets. Under conditions of partial denaturation (DNA breathing), the oligonucleotide probes enhance double-stranded DNA-specific nuclease digestion at the selected targets, with high preference toward WT over mutant DNA. To validate ND-NaME-PrO, we used multiplexed HRM, digital PCR, and MiSeq targeted resequencing of mutated genomic DNA and cfDNA. Serial dilution of KRAS mutation-containing DNA shows mutation enrichment by 10- to 120-fold and detection of allelic fractions down to 0.01%. Multiplexed ND-NaME-PrO combined with multiplexed PCR-HRM showed mutation scanning of 10-20 DNA amplicons simultaneously. ND-NaME-PrO applied on cfDNA from clinical samples enables mutation enrichment and HRM scanning over 10 DNA targets. cfDNA mutations were enriched up to approximately 100-fold (average approximately 25-fold) and identified via targeted resequencing. Closed-tube homogeneous ND-NaME-PrO combined with multiplexed HRM is a convenient approach to efficiently enrich for mutations on multiple DNA targets and to enable prescreening before targeted resequencing. © 2017 American Association for Clinical Chemistry.

Multiple Resistances and Complex Mechanisms of Anopheles sinensis Mosquito: A Major Obstacle to Mosquito-Borne Diseases Control and Elimination in China

PubMed Central

Fang, Qiang; Hartsel, Joshua; Zhou, Guofa; Shi, Linna; Fang, Fujin; Zhu, Changliang; Yan, Guiyun

2014-01-01

Malaria, dengue fever, and filariasis are three of the most common mosquito-borne diseases worldwide. Malaria and lymphatic filariasis can occur as concomitant human infections while also sharing common mosquito vectors. The overall prevalence and health significance of malaria and filariasis have made them top priorities for global elimination and control programmes. Pyrethroid resistance in anopheline mosquito vectors represents a highly significant problem to malaria control worldwide. Several methods have been proposed to mitigate insecticide resistance, including rotational use of insecticides with different modes of action. Anopheles sinensis, an important malaria and filariasis vector in Southeast Asia, represents an interesting mosquito species for examining the consequences of long-term insecticide rotation use on resistance. We examined insecticide resistance in two An. Sinensis populations from central and southern China against pyrethroids, organochlorines, organophosphates, and carbamates, which are the major classes of insecticides recommended for indoor residual spray. We found that the mosquito populations were highly resistant to the four classes of insecticides. High frequency of kdr mutation was revealed in the central population, whereas no kdr mutation was detected in the southern population. The frequency of G119S mutation in the ace-1 gene was moderate in both populations. The classification and regression trees (CART) statistical analysis found that metabolic detoxification was the most important resistance mechanism, whereas target site insensitivity of L1014 kdr mutation played a less important role. Our results indicate that metabolic detoxification was the dominant mechanism of resistance compared to target site insensitivity, and suggests that long-term rotational use of various insecticides has led An. sinensis to evolve a high insecticide resistance. This study highlights the complex network of mechanisms conferring multiple resistances to chemical insecticides in mosquito vectors and it has important implication for designing and implementing vector resistance management strategies. PMID:24852174

Evolutionary Analysis Predicts Sensitive Positions of MMP20 and Validates Newly- and Previously-Identified MMP20 Mutations Causing Amelogenesis Imperfecta

PubMed Central

Gasse, Barbara; Prasad, Megana; Delgado, Sidney; Huckert, Mathilde; Kawczynski, Marzena; Garret-Bernardin, Annelyse; Lopez-Cazaux, Serena; Bailleul-Forestier, Isabelle; Manière, Marie-Cécile; Stoetzel, Corinne; Bloch-Zupan, Agnès; Sire, Jean-Yves

2017-01-01

Amelogenesis imperfecta (AI) designates a group of genetic diseases characterized by a large range of enamel disorders causing important social and health problems. These defects can result from mutations in enamel matrix proteins or protease encoding genes. A range of mutations in the enamel cleavage enzyme matrix metalloproteinase-20 gene (MMP20) produce enamel defects of varying severity. To address how various alterations produce a range of AI phenotypes, we performed a targeted analysis to find MMP20 mutations in French patients diagnosed with non-syndromic AI. Genomic DNA was isolated from saliva and MMP20 exons and exon-intron boundaries sequenced. We identified several homozygous or heterozygous mutations, putatively involved in the AI phenotypes. To validate missense mutations and predict sensitive positions in the MMP20 sequence, we evolutionarily compared 75 sequences extracted from the public databases using the Datamonkey webserver. These sequences were representative of mammalian lineages, covering more than 150 million years of evolution. This analysis allowed us to find 324 sensitive positions (out of the 483 MMP20 residues), pinpoint functionally important domains, and build an evolutionary chart of important conserved MMP20 regions. This is an efficient tool to identify new- and previously-identified mutations. We thus identified six functional MMP20 mutations in unrelated families, finding two novel mutated sites. The genotypes and phenotypes of these six mutations are described and compared. To date, 13 MMP20 mutations causing AI have been reported, making these genotypes and associated hypomature enamel phenotypes the most frequent in AI. PMID:28659819

Evolutionary Analysis Predicts Sensitive Positions of MMP20 and Validates Newly- and Previously-Identified MMP20 Mutations Causing Amelogenesis Imperfecta.

PubMed

Gasse, Barbara; Prasad, Megana; Delgado, Sidney; Huckert, Mathilde; Kawczynski, Marzena; Garret-Bernardin, Annelyse; Lopez-Cazaux, Serena; Bailleul-Forestier, Isabelle; Manière, Marie-Cécile; Stoetzel, Corinne; Bloch-Zupan, Agnès; Sire, Jean-Yves

2017-01-01

Amelogenesis imperfecta (AI) designates a group of genetic diseases characterized by a large range of enamel disorders causing important social and health problems. These defects can result from mutations in enamel matrix proteins or protease encoding genes. A range of mutations in the enamel cleavage enzyme matrix metalloproteinase-20 gene ( MMP20 ) produce enamel defects of varying severity. To address how various alterations produce a range of AI phenotypes, we performed a targeted analysis to find MMP20 mutations in French patients diagnosed with non-syndromic AI. Genomic DNA was isolated from saliva and MMP20 exons and exon-intron boundaries sequenced. We identified several homozygous or heterozygous mutations, putatively involved in the AI phenotypes. To validate missense mutations and predict sensitive positions in the MMP20 sequence, we evolutionarily compared 75 sequences extracted from the public databases using the Datamonkey webserver. These sequences were representative of mammalian lineages, covering more than 150 million years of evolution. This analysis allowed us to find 324 sensitive positions (out of the 483 MMP20 residues), pinpoint functionally important domains, and build an evolutionary chart of important conserved MMP20 regions. This is an efficient tool to identify new- and previously-identified mutations. We thus identified six functional MMP20 mutations in unrelated families, finding two novel mutated sites. The genotypes and phenotypes of these six mutations are described and compared. To date, 13 MMP20 mutations causing AI have been reported, making these genotypes and associated hypomature enamel phenotypes the most frequent in AI.

Synergistic Effects of Targeted PI3K Signaling Inhibition and Chemotherapy in Liposarcoma

PubMed Central

Guo, Shang; Lopez-Marquez, Hector; Fan, Kenneth C.; Choy, Edwin; Cote, Gregory; Harmon, David; Nielsen, G. Petur; Yang, Cao; Zhang, Changqing; Mankin, Henry; Hornicek, Francis J.; Borger, Darrell R.; Duan, Zhenfeng

2014-01-01

While liposarcoma is the second most common soft tissue malignant tumor, the molecular pathogenesis in this malignancy is poorly understood. Our goal was therefore to expand the understanding of molecular mechanisms that drive liposarcoma and identify therapeutically-susceptible genetic alterations. We studied a cohort of high-grade liposarcomas and benign lipomas across multiple disease sites, as well as two liposarcoma cell lines, using multiplexed mutational analysis. Nucleic acids extracted from diagnostic patient tissue were simultaneously interrogated for 150 common mutations across 15 essential cancer genes using a clinically-validated platform for cancer genotyping. Western blot analysis was implemented to detect activation of downstream pathways. Liposarcoma cell lines were used to determine the effects of PI3K targeted drug treatment with or without chemotherapy. We identified mutations in the PIK3CA gene in 4 of 18 human liposarcoma patients (22%). No PIK3CA mutations were identified in benign lipomas. Western blot analysis confirmed downstream activation of AKT in both PIK3CA mutant and non-mutant liposarcoma samples. PI-103, a dual PI3K/mTOR inhibitor, effectively inhibited the activation of the PI3K/AKT in liposarcoma cell lines and induced apoptosis. Importantly, combination with PI-103 treatment strongly synergized the growth-inhibitory effects of the chemotherapy drugs doxorubicin and cisplatin in liposarcoma cells. Taken together, these findings suggest that activation of the PI3K/AKT pathway is an important cancer mechanism in liposarcoma. Targeting the PI3K/AKT/pathway with small molecule inhibitors in combination with chemotherapy could be exploited as a novel strategy in the treatment of liposarcoma. PMID:24695632

HCK is a survival determinant transactivated by mutated MYD88, and a direct target of ibrutinib.

PubMed

Yang, Guang; Buhrlage, Sara J; Tan, Li; Liu, Xia; Chen, Jie; Xu, Lian; Tsakmaklis, Nicholas; Chen, Jiaji G; Patterson, Christopher J; Brown, Jennifer R; Castillo, Jorge J; Zhang, Wei; Zhang, Xiaofeng; Liu, Shuai; Cohen, Philip; Hunter, Zachary R; Gray, Nathanael; Treon, Steven P

2016-06-23

Activating mutations in MYD88 are present in ∼95% of patients with Waldenström macroglobulinemia (WM), as well as other B-cell malignancies including activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL). In WM, mutated MYD88 triggers activation of Bruton tyrosine kinase (BTK). Ibrutinib, a pleiotropic kinase inhibitor that targets BTK, is highly active in patients with mutated MYD88. We observed that mutated MYD88 WM and ABC DLBCL cell lines, as well as primary WM cells show enhanced hematopoietic cell kinase (HCK) transcription and activation, and that HCK is activated by interleukin 6 (IL-6). Over-expression of mutated MYD88 triggers HCK and IL-6 transcription, whereas knockdown of HCK reduced survival and attenuated BTK, phosphoinositide 3-kinase/AKT, and mitogen-activated protein kinase/extracellular signal-regulated kinase signaling in mutated MYD88 WM and/or ABC DLBCL cells. Ibrutinib and the more potent HCK inhibitor A419259, blocked HCK activation and induced apoptosis in mutated MYD88 WM and ABC DLBCL cells. Docking and pull-down studies confirmed that HCK was a target of ibrutinib. Ibrutinib and A419259 also blocked adenosine triphosphate binding to HCK, whereas transduction of mutated MYD88 expressing WM cells with a mutated HCK gatekeeper greatly increased the half maximal effective concentration for ibrutinib and A419259. The findings support that HCK expression and activation is triggered by mutated MYD88, supports the growth and survival of mutated MYD88 WM and ABC DLBCL cells, and is a direct target of ibrutinib. HCK represents a novel target for therapeutic development in MYD88-mutated WM and ABC DLBCL, and possibly other diseases driven by mutated MYD88. © 2016 by The American Society of Hematology.

DNA targeting specificity of RNA-guided Cas9 nucleases.

PubMed

Hsu, Patrick D; Scott, David A; Weinstein, Joshua A; Ran, F Ann; Konermann, Silvana; Agarwala, Vineeta; Li, Yinqing; Fine, Eli J; Wu, Xuebing; Shalem, Ophir; Cradick, Thomas J; Marraffini, Luciano A; Bao, Gang; Zhang, Feng

2013-09-01

The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.

Infants Infected with Respiratory Syncytial Virus Generate Potent Neutralizing Antibodies that Lack Somatic Hypermutation

PubMed Central

Goodwin, Eileen; Gilman, Morgan S. A.; Wrapp, Daniel; Chen, Man; Ngwuta, Joan O.; Moin, Syed M.; Bai, Patricia; Sivasubramanian, Arvind; Connor, Ruth I.; Wright, Peter F.; Graham, Barney S.; McLellan, Jason S.; Walker, Laura M.

2018-01-01

SUMMARY Respiratory syncytial virus (RSV) is a leading cause of infant mortality, and there are currently no licensed vaccines to protect this vulnerable population. A comprehensive understanding of infant antibody responses to natural RSV infection would facilitate vaccine development. Here, we isolated over 450 RSV fusion glycoprotein (F)-specific antibodies from seven RSV-infected infants and found that half of the antibodies recognized only two antigenic sites. Antibodies targeting both sites showed convergent sequence features, and structural studies revealed the molecular basis for their recognition of RSV F. A subset of antibodies targeting one of these sites displayed potent neutralizing activity despite lacking somatic mutations, and similar antibodies were detected in RSV-naïve B cell repertoires, suggesting that expansion of these B cells in infants may be possible with suitably designed vaccine antigens. Collectively, our results provide fundamental insights into infant antibody responses and a framework for the rational design of age-specific RSV vaccines. PMID:29396163

I-SceI-Induced Gene Replacement at a Natural Locus in Embryonic Stem Cells

PubMed Central

Cohen-Tannoudji, Michel; Robine, Sylvie; Choulika, André; Pinto, Daniel; El Marjou, Fatima; Babinet, Charles; Louvard, Daniel; Jaisser, Frédéric

1998-01-01

Gene targeting is a very powerful tool for studying mammalian development and physiology and for creating models of human diseases. In many instances, however, it is desirable to study different modifications of a target gene, but this is limited by the generally low frequency of homologous recombination in mammalian cells. We have developed a novel gene-targeting strategy in mouse embryonic stem cells that is based on the induction of endogenous gap repair processes at a defined location within the genome by induction of a double-strand break (DSB) in the gene to be mutated. This strategy was used to knock in an NH2-ezrin mutant in the villin gene, which encodes an actin-binding protein expressed in the brush border of the intestine and the kidney. To induce the DSB, an I-SceI yeast meganuclease restriction site was first introduced by gene targeting to the villin gene, followed by transient expression of I-SceI. The repair of the ensuing DSB was achieved with high efficiency (6 × 10−6) by a repair shuttle vector sharing only a 2.8-kb region of homology with the villin gene and no negative selection marker. Compared to conventional gene-targeting experiments at the villin locus, this represents a 100-fold stimulation of gene-targeting frequency, notwithstanding a much lower length of homology. This strategy will be very helpful in facilitating the targeted introduction of several types of mutations within a gene of interest. PMID:9488460

Seamless editing of the chloroplast genome in plants.

PubMed

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

2016-07-29

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

The contribution of de novo coding mutations to autism spectrum disorder

PubMed Central

Iossifov, Ivan; O’Roak, Brian J.; Sanders, Stephan J.; Ronemus, Michael; Krumm, Niklas; Levy, Dan; Stessman, Holly A.; Witherspoon, Kali; Vives, Laura; Patterson, Karynne E.; Smith, Joshua D.; Paeper, Bryan; Nickerson, Deborah A.; Dea, Jeanselle; Dong, Shan; Gonzalez, Luis E.; Mandell, Jefferey D.; Mane, Shrikant M.; Murtha, Michael T.; Sullivan, Catherine A.; Walker, Michael F.; Waqar, Zainulabedin; Wei, Liping; Willsey, A. Jeremy; Yamrom, Boris; Lee, Yoon-ha; Grabowska, Ewa; Dalkic, Ertugrul; Wang, Zihua; Marks, Steven; Andrews, Peter; Leotta, Anthony; Kendall, Jude; Hakker, Inessa; Rosenbaum, Julie; Ma, Beicong; Rodgers, Linda; Troge, Jennifer; Narzisi, Giuseppe; Yoon, Seungtai; Schatz, Michael C.; Ye, Kenny; McCombie, W. Richard; Shendure, Jay; Eichler, Evan E.; State, Matthew W.; Wigler, Michael

2015-01-01

We sequenced exomes from more than 2,500 simplex families each having a child with an autistic spectrum disorder (ASD). By comparing affected to unaffected siblings, we estimate that 13% of de novo (DN) missense mutations and 42% of DN likely gene-disrupting (LGD) mutations contribute to 12% and 9% of diagnoses, respectively. Including copy number variants, coding DN mutations contribute to about 30% of all simplex and 45% of female diagnoses. Virtually all LGD mutations occur opposite wild-type alleles. LGD targets in affected females significantly overlap the targets in males of lower IQ, but neither overlaps significantly with targets in males of higher IQ. We estimate that LGD mutation in about 400 genes can contribute to the joint class of affected females and males of lower IQ, with an overlapping and similar number of genes vulnerable to causative missense mutation. LGD targets in the joint class overlap with published targets for intellectual disability and schizophrenia, and are enriched for chromatin modifiers, FMRP-associated genes and embryonically expressed genes. Virtually all significance for the latter comes from affected females. PMID:25363768

Understanding the Basis of Drug Resistance of the Mutants of αβ-Tubulin Dimer via Molecular Dynamics Simulations

PubMed Central

Natarajan, Kathiresan; Senapati, Sanjib

2012-01-01

The vital role of tubulin dimer in cell division makes it an attractive drug target. Drugs that target tubulin showed significant clinical success in treating various cancers. However, the efficacy of these drugs is attenuated by the emergence of tubulin mutants that are unsusceptible to several classes of tubulin binding drugs. The molecular basis of drug resistance of the tubulin mutants is yet to be unraveled. Here, we employ molecular dynamics simulations, protein-ligand docking, and MMPB(GB)SA analyses to examine the binding of anticancer drugs, taxol and epothilone to the reported point mutants of tubulin - T274I, R282Q, and Q292E. Results suggest that the mutations significantly alter the tubulin structure and dynamics, thereby weaken the interactions and binding of the drugs, primarily by modifying the M loop conformation and enlarging the pocket volume. Interestingly, these mutations also affect the tubulin distal sites that are associated with microtubule building processes. PMID:22879949

Targeted inhibition of mutant IDH2 in leukemia cells induces cellular differentiation.

PubMed

Wang, Fang; Travins, Jeremy; DeLaBarre, Byron; Penard-Lacronique, Virginie; Schalm, Stefanie; Hansen, Erica; Straley, Kimberly; Kernytsky, Andrew; Liu, Wei; Gliser, Camelia; Yang, Hua; Gross, Stefan; Artin, Erin; Saada, Veronique; Mylonas, Elena; Quivoron, Cyril; Popovici-Muller, Janeta; Saunders, Jeffrey O; Salituro, Francesco G; Yan, Shunqi; Murray, Stuart; Wei, Wentao; Gao, Yi; Dang, Lenny; Dorsch, Marion; Agresta, Sam; Schenkein, David P; Biller, Scott A; Su, Shinsan M; de Botton, Stephane; Yen, Katharine E

2013-05-03

A number of human cancers harbor somatic point mutations in the genes encoding isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2). These mutations alter residues in the enzyme active sites and confer a gain-of-function in cancer cells, resulting in the accumulation and secretion of the oncometabolite (R)-2-hydroxyglutarate (2HG). We developed a small molecule, AGI-6780, that potently and selectively inhibits the tumor-associated mutant IDH2/R140Q. A crystal structure of AGI-6780 complexed with IDH2/R140Q revealed that the inhibitor binds in an allosteric manner at the dimer interface. The results of steady-state enzymology analysis were consistent with allostery and slow-tight binding by AGI-6780. Treatment with AGI-6780 induced differentiation of TF-1 erythroleukemia and primary human acute myelogenous leukemia cells in vitro. These data provide proof-of-concept that inhibitors targeting mutant IDH2/R140Q could have potential applications as a differentiation therapy for cancer.

[Gene mutation analysis of X-linked hypophosphatemic rickets].

PubMed

Song, Ying; Ma, Hong-Wei; Li, Fang; Hu, Man; Ren, Shuang; Yu, Ya-Fen; Zhao, Gui-Jie

2013-11-01

To investigate the frequency and type of PHEX gene mutations in children with X-linked hypophosphatemic rickets (XLH), the possible presence of mutational hot spots, and the relationship between genotype and clinical phenotype. Clinical data of 10 children with XLH was retrospectively reviewed. The relationship between gene mutation type and severity of XLH was evaluated. PHEX gene mutations were detected in all 10 children with XLH, including 6 cases of missense mutation, 2 cases of splice site mutation, 1 case of frameshift mutation, and 1 case of nonsense mutation. Two new mutations, c.2048T>C and IVS14+1delAG, were found. The type of PHEX gene mutation was not associated with the degree of short stature and leg deformity (P=0.571 and 0.467), and the mutation site was also not associated with the degree of short stature and leg deformity (P=0.400 and 1.000). Missense mutation is the most common type of PHEX gene mutation in children with XLH, and c.2048T>C and IVS14+1delAG are two new PHEX gene mutations. The type and site of PHEX gene mutation are not associated with the severity of XLH.

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

PubMed Central

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

2015-01-01

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

Drug resistance conferred by mutations outside the active site through alterations in the dynamic and structural ensemble of HIV-1 protease.

