Evidence that the recA441 (tif-1) mutant of Escherichia coli K-12 contains a thermosensitive intragenic suppressor of RecA constitutive protease activity.

PubMed Central

Wang, W B; Tessman, E S

1985-01-01

The recA441 mutant of Escherichia coli, which has been thought to have thermoinducible constitutive RecA protease activity, is known to have two mutations within recA. We show here that the mutation that alters codon 38 actually confers temperature-independent constitutive protease activity; the second mutation in recA441, which is at codon 298, appears to be acting as a temperature-sensitive suppressor of the protease activity. Images PMID:3891740

Evidence that the recA441 (tif-1) mutant of Escherichia coli K-12 contains a thermosensitive intragenic suppressor of RecA constitutive protease activity.

PubMed

Wang, W B; Tessman, E S

1985-07-01

The recA441 mutant of Escherichia coli, which has been thought to have thermoinducible constitutive RecA protease activity, is known to have two mutations within recA. We show here that the mutation that alters codon 38 actually confers temperature-independent constitutive protease activity; the second mutation in recA441, which is at codon 298, appears to be acting as a temperature-sensitive suppressor of the protease activity.

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

PubMed Central

Nishiyama, Shoko; Ikegami, Tetsuro

2015-01-01

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to the African continent. RVF is characterized by high rate of abortions in ruminants and hemorrhagic fever, encephalitis, or blindness in humans. RVF is caused by the Rift Valley fever virus (RVFV: genus Phlebovirus, family Bunyaviridae). Vaccination is the only known effective strategy to prevent the disease, but there are no licensed RVF vaccines available for humans. A live-attenuated vaccine candidate derived from the wild-type pathogenic Egyptian ZH548 strain, MP-12, has been conditionally licensed for veterinary use in the U.S. MP-12 displays a temperature-sensitive (ts) phenotype and does not replicate at 41°C. The ts mutation limits viral replication at a specific body temperature and may lead to an attenuation of the virus. Here we will review well-characterized ts mutations for RNA viruses, and further discuss the potential in designing novel live-attenuated vaccines for RVF. PMID:26322023

A temperature-sensitive mutation in the nodal-related gene cyclops reveals that the floor plate is induced during gastrulation in zebrafish.

PubMed

Tian, Jing; Yam, Caleb; Balasundaram, Gayathri; Wang, Hui; Gore, Aniket; Sampath, Karuna

2003-07-01

The floor plate, a specialized group of cells in the ventral midline of the neural tube of vertebrates, plays crucial roles in patterning the central nervous system. Recent work from zebrafish, chick, chick-quail chimeras and mice to investigate the development of the floor plate have led to several models of floor-plate induction. One model suggests that the floor plate is formed by inductive signalling from the notochord to the overlying neural tube. The induction is thought to be mediated by notochord-derived Sonic hedgehog (Shh), a secreted protein, and requires direct cellular contact between the notochord and the neural tube. Another model proposes a role for the organizer in generating midline precursor cells that produce floor plate cells independent of notochord specification, and proposes that floor plate specification occurs early, during gastrulation. We describe a temperature-sensitive mutation that affects the zebrafish Nodal-related secreted signalling factor, Cyclops, and use it to address the issue of when the floor plate is induced in zebrafish. Zebrafish cyclops regulates the expression of shh in the ventral neural tube. Although null mutations in cyclops result in the lack of the medial floor plate, embryos homozygous for the temperature-sensitive mutation have floor plate cells at the permissive temperature and lack floor plate cells at the restrictive temperature. We use this mutant allele in temperature shift-up and shift-down experiments to answer a central question pertaining to the timing of vertebrate floor plate induction. Abrogation of Cyc/Nodal signalling in the temperature-sensitive mutant embryos at various stages indicates that the floor plate in zebrafish is induced early in development, during gastrulation. In addition, continuous Cyclops signalling is required through gastrulation for a complete ventral neural tube throughout the length of the neuraxis. Finally, by modulation of Nodal signalling levels in mutants and in ectopic overexpression experiments, we show that, similar to the requirements for prechordal plate mesendoderm fates, uninterrupted and high levels of Cyclops signalling are required for induction and specification of a complete ventral neural tube.

A mutational analysis of the yeast proliferating cell nuclear antigen indicates distinct roles in DNA replication and DNA repair.

PubMed Central

Ayyagari, R; Impellizzeri, K J; Yoder, B L; Gary, S L; Burgers, P M

1995-01-01

The saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA), encoded by the POL30 gene, is essential for DNA replication and DNA repair processes. Twenty-one site-directed mutations were constructed in the POL30 gene, each mutation changing two adjacently located charged amino acids to alanines. Although none of the mutant strains containing these double-alanine mutations as the sole source of PCNA were temperature sensitive or cold sensitive for growth, about a third of the mutants showed sensitivity to UV light. Some of those UV-sensitive mutants had elevated spontaneous mutation rates. In addition, several mutants suppressed a cold-sensitive mutation in the CDC44 gene, which encodes the large subunit of replication factor C. A cold-sensitive mutant, which was isolated by random mutagenesis, showed a terminal phenotype at the restrictive temperature consistent with a defect in DNA replication. Several mutant PCNAs were expressed and purified from Escherichia coli, and their in vitro properties were determined. The cold-sensitive mutant (pol30-52, S115P) was a monomer, rather than a trimer, in solution. This mutant was deficient for DNA synthesis in vitro. Partial restoration of DNA polymerase delta holoenzyme activity was achieved at 37 degrees C but not at 14 degrees C by inclusion of the macromolecular crowding agent polyethylene glycol in the assay. The only other mutant (pol30-6, DD41,42AA) that showed a growth defect was partially defective for interaction with replication factor C and DNA polymerase delta but completely defective for interaction with DNA polymerase epsilon. Two other mutants sensitive to DNA damage showed no defect in vitro. These results indicate that the latter mutants are specifically impaired in one or more DNA repair processes whereas pol30-6 and pol30-52 mutants show their primary defects in the basic DNA replication machinery with probable associated defects in DNA repair. Therefore, DNA repair requires interactions between repair-specific protein(s) and PCNA, which are distinct from those required for DNA replication. PMID:7623835

Arabidopsis HSP90 protein modulates RPP4-mediated temperature-dependent cell death and defense responses.

PubMed

Bao, Fei; Huang, Xiaozhen; Zhu, Chipan; Zhang, Xiaoyan; Li, Xin; Yang, Shuhua

2014-06-01

Plant defense responses are regulated by temperature. In Arabidopsis, the chilling-sensitive mutant chs2-1 (rpp4-1d) contains a gain-of-function mutation in the TIR-NB-LRR (Toll and interleukin 1 receptor-nucleotide binding-leucine-rich repeat) gene, RPP4 (RECOGNITION OF PERONOSPORA PARASITICA 4), which leads to constitutive activation of the defense response at low temperatures. Here, we identified and characterized two suppressors of rpp4-1d from a genetic screen, hsp90.2 and hsp90.3, which carry point mutations in the cytosolic heat shock proteins HSP90.2 and HSP90.3, respectively. The hsp90 mutants suppressed the chilling sensitivity of rpp4-1d, including seedling lethality, activation of the defense responses and cell death under chilling stress. The hsp90 mutants exhibited compromised RPM1 (RESISTANCE TO PSEUDOMONAS MACULICOLA 1)-, RPS4 (RESISTANCE TO P. SYRINGAE 4)- and RPP4-mediated pathogen resistance. The wild-type RPP4 and the mutated form rpp4 could interact with HSP90 to form a protein complex. Furthermore, RPP4 and rpp4 proteins accumulated in the cytoplasm and nucleus at normal temperatures, whereas the nuclear accumulation of the mutated rpp4 was decreased at low temperatures. Genetic analysis of the intragenic suppressors of rpp4-1d revealed the important functions of the NB-ARC and LRR domains of RPP4 in temperature-dependent defense signaling. In addition, the rpp4-1d-induced chilling sensitivity was largely independent of the WRKY70 or MOS (modifier of snc1) genes. [Correction added after online publication 11 March 2013: the expansions of TIR-NB-LRR and RPS4 were amended] This study reveals that Arabidopsis HSP90 regulates RPP4-mediated temperature-dependent cell death and defense responses. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

Genetic stability of genome-scale deoptimized RNA virus vaccine candidates under selective pressure

PubMed Central

Le Nouën, Cyril; McCarty, Thomas; Brown, Michael; Smith, Melissa Laird; Lleras, Roberto; Dolan, Michael A.; Mehedi, Masfique; Yang, Lijuan; Luongo, Cindy; Liang, Bo; Munir, Shirin; DiNapoli, Joshua M.; Mueller, Steffen; Wimmer, Eckard; Collins, Peter L.; Buchholz, Ursula J.

2017-01-01

Recoding viral genomes by numerous synonymous but suboptimal substitutions provides live attenuated vaccine candidates. These vaccine candidates should have a low risk of deattenuation because of the many changes involved. However, their genetic stability under selective pressure is largely unknown. We evaluated phenotypic reversion of deoptimized human respiratory syncytial virus (RSV) vaccine candidates in the context of strong selective pressure. Codon pair deoptimized (CPD) versions of RSV were attenuated and temperature-sensitive. During serial passage at progressively increasing temperature, a CPD RSV containing 2,692 synonymous mutations in 9 of 11 ORFs did not lose temperature sensitivity, remained genetically stable, and was restricted at temperatures of 34 °C/35 °C and above. However, a CPD RSV containing 1,378 synonymous mutations solely in the polymerase L ORF quickly lost substantial attenuation. Comprehensive sequence analysis of virus populations identified many different potentially deattenuating mutations in the L ORF as well as, surprisingly, many appearing in other ORFs. Phenotypic analysis revealed that either of two competing mutations in the virus transcription antitermination factor M2-1, outside of the CPD area, substantially reversed defective transcription of the CPD L gene and substantially restored virus fitness in vitro and in case of one of these two mutations, also in vivo. Paradoxically, the introduction into Min L of one mutation each in the M2-1, N, P, and L proteins resulted in a virus with increased attenuation in vivo but increased immunogenicity. Thus, in addition to providing insights on the adaptability of genome-scale deoptimized RNA viruses, stability studies can yield improved synthetic RNA virus vaccine candidates. PMID:28049853

Genetic manipulation of membrane phospholipid composition in Escherichia coli: pgsA mutants defective in phosphatidylglycerol synthesis.

PubMed Central

Miyazaki, C; Kuroda, M; Ohta, A; Shibuya, I

1985-01-01

Unique mutants of Escherichia coli K-12, defective in phosphatidylglycerol synthesis, have been isolated from a temperature-sensitive strain incubated at its nonpermissive temperature. The parent strain had excess phosphatidylglycerol by harboring both the pss-1 allele [coding for a temperature-sensitive phosphatidylserine synthase (EC 2.7.8.8)] and the cls- allele (responsible for a defective cardiolipin synthase). The newly acquired mutations caused better growth at higher temperatures. One of the mutations (pgsA3) has been identified in the structural gene for phosphatidylglycerophosphate synthase [glycerophosphate phosphatidyltransferase (EC 2.7.8.5)]. Phospholipid compositions of these mutants were remarkable; phosphatidylethanolamine was the sole major lipid. In media with low osmotic pressures, these cells grew more slowly than the wild-type cells. They grew normally without recovering from the phospholipid abnormality in media appropriately supplemented with sucrose and MgCl2. Formation of cardiolipin and phosphoglycerol derivatives of membrane-derived oligosaccharides was reduced in a pgsA3 mutant. E. coli strains having the pgsA3, pss-1, and cls- mutations, either individually or in combination, constitute an empirical system in which the molar ratio of three major membrane phospholipids can be variously altered. Images PMID:2999767

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

PubMed

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

2017-07-15

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

Isolation of Temperature-Sensitive Mutants of Arabidopsis thaliana That Are Defective in the Redifferentiation of Shoots.

PubMed Central

Yasutani, I.; Ozawa, S.; Nishida, T.; Sugiyama, M.; Komamine, A.

1994-01-01

Three temperature-sensitive mutants of Arabidopsis thaliana that were defective in the redifferentiation of shoots were isolated as tools for the study of organogenesis. M3 lines were constructed by harvesting M3 seeds separately from each M2 plant. Comparative examination of shoot redifferentiation in root explants of 2700 M3 lines at 22[deg]C (permissive temperature) and at 27[deg]C (restrictive temperature) led to the identification of seven temperature-sensitive mutant lines. Genetic tests of three of the seven mutant lines indicated that temperature-sensitive redifferentiation of shoots in these three lines resulted from single, nuclear, recessive mutations in three different genes, designated SRD1, SRD2, and SRD3. The morphology of root explants of srd mutants cultured at the restrictive temperature suggests that the products of these SRD genes function at different stages of the redifferentiation of shoots. PMID:12232244

Isolation of temperature-sensitive mutants of 16 S rRNA in Escherichia coli.

PubMed

Triman, K; Becker, E; Dammel, C; Katz, J; Mori, H; Douthwaite, S; Yapijakis, C; Yoast, S; Noller, H F

1989-10-20

Temperature-sensitive mutants have been isolated following hydroxylamine mutagenesis of a plasmid containing Escherichia coli rRNA genes carrying selectable markers for spectinomycin resistance (U1192 in 16 S rRNA) and erythromycin resistance (G2058 in 23 S rRNA). These antibiotic resistance alleles, originally identified by Morgan and co-workers, enable us to follow expression of cloned rRNA genes in vivo. Recessive mutations causing the loss of expression of the cloned 16 S rRNA gene were identified by the loss of the ability of cells to survive on media containing spectinomycin. The mutations were localized by in vitro restriction fragment replacement followed by in vivo marker rescue and were identified by DNA sequence analysis. We report here seven single-base alterations in 16 S rRNA (A146, U153, A350, A359, A538, A1292 and U1293), five of which produce temperature-sensitive spectinomycin resistance and two that produce unconditional loss of resistance. In each case, loss of ribosomal function can be accounted for by disruption of base-pairing in the secondary structure of 16 S rRNA. For the temperature-sensitive mutants, there is a lag period of about two generations between a shift to the restrictive temperature and cessation of growth, implying that the structural defects cause impairment of ribosome assembly.

A branch-migration based fluorescent probe for straightforward, sensitive and specific discrimination of DNA mutations

PubMed Central

Xiao, Xianjin; Wu, Tongbo; Xu, Lei; Chen, Wei

2017-01-01

Abstract Genetic mutations are important biomarkers for cancer diagnostics and surveillance. Preferably, the methods for mutation detection should be straightforward, highly specific and sensitive to low-level mutations within various sequence contexts, fast and applicable at room-temperature. Though some of the currently available methods have shown very encouraging results, their discrimination efficiency is still very low. Herein, we demonstrate a branch-migration based fluorescent probe (BM probe) which is able to identify the presence of known or unknown single-base variations at abundances down to 0.3%-1% within 5 min, even in highly GC-rich sequence regions. The discrimination factors between the perfect-match target and single-base mismatched target are determined to be 89–311 by measurement of their respective branch-migration products via polymerase elongation reactions. The BM probe not only enabled sensitive detection of two types of EGFR-associated point mutations located in GC-rich regions, but also successfully identified the BRAF V600E mutation in the serum from a thyroid cancer patient which could not be detected by the conventional sequencing method. The new method would be an ideal choice for high-throughput in vitro diagnostics and precise clinical treatment. PMID:28201758

Genetic Characterization of Escherichia coli Type 1 Pilus Adhesin Mutants and Identification of a Novel Binding Phenotype

PubMed Central

Hamrick, Terri S.; Harris, Sandra L.; Spears, Patricia A.; Havell, Edward A.; Horton, John R.; Russell, Perry W.; Orndorff, Paul E.

2000-01-01

Five Escherichia coli type 1 pilus mutants that had point mutations in fimH, the gene encoding the type 1 pilus adhesin FimH, were characterized. FimH is a minor component of type 1 pili that is required for the pili to bind and agglutinate guinea pig erythrocytes in a mannose-inhibitable manner. Point mutations were located by DNA sequencing and deletion mapping. All mutations mapped within the signal sequence or in the first 28% of the predicted mature protein. All mutations were missense mutations except for one, a frameshift lesion that was predicted to cause the loss of approximately 60% of the mature FimH protein. Bacterial agglutination tests with polyclonal antiserum raised to a LacZ-FimH fusion protein failed to confirm that parental amounts of FimH cross-reacting material were expressed in four of the five mutants. The remaining mutant, a temperature-sensitive (ts) fimH mutant that agglutinated guinea pig erythrocytes after growth at 31°C but not at 42°C, reacted with antiserum at both temperatures in a manner similar to the parent. Consequently, this mutant was chosen for further study. Temperature shift experiments revealed that new FimH biosynthesis was required for the phenotypic change. Guinea pig erythrocyte and mouse macrophage binding experiments using the ts mutant grown at the restrictive and permissive temperatures revealed that whereas erythrocyte binding was reduced to a level comparable to that of a fimH insertion mutant at the restrictive temperature, mouse peritoneal macrophages were bound with parental efficiency at both the permissive and restrictive temperatures. Also, macrophage binding by the ts mutant was insensitive to mannose inhibition after growth at 42°C but sensitive after growth at 31°C. The ts mutant thus binds macrophages with one receptor specificity at 31°C and another at 42°C. PMID:10869080

A nuclear mutation defective in mitochondrial recombination in yeast.

PubMed

Ling, F; Makishima, F; Morishima, N; Shibata, T

1995-08-15

Homologous recombination (crossing over and gene conversion) is generally essential for heritage and DNA repair, and occasionally causes DNA aberrations, in nuclei of eukaryotes. However, little is known about the roles of homologous recombination in the inheritance and stability of mitochondrial DNA which is continuously damaged by reactive oxygen species, by-products of respiration. Here, we report the first example of a nuclear recessive mutation which suggests an essential role for homologous recombination in the stable inheritance of mitochondrial DNA. For the detection of this class of mutants, we devised a novel procedure, 'mitochondrial crossing in haploid', which has enabled us to examine many mutant clones. Using this procedure, we examined mutants of Saccharomyces cerevisiae that showed an elevated UV induction of respiration-deficient mutations. We obtained a mutant that was defective in both the omega-intron homing and Endo.SceI-induced homologous gene conversion. We found that the mutant cells are temperature sensitive in the maintenance of mitochondrial DNA. A tetrad analysis indicated that elevated UV induction of respiration-deficient mutations, recombination deficiency and temperature sensitivity are all caused by a single nuclear mutation (mhr1) on chromosome XII. The pleiotropic characteristics of the mutant suggest an essential role for the MHR1 gene in DNA repair, recombination and the maintenance of DNA in mitochondria.

Novel HCN2 mutation contributes to febrile seizures by shifting the channel's kinetics in a temperature-dependent manner.

PubMed

Nakamura, Yuki; Shi, Xiuyu; Numata, Tomohiro; Mori, Yasuo; Inoue, Ryuji; Lossin, Christoph; Baram, Tallie Z; Hirose, Shinichi

2013-01-01

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel-mediated currents, known as I h, are involved in the control of rhythmic activity in neuronal circuits and in determining neuronal properties including the resting membrane potential. Recent studies have shown that HCN channels play a role in seizure susceptibility and in absence and limbic epilepsy including temporal lobe epilepsy following long febrile seizures (FS). This study focused on the potential contributions of abnormalities in the HCN2 isoform and their role in FS. A novel heterozygous missense mutation in HCN2 exon 1 leading to p.S126L was identified in two unrelated patients with FS. The mutation was inherited from the mother who had suffered from FS in a pedigree. To determine the effect of this substitution we conducted whole-cell patch clamp electrophysiology. We found that mutant channels had elevated sensitivity to temperature. More specifically, they displayed faster kinetics at higher temperature. Kinetic shift by change of temperature sensitivity rather than the shift of voltage dependence led to increased availability of I h in conditions promoting FS. Responses to cyclic AMP did not differ between wildtype and mutant channels. Thus, mutant HCN2 channels cause significant cAMP-independent enhanced availability of I h during high temperatures, which may contribute to hyperthermia-induced neuronal hyperexcitability in some individuals with FS.

Application of temperature-gradient gel electrophoresis for detection of prion protein gene polymorphisms in Polish Swiniarka sheep.

PubMed

Jasik, Agnieszka; Reichert, Michal

2006-05-01

This study presents preliminary data on the polymorphism in the prion protein gene of Swiniarka sheep using temperature gradient gel electrophoresis (TGGE). Available data indicate that sensitivity to scrapie is associated with polymorphisms in three codons of prion protein gene: 136,154, and 171. The TGGE method was used to detect point mutations in these codons responsible for sensitivity or resistance to scrapie. This study revealed presence of an allele encoding valine (V) in codon 136, which is associated with high sensitivity to scrapie and occurred in the form of heterozygous allele together with alanine (AV). The highest variability was observed in codon 171, with presence of arginine (R) and glutamine (Q) in the homozygous (RR or QQ) as well as the heterozygous form (RQ). The results of examination of fifty sheep DNA samples with mutations in codons 136, 154, and 171 demonstrated that TGGE can be used as a simple and rapid method to detect mutations in the PrP gene of sheep. Several samples can be run at the same time, making TGGE ideal for the screening of large numbers of samples.

A Temperature-Sensitive Lesion in the N-Terminal Domain of the Rotavirus Polymerase Affects Its Intracellular Localization and Enzymatic Activity

PubMed Central

McKell, Allison O.; LaConte, Leslie E. W.

2017-01-01

ABSTRACT Temperature-sensitive (ts) mutants of simian rotavirus (RV) strain SA11 have been previously created to investigate the functions of viral proteins during replication. One mutant, SA11-tsC, has a mutation that maps to the gene encoding the VP1 polymerase and shows diminished growth and RNA synthesis at 39°C compared to that at 31°C. In the present study, we sequenced all 11 genes of SA11-tsC, confirming the presence of an L138P mutation in the VP1 N-terminal domain and identifying 52 additional mutations in four other viral proteins (VP4, VP7, NSP1, and NSP2). To investigate whether the L138P mutation induces a ts phenotype in VP1 outside the SA11-tsC genetic context, we employed ectopic expression systems. Specifically, we tested whether the L138P mutation affects the ability of VP1 to localize to viroplasms, which are the sites of RV RNA synthesis, by expressing the mutant form as a green fluorescent protein (GFP) fusion protein (VP1L138P-GFP) (i) in wild-type SA11-infected cells or (ii) in uninfected cells along with viroplasm-forming proteins NSP2 and NSP5. We found that VP1L138P-GFP localized to viroplasms and interacted with NSP2 and/or NSP5 at 31°C but not at 39°C. Next, we tested the enzymatic activity of a recombinant mutant polymerase (rVP1L138P) in vitro and found that it synthesized less RNA at 39°C than at 31°C, as well as less RNA than the control at all temperatures. Together, these results provide a mechanistic basis for the ts phenotype of SA11-tsC and raise important questions about the role of leucine 138 in supporting key protein interactions and the catalytic function of the VP1 polymerase. IMPORTANCE RVs cause diarrhea in the young of many animal species, including humans. Despite their medical and economic importance, gaps in knowledge exist about how these viruses replicate inside host cells. Previously, a mutant simian RV (SA11-tsC) that replicates worse at higher temperatures was identified. This virus has an amino acid mutation in VP1, which is the enzyme responsible for copying the viral RNA genome. The mutation is located in a poorly understood region of the polymerase called the N-terminal domain. In this study, we determined that the mutation reduces the ability of VP1 to properly localize within infected cells at high temperatures, as well as reduced the ability of the enzyme to copy viral RNA in a test tube. The results of this study explain the temperature sensitivity of SA11-tsC and shed new light on functional protein-protein interaction sites of VP1. PMID:28100623

Temperature Sensitivity Conferred by ligA Alleles from Psychrophilic Bacteria upon Substitution in Mesophilic Bacteria and a Yeast Species

PubMed Central

Pankowski, Jarosław A.; Puckett, Stephanie M.