PubMed

Ragland, Debra A; Nalivaika, Ellen A; Nalam, Madhavi N L; Prachanronarong, Kristina L; Cao, Hong; Bandaranayake, Rajintha M; Cai, Yufeng; Kurt-Yilmaz, Nese; Schiffer, Celia A

2014-08-27

HIV-1 protease inhibitors are part of the highly active antiretroviral therapy effectively used in the treatment of HIV infection and AIDS. Darunavir (DRV) is the most potent of these inhibitors, soliciting drug resistance only when a complex combination of mutations occur both inside and outside the protease active site. With few exceptions, the role of mutations outside the active site in conferring resistance remains largely elusive. Through a series of DRV-protease complex crystal structures, inhibition assays, and molecular dynamics simulations, we find that single and double site mutations outside the active site often associated with DRV resistance alter the structure and dynamic ensemble of HIV-1 protease active site. These alterations correlate with the observed inhibitor binding affinities for the mutants, and suggest a network hypothesis on how the effect of distal mutations are propagated to pivotal residues at the active site and may contribute to conferring drug resistance.

L1014F-kdr Mutation in Indian Anopheles subpictus (Diptera: Culicidae) Arising From Two Alternative Transversions in the Voltage-Gated Sodium Channel and a Single PIRA-PCR for Their Detection.

PubMed

Singh, O P; Dykes, C L; Sharma, G; Das, M K

2015-01-01

Leucine-to-phenylalanine substitution at residue L1014 in the voltage-gated sodium channel, target site of action for dichlorodiphenyltrichloroethane (DDT) and pyrethroids, is the most common knockdown resistance (kdr) mutation reported in several insects conferring resistance against DDT and pyrethroids. Here, we report presence of two coexisting alternative transversions, A>T and A>C, on the third codon position of L1014 residue in malaria vector Anopheles subpictus Grassi (species A) from Jamshedpur (India), both leading to the same amino acid substitution of Leu-to-Phe with allelic frequencies of 19 and 67%, respectively. A single primer-introduced restriction analysis-polymerase chain reaction (PIRA-PCR) was devised for the identification of L1014F-kdr mutation in An. subpictus resulting from either type of point mutation. Genotyping of samples with PIRA-PCR revealed high frequency (82%) of L1014F-kdr mutation in the study area. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants

PubMed Central

Hehle, Verena K.; Paul, Matthew J.; Roberts, Victoria A.; van Dolleweerd, Craig J.; Ma, Julian K.-C.

2016-01-01

This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformed Nicotiana tabacum. Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy’s 13 antibody heavy and light chain mutant combinations were expressed transiently in N. tabacum and demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.—Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. PMID:26712217

Phosphorylation promotes activation-induced cytidine deaminase activity at the Myc oncogene

PubMed Central

2017-01-01

Activation-induced cytidine deaminase (AID) is a mutator enzyme that targets immunoglobulin (Ig) genes to initiate antibody somatic hypermutation (SHM) and class switch recombination (CSR). Off-target AID association also occurs, which causes oncogenic mutations and chromosome rearrangements. However, AID occupancy does not directly correlate with DNA damage, suggesting that factors beyond AID association contribute to mutation targeting. CSR and SHM are regulated by phosphorylation on AID serine38 (pS38), but the role of pS38 in off-target activity has not been evaluated. We determined that lithium, a clinically used therapeutic, induced high AID pS38 levels. Using lithium and an AID-S38 phospho mutant, we compared the role of pS38 in AID activity at the Ig switch region and off-target Myc gene. We found that deficient pS38 abated AID chromatin association and CSR but not mutation at Myc. Enhanced pS38 elevated Myc translocation and mutation frequency but not CSR or Ig switch region mutation. Thus, AID activity can be differentially targeted by phosphorylation to induce oncogenic lesions. PMID:29122947

Pathogenicity in POLG syndromes: DNA polymerase gamma pathogenicity prediction server and database.

PubMed

Nurminen, Anssi; Farnum, Gregory A; Kaguni, Laurie S

2017-06-01

DNA polymerase gamma (POLG) is the replicative polymerase responsible for maintaining mitochondrial DNA (mtDNA). Disorders related to its functionality are a major cause of mitochondrial disease. The clinical spectrum of POLG syndromes includes Alpers-Huttenlocher syndrome (AHS), childhood myocerebrohepatopathy spectrum (MCHS), myoclonic epilepsy myopathy sensory ataxia (MEMSA), the ataxia neuropathy spectrum (ANS) and progressive external ophthalmoplegia (PEO). We have collected all publicly available POLG-related patient data and analyzed it using our pathogenic clustering model to provide a new research and clinical tool in the form of an online server. The server evaluates the pathogenicity of both previously reported and novel mutations. There are currently 176 unique point mutations reported and found in mitochondrial patients in the gene encoding the catalytic subunit of POLG, POLG . The mutations are distributed nearly uniformly along the length of the primary amino acid sequence of the gene. Our analysis shows that most of the mutations are recessive, and that the reported dominant mutations cluster within the polymerase active site in the tertiary structure of the POLG enzyme. The POLG Pathogenicity Prediction Server (http://polg.bmb.msu.edu) is targeted at clinicians and scientists studying POLG disorders, and aims to provide the most current available information regarding the pathogenicity of POLG mutations.

Therapeutic strategies based on modified U1 snRNAs and chaperones for Sanfilippo C splicing mutations.

PubMed

Matos, Liliana; Canals, Isaac; Dridi, Larbi; Choi, Yoo; Prata, Maria João; Jordan, Peter; Desviat, Lourdes R; Pérez, Belén; Pshezhetsky, Alexey V; Grinberg, Daniel; Alves, Sandra; Vilageliu, Lluïsa

2014-12-10

Mutations affecting RNA splicing represent more than 20% of the mutant alleles in Sanfilippo syndrome type C, a rare lysosomal storage disorder that causes severe neurodegeneration. Many of these mutations are localized in the conserved donor or acceptor splice sites, while few are found in the nearby nucleotides. In this study we tested several therapeutic approaches specifically designed for different splicing mutations depending on how the mutations affect mRNA processing. For three mutations that affect the donor site (c.234 + 1G > A, c.633 + 1G > A and c.1542 + 4dupA), different modified U1 snRNAs recognizing the mutated donor sites, have been developed in an attempt to rescue the normal splicing process. For another mutation that affects an acceptor splice site (c.372-2A > G) and gives rise to a protein lacking four amino acids, a competitive inhibitor of the HGSNAT protein, glucosamine, was tested as a pharmacological chaperone to correct the aberrant folding and to restore the normal trafficking of the protein to the lysosome. Partial correction of c.234 + 1G > A mutation was achieved with a modified U1 snRNA that completely matches the splice donor site suggesting that these molecules may have a therapeutic potential for some splicing mutations. Furthermore, the importance of the splice site sequence context is highlighted as a key factor in the success of this type of therapy. Additionally, glucosamine treatment resulted in an increase in the enzymatic activity, indicating a partial recovery of the correct folding. We have assayed two therapeutic strategies for different splicing mutations with promising results for the future applications.

Prognostic value of plasma EGFR ctDNA in NSCLC patients treated with EGFR-TKIs.

PubMed

Zhang, Chengjuan; Wei, Bing; Li, Peng; Yang, Ke; Wang, Zhizhong; Ma, Jie; Guo, Yongjun

2017-01-01

Epidermal growth factor receptor (EGFR) specific mutations have been known to improve survival of patients with non-small-cell lung carcinoma (NSCLC). However, whether there are any changes of EGFR mutations after targeted therapy and its clinical significance is unclear. This study was to identify the status of EGFR mutations after targeted therapy and predict the prognostic significance for NSCLC patients. A total of forty-five (45) NSCLC patients who received EGFR-TKI therapy were enrolled. We identified the changes of EGFR mutations in plasma ctDNA by Amplification Refractory Mutation System (ARMS) PCR technology. In the 45 cases of NSCLC with EGFR mutations, the EGFR mutation status changed in 26 cases, in which, 12 cases (26.7%) from positive to negative, and 14 cases (31.1%) from T790M mutation negative to positive after TKI targeted therapy. The T790M occurance group had a shorter Progression -Free-Survival (PFS) than the groups of EGFR mutation undetected and EGFR mutation turned out to have no change after EGFR-TKI therapy (p < 0.05). According to this study, it's necessary to closely monitor EGFR mutations during follow-up to predict the prognosis of NSCLC patients who are to receive the TKI targeted therapy.

Novel splice site mutation in the growth hormone receptor gene in Turkish patients with Laron-type dwarfism.

PubMed

Arman, Ahmet; Ozon, Alev; Isguven, Pinar S; Coker, Ajda; Peker, Ismail; Yordam, Nursen

2008-01-01

Growth hormone (GH) is involved in growth, and fat and carbohydrate metabolism. Interaction of GH with the GH receptor (GHR) is necessary for systemic and local production of insulin-like growth factor-I (IGF-I) which mediates GH actions. Mutations in the GHR cause severe postnatal growth failure; the disorder is an autosomal recessive genetic disease resulting in GH insensitivity, called Laron syndrome. It is characterized by dwarfism with elevated serum GH and low levels of IGF-I. We analyzed the GHR gene for mutations and polymorphisms in eight patients with Laron-type dwarfism from six families. We found three missense mutations (S40L, V125A, I526L), one nonsense mutation (W157X), and one splice site mutation in the extracellular domain of GHR. Furthermore, G168G and exon 3 deletion polymorphisms were detected in patients with Laron syndrome. The splice site mutation, which is a novel mutation, was located at the donor splice site of exon 2/ intron 2 within GHR. Although this mutation changed the highly conserved donor splice site consensus sequence GT to GGT by insertion of a G residue, the intron splicing between exon 2 and exon 3 was detected in the patient. These results imply that the splicing occurs arthe GT site in intron 2, leaving the extra inserted G residue at the end of exon 2, thus changing the open reading frame of GHR resulting in a premature termination codon in exon 3.

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

PubMed

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 (P<0.05). No statistically significant difference was observed in the mutations among the mild, moderate and severe OSAHS groups (P>0.05). 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.

Trafficking defects and loss of ligand binding are the underlying causes of all reported DDR2 missense mutations found in SMED-SL patients.

PubMed

Ali, Bassam R; Xu, Huifang; Akawi, Nadia A; John, Anne; Karuvantevida, Noushad S; Langer, Ruth; Al-Gazali, Lihadh; Leitinger, Birgit

2010-06-01

Spondylo-meta-epiphyseal dysplasia (SMED) with short limbs and abnormal calcifications (SMED-SL) is a rare, autosomal recessive human growth disorder, characterized by disproportionate short stature, short limbs, short broad fingers, abnormal metaphyses and epiphyses, platyspondyly and premature calcifications. Recently, three missense mutations and one splice-site mutation in the DDR2 gene were identified as causative genetic defects for SMED-SL, but the underlying cellular and biochemical mechanisms were not explored. Here we report a novel DDR2 missense mutation, c.337G>A (p.E113K), that causes SMED-SL in two siblings in the United Arab Emirates. Another DDR2 missense mutation, c.2254C>T (p.R752C), matching one of the previously reported SMED-SL mutations, was found in a second affected family. DDR2 is a plasma membrane receptor tyrosine kinase that functions as a collagen receptor. We expressed DDR2 constructs with the identified point mutations in human cell lines and evaluated their localization and functional properties. We found that all SMED-SL missense mutants were defective in collagen-induced receptor activation and that the three previously reported mutants (p.T713I, p.I726R and p.R752C) were retained in the endoplasmic reticulum. The novel mutant (p.E113K), in contrast, trafficked normally, like wild-type DDR2, but failed to bind collagen. This finding is in agreement with our recent structural data identifying Glu113 as an important amino acid in the DDR2 ligand-binding site. Our data thus demonstrate that SMED-SL can result from at least two different loss-of-function mechanisms: namely defects in DDR2 targeting to the plasma membrane or the loss of its ligand-binding activity.

Trafficking defects and loss of ligand binding are the underlying causes of all reported DDR2 missense mutations found in SMED-SL patients

PubMed Central

Ali, Bassam R.; Xu, Huifang; Akawi, Nadia A.; John, Anne; Karuvantevida, Noushad S.; Langer, Ruth; Al-Gazali, Lihadh; Leitinger, Birgit

2010-01-01

Spondylo-meta-epiphyseal dysplasia (SMED) with short limbs and abnormal calcifications (SMED-SL) is a rare, autosomal recessive human growth disorder, characterized by disproportionate short stature, short limbs, short broad fingers, abnormal metaphyses and epiphyses, platyspondyly and premature calcifications. Recently, three missense mutations and one splice-site mutation in the DDR2 gene were identified as causative genetic defects for SMED-SL, but the underlying cellular and biochemical mechanisms were not explored. Here we report a novel DDR2 missense mutation, c.337G>A (p.E113K), that causes SMED-SL in two siblings in the United Arab Emirates. Another DDR2 missense mutation, c.2254C>T (p.R752C), matching one of the previously reported SMED-SL mutations, was found in a second affected family. DDR2 is a plasma membrane receptor tyrosine kinase that functions as a collagen receptor. We expressed DDR2 constructs with the identified point mutations in human cell lines and evaluated their localization and functional properties. We found that all SMED-SL missense mutants were defective in collagen-induced receptor activation and that the three previously reported mutants (p.T713I, p.I726R and p.R752C) were retained in the endoplasmic reticulum. The novel mutant (p.E113K), in contrast, trafficked normally, like wild-type DDR2, but failed to bind collagen. This finding is in agreement with our recent structural data identifying Glu113 as an important amino acid in the DDR2 ligand-binding site. Our data thus demonstrate that SMED-SL can result from at least two different loss-of-function mechanisms: namely defects in DDR2 targeting to the plasma membrane or the loss of its ligand-binding activity. PMID:20223752

Identification of essential histidine residues in the active site of Escherichia coli xylose (glucose) isomerase.

PubMed Central

Batt, C A; Jamieson, A C; Vandeyar, M A

1990-01-01

Two conserved histidine residues (His-101 and His-271) appear to be essential components in the active site of the enzyme xylose (glucose) isomerase (EC 5.3.1.5). These amino acid residues were targeted for mutagenesis on the basis of sequence homology among xylose isomerases isolated from Escherichia coli, Bacillus subtilis, Ampullariella sp. strain 3876, and Streptomyces violaceus-niger. Each residue was selectively replaced by site-directed mutagenesis and shown to be essential for activity. No measurable activity was observed for any mutations replacing either His-101 or His-271. Circular dichroism measurements revealed no significant change in the overall conformation of the mutant enzymes, and all formed dimers similar to the wild-type enzyme. Mutations at His-271 could be distinguished from those at His-101, since the former resulted in a thermolabile protein whereas no significant change in heat stability was observed for the latter. Based upon these results and structural data recently reported, we speculate that His-101 is the catalytic base mediating the reaction. Replacement of His-271 may render the enzyme thermolabile, since this residue appears to be a ligand for one of the metal ions in the active site of the enzyme. Images PMID:2405386

Quantitative targeted proteomic analysis of potential markers of tyrosine kinase inhibitor (TKI) sensitivity in EGFR mutated lung adenocarcinoma.

PubMed

Awasthi, Shivangi; Maity, Tapan; Oyler, Benjamin L; Qi, Yue; Zhang, Xu; Goodlett, David R; Guha, Udayan

2018-04-13

Lung cancer causes the highest mortality among all cancers. Patients harboring kinase domain mutations in the epidermal growth factor receptor (EGFR) respond to EGFR tyrosine kinase inhibitors (TKIs), however, acquired resistance always develops. Moreover, 30-40% of patients with EGFR mutations exhibit primary resistance. Hence, there is an unmet need for additional biomarkers of TKI sensitivity that complement EGFR mutation testing and predict treatment response. We previously identified phosphopeptides whose phosphorylation is inhibited upon treatment with EGFR TKIs, erlotinib and afatinib in TKI sensitive cells, but not in resistant cells. These phosphosites are potential biomarkers of TKI sensitivity. Here, we sought to develop modified immuno-multiple reaction monitoring (immuno-MRM)-based quantitation assays for select phosphosites including EGFR-pY1197, pY1172, pY998, AHNAK-pY160, pY715, DAPP1-pY139, CAV1-pY14, INPPL1-pY1135, NEDD9-pY164, NF1-pY2579, and STAT5A-pY694. These sites were significantly hypophosphorylated by erlotinib and a 3rd generation EGFR TKI, osimertinib, in TKI-sensitive H3255 cells, which harbor the TKI-sensitizing EGFR L858R mutation. However, in H1975 cells, which harbor the TKI-resistant EGFR L858R/T790M mutant, osimertinib, but not erlotinib, could significantly inhibit phosphorylation of EGFR-pY-1197, STAT5A-pY694 and CAV1-pY14, suggesting these sites also predict response in TKI-resistant cells. We could further validate EGFR-pY-1197 as a biomarker of TKI sensitivity by developing a calibration curve-based modified immuno-MRM assay. In this report, we have shown the development and optimization of MRM assays coupled with global phosphotyrosine enrichment (modified immuno-MRM) for a list of 11 phosphotyrosine peptides. Our optimized assays identified the targets reproducibly in biological samples with good selectivity. We also developed and characterized quantitation methods to determine endogenous abundance of these targets and correlated the results of the relative quantification with amounts estimated from the calibration curves. This approach represents a way to validate and verify biomarker candidates discovered from large-scale global phospho-proteomics analysis. The application of these modified immuno-MRM assays in lung adenocarcinoma cells provides proof-of concept for the feasibility of clinical applications. These assays may be used in prospective clinical studies of EGFR TKI treatment of EGFR mutant lung cancer to correlate treatment response and other clinical endpoints. Copyright © 2018. Published by Elsevier B.V.

Clinical and functional characterization of a patient carrying a compound heterozygous pericentrin mutation and a heterozygous IGF1 receptor mutation.

PubMed

Müller, Eva; Dunstheimer, Desiree; Klammt, Jürgen; Friebe, Daniela; Kiess, Wieland; Kratzsch, Jürgen; Kruis, Tassilo; Laue, Sandy; Pfäffle, Roland; Wallborn, Tillmann; Heidemann, Peter H

2012-01-01

Intrauterine and postnatal longitudinal growth is controlled by a strong genetic component that regulates a complex network of endocrine factors integrating them with cellular proliferation, differentiation and apoptotic processes in target tissues, particularly the growth centers of the long bones. Here we report on a patient born small for gestational age (SGA) with severe, proportionate postnatal growth retardation, discreet signs of skeletal dysplasia, microcephaly and moyamoya disease. Initial genetic evaluation revealed a novel heterozygous IGF1R p.Leu1361Arg mutation affecting a highly conserved residue with the insulin-like growth factor type 1 receptor suggestive for a disturbance within the somatotropic axis. However, because the mutation did not co-segregate with the phenotype and functional characterization did not reveal an obvious impairment of the ligand depending major IGF1R signaling capabilities a second-site mutation was assumed. Mutational screening of components of the somatotropic axis, constituents of the IGF signaling system and factors involved in cellular proliferation, which are described or suggested to provoke syndromic dwarfism phenotypes, was performed. Two compound heterozygous PCNT mutations (p.[Arg585X];[Glu1774X]) were identified leading to the specification of the diagnosis to MOPD II. These investigations underline the need for careful assessment of all available information to derive a firm diagnosis from a sequence aberration.

Clinical and Functional Characterization of a Patient Carrying a Compound Heterozygous Pericentrin Mutation and a Heterozygous IGF1 Receptor Mutation

PubMed Central

Klammt, Jürgen; Friebe, Daniela; Kiess, Wieland; Kratzsch, Jürgen; Kruis, Tassilo; Laue, Sandy; Pfäffle, Roland; Wallborn, Tillmann; Heidemann, Peter H.

2012-01-01

Intrauterine and postnatal longitudinal growth is controlled by a strong genetic component that regulates a complex network of endocrine factors integrating them with cellular proliferation, differentiation and apoptotic processes in target tissues, particularly the growth centers of the long bones. Here we report on a patient born small for gestational age (SGA) with severe, proportionate postnatal growth retardation, discreet signs of skeletal dysplasia, microcephaly and moyamoya disease. Initial genetic evaluation revealed a novel heterozygous IGF1R p.Leu1361Arg mutation affecting a highly conserved residue with the insulin-like growth factor type 1 receptor suggestive for a disturbance within the somatotropic axis. However, because the mutation did not co-segregate with the phenotype and functional characterization did not reveal an obvious impairment of the ligand depending major IGF1R signaling capabilities a second-site mutation was assumed. Mutational screening of components of the somatotropic axis, constituents of the IGF signaling system and factors involved in cellular proliferation, which are described or suggested to provoke syndromic dwarfism phenotypes, was performed. Two compound heterozygous PCNT mutations (p.[Arg585X];[Glu1774X]) were identified leading to the specification of the diagnosis to MOPD II. These investigations underline the need for careful assessment of all available information to derive a firm diagnosis from a sequence aberration. PMID:22693602

SRSF2 mutations drive oncogenesis by activating a global program of aberrant alternative splicing in hematopoietic cells.