2016-01-01

We have assembled a collection of 13 psychrophilic ligA alleles that can serve as genetic elements for engineering mesophiles to a temperature-sensitive (TS) phenotype. When these ligA alleles were substituted into Francisella novicida, they conferred a TS phenotype with restrictive temperatures between 33 and 39°C. When the F. novicida ligA hybrid strains were plated above their restrictive temperatures, eight of them generated temperature-resistant variants. For two alleles, the mutations that led to temperature resistance clustered near the 5′ end of the gene, and the mutations increased the predicted strength of the ribosome binding site at least 3-fold. Four F. novicida ligA hybrid strains generated no temperature-resistant variants at a detectable level. These results suggest that multiple mutations are needed to create temperature-resistant variants of these ligA gene products. One ligA allele was isolated from a Colwellia species that has a maximal growth temperature of 12°C, and this allele supported growth of F. novicida only as a hybrid between the psychrophilic and the F. novicida ligA genes. However, the full psychrophilic gene alone supported the growth of Salmonella enterica, imparting a restrictive temperature of 27°C. We also tested two ligA alleles from two Pseudoalteromonas strains for their ability to support the viability of a Saccharomyces cerevisiae strain that lacked its essential gene, CDC9, encoding an ATP-dependent DNA ligase. In both cases, the psychrophilic bacterial alleles supported yeast viability and their expression generated TS phenotypes. This collection of ligA alleles should be useful in engineering bacteria, and possibly eukaryotic microbes, to predictable TS phenotypes. PMID:26773080

The L, M, and S Segments of Rift Valley Fever Virus MP-12 Vaccine Independently Contribute to a Temperature-Sensitive Phenotype

PubMed Central

Nishiyama, Shoko; Lokugamage, Nandadeva

2016-01-01

ABSTRACT Rift Valley fever (RVF) is endemic to Africa, and the mosquito-borne disease is characterized by “abortion storms” in ruminants and by hemorrhagic fever, encephalitis, and blindness in humans. Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) has a tripartite negative-stranded RNA genome (L, M, and S segments). A live-attenuated vaccine for RVF, the MP-12 vaccine, is conditionally licensed for veterinary use in the United States. MP-12 is fully attenuated by the combination of the partially attenuated L, M, and S segments. Temperature sensitivity (ts) limits viral replication at a restrictive temperature and may be involved with viral attenuation. In this study, we aimed to characterize the ts mutations for MP-12. The MP-12 vaccine showed restricted replication at 38°C and replication shutoff (100-fold or greater reduction in virus titer compared to that at 37°C) at 39°C in Vero and MRC-5 cells. Using rZH501 reassortants with either the MP-12 L, M, or S segment, we found that all three segments encode a temperature-sensitive phenotype. However, the ts phenotype of the S segment was weaker than that of the M or L segment. We identified Gn-Y259H, Gc-R1182G, L-V172A, and L-M1244I as major ts mutations for MP-12. The ts mutations in the L segment decreased viral RNA synthesis, while those in the M segment delayed progeny production from infected cells. We also found that a lack of NSs and/or 78kD/NSm protein expression minimally affected the ts phenotype. Our study revealed that MP-12 is a unique vaccine carrying ts mutations in the L, M, and S segments. IMPORTANCE Rift Valley fever (RVF) is a mosquito-borne viral disease endemic to Africa, characterized by high rates of abortion in ruminants and severe diseases in humans. Vaccination is important to prevent the spread of disease, and a live-attenuated MP-12 vaccine is currently the only vaccine with a conditional license in the United States. This study determined the temperature sensitivity (ts) of MP-12 vaccine to understand virologic characteristics. Our study revealed that MP-12 vaccine contains ts mutations independently in the L, M, and S segments and that MP-12 displays a restrictive replication at 38°C. PMID:26819307

The L, M, and S Segments of Rift Valley Fever Virus MP-12 Vaccine Independently Contribute to a Temperature-Sensitive Phenotype.

PubMed

Nishiyama, Shoko; Lokugamage, Nandadeva; Ikegami, Tetsuro

2016-01-27

Rift Valley fever (RVF) is endemic to Africa, and the mosquito-borne disease is characterized by "abortion storms" in ruminants and by hemorrhagic fever, encephalitis, and blindness in humans. Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) has a tripartite negative-stranded RNA genome (L, M, and S segments). A live-attenuated vaccine for RVF, the MP-12 vaccine, is conditionally licensed for veterinary use in the United States. MP-12 is fully attenuated by the combination of the partially attenuated L, M, and S segments. Temperature sensitivity (ts) limits viral replication at a restrictive temperature and may be involved with viral attenuation. In this study, we aimed to characterize the ts mutations for MP-12. The MP-12 vaccine showed restricted replication at 38°C and replication shutoff (100-fold or greater reduction in virus titer compared to that at 37°C) at 39°C in Vero and MRC-5 cells. Using rZH501 reassortants with either the MP-12 L, M, or S segment, we found that all three segments encode a temperature-sensitive phenotype. However, the ts phenotype of the S segment was weaker than that of the M or L segment. We identified Gn-Y259H, Gc-R1182G, L-V172A, and L-M1244I as major ts mutations for MP-12. The ts mutations in the L segment decreased viral RNA synthesis, while those in the M segment delayed progeny production from infected cells. We also found that a lack of NSs and/or 78kD/NSm protein expression minimally affected the ts phenotype. Our study revealed that MP-12 is a unique vaccine carrying ts mutations in the L, M, and S segments. Rift Valley fever (RVF) is a mosquito-borne viral disease endemic to Africa, characterized by high rates of abortion in ruminants and severe diseases in humans. Vaccination is important to prevent the spread of disease, and a live-attenuated MP-12 vaccine is currently the only vaccine with a conditional license in the United States. This study determined the temperature sensitivity (ts) of MP-12 vaccine to understand virologic characteristics. Our study revealed that MP-12 vaccine contains ts mutations independently in the L, M, and S segments and that MP-12 displays a restrictive replication at 38°C. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Temperature-dependent sex-reversal by a transformer-2 gene-edited mutation in the spotted wing drosophila, Drosophila suzukii.

PubMed

Li, Jianwei; Handler, Alfred M

2017-09-28

Female to male sex reversal was achieved in an emerging agricultural insect pest, Drosophila suzukii, by creating a temperature-sensitive point mutation in the sex-determination gene, transformer-2 (tra-2), using CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) homology-directed repair gene-editing. Ds-tra-2 ts2 mutants developed as normal fertile XX and XY adults at permissive temperatures below 20 °C, but at higher restrictive temperatures (26 to 29 °C) chromosomal XX females developed as sterile intersexuals with a predominant male phenotype, while XY males developed with normal morphology, but were sterile. The temperature-dependent function of the Ds-TRA-2 ts2 protein was also evident by the up- and down-regulation of female-specific Ds-Yolk protein 1 (Ds-Yp1) gene expression by temperature shifts during adulthood. This study confirmed the temperature-dependent function of a gene-edited mutation and provides a new method for the more general creation of conditional mutations for functional genomic analysis in insects, and other organisms. Furthermore, it provides a temperature-dependent system for creating sterile male populations useful for enhancing the efficacy of biologically-based programs, such as the sterile insect technique (SIT), to control D. suzukii and other insect pest species of agricultural and medical importance.

Bathing suit ichthyosis is caused by transglutaminase-1 deficiency: evidence for a temperature-sensitive phenotype.

PubMed

Oji, Vinzenz; Hautier, Juliette Mazereeuw; Ahvazi, Bijan; Hausser, Ingrid; Aufenvenne, Karin; Walker, Tatjana; Seller, Natalia; Steijlen, Peter M; Küster, Wolfgang; Hovnanian, Alain; Hennies, Hans Christian; Traupe, Heiko

2006-11-01

Bathing suit ichthyosis (BSI) is a striking and unique clinical form of autosomal recessive congenital ichthyosis characterized by pronounced scaling on the bathing suit areas but sparing of the extremities and the central face. Here we report on a series of 10 BSI patients. Our genetic, ultrastructural and biochemical investigations show that BSI is caused by transglutaminase-1 (TGase-1) deficiency. Altogether, we identified 13 mutations in TGM1-among them seven novel missense mutations and one novel nonsense mutation. Structural modeling for the Tyr276Asn mutation reveals that the residue is buried in the hydrophobic interior of the enzyme and that the hydroxyl side chain of Tyr276 is exposed to solvent in a cavity of the enzyme. Cryosections of healthy skin areas demonstrated an almost normal TGase activity, in contrast to the affected BSI skin, which only showed a cytoplasmic and clearly reduced TGase-1 activity. The distribution of TGase-1 substrates in the epidermis of affected skin corresponded to the situation in TGase-1 deficiency. Interestingly, the expression of TGase-3 and cathepsin D was reduced. Digital thermography validated a striking correlation between warmer body areas and presence of scaling in patients suggesting a decisive influence of the skin temperature. In situ TGase testing in skin of BSI patients demonstrated a marked decrease of enzyme activity when the temperature was increased from 25 to 37 degrees C. We conclude that BSI is caused by TGase-1 deficiency and suggest that it is a temperature-sensitive phenotype.

Novel methods to enhance single strand conformation polymorphism (SSCP) senstivity and efficiency: Application to mutation detection in cystic fibrosis (CF)

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

Hagstrom, D.J.; Snow, K.; Yuan, Z.

1994-09-01

For single gene defects in which there are a variety of mutations with significant frequencies, it is a challenge to find an efficient and sensitive method for mutation detection. For example, although 70% to 75% of CF chromosomes in a North American Caucasian population have the mutation {delta}F508, more than 400 mutations (mostly single base pair substitutions) are represented on the remaining chromosomes. SSCP analysis is a relatively straightforward procedure and therefore suitable for routine use in a clinical laboratory. However, previous reports have demonstrated suboptimal sensitivity rates in screening for mutations. We have developed a novel set of conditionsmore » which greatly enhances sensitivity and efficiency of SSCP. Our protocol incorporates multiplex PCR, stepping of wattages during electrophoresis and increased salt concentration at the anode relative to the gel. To screen for mutations in the CFTR gene, three multiplex PCR reactions are performed using identical thermocycler parameters. Sizes of PCR products range from 441 bp to 196 bp: size differences of > 30 bp are necessary to ensure separation during electrophoresis. All PCR products are separated by electrophoresis at room temperature on a single gel containing 8% (37.5:1) polyacrylamide, 5% glycerol and 1x TBE. Using an anode buffer with increased salt (2x TBE) sharpens smaller sized bands, and stepping watts from 5W to 20W during electrophoresis enhances sensitivity. Positive controls were used to demonstrate that mutations could be detected. Other mutations or polymorphisms were verified by cycle sequencing of PCR products or by alternative PCR-based assays for the more common mutations. Thus, using 3 PCR reactions per patient and one gel condition, we are able to achieve a CF mutation detection rate of approximately 90% in a North American Caucasian population.« less

Tolerance of an Antarctic Bacterium to Multiple Environmental Stressors.

PubMed

Sengupta, Dipanwita; Sangu, Kavya; Shivaji, Sisinthy; Chattopadhyay, Madhab K

2015-10-01

A population of cold-tolerant Antarctic bacteria was screened for their ability to tolerate other environmental stress factors. Besides low temperature, they were predominantly found to be tolerant to alkali. Attempt was also made to postulate a genetic basis of their multistress-tolerance. Transposon mutagenesis of an isolate Pseudomonas syringae Lz4W was performed, and mutants with delayed growth at low temperature were further screened for sensitivity to some other stress factors. A number of multistress-sensitive mutants were isolated. The mutated gene in one of the mutants sensitive to low temperature, acid and alkali was found to encode citrate synthase. Possible role of citrate synthase in conferring multistress-tolerance was postulated.

Characterization and Use of Temperature-Sensitive Mutations of BRCA1 for the Study of BRCA1 Function

DTIC Science & Technology

2005-01-01

Transcriptional activation by BRCA1. Nature 1996; 382:678-9. tion activation by BRCA1. The mutation would cause a marked 10. Hayes F, Cayanan C, Barilla D...cells was even higher than the Rev Mol Cell Biol 2002; 3:41-9. 20. Hayes F, Cayanan C, Barilla D, Monteiro AN. Functional assay for BRCAI

In Hyperthermia Increased ERK and WNT Signaling Suppress Colorectal Cancer Cell Growth

PubMed Central

Bordonaro, Michael; Shirasawa, Senji; Lazarova, Darina L.

2016-01-01

Although neoplastic cells exhibit relatively higher sensitivity to hyperthermia than normal cells, hyperthermia has had variable success as an anti-cancer therapy. This variable outcome might be due to the fact that cancer cells themselves have differential degrees of sensitivity to high temperature. We hypothesized that the varying sensitivity of colorectal cancer (CRC) cells to hyperthermia depends upon the differential induction of survival pathways. Screening of such pathways revealed that Extracellular Signal-Regulated Kinase (ERK) signaling is augmented by hyperthermia, and the extent of this modulation correlates with the mutation status of V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS). Through clonal growth assays, apoptotic analyses and transcription reporter assays of CRC cells that differ only in KRAS mutation status we established that mutant KRAS cells are more sensitive to hyperthermia, as they exhibit sustained ERK signaling hyperactivation and increased Wingless/Integrated (WNT)/beta-catenin signaling. We propose that whereas increased levels of WNT and ERK signaling and a positive feedback between the two pathways is a major obstacle in anti-cancer therapy today, under hyperthermia the hyperinduction of the pathways and their positive crosstalk contribute to CRC cell death. Ascertaining the causative association between types of mutations and hyperthermia sensitivity may allow for a mutation profile-guided application of hyperthermia as an anti-cancer therapy. Since KRAS and WNT signaling mutations are prevalent in CRC, our results suggest that hyperthermia-based therapy might benefit a significant number, but not all, CRC patients. PMID:27187477

A Temperature-Sensitive Lesion in the N-Terminal Domain of the Rotavirus Polymerase Affects Its Intracellular Localization and Enzymatic Activity.

PubMed

McKell, Allison O; LaConte, Leslie E W; McDonald, Sarah M

2017-04-01

Temperature-sensitive ( ts ) mutants of simian rotavirus (RV) strain SA11 have been previously created to investigate the functions of viral proteins during replication. One mutant, SA11- ts C, has a mutation that maps to the gene encoding the VP1 polymerase and shows diminished growth and RNA synthesis at 39°C compared to that at 31°C. In the present study, we sequenced all 11 genes of SA11- ts C, confirming the presence of an L138P mutation in the VP1 N-terminal domain and identifying 52 additional mutations in four other viral proteins (VP4, VP7, NSP1, and NSP2). To investigate whether the L138P mutation induces a ts phenotype in VP1 outside the SA11- ts C genetic context, we employed ectopic expression systems. Specifically, we tested whether the L138P mutation affects the ability of VP1 to localize to viroplasms, which are the sites of RV RNA synthesis, by expressing the mutant form as a green fluorescent protein (GFP) fusion protein (VP1 L138P -GFP) (i) in wild-type SA11-infected cells or (ii) in uninfected cells along with viroplasm-forming proteins NSP2 and NSP5. We found that VP1 L138P -GFP localized to viroplasms and interacted with NSP2 and/or NSP5 at 31°C but not at 39°C. Next, we tested the enzymatic activity of a recombinant mutant polymerase (rVP1 L138P ) in vitro and found that it synthesized less RNA at 39°C than at 31°C, as well as less RNA than the control at all temperatures. Together, these results provide a mechanistic basis for the ts phenotype of SA11- ts C and raise important questions about the role of leucine 138 in supporting key protein interactions and the catalytic function of the VP1 polymerase. IMPORTANCE RVs cause diarrhea in the young of many animal species, including humans. Despite their medical and economic importance, gaps in knowledge exist about how these viruses replicate inside host cells. Previously, a mutant simian RV (SA11- ts C) that replicates worse at higher temperatures was identified. This virus has an amino acid mutation in VP1, which is the enzyme responsible for copying the viral RNA genome. The mutation is located in a poorly understood region of the polymerase called the N-terminal domain. In this study, we determined that the mutation reduces the ability of VP1 to properly localize within infected cells at high temperatures, as well as reduced the ability of the enzyme to copy viral RNA in a test tube. The results of this study explain the temperature sensitivity of SA11- ts C and shed new light on functional protein-protein interaction sites of VP1. Copyright © 2017 American Society for Microbiology.

A dynamic sandwich assay on magnetic beads for selective detection of single-nucleotide mutations at room temperature.

PubMed

Wang, Junxiu; Xiong, Guoliang; Ma, Liang; Wang, Shihui; Zhou, Xu; Wang, Lei; Xiao, Lehui; Su, Xin; Yu, Changyuan

2017-08-15

Single-nucleotide mutation (SNM) has proven to be associated with a variety of human diseases. Development of reliable methods for the detection of SNM is crucial for molecular diagnosis and personalized medicine. The sandwich assays are widely used tools for detecting nucleic acid biomarkers due to their low cost and rapid signaling. However, the poor hybridization specificity of signal probe at room temperature hampers the discrimination of mutant and wild type. Here, we demonstrate a dynamic sandwich assay on magnetic beads for SNM detection based on the transient binding between signal probe and target. By taking the advantage of mismatch sensitive thermodynamics of transient DNA binding, the dynamic sandwich assay exhibits high discrimination factor for mutant with a broad range of salt concentration at room temperature. The beads used in this assay serve as a tool for separation, and might be helpful to enhance SNM selectivity. Flexible design of signal probe and facile magnetic separation allow multiple-mode downstream analysis including colorimetric detection and isothermal amplification. With this method, BRAF mutations in the genomic DNA extracted from cancer cell lines were tested, allowing sensitive detection of SNM at very low abundances (0.1-0.5% mutant/wild type). Copyright © 2017 Elsevier B.V. All rights reserved.

Multiplex Amplification Coupled with COLD-PCR and High Resolution Melting Enables Identification of Low-Abundance Mutations in Cancer Samples with Low DNA Content

PubMed Central

Milbury, Coren A.; Chen, Clark C.; Mamon, Harvey; Liu, Pingfang; Santagata, Sandro; Makrigiorgos, G. Mike

2011-01-01

Thorough screening of cancer-specific biomarkers, such as DNA mutations, can require large amounts of genomic material; however, the amount of genomic material obtained from some specimens (such as biopsies, fine-needle aspirations, circulating-DNA or tumor cells, and histological slides) may limit the analyses that can be performed. Furthermore, mutant alleles may be at low-abundance relative to wild-type DNA, reducing detection ability. We present a multiplex-PCR approach tailored to amplify targets of interest from small amounts of precious specimens, for extensive downstream detection of low-abundance alleles. Using 3 ng of DNA (1000 genome-equivalents), we amplified the 1 coding exons (2-11) of TP53 via multiplex-PCR. Following multiplex-PCR, we performed COLD-PCR (co-amplification of major and minor alleles at lower denaturation temperature) to enrich low-abundance variants and high resolution melting (HRM) to screen for aberrant melting profiles. Mutation-positive samples were sequenced. Evaluation of mutation-containing dilutions revealed improved sensitivities after COLD-PCR over conventional-PCR. COLD-PCR improved HRM sensitivity by approximately threefold to sixfold. Similarly, COLD-PCR improved mutation identification in sequence-chromatograms over conventional PCR. In clinical specimens, eight mutations were detected via conventional-PCR-HRM, whereas 12 were detected by COLD-PCR-HRM, yielding a 33% improvement in mutation detection. In summary, we demonstrate an efficient approach to increase screening capabilities from limited DNA material via multiplex-PCR and improve mutation detection sensitivity via COLD-PCR amplification. PMID:21354058

Identification and Characterization of Genes That Interact with Lin-12 in Caenorhabditis Elegans

PubMed Central

Tax, F. E.; Thomas, J. H.; Ferguson, E. L.; Horvitz, H. R.

1997-01-01

We identified and characterized 14 extragenic mutations that suppressed the dominant egg-laying defect of certain lin-12 gain-of-function mutations. These suppressors defined seven genes: sup-17, lag-2, sel-4, sel-5, sel-6, sel-7 and sel-8. Mutations in six of the genes are recessive suppressors, whereas the two mutations that define the seventh gene, lag-2, are semi-dominant suppressors. These suppressor mutations were able to suppress other lin-12 gain-of-function mutations. The suppressor mutations arose at a very low frequency per gene, 10-50 times below the typical loss-of-function mutation frequency. The suppressor mutations in sup-17 and lag-2 were shown to be rare non-null alleles, and we present evidence that null mutations in these two genes cause lethality. Temperature-shift studies for two suppressor genes, sup-17 and lag-2, suggest that both genes act at approximately the same time as lin-12 in specifying a cell fate. Suppressor alleles of six of these genes enhanced a temperature-sensitive loss-of-function allele of glp-1, a gene related to lin-12 in structure and function. Our analysis of these suppressors suggests that the majority of these genes are part of a shared lin-12/glp-1 signal transduction pathway, or act to regulate the expression or stability of lin-12 and glp-1. PMID:9409830

Varicella-zoster virus complements herpes simplex virus type 1 temperature-sensitive mutants.

PubMed Central

Felser, J M; Straus, S E; Ostrove, J M

1987-01-01

Varicella-zoster virus (VZV) can complement temperature-sensitive mutants of herpes simplex virus. Of seven mutants tested, two, carrying mutations in the immediate-early ICP4 and ICP27 proteins, were complemented. This complementation was not seen in coinfections with adenovirus type 5 or cytomegalovirus. Following transfection into CV-1 cells, a DNA fragment containing the VZV short repeat sequence complemented the ICP4 mutant. These data demonstrate a functional relationship between VZV and herpes simplex virus and have allowed localization of a putative VZV immediate-early gene. PMID:3023701

Massive Thermal Acceleration of the Emergence of Primordial Chemistry, the Incidence of Spontaneous Mutation, and the Evolution of Enzymes*

PubMed Central

Wolfenden, Richard

2014-01-01

Kelvin considered it unlikely that sufficient time had elapsed on the earth for life to have reached its present level of complexity. In the warm surroundings in which life first appeared, however, elevated temperatures would have reduced the kinetic barriers to reaction. Recent experiments disclose the profound extent to which very slow reactions are accelerated by elevated temperatures, collapsing the time that would have been required for early events in primordial chemistry before the advent of enzymes. If a primitive enzyme, like model catalysts and most modern enzymes, accelerated a reaction by lowering its enthalpy of activation, then the rate enhancement that it produced would have increased automatically as the environment cooled, quite apart from any improvements in catalytic activity that arose from mutation and natural selection. The chemical events responsible for spontaneous mutation are also highly sensitive to temperature, furnishing an independent mechanism for accelerating evolution. PMID:25210030

Different Mutations Endowing Resistance to Acetyl-CoA Carboxylase Inhibitors Results in Changes in Ecological Fitness of Lolium rigidum Populations

PubMed Central

Matzrafi, Maor; Gerson, Ofri; Rubin, Baruch; Peleg, Zvi

2017-01-01

Various mutations altering the herbicide target site (TS), can lead to structural modifications that decrease binding efficiency and results in herbicide resistant weed. In most cases, such a mutation will be associated with ecological fitness penalty under herbicide free environmental conditions. Here we describe the effect of various mutations, endowing resistance to acetyl-CoA carboxylase (ACCase) inhibitors, on the ecological fitness penalty of Lolium rigidum populations. The TS resistant populations, MH (substitution of isoleucine 1781 to leucine) and NO (cysteine 2088 to arginine), were examined and compared to a sensitive population (AL). Grain weight (GW) characterization of individual plants from both MH and NO populations, showed that resistant individuals had significantly lower GW compared with sensitive ones. Under high temperatures, both TS resistant populations exhibited lower germination rate as compared with the sensitive (AL) population. Likewise, early vigor of plants from both TS resistant populations was significantly lower than the one measured in plants of the sensitive population. Under crop-weed intra-species competition, we found an opposite trend in the response of plants from different populations. Relatively to inter-population competition conditions, plants of MH population were less affected and presented higher reproduction abilities compared to plants from both AL and NO populations. On the basis of our results, a non-chemical approach can be taken to favor the sensitive individuals, eventually leading to a decline in resistant individuals in the population. PMID:28690621

A New Microarray Substrate for Ultra-Sensitive Genotyping of KRAS and BRAF Gene Variants in Colorectal Cancer

PubMed Central

Pinzani, Pamela; Mancini, Irene; Vinci, Serena; Chiari, Marcella; Orlando, Claudio; Cremonesi, Laura; Ferrari, Maurizio

2013-01-01

Molecular diagnostics of human cancers may increase accuracy in prognosis, facilitate the selection of the optimal therapeutic regimen, improve patient outcome, reduce costs of treatment and favour development of personalized approaches to patient care. Moreover sensitivity and specificity are fundamental characteristics of any diagnostic method. We developed a highly sensitive microarray for the detection of common KRAS and BRAF oncogenic mutations. In colorectal cancer, KRAS and BRAF mutations have been shown to identify a cluster of patients that does not respond to anti-EGFR therapies; the identification of these mutations is therefore clinically extremely important. To verify the technical characteristics of the microarray system for the correct identification of the KRAS mutational status at the two hotspot codons 12 and 13 and of the BRAFV600E mutation in colorectal tumor, we selected 75 samples previously characterized by conventional and CO-amplification at Lower Denaturation temperature-PCR (COLD-PCR) followed by High Resolution Melting analysis and direct sequencing. Among these samples, 60 were collected during surgery and immediately steeped in RNAlater while the 15 remainders were formalin-fixed and paraffin-embedded (FFPE) tissues. The detection limit of the proposed method was different for the 7 KRAS mutations tested and for the V600E BRAF mutation. In particular, the microarray system has been able to detect a minimum of about 0.01% of mutated alleles in a background of wild-type DNA. A blind validation displayed complete concordance of results. The excellent agreement of the results showed that the new microarray substrate is highly specific in assigning the correct genotype without any enrichment strategy. PMID:23536897

Co-amplification at lower denaturation-temperature PCR combined with unlabled-probe high-resolution melting to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma cases.

PubMed

Wu, Jiong; Zhou, Yan; Zhang, Chun-Yan; Song, Bin-Bin; Wang, Bei-Li; Pan, Bai-Shen; Lou, Wen-Hui; Guo, Wei

2014-01-01

The aim of our study was to establish COLD-PCR combined with an unlabeled-probe HRM approach for detecting KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma (PA) cases as a novel and effective diagnostic technique. We tested the sensitivity and specificity of this approach with dilutions of known mutated cell lines. We screened 36 plasma-circulating DNA samples, 24 from the disease control group and 25 of a healthy group, to be subsequently sequenced to confirm mutations. Simultaneously, we tested the specimens using conventional PCR followed by HRM and then used target-DNA cloning and sequencing for verification. The ROC and respective AUC were calculated for KRAS mutations and/or serum CA 19-9. It was found that the sensitivity of Sanger reached 0.5% with COLD- PCR, whereas that obtained after conventional PCR did 20%; that of COLD-PCR based on unlabeled-probe HRM, 0.1%. KRAS mutations were identified in 26 of 36 PA cases (72.2%), while none were detected in the disease control and/or healthy group. KRAS mutations were identified both in 26 PA tissues and plasma samples. The AUC of COLD-PCR based unlabeled probe HRM turned out to be 0.861, which when combined with CA 19-9 increased to 0.934. It was concluded that COLD-PCR with unlabeled-probe HRM can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA for diagnosing and treating PA.

Structure-Based Systematic Isolation of Conditional-Lethal Mutations in the Single Yeast Calmodulin Gene

PubMed Central

Ohya, Y.; Botstein, D.

1994-01-01

Conditional-lethal mutations of the single calmodulin gene in Saccharomyces cerevisiae have been very difficult to isolate by random and systematic methods, despite the fact that deletions cause recessive lethality. We report here the isolation of numerous conditional-lethal mutants that were recovered by systematically altering phenylalanine residues. The phenylalanine residues of calmodulin were implicated in function both by structural studies of calmodulin bound to target peptides and by their extraordinary conservation in evolution. Seven single and 26 multiple Phe -> Ala mutations were constructed. Mutant phenotypes were examined in a haploid cmd1 disrupted strain under three conditions: single copy, low copy, and overexpressed. Whereas all but one of the single mutations caused no obvious phenotype, most of the multiple mutations caused obvious growth phenotypes. Five were lethal, 6 were lethal only in synthetic medium, 13 were temperature-sensitive lethal and 2 had no discernible phenotypic consequences. Overexpression of some of the mutant genes restored the phenotype to nearly wild type. Several temperature-sensitive calmodulin mutations were suppressed by elevated concentration of CaCl(2) in the medium. Mutant calmodulin protein was detected at normal levels in extracts of most of the lethal mutant cells, suggesting that the deleterious phenotypes were due to loss of the calmodulin function and not protein instability. Analysis of diploid strains heterozygous for all combinations of cmd1-ts alleles revealed four intragenic complementation groups. The contributions of individual phe->ala changes to mutant phenotypes support the idea of internal functional redundancy in the symmetrical calmodulin protein molecule. These results suggest that the several phenylalanine residues in calmodulin are required to different extents in different combinations in order to carry out each of the several essential tasks. PMID:7896089

Faithful chromosome transmission requires Spt4p, a putative regulator of chromatin structure in Saccharomyces cerevisiae.

PubMed Central

Basrai, M A; Kingsbury, J; Koshland, D; Spencer, F; Hieter, P

1996-01-01

A chromosome transmission fidelity (ctf) mutant, s138, of Saccharomyces cerevisiae was identified by its centromere (CEN) transcriptional readthrough phenotype, suggesting perturbed kinetochore integrity in vivo. The gene complementing the s138 mutation was found to be identical to the S. cerevisiae SPT4 gene. The s138 mutation is a missense mutation in the second of four conserved cysteine residues positioned similarly to those of zinc finger proteins, and we henceforth refer to the mutation of spt4-138. Both spt4-138 and spt4 delta strains missegregate a chromosome fragment at the permissive temperature, are temperature sensitive for growth at 37 degrees C, and upon a shift to the nonpermissive temperature show an accumulation of large budded cells, each with a nucleus. Previous studies suggest that Spt4p functions in a complex with Spt5p and Spt6p, and we determined that spt6-140 also causes missegregation of a chromosome fragment. Double mutants carrying spt4 delta 2::HIS3 and kinetochore mutation ndc10-42 or ctf13-30 show a synthetic conditional phenotype. Both spt4-138 and spt4 delta strains exhibit synergistic chromosome instability in combination with CEN DNA mutations and show in vitro defects in microtubule binding to minichromosomes. These results indicate that Spt4p plays a role in chromosome segregation. The results of in vivo genetic interactions with mutations in kinetochore proteins and CEN DNA and of in vitro biochemical assays suggest that Spt4p is important for kinetochore function. PMID:8649393

COLD-PCR Technologies in the Area of Personalized Medicine: Methodology and Applications.

PubMed

Mauger, Florence; How-Kit, Alexandre; Tost, Jörg

2017-06-01

Somatic mutations bear great promise for use as biomarkers for personalized medicine, but are often present only in low abundance in biological material and are therefore difficult to detect. Many assays for mutation analysis in cancer-related genes (hotspots) have been developed to improve diagnosis, prognosis, prediction of drug resistance, and monitoring of the response to treatment. Two major approaches have been developed: mutation-specific amplification methods and methods that enrich and detect mutations without prior knowledge on the exact location and identity of the mutation. CO-amplification at Lower Denaturation temperature Polymerase Chain Reaction (COLD-PCR) methods such as full-, fast-, ice- (improved and complete enrichment), enhanced-ice, and temperature-tolerant COLD-PCR make use of a critical temperature in the polymerase chain reaction to selectively denature wild-type-mutant heteroduplexes, allowing the enrichment of rare mutations. Mutations can subsequently be identified using a variety of laboratory technologies such as high-resolution melting, digital polymerase chain reaction, pyrosequencing, Sanger sequencing, or next-generation sequencing. COLD-PCR methods are sensitive, specific, and accurate if appropriately optimized and have a short time to results. A large variety of clinical samples (tumor DNA, circulating cell-free DNA, circulating cell-free fetal DNA, and circulating tumor cells) have been studied using COLD-PCR in many different applications including the detection of genetic changes in cancer and infectious diseases, non-invasive prenatal diagnosis, detection of microorganisms, or DNA methylation analysis. In this review, we describe in detail the different COLD-PCR approaches, highlighting their specificities, advantages, and inconveniences and demonstrating their use in different fields of biological and biomedical research.

Analysis of yeast prp20 mutations and functional complementation by the human homologue RCC1, a protein involved in the control of chromosome condensation.

PubMed

Fleischmann, M; Clark, M W; Forrester, W; Wickens, M; Nishimoto, T; Aebi, M

1991-07-01

Mutations in the PRP20 gene of yeast show a pleiotropic phenotype, in which both mRNA metabolism and nuclear structure are affected. srm1 mutants, defective in the same gene, influence the signal transduction pathway for the pheromone response. The yeast PRP20/SRM1 protein is highly homologous to the RCC1 protein of man, hamster and frog. In mammalian cells, this protein is a negative regulator for initiation of chromosome condensation. We report the analysis of two, independently isolated, recessive temperature-sensitive prp20 mutants. They have identical G to A transitions, leading to the alteration of a highly conserved glycine residue to glutamic acid. By immunofluorescence microscopy the PRP20 protein was localized in the nucleus. Expression of the RCC1 protein can complement the temperature-sensitive phenotype of prp20 mutants, demonstrating the functional similarity of the yeast and mammalian proteins.

Thermoresistant revertants of an Escherichia coli strain carrying tif-1 and ruv mutations: non-suppressibility of ruv by sfi.

PubMed Central

Otsuji, N; Iyehara-Ogawa, H

1979-01-01

Spontaneous thermoresistant revertants were isolated from Tif1 Ruv- and Tif1 Ruv+ strains of Escherichia coli K-12. They were divided into five groups; backmutants to tif+ and recA structural gene mutants accounted for at least two of these groups. Mutations with an unconditional RecA- phenyotype were detected at a higher frequency in the Tif1 Ruv- strains (65%) than in the Tif1 Ruv+ strains (25%). A third group consisted of revertants exhibiting a RecA- phenotype at low temperature. Revertants with normal recombination ability and UV resistance, but with a thermosensitive defect in propagating lambda bio11 phage, were also isolated (group 4). The alleles responsible for this property were cotransducible with the srl gene, suggesting that they are located at the recA locus. Other revertants, which might carry lex, LEXB, or zab mutations, were UV sensitive and were able to propagate lambda bio11 phage (group 5). The sfi mutation, which suppresses filamentation in the Tif1 and UV-sensitive Lon- strains, does not restore UV resistance of the Ruv- mutant. PMID:374356

Distinct mutations in yeast TAF(II)25 differentially affect the composition of TFIID and SAGA complexes as well as global gene expression patterns.

PubMed

Kirschner, Doris B; vom Baur, Elmar; Thibault, Christelle; Sanders, Steven L; Gangloff, Yann-Gaël; Davidson, Irwin; Weil, P Anthony; Tora, Làszlò

2002-05-01

The RNA polymerase II transcription factor TFIID, composed of the TATA-binding protein (TBP) and TBP-associated factors (TAF(II)s), nucleates preinitiation complex formation at protein-coding gene promoters. SAGA, a second TAF(II)-containing multiprotein complex, is involved in transcription regulation in Saccharomyces cerevisiae. One of the essential protein components common to SAGA and TFIID is yTAF(II)25. We define a minimal evolutionarily conserved 91-amino-acid region of TAF(II)25 containing a histone fold domain that is necessary and sufficient for growth in vivo. Different temperature-sensitive mutations of yTAF(II)25 or chimeras with the human homologue TAF(II)30 arrested cell growth at either the G(1) or G(2)/M cell cycle phase and displayed distinct phenotypic changes and gene expression patterns. Immunoprecipitation studies revealed that TAF(II)25 mutation-dependent gene expression and phenotypic changes correlated at least partially with the integrity of SAGA and TFIID. Genome-wide expression analysis revealed that the five TAF(II)25 temperature-sensitive mutant alleles individually affect the expression of between 18 and 33% of genes, whereas taken together they affect 64% of all class II genes. Thus, different yTAF(II)25 mutations induce distinct phenotypes and affect the regulation of different subsets of genes, demonstrating that no individual TAF(II) mutant allele reflects the full range of its normal functions.

Loss of function of Saccharomyces cerevisiae kinesin-related CIN8 and KIP1 is suppressed by KAR3 motor domain mutations.

PubMed

Hoyt, M A; He, L; Totis, L; Saunders, W S

1993-09-01

The kinesin-related products of the CIN8 and KIP1 genes of Saccharomyces cerevisiae redundantly perform an essential function in mitosis. The action of either gene-product is required for an outwardly directed force that acts upon the spindle poles. We have selected mutations that suppress the temperature-sensitivity of a cin8-temperature-sensitive kip1-delta strain. The extragenic suppressors analyzed were all found to be alleles of the KAR3 gene. KAR3 encodes a distinct kinesin-related protein whose action antagonizes Cin8p/Kip1p function. All seven alleles analyzed were altered within the region of KAR3 that encodes the putative force-generating (or "motor") domain. These mutations also suppressed the inviability associated with the cin8-delta kip1-delta genotype, a property not shared by a deletion of KAR3. Other properties of the suppressing alleles revealed that they were not null for function. Six of the seven were unaffected for the essential karyogamy and meiosis properties of KAR3 and the seventh was dominant for the suppressing trait. Our findings suggest that despite an antagonistic relationship between Cin8p/Kip1p and Kar3p, aspects of their mitotic roles may be similar.

Highly sensitive chemiluminescent point mutation detection by circular strand-displacement amplification reaction.

PubMed

Shi, Chao; Ge, Yujie; Gu, Hongxi; Ma, Cuiping

2011-08-15

Single nucleotide polymorphism (SNP) genotyping is attracting extensive attentions owing to its direct connections with human diseases including cancers. Here, we have developed a highly sensitive chemiluminescence biosensor based on circular strand-displacement amplification and the separation by magnetic beads reducing the background signal for point mutation detection at room temperature. This method took advantage of both the T4 DNA ligase recognizing single-base mismatch with high selectivity and the strand-displacement reaction of polymerase to perform signal amplification. The detection limit of this method was 1.3 × 10(-16)M, which showed better sensitivity than that of most of those reported detection methods of SNP. Additionally, the magnetic beads as carrier of immobility was not only to reduce the background signal, but also may have potential apply in high through-put screening of SNP detection in human genome. Copyright © 2011 Elsevier B.V. All rights reserved.

Alteration in the contents of unsaturated fatty acids in dnaA mutants of Escherichia coli.

PubMed

Suzuki, E; Kondo, T; Makise, M; Mima, S; Sakamoto, K; Tsuchiya, T; Mizushima, T

1998-04-01

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, has a high affinity for acidic phospholipids containing unsaturated fatty acids. We have examined here the fatty acid composition of phospholipids in dnaA mutants. A temperature-sensitive dnaA46 mutant showed a lower level of unsaturation of fatty acids (ratio of unsaturated to saturated fatty acids) at 42 degrees C (non-permissive temperature) and at 37 degrees C (semi-permissive temperature), but not at 28 degrees C (permissive temperature), compared with the wild-type strain. Plasmid complementation analysis revealed that the dnaA46 mutation is responsible for the phenotype. Other temperature-sensitive dnaA mutants showed similar results. On the other hand, a cold-sensitive dnaAcos mutant, in which over-initiation of DNA replication occurs at low temperature (28 degrees C), showed a higher level of unsaturation of fatty acids at 28 degrees C. Based on these observations, we discuss the role of phospholipids in the regulation of the activity of DnaA protein.

An Activity-Staining Method on Filtration Paper Enables High-Throughput Screening of Temperature-Sensitive and Inactive Mutations of Rice α-Amylase for Improvement of Rice Grain Quality.

PubMed

Yamakawa, Hiromoto; Hirai-Kimura, Rieko; Nakata, Yuriko; Nakata, Masaru; Kuroda, Masaharu; Yamaguchi, Takeshi

2017-04-01

α-Amylase is a starch-hydrolyzing enzyme (EC 3.2.1.1) indispensable for germination of cereal seeds, but it is also expressed during the ripening stage. Previous studies demonstrated that the enzyme is activated in developing rice seeds under extremely hot weather and triggers a loss of grain quality by hindering the accumulation of storage starch in the endosperm. Since inactive or, preferably, heat-labile α-amylases are preferable for breeding premium rice, we developed a method for rapid screening of inactive and temperature-sensitive mutants of the enzyme by combining the random mutagenesis by error-prone PCR and an on-filter activity test of the recombinant enzyme expressed by Escherichia coli. This technique was applied to a major α-amylase in the developing seed, Amy3D, and the activity of the isolated mutant enzymes was verified with both the bacteria-expressed recombinant proteins and the extract from the endosperm overexpressing each of them. Then, we identified several substitutions leading to loss of the activity of amino acid residues (Leu28, Asp112, Cys149, Trp201, Asp204, Gly295, Leu300 and Cys342), as well as a variety of heat-sensitive substitutions of Asp83, Asp187 and Glu252. Furthermore, variations of the heat-labile enzymes were created by combining these heat-sensitive mutations. The effects of the respective mutations and their relationship to the structure of the enzyme molecule are discussed. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Isolation of an essential Schizosaccharomyces pombe gene, prp31+, that links splicing and meiosis

PubMed Central

Bishop, Danielle T.; McDonald, W. Hayes; Gould, Kathleen L.; Forsburg, Susan L.

2000-01-01

We carried out a screen for mutants that arrest prior to premeiotic S phase. One of the strains we isolated contains a temperature-sensitive allele mutation in the fission yeast prp31+ gene. The prp31-E1 mutant is defective in vegetative cell growth and in meiotic progression. It is synthetically lethal with prp6 and displays a pre-mRNA splicing defect at the restrictive temperature. We cloned the wild-type gene by complementation of the temperature-sensitive mutant phenotype. Prp31p is closely related to human and budding yeast PRP31 homologs and is likely to function as a general splicing factor in both vegetative growth and sexual differentiation. PMID:10871341

Alteration of phospholipid composition by combined defects in phosphatidylserine and cardiolipin synthases and physiological consequences in Escherichia coli.

PubMed Central

Shibuya, I; Miyazaki, C; Ohta, A

1985-01-01

Escherichia coli K-12 derivatives with a common genetic background carrying, either alone or in combination, the pss-1 allele coding for a temperature-sensitive phosphatidylserine synthase (A. Ohta and I. Shibuya, J. Bacteriol. 132:434-443, 1977) and cls- for a defective cardiolipin synthase (G. Pluschke et al., J. Biol. Chem. 253:5048-5055, 1978) were constructed. The phospholipid polar headgroup compositions of these strains were significantly different from each other depending on their genotypes and growth temperature, whereas other membrane characteristics such as the total phospholipid content, fatty acid composition, membrane protein profile, and lipopolysaccharide content were practically the same, suggesting that the phenotypes of these strains were the direct consequences of abnormalities in membrane phospholipid composition. The cls pss-1 double mutation caused an unusual accumulation of phosphatidylglycerol with an extremely low content of cardiolipin. The cls mutation alone was found to give a growth defect, and its introduction into a pss-1 mutant resulted in an enhanced temperature sensitivity of growth. Addition to a broth medium of a proper concentration of sucrose, NaCl, Mg2+, or Ca2+ allowed the growth of a pss-1 mutant at otherwise nonpermissive temperature, but a pss-1 cls double mutant required the combined addition of sucrose or NaCl and MgCl2 for full growth at 42 degrees C. The possible mechanisms for these physiological consequences of the mutations are discussed on a molecular basis. The remedial effects of culture supplements allowed the pss-1 mutants to grow at 42 degrees C resulting in enhanced abnormalities of membrane phospholipid composition. Images PMID:2982784

CHO-cell mutant with a defect in cytokinesis

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

Thompson, L.H.; Lindl, P.A.

1976-01-01

In a selection procedure designed to enrich for temperature-sensitive mutant cells blocked in mitosis a CHO-cell mutant was isolated which has a defect in cytokinesis as the basis of its temperature-sensitive phenotype. Cultures of the mutant had an abnormally high percentage (ie, 34 percent) of polyploid cells at the permissive temperature of 34/sup 0/C and showed further increased frequencies of polyploidy as well as many multinucleated cells at 38.5/sup 0/ and 39.5/sup 0/. When the mutant cells were synchronized in metaphase by Colcemid arrest and then placed into fresh medium at nonpermissive temperature, they did not divide although the completionmore » of mitosis appeared cytologically normal. Ultrastructural examination by electron microscopy of such synchronized cells at telophase revealed no specific defects in cellular components other than failure of development of a normal midbody. The sensitivity of the mutant to cytochalasin B and to Colcemid was the same as for wild-type cells. This mutation behaved as recessive in tetraploid cell hybrids constructed by fusing the mutant with a CHO strain which was wild-type with respect to temperature sensitivity.« less

A novel COLD-PCR/FMCA assay enhances the detection of low-abundance IDH1 mutations in gliomas.

PubMed

Pang, Brendan; Durso, Mary B; Hamilton, Ronald L; Nikiforova, Marina N

2013-03-01

Point mutations in isocitrate dehydrogenase 1 (IDH1) have been identified in many gliomas. The detection of IDH1 mutations becomes challenging on suboptimal glioma biopsies when a limited number of tumor cells is available for analysis. Coamplification at lower denaturing-polymerase chain reaction (COLD-PCR) is a PCR technique that deliberately lowers the denaturing cycle temperature to selectively favor amplification of mutant alleles, allowing for the sensitive detection of low-abundance mutations. We developed a novel COLD-PCR assay on the LightCycler platform (Roche, Applied Science, Indianapolis, IN), using post-PCR fluorescent melting curve analysis (FMCA) for the detection of mutant IDH1 with a detection limit of 1%. Thirty-five WHO grade I to IV gliomas and 9 non-neoplastic brain and spinal cord biopsies were analyzed with this technique and the results were compared with the conventional real-time PCR and the Sanger sequencing analysis. COLD-PCR/FMCA was able to detect the most common IDH1 R132H mutation and rare mutation types including R132H, R132C, R132L, R132S, and R132G mutations. Twenty-five glioma cases were positive for IDH1 mutations by COLD-PCR/FMCA, and 23 gliomas were positive by the conventional real-time PCR and Sanger sequencing. A pilocytic astrocytoma (PA I) and a glioblastoma multiforme (GBM IV) showed low-abundance IDH1 mutations detected by COLD-PCR/FMCA. The remaining 10 glioma and 9 non-neoplastic samples were negative by all the 3 methods. In summary, we report a novel COLD-PCR/FMCA method that provides rapid and sensitive detection of IDH1 mutations in formalin-fixed paraffin-embedded tissue and can be used in the clinical setting to assess the small brain biopsies.

Courtship and other behaviors affected by a heat-sensitive, molecularly novel mutation in the cacophony calcium-channel gene of Drosophila.

PubMed Central

Chan, Betty; Villella, Adriana; Funes, Pablo; Hall, Jeffrey C

2002-01-01

The cacophony (cac) locus of Drosophila melanogaster, which encodes a calcium-channel subunit, has been mutated to cause courtship-song defects or abnormal responses to visual stimuli. However, the most recently isolated cac mutant was identified as an enhancer of a comatose mutation's effects on general locomotion. We analyzed the cac(TS2) mutation in terms of its intragenic molecular change and its effects on behaviors more complex than the fly's elementary ability to move. The molecular etiology of this mutation is a nucleotide substitution that causes a proline-to-serine change in a region of the polypeptide near its EF hand. Given that this motif is involved in channel inactivation, it was intriguing that cac(TS2) males generate song pulses containing larger-than-normal numbers of cycles--provided that such males are exposed to an elevated temperature. Similar treatments caused only mild visual-response abnormalities and generic locomotor sluggishness. These results are discussed in the context of calcium-channel functions that subserve certain behaviors and of defects exhibited by the original cacophony mutant. Despite its different kind of amino-acid substitution, compared with that of cac(TS2), cac(S) males sing abnormally in a manner that mimics the new mutant's heat-sensitive song anomaly. PMID:12242229

Gain-of-function mutations of fem-3, a sex-determination gene in Caenorhabditis elegans

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

Barton, M.K.; Schedl, T.B.; Kimble, J.

1987-01-01

The authors have isolated nine gain-of-function (gf) alleles of the sex-determination gene fem-3 as suppressors of feminizing mutations in fem-1 and fem-2. The wild type fem-3 gene is needed for spermatogenesis in XX self-fertilizing hermaphrodites and for male development in both soma and germ line of XO animals. Loss-of-function alleles of fem-3 transform XX and XO animals into females (spermless hermaphrodites). In contrast, fem-3 (gf) alleles masculinize only one tissue, the hermaphrodite germ line. Thus, XX fem-3 (gf) mutant animals have a normal hermaphrodite soma, but the germ line produces a vast excess of sperm and no oocytes. All ninemore » fem-3 (gf) alleles are temperature sensitive. The temperature-sensitive period is from late L4 to early adult, a period just preceding the first signs of oogenesis. The finding of gain-of-function alleles which confer a phenotype opposite to that of loss-of-function alleles supports the idea that fem-3 plays a critical role in germ-line sex determination. Furthermore, the germ-line specificity of the fem-3 (gf) mutant phenotype and the late temperature-sensitive period suggest that, in the wild-type XX hermaphrodite, fem-3 is negatively regulated so that the hermaphrodite stops making sperm and starts making oocytes. Temperature shift experiments also show that, in the germ line, sexual commitment appears to be a continuing process. Spermatogenesis can resume even after oogenesis has begun, and oogenesis can be initiated much later than normal.« less

Phenotypic characterization of adenovirus type 12 temperature-sensitive mutants in productive infection and transformation.