PubMed

Liang, Yang; Tebaldi, Toma; Rejeski, Kai; Joshi, Poorval; Stefani, Giovanni; Taylor, Ashley; Song, Yuanbin; Vasic, Radovan; Maziarz, Jamie; Balasubramanian, Kunthavai; Ardasheva, Anastasia; Ding, Alicia; Quattrone, Alessandro; Halene, Stephanie

2018-06-01

Recurrent mutations in the splicing factor SRSF2 are associated with poor clinical outcomes in myelodysplastic syndromes (MDS). Their high frequency suggests these mutations drive oncogenesis, yet the molecular explanation for this process is unclear. SRSF2 mutations could directly affect pre-mRNA splicing of a vital gene product; alternatively, a whole network of gene products could be affected. Here we determine how SRSF2 mutations globally affect RNA binding and splicing in vivo using HITS-CLIP. Remarkably, the majority of differential binding events do not translate into alternative splicing of exons with SRSF2 P95H binding sites. Alternative splice alterations appear to be dominated by indirect effects. Importantly, SRSF2 P95H targets are enriched in RNA processing and splicing genes, including several members of the hnRNP and SR families of proteins, suggesting a "splicing-cascade" phenotype wherein mutation of a single splicing factor leads to widespread modifications in multiple RNA processing and splicing proteins. We show that splice alteration of HNRNPA2B1, a splicing factor differentially bound and spliced by SRSF2 P95H , impairs hematopoietic differentiation in vivo. Our data suggests a model whereby the recurrent mutations in splicing factors set off a cascade of gene regulatory events that together affect hematopoiesis and drive cancer.

Nationwide genetic analysis for molecularly unresolved cystic fibrosis patients in a multiethnic society: implications for preconception carrier screening.

PubMed

Behar, Doron M; Inbar, Ori; Shteinberg, Michal; Gur, Michal; Mussaffi, Huda; Shoseyov, David; Ashkenazi, Moshe; Alkrinawi, Soliman; Bormans, Concetta; Hakim, Fahed; Mei-Zahav, Meir; Cohen-Cymberknoh, Malena; Dagan, Adi; Prais, Dario; Sarouk, Ifat; Stafler, Patrick; Bar Aluma, Bat El; Akler, Gidon; Picard, Elie; Aviram, Micha; Efrati, Ori; Livnat, Galit; Rivlin, Joseph; Bentur, Lea; Blau, Hannah; Kerem, Eitan; Singer, Amihood

2017-05-01

Preconception carrier screening for cystic fibrosis (CF) is usually performed using ethnically targeted panels of selected mutations. This has been recently challenged by the use of expanded, ethnically indifferent, pan-population panels. Israel is characterized by genetically heterogeneous populations carrying a wide range of CFTR mutations. To assess the potential of expanding the current Israeli preconception screening program, we sought the subset of molecularly unresolved CF patients listed in the Israeli CF data registry comprising ~650 patients. An Israeli nationwide genotyping of 152 CF cases, representing 176 patients lacking molecular diagnosis, was conducted. Molecular analysis included Sanger sequencing for all exons and splice sites, multiplex ligation probe amplification (MLPA), and next-generation sequencing of the poly-T/TG tracts. We identified 54 different mutations, of which only 16 overlapped the 22 mutations included in the Israeli preconception screening program. A total of 29/54 (53.7%) mutations were already listed as CF causing by the CFTR2 database, and only 4/54 (7.4%) were novel. Molecular diagnosis was reached in 78/152 (51.3%) cases. Prenatal diagnosis of 24/78 (30.8%) cases could have been achieved by including all CFTR2-causing mutations in the Israeli panel. Our data reveal an overwhelming hidden abundance of CFTR gene mutations suggesting that expanded preconception carrier screening might achieve higher preconception detection rates.

The diagnosis and treatment of brain metastases in EGFR mutant lung cancer.

PubMed

Minchom, Anna; Yu, Ken C; Bhosle, Jaishree; O'Brien, Mary

2014-05-01

The epidemiology of non-small-cell lung cancer (NSCLC) has changed with a new pattern of disease emerging - a form of adenocarcinoma in mostly younger female patients, who are never or light smokers and more frequently in East Asian populations. Description of EGF receptor (EGFR) mutations has allowed new management strategies to evolve. Oral targeted therapies have broadened the treatment options in the advanced setting with the potential for periods of long term response. The brain is a common site of metastases with EGFR mutated lung cancer typically displaying asymptomatic, small volume, multiple lesions that respond to treatment. We explore the role of local and system therapies for brain metastases in this disease including the role of EGFR inhibitors.

Disruption of a long-range cis-acting regulator for Shh causes preaxial polydactyly

PubMed Central

Lettice, Laura A.; Horikoshi, Taizo; Heaney, Simon J. H.; van Baren, Marijke J.; van der Linde, Herma C.; Breedveld, Guido J.; Joosse, Marijke; Akarsu, Nurten; Oostra, Ben A.; Endo, Naoto; Shibata, Minoru; Suzuki, Mikio; Takahashi, Eiichi; Shinka, Toshikatsu; Nakahori, Yutaka; Ayusawa, Dai; Nakabayashi, Kazuhiko; Scherer, Stephen W.; Heutink, Peter; Hill, Robert E.; Noji, Sumihare

2002-01-01

Preaxial polydactyly (PPD) is a common limb malformation in human. A number of polydactylous mouse mutants indicate that misexpression of Shh is a common requirement for generating extra digits. Here we identify a translocation breakpoint in a PPD patient and a transgenic insertion site in the polydactylous mouse mutant sasquatch (Ssq). The genetic lesions in both lie within the same respective intron of the LMBR1/Lmbr1 gene, which resides ≈1 Mb away from Shh. Genetic analysis of Ssq reveals that the Lmbr1 gene is incidental to the phenotype and that the mutation directly interrupts a cis-acting regulator of Shh. This regulator is most likely the target for generating PPD mutations in human. PMID:12032320

eSensor®: A Microarray Technology Based on Electrochemical Detection of Nucleic Acids and Its Application to Cystic Fibrosis Carrier Screening

NASA Astrophysics Data System (ADS)

Reed, Michael R.; Coty, William A.

We have developed a test for identification of carriers for cystic fibrosis using the eSensor® DNA detection technology. Oligonucleotide probes are deposited within self-assembled monolayers on gold electrodes arrayed upon printed circuit boards. These probes allow sequence-specific capture of amplicons containing a panel of mutation sites associated with cystic fibrosis. DNA targets are detected and mutations genotyped using a “sandwich” assay methodology employing electrochemical detection of ferrocene-labeled oligonucleotides for discrimination of carrier and non-carrier alleles. Performance of the cystic fibrosis application demonstrates sufficient accuracy and reliability for clinical diagnostic use, and the procedure can be performed by trained medical technologists available in the hospital laboratory.

Bone Metastasis in Prostate Cancer: Recurring Mitochondrial DNA Mutation Reveals Selective Pressure Exerted by the Bone Microenvironment

PubMed Central

Arnold, Rebecca S.; Fedewa, Stacey A.; Goodman, Michael; Osunkoya, Adeboye O.; Kissick, Haydn T.; Morrissey, Colm; True, Lawrence D.; Petros, John A.

2015-01-01

Background Cancer progression and metastasis occurs such that cells with acquired mutations enhancing growth and survival (or inhibiting cell death) increase in number, a concept that has been recognized as analogous to Darwinian evolution of species since Peter C. Nowell’s description in 1976. Selective forces include those intrinsic to the host (including metastatic site) as well as those resulting from anti-cancer therapies. By examining the mutational status of multiple tumor sites within an individual patient some insight may be gained into those genetic variants that enhance site-specific metastasis. By comparing these data across multiple individuals, recurrent patterns may identify alterations that are fundamental to successful site-specific metastasis. Methods We sequenced the mitochondrial genome in 10 prostate cancer patients with bone metastases enrolled in a rapid autopsy program. Patients had late stage disease and received androgen ablation and frequently other systemic therapies. For each of 9 patients, 4 separate tissues were sequenced: the primary prostate cancer, a soft tissue metastasis, a bone metastasis and an uninvolved normal tissue that served as the non-cancerous control. An additional (10th) patient had no primary prostate available for sequencing but had both metastatic sites (and control DNA) sequenced. We then examined the number and location of somatically acquired mitochondrial DNA (mtDNA) mutations in the primary and two metastatic sites in each individual patient. Finally, we compared patients with each other to determine any common patterns of somatic mutation. Results Somatic mutations were significantly more numerous in bone compared to either the primary tumor or soft tissue metastases. A missense mutation at nucleotide position (np) 10398 (A10398G; Thr114Ala) in the respiratory complex I gene ND3 was the most common (7 of 10 patients) and was detected only in bone. Other notable somatic mutations that occurred in more than one patient include a tRNA Arg mutation at np 10436 and a tRNA Thr mutation at np 15928. The tRNA Arg mutation was restricted to bone metastases and occurred in three of 10 patients (30%). Somatic mutation at 15928 was not restricted to bone and also occurred in three patients. Conclusions Mitochondrial genomic variation was greater in metastatic sites than the primary tumor and bone metastases had statistically significantly greater numbers of somatic mutations than either the primary or the soft tissue metastases. The genome was not mutated randomly. At least one mutational “hot-spot” was identified at the individual base level (nucleotide position 10398 in bone metastases) indicating a pervasive selective pressure for bone metastatic cells that had acquired the 10398 mtDNA mutation. Two additional recurrent mutations (tRNA Arg and tRNA Thr) support the concept of bone site-specific “survival of the fittest” as revealed by variation in the mitochondrial genome and selective pressure exerted by the metastatic site. PMID:25952970

Bone metastasis in prostate cancer: Recurring mitochondrial DNA mutation reveals selective pressure exerted by the bone microenvironment.

PubMed

Arnold, Rebecca S; Fedewa, Stacey A; Goodman, Michael; Osunkoya, Adeboye O; Kissick, Haydn T; Morrissey, Colm; True, Lawrence D; Petros, John A

2015-09-01

Cancer progression and metastasis occur such that cells with acquired mutations enhancing growth and survival (or inhibiting cell death) increase in number, a concept that has been recognized as analogous to Darwinian evolution of species since Peter C. Nowell's description in 1976. Selective forces include those intrinsic to the host (including metastatic site) as well as those resulting from anti-cancer therapies. By examining the mutational status of multiple tumor sites within an individual patient some insight may be gained into those genetic variants that enhance site-specific metastasis. By comparing these data across multiple individuals, recurrent patterns may identify alterations that are fundamental to successful site-specific metastasis. We sequenced the mitochondrial genome in 10 prostate cancer patients with bone metastases enrolled in a rapid autopsy program. Patients had late stage disease and received androgen ablation and frequently other systemic therapies. For each of 9 patients, 4 separate tissues were sequenced: the primary prostate cancer, a soft tissue metastasis, a bone metastasis and an uninvolved normal tissue that served as the non-cancerous control. An additional (10th) patient had no primary prostate available for sequencing but had both metastatic sites (and control DNA) sequenced. We then examined the number and location of somatically acquired mitochondrial DNA (mtDNA) mutations in the primary tumor and two metastatic sites in each individual patient. Finally, we compared patients with each other to determine any common patterns of somatic mutation. Somatic mutations were significantly more numerous in the bone compared to either the primary tumor or soft tissue metastases. A missense mutation at nucleotide position (n.p.) 10398 (A10398G; Thr114Ala) in the respiratory complex I gene ND3 was the most common (7 of 10 patients) and was detected only in the bone. Other notable somatic mutations that occurred in more than one patient include a tRNA Arg mutation at n.p. 10436 and a tRNA Thr mutation at n.p. 15928. The tRNA Arg mutation was restricted to bone metastases and occurred in three of 10 patients (30%). Somatic mutation at 15928 was not restricted to the bone and also occurred in three patients. Mitochondrial genomic variation was greater in metastatic sites than in the primary tumor and bone metastases had statistically significantly greater numbers of somatic mutations than either the primary or the soft tissue metastases. The genome was not mutated randomly. At least one mutational "hot-spot" was identified at the individual base level (nucleotide position 10398 in bone metastases) indicating a pervasive selective pressure for bone metastatic cells that had acquired the 10398 mtDNA mutation. Two additional recurrent mutations (tRNA Arg and tRNA Thr) support the concept of bone site-specific "survival of the fittest" as revealed by variation in the mitochondrial genome and selective pressure exerted by the metastatic site. Published by Elsevier Inc.

HER2 activating mutations are targets for colorectal cancer treatment

PubMed Central

Kavuri, Shyam M.; Jain, Naveen; Galimi, Francesco; Cottino, Francesca; Leto, Simonetta M.; Migliardi, Giorgia; Searleman, Adam C.; Shen, Wei; Monsey, John; Trusolino, Livio; Jacobs, Samuel A.; Bertotti, Andrea; Bose, Ron

2015-01-01

The Cancer Genome Atlas project identified HER2 somatic mutations and gene amplification in 7% of colorectal cancer patients. Introduction of the HER2 mutations, S310F, L755S, V777L, V842I, and L866M, into colon epithelial cells increased signaling pathways and anchorage-independent cell growth, indicating that they are activating mutations. Introduction of these HER2 activating mutations into colorectal cancer cell lines produced resistance to cetuximab and panitumumab by sustaining MAPK phosphorylation. HER2 mutations are potently inhibited by low nanomolar doses of the irreversible tyrosine kinase inhibitors, neratinib and afatinib. HER2 gene sequencing of 48 cetuximab resistant, quadruple (KRAS, NRAS, BRAF, and PIK3CA) WT colorectal cancer patient-derived xenografts (PDX’s) identified 4 PDX’s with HER2 mutations. HER2 targeted therapies were tested on two PDX’s. Treatment with a single HER2 targeted drug (trastuzumab, neratinib, or lapatinib) delayed tumor growth, but dual HER2 targeted therapy with trastuzumab plus tyrosine kinase inhibitors produced regression of these HER2 mutated PDX’s. PMID:26243863

Genetic mutations in human rectal cancers detected by targeted sequencing.

PubMed

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

2015-10-01

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

Mapping mutational effects along the evolutionary landscape of HIV envelope.

PubMed

Haddox, Hugh K; Dingens, Adam S; Hilton, Sarah K; Overbaugh, Julie; Bloom, Jesse D

2018-03-28

The immediate evolutionary space accessible to HIV is largely determined by how single amino acid mutations affect fitness. These mutational effects can shift as the virus evolves. However, the prevalence of such shifts in mutational effects remains unclear. Here, we quantify the effects on viral growth of all amino acid mutations to two HIV envelope (Env) proteins that differ at [Formula: see text]100 residues. Most mutations similarly affect both Envs, but the amino acid preferences of a minority of sites have clearly shifted. These shifted sites usually prefer a specific amino acid in one Env, but tolerate many amino acids in the other. Surprisingly, shifts are only slightly enriched at sites that have substituted between the Envs-and many occur at residues that do not even contact substitutions. Therefore, long-range epistasis can unpredictably shift Env's mutational tolerance during HIV evolution, although the amino acid preferences of most sites are conserved between moderately diverged viral strains. © 2018, Haddox et al.

A three amino acid deletion in the transmembrane domain of the nicotinic acetylcholine receptor α6 subunit confers high-level resistance to spinosad in Plutella xylostella.

PubMed

Wang, Jing; Wang, Xingliang; Lansdell, Stuart J; Zhang, Jianheng; Millar, Neil S; Wu, Yidong

2016-04-01

Spinosad is a macrocyclic lactone insecticide that acts primarily at the nicotinic acetylcholine receptors (nAChRs) of target insects. Here we describe evidence that high levels of resistance to spinosad in the diamondback moth (Plutella xylostella) are associated with a three amino acid (3-aa) deletion in the fourth transmembrane domain (TM4) of the nAChR α6 subunit (Pxα6). Following laboratory selection with spinosad, the SZ-SpinR strain of P. xylostella exhibited 940-fold resistance to spinosad. In addition, the selected insect population had 1060-fold cross-resistance to spinetoram but, in contrast, no cross-resistance to abamectin was observed. Genetic analysis indicates that spinosad resistance in SZ-SpinR is inherited as a recessive and autosomal trait, and that the 3-aa deletion (IIA) in TM4 of Pxα6 is tightly linked to spinosad resistance. Because of well-established difficulties in functional expression of cloned insect nAChRs, the analogous resistance-associated deletion mutation was introduced into a prototype nAChR (the cloned human α7 subunit). Two-electrode voltage-clamp recording with wild-type and mutated nAChRs expressed in Xenopus laevis oocytes indicated that the mutation causes a complete loss of agonist activation. In addition, radioligand binding studies indicated that the 3-aa deletion resulted in significantly lower-affinity binding of the extracellular neurotransmitter-binding site. These findings are consistent with the 3-amino acid (IIA) deletion within the transmembrane domain of Pxα6 being responsible for target-site resistance to spinosad in the SZ-SpinR strain of P. xylostella. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

A three amino acid deletion in the transmembrane domain of the nicotinic acetylcholine receptor α6 subunit confers high-level resistance to spinosad in Plutella xylostella

PubMed Central

Wang, Jing; Wang, Xingliang; Lansdell, Stuart J.; Zhang, Jianheng; Millar, Neil S.; Wu, Yidong

2016-01-01

Spinosad is a macrocyclic lactone insecticide that acts primarily at the nicotinic acetylcholine receptors (nAChRs) of target insects. Here we describe evidence that high levels of resistance to spinosad in the diamondback moth (Plutella xylostella) are associated with a three amino acid (3-aa) deletion in the fourth transmembrane domain (TM4) of the nAChR α6 subunit (Pxα6). Following laboratory selection with spinosad, the SZ-SpinR strain of P. xylostella exhibited 940-fold resistance to spinosad. In addition, the selected insect population had 1060-fold cross-resistance to spinetoram but, in contrast, no cross-resistance to abamectin was observed. Genetic analysis indicates that spinosad resistance in SZ-SpinR is inherited as a recessive and autosomal trait, and that the 3-aa deletion (IIA) in TM4 of Pxα6 is tightly linked to spinosad resistance. Because of well-established difficulties in functional expression of cloned insect nAChRs, the analogous resistance-associated deletion mutation was introduced into a prototype nAChR (the cloned human α7 subunit). Two-electrode voltage-clamp recording with wild-type and mutated nAChRs expressed in Xenopus laevis oocytes indicated that the mutation causes a complete loss of agonist activation. In addition, radioligand binding studies indicated that the 3-aa deletion resulted in significantly lower-affinity binding of the extracellular neurotransmitter-binding site. These findings are consistent with the 3-amino acid (IIA) deletion within the transmembrane domain of Pxα6 being responsible for target-site resistance to spinosad in the SZ-SpinR strain of P. xylostella. PMID:26855198

Structure-Function Analysis of Chloroplast Proteins via Random Mutagenesis Using Error-Prone PCR.

PubMed

Dumas, Louis; Zito, Francesca; Auroy, Pascaline; Johnson, Xenie; Peltier, Gilles; Alric, Jean

2018-06-01

Site-directed mutagenesis of chloroplast genes was developed three decades ago and has greatly advanced the field of photosynthesis research. Here, we describe a new approach for generating random chloroplast gene mutants that combines error-prone polymerase chain reaction of a gene of interest with chloroplast complementation of the knockout Chlamydomonas reinhardtii mutant. As a proof of concept, we targeted a 300-bp sequence of the petD gene that encodes subunit IV of the thylakoid membrane-bound cytochrome b 6 f complex. By sequencing chloroplast transformants, we revealed 149 mutations in the 300-bp target petD sequence that resulted in 92 amino acid substitutions in the 100-residue target subunit IV sequence. Our results show that this method is suited to the study of highly hydrophobic, multisubunit, and chloroplast-encoded proteins containing cofactors such as hemes, iron-sulfur clusters, and chlorophyll pigments. Moreover, we show that mutant screening and sequencing can be used to study photosynthetic mechanisms or to probe the mutational robustness of chloroplast-encoded proteins, and we propose that this method is a valuable tool for the directed evolution of enzymes in the chloroplast. © 2018 American Society of Plant Biologists. All rights reserved.

Mcl-1–Bim complexes accommodate surprising point mutations via minor structural changes

PubMed Central

Fire, Emiko; Gullá, Stefano V; Grant, Robert A; Keating, Amy E

2010-01-01

Mcl-1 is an antiapoptotic Bcl-2-family protein that protects cells against death. Structures of Mcl-1, and of other anti-apoptotic Bcl-2 proteins, reveal a surface groove into which the α-helical BH3 regions of certain proapoptotic proteins can bind. Despite high overall structural conservation, differences in this groove afford binding specificity that is important for the mechanism of Bcl-2 family function. We report the crystal structure of human Mcl-1 bound to a BH3 peptide derived from human Bim and the structures for three complexes that accommodate large physicochemical changes at conserved Bim sites. The mutations had surprisingly modest effects on complex stability, and the structures show that Mcl-1 can undergo small changes to accommodate the mutant ligands. For example, a shift in a leucine side chain fills a hole left by an isoleucine-to-alanine mutation at the first hydrophobic buried position of Bim BH3. Larger changes are also observed, with shifting of helix α3 accommodating an isoleucine-to-tyrosine mutation at this same position. We surveyed the variation in available Mcl-1 and Bcl-xL structures and observed moderate flexibility that is likely critical for facilitating interactions of diverse BH3-only proteins with Mcl-1. With the antiapoptotic Bcl-2 family members attracting significant attention as therapeutic targets, these structures contribute to our growing understanding of how specificity is achieved and can help to guide the design of novel inhibitors that target Mcl-1. PMID:20066663

Chromosomal aberrations and delays in cell progression induced by x-rays in Tradescantia clone 02 meristems

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

Geard, C.R.

1983-01-01

In root meristems of Tradescantia clone 02 (developed by Sparrow and his colleagues for mutation studies), X-rays interfere with the progression of cells through the cell cycle and induce chromosomal aberrations in a dose-dependent manner consistent with linear-quadratic kinetics. Sequential mitotic cell accumulations after irradiation indicate that sensitivity to aberrration induction is probably greatest in cells from late S to early G2, with chromatid interchanges the most frequent aberration type and all aberrations consistent with intiation from the interaction between two lesions. The ratio of the coefficients in the linear (..cap alpha..) and the quadratic (..beta..) terms (..cap alpha../..beta..) ismore » equal to the dose average of specific energy produced by individual particles in the site where interaction takes place. The ratio ..cap alpha../..beta.. for chromosomal aberrations is similar to that previously found for X-ray-induced mutation in Tradescantia stamen hairs, supporting the proposal that radiation-induced mutational events are due to chromosomal aberrations with interaction distances of about 1 ..mu..m. Abrahmson and co-workers have noted that both ..cap alpha../..beta.. ratios appear to be related to nuclear target size and are similar for chromosomal and mutational endpoints in the same organism. These findings support this concept; however, it is apparent that any situation which diminishes yield at high doses (e.g., mitotic delay) will primarily affect the ..beta.. component, resulting in low assessments of interaction site diameters.« less

Interplay between DMD Point Mutations and Splicing Signals in Dystrophinopathy Phenotypes

PubMed Central

Juan-Mateu, Jonàs; González-Quereda, Lidia; Rodríguez, Maria José; Verdura, Edgard; Lázaro, Kira; Jou, Cristina; Nascimento, Andrés; Jiménez-Mallebrera, Cecilia; Colomer, Jaume; Monges, Soledad; Lubieniecki, Fabiana; Foncuberta, Maria Eugenia; Pascual-Pascual, Samuel Ignacio; Molano, Jesús; Baiget, Montserrat; Gallano, Pia

2013-01-01

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. PMID:23536893

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

PubMed

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

2018-01-01

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

Elucidating the Interdependence of Drug Resistance from Combinations of Mutations.

PubMed

Ragland, Debra A; Whitfield, Troy W; Lee, Sook-Kyung; Swanstrom, Ronald; Zeldovich, Konstantin B; Kurt-Yilmaz, Nese; Schiffer, Celia A

2017-11-14

HIV-1 protease is responsible for the cleavage of 12 nonhomologous sites within the Gag and Gag-Pro-Pol polyproteins in the viral genome. Under the selective pressure of protease inhibition, the virus evolves mutations within (primary) and outside of (secondary) the active site, allowing the protease to process substrates while simultaneously countering inhibition. The primary protease mutations impede inhibitor binding directly, while the secondary mutations are considered accessory mutations that compensate for a loss in fitness. However, the role of secondary mutations in conferring drug resistance remains a largely unresolved topic. We have shown previously that mutations distal to the active site are able to perturb binding of darunavir (DRV) via the protein's internal hydrogen-bonding network. In this study, we show that mutations distal to the active site, regardless of context, can play an interdependent role in drug resistance. Applying eigenvalue decomposition to collections of hydrogen bonding and van der Waals interactions from a series of molecular dynamics simulations of 15 diverse HIV-1 protease variants, we identify sites in the protease where amino acid substitutions lead to perturbations in nonbonded interactions with DRV and/or the hydrogen-bonding network of the protease itself. While primary mutations are known to drive resistance in HIV-1 protease, these findings delineate the significant contributions of accessory mutations to resistance. Identifying the variable positions in the protease that have the greatest impact on drug resistance may aid in future structure-based design of inhibitors.

Mutations in MexB that affect the efflux of antibiotics with cytoplasmic targets.

PubMed

Ohene-Agyei, Thelma; Lea, Jon D; Venter, Henrietta

2012-08-01

Drug efflux pumps such as MexAB-OprM from Pseudomonas aeruginosa confer resistance to a wide range of chemically different compounds. Within the tripartite assembly, the inner membrane protein MexB is mainly responsible for substrate recognition. Recently, considerable advances have been made in elucidating the drug efflux pathway through the large periplasmic domains of resistance-nodulation-division (RND) transporters. However, little is known about the role of amino acids in other parts of the protein. We have investigated the role of two conserved phenylalanine residues that are aligned around the cytoplasmic side of the central cavity of MexB. The two conserved phenylalanine residues have been mutated to alanine residues (FAFA MexB). The interaction of the wild-type and mutant proteins with a variety of drugs from different classes was investigated by assays of cytotoxicity and drug transport. The FAFA mutation affected the efflux of compounds that have targets inside the cell, but antibiotics that act on cell wall synthesis and membrane probes were unaffected. Combined, our results indicate the presence of a hitherto unidentified cytoplasmic-binding site in RND drug transporters and enhance our understanding of the molecular mechanisms that govern drug resistance in Gram-negative pathogens. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Generation of biallelic knock-out sheep via gene-editing and somatic cell nuclear transfer

PubMed Central

Li, Honghui; Wang, Gui; Hao, Zhiqiang; Zhang, Guozhong; Qing, Yubo; Liu, Shuanghui; Qing, Lili; Pan, Weirong; Chen, Lei; Liu, Guichun; Zhao, Ruoping; Jia, Baoyu; Zeng, Luyao; Guo, Jianxiong; Zhao, Lixiao; Zhao, Heng; Lv, Chaoxiang; Xu, Kaixiang; Cheng, Wenmin; Li, Hushan; Zhao, Hong-Ye; Wang, Wen; Wei, Hong-Jiang

2016-01-01

Transgenic sheep can be used to achieve genetic improvements in breeds and as an important large-animal model for biomedical research. In this study, we generated a TALEN plasmid specific for ovine MSTN and transfected it into fetal fibroblast cells of STH sheep. MSTN biallelic-KO somatic cells were selected as nuclear donor cells for SCNT. In total, cloned embryos were transferred into 37 recipient gilts, 28 (75.7%) becoming pregnant and 15 delivering, resulting in 23 lambs, 12 of which were alive. Mutations in the lambs were verified via sequencing and T7EI assay, and the gene mutation site was consistent with that in the donor cells. Off-target analysis was performed, and no off-target mutations were detected. MSTN KO affected the mRNA expression of MSTN relative genes. The growth curve for the resulting sheep suggested that MSTN KO caused a remarkable increase in body weight compared with those of wild-type sheep. Histological analyses revealed that MSTN KO resulted in muscle fiber hypertrophy. These findings demonstrate the successful generation of MSTN biallelic-KO STH sheep via gene editing in somatic cells using TALEN technology and SCNT. These MSTN mutant sheep developed and grew normally, and exhibited increased body weight and muscle growth. PMID:27654750

Effect of herbicide resistance endowing Ile-1781-Leu and Asp-2078-Gly ACCase gene mutations on ACCase kinetics and growth traits in Lolium rigidum.

PubMed

Vila-Aiub, Martin M; Yu, Qin; Han, Heping; Powles, Stephen B

2015-08-01

The rate of herbicide resistance evolution in plants depends on fitness traits endowed by alleles in both the presence and absence (resistance cost) of herbicide selection. The effect of two Lolium rigidum spontaneous homozygous target-site resistance-endowing mutations (Ile-1781-Leu, Asp-2078-Gly) on both ACCase activity and various plant growth traits have been investigated here. Relative growth rate (RGR) and components (net assimilation rate, leaf area ratio), resource allocation to different organs, and growth responses in competition with a wheat crop were assessed. Unlike plants carrying the Ile-1781-Leu resistance mutation, plants homozygous for the Asp-2078-Gly mutation exhibited a significantly lower RGR (30%), which translated into lower allocation of biomass to roots, shoots, and leaves, and poor responses to plant competition. Both the negligible and significant growth reductions associated, respectively, with the Ile-1781-Leu and Asp-2078-Gly resistance mutations correlated with their impact on ACCase activity. Whereas the Ile-1781-Leu mutation showed no pleiotropic effects on ACCase kinetics, the Asp-2078-Gly mutation led to a significant reduction in ACCase activity. The impaired growth traits are discussed in the context of resistance costs and the effects of each resistance allele on ACCase activity. Similar effects of these two particular ACCase mutations on the ACCase activity of Alopecurus myosuroides were also confirmed. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Correction of β-thalassemia mutant by base editor in human embryos.

PubMed

Liang, Puping; Ding, Chenhui; Sun, Hongwei; Xie, Xiaowei; Xu, Yanwen; Zhang, Xiya; Sun, Ying; Xiong, Yuanyan; Ma, Wenbin; Liu, Yongxiang; Wang, Yali; Fang, Jianpei; Liu, Dan; Songyang, Zhou; Zhou, Canquan; Huang, Junjiu

2017-11-01

β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB -28 (A>G) mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor (BE) system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB -28 (A>G) mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB -28 (A>G) homozygous mutation. Data showed that base editor could precisely correct HBB -28 (A>G) mutation in the patient's primary cells. To model homozygous mutation disease embryos, we constructed nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured (IVM) oocytes. Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB -28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.

Development of a Targeted Next-Generation Sequencing Assay to Detect Diagnostically Relevant Mutations of JAK2, CALR, and MPL in Myeloproliferative Neoplasms.

PubMed

Frawley, Thomas; O'Brien, Cathal P; Conneally, Eibhlin; Vandenberghe, Elisabeth; Percy, Melanie; Langabeer, Stephen E; Haslam, Karl

2018-02-01

The classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), consisting of polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are a heterogeneous group of neoplasms that harbor driver mutations in the JAK2, CALR, and MPL genes. The detection of mutations in these genes has been incorporated into the recent World Health Organization (WHO) diagnostic criteria for MPN. Given a pressing clinical need to screen for mutations in these genes in a routine diagnostic setting, a targeted next-generation sequencing (NGS) assay for the detection of MPN-associated mutations located in JAK2 exon 14, JAK2 exon 12, CALR exon 9, and MPL exon 10 was developed to provide a single platform alternative to reflexive, stepwise diagnostic algorithms. Polymerase chain reaction (PCR) primers were designed to target mutation hotspots in JAK2 exon 14, JAK2 exon 12, MPL exon 10, and CALR exon 9. Multiplexed PCR conditions were optimized by using qualitative PCR followed by NGS. Diagnostic genomic DNA from 35 MPN patients, known to harbor driver mutations in one of the target genes, was used to validate the assay. One hundred percent concordance was observed between the previously-identified mutations and those detected by NGS, with no false positives, nor any known mutations missed (specificity = 100%, CI = 0.96, sensitivity = 100%, CI = 0.89). Improved resolution of mutation sequences was also revealed by NGS analysis. Detection of diagnostically relevant driver mutations of MPN is enhanced by employing a targeted multiplex NGS approach. This assay presents a robust solution to classical MPN mutation screening, providing an alternative to time-consuming sequential analyses.

Inhalational anaesthetics and n-alcohols share a site of action in the neuronal Shaw2 Kv channel

PubMed Central

Bhattacharji, Aditya; Klett, Nathan; Go, Ramon Christopher V; Covarrubias, Manuel

2010-01-01

Background and purpose: Neuronal ion channels are key targets of general anaesthetics and alcohol, and binding of these drugs to pre-existing and relatively specific sites is thought to alter channel gating. However, the underlying molecular mechanisms of this action are still poorly understood. Here, we investigated the neuronal Shaw2 voltage-gated K+ (Kv) channel to ask whether the inhalational anaesthetic halothane and n-alcohols share a binding site near the activation gate of the channel. Experimental approach: Focusing on activation gate mutations that affect channel modulation by n-alcohols, we investigated n-alcohol-sensitive and n-alcohol-resistant Kv channels heterologously expressed in Xenopus oocytes to probe the functional modulation by externally applied halothane using two-electrode voltage clamping and a gas-tight perfusion system. Key results: Shaw2 Kv channels are reversibly inhibited by halothane in a dose-dependent and saturable manner (K0.5= 400 µM; nH= 1.2). Also, discrete mutations in the channel's S4S5 linker are sufficient to reduce or confer inhibition by halothane (Shaw2-T330L and Kv3.4-G371I/T378A respectively). Furthermore, a point mutation in the S6 segment of Shaw2 (P410A) converted the halothane-induced inhibition into halothane-induced potentiation. Lastly, the inhibition resulting from the co-application of n-butanol and halothane is consistent with the presence of overlapping binding sites for these drugs and weak binding cooperativity. Conclusions and implications: These observations strongly support a molecular model of a general anaesthetic binding site in the Shaw2 Kv channel. This site may involve the amphiphilic interface between the S4S5 linker and the S6 segment, which plays a pivotal role in Kv channel activation. PMID:20136839

Conformational Dynamics, Ligand Binding and Effects of Mutations in NirE an S-Adenosyl-L-Methionine Dependent Methyltransferase

NASA Astrophysics Data System (ADS)

Singh, Warispreet; Karabencheva-Christova, Tatyana G.; Black, Gary W.; Ainsley, Jon; Dover, Lynn; Christov, Christo Z.

2016-01-01

Heme d1, a vital tetrapyrrol involved in the denitrification processes is synthesized from its precursor molecule precorrin-2 in a chemical reaction catalysed by an S-adenosyl-L-methionine (SAM) dependent Methyltransferase (NirE). The NirE enzyme catalyses the transfer of a methyl group from the SAM to uroporphyrinogen III and serves as a novel potential drug target for the pharmaceutical industry. An important insight into the structure-activity relationships of NirE has been revealed by elucidating its crystal structure, but there is still no understanding about how conformational flexibility influences structure, cofactor and substrate binding by the enzyme as well as the structural effects of mutations of residues involved in binding and catalysis. In order to provide this missing but very important information we performed a comprehensive atomistic molecular dynamics study which revealed that i) the binding of the substrate contributes to the stabilization of the structure of the full complex; ii) conformational changes influence the orientation of the pyrrole rings in the substrate, iii) more open conformation of enzyme active site to accommodate the substrate as an outcome of conformational motions; and iv) the mutations of binding and active site residues lead to sensitive structural changes which influence binding and catalysis.

Direct induction of T lymphocyte-specific gene expression by the mammalian Notch signaling pathway

PubMed Central

Reizis, Boris; Leder, Philip

2002-01-01

The Notch signaling pathway regulates the commitment and early development of T lymphocytes. We studied Notch-mediated induction of the pre-T cell receptor α (pTa) gene, a T-cell-specific transcriptional target of Notch. The pTa enhancer was activated by Notch signaling and contained binding sites for its nuclear effector, CSL. Mutation of the CSL-binding sites abolished enhancer induction by Notch and delayed the up-regulation of pTa transgene expression during T cell lineage commitment. These results show a direct mechanism of stage- and tissue-specific gene induction by the mammalian Notch/CSL signaling pathway. PMID:11825871

Hitchhiking and epistasis give rise to cohort dynamics in adapting populations

PubMed Central

Buskirk, Sean W.; Peace, Ryan Emily; Lang, Gregory I.

2017-01-01

Beneficial mutations are the driving force of adaptive evolution. In asexual populations, the identification of beneficial alleles is confounded by the presence of genetically linked hitchhiker mutations. Parallel evolution experiments enable the recognition of common targets of selection; yet these targets are inherently enriched for genes of large target size and mutations of large effect. A comprehensive study of individual mutations is necessary to create a realistic picture of the evolutionarily significant spectrum of beneficial mutations. Here we use a bulk-segregant approach to identify the beneficial mutations across 11 lineages of experimentally evolved yeast populations. We report that nearly 80% of detected mutations have no discernible effects on fitness and less than 1% are deleterious. We determine the distribution of driver and hitchhiker mutations in 31 mutational cohorts, groups of mutations that arise synchronously from low frequency and track tightly with one another. Surprisingly, we find that one-third of cohorts lack identifiable driver mutations. In addition, we identify intracohort synergistic epistasis between alleles of hsl7 and kel1, which arose together in a low-frequency lineage. PMID:28720700

Splice Site Mutations in the ATP7A Gene

PubMed Central

Møller, Lisbeth Birk

2011-01-01

Menkes disease (MD) is caused by mutations in the ATP7A gene. We describe 33 novel splice site mutations detected in patients with MD or the milder phenotypic form, Occipital Horn Syndrome. We review these 33 mutations together with 28 previously published splice site mutations. We investigate 12 mutations for their effect on the mRNA transcript in vivo. Transcriptional data from another 16 mutations were collected from the literature. The theoretical consequences of splice site mutations, predicted with the bioinformatics tool Human Splice Finder, were investigated and evaluated in relation to in vivo results. Ninety-six percent of the mutations identified in 45 patients with classical MD were predicted to have a significant effect on splicing, which concurs with the absence of any detectable wild-type transcript in all 19 patients investigated in vivo. Sixty-seven percent of the mutations identified in 12 patients with milder phenotypes were predicted to have no significant effect on splicing, which concurs with the presence of wild-type transcript in 7 out of 9 patients investigated in vivo. Both the in silico predictions and the in vivo results support the hypothesis previously suggested by us and others, that the presence of some wild-type transcript is correlated to a milder phenotype. PMID:21494555

The contribution of de novo coding mutations to autism spectrum disorder.

PubMed

Iossifov, Ivan; O'Roak, Brian J; Sanders, Stephan J; Ronemus, Michael; Krumm, Niklas; Levy, Dan; Stessman, Holly A; Witherspoon, Kali T; Vives, Laura; Patterson, Karynne E; Smith, Joshua D; Paeper, Bryan; Nickerson, Deborah A; Dea, Jeanselle; Dong, Shan; Gonzalez, Luis E; Mandell, Jeffrey D; Mane, Shrikant M; Murtha, Michael T; Sullivan, Catherine A; Walker, Michael F; Waqar, Zainulabedin; Wei, Liping; Willsey, A Jeremy; Yamrom, Boris; Lee, Yoon-ha; Grabowska, Ewa; Dalkic, Ertugrul; Wang, Zihua; Marks, Steven; Andrews, Peter; Leotta, Anthony; Kendall, Jude; Hakker, Inessa; Rosenbaum, Julie; Ma, Beicong; Rodgers, Linda; Troge, Jennifer; Narzisi, Giuseppe; Yoon, Seungtai; Schatz, Michael C; Ye, Kenny; McCombie, W Richard; Shendure, Jay; Eichler, Evan E; State, Matthew W; Wigler, Michael

2014-11-13

Whole exome sequencing has proven to be a powerful tool for understanding the genetic architecture of human disease. Here we apply it to more than 2,500 simplex families, each having a child with an autistic spectrum disorder. By comparing affected to unaffected siblings, we show that 13% of de novo missense mutations and 43% of de novo likely gene-disrupting (LGD) mutations contribute to 12% and 9% of diagnoses, respectively. Including copy number variants, coding de novo mutations contribute to about 30% of all simplex and 45% of female diagnoses. Almost all LGD mutations occur opposite wild-type alleles. LGD targets in affected females significantly overlap the targets in males of lower intelligence quotient (IQ), but neither overlaps significantly with targets in males of higher IQ. We estimate that LGD mutation in about 400 genes can contribute to the joint class of affected females and males of lower IQ, with an overlapping and similar number of genes vulnerable to contributory missense mutation. LGD targets in the joint class overlap with published targets for intellectual disability and schizophrenia, and are enriched for chromatin modifiers, FMRP-associated genes and embryonically expressed genes. Most of the significance for the latter comes from affected females.

Levels of DNA Methylation Vary at CpG Sites across the BRCA1 Promoter, and Differ According to Triple Negative and “BRCA-Like” Status, in Both Blood and Tumour DNA

PubMed Central

Burghel, George J.; Chambers, Philip; Al-Baba, Shadi; Connley, Daniel D.; Brock, Ian W.; Cramp, Helen E.; Dotsenko, Olena; Wilks, Octavia; Wyld, Lynda; Cross, Simon S.; Cox, Angela

2016-01-01

Triple negative breast cancer is typically an aggressive and difficult to treat subtype. It is often associated with loss of function of the BRCA1 gene, either through mutation, loss of heterozygosity or methylation. This study aimed to measure methylation of the BRCA1 gene promoter at individual CpG sites in blood, tumour and normal breast tissue, to assess whether levels were correlated between different tissues, and with triple negative receptor status, histopathological scoring for BRCA-like features and BRCA1 protein expression. Blood DNA methylation levels were significantly correlated with tumour methylation at 9 of 11 CpG sites examined (p<0.0007). The levels of tumour DNA methylation were significantly higher in triple negative tumours, and in tumours with high BRCA-like histopathological scores (10 of 11 CpG sites; p<0.01 and p<0.007 respectively). Similar results were observed in blood DNA (6 of 11 CpG sites; p<0.03 and 7 of 11 CpG sites; p<0.02 respectively). This study provides insight into the pattern of CpG methylation across the BRCA1 promoter, and supports previous studies suggesting that tumours with BRCA1 promoter methylation have similar features to those with BRCA1 mutations, and therefore may be suitable for the same targeted therapies. PMID:27463681

Target Acquired: Progress and Promise of Targeted Therapeutics in the Treatment of Prostate Cancer.

PubMed

Stuchbery, Ryan; Kurganovs, Natalie J; McCoy, Patrick J; Nelson, Colleen C; Hayes, Vanessa M; Corcoran, Niall M; Hovens, Christopher M

2015-01-01

Cancer is fundamentally a genomic disease caused by mutations or rearrangements in the DNA or epigenetic machinery of a patient. An emerging field in cancer treatment targets key aberrations arising from the mutational landscape of an individual patient's disease rather than employing a cancer-wide cytotoxic therapy approach. In prostate cancer in particular, where there is an observed variation in response to standard treatments between patients with disease of a similar pathological stage and grade, mutationdirected treatment may grow to be a viable tool for clinicians to tailor more effective treatments. This review will describe a number of mutations across multiple forms of cancer that have been successfully antagonised by targeted therapeutics including their identification, the development of targeted compounds to combat them and the development of resistance to these therapies. This review will continue to examine these same mutations in the treatment and management of prostate cancer; the prevalence of targetable mutations in prostate cancer, recent clinical trials of targeted-agents and the potential or limitations for their use.