PubMed

Hama, S; Kimura, G

1980-01-01

Eleven temperature-sensitive mutants of adenovirus type 12, capable of forming plaques in human cells at 33 C but not at 39.5 C, were isolated from a stock of a wild-type strain after treatment with either nitrous acid or hydroxylamine. Complementation tests in doubly infected human cells permitted a tentative assignment of eight of these mutants to six complementation groups. Temperature-shift experiments revealed that one mutant is affected early and most of the other mutants are affected late. Only the early mutant, H12ts505, was temperature sensitive in viral DNA replication. Infectious virions of all the mutants except H12ts505 and two of the late mutants produced at 33 C, appeared to be more heat labile than those of the wild type. Only H12ts505 was temperature sensitive for the establishment of transformation of rat 3Y1 cells. One of the late mutants (H12ts504) had an increased transforming ability at the permissive temperature. Results of temperature-shift transformation experiments suggest that a viral function affected in H12ts505 is required for "initiation" of transformation. Some of the growth properties of H12ts505-transformed cells were also temperature dependent, suggesting that a functional expression of a gene mutation in H12ts505 is required to maintain at least some aspects of the transformed state.

Mutations in the Drosophila neuroglian cell adhesion molecule affect motor neuron pathfinding and peripheral nervous system patterning.

PubMed

Hall, S G; Bieber, A J

1997-03-01

We have identified and characterized three embryonic lethal mutations that alter or abolish expression of Drosophila Neuroglian and have used these mutations to analyze Neuroglian function during development. Neuroglian is a member of the immunoglobulin superfamily. It is expressed by a variety of cell types during embryonic development, including expression on motoneurons and the muscle cells that they innervate. Examination of the nervous systems of neuroglian mutant embryos reveals that motoneurons have altered pathfinding trajectories. Additionally, the sensory cell bodies of the peripheral nervous system display altered morphology and patterning. Using a temperature-sensitive mutation, the phenocritical period for Neuroglian function was determined to occur during late embryogenesis, an interval which coincides with the period during which neuromuscular connections and the peripheral nervous system pattern are established.

Fitness, Competitive Ability, and Mutation Stability of Isolates of Colletotrichum acutatum from Strawberry Resistant to QoI Fungicides.

PubMed

Forcelini, Bruna B; Rebello, Carolina S; Wang, Nan-Yi; Peres, Natalia A

2018-04-01

Quinone-outside inhibitor (QoI) fungicides are used to manage anthracnose of strawberry, caused by Colletotrichum acutatum. However, selection for resistance to QoI fungicides was first reported in 2013 in Florida and, subsequently, in strawberry nurseries and production areas across the United States and Canada. C. acutatum resistance to QoIs is associated with the G143A point mutation in the cytochrome b gene. This mutation is known to be associated with field resistance even at high rates of QoI. In this study, we investigated the relative fitness and competitive ability of QoI-resistant and -sensitive C. acutatum isolates. A fitness comparison did not indicate any difference between resistant and sensitive isolates in aggressiveness, spore production, and mycelial growth at different temperatures. Additionally, in the absence of selection pressure, resistant and sensitive isolates were equally competitive. Cultivation of QoI-resistant and QoI-sensitive isolates for four culture cycles in vitro in the absence of azoxystrobin showed that QoI resistance was stable. The observed lack of fitness penalties and stability of the G143A mutation in QoI-resistant C. acutatum populations suggest that the interruption and further reintroduction of QoI fungicides might not be an option for strawberry nurseries and fruit production areas. Further investigation of alternative chemical and nonchemical C. acutatum control practices, in addition to the integration of multisite fungicides, is needed to reduce the occurrence and distribution of QoI-resistant populations in strawberry fields.

DNA Variations in Oculocutaneous Albinism: An Updated Mutation List and Current Outstanding Issues in Molecular Diagnostics

PubMed Central

Simeonov, Dimitre R.; Wang, Xinjing; Wang, Chen; Sergeev, Yuri; Dolinska, Monika; Bower, Matthew; Fischer, Roxanne; Winer, David; Dubrovsky, Genia; Balog, Joan Z.; Huizing, Marjan; Hart, Rachel; Zein, Wadih M.; Gahl, William A.; Brooks, Brian P.; Adams, David R.

2014-01-01

Oculocutaneous albinism (OCA) is a rare genetic disorder of melanin synthesis that results in hypopigmented hair, skin, and eyes. There are four types of OCA, caused by mutations in TYR (OCA-1), OCA2 (OCA-2), TYRP1 (OCA-3), or SLC45A2 (OCA-4). Here we report 22 novel mutations; 14 from a cohort of 61 patients seen as part of the NIH OCA Natural History Study and 8 from a prior study at the University of Minnesota. We also include a comprehensive list of almost 600 previously reported OCA mutations, along with ethnicity information, carrier frequencies, and in silico pathogenicity predictions. In addition to discussing the clinical and molecular features of OCA, we address the cases of apparent missing heritability. In our cohort, 25% of patients did not have two mutations in a single OCA gene. We demonstrate the utility of multiple detection methods to reveal mutations missed by Sanger sequencing. Finally, we review the TYR p.R402Q temperature sensitive variant and confirm its association with cases of albinism with only one identifiable TYR mutation. PMID:23504663

West Nile Virus Temperature Sensitivity and Avian Virulence Are Modulated by NS1-2B Polymorphisms.

PubMed

Dietrich, Elizabeth A; Langevin, Stanley A; Huang, Claire Y-H; Maharaj, Payal D; Delorey, Mark J; Bowen, Richard A; Kinney, Richard M; Brault, Aaron C

2016-08-01

West Nile virus (WNV) replicates in a wide variety of avian species, which serve as reservoir and amplification hosts. WNV strains isolated in North America, such as the prototype strain NY99, elicit a highly pathogenic response in certain avian species, notably American crows (AMCRs; Corvus brachyrhynchos). In contrast, a closely related strain, KN3829, isolated in Kenya, exhibits a low viremic response with limited mortality in AMCRs. Previous work has associated the difference in pathogenicity primarily with a single amino acid mutation at position 249 in the helicase domain of the NS3 protein. The NY99 strain encodes a proline residue at this position, while KN3829 encodes a threonine. Introduction of an NS3-T249P mutation in the KN3829 genetic background significantly increased virulence and mortality; however, peak viremia and mortality were lower than those of NY99. In order to elucidate the viral genetic basis for phenotype variations exclusive of the NS3-249 polymorphism, chimeric NY99/KN3829 viruses were created. We show herein that differences in the NS1-2B region contribute to avian pathogenicity in a manner that is independent of and additive with the NS3-249 mutation. Additionally, NS1-2B residues were found to alter temperature sensitivity when grown in avian cells.

An alphavirus temperature-sensitive capsid mutant reveals stages of nucleocapsid assembly

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

Zheng, Yan, E-mail: yzheng15@students.kgi.edu; Kielian, Margaret, E-mail: margaret.kielian@einstein.yu.edu

2015-10-15

Alphaviruses have a nucleocapsid core composed of the RNA genome surrounded by an icosahedral lattice of capsid protein. An insertion after position 186 in the capsid protein produced a strongly temperature-sensitive growth phenotype. Even when the structural proteins were synthesized at the permissive temperature (28 °C), subsequent incubation of the cells at the non-permissive temperature (37 °C) dramatically decreased mutant capsid protein stability and particle assembly. Electron microscopy confirmed the presence of cytoplasmic nucleocapsids in mutant-infected cells cultured at the permissive temperature, but these nucleocapsids were not stable to sucrose gradient separation. In contrast, nucleocapsids isolated from mutant virus particlesmore » had similar stability to that of wildtype virus. Our data support a model in which cytoplasmic nucleocapsids go through a maturation step during packaging into virus particles. The insertion site lies in the interface between capsid proteins in the assembled nucleocapsid, suggesting the region where such a stabilizing transition occurs. - Highlights: • We characterize an alphavirus capsid insertion mutation. • These capsid mutants are highly temperature sensitive for growth. • The insertion affects nucleocapsid stability. • Results suggest that the nucleocapsid is stabilized during virus budding.« less

Cold-Adapted Viral Attenuation (CAVA): Highly Temperature Sensitive Polioviruses as Novel Vaccine Strains for a Next Generation Inactivated Poliovirus Vaccine

PubMed Central

Sanders, Barbara P.; de los Rios Oakes, Isabel; van Hoek, Vladimir; Bockstal, Viki; Kamphuis, Tobias; Uil, Taco G.; Song, Yutong; Cooper, Gillian; Crawt, Laura E.; Martín, Javier; Zahn, Roland; Lewis, John; Wimmer, Eckard; Custers, Jerome H. H. V.; Schuitemaker, Hanneke; Cello, Jeronimo; Edo-Matas, Diana

2016-01-01

The poliovirus vaccine field is moving towards novel vaccination strategies. Withdrawal of the Oral Poliovirus Vaccine and implementation of the conventional Inactivated Poliovirus Vaccine (cIPV) is imminent. Moreover, replacement of the virulent poliovirus strains currently used for cIPV with attenuated strains is preferred. We generated Cold-Adapted Viral Attenuation (CAVA) poliovirus strains by serial passage at low temperature and subsequent genetic engineering, which contain the capsid sequences of cIPV strains combined with a set of mutations identified during cold-adaptation. These viruses displayed a highly temperature sensitive phenotype with no signs of productive infection at 37°C as visualized by electron microscopy. Furthermore, decreases in infectious titers, viral RNA, and protein levels were measured during infection at 37°C, suggesting a block in the viral replication cycle at RNA replication, protein translation, or earlier. However, at 30°C, they could be propagated to high titers (9.4–9.9 Log10TCID50/ml) on the PER.C6 cell culture platform. We identified 14 mutations in the IRES and non-structural regions, which in combination induced the temperature sensitive phenotype, also when transferred to the genomes of other wild-type and attenuated polioviruses. The temperature sensitivity translated to complete absence of neurovirulence in CD155 transgenic mice. Attenuation was also confirmed after extended in vitro passage at small scale using conditions (MOI, cell density, temperature) anticipated for vaccine production. The inability of CAVA strains to replicate at 37°C makes reversion to a neurovirulent phenotype in vivo highly unlikely, therefore, these strains can be considered safe for the manufacture of IPV. The CAVA strains were immunogenic in the Wistar rat potency model for cIPV, inducing high neutralizing antibody titers in a dose-dependent manner in response to D-antigen doses used for cIPV. In combination with the highly productive PER.C6 cell culture platform, the stably attenuated CAVA strains may serve as an attractive low-cost and (bio)safe option for the production of a novel next generation IPV. PMID:27032093

Cold-Adapted Viral Attenuation (CAVA): Highly Temperature Sensitive Polioviruses as Novel Vaccine Strains for a Next Generation Inactivated Poliovirus Vaccine.

PubMed

Sanders, Barbara P; de Los Rios Oakes, Isabel; van Hoek, Vladimir; Bockstal, Viki; Kamphuis, Tobias; Uil, Taco G; Song, Yutong; Cooper, Gillian; Crawt, Laura E; Martín, Javier; Zahn, Roland; Lewis, John; Wimmer, Eckard; Custers, Jerome H H V; Schuitemaker, Hanneke; Cello, Jeronimo; Edo-Matas, Diana

2016-03-01

The poliovirus vaccine field is moving towards novel vaccination strategies. Withdrawal of the Oral Poliovirus Vaccine and implementation of the conventional Inactivated Poliovirus Vaccine (cIPV) is imminent. Moreover, replacement of the virulent poliovirus strains currently used for cIPV with attenuated strains is preferred. We generated Cold-Adapted Viral Attenuation (CAVA) poliovirus strains by serial passage at low temperature and subsequent genetic engineering, which contain the capsid sequences of cIPV strains combined with a set of mutations identified during cold-adaptation. These viruses displayed a highly temperature sensitive phenotype with no signs of productive infection at 37°C as visualized by electron microscopy. Furthermore, decreases in infectious titers, viral RNA, and protein levels were measured during infection at 37°C, suggesting a block in the viral replication cycle at RNA replication, protein translation, or earlier. However, at 30°C, they could be propagated to high titers (9.4-9.9 Log10TCID50/ml) on the PER.C6 cell culture platform. We identified 14 mutations in the IRES and non-structural regions, which in combination induced the temperature sensitive phenotype, also when transferred to the genomes of other wild-type and attenuated polioviruses. The temperature sensitivity translated to complete absence of neurovirulence in CD155 transgenic mice. Attenuation was also confirmed after extended in vitro passage at small scale using conditions (MOI, cell density, temperature) anticipated for vaccine production. The inability of CAVA strains to replicate at 37°C makes reversion to a neurovirulent phenotype in vivo highly unlikely, therefore, these strains can be considered safe for the manufacture of IPV. The CAVA strains were immunogenic in the Wistar rat potency model for cIPV, inducing high neutralizing antibody titers in a dose-dependent manner in response to D-antigen doses used for cIPV. In combination with the highly productive PER.C6 cell culture platform, the stably attenuated CAVA strains may serve as an attractive low-cost and (bio)safe option for the production of a novel next generation IPV.

EGFR mutation detection in ctDNA from NSCLC patient plasma: A cross-platform comparison of leading technologies to support the clinical development of AZD9291.

PubMed

Thress, Kenneth S; Brant, Roz; Carr, T Hedley; Dearden, Simon; Jenkins, Suzanne; Brown, Helen; Hammett, Tracey; Cantarini, Mireille; Barrett, J Carl

2015-12-01

To assess the ability of different technology platforms to detect epidermal growth factor receptor (EGFR) mutations, including T790M, from circulating tumor DNA (ctDNA) in advanced non-small cell lung cancer (NSCLC) patients. A comparison of multiple platforms for detecting EGFR mutations in plasma ctDNA was undertaken. Plasma samples were collected from patients entering the ongoing AURA trial (NCT01802632), investigating the safety, tolerability, and efficacy of AZD9291 in patients with EGFR-sensitizing mutation-positive NSCLC. Plasma was collected prior to AZD9291 dosing but following clinical progression on a previous EGFR-tyrosine kinase inhibitor (TKI). Extracted ctDNA was analyzed using two non-digital platforms (cobas(®) EGFR Mutation Test and therascreen™ EGFR amplification refractory mutation system assay) and two digital platforms (Droplet Digital™ PCR and BEAMing digital PCR [dPCR]). Preliminary assessment (38 samples) was conducted using all four platforms. For EGFR-TKI-sensitizing mutations, high sensitivity (78-100%) and specificity (93-100%) were observed using tissue as a non-reference standard. For the T790M mutation, the digital platforms outperformed the non-digital platforms. Subsequent assessment using 72 additional baseline plasma samples was conducted using the cobas(®) EGFR Mutation Test and BEAMing dPCR. The two platforms demonstrated high sensitivity (82-87%) and specificity (97%) for EGFR-sensitizing mutations. For the T790M mutation, the sensitivity and specificity were 73% and 67%, respectively, with the cobas(®) EGFR Mutation Test, and 81% and 58%, respectively, with BEAMing dPCR. Concordance between the platforms was >90%, showing that multiple platforms are capable of sensitive and specific detection of EGFR-TKI-sensitizing mutations from NSCLC patient plasma. The cobas(®) EGFR Mutation Test and BEAMing dPCR demonstrate a high sensitivity for T790M mutation detection. Genomic heterogeneity of T790M-mediated resistance may explain the reduced specificity observed with plasma-based detection of T790M mutations versus tissue. These data support the use of both platforms in the AZD9291 clinical development program. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Detection of low-level DNA mutation by ARMS-blocker-Tm PCR.

PubMed

Qu, Shoufang; Liu, Licheng; Gan, Shuzhen; Feng, Huahua; Zhao, Jingyin; Zhao, Jing; Liu, Qi; Gao, Shangxiang; Chen, Weijun; Wang, Mengzhao; Jiang, Yongqiang; Huang, Jie

2016-02-01

Low-level DNA mutations play important roles in cancer prognosis and treatment. However, most existing methods for the detection of low-level DNA mutations are insufficient for clinical applications because of the high background of wild-type DNA. In this study, a novel assay based on Tm-dependent inhibition of wild type template amplification was developed. The defining characteristic of this assay is an additional annealing step was introduced into the ARMS-blocker PCR. The temperature of this additional annealing step is equal to the Tm of the blocker. Due to this additional annealing step, the blocker can preferentially and specifically bind the wild-type DNA. Thus, the inhibition of wild type template is realized and the mutant DNA is enriched. The sensitivity of this assay was between 10(-4) and 10(-5), which is approximately 5 to 10 times greater than the sensitivity of the assay without the additional annealing step. To evaluate the performance of this assay in detecting K-ras mutation, we analyzed 100 formalin-fixed paraffin-embedded (FFPE) specimens from colorectal cancer patients using this new assay and Sanger sequencing. Of the clinical samples, 27 samples were positive for K-ras mutation by both methods. Our results indicated that this new assay is a highly selective, convenient, and economical method for detecting rare mutations in the presence of higher concentrations of wild-type DNA. Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Ultraviolet-Sensitive Mutator Strain of Escherichia coli K-12

PubMed Central

Siegel, Eli C.

1973-01-01

An ultraviolet (UV)-sensitive mutator gene, mutU, was identified in Escherichia coli K-12. The mutation mutU4 is very close to uvrD, between metE and ilv, on the E. coli chromosome. It was recessive as a mutator and as a UV-sensitive mutation. The frequency of reversion of trpA46 on an F episome was increased by mutU4 on the chromosome. The mutator gene did not increase mutation frequencies in virulent phages or in lytically grown phage λ. The mutU4 mutation predominantly induced transitional base changes. Mutator strains were normal for recombination and host-cell reactivation of UV-irradiated phage T1. They were normally resistant to methyl methanesulfonate and were slightly more sensitive to gamma irradiation than Mut+ strains. UV irradiation induced mutations in a mutU4 strain, and phage λ was UV-inducible. Double mutants containing mutU4 and recA, B, or C were extremely sensitive to UV irradiation; a mutU4 uvrA6 double mutant was only slightly more sensitive than a uvrA6 strain. The mutU4 uvrA6 and mutU4 recA, B, or C double mutants had mutation rates similar to that of a mutU4 strain. Two UV-sensitive mutators, mut-9 and mut-10, isolated by Liberfarb and Bryson in E. coli B/UV, were found to be co-transducible with ilv in the same general region as mutU4. PMID:4345920

Genetic analysis of an Escherichia coli syndrome.

PubMed

Lennette, E T; Apirion, D

1971-12-01

A mutant strain of Escherichia coli that fails to recover from prolonged (72 hr) starvation also fails to grow at 43 C. Extracts of this mutant strain show an increased ribonuclease II activity as compared to extracts of the parental strain, and stable ribonucleic acid is degraded to a larger extent in this strain during starvation. Ts(+) transductants and revertants were tested for all the above-mentioned phenotypes. All the Ts(+) transductants and revertants tested behaved like the Ts(+) parental strain, which suggests that all the observed phenotypes are caused by a single sts (starvation-temperature sensitivity) mutation. The reversion rate from sts(-) to sts(+) is rather low but is within the range of reversion rates for other single-site mutations. Three-point transduction crosses located this sts mutation between the ilv and rbs genes. The properties of sts(+)/sts(-) merozygotes suggested that the Ts(-) phenotype of this mutation is recessive.

Alteration in levels of unsaturated fatty acids in mutants of Escherichia coli defective in DNA replication.

PubMed

Suzuki, E; Kondo, T; Makise, M; Mima, S; Sakamoto, K; Tsuchiya, T; Mizushima, T

1998-07-01

We previously reported that mutations in the dnaA gene which encodes the initiator of chromosomal DNA replication in Escherichia coli caused an alteration in the levels of unsaturated fatty acids of phospholipids in membranes. In this study, we examined fatty acid compositions in other mutants which are defective in DNA replication. As in the case of temperature-sensitive dnaA mutants, temperature-sensitive dnaC and dnaE mutants, which have defects in initiation and elongation, respectively, of DNA replication showed a lower level of unsaturation of fatty acids (ratio of unsaturated to saturated fatty acids) compared with the wild-type strain, especially at high temperatures. On the other hand, temperature-sensitive mutants defective in cellular processes other than DNA replication, such as RNA synthesis and cell division, did not show a lower level of unsaturation of fatty acids compared with the wild-type strain. These results suggest that the inhibition of DNA replication causes a lower level of unsaturation of fatty acids in Escherichia coli cells.

A Fast Microfluidic Temperature Control Device for Studying Microtubule Dynamics in Fission Yeast

PubMed Central

Velve-Casquillas, Guilhem; Costa, Judite; Carlier-Grynkorn, Frédérique; Mayeux, Adeline; Tran, Phong T.

2010-01-01

Recent development in soft lithography and microfluidics enables biologists to create tools to control the cellular microenvironment. One such control is the ability to quickly change the temperature of the cells. Genetic model organism such as fission yeast has been useful for studies of the cell cytoskeleton. In particular, the dynamic microtubule cytoskeleton responds to changes in temperature. In addition, there are temperature-sensitive mutations of cytoskeletal proteins. We describe here the fabrication and use of a microfluidic device to quickly and reversibly change cellular temperature between 2°C and 50°C. We demonstrate the use of this device while imaging at high-resolution microtubule dynamics in fission yeast. PMID:20719272

Mutations in STX1B, encoding a presynaptic protein, cause fever-associated epilepsy syndromes.

PubMed

Schubert, Julian; Siekierska, Aleksandra; Langlois, Mélanie; May, Patrick; Huneau, Clément; Becker, Felicitas; Muhle, Hiltrud; Suls, Arvid; Lemke, Johannes R; de Kovel, Carolien G F; Thiele, Holger; Konrad, Kathryn; Kawalia, Amit; Toliat, Mohammad R; Sander, Thomas; Rüschendorf, Franz; Caliebe, Almuth; Nagel, Inga; Kohl, Bernard; Kecskés, Angela; Jacmin, Maxime; Hardies, Katia; Weckhuysen, Sarah; Riesch, Erik; Dorn, Thomas; Brilstra, Eva H; Baulac, Stephanie; Møller, Rikke S; Hjalgrim, Helle; Koeleman, Bobby P C; Jurkat-Rott, Karin; Lehman-Horn, Frank; Roach, Jared C; Glusman, Gustavo; Hood, Leroy; Galas, David J; Martin, Benoit; de Witte, Peter A M; Biskup, Saskia; De Jonghe, Peter; Helbig, Ingo; Balling, Rudi; Nürnberg, Peter; Crawford, Alexander D; Esguerra, Camila V; Weber, Yvonne G; Lerche, Holger

2014-12-01

Febrile seizures affect 2-4% of all children and have a strong genetic component. Recurrent mutations in three main genes (SCN1A, SCN1B and GABRG2) have been identified that cause febrile seizures with or without epilepsy. Here we report the identification of mutations in STX1B, encoding syntaxin-1B, that are associated with both febrile seizures and epilepsy. Whole-exome sequencing in independent large pedigrees identified cosegregating STX1B mutations predicted to cause an early truncation or an in-frame insertion or deletion. Three additional nonsense or missense mutations and a de novo microdeletion encompassing STX1B were then identified in 449 familial or sporadic cases. Video and local field potential analyses of zebrafish larvae with antisense knockdown of stx1b showed seizure-like behavior and epileptiform discharges that were highly sensitive to increased temperature. Wild-type human syntaxin-1B but not a mutated protein rescued the effects of stx1b knockdown in zebrafish. Our results thus implicate STX1B and the presynaptic release machinery in fever-associated epilepsy syndromes.

Cloning of the altered mRNA stability (ams) gene of Escherichia coli K-12.

PubMed Central

Claverie-Martin, F; Diaz-Torres, M R; Yancey, S D; Kushner, S R

1989-01-01

A temperature-sensitive mutation in the ams gene of Escherichia coli causes an increase in the chemical half-life of pulse-labeled RNA at the nonpermissive temperature. Using lambda clones containing DNA fragments from the 23- to 24-min region on the E. coli chromosome, we have isolated a 5.8-kilobase DNA fragment which, when present in a low-copy-number plasmid, complements the conditional lethality and increased mRNA stability associated with the ams-1 mutation. The approximate initiation site and the direction of transcription of the ams gene were determined from the size of truncated polypeptides produced by Tn1000 insertions and Bal 31 deletions. Overexpression of the ams locus by using a T7 RNA polymerase-promoter system permitted the identification of an ams-encoded polypeptide of 110 kilodaltons. Images PMID:2477358

Correlation of RNA secondary structure and attenuation of Sabin vaccine strains of poliovirus in tissue culture.