An anthrax toxin variant with an improved activity in tumor targeting

PubMed Central

Wein, Alexander N.; Peters, Diane E.; Valivullah, Zaheer; Hoover, Benjamin J.; Tatineni, Aparna; Ma, Qian; Fattah, Rasem; Bugge, Thomas H.; Leppla, Stephen H.; Liu, Shihui

2015-01-01

Anthrax lethal toxin (LT) is an A-B type toxin secreted by Bacillus anthracis, consisting of the cellular binding moiety, protective antigen (PA), and the catalytic moiety, lethal factor (LF). To target cells, PA binds to cell-surface receptors and is then proteolytically processed forming a LF-binding competent PA oligomer where each LF binding site is comprised of three subsites on two adjacent PA monomers. We previously generated PA-U2-R200A, a urokinase-activated PA variant with LF-binding subsite II residue Arg200 mutated to Ala, and PA-L1-I210A, a matrix metalloproteinase-activated PA variant with subsite III residue Ile210 mutated to Ala. PA-U2-R200A and PA-L1-I210A displayed reduced cytotoxicity when used singly. However, when combined, they formed LF-binding competent heterogeneous oligomers by intermolecular complementation, and achieved high specificity in tumor targeting. Nevertheless, each of these proteins, in particular PA-L1-I210A, retained residual LF-binding ability. In this work, we screened a library containing all possible amino acid substitutions for LF-binding site to find variants with activity strictly dependent upon intermolecular complementation. PA-I207R was identified as an excellent replacement for the original clockwise-side variant, PA-I210A. Consequently, the new combination of PA-L1-I207R and PA-U2-R200A showed potent anti-tumor activity and low toxicity, exceeding the performance of the original combination, and warranting further investigation. PMID:26584669

The dynamics of pyrethroid resistance in Anopheles arabiensis from Zanzibar and an assessment of the underlying genetic basis

PubMed Central

2013-01-01

Background The emergence of pyrethroid resistance in the malaria vector, Anopheles arabiensis, threatens to undermine the considerable gains made towards eliminating malaria on Zanzibar. Previously, resistance was restricted to the island of Pemba while mosquitoes from Unguja, the larger of the two islands of Zanzibar, were susceptible. Here, we characterised the mechanism(s) responsible for resistance on Zanzibar using a combination of gene expression and target-site mutation assays. Methods WHO resistance bioassays were conducted using 1-5d old adult Anopheles gambiae s.l. collected between 2011 and 2013 across the archipelago. Synergist assays with the P450 inhibitor piperonyl-butoxide were performed in 2013. Members of the An. gambiae complex were PCR-identified and screened for target-site mutations (kdr and Ace-1). Gene expression in pyrethroid resistant An. arabiensis from Pemba was analysed using whole-genome microarrays. Results Pyrethroid resistance is now present across the entire Zanzibar archipelago. Survival to the pyrethroid lambda-cyhalothrin in bioassays conducted in 2013 was 23.5-54.3% on Unguja and 32.9-81.7% on Pemba. We present evidence that resistance is mediated, in part at least, by elevated P450 monoxygenases. Whole-genome microarray scans showed that the most enriched gene terms in resistant An. arabiensis from Pemba were associated with P450 activity and synergist assays with PBO completely restored susceptibility to pyrethroids in both islands. CYP4G16 was the most consistently over-expressed gene in resistant mosquitoes compared with two susceptible strains from Unguja and Dar es Salaam. Expression of this P450 is enriched in the abdomen and it is thought to play a role in hydrocarbon synthesis. Microarray and qPCR detected several additional genes putatively involved in this pathway enriched in the Pemba pyrethroid resistant population and we hypothesise that resistance may be, in part, related to alterations in the structure of the mosquito cuticle. None of the kdr target-site mutations, associated with pyrethroid/DDT resistance in An. gambiae elsewhere in Africa, were found on the islands. Conclusion The consequences of this resistance phenotype are discussed in relation to future vector control strategies on Zanzibar to support the ongoing malaria elimination efforts on the islands. PMID:24314005

NCI-MATCH Trial Links Targeted Drugs to Mutations

Cancer.gov

Investigators for the nationwide trial, NCI-MATCH: Molecular Analysis for Therapy Choice, announced that the trial will seek to determine whether targeted therapies for people whose tumors have specific gene mutations will be effective regardless of their cancer type. NCI-MATCH will incorporate more than 20 different study drugs or drug combinations, each targeting a specific gene mutation, in order to match each patient in the trial with a therapy that targets a molecular abnormality in their tumor.

Unnecessary antiretroviral treatment switches and accumulation of HIV resistance mutations; two arguments for viral load monitoring in Africa.

PubMed

Sigaloff, Kim C E; Hamers, Raph L; Wallis, Carole L; Kityo, Cissy; Siwale, Margaret; Ive, Prudence; Botes, Mariette E; Mandaliya, Kishor; Wellington, Maureen; Osibogun, Akin; Stevens, Wendy S; van Vugt, Michèle; de Wit, Tobias F Rinke

2011-09-01

This study aimed to investigate the consequences of using clinicoimmunological criteria to detect antiretroviral treatment (ART) failure and guide regimen switches in HIV-infected adults in sub-Saharan Africa. Frequencies of unnecessary switches, patterns of HIV drug resistance, and risk factors for the accumulation of nucleoside reverse transcriptase inhibitor (NRTI)-associated mutations were evaluated. Cross-sectional analysis of adults switching ART regimens at 13 clinical sites in 6 African countries was performed. Two types of failure identification were compared: diagnosis of clinicoimmunological failure without viral load testing (CIF only) or CIF with local targeted viral load testing (targeted VL). After study enrollment, reference HIV RNA and genotype were determined retrospectively. Logistic regression assessed factors associated with multiple thymidine analogue mutations (TAMs) and NRTI cross-resistance (≥2 TAMs or Q151M or K65R/K70E). Of 250 patients with CIF switching to second-line ART, targeted VL was performed in 186. Unnecessary switch at reference HIV RNA <1000 copies per milliliter occurred in 46.9% of CIF only patients versus 12.4% of patients with targeted VL (P < 0.001). NRTI cross-resistance was observed in 48.0% of 183 specimens available for genotypic analysis, comprising ≥2 TAMs (37.7%), K65R (7.1%), K70E (3.3%), or Q151M (3.3%). The presence of NRTI cross-resistance was associated with the duration of ART exposure and zidovudine use. Clinicoimmunological monitoring without viral load testing resulted in frequent unnecessary regimen switches. Prolonged treatment failure was indicated by extensive NRTI cross-resistance. Access to virological monitoring should be expanded to prevent inappropriate switches, enable early failure detection and preserve second-line treatment options in Africa.

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

NASA Astrophysics Data System (ADS)

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

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

Modeling the ferrochelatase c.315-48C modifier mutation for erythropoietic protoporphyria (EPP) in mice

PubMed Central

Bansode, Vijay B.; Koentgen, Frank; Trüb, Judith; Pelczar, Pawel; Schneider-Yin, Xiaoye; Minder, Elisabeth I.

2017-01-01

ABSTRACT Erythropoietic protoporphyria (EPP) is caused by deficiency of ferrochelatase (FECH), which incorporates iron into protoporphyrin IX (PPIX) to form heme. Excitation of accumulated PPIX by light generates oxygen radicals that evoke excessive pain and, after longer light exposure, cause ulcerations in exposed skin areas of individuals with EPP. Moreover, ∼5% of the patients develop a liver dysfunction as a result of PPIX accumulation. Most patients (∼97%) have a severe FECH mutation (Mut) in trans to an intronic polymorphism (c.315-48C), which reduces ferrochelatase synthesis by stimulating the use of an aberrant 3′ splice site 63 nt upstream of the normal site for exon 4. In contrast, with the predominant c.315-48T allele, the correct splice site is mostly used, and individuals with a T/Mut genotype do not develop EPP symptoms. Thus, the C allele is a potential target for therapeutic approaches that modify this splicing decision. To provide a model for pre-clinical studies of such approaches, we engineered a mouse containing a partly humanized Fech gene with the c.315-48C polymorphism. F1 hybrids obtained by crossing these mice with another inbred line carrying a severe Fech mutation (named m1Pas) show a very strong EPP phenotype that includes elevated PPIX in the blood, enlargement of liver and spleen, anemia, as well as strong pain reactions and skin lesions after a short period of light exposure. In addition to the expected use of the aberrant splice site, the mice also show a strong skipping of the partly humanized exon 3. This will limit the use of this model for certain applications and illustrates that engineering of a hybrid gene may have unforeseeable consequences on its splicing. PMID:28093505

Detection of First-Line Drug Resistance Mutations and Drug-Protein Interaction Dynamics from Tuberculosis Patients in South India.

PubMed

Nachappa, Somanna Ajjamada; Neelambike, Sumana M; Amruthavalli, Chokkanna; Ramachandra, Nallur B

2018-05-01

Diagnosis of drug-resistant tuberculosis predominantly relies on culture-based drug susceptibility testing, which take weeks to produce a result and a more time-efficient alternative method is multiplex allele-specific PCR (MAS-PCR). Also, understanding the role of mutations in causing resistance helps better drug designing. To evaluate the ability of MAS-PCR in the detection of drug resistance and to understand the mechanism of interaction of drugs with mutant proteins in Mycobacterium tuberculosis. Detection of drug-resistant mutations using MAS-PCR and validation through DNA sequencing. MAS-PCR targeted five loci on three genes, katG 315 and inhA -15 for the drug isoniazid (INH), and rpoB 516, 526, and 531 for rifampicin (RIF). Furthermore, the sequence data were analyzed to study the effect on interaction of the anti-TB drug molecule with the target protein using in silico docking. We identified drug-resistant mutations in 8 out of 114 isolates with 2 of them as multidrug-resistant TB using MAS-PCR. DNA sequencing confirmed only six of these, recording a sensitivity of 85.7% and specificity of 99.3% for MAS-PCR. Molecular docking showed estimated free energy of binding (ΔG) being higher for RIF binding with RpoB S531L mutant. Codon 315 in KatG does not directly interact with INH but blocks the drug access to active site. We propose DNA sequencing-based drug resistance detection for TB, which is more accurate than MAS-PCR. Understanding the action of resistant mutations in disrupting the normal drug-protein interaction aids in designing effective drug alternatives.

Single molecule molecular inversion probes for targeted, high-accuracy detection of low-frequency variation.

PubMed

Hiatt, Joseph B; Pritchard, Colin C; Salipante, Stephen J; O'Roak, Brian J; Shendure, Jay

2013-05-01

The detection and quantification of genetic heterogeneity in populations of cells is fundamentally important to diverse fields, ranging from microbial evolution to human cancer genetics. However, despite the cost and throughput advances associated with massively parallel sequencing, it remains challenging to reliably detect mutations that are present at a low relative abundance in a given DNA sample. Here we describe smMIP, an assay that combines single molecule tagging with multiplex targeted capture to enable practical and highly sensitive detection of low-frequency or subclonal variation. To demonstrate the potential of the method, we simultaneously resequenced 33 clinically informative cancer genes in eight cell line and 45 clinical cancer samples. Single molecule tagging facilitated extremely accurate consensus calling, with an estimated per-base error rate of 8.4 × 10(-6) in cell lines and 2.6 × 10(-5) in clinical specimens. False-positive mutations in the single molecule consensus base-calls exhibited patterns predominantly consistent with DNA damage, including 8-oxo-guanine and spontaneous deamination of cytosine. Based on mixing experiments with cell line samples, sensitivity for mutations above 1% frequency was 83% with no false positives. At clinically informative sites, we identified seven low-frequency point mutations (0.2%-4.7%), including BRAF p.V600E (melanoma, 0.2% alternate allele frequency), KRAS p.G12V (lung, 0.6%), JAK2 p.V617F (melanoma, colon, two lung, 0.3%-1.4%), and NRAS p.Q61R (colon, 4.7%). We anticipate that smMIP will be broadly adoptable as a practical and effective method for accurately detecting low-frequency mutations in both research and clinical settings.

Single molecule molecular inversion probes for targeted, high-accuracy detection of low-frequency variation

PubMed Central

Hiatt, Joseph B.; Pritchard, Colin C.; Salipante, Stephen J.; O'Roak, Brian J.; Shendure, Jay

2013-01-01

The detection and quantification of genetic heterogeneity in populations of cells is fundamentally important to diverse fields, ranging from microbial evolution to human cancer genetics. However, despite the cost and throughput advances associated with massively parallel sequencing, it remains challenging to reliably detect mutations that are present at a low relative abundance in a given DNA sample. Here we describe smMIP, an assay that combines single molecule tagging with multiplex targeted capture to enable practical and highly sensitive detection of low-frequency or subclonal variation. To demonstrate the potential of the method, we simultaneously resequenced 33 clinically informative cancer genes in eight cell line and 45 clinical cancer samples. Single molecule tagging facilitated extremely accurate consensus calling, with an estimated per-base error rate of 8.4 × 10−6 in cell lines and 2.6 × 10−5 in clinical specimens. False-positive mutations in the single molecule consensus base-calls exhibited patterns predominantly consistent with DNA damage, including 8-oxo-guanine and spontaneous deamination of cytosine. Based on mixing experiments with cell line samples, sensitivity for mutations above 1% frequency was 83% with no false positives. At clinically informative sites, we identified seven low-frequency point mutations (0.2%–4.7%), including BRAF p.V600E (melanoma, 0.2% alternate allele frequency), KRAS p.G12V (lung, 0.6%), JAK2 p.V617F (melanoma, colon, two lung, 0.3%–1.4%), and NRAS p.Q61R (colon, 4.7%). We anticipate that smMIP will be broadly adoptable as a practical and effective method for accurately detecting low-frequency mutations in both research and clinical settings. PMID:23382536

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

PubMed

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

2018-04-19

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

Effects of mutations on active site conformation and dynamics of RNA-dependent RNA polymerase from Coxsackievirus B3.

PubMed

Shen, Hujun; Deng, Mingsen; Zhang, Yachao

2017-10-01

Recent crystal structures of RNA-dependent RNA polymerase (3D pol ) from Coxsackievirus B3 (CVB3) revealed that a tyrosine mutation at Phe364 (F364Y) resulted in structures with open active site whereas a hydrophobic mutation at Phe364 (F364A) led to conformations with closed active site. Besides, the crystal structures showed that the F364W mutation had no preference between the open and closed active sites, similar to wild-type. In this paper, we present a molecular dynamics (MD) study on CVB3 3D pol in order to address some important questions raised by experiments. First, MD simulations of F364Y and F364A were carried out to explore how these mutations at Phe364 influence active site dynamics and conformations. Second, MD simulations of wild-type and mutants were performed to discover the connection between active site dynamics and polymerase function. MD simulations reveal that the effect of mutations on active site dynamics is associated with the interaction between the structural motifs A and D in CVB3 3D pol . Interestingly, we discover that the active site state is influenced by the formation of a hydrogen bond between backbone atoms of Ala231 (in motif A) and Ala358 (in motif D), which has never been revealed before. Copyright © 2017 Elsevier Inc. All rights reserved.

Genetic alterations in seborrheic keratoses

PubMed Central

Heidenreich, Barbara; Denisova, Evygenia; Rachakonda, Sivaramakrishna; Sanmartin, Onofre; Dereani, Timo; Hosen, Ismail; Nagore, Eduardo; Kumar, Rajiv

2017-01-01

Seborrheic keratoses are common benign epidermal lesions that are associated with increased age and sun-exposure. Those lesions despite harboring multiple somatic alterations in contrast to malignant tumors appear to be genetically stable. In order to investigate and characterize the presence of recurrent mutations, we performed exome sequencing on DNA from one seborrheic keratosis lesion and corresponding blood cells from the same patients with follow up investigation of alterations identified by exome sequencing in 24 additional lesions from as many patients. In addition we investigated alterations in all lesions at specific genes loci that included FGFR3, PIK3CA, HRAS, BRAF, CDKN2A and TERT and DHPH3 promoters. The exome sequencing data indicated three mutations per Mb of the targeted sequence. The mutational pattern depicted typical UV signature with majority of alterations being C>T and CC>TT base changes at dipyrimidinic sites. The FGFR3 mutations were the most frequent, detected in 12 of 25 (48%) lesions, followed by the PIK3CA (32%), TERT promoter (24%) and DPH3 promoter mutations (24%). TERT promoter mutations associated with increased age and were present mainly in the lesions excised from head and neck. Three lesions also carried alterations in CDKN2A. FGFR3, TERT and DPH3 expression did not correlate with mutations in the respective genes and promoters; however, increased FGFR3 transcript levels were associated with increased FOXN1 levels, a suggested positive feedback loop that stalls malignant progression. Thus, in this study we report overall mutation rate through exome sequencing and show the most frequent mutations seborrheic keratosis. PMID:28410231

Drug Resistance Missense Mutations in Cancer Are Subject to Evolutionary Constraints

PubMed Central

Friedman, Ran

2013-01-01

Several tumour types are sensitive to deactivation of just one or very few genes that are constantly active in the cancer cells, a phenomenon that is termed ‘oncogene addiction’. Drugs that target the products of those oncogenes can yield a temporary relief, and even complete remission. Unfortunately, many patients receiving oncogene-targeted therapies relapse on treatment. This often happens due to somatic mutations in the oncogene (‘resistance mutations’). ‘Compound mutations’, which in the context of cancer drug resistance are defined as two or more mutations of the drug target in the same clone may lead to enhanced resistance against the most selective inhibitors. Here, it is shown that the vast majority of the resistance mutations occurring in cancer patients treated with tyrosin kinase inhibitors aimed at three different proteins follow an evolutionary pathway. Using bioinformatic analysis tools, it is found that the drug-resistance mutations in the tyrosine kinase domains of Abl1, ALK and exons 20 and 21 of EGFR favour transformations to residues that can be identified in similar positions in evolutionary related proteins. The results demonstrate that evolutionary pressure shapes the mutational landscape in the case of drug-resistance somatic mutations. The constraints on the mutational landscape suggest that it may be possible to counter single drug-resistance point mutations. The observation of relatively many resistance mutations in Abl1, but not in the other genes, is explained by the fact that mutations in Abl1 tend to be biochemically conservative, whereas mutations in EGFR and ALK tend to be radical. Analysis of Abl1 compound mutations suggests that such mutations are more prevalent than hitherto reported and may be more difficult to counter. This supports the notion that such mutations may provide an escape route for targeted cancer drug resistance. PMID:24376513

Cross-resistance to herbicides of five ALS-inhibiting groups and sequencing of the ALS gene in Cyperus difformis L.

PubMed

Merotto, Aldo; Jasieniuk, Marie; Osuna, Maria D; Vidotto, Francesco; Ferrero, Aldo; Fischer, Albert J

2009-02-25

Resistance to ALS-inhibiting herbicides in Cyperus difformis has evolved rapidly in many rice areas worldwide. This study identified the mechanism of resistance, assessed cross-resistance patterns to all five chemical groups of ALS-inhibiting herbicides in four C. difformis biotypes, and attempted to sequence the ALS gene. Whole-plant and ALS enzyme activity dose-response assays indicated that the WA biotype was resistant to all ALS-inhibiting herbicides evaluated. The IR biotype was resistant to bensulfuron-methyl, orthosulfamuron, imazethapyr, and propoxycarbazone-sodium and less resistant to bispyribac-sodium and halosulfuron-methyl, and susceptible to penoxsulam. ALS enzyme activity assays indicated that resistance is due to an altered target site yet mutations previously found to endow target-site resistance in weeds were not detected in the sequences obtained. The inability to detect resistance mutations in C. difformis may result from the presence of additional ALS genes, which were not amplified by the primers used. This study reports the first ALS gene sequence from Cyperus difformis. Certain ALS-inhibiting herbicides can still be used to control some resistant C. difformis biotypes. However, because cross-resistance to all five classes of ALS-inhibitors was detected in other resistant biotypes, these herbicides should only be used within an integrated weed management program designed to delay the evolution of herbicide resistance.

Optimization of Benzoisothiazole dioxide inhibitory activity of the NS5B polymerase of HCV genotype 4 using ligand-steered homological modeling, reaction-driven scaffold-hopping and Enovo workflow.

PubMed

Mahmoud, Amr Hamed; Mohamed Abouzid, Khaled Abouzid; El Ella, Dalal Abd El Rahman Abou; Hamid Ismail, Mohamed Abdel

2011-01-01

Infection caused by hepatitis C virus (HCV) is a significant world health problem for which novel therapies are in urgent demand. The virus is highly prevalent in the Middle East and Africa particularly Egypt with more than 90% of infections due to genotype 4. Nonstructural (NS5B) viral proteins have emerged as an attractive target for HCV antivirals discovery. A potent class of inhibitors having benzisothiazole dioxide scaffold has been identified on this target, however they were mainly active on genotype 1 while exhibiting much lowered activity on other genotypes due to the high degree of mutation of its binding site. Based on this fact, we employed a novel strategy to optimize this class on genotype 4. This strategy depends on using a refined ligand-steered homological model of this genotype to study the mutation binding energies of the binding site amino acid residues, the essential features for interaction and provide a structure-based pharmacophore model that can aid optimization. This model was applied on a focused library which was generated using a reaction-driven scaffold-hopping strategy. The hits retrieved were subjected to Enovo pipeline pilot optimization workflow that employs R-group enumeration, core-constrained protein docking using modified CDOCKER and finally ranking of poses using an accurate molecular mechanics generalized Born with surface area method.

Covalent ISG15 conjugation positively regulates the ubiquitin E3 ligase activity of parkin

PubMed Central

Im, Eunju; Yoo, Lang; Hyun, Minju; Shin, Woo Hyun

2016-01-01

Parkinson's disease (PD) is characterized by selective loss of dopaminergic neurons in the pars compacta of the substantia nigra and accumulation of ubiquitinated proteins in aggregates called Lewy bodies. Several mutated genes have been found in familial PD patients, including SNCA (α-synuclein), PARK2 (parkin), PINK1, PARK7 (DJ-1), LRRK2 and ATP13A2. Many pathogenic mutations of PARK2, which encodes the ubiquitin E3 ligase parkin, result in loss of function, leading to accumulation of parkin substrates and consequently contributing to dopaminergic cell death. ISG15 is a member of the ubiquitin-like modifier family and is induced by stimulation with type I interferons. Similar to ubiquitin and ubiquitination, covalent conjugation of ISG15 to target proteins (ISGylation) regulates their biochemical properties. In this study, we identified parkin as a novel target of ISGylation specifically mediated by the ISG15-E3 ligase HERC5. In addition, we identified two ISGylation sites, Lys-349 and Lys-369, in the in-between-ring domain of parkin. ISGylation of these sites promotes parkin's ubiquitin E3 ligase activity by suppressing the intramolecular interaction that maintains its autoinhibited conformation and increases its cytoprotective effect. In conclusion, covalent ISG15 conjugation is a novel mode of modulating parkin activity, and alteration in this pathway may be associated with PD pathogenesis. PMID:27534820

Covalent ISG15 conjugation positively regulates the ubiquitin E3 ligase activity of parkin.

PubMed

Im, Eunju; Yoo, Lang; Hyun, Minju; Shin, Woo Hyun; Chung, Kwang Chul

2016-08-01

Parkinson's disease (PD) is characterized by selective loss of dopaminergic neurons in the pars compacta of the substantia nigra and accumulation of ubiquitinated proteins in aggregates called Lewy bodies. Several mutated genes have been found in familial PD patients, including SNCA (α-synuclein), PARK2 (parkin), PINK1, PARK7 (DJ-1), LRRK2 and ATP13A2 Many pathogenic mutations of PARK2, which encodes the ubiquitin E3 ligase parkin, result in loss of function, leading to accumulation of parkin substrates and consequently contributing to dopaminergic cell death. ISG15 is a member of the ubiquitin-like modifier family and is induced by stimulation with type I interferons. Similar to ubiquitin and ubiquitination, covalent conjugation of ISG15 to target proteins (ISGylation) regulates their biochemical properties. In this study, we identified parkin as a novel target of ISGylation specifically mediated by the ISG15-E3 ligase HERC5. In addition, we identified two ISGylation sites, Lys-349 and Lys-369, in the in-between-ring domain of parkin. ISGylation of these sites promotes parkin's ubiquitin E3 ligase activity by suppressing the intramolecular interaction that maintains its autoinhibited conformation and increases its cytoprotective effect. In conclusion, covalent ISG15 conjugation is a novel mode of modulating parkin activity, and alteration in this pathway may be associated with PD pathogenesis. © 2016 The Authors.

K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions

PubMed Central

Ostrem, Jonathan M.; Peters, Ulf; Sos, Martin L.; Wells, James A.; Shokat, Kevan M.

2014-01-01

Somatic mutations in the small GTPase K-Ras are the most common activating lesions found in human cancer, and are generally associated with poor response to standard therapies1–3. Efforts to target this oncogene directly have faced difficulties owing to its picomolar affinity for GTP/GDP4 and the absence of known allosteric regulatory sites. Oncogenic mutations result in functional activation of Ras family proteins by impairing GTP hydrolysis5,6. With diminished regulation by GTPase activity, the nucleotide state of Ras becomes more dependent on relative nucleotide affinity and concentration. This gives GTP an advantage over GDP7 and increases the proportion of active GTP-bound Ras. Here we report the development of small molecules that irreversibly bind to a common oncogenic mutant, K-Ras(G12C). These compounds rely on the mutant cysteine for binding and therefore do not affect the wild-type protein. Crystallographic studies reveal the formation of a new pocket that is not apparent in previous structures of Ras, beneath the effector binding switch-II region. Binding of these inhibitors to K-Ras(G12C) disrupts both switch-I and switch-II, subverting the native nucleotide preference to favour GDP over GTP and impairing binding to Raf. Our data provide structure-based validation of a new allosteric regulatory site on Ras that is targetable in a mutant-specific manner. PMID:24256730

Hes repressors are essential regulators of hematopoietic stem cell development downstream of Notch signaling

PubMed Central

Guiu, Jordi; Shimizu, Ritsuko; D’Altri, Teresa; Fraser, Stuart T.; Hatakeyama, Jun; Bresnick, Emery H.; Kageyama, Ryoichiro; Dzierzak, Elaine; Yamamoto, Masayuki; Espinosa, Lluis

2013-01-01

Previous studies have identified Notch as a key regulator of hematopoietic stem cell (HSC) development, but the underlying downstream mechanisms remain unknown. The Notch target Hes1 is widely expressed in the aortic endothelium and hematopoietic clusters, though Hes1-deficient mice show no overt hematopoietic abnormalities. We now demonstrate that Hes is required for the development of HSC in the mouse embryo, a function previously undetected as the result of functional compensation by de novo expression of Hes5 in the aorta/gonad/mesonephros (AGM) region of Hes1 mutants. Analysis of embryos deficient for Hes1 and Hes5 reveals an intact arterial program with overproduction of nonfunctional hematopoietic precursors and total absence of HSC activity. These alterations were associated with increased expression of the hematopoietic regulators Runx1, c-myb, and the previously identified Notch target Gata2. By analyzing the Gata2 locus, we have identified functional RBPJ-binding sites, which mutation results in loss of Gata2 reporter expression in transgenic embryos, and functional Hes-binding sites, which mutation leads to specific Gata2 up-regulation in the hematopoietic precursors. Together, our findings show that Notch activation in the AGM triggers Gata2 and Hes1 transcription, and next HES-1 protein represses Gata2, creating an incoherent feed-forward loop required to restrict Gata2 expression in the emerging HSCs. PMID:23267012

In situ mutation detection and visualization of intratumor heterogeneity for cancer research and diagnostics

PubMed Central

Grundberg, Ida; Kiflemariam, Sara; Mignardi, Marco; Imgenberg-Kreuz, Juliana; Edlund, Karolina; Micke, Patrick; Sundström, Magnus; Sjöblom, Tobias

2013-01-01

Current assays for somatic mutation analysis are based on extracts from tissue sections that often contain morphologically heterogeneous neoplastic regions with variable contents of genetically normal stromal and inflammatory cells, obscuring the results of the assays. We have developed an RNA-based in situ mutation assay that targets oncogenic mutations in a multiplex fashion that resolves the heterogeneity of the tissue sample. Activating oncogenic mutations are targets for a new generation of cancer drugs. For anti-EGFR therapy prediction, we demonstrate reliable in situ detection of KRAS mutations in codon 12 and 13 in colon and lung cancers in three different types of routinely processed tissue materials. High-throughput screening of KRAS mutation status was successfully performed on a tissue microarray. Moreover, we show how the patterns of expressed mutated and wild-type alleles can be studied in situ in tumors with complex combinations of mutated EGFR, KRAS and TP53. This in situ method holds great promise as a tool to investigate the role of somatic mutations during tumor progression and for prediction of response to targeted therapy. PMID:24280411

N-terminal dual lipidation-coupled molecular targeting into the primary cilium.

PubMed

Kumeta, Masahiro; Panina, Yulia; Yamazaki, Hiroya; Takeyasu, Kunio; Yoshimura, Shige H

2018-06-13

The primary cilium functions as an "antenna" for cell signaling, studded with characteristic transmembrane receptors and soluble protein factors, raised above the cell surface. In contrast to the transmembrane proteins, targeting mechanisms of nontransmembrane ciliary proteins are poorly understood. We focused on a pathogenic mutation that abolishes ciliary localization of retinitis pigmentosa 2 protein and revealed a dual acylation-dependent ciliary targeting pathway. Short N-terminal sequences which contain myristoylation and palmitoylation sites are sufficient to target a marker protein into the cilium in a palmitoylation-dependent manner. A Golgi-localized palmitoyltransferase DHHC-21 was identified as the key enzyme controlling this targeting pathway. Rapid turnover of the targeted protein was ensured by cholesterol-dependent membrane fluidity, which balances highly and less-mobile populations of the molecules within the cilium. This targeting signal was found in a set of signal transduction molecules, suggesting a general role of this pathway in proper ciliary organization, and dysfunction in ciliary disorders. © 2018 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Structural characterization and mutational assessment of podocin - a novel drug target to nephrotic syndrome - an in silico approach.

PubMed

Tabassum, Asra; Rajeshwari, Tadigadapa; Soni, Nidhi; Raju, D S B; Yadav, Mukesh; Nayarisseri, Anuraj; Jahan, Parveen

2014-03-01

Non-synonymous single nucleotide changes (nSNC) are coding variants that introduce amino acid changes in their corresponding proteins. They can affect protein function; they are believed to have the largest impact on human health compared with SNCs in other regions of the genome. Such a sequence alteration directly affects their structural stability through conformational changes. Presence of these conformational changes near catalytic site or active site may alter protein function and as a consequence receptor-ligand complex interactions. The present investigation includes assessment of human podocin mutations (G92C, P118L, R138Q, and D160G) on its structure. Podocin is an important glomerular integral membrane protein thought to play a key role in steroid resistant nephrotic syndrome. Podocin has a hairpin like structure with 383 amino acids, it is an integral protein homologous to stomatin, and acts as a molecular link in a stretch-sensitive system. We modeled 3D structure of podocin by means of Modeller and validated via PROCHECK to get a Ramachandran plot (88.5% in most favored region), main chain, side chain, bad contacts, gauche and pooled standard deviation. Further, a protein engineering tool Triton was used to induce mutagenesis corresponding to four variants G92C, P118L, R138Q and D160G in the wild type. Perusal of energies of wild and mutated type of podocin structures confirmed that mutated structures were thermodynamically more stable than wild type and therefore biological events favored synthesis of mutated forms of podocin than wild type. As a conclusive part, two mutations G92C (-8179.272 kJ/mol) and P118L (-8136.685 kJ/mol) are more stable and probable to take place in podocin structure over wild podocin structure (-8105.622 kJ/mol). Though there is lesser difference in mutated and wild type (approximately, 74 and 35 kJ/mol), it may play a crucial role in deciding why mutations are favored and occur at the genetic level.

Mathematical models of tissue stem and transit target cell divisions and the risk of radiation- or smoking-associated cancer

PubMed Central

Hendry, Jolyon H.

2017-01-01

There is compelling biological data to suggest that cancer arises from a series of mutations in single target cells, resulting in defects in cell renewal and differentiation processes which lead to malignancy. Because much mutagenic damage is expressed following cell division, more-rapidly renewing tissues could be at higher risk because of the larger number of cell replications. Cairns suggested that renewing tissues may reduce cancer risk by partitioning the dividing cell populations into lineages comprising infrequently-dividing long-lived stem cells and frequently-dividing short-lived daughter transit cells. We develop generalizations of three recent cancer-induction models that account for the joint maintenance and renewal of stem and transit cells, also competing processes of partially transformed cell proliferation and differentiation/apoptosis. We are particularly interested in using these models to separately assess the probabilities of mutation and development of cancer associated with “spontaneous” processes and with those linked to a specific environmental mutagen, specifically ionizing radiation or cigarette smoking. All three models demonstrate substantial variation in cancer risks, by at least 20 orders of magnitude, depending on the assumed number of critical mutations required for cancer, and the stem-cell and transition-cell mutation rates. However, in most cases the conditional probabilities of cancer being mutagen-induced range between 7–96%. The relative risks associated with mutagen exposure compared to background rates are also stable, ranging from 1.0–16.0. Very few cancers, generally <0.5%, arise from mutations occurring solely in stem cells rather than in a combination of stem and transit cells. However, for cancers with 2 or 3 critical mutations, a substantial proportion of cancers, in some cases 100%, have at least one mutation derived from a mutated stem cell. Little difference is made to relative risks if competing processes of proliferation and differentiation in the partially transformed stem and transit cell population are allowed for, nor is any difference made if one assumes that transit cells require an extra mutation to confer malignancy from the number required by stem cells. The probability of a cancer being mutagen-induced correlates across cancer sites with the estimated cumulative number of stem cell divisions in the associated tissue (p<0.05), although in some cases there is sensitivity of findings to removal of high-leverage outliers and in some cases only modest variation in probability, but these issues do not affect the validity of the findings. There are no significant correlations (p>0.3) between lifetime cancer-site specific radiation risk and the probability of that cancer being mutagen-induced. These results do not depend on the assumed critical number of mutations leading to cancer, or on the assumed mutagen-associated mutation rate, within the generally-accepted ranges tested. However, there are borderline significant negative correlations (p = 0.08) between the smoking-associated mortality rate difference (current vs former smokers) and the probability of cancer being mutagen-induced. This is only the case where values of the critical number of mutations leading to cancer, k, is 3 or 4 and not for smaller values (1 or 2), but does not strongly depend on the assumed mutagen-associated mutation rate. PMID:28196079

Mathematical models of tissue stem and transit target cell divisions and the risk of radiation- or smoking-associated cancer.

PubMed

Little, Mark P; Hendry, Jolyon H

2017-02-01

There is compelling biological data to suggest that cancer arises from a series of mutations in single target cells, resulting in defects in cell renewal and differentiation processes which lead to malignancy. Because much mutagenic damage is expressed following cell division, more-rapidly renewing tissues could be at higher risk because of the larger number of cell replications. Cairns suggested that renewing tissues may reduce cancer risk by partitioning the dividing cell populations into lineages comprising infrequently-dividing long-lived stem cells and frequently-dividing short-lived daughter transit cells. We develop generalizations of three recent cancer-induction models that account for the joint maintenance and renewal of stem and transit cells, also competing processes of partially transformed cell proliferation and differentiation/apoptosis. We are particularly interested in using these models to separately assess the probabilities of mutation and development of cancer associated with "spontaneous" processes and with those linked to a specific environmental mutagen, specifically ionizing radiation or cigarette smoking. All three models demonstrate substantial variation in cancer risks, by at least 20 orders of magnitude, depending on the assumed number of critical mutations required for cancer, and the stem-cell and transition-cell mutation rates. However, in most cases the conditional probabilities of cancer being mutagen-induced range between 7-96%. The relative risks associated with mutagen exposure compared to background rates are also stable, ranging from 1.0-16.0. Very few cancers, generally <0.5%, arise from mutations occurring solely in stem cells rather than in a combination of stem and transit cells. However, for cancers with 2 or 3 critical mutations, a substantial proportion of cancers, in some cases 100%, have at least one mutation derived from a mutated stem cell. Little difference is made to relative risks if competing processes of proliferation and differentiation in the partially transformed stem and transit cell population are allowed for, nor is any difference made if one assumes that transit cells require an extra mutation to confer malignancy from the number required by stem cells. The probability of a cancer being mutagen-induced correlates across cancer sites with the estimated cumulative number of stem cell divisions in the associated tissue (p<0.05), although in some cases there is sensitivity of findings to removal of high-leverage outliers and in some cases only modest variation in probability, but these issues do not affect the validity of the findings. There are no significant correlations (p>0.3) between lifetime cancer-site specific radiation risk and the probability of that cancer being mutagen-induced. These results do not depend on the assumed critical number of mutations leading to cancer, or on the assumed mutagen-associated mutation rate, within the generally-accepted ranges tested. However, there are borderline significant negative correlations (p = 0.08) between the smoking-associated mortality rate difference (current vs former smokers) and the probability of cancer being mutagen-induced. This is only the case where values of the critical number of mutations leading to cancer, k, is 3 or 4 and not for smaller values (1 or 2), but does not strongly depend on the assumed mutagen-associated mutation rate.

Fitness-Balanced Escape Determines Resolution of Dynamic Founder Virus Escape Processes in HIV-1 Infection

PubMed Central

Sunshine, Justine E.; Larsen, Brendan B.; Maust, Brandon; Casey, Ellie; Deng, Wenje; Chen, Lennie; Westfall, Dylan H.; Kim, Moon; Zhao, Hong; Ghorai, Suvankar; Lanxon-Cookson, Erinn; Rolland, Morgane; Collier, Ann C.; Maenza, Janine; Mullins, James I.

2015-01-01

ABSTRACT To understand the interplay between host cytotoxic T-lymphocyte (CTL) responses and the mechanisms by which HIV-1 evades them, we studied viral evolutionary patterns associated with host CTL responses in six linked transmission pairs. HIV-1 sequences corresponding to full-length p17 and p24 gag were generated by 454 pyrosequencing for all pairs near the time of transmission, and seroconverting partners were followed for a median of 847 days postinfection. T-cell responses were screened by gamma interferon/interleukin-2 (IFN-γ/IL-2) FluoroSpot using autologous peptide sets reflecting any Gag variant present in at least 5% of sequence reads in the individual's viral population. While we found little evidence for the occurrence of CTL reversions, CTL escape processes were found to be highly dynamic, with multiple epitope variants emerging simultaneously. We found a correlation between epitope entropy and the number of epitope variants per response (r = 0.43; P = 0.05). In cases in which multiple escape mutations developed within a targeted epitope, a variant with no fitness cost became fixed in the viral population. When multiple mutations within an epitope achieved fitness-balanced escape, these escape mutants were each maintained in the viral population. Additional mutations found to confer escape but undetected in viral populations incurred high fitness costs, suggesting that functional constraints limit the available sites tolerable to escape mutations. These results further our understanding of the impact of CTL escape and reversion from the founder virus in HIV infection and contribute to the identification of immunogenic Gag regions most vulnerable to a targeted T-cell attack. IMPORTANCE Rapid diversification of the viral population is a hallmark of HIV-1 infection, and understanding the selective forces driving the emergence of viral variants can provide critical insight into the interplay between host immune responses and viral evolution. We used deep sequencing to comprehensively follow viral evolution over time in six linked HIV transmission pairs. We then mapped T-cell responses to explore if mutations arose due to adaption to the host and found that escape processes were often highly dynamic, with multiple mutations arising within targeted epitopes. When we explored the impact of these mutations on replicative capacity, we found that dynamic escape processes only resolve with the selection of mutations that conferred escape with no fitness cost to the virus. These results provide further understanding of the complicated viral-host interactions that occur during early HIV-1 infection and may help inform the design of future vaccine immunogens. PMID:26223634

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

PubMed Central

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

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 Zn2+-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. PMID:29892505

Systematic Analysis of Splice-Site-Creating Mutations in Cancer

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

Jayasinghe, Reyka G.; Cao, Song; Gao, Qingsong

For the past decade, cancer genomic studies have focused on mutations leading to splice-site disruption, overlooking those having splice-creating potential. Here, we applied a bioinformatic tool, MiSplice, for the large-scale discovery of splice-site-creating mutations (SCMs) across 8,656 TCGA tumors. We report 1,964 originally mis-annotated mutations having clear evidence of creating alternative splice junctions. TP53 and GATA3 have 26 and 18 SCMs, respectively, and ATRX has 5 from lower-grade gliomas. Mutations in 11 genes, including PARP1, BRCA1, and BAP1, were experimentally validated for splice-site-creating function. Notably, we found that neoantigens induced by SCMs are likely several folds more immunogenic compared tomore » missense mutations, exemplified by the recurrent GATA3 SCM. Further, high expression of PD-1 and PD-L1 was observed in tumors with SCMs, suggesting candidates for immune blockade therapy. Finally, our work highlights the importance of integrating DNA and RNA data for understanding the functional and the clinical implications of mutations in human diseases.« less

Systematic Analysis of Splice-Site-Creating Mutations in Cancer.

PubMed

Jayasinghe, Reyka G; Cao, Song; Gao, Qingsong; Wendl, Michael C; Vo, Nam Sy; Reynolds, Sheila M; Zhao, Yanyan; Climente-González, Héctor; Chai, Shengjie; Wang, Fang; Varghese, Rajees; Huang, Mo; Liang, Wen-Wei; Wyczalkowski, Matthew A; Sengupta, Sohini; Li, Zhi; Payne, Samuel H; Fenyö, David; Miner, Jeffrey H; Walter, Matthew J; Vincent, Benjamin; Eyras, Eduardo; Chen, Ken; Shmulevich, Ilya; Chen, Feng; Ding, Li

2018-04-03

For the past decade, cancer genomic studies have focused on mutations leading to splice-site disruption, overlooking those having splice-creating potential. Here, we applied a bioinformatic tool, MiSplice, for the large-scale discovery of splice-site-creating mutations (SCMs) across 8,656 TCGA tumors. We report 1,964 originally mis-annotated mutations having clear evidence of creating alternative splice junctions. TP53 and GATA3 have 26 and 18 SCMs, respectively, and ATRX has 5 from lower-grade gliomas. Mutations in 11 genes, including PARP1, BRCA1, and BAP1, were experimentally validated for splice-site-creating function. Notably, we found that neoantigens induced by SCMs are likely several folds more immunogenic compared to missense mutations, exemplified by the recurrent GATA3 SCM. Further, high expression of PD-1 and PD-L1 was observed in tumors with SCMs, suggesting candidates for immune blockade therapy. Our work highlights the importance of integrating DNA and RNA data for understanding the functional and the clinical implications of mutations in human diseases. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Systematic Analysis of Splice-Site-Creating Mutations in Cancer

DOE PAGES

Jayasinghe, Reyka G.; Cao, Song; Gao, Qingsong; ...