PubMed

Macadam, A J; Ferguson, G; Burlison, J; Stone, D; Skuce, R; Almond, J W; Minor, P D

1992-08-01

Part of the 5' noncoding regions of all three Sabin vaccine strains of poliovirus contains determinants of attenuation that are shown here to influence the ability of these strains to grow at elevated temperatures in BGM cells. The predicted RNA secondary structure of this region (nt 464-542 in P3/Sabin) suggests that both phenotypes are due to perturbation of base-paired stems. Ts phenotypes of site-directed mutants with defined changes in this region correlated well with predicted secondary structure stabilities. Reversal of base-pair orientation had little effect whereas stem disruption led to marked increases in temperature sensitivity. Phenotypic revertants of such viruses displayed mutations on either side of the stem. Mutations destabilizing stems led to intermediate phenotypes. These results provided evidence for the biological significance of the predicted RNA secondary structure.

Distinct Roles of the DmNav and DSC1 Channels in the Action of DDT and Pyrethroids

PubMed Central

Rinkevich, Frank D.; Du, Yuzhe; Tolinski, Josh; Ueda, Atsushi; Wu, Chun-Fang; Zhorov, Boris S.; Dong, Ke

2015-01-01

Voltage-gated sodium channels (Nav channels) are critical for electrical signaling in the nervous system and are the primary targets of the insecticides DDT and pyrethroids. In Drosophila melanogaster, besides the canonical Nav channel, Para (also called DmNav), there is a sodium channel-like cation channel called DSC1 (Drosophila sodium channel 1). Temperature-sensitive paralytic mutations in DmNav (parats) confer resistance to DDT and pyrethroids, whereas DSC1 knockout flies exhibit enhanced sensitivity to pyrethroids. To further define the roles and interaction of DmNav and DSC1 channels in DDT and pyrethroid neurotoxicology, we generated a DmNav/DSC1 double mutant line by introducing a parats1 allele (carrying the I265N mutation) into a DSC1 knockout line. We confirmed that the I265N mutation reduced the sensitivity to two pyrethroids, permethrin and deltamethrin of a DmNav variant expressed in Xenopus oocytes. Computer modeling predicts that the I265N mutation confers pyrethroid resistance by allosterically altering the second pyrethroid receptor site on the DmNav channel. Furthermore, we found that I265N-mediated pyrethroid resistance in parats1 mutant flies was almost completely abolished in parats1;DSC1−/− double mutant flies. Unexpectedly, however, the DSC1 knockout flies were less sensitive to DDT, compared to the control flies (w1118A), and the parats1;DSC1−/− double mutant flies were even more resistant to DDT compared to the DSC1 knockout or parats1 mutant. Our findings revealed distinct roles of the DmNav and DSC1 channels in the neurotoxicology of DDT vs. pyrethroids and implicate the exciting possibility of using DSC1 channel blockers or modifiers in the management of pyrethroid resistance. PMID:25687544

Engineering Temperature Sensitive Live Attenuated Influenza Vaccines from Emerging Viruses

PubMed Central

Zhou, Bin; Li, Yan; Speer, Scott D.; Subba, Anju; Lin, Xudong; Wentworth, David E.

2012-01-01

The licensed live attenuated influenza A vaccine (LAIV) in the United States is created by making a reassortant containing six internal genes from a cold-adapted master donor strain (ca A/AA/6/60) and two surface glycoprotein genes from a circulating/emerging strain (e.g., A/CA/7/09 for the 2009/2010 H1N1 pandemic). Technologies to rapidly create recombinant viruses directly from patient specimens were used to engineer alternative LAIV candidates that have genomes composed entirely of vRNAs from pandemic or seasonal strains. Multiple mutations involved in the temperature-sensitive (ts) phenotype of the ca A/AA/6/60 master donor strain were introduced into a 2009 H1N1 pandemic strain rA/New York/1682/2009 (rNY1682-WT) to create rNY1682-TS1, and additional mutations identified in other ts viruses were added to rNY1682-TS1 to create rNY1682-TS2. Both rNY1682-TS1 and rNY1682-TS2 replicated efficiently at 30°C and 33°C. However, rNY1682-TS1 was partially restricted, and rNY1682-TS2 was completely restricted at 39°C. Additionally, engineering the TS1 or TS2 mutations into a distantly related human seasonal H1N1 influenza A virus also resulted pronounced restriction of replication in vitro. Clinical symptoms and virus replication in the lungs of mice showed that although rNY1682-TS2 and the licensed FluMist®-H1N1pdm LAIV that was used to combat the 2009/2010 pandemic were similarly attenuated, the rNY1682-TS2 was more protective upon challenge with a virulent mutant of pandemic H1N1 virus or a heterologous H1N1 (A/PR/8/1934) virus. This study demonstrates that engineering key temperature sensitive mutations (PB1-K391E, D581G, A661T; PB2-P112S, N265S, N556D, Y658H) into the genomes of influenza A viruses attenuates divergent human virus lineages and provides an alternative strategy for the generation of LAIVs. PMID:22449422

Mutations in the Prokaryotic Pathway Rescue the fatty acid biosynthesis1 Mutant in the Cold.

PubMed

Gao, Jinpeng; Wallis, James G; Browse, John

2015-09-01

The Arabidopsis (Arabidopsis thaliana) fatty acid biosynthesis1 (fab1) mutant has increased levels of the saturated fatty acid 16:0 due to decreased activity of 3-ketoacyl-acyl carrier protein (ACP) synthase II. In fab1 leaves, phosphatidylglycerol, the major chloroplast phospholipid, contains up to 45% high-melting-point molecular species (molecules that contain only 16:0, 16:1-trans, and 18:0), a trait associated with chilling-sensitive plants, compared with less than 10% in wild-type Arabidopsis. Although they do not exhibit typical chilling sensitivity, when exposed to low temperatures (2°C-6°C) for long periods, fab1 plants do suffer collapse of photosynthesis, degradation of chloroplasts, and eventually death. A screen for suppressors of this low-temperature phenotype has identified 11 lines, some of which contain additional alterations in leaf-lipid composition relative to fab1. Here, we report the identification of two suppressor mutations, one in act1, which encodes the chloroplast acyl-ACP:glycerol-3-phosphate acyltransferase, and one in lpat1, which encodes the chloroplast acyl-ACP:lysophosphatidic acid acyltransferase. These enzymes catalyze the first two steps of the prokaryotic pathway for glycerolipid synthesis, so we investigated whether other mutations in this pathway would rescue the fab1 phenotype. Both the gly1 mutation, which reduces glycerol-3-phosphate supply to the prokaryotic pathway, and fad6, which is deficient in the chloroplast 16:1/18:1 fatty acyl desaturase, were discovered to be suppressors. Analyses of leaf-lipid compositions revealed that mutations at all four of the suppressor loci result in reductions in the proportion of high-melting-point molecular species of phosphatidylglycerol relative to fab1. We conclude that these reductions are likely the basis for the suppressor phenotypes. © 2015 American Society of Plant Biologists. All Rights Reserved.

Nucleotide sequences of Herpes Simplex Virus type 1 (HSV-1) affecting virus entry, cell fusion, and production of glycoprotein gB (VP7)

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

DeLuca, N.; Bzik, D.J.; Bond, V.C.

1982-10-30

The tsB5 strain of Herpes Simplex Virus type 1 (HSV-1) contains at least two mutations; one mutation specifies the syncytial phenotype and the other confers temperature sensitivity for virus growth. These functions are known to be located between the prototypic map coordinates 0.30 and 0.42. In this study it was demonstrated that tsB5 enters human embryonic lung (HEL) cells more rapidly than KOS, another strain of HSV-1. The EcoRI restriction fragment F from the KOS strain (map coordinates 0.315 to 0.421) was mapped with eight restriction endonucleases, and 16 recombinant plasmids were constructed which contained varying portions of the KOSmore » genome. Recombinant viruses were generated by marker-rescue and marker-transfer cotransfection procedures, using intact DNA from one strain and a recombinant plasmid containing DNA from the other strain. The region of the crossover between the two nonisogenic strains was inferred by the identification of restriction sites in the recombinants that were characteristic of the parental strains. The recombinants were subjected to phenotypic analysis. Syncytium formation, rate of virus entry, and the production of gB were all separable by the crossovers that produced the recombinants. The KOS sequences which rescue the syncytial phenotype of tsB5 were localized to 1.5 kb (map coordinates 0.345 to 0.355), and the temperature-sensitive mutation was localized to 1.2 kb (0.360 to 0.368), giving an average separation between the mutations of 2.5 kb on the 150-kb genome. DNA sequences that specify a functional domain for virus entry were localized to the nucleotide sequences between the two mutations. All three functions could be encoded by the virus gene specifying the gB glycoprotein.« less

DNA packaging and the pathway of bacteriophage T4 head assembly.

PubMed Central

Hsiao, C L; Black, L W

1977-01-01

A cold-sensitive mutation in the structural gene for a minor phage T4 capsid protein (p20) leads to formation of heads containing p20 and cleaved head proteins and empty of DNA. Such heads can be filled with DNA and converted to active phages in vivo uponshift to high temperature. It appears that p20 has two distinct roles in head assembly: first, in construction of the prehead shell (blocked by ts and am mutation) and, second,in DNA packaging (blocked by cs mutation). The latter function is closely associated with gene 17 product, previously known to be required for DNA packagaing. Temperature shift studies of cs-ts double mutants and other observations allow determination of phage function required for DNA packaging. Contrary to previous proposals, we find that T4 DNA packaging is not directly coupled to and can follow DNA synthesis, protein cleavage, prehead core removal, and gene 21-mediated cleavage-induced increase in head volume. Our evidence suggests that an altered head assembly pathway exists and that DNA packaging is probably initiated by DNA-capsid (p20) interaction. Images PMID:269421

IgE sensitization in relation to preschool eczema and filaggrin mutation.

PubMed

Johansson, Emma Kristin; Bergström, Anna; Kull, Inger; Lind, Tomas; Söderhäll, Cilla; van Hage, Marianne; Wickman, Magnus; Ballardini, Natalia; Wahlgren, Carl-Fredrik

2017-12-01

Eczema (atopic dermatitis) is associated with an increased risk of having IgE antibodies. IgE sensitization can occur through an impaired skin barrier. Filaggrin gene (FLG) mutation is associated with eczema and possibly also with IgE sensitization. We sought to explore the longitudinal relation between preschool eczema (PSE), FLG mutation, or both and IgE sensitization in childhood. A total of 3201 children from the BAMSE (Children Allergy Milieu Stockholm Epidemiology) birth cohort recruited from the general population were included. Regular parental questionnaires identified children with eczema. Blood samples were collected at 4, 8, and 16 years of age for analysis of specific IgE. FLG mutation analysis was performed on 1890 of the children. PSE was associated with IgE sensitization to both food allergens and aeroallergens up to age 16 years (overall adjusted odds ratio, 2.30; 95% CI, 2.00-2.66). This association was even stronger among children with persistent PSE. FLG mutation was associated with IgE sensitization to peanut at age 4 years (adjusted odds ratio, 1.88; 95% CI, 1.03-3.44) but not to other allergens up to age 16 years. FLG mutation and PSE were not effect modifiers for the association between IgE sensitization and PSE or FLG mutation, respectively. Sensitized children with PSE were characterized by means of polysensitization, but no other specific IgE sensitization patterns were found. PSE is associated with IgE sensitization to both food allergens and aeroallergens up to 16 years of age. FLG mutation is associated with IgE sensitization to peanut but not to other allergens. Sensitized children with preceding PSE are more often polysensitized. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Cloning-Independent and Counterselectable Markerless Mutagenesis System in Streptococcus mutans▿

PubMed Central

Xie, Zhoujie; Okinaga, Toshinori; Qi, Fengxia; Zhang, Zhijun; Merritt, Justin

2011-01-01

Insertion duplication mutagenesis and allelic replacement mutagenesis are among the most commonly utilized approaches for targeted mutagenesis in bacteria. However, both techniques are limited by a variety of factors that can complicate mutant phenotypic studies. To circumvent these limitations, multiple markerless mutagenesis techniques have been developed that utilize either temperature-sensitive plasmids or counterselectable suicide vectors containing both positive- and negative-selection markers. For many species, these techniques are not especially useful due to difficulties of cloning with Escherichia coli and/or a lack of functional negative-selection markers. In this study, we describe the development of a novel approach for the creation of markerless mutations. This system employs a cloning-independent methodology and should be easily adaptable to a wide array of Gram-positive and Gram-negative bacterial species. The entire process of creating both the counterselection cassette and mutation constructs can be completed using overlapping PCR protocols, which allows extremely quick assembly and eliminates the requirement for either temperature-sensitive replicons or suicide vectors. As a proof of principle, we used Streptococcus mutans reference strain UA159 to create markerless in-frame deletions of 3 separate bacteriocin genes as well as triple mutants containing all 3 deletions. Using a panel of 5 separate wild-type S. mutans strains, we further demonstrated that the procedure is nearly 100% efficient at generating clones with the desired markerless mutation, which is a considerable improvement in yield compared to existing approaches. PMID:21948849

Hypomorphic Temperature-Sensitive Alleles of NSDHL Cause CK Syndrome

PubMed Central

McLarren, Keith W.; Severson, Tesa M.; du Souich, Christèle; Stockton, David W.; Kratz, Lisa E.; Cunningham, David; Hendson, Glenda; Morin, Ryan D.; Wu, Diane; Paul, Jessica E.; An, Jianghong; Nelson, Tanya N.; Chou, Athena; DeBarber, Andrea E.; Merkens, Louise S.; Michaud, Jacques L.; Waters, Paula J.; Yin, Jingyi; McGillivray, Barbara; Demos, Michelle; Rouleau, Guy A.; Grzeschik, Karl-Heinz; Smith, Raffaella; Tarpey, Patrick S.; Shears, Debbie; Schwartz, Charles E.; Gecz, Jozef; Stratton, Michael R.; Arbour, Laura; Hurlburt, Jane; Van Allen, Margot I.; Herman, Gail E.; Zhao, Yongjun; Moore, Richard; Kelley, Richard I.; Jones, Steven J.M.; Steiner, Robert D.; Raymond, F. Lucy; Marra, Marco A.; Boerkoel, Cornelius F.

2010-01-01

CK syndrome (CKS) is an X-linked recessive intellectual disability syndrome characterized by dysmorphism, cortical brain malformations, and an asthenic build. Through an X chromosome single-nucleotide variant scan in the first reported family, we identified linkage to a 5 Mb region on Xq28. Sequencing of this region detected a segregating 3 bp deletion (c.696_698del [p.Lys232del]) in exon 7 of NAD(P) dependent steroid dehydrogenase-like (NSDHL), a gene that encodes an enzyme in the cholesterol biosynthesis pathway. We also found that males with intellectual disability in another reported family with an NSDHL mutation (c.1098 dup [p.Arg367SerfsX33]) have CKS. These two mutations, which alter protein folding, show temperature-sensitive protein stability and complementation in Erg26-deficient yeast. As described for the allelic disorder CHILD syndrome, cells and cerebrospinal fluid from CKS patients have increased methyl sterol levels. We hypothesize that methyl sterol accumulation, not only cholesterol deficiency, causes CKS, given that cerebrospinal fluid cholesterol, plasma cholesterol, and plasma 24S-hydroxycholesterol levels are normal in males with CKS. In summary, CKS expands the spectrum of cholesterol-related disorders and insight into the role of cholesterol in human development. PMID:21129721

RADIATION-INDUCED MUTATIONS FOR STEM RUST RESISTANCE IN OATS

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

Konzak, C.F.

1959-01-01

Stem rust rcsistant viriants from earlier experiments on the induction or resistance in oats by radiation were found to result from natural field hybridization. Recent controlled experiments did, however, yield new variants at a low frequency in one instance. and no variants in another. Both experiments included over 3,000 lines from irradiated seeds. One previously unknown type of rust resistance reaction was obtained in a mutant plant. This mutant shows a temperature sensitive response for resistance to race 7A of Puccinia graminis avenae. It was suggested that some, as yet unknown, mcdifying factors mav limit the development of induced changesmore » into mutations. (auth)« less

The use of Taqman genotyping assays for rapid confirmation of β-thalassaemia mutations in the Malays: accurate diagnosis with low DNA concentrations.

PubMed

Teh, L-K; Lee, T-Y; Tan, J A M A; Lai, M-I; George, E

2015-02-01

In Malaysia, β-thalassaemia is a common inherited blood disorder in haemoglobin synthesis with a carrier rate of 4.5%. Currently, PCR-incorporating techniques such as amplification refractory mutation system (ARMS) or reverse dot blot hybridization (RDBH) are used in β-thalassaemia mutation detection. ARMS allows single-mutation identification using two reactions, one for wild type and another for mutant alleles. RDBH requires probe immobilization and optimization of hybridization and washing temperatures which is time consuming. The aim of our study was to investigate whether β-thalassaemia mutations can be identified in samples with low DNA concentrations. Genotype identification of common β-thalassaemia mutations in Malays was carried out using Taqman genotyping assays. Results show that the Taqman assays allow mutation detection with DNA template concentrations as low as 2-100 ng. In addition, consistent reproducibility was observed in the Taqman assays when repeated eight times and at different time intervals. The developed sensitive Taqman assays allow molecular characterization of β-thalassaemia mutations in samples with low DNA concentrations. The Taqman genotyping assays have potential as a diagnostic tool for foetal blood, chorionic villi or pre-implantation genetic diagnosis where DNA is limited and precious. © 2014 John Wiley & Sons Ltd.

Computational Analysis of Epidermal Growth Factor Receptor Mutations Predicts Differential Drug Sensitivity Profiles toward Kinase Inhibitors.

PubMed

Akula, Sravani; Kamasani, Swapna; Sivan, Sree Kanth; Manga, Vijjulatha; Vudem, Dashavantha Reddy; Kancha, Rama Krishna

2018-05-01

A significant proportion of patients with lung cancer carry mutations in the EGFR kinase domain. The presence of a deletion mutation in exon 19 or L858R point mutation in the EGFR kinase domain has been shown to cause enhanced efficacy of inhibitor treatment in patients with NSCLC. Several less frequent (uncommon) mutations in the EGFR kinase domain with potential implications in treatment response have also been reported. The role of a limited number of uncommon mutations in drug sensitivity was experimentally verified. However, a huge number of these mutations remain uncharacterized for inhibitor sensitivity or resistance. A large-scale computational analysis of clinically reported 298 point mutants of EGFR kinase domain has been performed, and drug sensitivity profiles for each mutant toward seven kinase inhibitors has been determined by molecular docking. In addition, the relative inhibitor binding affinity toward each drug as compared with that of adenosine triphosphate was calculated for each mutant. The inhibitor sensitivity profiles predicted in this study for a set of previously characterized mutants correlated well with the published clinical, experimental, and computational data. Both the single and compound mutations displayed differential inhibitor sensitivity toward first- and next-generation kinase inhibitors. The present study provides predicted drug sensitivity profiles for a large panel of uncommon EGFR mutations toward multiple inhibitors, which may help clinicians in deciding mutant-specific treatment strategies. Copyright © 2018 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors

PubMed Central

Ban, Hiroshi; Nishishita, Naoki; Fusaki, Noemi; Tabata, Toshiaki; Saeki, Koichi; Shikamura, Masayuki; Takada, Nozomi; Inoue, Makoto; Hasegawa, Mamoru; Kawamata, Shin; Nishikawa, Shin-Ichi

2011-01-01

After the first report of induced pluripotent stem cells (iPSCs), considerable efforts have been made to develop more efficient methods for generating iPSCs without foreign gene insertions. Here we show that Sendai virus vector, an RNA virus vector that carries no risk of integrating into the host genome, is a practical solution for the efficient generation of safer iPSCs. We improved the Sendai virus vectors by introducing temperature-sensitive mutations so that the vectors could be easily removed at nonpermissive temperatures. Using these vectors enabled the efficient production of viral/factor-free iPSCs from both human fibroblasts and CD34+ cord blood cells. Temperature-shift treatment was more effective in eliminating remaining viral vector-related genes. The resulting iPSCs expressed human embryonic stem cell markers and exhibited pluripotency. We suggest that generation of transgene-free iPSCs from cord blood cells should be an important step in providing allogeneic iPSC-derived therapy in the future. PMID:21821793

[Combustion temperature measurement of pyrotechnic composition using remote sensing Fourier transform infrared spectrometry].

PubMed

Zhou, Xin-li; Li, Yan; Liu, Zu-liang; Zhu, Chang-jiang; Wang, Jun-de; Lu, Chun-xu

2002-10-01

In this paper, combustion characterization of pyrotechnic composition is investigated using a remote sensing Fourier transform infrared spectrometry. The emission spectra have been recorded between 4,700 and 740 cm-1 with a spectral resolution of 4 cm-1. The combustion temperature can be determined remotely from spectral line intensity distribution of the fine structure of the emission fundamental band of gaseous products such as HF. The relationship between combustion temperature and combustion time has been given. Results show that there is a violent mutative temperature field with bigger temperature gradient near combustion surface. It reveals that the method of temperature measurement using remote sensing FTIR for flame temperature of unstable, violent and short time combustion on real time is a rapid, accurate and sensitive technique without interference the flame temperature field. Potential prospects of temperature measurement, gas product concentration measurement and combustion mechanism are also revealed.

Detection of IDH1 mutation in the plasma of patients with glioma.

PubMed

Boisselier, Blandine; Gállego Pérez-Larraya, Jaime; Rossetto, Marta; Labussière, Marianne; Ciccarino, Pietro; Marie, Yannick; Delattre, Jean-Yves; Sanson, Marc

2012-10-16

The IDH1(R132H) mutation is both a strong prognostic predictor and a diagnostic hallmark of gliomas and therefore has major clinical relevance. Here, we developed a new technique to detect the IDH1(R132H) mutation in the plasma of patients with glioma. Small-size DNA (150-250 base pairs) was extracted from the plasma of 31 controls and 80 patients with glioma with known IDH1(R132H) status and correlated with MRI data. The IDH1(R132H) mutation was detected by a combination of coamplification at lower denaturation temperature and digital PCR. The small size DNA concentration was 1.2 ng/mL (range 0.1-6.6) in controls vs 1.2 ng/mL (range 0.1-50.3) in patients with glioma (p = not significant) and 0.9 ng/mL (0.0-3.0) in low-grade gliomas vs 1.5 ng/mL in high-grade gliomas (p < 0.01). The small size DNA concentration correlated with enhancing tumor volume (1.6 ng/mL [0.4-24.9] when <10 cm(3) and 14.0 ng/mL [0.6-50.3] when ≥10 cm(3)). The IDH1(R132H) mutation was detected in 15 out of 25 plasma DNA mixtures (60%) from patients with mutated tumors and in none of the 14 patients with a nonmutated tumor. The sensitivity increased with enhancing tumor volume (3/9 in nonenhancing tumors, 6/10 for enhancing volume <10 cm(3), and 6/6 for enhancing volume ≥10 cm(3)). With a specificity of 100% and a sensitivity related to the tumor volume and contrast enhancement, IDH1(R132H) identification has a valuable diagnostic accuracy in patients not amenable to biopsy.

Highly sensitive detection of ESR1 mutations in cell-free DNA from patients with metastatic breast cancer using molecular barcode sequencing.

PubMed

Masunaga, Nanae; Kagara, Naofumi; Motooka, Daisuke; Nakamura, Shota; Miyake, Tomohiro; Tanei, Tomonori; Naoi, Yasuto; Shimoda, Masafumi; Shimazu, Kenzo; Kim, Seung Jin; Noguchi, Shinzaburo

2018-01-01

We aimed to develop a highly sensitive method to detect ESR1 mutations in cell-free DNA (cfDNA) using next-generation sequencing with molecular barcode (MB-NGS) targeting the hotspot segment (c.1600-1713). The sensitivity of MB-NGS was tested using serially diluted ESR1 mutant DNA and then cfDNA samples from 34 patients with metastatic breast cancer were analyzed with MB-NGS. The results of MB-NGS were validated in comparison with conventional NGS and droplet digital PCR (ddPCR). MB-NGS showed a higher sensitivity (0.1%) than NGS without barcode (1%) by reducing background errors. Of the cfDNA samples from 34 patients with metastatic breast cancer, NGS without barcode revealed seven mutations in six patients (17.6%) and MB-NGS revealed six additional mutations including three mutations not reported in the COSMIC database of breast cancer, resulting in total 13 ESR1 mutations in ten patients (29.4%). Regarding the three hotspot mutations, all the patients with mutations detected by MB-NGS had identical mutations detected by droplet digital PCR (ddPCR), and mutant allele frequency correlated very well between both (r = 0.850, p < 0.01). Moreover, all the patients without these mutations by MB-NGS were found to have no mutations by ddPCR. In conclusion, MB-NGS could successfully detect ESR1 mutations in cfDNA with a higher sensitivity of 0.1% than conventional NGS and was considered as clinically useful as ddPCR.