2018-04-05

For the past decade, cancer genomic studies have focused on mutations leading to splice-site disruption, overlooking those having splice-creating potential. Here, we applied a bioinformatic tool, MiSplice, for the large-scale discovery of splice-site-creating mutations (SCMs) across 8,656 TCGA tumors. We report 1,964 originally mis-annotated mutations having clear evidence of creating alternative splice junctions. TP53 and GATA3 have 26 and 18 SCMs, respectively, and ATRX has 5 from lower-grade gliomas. Mutations in 11 genes, including PARP1, BRCA1, and BAP1, were experimentally validated for splice-site-creating function. Notably, we found that neoantigens induced by SCMs are likely several folds more immunogenic compared tomore » missense mutations, exemplified by the recurrent GATA3 SCM. Further, high expression of PD-1 and PD-L1 was observed in tumors with SCMs, suggesting candidates for immune blockade therapy. Finally, our work highlights the importance of integrating DNA and RNA data for understanding the functional and the clinical implications of mutations in human diseases.« less

RAS mutations affect pattern of metastatic spread and increase propensity for brain metastasis in colorectal cancer.

PubMed

Yaeger, Rona; Cowell, Elizabeth; Chou, Joanne F; Gewirtz, Alexandra N; Borsu, Laetitia; Vakiani, Efsevia; Solit, David B; Rosen, Neal; Capanu, Marinela; Ladanyi, Marc; Kemeny, Nancy

2015-04-15

RAS and PIK3CA mutations in metastatic colorectal cancer (mCRC) have been associated with worse survival. We sought to evaluate the impact of RAS and PIK3CA mutations on cumulative incidence of metastasis to potentially curable sites of liver and lung and other sites such as bone and brain. We performed a computerized search of the electronic medical record of our institution for mCRC cases genotyped for RAS or PIK3CA mutations from 2008 to 2012. Cases were reviewed for patient characteristics, survival, and site-specific metastasis. Among the 918 patients identified, 477 cases were RAS wild type, and 441 cases had a RAS mutation (394 at KRAS exon 2, 29 at KRAS exon 3 or 4, and 18 in NRAS). RAS mutation was significantly associated with shorter median overall survival (OS) and on multivariate analysis independently predicted worse OS (HR, 1.6; P < .01). RAS mutant mCRC exhibited a significantly higher cumulative incidence of lung, bone, and brain metastasis and on multivariate analysis was an independent predictor of involvement of these sites (HR, 1.5, 1.6, and 3.7, respectively). PIK3CA mutations occurred in 10% of the 786 cases genotyped, did not predict for worse survival, and did not exhibit a site-specific pattern of metastatic spread. The metastatic potential of CRC varies with the presence of RAS mutation. RAS mutation is associated with worse OS and increased incidence of lung, bone, and brain metastasis. An understanding of this site-specific pattern of spread may help to inform physicians' assessment of symptoms in patients with mCRC. © 2014 American Cancer Society.

X-linked Alport syndrome caused by splicing mutations in COL4A5.

PubMed

Nozu, Kandai; Vorechovsky, Igor; Kaito, Hiroshi; Fu, Xue Jun; Nakanishi, Koichi; Hashimura, Yuya; Hashimoto, Fusako; Kamei, Koichi; Ito, Shuichi; Kaku, Yoshitsugu; Imasawa, Toshiyuki; Ushijima, Katsumi; Shimizu, Junya; Makita, Yoshio; Konomoto, Takao; Yoshikawa, Norishige; Iijima, Kazumoto

2014-11-07

X-linked Alport syndrome is caused by mutations in the COL4A5 gene. Although many COL4A5 mutations have been detected, the mutation detection rate has been unsatisfactory. Some men with X-linked Alport syndrome show a relatively mild phenotype, but molecular basis investigations have rarely been conducted to clarify the underlying mechanism. In total, 152 patients with X-linked Alport syndrome who were suspected of having Alport syndrome through clinical and pathologic investigations and referred to the hospital for mutational analysis between January of 2006 and January of 2013 were genetically diagnosed. Among those patients, 22 patients had suspected splice site mutations. Transcripts are routinely examined when suspected splice site mutations for abnormal transcripts are detected; 11 of them showed expected exon skipping, but others showed aberrant splicing patterns. The mutation detection strategy had two steps: (1) genomic DNA analysis using PCR and direct sequencing and (2) mRNA analysis using RT-PCR to detect RNA processing abnormalities. Six splicing consensus site mutations resulting in aberrant splicing patterns, one exonic mutation leading to exon skipping, and four deep intronic mutations producing cryptic splice site activation were identified. Interestingly, one case produced a cryptic splice site with a single nucleotide substitution in the deep intron that led to intronic exonization containing a stop codon; however, the patient showed a clearly milder phenotype for X-linked Alport syndrome in men with a truncating mutation. mRNA extracted from the kidney showed both normal and abnormal transcripts, with the normal transcript resulting in the milder phenotype. This novel mechanism leads to mild clinical characteristics. This report highlights the importance of analyzing transcripts to enhance the mutation detection rate and provides insight into genotype-phenotype correlations. This approach can clarify the cause of atypically mild phenotypes in X-linked Alport syndrome. Copyright © 2014 by the American Society of Nephrology.

Transcriptome Meta-Analysis of Lung Cancer Reveals Recurrent Aberrations in NRG1 and Hippo Pathway Genes

PubMed Central

Dhanasekaran, Saravana M.; Balbin, O. Alejandro; Chen, Guoan; Nadal, Ernest; Kalyana-Sundaram, Shanker; Pan, Jincheng; Veeneman, Brendan; Cao, Xuhong; Malik, Rohit; Vats, Pankaj; Wang, Rui; Huang, Stephanie; Zhong, Jinjie; Jing, Xiaojun; Iyer, Matthew; Wu, Yi-Mi; Harms, Paul W.; Lin, Jules; Reddy, Rishindra; Brennan, Christine; Palanisamy, Nallasivam; Chang, Andrew C.; Truini, Anna; Truini, Mauro; Robinson, Dan R.; Beer, David G.; Chinnaiyan, Arul M.

2014-01-01

Lung cancer is emerging as a paradigm for disease molecular subtyping, facilitating targeted therapy based on driving somatic alterations. Here, we perform transcriptome analysis of 153 samples representing lung adenocarcinomas, squamous cell carcinomas, large cell lung cancer, adenoid cystic carcinomas and cell lines. By integrating our data with The Cancer Genome Atlas and published sources, we analyze 753 lung cancer samples for gene fusions and other transcriptomic alterations. We show that higher numbers of gene fusions is an independent prognostic factor for poor survival in lung cancer. Our analysis confirms the recently reported CD74-NRG1 fusion and suggests that NRG1, NF1 and Hippo pathway fusions may play important roles in tumors without known driver mutations. In addition, we observe exon skipping events in c-MET, which are attributable to splice site mutations. These classes of genetic aberrations may play a significant role in the genesis of lung cancers lacking known driver mutations. PMID:25531467

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

PubMed Central

Creixell, Pau; Schoof, Erwin M.; Simpson, Craig D.; Longden, James; Miller, Chad J.; Lou, Hua Jane; Perryman, Lara; Cox, Thomas R.; Zivanovic, Nevena; Palmeri, Antonio; Wesolowska-Andersen, Agata; Helmer-Citterich, Manuela; Ferkinghoff-Borg, Jesper; Itamochi, Hiroaki; Bodenmiller, Bernd; Erler, Janine T.; Turk, Benjamin E.; Linding, Rune

2015-01-01

Summary Cancer cells acquire pathological phenotypes through accumulation of mutations that perturb signaling networks. However, global analysis of these events is currently limited. Here, we identify six types of network-attacking mutations (NAMs), including changes in kinase and SH2 modulation, network rewiring, and the genesis and extinction of phosphorylation sites. We developed a computational platform (ReKINect) to identify NAMs and systematically interpreted the exomes and quantitative (phospho-)proteomes of five ovarian cancer cell lines and the global cancer genome repository. We identified and experimentally validated several NAMs, including PKCγ M501I and PKD1 D665N, which encode specificity switches analogous to the appearance of kinases de novo within the kinome. We discover mutant molecular logic gates, a drift toward phospho-threonine signaling, weakening of phosphorylation motifs, and kinase-inactivating hotspots in cancer. Our method pinpoints functional NAMs, scales with the complexity of cancer genomes and cell signaling, and may enhance our capability to therapeutically target tumor-specific networks. PMID:26388441

Tracking the origins and drivers of subclonal metastatic expansion in prostate cancer

DOE PAGES

Hong, Matthew K. H.; Macintyre, Geoff; Wedge, David C.; ...

2015-04-01

Tumour heterogeneity in primary prostate cancer is a well-established phenomenon. However, how the subclonal diversity of tumours changes during metastasis and progression to lethality is poorly understood. Here we reveal the precise direction of metastatic spread across four lethal prostate cancer patients using whole-genome and ultra-deep targeted sequencing of longitudinally collected primary and metastatic tumours. We find one case of metastatic spread to the surgical bed causing local recurrence, and another case of cross-metastatic site seeding combining with dynamic remoulding of subclonal mixtures in response to therapy. By ultra-deep sequencing end-stage blood, we detect both metastatic and primary tumour clones,more » even years after removal of the prostate. As a result, analysis of mutations associated with metastasis reveals an enrichment of TP53 mutations, and additional sequencing of metastases from 19 patients demonstrates that acquisition of TP53 mutations is linked with the expansion of subclones with metastatic potential which we can detect in the blood.« less

Tracking the origins and drivers of subclonal metastatic expansion in prostate cancer.

PubMed

Hong, Matthew K H; Macintyre, Geoff; Wedge, David C; Van Loo, Peter; Patel, Keval; Lunke, Sebastian; Alexandrov, Ludmil B; Sloggett, Clare; Cmero, Marek; Marass, Francesco; Tsui, Dana; Mangiola, Stefano; Lonie, Andrew; Naeem, Haroon; Sapre, Nikhil; Phal, Pramit M; Kurganovs, Natalie; Chin, Xiaowen; Kerger, Michael; Warren, Anne Y; Neal, David; Gnanapragasam, Vincent; Rosenfeld, Nitzan; Pedersen, John S; Ryan, Andrew; Haviv, Izhak; Costello, Anthony J; Corcoran, Niall M; Hovens, Christopher M

2015-04-01

Tumour heterogeneity in primary prostate cancer is a well-established phenomenon. However, how the subclonal diversity of tumours changes during metastasis and progression to lethality is poorly understood. Here we reveal the precise direction of metastatic spread across four lethal prostate cancer patients using whole-genome and ultra-deep targeted sequencing of longitudinally collected primary and metastatic tumours. We find one case of metastatic spread to the surgical bed causing local recurrence, and another case of cross-metastatic site seeding combining with dynamic remoulding of subclonal mixtures in response to therapy. By ultra-deep sequencing end-stage blood, we detect both metastatic and primary tumour clones, even years after removal of the prostate. Analysis of mutations associated with metastasis reveals an enrichment of TP53 mutations, and additional sequencing of metastases from 19 patients demonstrates that acquisition of TP53 mutations is linked with the expansion of subclones with metastatic potential which we can detect in the blood.

Toward the treatment and prevention of Alzheimer's disease: rational strategies and recent progress.

PubMed

Gandy, Sam; DeKosky, Steven T

2013-01-01

Alzheimer's disease (AD) is the major cause of late-life brain failure. In the past 25 years, autosomal dominant forms of AD were found to be primariy attributable to mutations in one of two presenilins, polytopic proteins that contain the catalytic site of the γ-secretase protease that releases the amyloid beta (Aβ) peptide. Some familial AD is also due to mutations in the amyloid precursor protein (APP), but recently a mutation in APP was discovered that reduces Aβ generation and is protective against AD, further implicating amyloid metabolism. Prion-like seeding of amyloid fibrils and neurofibrillary tangles has been invoked to explain the stereotypical spread of AD within the brain. Treatment trials with anti-Aβ antibodies have shown target engagement, if not significant treatment effects. Attention is increasingly focused on presymptomatic intervention, because Aβ mismetabolism begins up to 25 years before symptoms begin. AD trials deriving from new biological information involve extraordinary international collaboration and may hold the best hope for success in the fight against AD.

Activation of Ftz-F1-Responsive Genes through Ftz/Ftz-F1 Dependent Enhancers

PubMed Central

Field, Amanda; Xiang, Jie; Anderson, W. Ray; Graham, Patricia; Pick, Leslie

2016-01-01

The orphan nuclear receptor Ftz-F1 is expressed in all somatic nuclei in Drosophila embryos, but mutations result in a pair-rule phenotype. This was explained by the interaction of Ftz-F1 with the homeodomain protein Ftz that is expressed in stripes in the primordia of segments missing in either ftz-f1 or ftz mutants. Ftz-F1 and Ftz were shown to physically interact and coordinately activate the expression of ftz itself and engrailed by synergistic binding to composite Ftz-F1/Ftz binding sites. However, attempts to identify additional target genes on the basis of Ftz-F1/ Ftz binding alone has met with only limited success. To discern rules for Ftz-F1 target site selection in vivo and to identify additional target genes, a microarray analysis was performed comparing wildtype and ftz-f1 mutant embryos. Ftz-F1-responsive genes most highly regulated included engrailed and nine additional genes expressed in patterns dependent on both ftz and ftz-f1. Candidate enhancers for these genes were identified by combining BDTNP Ftz ChIP-chip data with a computational search for Ftz-F1 binding sites. Of eight enhancer reporter genes tested in transgenic embryos, six generated expression patterns similar to the corresponding endogenous gene and expression was lost in ftz mutants. These studies identified a new set of Ftz-F1 targets, all of which are co-regulated by Ftz. Comparative analysis of enhancers containing Ftz/Ftz-F1 binding sites that were or were not bona fide targets in vivo suggested that GAF negatively regulates enhancers that contain Ftz/Ftz-F1 binding sites but are not actually utilized. These targets include other regulatory factors as well as genes involved directly in morphogenesis, providing insight into how pair-rule genes establish the body pattern. PMID:27723822

MicroRNA-339-5p down-regulates protein expression of β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) in human primary brain cultures and is reduced in brain tissue specimens of Alzheimer disease subjects.

PubMed

Long, Justin M; Ray, Balmiki; Lahiri, Debomoy K

2014-02-21

Alzheimer disease (AD) results, in part, from the excess accumulation of the amyloid-β (Aβ) peptide as neuritic plaques in the brain. The short Aβ peptide is derived from the large transmembrane Aβ precursor protein (APP). The rate-limiting step in the production of Aβ from APP is mediated by the β-site APP-cleaving enzyme 1 (BACE1). Dysregulation of BACE1 levels leading to excess Aβ deposition is implicated in sporadic AD. Thus, elucidating the full complement of regulatory pathways that control BACE1 expression is key to identifying novel drug targets central to the Aβ-generating process. MicroRNAs (miRNAs) are expected to participate in this molecular network. Here, we identified a known miRNA, miR-339-5p, as a key contributor to this regulatory network. Two distinct miR-339-5p target sites were predicted in the BACE1 3'-UTR by in silico analyses. Co-transfection of miR-339-5p with a BACE1 3'-UTR reporter construct resulted in significant reduction in reporter expression. Mutation of both target sites eliminated this effect. Delivery of the miR-339-5p mimic also significantly inhibited expression of BACE1 protein in human glioblastoma cells and human primary brain cultures. Delivery of target protectors designed against the miR-339-5p BACE1 3'-UTR target sites in primary human brain cultures significantly elevated BACE1 expression. Finally, miR-339-5p levels were found to be significantly reduced in brain specimens isolated from AD patients as compared with age-matched controls. Therefore, miR-339-5p regulates BACE1 expression in human brain cells and is most likely dysregulated in at least a subset of AD patients making this miRNA a novel drug target.

MicroRNA-339-5p Down-regulates Protein Expression of β-Site Amyloid Precursor Protein-Cleaving Enzyme 1 (BACE1) in Human Primary Brain Cultures and Is Reduced in Brain Tissue Specimens of Alzheimer Disease Subjects*

PubMed Central

Long, Justin M.; Ray, Balmiki; Lahiri, Debomoy K.

2014-01-01

Alzheimer disease (AD) results, in part, from the excess accumulation of the amyloid-β (Aβ) peptide as neuritic plaques in the brain. The short Aβ peptide is derived from the large transmembrane Aβ precursor protein (APP). The rate-limiting step in the production of Aβ from APP is mediated by the β-site APP-cleaving enzyme 1 (BACE1). Dysregulation of BACE1 levels leading to excess Aβ deposition is implicated in sporadic AD. Thus, elucidating the full complement of regulatory pathways that control BACE1 expression is key to identifying novel drug targets central to the Aβ-generating process. MicroRNAs (miRNAs) are expected to participate in this molecular network. Here, we identified a known miRNA, miR-339-5p, as a key contributor to this regulatory network. Two distinct miR-339-5p target sites were predicted in the BACE1 3′-UTR by in silico analyses. Co-transfection of miR-339-5p with a BACE1 3′-UTR reporter construct resulted in significant reduction in reporter expression. Mutation of both target sites eliminated this effect. Delivery of the miR-339-5p mimic also significantly inhibited expression of BACE1 protein in human glioblastoma cells and human primary brain cultures. Delivery of target protectors designed against the miR-339-5p BACE1 3′-UTR target sites in primary human brain cultures significantly elevated BACE1 expression. Finally, miR-339-5p levels were found to be significantly reduced in brain specimens isolated from AD patients as compared with age-matched controls. Therefore, miR-339-5p regulates BACE1 expression in human brain cells and is most likely dysregulated in at least a subset of AD patients making this miRNA a novel drug target. PMID:24352696

HLA-B27 Selects for Rare Escape Mutations that Significantly Impair Hepatitis C Virus Replication and Require Compensatory Mutations

PubMed Central

Neumann-Haefelin, Christoph; Oniangue-Ndza, Cesar; Kuntzen, Thomas; Schmidt, Julia; Nitschke, Katja; Sidney, John; Caillet-Saguy, Célia; Binder, Marco; Kersting, Nadine; Kemper, Michael W.; Power, Karen A.; Ingber, Susan; Reyor, Laura L.; Hills-Evans, Kelsey; Kim, Arthur Y.; Lauer, Georg M.; Lohmann, Volker; Sette, Alessandro; Henn, Matthew R.; Bressanelli, Stéphane; Thimme, Robert; Allen, Todd M.

2011-01-01

HLA-B27 is associated with spontaneous viral clearance in hepatitis C virus (HCV) infection. Viral escape within the immunodominant HLA-B27 restricted HCV-specific CD8+ T cell epitope NS5B2841-2849 (ARMILMTHF) has been shown to be limited by viral fitness costs as well as broad T cell cross-recognition, suggesting a potential mechanism of protection by HLA-B27. Here, we studied the subdominant HLA-B27 restricted epitope NS5B2936-2944 (GRAAICGKY) in order to further define the mechanisms of protection by HLA-B27. We identified a unique pattern of escape mutations within this epitope in a large cohort of HCV genotype 1a infected patients. The predominant escape mutations represented conservative substitutions at the main HLA-B27 anchor residue or a T cell receptor contact site, neither of which impaired viral replication capacity as assessed in a subgenomic HCV replicon system. In contrast, however, in a subset of HLA-B27+ subjects rare escape mutations arose at the HLA-B27 anchor residue R2937, which nearly abolished viral replication. Notably, these rare mutations only occurred in conjunction with the selection of two equally rare, and structurally proximal, upstream mutations. Co-expression of these upstream mutations with the rare escape mutations dramatically restored viral replication capacity from <5% to ≥70% of wild-type levels. Conclusion The selection of rare CTL escape mutations in this HLA-B27 restricted epitope dramatically impairs viral replicative fitness unless properly compensated. These data support a role for the targeting of highly-constrained regions by HLA-B27 in its ability to assert immune control of HCV and other highly variable pathogens. PMID:22006856

Small Cell Lung Cancer Exhibits Frequent Inactivating Mutations in the Histone Methyltransferase KMT2D/MLL2: CALGB 151111 (Alliance)

PubMed Central

Augert, Arnaud; Zhang, Qing; Bates, Breanna; Cui, Min; Wang, Xiaofei; Wildey, Gary; Dowlati, Afshin; MacPherson, David

2017-01-01

Introduction SCLC is a lethal neuroendocrine tumor type that is highly prone to metastasis. There is an urgency to understand the mutated genes that promote SCLC, as there are no approved targeted therapies yet available. SCLC is rarely resected, limiting the number of samples available for genomic analyses of somatic mutations. Methods To identify potential driver mutations in human SCLC we sequenced the whole exomes of 18 primary SCLCs and seven cell lines along with matched normal controls. We extended these data by resequencing a panel of genes across 40 primary SCLCs and 48 cell lines. Results We report frequent mutations in the lysine methyltransferase 2D gene (KMT2D) (also known as MLL2), a key regulator of transcriptional enhancer function. KMT2D exhibited truncating nonsense/frameshift/splice site mutations in 8% of SCLC tumors and 17% of SCLC cell lines. We found that KMT2D mutation in human SCLC cell lines was associated with reduced lysine methyltransferase 2D protein levels and reduced monomethylation of histone H3 lysine 4, a mark associated with transcriptional enhancers. We also found mutations in other genes associated with transcriptional enhancer control, including CREB binding protein gene (CREBBP), E1A binding protein p300 gene (EP300), and chromodomain helicase DNA binding protein 7 gene (CHD7), and we report mutations in additional chromatin remodeling genes such as polybromo 1 gene (PBRM1). Conclusions These data indicate that KMT2D is one of the major mutated genes in SCLC, and they point to perturbation of transcriptional enhancer control as potentially contributing to SCLC. PMID:28007623

Rapid development of stable transgene CHO cell lines by CRISPR/Cas9-mediated site-specific integration into C12orf35.