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

PubMed

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

2017-07-18

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

A genetic screen for temperature-sensitive cell-division mutants of Caenorhabditis elegans.

PubMed Central

O'Connell, K F; Leys, C M; White, J G

1998-01-01

A novel screen to isolate conditional cell-division mutants in Caenorhabditis elegans has been developed. The screen is based on the phenotypes associated with existing cell-division mutations: some disrupt postembryonic divisions and affect formation of the gonad and ventral nerve cord-resulting in sterile, uncoordinated animals-while others affect embryonic divisions and result in lethality. We obtained 19 conditional mutants that displayed these phenotypes when shifted to the restrictive temperature at the appropriate developmental stage. Eighteen of these mutations have been mapped; 17 proved to be single alleles of newly identified genes, while 1 proved to be an allele of a previously identified gene. Genetic tests on the embryonic lethal phenotypes indicated that for 13 genes, embryogenesis required maternal expression, while for 6, zygotic expression could suffice. In all cases, maternal expression of wild-type activity was found to be largely sufficient for embryogenesis. Cytological analysis revealed that 10 mutants possessed embryonic cell-division defects, including failure to properly segregate DNA, failure to assemble a mitotic spindle, late cytokinesis defects, prolonged cell cycles, and improperly oriented mitotic spindles. We conclude that this approach can be used to identify mutations that affect various aspects of the cell-division cycle. PMID:9649522

Hypomorphic temperature-sensitive alleles of NSDHL cause CK syndrome.

PubMed

McLarren, Keith W; Severson, Tesa M; du Souich, Christèle; Stockton, David W; Kratz, Lisa E; Cunningham, David; Hendson, Glenda; Morin, Ryan D; Wu, Diane; Paul, Jessica E; An, Jianghong; Nelson, Tanya N; Chou, Athena; DeBarber, Andrea E; Merkens, Louise S; Michaud, Jacques L; Waters, Paula J; Yin, Jingyi; McGillivray, Barbara; Demos, Michelle; Rouleau, Guy A; Grzeschik, Karl-Heinz; Smith, Raffaella; Tarpey, Patrick S; Shears, Debbie; Schwartz, Charles E; Gecz, Jozef; Stratton, Michael R; Arbour, Laura; Hurlburt, Jane; Van Allen, Margot I; Herman, Gail E; Zhao, Yongjun; Moore, Richard; Kelley, Richard I; Jones, Steven J M; Steiner, Robert D; Raymond, F Lucy; Marra, Marco A; Boerkoel, Cornelius F

2010-12-10

CK syndrome (CKS) is an X-linked recessive intellectual disability syndrome characterized by dysmorphism, cortical brain malformations, and an asthenic build. Through an X chromosome single-nucleotide variant scan in the first reported family, we identified linkage to a 5 Mb region on Xq28. Sequencing of this region detected a segregating 3 bp deletion (c.696_698del [p.Lys232del]) in exon 7 of NAD(P) dependent steroid dehydrogenase-like (NSDHL), a gene that encodes an enzyme in the cholesterol biosynthesis pathway. We also found that males with intellectual disability in another reported family with an NSDHL mutation (c.1098 dup [p.Arg367SerfsX33]) have CKS. These two mutations, which alter protein folding, show temperature-sensitive protein stability and complementation in Erg26-deficient yeast. As described for the allelic disorder CHILD syndrome, cells and cerebrospinal fluid from CKS patients have increased methyl sterol levels. We hypothesize that methyl sterol accumulation, not only cholesterol deficiency, causes CKS, given that cerebrospinal fluid cholesterol, plasma cholesterol, and plasma 24S-hydroxycholesterol levels are normal in males with CKS. In summary, CKS expands the spectrum of cholesterol-related disorders and insight into the role of cholesterol in human development. Copyright © 2010 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Insight on Mutation-Induced Resistance from Molecular Dynamics Simulations of the Native and Mutated CSF-1R and KIT

PubMed Central

Da Silva Figueiredo Celestino Gomes, Priscila; Chauvot De Beauchêne, Isaure; Panel, Nicolas; Lopez, Sophie; De Sepulveda, Paulo; Geraldo Pascutti, Pedro; Solary, Eric; Tchertanov, Luba

2016-01-01

The receptors tyrosine kinases (RTKs) for the colony stimulating factor-1, CSF-1R, and for the stem cell factor, SCFR or KIT, are important mediators of signal transduction. The abnormal function of these receptors, promoted by gain-of-function mutations, leads to their constitutive activation, associated with cancer or other proliferative diseases. A secondary effect of the mutations is the alteration of receptors’ sensitivity to tyrosine kinase inhibitors, compromising effectiveness of these molecules in clinical treatment. In particular, the mutation V560G in KIT increases its sensitivity to Imatinib, while the D816V in KIT, and D802V in CSF-1R, triggers resistance to the drug. We analyzed the Imatinib binding affinity to the native and mutated KIT (mutations V560G, S628N and D816V) and CSF-1R (mutation D802V) by using molecular dynamics simulations and energy calculations of Imatinib•target complexes. Further, we evaluated the sensitivity of the studied KIT receptors to Imatinib by measuring the inhibition of KIT phosphorylation. Our study showed that (i) the binding free energy of Imatinib to the targets is highly correlated with their experimentally measured sensitivity; (ii) the electrostatic interactions are a decisive factor affecting the binding energy; (iii) the most deleterious impact to the Imatinib sensitivity is promoted by D802V (CSF-1R) and D816V (KIT) mutations; (iv) the role of the juxtamembrane region, JMR, in the imatinib binding is accessory. These findings contribute to a better description of the mutation-induced effects alternating the targets sensitivity to Imatinib. PMID:27467080

Insight on Mutation-Induced Resistance from Molecular Dynamics Simulations of the Native and Mutated CSF-1R and KIT.

PubMed

Da Silva Figueiredo Celestino Gomes, Priscila; Chauvot De Beauchêne, Isaure; Panel, Nicolas; Lopez, Sophie; De Sepulveda, Paulo; Geraldo Pascutti, Pedro; Solary, Eric; Tchertanov, Luba

2016-01-01

The receptors tyrosine kinases (RTKs) for the colony stimulating factor-1, CSF-1R, and for the stem cell factor, SCFR or KIT, are important mediators of signal transduction. The abnormal function of these receptors, promoted by gain-of-function mutations, leads to their constitutive activation, associated with cancer or other proliferative diseases. A secondary effect of the mutations is the alteration of receptors' sensitivity to tyrosine kinase inhibitors, compromising effectiveness of these molecules in clinical treatment. In particular, the mutation V560G in KIT increases its sensitivity to Imatinib, while the D816V in KIT, and D802V in CSF-1R, triggers resistance to the drug. We analyzed the Imatinib binding affinity to the native and mutated KIT (mutations V560G, S628N and D816V) and CSF-1R (mutation D802V) by using molecular dynamics simulations and energy calculations of Imatinib•target complexes. Further, we evaluated the sensitivity of the studied KIT receptors to Imatinib by measuring the inhibition of KIT phosphorylation. Our study showed that (i) the binding free energy of Imatinib to the targets is highly correlated with their experimentally measured sensitivity; (ii) the electrostatic interactions are a decisive factor affecting the binding energy; (iii) the most deleterious impact to the Imatinib sensitivity is promoted by D802V (CSF-1R) and D816V (KIT) mutations; (iv) the role of the juxtamembrane region, JMR, in the imatinib binding is accessory. These findings contribute to a better description of the mutation-induced effects alternating the targets sensitivity to Imatinib.

Distinct roles of the DmNav and DSC1 channels in the action of DDT and pyrethroids.

PubMed

Rinkevich, Frank D; Du, Yuzhe; Tolinski, Josh; Ueda, Atsushi; Wu, Chun-Fang; Zhorov, Boris S; Dong, Ke

2015-03-01

Voltage-gated sodium channels (Nav channels) are critical for electrical signaling in the nervous system and are the primary targets of the insecticides DDT and pyrethroids. In Drosophila melanogaster, besides the canonical Nav channel, Para (also called DmNav), there is a sodium channel-like cation channel called DSC1 (Drosophila sodium channel 1). Temperature-sensitive paralytic mutations in DmNav (para(ts)) confer resistance to DDT and pyrethroids, whereas DSC1 knockout flies exhibit enhanced sensitivity to pyrethroids. To further define the roles and interaction of DmNav and DSC1 channels in DDT and pyrethroid neurotoxicology, we generated a DmNav/DSC1 double mutant line by introducing a para(ts1) allele (carrying the I265N mutation) into a DSC1 knockout line. We confirmed that the I265N mutation reduced the sensitivity to two pyrethroids, permethrin and deltamethrin of a DmNav variant expressed in Xenopus oocytes. Computer modeling predicts that the I265N mutation confers pyrethroid resistance by allosterically altering the second pyrethroid receptor site on the DmNav channel. Furthermore, we found that I265N-mediated pyrethroid resistance in para(ts1) mutant flies was almost completely abolished in para(ts1);DSC1(-/-) double mutant flies. Unexpectedly, however, the DSC1 knockout flies were less sensitive to DDT, compared to the control flies (w(1118A)), and the para(ts1);DSC1(-/-) double mutant flies were even more resistant to DDT compared to the DSC1 knockout or para(ts1) mutant. Our findings revealed distinct roles of the DmNav and DSC1 channels in the neurotoxicology of DDT vs. pyrethroids and implicate the exciting possibility of using DSC1 channel blockers or modifiers in the management of pyrethroid resistance. Copyright © 2015 Elsevier Inc. All rights reserved.

Evaluation of digital PCR for detecting low-level EGFR mutations in advanced lung adenocarcinoma patients: a cross-platform comparison study

PubMed Central

Liu, Bing; Li, Lei; Huang, Lixia; Li, Shaoli; Rao, Guanhua; Yu, Yang; Zhou, Yanbin

2017-01-01

Emerging evidence has indicated that circulating tumor DNA (ctDNA) from plasma could be used to analyze EGFR mutation status for NSCLC patients; however, due to the low level of ctDNA in plasma, highly sensitive approaches are required to detect low frequency mutations. In addition, the cutoff for the mutation abundance that can be detected in tumor tissue but cannot be detected in matched ctDNA is still unknown. To assess a highly sensitive method, we evaluated the use of digital PCR in the detection of EGFR mutations in tumor tissue from 47 advanced lung adenocarcinoma patients through comparison with NGS and ARMS. We determined the degree of concordance between tumor tissue DNA and paired ctDNA and analyzed the mutation abundance relationship between them. Digital PCR and Proton had a high sensitivity (96.00% vs. 100%) compared with that of ARMS in the detection of mutations in tumor tissue. Digital PCR outperformed Proton in identifying more low abundance mutations. The ctDNA detection rate of digital PCR was 87.50% in paired tumor tissue with a mutation abundance above 5% and 7.59% in paired tumor tissue with a mutation abundance below 5%. When the DNA mutation abundance of tumor tissue was above 3.81%, it could identify mutations in paired ctDNA with a high sensitivity. Digital PCR will help identify alternative methods for detecting low abundance mutations in tumor tissue DNA and plasma ctDNA. PMID:28978074

Comparison of next-generation sequencing mutation profiling with BRAF and IDH1 mutation-specific immunohistochemistry.

PubMed

Jabbar, Kausar J; Luthra, Rajalakshmi; Patel, Keyur P; Singh, Rajesh R; Goswami, Rashmi; Aldape, Ken D; Medeiros, L Jeffrey; Routbort, Mark J

2015-04-01

Mutation-specific antibodies for BRAF V600E and IDH1 R132H offer convenient immunohistochemical (IHC) assays to detect these mutations in tumors. Previous studies using these antibodies have shown high sensitivity and specificity, but use in routine diagnosis with qualitative assessment has not been well studied. In this retrospective study, we reviewed BRAF and IDH1 mutation-specific IHC results compared with separately obtained clinical next-generation sequencing results. For 67 tumors with combined IDH1 IHC and mutation data, IHC was unequivocally reported as positive or negative in all cases. Sensitivity of IHC for IDH1 R132H was 98% and specificity was 100% compared with mutation status. Four IHC-negative samples showed non-R132H IDH1 mutations including R132C, R132G, and P127T. For 128 tumors with combined BRAF IHC and mutation data, IHC was positive in 33, negative in 82, and equivocal in 13 tumors. The sensitivity of IHC was 97% and specificity was 99% when including only unequivocally positive or negative results. If equivocal IHC cases were included in the analysis as negative, sensitivity fell to 81%. If equivocal cases were classified as positive, specificity dropped to 91%. Eight IHC-negative samples showed non-V600E BRAF mutations including V600K, N581I, V600M, and K601E. We conclude that IHC for BRAF V600E and IDH1 R132H is relatively sensitive and specific, but there is a discordance rate that is not trivial. In addition, a significant proportion of patients harbor BRAF non-V600E or IDH1 non-R132H mutations not detectable by IHC, potentially limiting utility of IHC screening for BRAF and IDH1 mutations.

Filaggrin mutations increase allergic airway disease in childhood and adolescence through interactions with eczema and aeroallergen sensitization.

PubMed

Chan, Adrian; Terry, William; Zhang, Hongmei; Karmaus, Wilfried; Ewart, Susan; Holloway, John W; Roberts, Graham; Kurukulaaratchy, Ramesh; Arshad, Syed Hasan

2018-02-01

Filaggrin loss-of-function (FLG-LOF) mutations are an established genetic cause of eczema. These mutations have subsequently been reported to increase the risk of aeroallergen sensitization and allergic airway disease. However, it is unclear whether FLG variants require both eczema and aeroallergen sensitization to influence airway disease development long-term outcomes. To examine the effects of FLG-LOF mutations on allergic airway disease outcomes, with eczema and aeroallergen sensitization as intermediate variables, using the Isle of Wight birth cohort. Study participants were evaluated at ages 1, 2, 4, 10 and 18 years to ascertain the development of allergic diseases (eczema, asthma and allergic rhinitis) and aeroallergen sensitization (determined by skin prick tests). FLG-LOF mutations were genotyped in 1150 subjects. To understand the complex associations between FLG mutations, intermediate variables (eczema and aeroallergen sensitization) and airway disease, path analysis was performed. There were significant total effects of FLG-LOF mutations on both asthma and allergic rhinitis at all ages as well as on aeroallergen sensitization up till 10 years old. In the filaggrin-asthma analysis, a direct effect of FLG-LOF mutations was observed on early childhood eczema (age 1 and 2 years) (relative risk (RR) 2.01, 95% CI: 1.74-2.31, P < .001), and all significant indirect pathways on asthma outcomes passed through eczema at these ages. In contrast, for the filaggrin-rhinitis model, FLG-LOF mutations exerted significant direct effects on early eczema as well as rhinitis at 10 years (RR 1.99; 95% CI: 1.72-2.29, P = .002). FLG-LOF mutations are a significant risk factor for later childhood asthma and rhinitis. However, the pathway to asthma is only through early childhood eczema while a direct effect was observed for childhood rhinitis. © 2017 John Wiley & Sons Ltd.

Thermo-labile stability of sigmaH (Spo0H) in temperature-sensitive spo0H mutants of Bacillus subtilis can be suppressed by mutations in RNA polymerase beta subunit.

PubMed

Ohashi, Y; Sugimaru, K; Nanamiya, H; Sebata, T; Asai, K; Yoshikawa, H; Kawamura, F

1999-03-18

We isolated novel temperature-sensitive mutants of spo0H, spo0H1 and spo0H5, having E61K and G30E amino-acid substitutions within the sigmaH protein, respectively, and located in the highly conserved region, "2", among prokaryotic sigma factors that participates in binding to core enzyme of RNA polymerase. These mutants showed a sporulation-deficient phenotype at 43 degrees C. Moreover, we successfully isolated suppressor mutants that were spontaneously generated from the spo0H mutants. Our genetic analysis of these suppressor mutations revealed that the suppressor mutations are within the rpoB gene coding for the beta subunit of RNA polymerase. The mutations caused single amino-acid substitutions, E857A and P1055S, in rpoB18 and rpoB532 mutants that were generated from spo0H1 and spo0H5, respectively. Whereas the sigmaH-dependent expression of a spo0A-bgaB fusion was greatly reduced in both spo0H mutants, their expression was partially restored in the suppressor mutants at 43 degrees C. Western blot analysis showed that the level of sigmaH protein in the wild type increased between T0 and T2 and decreased after T3, while the level of sigmaH protein in spo0H mutants was greatly reduced throughout growth, indicating that the mutant sigmaH proteins were rapidly degraded by some unknown proteolytic enzyme(s). The analysis of the half-life of sigmaH protein showed that the short life of sigmaH in spo0H mutants is prolonged in the suppressor mutants. These findings suggest that, at least to some extent, the process of E-sigmaH formation may be involved in stabilization of sigmaH at the onset of sporulation.

COLD-PCR and microarray: two independent highly sensitive approaches allowing the identification of fetal paternally inherited mutations in maternal plasma.

PubMed

Galbiati, Silvia; Monguzzi, Alessandra; Damin, Francesco; Soriani, Nadia; Passiu, Marianna; Castellani, Carlo; Natacci, Federica; Curcio, Cristina; Seia, Manuela; Lalatta, Faustina; Chiari, Marcella; Ferrari, Maurizio; Cremonesi, Laura

2016-07-01

Until now, non-invasive prenatal diagnosis of genetic diseases found only limited routine applications. In autosomal recessive diseases, it can be used to determine the carrier status of the fetus through the detection of a paternally inherited disease allele in cases where maternal and paternal mutated alleles differ. Conditions for non-invasive identification of fetal paternally inherited mutations in maternal plasma were developed by two independent approaches: coamplification at lower denaturation temperature-PCR (COLD-PCR) and highly sensitive microarrays. Assays were designed for identifying 14 mutations, 7 causing β-thalassaemia and 7 cystic fibrosis. In total, 87 non-invasive prenatal diagnoses were performed by COLD-PCR in 75 couples at risk for β-thalassaemia and 12 for cystic fibrosis. First, to identify the more appropriate methodology for the analysis of minority mutated fetal alleles in maternal plasma, both fast and full COLD-PCR protocols were developed for the most common Italian β-thalassaemia Cd39 and IVSI.110 mutations. In 5 out of 31 samples, no enrichment was obtained with the fast protocol, while full COLD-PCR provided the correct fetal genotypes. Thus, full COLD-PCR protocols were developed for all the remaining mutations and all analyses confirmed the fetal genotypes obtained by invasive prenatal diagnosis. Microarray analysis was performed on 40 samples from 28 couples at risk for β-thalassaemia and 12 for cystic fibrosis. Results were in complete concordance with those obtained by both COLD-PCR and invasive procedures. COLD-PCR and microarray approaches are not expensive, simple to handle, fast and can be easily set up in specialised clinical laboratories where prenatal diagnosis is routinely performed. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Seven functional classes of Barth syndrome mutation.

PubMed

Whited, Kevin; Baile, Matthew G; Currier, Pamela; Claypool, Steven M

2013-02-01

Patients with Barth syndrome (BTHS), a rare X-linked disease, suffer from skeletal and cardiomyopathy and bouts of cyclic neutropenia. The causative gene encodes tafazzin, a transacylase, which is the major determinant of the final acyl chain composition of the mitochondrial-specific phospholipid, CL. In addition to numerous frame shift and splice-site mutations, 36 missense mutations have been associated with BTHS. Previously, we established a BTHS-mutant panel in the yeast Saccharomyces cerevisiae that successfully models 18/21 conserved pathogenic missense mutations and defined the loss-of-function mechanism associated with a subset of the mutant tafazzins. Here, we report the biochemical and cell biological characterization of the rest of the yeast BTHS-mutant panel and in so doing identify three additional modes of tafazzin dysfunction. The largest group of mutant tafazzins is catalytically null, two mutants encode hypomorphic alleles, and another two mutants are temperature sensitive. Additionally, we have expanded the defects associated with previously characterized matrix-mislocalized-mutant tafazzins to include the rapid degradation of aggregation-prone polypeptides that correctly localize to the mitochondrial IMS. In sum, our in-depth characterization of the yeast BTHS-mutant panel has identified seven functional classes of BTHS mutation.

ctDNA Determination of EGFR Mutation Status in European and Japanese Patients with Advanced NSCLC: The ASSESS Study.

PubMed

Reck, Martin; Hagiwara, Koichi; Han, Baohui; Tjulandin, Sergei; Grohé, Christian; Yokoi, Takashi; Morabito, Alessandro; Novello, Silvia; Arriola, Edurne; Molinier, Olivier; McCormack, Rose; Ratcliffe, Marianne; Normanno, Nicola

2016-10-01

To offer patients with EGFR mutation-positive advanced NSCLC appropriate EGFR tyrosine kinase inhibitor treatment, mutation testing of tumor samples is required. However, tissue/cytologic samples are not always available or evaluable. The large, noninterventional diagnostic ASSESS study (NCT01785888) evaluated the utility of circulating free tumor-derived DNA (ctDNA) from plasma for EGFR mutation testing. ASSESS was conducted in 56 centers (in Europe and Japan). Eligible patients (with newly diagnosed locally advanced/metastatic treatment-naive advanced NSCLC) provided diagnostic tissue/cytologic and plasma samples. DNA extracted from tissue/cytologic samples was subjected to EGFR mutation testing using local practices; designated laboratories performed DNA extraction/mutation testing of blood samples. The primary end point was level of concordance of EGFR mutation status between matched tissue/cytologic and plasma samples. Of 1311 patients enrolled, 1288 were eligible. Concordance of mutation status in 1162 matched samples was 89% (sensitivity 46%, specificity 97%, positive predictive value 78%, and negative predictive value 90%). A group of 25 patients with apparent false-positive plasma results was overrepresented for cytologic samples, use of less sensitive tissue testing methodologies, and smoking habits associated with high EGFR mutation frequency, indicative of false-negative tumor results. In cases in which plasma and tumor samples were tested with identical highly sensitive methods, positive predictive value/sensitivity were generally improved. These real-world data suggest that ctDNA is a feasible sample for EGFR mutation analysis. It is important to conduct mutation testing of both tumor and plasma samples in specialized laboratories, using robust/sensitive methods to ensure that patients receive appropriate treatments that target the molecular features of their disease. Copyright © 2016 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

A mutation in the 14 alpha-demethylase gene of Uncinula necator that correlates with resistance to a sterol biosynthesis inhibitor.

PubMed Central

Délye, C; Laigret, F; Corio-Costet, M F

1997-01-01

We investigated the molecular basis of resistance of the obligate biotrophic grape powdery mildew fungus Uncinula necator to sterol demethylation-inhibiting fungicides (DMIs). The sensitivity of 91 single-spore field isolates of U. necator to triadimenol was assessed by using a leaf disc assay. Resistance factors (RF) ranged from 1.8 to 26.0. The gene encoding the target of DMIs (eburicol 14 alpha-demethylase) from five sensitive and seven resistant isolates was cloned and sequenced. A single mutation, leading to the substitution of a phenylalanine residue for a tyrosine residue at position 136, was found in all isolates exhibiting an RF higher than 5. No mutation was found in sensitive or weakly resistant (RF, < 5) isolates. An allele-specific PCR assay was developed to detect the mutation. Among the 91 isolates tested, only isolates with RF higher than 5 carried the mutation. Three of the 19 resistant isolates and all sensitive and weakly resistant isolates did not possess the mutation. The mutation at codon 136 is thus clearly associated with high levels of resistance to triadimenol. PMID:9251183

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

PubMed

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

2013-05-24

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

Genetic Map of Bacteriophage φX174

PubMed Central

Benbow, R. M.; Hutchison, C. A.; Fabricant, J. D.; Sinsheimer, R. L.

1971-01-01

Bacteriophage φX174 temperature-sensitive and nonsense mutations in eight cistrons were mapped by using two-, three-, and four-factor genetic crosses. The genetic map is circular with a total length of 24 × 10−4wt recombinants per progeny phage. The cistron order is D-E-F-G-H-A-B-C. High negative interference is seen, consistent with a small closed circular deoxyribonucleic acid molecule as a genome. PMID:16789129

Muver, a computational framework for accurately calling accumulated mutations.

PubMed

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

2018-05-09

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

Enhancement of the safety of live influenza vaccine by attenuating mutations from cold-adapted hemagglutinin

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

Lee, Yoon Jae; Laboratory of Molecular Medicine, Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul; Vaccine Translational Research Center, Yonsei University, Seoul

In our previous study, X-31ca-based H5N1 LAIVs, in particular, became more virulent in mice than the X-31ca MDV, possibly by the introduction of the surface antigens of highly pathogenic H5N1 influenza virus, implying that additional attenuation is needed in this cases to increase the safety level of the vaccine. In this report we suggest an approach to further increase the safety of LAIV through additional cold-adapted mutations in the hemagglutinin. The cold-adaptation of X-31 virus resulted in four amino acid mutations in the HA. We generated a panel of 7:1 reassortant viruses each carrying the hemagglutinins with individual single aminomore » acid mutations. We examined their phenotypes and found a major attenuating mutation, N81K. This attenuation marker conferred additional temperature-sensitive and attenuation phenotype to the LAIV. Our data indicate that the cold-adapted mutation in the HA confers additional attenuation to the LAIV strain, without compromising its productivity and immune response. - Highlights: • Cold-adaptation process induced four amino acid mutations in the HA of X-31 virus. • The four mutations in the HA also contributed to attenuation of the X-31ca virus • N81K mutation was the most significant marker for the attenuation of X-31ca virus. • Introduction of N81K mutation into H3N2 LAIV further attenuated the vaccine. • This approach provides a useful guideline for enhancing the safety of the LAIVs.« less

Analysis of ESR1 mutation in circulating tumor DNA demonstrates evolution during therapy for metastatic breast cancer

PubMed Central

Schiavon, Gaia; Hrebien, Sarah; Garcia-Murillas, Isaac; Cutts, Rosalind J; Pearson, Alex; Tarazona, Noelia; Fenwick, Kerry; Kozarewa, Iwanka; Lopez-Knowles, Elena; Ribas, Ricardo; Nerurkar, Ashutosh; Osin, Peter; Chandarlapaty, Sarat; Martin, Lesley-Ann; Dowsett, Mitch; Smith, Ian E; Turner, Nicholas C.

2016-01-01

Acquired ESR1 mutations are a major mechanism of resistance to aromatase inhibitors (AI). We developed ultra-high sensitivity multiplexed digital PCR assays for ESR1 mutations in circulating tumor DNA (ctDNA) and used these to investigate the clinical relevance and origin of ESR1 mutations in a cohort of 171 women with advanced breast cancer. ESR1 mutation status in ctDNA showed high concordance with contemporaneous tumor biopsies, and could be assessed in samples shipped at room temperature in preservative tubes without loss of accuracy. ESR1 mutations were found exclusively in patients with estrogen receptor positive breast cancer previously exposed to AI. Patients with ESR1 mutations had a substantially shorter progression-free survival on subsequent AI-based therapy (HR 3.1, 95%CI 1.9-23.1, log rank p=0.0041). ESR1 mutation prevalence differed markedly between patients that were first exposed to AI during the adjuvant and metastatic settings (5.8% (3/52) vs 36.4% (16/44) respectively, p=0.0002). In an independent cohort, ESR1 mutations were identified in 0% (0/32, 95%CI 0-10.9%) tumor biopsies taken after progression on adjuvant AI. In a patient with serial samples taken during metastatic treatment, ESR1 mutation was selected during metastatic AI therapy, to become the dominant clone in the cancer. ESR1 mutations can be robustly identified with ctDNA analysis and predict for resistance to subsequent AI therapy. ESR1 mutations are rarely acquired during adjuvant AI therapy, but are commonly selected by therapy for metastatic disease, providing evidence that the mechanisms of resistance to targeted therapy may be substantially different between the treatment of micro-metastatic and overt metastatic cancer. PMID:26560360

Analysis of ESR1 mutation in circulating tumor DNA demonstrates evolution during therapy for metastatic breast cancer.

PubMed

Schiavon, Gaia; Hrebien, Sarah; Garcia-Murillas, Isaac; Cutts, Rosalind J; Pearson, Alex; Tarazona, Noelia; Fenwick, Kerry; Kozarewa, Iwanka; Lopez-Knowles, Elena; Ribas, Ricardo; Nerurkar, Ashutosh; Osin, Peter; Chandarlapaty, Sarat; Martin, Lesley-Ann; Dowsett, Mitch; Smith, Ian E; Turner, Nicholas C

2015-11-11

Acquired ESR1 mutations are a major mechanism of resistance to aromatase inhibitors (AIs). We developed ultra high-sensitivity multiplex digital polymerase chain reaction assays for ESR1 mutations in circulating tumor DNA (ctDNA) and investigated the clinical relevance and origin of ESR1 mutations in 171 women with advanced breast cancer. ESR1 mutation status in ctDNA showed high concordance with contemporaneous tumor biopsies and was accurately assessed in samples shipped at room temperature in preservative tubes. ESR1 mutations were found exclusively in estrogen receptor-positive breast cancer patients previously exposed to AI. Patients with ESR1 mutations had a substantially shorter progression-free survival on subsequent AI-based therapy [hazard ratio, 3.1; 95% confidence interval (CI), 1.9 to 23.1; P = 0.0041]. ESR1 mutation prevalence differed markedly between patients who were first exposed to AI during the adjuvant and metastatic settings [5.8% (3 of 52) versus 36.4% (16 of 44), respectively; P = 0.0002]. In an independent cohort, ESR1 mutations were identified in 0% (0 of 32; 95% CI, 0 to 10.9) tumor biopsies taken after progression on adjuvant AI. In a patient with serial sampling, ESR1 mutation was selected during metastatic AI therapy to become the dominant clone in the cancer. ESR1 mutations can be robustly identified with ctDNA analysis and predict for resistance to subsequent AI therapy. ESR1 mutations are rarely acquired during adjuvant AI but are commonly selected by therapy for metastatic disease, providing evidence that mechanisms of resistance to targeted therapy may be substantially different between the treatment of micrometastatic and overt metastatic cancer. Copyright © 2015, American Association for the Advancement of Science.

A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis

NASA Technical Reports Server (NTRS)

Garbers, C.; DeLong, A.; Deruere, J.; Bernasconi, P.; Soll, D.; Evans, M. L. (Principal Investigator)

1996-01-01

The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.

Analytical validation of BRAF mutation testing from circulating free DNA using the amplification refractory mutation testing system.

PubMed

Aung, Kyaw L; Donald, Emma; Ellison, Gillian; Bujac, Sarah; Fletcher, Lynn; Cantarini, Mireille; Brady, Ged; Orr, Maria; Clack, Glen; Ranson, Malcolm; Dive, Caroline; Hughes, Andrew

2014-05-01

BRAF mutation testing from circulating free DNA (cfDNA) using the amplification refractory mutation testing system (ARMS) holds potential as a surrogate for tumor mutation testing. Robust assay validation is needed to establish the optimal clinical matrix for measurement and cfDNA-specific mutation calling criteria. Plasma- and serum-derived cfDNA samples from 221 advanced melanoma patients were analyzed for BRAF c.1799T>A (p.V600E) mutation using ARMS in two stages in a blinded fashion. cfDNA-specific mutation calling criteria were defined in stage 1 and validated in stage 2. cfDNA concentrations in serum and plasma, and the sensitivities and specificities of BRAF mutation detection in these two clinical matrices were compared. Sensitivity of BRAF c.1799T>A (p.V600E) mutation detection in cfDNA was increased by using mutation calling criteria optimized for cfDNA (these criteria were adjusted from those used for archival tumor biopsies) without compromising specificity. Sensitivity of BRAF mutation detection in serum was 44% (95% CI, 35% to 53%) and in plasma 52% (95% CI, 43% to 61%). Specificity was 96% (95% CI, 90% to 99%) in both matrices. Serum contains significantly higher total cfDNA than plasma, whereas the proportion of tumor-derived mutant DNA was significantly higher in plasma. Using mutation calling criteria optimized for cfDNA improves sensitivity of BRAF c.1799T>A (p.V600E) mutation detection. The proportion of tumor-derived cfDNA in plasma was significantly higher than in serum. Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

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

PubMed Central

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

2011-01-01

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

Resistance of Adenoviral DNA Replication to Aphidicolin Is Dependent on the 72-Kilodalton DNA-Binding Protein

PubMed Central

Foster, David A.; Hantzopoulos, Petros; Zubay, Geoffrey

1982-01-01

Aphidicolin is a highly specific inhibitor of DNA polymerase α and has been most useful for assessing the role of this enzyme in various replication processes (J. A. Huberman, Cell 23:647-648, 1981). Both nuclear DNA replication and simian virus 40 DNA replication are highly sensitive to this drug (Krokan et al., Biochemistry 18:4431-4443, 1979), whereas mitochondrial DNA synthesis is completely insensitive (Zimmerman et al., J. Biol. Chem. 255:11847-11852, 1980). Adenovirus DNA replication is sensitive to aphidicolin, but only at much higher concentrations. These patterns of sensitivity are seen both in vivo and in vitro (Krokan et al., Biochemistry 18:4431-4443, 1979). A temperature-sensitive mutant of adenovirus type 5 known as H5ts125 is able to complete but not initiate new rounds of replication at nonpermissive temperatures (P. C. van der Vliet and J. S. Sussenbach, Virology 67:415-426, 1975). When cells infected with H5ts125 were shifted from permissive (33°C) to nonpermissive (41°C) conditions, the residual DNA synthesis (elongation) showed a striking increase in sensitivity to aphidicolin. The temperature-sensitive mutation of H5ts125 is in the gene for the 72-kilodalton single-stranded DNA-binding protein. This demonstrated that the increased resistance to aphidicolin shown by adenovirus DNA replication was dependent on that protein. It also supports an elongation role for both DNA polymerase α and the 72-kilodalton single-stranded DNA-binding protein in adenovirus DNA replication. Further support for an elongation role of DNA polymerase α came from experiments with permissive temperature conditions and inhibiting levels of aphidicolin in which it was shown that newly initiated strands failed to elongate to completion. Images PMID:6809958

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

PubMed

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

2014-10-01

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

Suppression of the UV-sensitive phenotype of Escherichia coli recF mutants by recA(Srf) and recA(Tif) mutations requires recJ+.

PubMed Central

Thoms, B; Wackernagel, W

1988-01-01

Mutations in recA, such as recA801(Srf) (suppressor of RecF) or recA441(Tif) (temperature-induced filamentation) partially suppress the deficiency in postreplication repair of UV damage conferred by recF mutations. We observed that spontaneous recA(Srf) mutants accumulated in cultures of recB recC sbcB sulA::Mu dX(Ap lac) lexA51 recF cells because they grew faster than the parental strain. We show that in a uvrA recB+ recC+ genetic background there are two prerequisites for the suppression by recA(Srf) of the UV-sensitive phenotype of recF mutants. (i) The recA(Srf) protein must be provided in increased amounts either by SOS derepression or by a recA operator-constitutive mutation in a lexA(Ind) (no induction of SOS functions) genetic background. (ii) The gene recJ, which has been shown previously to be involved in the recF pathway of recombination and repair, must be functional. The level of expression of recJ in a lexA(Ind) strain suffices for full suppression. Suppression by recA441 at 30 degrees C also depends on recJ+. The hampered induction by UV of the SOS gene uvrA seen in a recF mutant was improved by a recA(Srf) mutation. This improvement did not require recJ+. We suggest that recA(Srf) and recA(Tif) mutant proteins can operate in postreplication repair independent of recF by using the recJ+ function. PMID:2841294

Mutations in proteins of the Conserved Oligomeric Golgi Complex affect polarity, cell wall structure, and glycosylation in the filamentous fungus Aspergillus nidulans.

PubMed

Gremillion, S K; Harris, S D; Jackson-Hayes, L; Kaminskyj, S G W; Loprete, D M; Gauthier, A C; Mercer, S; Ravita, A J; Hill, T W

2014-12-01

We have described two Aspergillus nidulans gene mutations, designated podB1 (polarity defective) and swoP1 (swollen cell), which cause temperature-sensitive defects during polarization. Mutant strains also displayed unevenness and abnormal thickness of cell walls. Un-polarized or poorly-polarized mutant cells were capable of establishing normal polarity after a shift to a permissive temperature, and mutant hyphae shifted from permissive to restrictive temperature show wall and polarity abnormalities in subsequent growth. The mutated genes (podB=AN8226.3; swoP=AN7462.3) were identified as homologues of COG2 and COG4, respectively, each predicted to encode a subunit of the multi-protein COG (Conserved Oligomeric Golgi) Complex involved in retrograde vesicle trafficking in the Golgi apparatus. Down-regulation of COG2 or COG4 resulted in abnormal polarization and cell wall staining. The GFP-tagged COG2 and COG4 homologues displayed punctate, Golgi-like localization. Lectin-blotting indicated that protein glycosylation was altered in the mutant strains compared to the wild type. A multicopy expression experiment showed evidence for functional interactions between the homologues COG2 and COG4 as well as between COG2 and COG3. To date, this work is the first regarding a functional role of the COG proteins in the development of a filamentous fungus. Copyright © 2014 Elsevier Inc. All rights reserved.

Clinical validation of a highly sensitive assay to detect EGFR mutations in plasma cell-free DNA from patients with advanced lung adenocarcinoma.

PubMed

Li, Yuping; Xu, Hanyan; Su, Shanshan; Ye, Junru; Chen, Junjie; Jin, Xuru; Lin, Quan; Zhang, Dongqing; Ye, Caier; Chen, Chengshui

2017-01-01

Circulating tumor DNA (ctDNA) is a promising biomarker for noninvasive epidermal growth factor receptor (EGFR) mutations detection in lung cancer patients, but the existing methods have limitations in sensitivity or in availability. In this study, we evaluated the performance of a novel assay called ADx-SuperARMS in detecting EGFR mutations in plasma cell-free DNA from patients with advanced lung adenocarcinoma. A total of 109 patients with metastatic advanced adenocarcinoma were recruited who provided both blood samples and matched tumor tissue samples. EGFR mutation status in plasma samples were tested with ADx-SuperARMS EGFR assay and tumor tissue samples were tested with ADx-ARMS EGFR assay. The clinical sensitivity, specificity, positive prediction value (PPV), and negative prediction value (NPV) of ADx-SuperARMS EGFR assay were calculated by using EGFR mutation status in tumor tissue as standard reference. A receiver operating characteristic (ROC) analysis was implemented and an area under the curve (AUC) was calculated to evaluate sensitivity and specificity of exon 19 deletion (E19Del) and L858R mutation detection. The objective response rate (ORR) were calculated according to the EGFR mutation status determined by ADx-superARMS as well. 0.2% analytical sensitivity and 100% specificity of the ADx-SuperARMS EGFR assays for EGFR E19Del, L858R, and T790M mutants were confirmed by using a series of diluted cell line DNA. In the clinical study, EGFR mutations were detected in 45.9% (50/109) of the plasma samples and in 56.9% (62/109) of the matched tumor tissue samples. The sensitivity, specificity, PPV and NPV of the ADx-SuperARMS EGFR assay for plasma EGFR mutation detection were 82.0% (50/61), 100% (48/48), 100% (50/50), and 81.4% (48/59), respectively. In ROC analysis, ADx-SuperARMS achieved sensitivity and specificity of 88% and 99% in E19Dels as well as sensitivity and specificity of 89% and 100% in L858R, respectively. Among the 35 patients who were plasma EGFR mutation positive and treated with first generation of EGFR-tyrosine kinase inhibitors (TKIs), 23 (65.7%) achieved partial response, 11 (31.4%) sustained disease, and 1 (2.9%) progressive disease. The ORR and disease control rate (DCR) were 65.7% and 97.1%, respectively. ADx-SuperARMS EGFR assay is likely to be a highly sensitive and specific method to noninvasively detect plasma EGFR mutations of patients with advanced lung adenocarcinoma. The EGFR mutations detected by ADx-SuperARMS EGFR assay could predict the efficacy of the treatment with first generation of EGFR-TKIs. Hence, EGFR blood testing with ADx-SuperARMS could address the unmet clinical needs.

Mutations of cellulose synthase (CESA1) phosphorylation sites modulate anisotropic cell expansion and bidirectional mobility of cellulose synthase.

PubMed

Chen, Shaolin; Ehrhardt, David W; Somerville, Chris R

2010-10-05

The CESA1 component of cellulose synthase is phosphorylated at sites clustered in two hypervariable regions of the protein. Mutations of the phosphorylated residues to Ala (A) or Glu (E) alter anisotropic cell expansion and cellulose synthesis in rapidly expanding roots and hypocotyls. Expression of T166E, S686E, or S688E mutants of CESA1 fully rescued the temperature sensitive cesA1-1 allele (rsw1) at a restrictive temperature whereas mutations to A at these positions caused defects in anisotropic cell expansion. However, mutations to E at residues surrounding T166 (i.e., S162, T165, and S167) caused opposite effects. Live-cell imaging of fluorescently labeled CESA showed close correlations between tissue or cell morphology and patterns of bidirectional motility of CESA complexes in the plasma membrane. In the WT, CESA complexes moved at similar velocities in both directions along microtubule tracks. By contrast, the rate of movement of CESA particles was directionally asymmetric in mutant lines that exhibited abnormal tissue or cell expansion, and the asymmetry was removed upon depolymerizing microtubules with oryzalin. This suggests that phosphorylation of CESA differentially affects a polar interaction with microtubules that may regulate the length or quantity of a subset of cellulose microfibrils and that this, in turn, alters microfibril structure in the primary cell wall resulting in or contributing to the observed defect in anisotropic cell expansion.

Mutations affecting gyrase in Haemophilus influenzae.

PubMed Central

Setlow, J K; Cabrera-Juárez, E; Albritton, W L; Spikes, D; Mutschler, A

1985-01-01

Mutants separately resistant to novobiocin, coumermycin, nalidixic acid, and oxolinic acid contained gyrase activity as measured in vitro that was resistant to the antibiotics, indicating that the mutations represented structural alterations of the enzyme. One Novr mutant contained an altered B subunit of the enzyme, as judged by the ability of a plasmid, pNov1, containing the mutation to complement a temperature-sensitive gyrase B mutation in Escherichia coli and to cause novobiocin resistance in that strain. Three other Novr mutations did not confer antibiotic resistance to the gyrase but appeared to increase the amount of active enzyme in the cell. One of these, novB1, could only act in cis, whereas a new mutation, novC, could act in trans. An RNA polymerase mutation partially substituted for the novB1 mutation, suggesting that novB1 may be a mutation in a promoter region for the B subunit gene. Growth responses of strains containing various combinations of mutations on plasmids or on the chromosome indicated that low-level resistance to novobiocin or coumermycin may have resulted from multiple copies of wild-type genes coding for the gyrase B subunit, whereas high-level resistance required a structural change in the gyrase B gene and was also dependent on alteration in a regulatory region. When there was mismatch at the novB locus, with the novB1 mutation either on a plasmid or the chromosome, and the corresponding wild-type gene present in trans, chromosome to plasmid recombination during transformation was much higher than when the genes matched, probably because plasmid to chromosome recombination, eliminating the plasmid, was inhibited by the mismatch. PMID:2997115

EGFR T790M mutation testing within the osimertinib AURA Phase I study.

PubMed

Dearden, Simon; Brown, Helen; Jenkins, Suzanne; Thress, Kenneth S; Cantarini, Mireille; Cole, Rebecca; Ranson, Malcolm; Jänne, Pasi A

2017-07-01

Reliable epidermal growth factor receptor (EGFR) mutation testing techniques are required to identify eligible patients with EGFR mutation/T790M positive advanced non-small cell lung cancer (NSCLC), for treatment with osimertinib (AZD9291), an oral, potent, irreversible EGFR tyrosine kinase inhibitor (TKI) selective for EGFR-TKI-sensitizing and T790M resistance mutations over wild-type EGFR. There is no current consensus regarding the best method to detect EGFR T790M mutations. The aim of this study was to describe the concordance between local testing, which used a variety of methods, and central testing, using the cobas ® EGFR Mutation Test, for EGFR-sensitizing mutations and the T790M resistance mutation. Tumor samples were obtained from all patients screened for inclusion onto the osimertinib Phase I expansion component of the AURA Phase I/II study (NCT01802632). Samples underwent central laboratory testing for EGFR-sensitizing mutations and T790M resistance mutation using the cobas ® EGFR Mutation Test. Results were compared with local laboratory test results, based on other testing methodologies including Sanger sequencing, therascreen ® , PNAClamp™, and Sequenom MassARRAY ® . Central laboratory testing was successful in 99% of samples passing histopathology review and testing success rates were comparable across the three central laboratories. Concordance between central and local testing for common sensitizing mutations was high (>98%) and concordance for the T790M mutation was also high (>90%). Tumor heterogeneity, along with other technical factors may have influenced this result. Within the osimertinib AURA Phase I study, EGFR mutation testing across three centralized laboratories using the cobas ® EGFR Mutation Test was feasible and successful, with strong concordance between local and central laboratory results, including for T790M. The cobas ® EGFR Mutation Test has subsequently been approved as the companion diagnostic test for osimertinib in the USA and Japan. Copyright © 2017 Elsevier B.V. All rights reserved.

A single base change in the acceptor stem of tRNA(3Leu) confers resistance upon Escherichia coli to the calmodulin inhibitor, 48/80.

PubMed Central

Chen, M X; Bouquin, N; Norris, V; Casarégola, S; Séror, S J; Holland, I B

1991-01-01

We have isolated several classes of spontaneous mutants resistant to the calmodulin inhibitor 48/80 which inhibits cell division in Escherichia coli K12. Several mutants were also temperature sensitive for growth and this property was exploited to clone a DNA fragment from an E. coli gene library restoring growth at 42 degrees C and drug sensitivity at 30 degrees C in one such mutant. Physical and genetic mapping confirmed that both the mutation and the cloned DNA were located at 15.5 min on the E. coli chromosome at a locus designated feeB. By subcloning, complementation analysis and sequencing, the feeB locus was identified as identical to the tRNA(CUALEU) gene. When the mutant locus was isolated and sequenced, the mutation was confirmed as a single base change, C to A, at position 77 in the acceptor stem of this rare Leu tRNA. In other studies we obtained evidence that this mutant tRNA, recognizing the rare Leu codon, CUA, was defective in translation at both permissive and non-permissive temperatures. The feeB1 mutant is defective in division and shows a reduced growth rate at non-permissive temperature. We discuss the possibility that the mutant tRNA(3Leu) is limiting for the synthesis of a polypeptide(s), requiring several CUA codons for translation which in turn regulates in some way the level or activity of the drug target, a putative cell cycle protein. Images PMID:1915285

Contribution of mammography to MRI screening in BRCA mutation carriers by BRCA status and age: individual patient data meta-analysis

PubMed Central

Phi, Xuan-Anh; Saadatmand, Sepideh; De Bock, Geertruida H; Warner, Ellen; Sardanelli, Francesco; Leach, Martin O; Riedl, Christopher C; Trop, Isabelle; Hooning, Maartje J; Mandel, Rodica; Santoro, Filippo; Kwan-Lim, Gek; Helbich, Thomas H; Tilanus-Linthorst, Madeleine MA; van den Heuvel, Edwin R; Houssami, Nehmat

2016-01-01

Background: We investigated the additional contribution of mammography to screening accuracy in BRCA1/2 mutation carriers screened with MRI at different ages using individual patient data from six high-risk screening trials. Methods: Sensitivity and specificity of MRI, mammography and the combination of these tests were compared stratified for BRCA mutation and age using generalised linear mixed models with random effect for studies. Number of screens needed (NSN) for additional mammography-only detected cancer was estimated. Results: In BRCA1/2 mutation carriers of all ages (BRCA1=1219 and BRCA2=732), adding mammography to MRI did not significantly increase screening sensitivity (increased by 3.9% in BRCA1 and 12.6% in BRCA2 mutation carriers, P>0.05). However, in women with BRCA2 mutation younger than 40 years, one-third of breast cancers were detected by mammography only. Number of screens needed for mammography to detect one breast cancer not detected by MRI was much higher for BRCA1 compared with BRCA2 mutation carriers at initial and repeat screening. Conclusions: Additional screening sensitivity from mammography above that from MRI is limited in BRCA1 mutation carriers, whereas mammography contributes to screening sensitivity in BRCA2 mutation carriers, especially those ⩽40 years. The evidence from our work highlights that a differential screening schedule by BRCA status is worth considering. PMID:26908327

Contribution of mammography to MRI screening in BRCA mutation carriers by BRCA status and age: individual patient data meta-analysis.