PubMed

Zhao, Menglin; Wang, Jiaxian; Luo, Manyu; Luo, Han; Zhao, Meiqi; Han, Lei; Zhang, Mengxiao; Yang, Hui; Xie, Yueqing; Jiang, Hua; Feng, Lei; Lu, Huili; Zhu, Jianwei

2018-07-01

Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for recombinant protein production. However, by conventional random integration strategy, development of a high-expressing and stable recombinant CHO cell line has always been a difficult task due to the heterogenic insertion and its caused requirement of multiple rounds of selection. Site-specific integration of transgenes into CHO hot spots is an ideal strategy to overcome these challenges since it can generate isogenic cell lines with consistent productivity and stability. In this study, we investigated three sites with potential high transcriptional activities: C12orf35, HPRT, and GRIK1, to determine the possible transcriptional hot spots in CHO cells, and further construct a reliable site-specific integration strategy to develop recombinant cell lines efficiently. Genes encoding representative proteins mCherry and anti-PD1 monoclonal antibody were targeted into these three loci respectively through CRISPR/Cas9 technology. Stable cell lines were generated successfully after a single round of selection. In comparison with a random integration control, all the targeted integration cell lines showed higher productivity, among which C12orf35 locus was the most advantageous in both productivity and cell line stability. Binding affinity and N-glycan analysis of the antibody revealed that all batches of product were of similar quality independent on integrated sites. Deep sequencing demonstrated that there was low level of off-target mutations caused by CRISPR/Cas9, but none of them contributed to the development process of transgene cell lines. Our results demonstrated the feasibility of C12orf35 as the target site for exogenous gene integration, and strongly suggested that C12orf35 targeted integration mediated by CRISPR/Cas9 is a reliable strategy for the rapid development of recombinant CHO cell lines.

Target Site Recognition by a Diversity-Generating Retroelement

PubMed Central

Guo, Huatao; Tse, Longping V.; Nieh, Angela W.; Czornyj, Elizabeth; Williams, Steven; Oukil, Sabrina; Liu, Vincent B.; Miller, Jeff F.

2011-01-01

Diversity-generating retroelements (DGRs) are in vivo sequence diversification machines that are widely distributed in bacterial, phage, and plasmid genomes. They function to introduce vast amounts of targeted diversity into protein-encoding DNA sequences via mutagenic homing. Adenine residues are converted to random nucleotides in a retrotransposition process from a donor template repeat (TR) to a recipient variable repeat (VR). Using the Bordetella bacteriophage BPP-1 element as a prototype, we have characterized requirements for DGR target site function. Although sequences upstream of VR are dispensable, a 24 bp sequence immediately downstream of VR, which contains short inverted repeats, is required for efficient retrohoming. The inverted repeats form a hairpin or cruciform structure and mutational analysis demonstrated that, while the structure of the stem is important, its sequence can vary. In contrast, the loop has a sequence-dependent function. Structure-specific nuclease digestion confirmed the existence of a DNA hairpin/cruciform, and marker coconversion assays demonstrated that it influences the efficiency, but not the site of cDNA integration. Comparisons with other phage DGRs suggested that similar structures are a conserved feature of target sequences. Using a kanamycin resistance determinant as a reporter, we found that transplantation of the IMH and hairpin/cruciform-forming region was sufficient to target the DGR diversification machinery to a heterologous gene. In addition to furthering our understanding of DGR retrohoming, our results suggest that DGRs may provide unique tools for directed protein evolution via in vivo DNA diversification. PMID:22194701

Doubling the referral rate of monogenic diabetes through a nationwide information campaign--update on glucokinase gene mutations in a Polish cohort.

PubMed

Borowiec, M; Fendler, W; Antosik, K; Baranowska, A; Gnys, P; Zmyslowska, A; Malecki, M; Mlynarski, W

2012-12-01

In order to improve recruitment efficiency of patients with monogenic diabetes in Poland, in September 2010 a nationwide advertising campaign was launched to inform multiple target groups interested or participating in pediatric diabetologic care. Promotional actions aimed at informing physicians, patients, parents and educators were carried out through nationwide newspapers, medical and patient-developed websites and educational conference presentations. Recruitment efficiency was compared between September 2010 (publication of the first report on project's results) and the following 12 months. The number of families and patients referred to genetic screening was increased by 92% and 96% respectively nearly reaching the numbers recruited throughout the initial 4 years of the project. Participation of non-academic centers was also significantly increased from 2.3% to 7.5% (p = 0.0005). DNA sequencing and Multiplex Ligation-dependant Probe Amplification of the glucokinase gene resulted in finding 50 different mutations. Among those mutations, 19 were novel variants, which included: 17 missense mutations (predicted to be pathogenic according to bioinformatic analysis), 1 nonsense mutation and 1 mutation affecting a consensus intronic splice site. Advertising actions directed at increasing recruitment efficiency are a powerful and possibly neglected tool in screening for rare genetic disorders with a clinically defined phenotype. © 2011 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

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

PubMed Central

Mancini, Irene; Lampronti, Ilaria; Salvatori, Francesca; Fabbri, Enrica; Zuccato, Cristina; Cosenza, Lucia C.; Montagner, Giulia; Borgatti, Monica; Altruda, Fiorella; Fagoonee, Sharmila; Carandina, Gianni; Aiello, Vincenzo; Breda, Laura; Rivella, Stefano; Gambari, Roberto

2015-01-01

Mouse models that carry mutations causing thalassemia represent a suitable tool to test in vivo new mutation-specific therapeutic approaches. Transgenic mice carrying the β-globin IVSI-6 mutation (the most frequent in Middle-Eastern regions and recurrent in Italy and Greece) are, at present, not available. We report the production and characterization of a transgenic mouse line (TG-β-IVSI-6) carrying the IVSI-6 thalassemia point mutation within the human β-globin gene. In the TG-β-IVSI-6 mouse (a) the transgenic integration region is located in mouse chromosome 7; (b) the expression of the transgene is tissue specific; (c) as expected, normally spliced human β-globin mRNA is produced, giving rise to β-globin production and formation of a human-mouse tetrameric chimeric hemoglobin mu α-globin2/hu β-globin2 and, more importantly, (d) the aberrant β-globin-IVSI-6 RNAs are present in blood cells. The TG-β-IVSI-6 mouse reproduces the molecular features of IVSI-6 β-thalassemia and might be used as an in vivo model to characterize the effects of antisense oligodeoxynucleotides targeting the cryptic sites responsible for the generation of aberrantly spliced β-globin RNA sequences, caused by the IVSI-6 mutation. These experiments are expected to be crucial for the development of a personalized therapy for β-thalassemia. PMID:26097845

A Recessive Mutation Resulting in a Disabling Amino Acid Substitution (T194R) in the LHX3 Homeodomain Causes Combined Pituitary Hormone Deficiency

PubMed Central

Bechtold-Dalla Pozza, Susanne; Hiedl, Stefan; Roeb, Julia; Lohse, Peter; Malik, Raleigh E.; Park, Soyoung; Durán-Prado, Mario; Rhodes, Simon J.

2012-01-01

Background/Aims Recessive mutations in the LHX3 ho-meodomain transcription factor gene are associated with developmental disorders affecting the pituitary and nervous system. We describe pediatric patients with combined pituitary hormone deficiency (CPHD) who harbor a novel mutation in LHX3. Methods Two female siblings from related parents were examined. Both patients had neonatal complications. The index patient had CPHD featuring deficiencies of GH, LH, FSH, PRL, and TSH, with later onset of ACTH deficiency. She also had a hypoplastic anterior pituitary, respiratory distress, hearing impairment, and limited neck rotation. The LHX3 gene was sequenced and the biochemical properties of the predicted altered proteins were characterized. Results A novel homozygous mutation predicted to change amino acid 194 from threonine to arginine (T194R) was detected in both patients. This amino acid is conserved in the DNA-binding homeodomain. Computer modeling predicted that the T194R change would alter the homeodomain structure. The T194R protein did not bind tested LHX3 DNA recognition sites and did not activate the α-glycoprotein and PRL target genes. Conclusion The T194R mutation affects a critical residue in the LHX3 protein. This study extends our understanding of the phenotypic features, molecular mechanism, and developmental course associated with mutations in the LHX3 gene. PMID:22286346

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

PubMed Central

Pritchard, Jonathan K.

2016-01-01

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

Antigenic Drift Defines a New D4 Subgenotype of Measles Virus.

PubMed

Muñoz-Alía, Miguel Ángel; Muller, Claude P; Russell, Stephen J

2017-06-01

The measles virus hemagglutinin (MeV-H) protein is the main target of protective neutralizing antibodies. Using a panel of monoclonal antibodies (MAbs) that recognize known major antigenic sites in MeV-H, we identified a D4 genotype variant that escapes neutralization by MAbs targeting the neutralizing epitope (NE) antigenic site. By site-directed mutagenesis, L249P was identified as the critical mutation disrupting the NE in this genotype D4 variant. Forty-two available D4 genotype gene sequences were subsequently analyzed and divided into 2 groups according to the presence or absence of the L249P MeV-H mutation. Further analysis of the MeV-N gene sequences of these 2 groups confirmed that they represent clearly definable, sequence-divergent D4 subgenotypes, which we named subgenotypes D4.1 and D4.2. The subgenotype D4.1 MeVs were isolated predominantly in Kenya and Ethiopia, whereas the MAb-resistant subgenotype D4.2 MeVs were isolated predominantly in France and Great Britain, countries with higher vaccine coverage rates. Interestingly, D4.2 subgenotype viruses showed a trend toward diminished susceptibility to neutralization by human sera pooled from approximately 60 to 80 North American donors. Escape from MAb neutralization may be a powerful epidemiological surveillance tool to monitor the evolution of new MeV subgenotypes. IMPORTANCE Measles virus is a paradigmatic RNA virus, as the antigenic composition of the vaccination has not needed to be updated since its discovery. The vaccine confers protection by inducing neutralizing antibodies that interfere with the function of the hemagglutinin protein. Viral strains are indistinguishable serologically, although characteristic nucleotide sequences differentiate 24 genotypes. In this work, we describe a distant evolutionary branch within genotype D4. Designated subgenotype D4.2, this virus is distinguishable by neutralization with vaccine-induced monoclonal antibodies that target the neutralizing epitope (NE). The subgenotype D4.2 viruses have a higher predominance in countries with intermediary levels of vaccine coverage. Our studies demonstrate that subgenotype D4.2 lacks epitopes associated with half of the known antigenic sites, which significantly impacts our understanding of measles virus evolution. Copyright © 2017 American Society for Microbiology.

Antigenic Drift Defines a New D4 Subgenotype of Measles Virus

PubMed Central

Muller, Claude P.

2017-01-01

ABSTRACT The measles virus hemagglutinin (MeV-H) protein is the main target of protective neutralizing antibodies. Using a panel of monoclonal antibodies (MAbs) that recognize known major antigenic sites in MeV-H, we identified a D4 genotype variant that escapes neutralization by MAbs targeting the neutralizing epitope (NE) antigenic site. By site-directed mutagenesis, L249P was identified as the critical mutation disrupting the NE in this genotype D4 variant. Forty-two available D4 genotype gene sequences were subsequently analyzed and divided into 2 groups according to the presence or absence of the L249P MeV-H mutation. Further analysis of the MeV-N gene sequences of these 2 groups confirmed that they represent clearly definable, sequence-divergent D4 subgenotypes, which we named subgenotypes D4.1 and D4.2. The subgenotype D4.1 MeVs were isolated predominantly in Kenya and Ethiopia, whereas the MAb-resistant subgenotype D4.2 MeVs were isolated predominantly in France and Great Britain, countries with higher vaccine coverage rates. Interestingly, D4.2 subgenotype viruses showed a trend toward diminished susceptibility to neutralization by human sera pooled from approximately 60 to 80 North American donors. Escape from MAb neutralization may be a powerful epidemiological surveillance tool to monitor the evolution of new MeV subgenotypes. IMPORTANCE Measles virus is a paradigmatic RNA virus, as the antigenic composition of the vaccination has not needed to be updated since its discovery. The vaccine confers protection by inducing neutralizing antibodies that interfere with the function of the hemagglutinin protein. Viral strains are indistinguishable serologically, although characteristic nucleotide sequences differentiate 24 genotypes. In this work, we describe a distant evolutionary branch within genotype D4. Designated subgenotype D4.2, this virus is distinguishable by neutralization with vaccine-induced monoclonal antibodies that target the neutralizing epitope (NE). The subgenotype D4.2 viruses have a higher predominance in countries with intermediary levels of vaccine coverage. Our studies demonstrate that subgenotype D4.2 lacks epitopes associated with half of the known antigenic sites, which significantly impacts our understanding of measles virus evolution. PMID:28356529

A novel high-throughput (HTP) cloning strategy for site-directed designed chimeragenesis and mutation using the Gateway cloning system

PubMed Central

Suzuki, Yasuhiro; Kagawa, Naoko; Fujino, Toru; Sumiya, Tsuyoshi; Andoh, Taichi; Ishikawa, Kumiko; Kimura, Rie; Kemmochi, Kiyokazu; Ohta, Tsutomu; Tanaka, Shigeo

2005-01-01

There is an increasing demand for easy, high-throughput (HTP) methods for protein engineering to support advances in the development of structural biology, bioinformatics and drug design. Here, we describe an N- and C-terminal cloning method utilizing Gateway cloning technology that we have adopted for chimeric and mutant genes production as well as domain shuffling. This method involves only three steps: PCR, in vitro recombination and transformation. All three processes consist of simple handling, mixing and incubation steps. We have characterized this novel HTP method on 96 targets with >90% success. Here, we also discuss an N- and C-terminal cloning method for domain shuffling and a combination of mutation and chimeragenesis with two types of plasmid vectors. PMID:16009811

Next-Generation EGFR Tyrosine Kinase Inhibitors for Treating EGFR-Mutant Lung Cancer beyond First Line

PubMed Central

Sullivan, Ivana; Planchard, David

2017-01-01

Tyrosine kinase inhibitors (TKIs) against the human epidermal growth factor receptor (EGFR) are now standard treatment in the clinic for patients with advanced EGFR mutant non-small-cell lung cancer (NSCLC). First-generation EGFR TKIs, binding competitively and reversibly to the ATP-binding site of the EGFR tyrosine kinase domain, have resulted in a significant improvement in outcome for NSCLC patients with activating EGFR mutations (L858R and Del19). However, after a median duration of response of ~12 months, all patients develop tumor resistance, and in over half of these patients this is due to the emergence of the EGFR T790M resistance mutation. The second-generation EGFR/HER TKIs were developed to treat resistant disease, targeting not only T790M but EGFR-activating mutations and wild-type EGFR. Although they exhibited promising anti-T790M activity in the laboratory, their clinical activity among T790M+ NSCLC was poor mainly because of dose-limiting toxicity due to simultaneous inhibition of wild-type EGFR. The third-generation EGFR TKIs selectively and irreversibly target EGFR T790M and activating EGFR mutations, showing promising efficacy in NSCLC resistant to the first- and second-generation EGFR TKIs. They also appear to have lower incidences of toxicity due to the limited inhibitory effect on wild-type EGFR. Currently, the first-generation gefitinib and erlotinib and second-generation afatinib have been approved for first-line treatment of metastatic NSCLC with activating EGFR mutations. Among the third-generation EGFR TKIs, osimertinib is today the only drug approved by the Food and Drug Administration and the European Medicines Agency to treat metastatic EGFR T790M NSCLC patients who have progressed on or after EGFR TKI therapy. In this review, we summarize the available post-progression therapies including third-generation EGFR inhibitors and combination treatment strategies for treating patients with NSCLC harboring EGFR mutations and address the known mechanisms of resistance. PMID:28149837

Oncometabolic mutation IDH1 R132H confers a metformin-hypersensitive phenotype.

PubMed

Cuyàs, Elisabet; Fernández-Arroyo, Salvador; Corominas-Faja, Bruna; Rodríguez-Gallego, Esther; Bosch-Barrera, Joaquim; Martin-Castillo, Begoña; De Llorens, Rafael; Joven, Jorge; Menendez, Javier A

2015-05-20

Metabolic flexibility might be particularly constrained in tumors bearing mutations in isocitrate dehydrogenase 1 (IDH1) leading to the production of the oncometabolite 2-hydroxygluratate (2HG). To test the hypothesis that IDH1 mutations could generate metabolic vulnerabilities for therapeutic intervention, we utilized an MCF10A cell line engineered with an arginine-to-histidine conversion at position 132 (R132H) in the catalytic site of IDH1, which equips the enzyme with a neomorphic α-ketoglutarate to 2HG reducing activity in an otherwise isogenic background. IDH1 R132H/+ and isogenic IDH1 +/+ parental cells were screened for their ability to generate energy-rich NADH when cultured in a standardized high-throughput Phenotype MicroArrayplatform comprising >300 nutrients. A radical remodeling of the metabotype occurred in cells carrying the R132H mutation since they presented a markedly altered ability to utilize numerous carbon catabolic fuels. A mitochondria toxicity-screening modality confirmed a severe inability of IDH1-mutated cells to use various carbon substrates that are fed into the electron transport chain at different points. The mitochondrial biguanide poisons, metformin and phenformin, further impaired the intrinsic weakness of IDH1-mutant cells to use certain carbon-energy sources. Additionally, metabolic reprogramming of IDH1-mutant cells increased their sensitivity to metformin in assays of cell proliferation, clonogenic potential, and mammosphere formation. Targeted metabolomics studies revealed that the ability of metformin to interfere with the anaplerotic entry of glutamine into the tricarboxylic acid cycle could explain the hypersensitivity of IDH1-mutant cells to biguanides. Moreover, synergistic interactions occurred when metformin treatment was combined with the selective R132H-IDH1 inhibitor AGI-5198. Together, these results suggest that therapy involving the simultaneous targeting of metabolic vulnerabilities with metformin, and 2HG overproduction with mutant-selective inhibitors (AGI-5198-related AG-120 [Agios]), might represent a worthwhile avenue of exploration in the treatment of IDH1-mutated tumors.

Oncometabolic mutation IDH1 R132H confers a metformin-hypersensitive phenotype

PubMed Central

Cuyàs, Elisabet; Fernández-Arroyo, Salvador; Corominas-Faja, Bruna; Rodríguez-Gallego, Esther; Bosch-Barrera, Joaquim; Martin-Castillo, Begoña; De Llorens, Rafael; Joven, Jorge; Menendez, Javier A.

2015-01-01

Metabolic flexibility might be particularly constrained in tumors bearing mutations in isocitrate dehydrogenase 1 (IDH1) leading to the production of the oncometabolite 2-hydroxygluratate (2HG). To test the hypothesis that IDH1 mutations could generate metabolic vulnerabilities for therapeutic intervention, we utilized an MCF10A cell line engineered with an arginine-to-histidine conversion at position 132 (R132H) in the catalytic site of IDH1, which equips the enzyme with a neomorphic α-ketoglutarate to 2HG reducing activity in an otherwise isogenic background. IDH1 R132H/+ and isogenic IDH1 +/+ parental cells were screened for their ability to generate energy-rich NADH when cultured in a standardized high-throughput Phenotype MicroArrayplatform comprising >300 nutrients. A radical remodeling of the metabotype occurred in cells carrying the R132H mutation since they presented a markedly altered ability to utilize numerous carbon catabolic fuels. A mitochondria toxicity-screening modality confirmed a severe inability of IDH1-mutated cells to use various carbon substrates that are fed into the electron transport chain at different points. The mitochondrial biguanide poisons, metformin and phenformin, further impaired the intrinsic weakness of IDH1-mutant cells to use certain carbon-energy sources. Additionally, metabolic reprogramming of IDH1-mutant cells increased their sensitivity to metformin in assays of cell proliferation, clonogenic potential, and mammosphere formation. Targeted metabolomics studies revealed that the ability of metformin to interfere with the anaplerotic entry of glutamine into the tricarboxylic acid cycle could explain the hypersensitivity of IDH1-mutant cells to biguanides. Moreover, synergistic interactions occurred when metformin treatment was combined with the selective R132H-IDH1 inhibitor AGI-5198. Together, these results suggest that therapy involving the simultaneous targeting of metabolic vulnerabilities with metformin, and 2HG overproduction with mutant-selective inhibitors (AGI-5198-related AG-120 [Agios]), might represent a worthwhile avenue of exploration in the treatment of IDH1-mutated tumors. PMID:25980580