PubMed

Phi, Xuan-Anh; Saadatmand, Sepideh; De Bock, Geertruida H; Warner, Ellen; Sardanelli, Francesco; Leach, Martin O; Riedl, Christopher C; Trop, Isabelle; Hooning, Maartje J; Mandel, Rodica; Santoro, Filippo; Kwan-Lim, Gek; Helbich, Thomas H; Tilanus-Linthorst, Madeleine M A; van den Heuvel, Edwin R; Houssami, Nehmat

2016-03-15

We investigated the additional contribution of mammography to screening accuracy in BRCA1/2 mutation carriers screened with MRI at different ages using individual patient data from six high-risk screening trials. Sensitivity and specificity of MRI, mammography and the combination of these tests were compared stratified for BRCA mutation and age using generalised linear mixed models with random effect for studies. Number of screens needed (NSN) for additional mammography-only detected cancer was estimated. In BRCA1/2 mutation carriers of all ages (BRCA1 = 1,219 and BRCA2 = 732), adding mammography to MRI did not significantly increase screening sensitivity (increased by 3.9% in BRCA1 and 12.6% in BRCA2 mutation carriers, P > 0.05). However, in women with BRCA2 mutation younger than 40 years, one-third of breast cancers were detected by mammography only. Number of screens needed for mammography to detect one breast cancer not detected by MRI was much higher for BRCA1 compared with BRCA2 mutation carriers at initial and repeat screening. Additional screening sensitivity from mammography above that from MRI is limited in BRCA1 mutation carriers, whereas mammography contributes to screening sensitivity in BRCA2 mutation carriers, especially those ⩽ 40 years. The evidence from our work highlights that a differential screening schedule by BRCA status is worth considering.

Highly Sensitive Droplet Digital PCR Method for Detection of EGFR-Activating Mutations in Plasma Cell-Free DNA from Patients with Advanced Non-Small Cell Lung Cancer.

PubMed

Zhu, Guanshan; Ye, Xin; Dong, Zhengwei; Lu, Ya Chao; Sun, Yun; Liu, Yi; McCormack, Rose; Gu, Yi; Liu, Xiaoqing

2015-05-01

Epidermal growth factor receptor (EGFR) mutation testing in plasma cell-free DNA from lung cancer patients is an emerging clinical tool. However, compared with tissue testing, the sensitivity of plasma testing is not yet satisfactory because of the highly fragmented nature of plasma cell-free DNA, low fraction of tumor DNA, and limitations of available detection technologies. We therefore developed a highly sensitive and specific droplet digital PCR method for plasma EGFR mutation (exon19 deletions and L858R) testing. Plasma from 86 EGFR-tyrosine kinase inhibitor-naive lung cancer patients was tested and compared with EGFR mutation status of matched tumor tissues tested by amplification refractory mutation system. By using EGFR mutation-positive cell DNA, we optimized the droplet digital PCR assays to reach 0.04% sensitivity. The plasma testing sensitivity and specificity, compared with the matched tumor tissues tested by amplification refractory mutation system, were 81.82% (95% CI, 59.72%-94.81%) and 98.44% (95% CI, 91.60%-99.96%), respectively, for exon19 deletions, with 94.19% concordance rate (κ = 0.840; 95% CI, 0.704-0.976; P < 0.0001), whereas they were 80.00% (95% CI, 51.91%-95.67%) and 95.77% (95% CI, 88.14%-99.12%), respectively, for L858R, with 93.02% concordance rate (κ = 0.758; 95% CI, 0.571-0.945; P < 0.0001). The reported highly sensitive and specific droplet digital PCR assays for EGFR mutation detection have potential in clinical blood testing. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

An automated microfluidic DNA microarray platform for genetic variant detection in inherited arrhythmic diseases.

PubMed

Huang, Shu-Hong; Chang, Yu-Shin; Juang, Jyh-Ming Jimmy; Chang, Kai-Wei; Tsai, Mong-Hsun; Lu, Tzu-Pin; Lai, Liang-Chuan; Chuang, Eric Y; Huang, Nien-Tsu

2018-03-12

In this study, we developed an automated microfluidic DNA microarray (AMDM) platform for point mutation detection of genetic variants in inherited arrhythmic diseases. The platform allows for automated and programmable reagent sequencing under precise conditions of hybridization flow and temperature control. It is composed of a commercial microfluidic control system, a microfluidic microarray device, and a temperature control unit. The automated and rapid hybridization process can be performed in the AMDM platform using Cy3 labeled oligonucleotide exons of SCN5A genetic DNA, which produces proteins associated with sodium channels abundant in the heart (cardiac) muscle cells. We then introduce a graphene oxide (GO)-assisted DNA microarray hybridization protocol to enable point mutation detection. In this protocol, a GO solution is added after the staining step to quench dyes bound to single-stranded DNA or non-perfectly matched DNA, which can improve point mutation specificity. As proof-of-concept we extracted the wild-type and mutant of exon 12 and exon 17 of SCN5A genetic DNA from patients with long QT syndrome or Brugada syndrome by touchdown PCR and performed a successful point mutation discrimination in the AMDM platform. Overall, the AMDM platform can greatly reduce laborious and time-consuming hybridization steps and prevent potential contamination. Furthermore, by introducing the reciprocating flow into the microchannel during the hybridization process, the total assay time can be reduced to 3 hours, which is 6 times faster than the conventional DNA microarray. Given the automatic assay operation, shorter assay time, and high point mutation discrimination, we believe that the AMDM platform has potential for low-cost, rapid and sensitive genetic testing in a simple and user-friendly manner, which may benefit gene screening in medical practice.

Diagnostic algorithm for detection of targetable driver mutations in lung adenocarcinomas: Comprehensive analyses of 205 cases with immunohistochemistry, real-time PCR and fluorescence in situ hybridization methods.

PubMed

Kao, Hua-Lin; Yeh, Yi-Chen; Lin, Chin-Hsuan; Hsu, Wei-Fang; Hsieh, Wen-Yu; Ho, Hsiang-Ling; Chou, Teh-Ying

2016-11-01

Analysis of the targetable driver mutations is now recommended in all patients with advanced lung adenocarcinoma. Molecular-based methods are usually adopted, however, along with the implementation of highly sensitive and/or mutation-specific antibodies, immunohistochemistry (IHC) has been considered an alternative method for identifying driver mutations in lung adenocarcinomas. A total of 205 lung adenocarcinomas were examined for EGFR mutations and ALK and ROS1 rearrangements using real-time PCR, fluorescence in situ hybridization (FISH) and IHC in parallel. The performance of different commercially available IHC antibody clones toward targetable driver mutations was evaluated. The association between these driver mutations and clinicopathological characteristics was also analyzed. In 205 cases we studied, 58.5% were found to harbor EGFR mutations, 6.3% ALK rearrangements and 1.0% ROS1 rearrangements. Compared to molecular-based methods, IHC of EGFR mutations showed an excellent specificity but the sensitivity is suboptimal, while IHC of ALK and ROS1 rearrangements demonstrated high sensitivity and specificity. No significant difference regarding the performance of different antibody clones toward these driver mutations was observed, except that clone SP125 showed a higher sensitivity than 43B2 in the detection of p.L858R of EGFR. In circumstances such as poor quality of nucleic acids or low content of tumor cells, IHC of EGFR mutation-specific antibodies could be used as an alternative method. Patients negative for EGFR mutations are subjected to further analysis on ALK and ROS1 rearrangements using IHC methods. Herein, we proposed a lung adenocarcinoma testing algorithm for the application of IHC in therapeutic diagnosis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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

PubMed

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

2013-04-08

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

Selective disruption of high sensitivity heat activation but not capsaicin activation of TRPV1 channels by pore turret mutations

PubMed Central

Cui, Yuanyuan; Yang, Fan; Cao, Xu; Yarov-Yarovoy, Vladimir

2012-01-01

The capsaicin receptor transient receptor potential vanilloid (TRPV)1 is a highly heat-sensitive ion channel. Although chemical activation and heat activation of TRPV1 elicit similar pungent, painful sensation, the molecular mechanism underlying synergistic activation remains mysterious. In particular, where the temperature sensor is located and whether heat and capsaicin share a common activation pathway are debated. To address these fundamental issues, we searched for channel mutations that selectively affected one form of activation. We found that deletion of the first 10 amino acids of the pore turret significantly reduced the heat response amplitude and shifted the heat activation threshold, whereas capsaicin activation remained unchanged. Removing larger portions of the turret disrupted channel function. Introducing an artificial sequence to replace the deleted region restored sensitive capsaicin activation in these nonfunctional channels. The heat activation, however, remained significantly impaired, with the current exhibiting diminishing heat sensitivity to a level indistinguishable from that of a voltage-gated potassium channel, Kv7.4. Our results demonstrate that heat and capsaicin activation of TRPV1 are structurally and mechanistically distinct processes, and the pore turret is an indispensible channel structure involved in the heat activation process but is not part of the capsaicin activation pathway. Synergistic effect of heat and capsaicin on TRPV1 activation may originate from convergence of the two pathways on a common activation gate. PMID:22412190

Efficacy and safety of cytotoxic drug chemotherapy after first-line EGFR-TKI treatment in elderly patients with non-small-cell lung cancer harboring sensitive EGFR mutations.

PubMed

Imai, Hisao; Minemura, Hiroyuki; Sugiyama, Tomohide; Yamada, Yutaka; Kaira, Kyoichi; Kanazawa, Kenya; Kasai, Takashi; Kaburagi, Takayuki; Minato, Koichi

2018-05-08

Epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is effective as first-line chemotherapy for patients with advanced non-small-cell lung cancer (NSCLC) harboring sensitive EGFR mutations. However, whether the efficacy of second-line cytotoxic drug chemotherapy after first-line EGFR-TKI treatment is similar to that of first-line cytotoxic drug chemotherapy in elderly patients aged ≥ 75 years harboring sensitive EGFR mutations is unclear. Therefore, we aimed to investigate the efficacy and safety of cytotoxic drug chemotherapy after first-line EGFR-TKI treatment in elderly patients with NSCLC harboring sensitive EGFR mutations. We retrospectively evaluated the clinical effects and safety profiles of second-line cytotoxic drug chemotherapy after first-line EGFR-TKI treatment in elderly patients with NSCLC harboring sensitive EGFR mutations (exon 19 deletion/exon 21 L858R mutation). Between April 2008 and December 2015, 78 elderly patients with advanced NSCLC harboring sensitive EGFR mutations received first-line EGFR-TKI at four Japanese institutions. Baseline characteristics, regimens, responses to first- and second-line treatments, whether or not patients received subsequent treatment, and if not, the reasons for non-administration were recorded. Overall, 20 patients with a median age of 79.5 years (range 75-85 years) were included in our analysis. The overall response, disease control, median progression-free survival, and overall survival rates were 15.0, 60.0%, 2.4, and 13.2 months, respectively. Common adverse events included leukopenia, neutropenia, anemia, thrombocytopenia, malaise, and anorexia. Major grade 3 or 4 toxicities included leukopenia (25.0%) and neutropenia (45.0%). No treatment-related deaths were noted. Second-line cytotoxic drug chemotherapy after first-line EGFR-TKI treatment among elderly patients with NSCLC harboring sensitive EGFR mutations was effective and safe and showed equivalent outcomes to first-line cytotoxic drug chemotherapy.

NS1 Protein Amino Acid Changes D189N and V194I Affect Interferon Responses, Thermosensitivity, and Virulence of Circulating H3N2 Human Influenza A Viruses

PubMed Central

Nogales, Aitor; Martinez-Sobrido, Luis

2016-01-01

ABSTRACT Influenza virus NS1 protein is a nonstructural, multifunctional protein that counteracts host innate immune responses, modulating virus pathogenesis. NS1 protein variability in subjects infected with H3N2 influenza A viruses (IAVs) during the 2010/2011 season was analyzed, and amino acid changes in residues 86, 189, and 194 were found. The consequences of these mutations for the NS1-mediated inhibition of IFN responses and the pathogenesis of the virus were evaluated, showing that NS1 mutations D189N and V194I impaired the ability of the NS1 protein to inhibit general gene expression, most probably because these mutations decreased the binding of NS1 to the cleavage and polyadenylation specificity factor 30 (CPSF30). A recombinant A/Puerto Rico/8/34 (PR8) H1N1 virus encoding the H3N2 NS1-D189N protein was slightly attenuated, whereas the virus encoding the H3N2 NS1-V194I protein was further attenuated in mice. The higher attenuation of this virus could not be explained by differences in the ability of the two NS1 proteins to counteract host innate immune responses, indicating that another factor must be responsible. In fact, we showed that the virus encoding the H3N2 NS1-V194I protein demonstrated a temperature-sensitive (ts) phenotype, providing a most likely explanation for the stronger attenuation observed. As far as we know, this is the first description of a mutation in NS1 residue 194 conferring a ts phenotype. These studies are relevant in order to identify new residues important for NS1 functions and in human influenza virus surveillance to assess mutations affecting the pathogenicity of circulating viruses. IMPORTANCE Influenza viral infections represent a serious public health problem, with influenza virus causing a contagious respiratory disease that is most effectively prevented through vaccination. The multifunctional nonstructural protein 1 (NS1) is the main viral factor counteracting the host antiviral response. Therefore, influenza virus surveillance to identify new mutations in the NS1 protein affecting the pathogenicity of the circulating viruses is highly important. In this work, we evaluated amino acid variability in the NS1 proteins from H3N2 human seasonal viruses and the effect of the mutations on innate immune responses and virus pathogenesis. NS1 mutations D189N and V194I impaired the ability of the NS1 protein to inhibit general gene expression, and recombinant viruses harboring these mutations were attenuated in a mouse model of influenza infection. Interestingly, a virus encoding the H3N2 NS1-V194I protein demonstrated a temperature-sensitive phenotype, further attenuating the virus in vivo. PMID:28003482

NS1 Protein Amino Acid Changes D189N and V194I Affect Interferon Responses, Thermosensitivity, and Virulence of Circulating H3N2 Human Influenza A Viruses.

PubMed

Nogales, Aitor; Martinez-Sobrido, Luis; Topham, David J; DeDiego, Marta L

2017-03-01

Influenza virus NS1 protein is a nonstructural, multifunctional protein that counteracts host innate immune responses, modulating virus pathogenesis. NS1 protein variability in subjects infected with H3N2 influenza A viruses (IAVs) during the 2010/2011 season was analyzed, and amino acid changes in residues 86, 189, and 194 were found. The consequences of these mutations for the NS1-mediated inhibition of IFN responses and the pathogenesis of the virus were evaluated, showing that NS1 mutations D189N and V194I impaired the ability of the NS1 protein to inhibit general gene expression, most probably because these mutations decreased the binding of NS1 to the cleavage and polyadenylation specificity factor 30 (CPSF30). A recombinant A/Puerto Rico/8/34 (PR8) H1N1 virus encoding the H3N2 NS1-D189N protein was slightly attenuated, whereas the virus encoding the H3N2 NS1-V194I protein was further attenuated in mice. The higher attenuation of this virus could not be explained by differences in the ability of the two NS1 proteins to counteract host innate immune responses, indicating that another factor must be responsible. In fact, we showed that the virus encoding the H3N2 NS1-V194I protein demonstrated a temperature-sensitive (ts) phenotype, providing a most likely explanation for the stronger attenuation observed. As far as we know, this is the first description of a mutation in NS1 residue 194 conferring a ts phenotype. These studies are relevant in order to identify new residues important for NS1 functions and in human influenza virus surveillance to assess mutations affecting the pathogenicity of circulating viruses. IMPORTANCE Influenza viral infections represent a serious public health problem, with influenza virus causing a contagious respiratory disease that is most effectively prevented through vaccination. The multifunctional nonstructural protein 1 (NS1) is the main viral factor counteracting the host antiviral response. Therefore, influenza virus surveillance to identify new mutations in the NS1 protein affecting the pathogenicity of the circulating viruses is highly important. In this work, we evaluated amino acid variability in the NS1 proteins from H3N2 human seasonal viruses and the effect of the mutations on innate immune responses and virus pathogenesis. NS1 mutations D189N and V194I impaired the ability of the NS1 protein to inhibit general gene expression, and recombinant viruses harboring these mutations were attenuated in a mouse model of influenza infection. Interestingly, a virus encoding the H3N2 NS1-V194I protein demonstrated a temperature-sensitive phenotype, further attenuating the virus in vivo . Copyright © 2017 American Society for Microbiology.

New Late Gene, dar, Involved in DNA Replication of Bacteriophage T4 I. Isolation, Characterization, and Genetic Location.

PubMed

Wu, J R; Yeh, Y C

1975-05-01

Suppressors of gene 59-defective mutants were isolated by screening spontaneous, temperature-sensitive (ts) revertants of the amber mutant, amC5, in gene 59. Six ts revertants were isolated. No gene 59-defective ts recombinant was obtained by crossing each ts revertant with the wild type, T4D. However, suppressors of gene 59-defective mutants were obtained from two of these ts revertants. These suppressor mutants are referred to as dar (DNA arrested restoration). dar mutants specifically restored the abnormalities, both in DNA synthesis and burst size, caused by gene 59-defective mutants to normal levels. It is unlikely that dar mutants are nonsense suppressors since theý failed to suppress amber mutations in 11 other genes investigated. The genetic expression of dar is controlled by gene 55; therefore, dar is a late gene. The genetic location of dar has been mapped between genes 24 and 25, a region contiguous to late genes. dar appears to be another nonessential gene of T4 since burst sizes of dar were almost identical to those of the wild type. Mutations in dar did not affect genetic recombination and repair of UV-damaged DNA, but caused a sensitivity to hydroxyurea in progeny formation. The effect of the dar mutation on host DNA degradation cannot account for its hydroxyurea sensitivity. dar mutant alleles were recessive to the wild-type allele as judged by restoration of arrested DNA synthesis. The possible mechanisms for the suppression of defects in gene 59 are discussed.

KRAS and BRAF mutation status in circulating colorectal tumor cells and their correlation with primary and metastatic tumor tissue.

PubMed

Mostert, Bianca; Jiang, Yuqiu; Sieuwerts, Anieta M; Wang, Haiying; Bolt-de Vries, Joan; Biermann, Katharina; Kraan, Jaco; Lalmahomed, Zarina; van Galen, Anne; de Weerd, Vanja; van der Spoel, Petra; Ramírez-Moreno, Raquel; Verhoef, Cornelis; Ijzermans, Jan N M; Wang, Yixin; Gratama, Jan-Willem; Foekens, John A; Sleijfer, Stefan; Martens, John W M

2013-07-01

Although anti-EGFR therapy has established efficacy in metastatic colorectal cancer, only 10-20% of unselected patients respond. This is partly due to KRAS and BRAF mutations, which are currently assessed in the primary tumor. To improve patient selection, assessing mutation status in circulating tumor cells (CTCs), which possibly better represent metastases than the primary tumor, could be advantageous. We investigated the feasibility of KRAS and BRAF mutation detection in colorectal CTCs by comparing three sensitive methods and compared mutation status in matching primary tumor, liver metastasis and CTCs. CTCs were isolated from blood drawn from 49 patients before liver resection using CellSearch™. DNA and RNA was isolated from primary tumors, metastases and CTCs. Mutations were assessed by co-amplification at lower denaturation temperature-PCR (Transgenomic™), real-time PCR (EntroGen™) and nested Allele-Specific Blocker (ASB-)PCR and confirmed by Sanger sequencing. In 43 of the 49 patients, tissue RNA and DNA was of sufficient quantity and quality. In these 43 patients, discordance between primary and metastatic tumor was 23% for KRAS and 7% for BRAF mutations. RNA and DNA from CTCs was available from 42 of the 43 patients, in which ASB-PCR was able to detect the most mutations. Inconclusive results in patients with low CTC counts limited the interpretation of discrepancies between tissue and CTCs. Determination of KRAS and BRAF mutations in CTCs is challenging but feasible. Of the tested methods, nested ASB-PCR, enabling detection of KRAS and BRAF mutations in patients with as little as two CTCs, seems to be superior. Copyright © 2012 UICC.

Ubc9 is required for damage-tolerance and damage-induced interchromosomal homologous recombination in S. cerevisiae.

PubMed

Maeda, Daisuke; Seki, Masayuki; Onoda, Fumitoshi; Branzei, Dana; Kawabe, Yoh-Ichi; Enomoto, Takemi

2004-03-04

Ubc9 is an enzyme involved in the conjugation of small ubiquitin related modifier (SUMO) to target proteins. A Saccharomyces cerevisiae ubc9 temperature sensitive (ts) mutant showed higher sensitivity to various DNA damaging agents such as methylmethanesulfonate (MMS) and UV at a semi-permissive temperature than wild-type cells. The sensitivity of ubc9ts cells was not suppressed by the introduction of a mutated UBC9 gene, UBC9-C93S, whose product is unable to covalently bind to SUMO and consequently fails to conjugate SUMO to target proteins. Diploid ubc9ts cells were more sensitive to various DNA damaging agents than haploid ubc9ts cells suggesting the involvement of homologous recombination in the sensitivity of ubc9ts cells. The frequency of interchromosomal recombination between heteroalleles, his1-1/his1-7 loci, in wild-type cells was remarkably increased upon exposure to MMS or UV. Although the frequency of spontaneous interchromosomal recombination between the heteroalleles in ubc9ts cells was almost the same as that of wild-type cells, no induction of interchromosomal recombination was observed in ubc9ts cells upon exposure to MMS or UV. Copyright 2003 Elsevier B.V.

Viral chimeras decrypt the role of enterovirus capsid proteins in viral tropism, acid sensitivity and optimal growth temperature

PubMed Central

Royston, Léna; Essaidi-Laziosi, Manel; Piuz, Isabelle; Geiser, Johan; Huang, Song; Kaiser, Laurent; Garcin, Dominique

2018-01-01

Despite their genetic similarities, enteric and respiratory enteroviruses (EVs) have highly heterogeneous biophysical properties and cause a vast diversity of human pathologies. In vitro differences include acid sensitivity, optimal growth temperature and tissue tropism, which reflect a preferential in vivo replication in the respiratory or gastrointestinal tract and are thus key determinants of EV virulence. To investigate the underlying cause of these differences, we generated chimeras at the capsid-level between EV-D68 (a respiratory EV) and EV-D94 (an enteric EV). Although some chimeras were nonfunctional, EV-D94 with both the capsid and 2A protease or the capsid only of EV-D68 were both viable. Using this latter construct, we performed several functional assays, which indicated that capsid proteins determine acid sensitivity and tropism in cell lines and in respiratory, intestinal and neural tissues. Additionally, capsid genes were shown to also participate in determining the optimal growth temperature, since EV-D94 temperature adaptation relied on single mutations in VP1, while constructs with EV-D68 capsid could not adapt to higher temperatures. Finally, we demonstrate that EV-D68 maintains residual binding-capacity after acid-treatment despite a loss of infectivity. In contrast, non-structural rather than capsid proteins modulate the innate immune response in tissues. These unique biophysical insights expose another layer in the phenotypic diversity of one of world’s most prevalent pathogens and could aid target selection for vaccine or antiviral development. PMID:29630666

Fine Structure Genetic and Physical Map of the Gene 3 to 10 Region of the Bacteriophage P22 Chromosome

PubMed Central

Casjens, S.; Eppler, K.; Sampson, L.; Parr, R.; Wyckoff, E.

1991-01-01

The mechanism by which dsDNA is packaged by viruses is not yet understood in any system. Bacteriophage P22 has been a productive system in which to study the molecular genetics of virus particle assembly and DNA packaging. Only five phage encoded proteins, the products of genes 3, 2, 1, 8 and 5, are required for packaging the virus chromosome inside the coat protein shell. We report here the construction of a detailed genetic and physical map of these genes, the neighboring gene 4 and a portion of gene 10, in which 289 conditional lethal amber, opal, temperature sensitive and cold sensitive mutations are mapped into 44 small (several hundred base pair) intervals of known sequence. Knowledge of missense mutant phenotypes and information on the location of these mutations allows us to begin the assignment of partial protein functions to portions of these genes. The map and mapping strains will be of use in the further genetic dissection of the P22 DNA packaging and prohead assembly processes. PMID:2029965

Validation and comparison of two NGS assays for the detection of EGFR T790M resistance mutation in liquid biopsies of NSCLC patients.

PubMed

Vollbrecht, Claudia; Lehmann, Annika; Lenze, Dido; Hummel, Michael

2018-04-06

Analysis of circulating cell-free DNA (cfDNA) derived from peripheral blood ("liquid biopsy") is an attractive alternative to identify non-small cell lung cancer (NSCLC) patients with the EGFR T790M mutation eligible for 3rd generation tyrosine kinase inhibitor therapy. We evaluated two PCR-based next generation sequencing (NGS) approaches, one including unique molecular identifiers (UMI), with focus on highly sensitive EGFR T790M mutation detection. Therefore, we extracted and sequenced cfDNA from synthetic plasma samples spiked with mutated DNA at decreasing allele frequencies and from 21 diagnostic NSCLC patients. Data evaluation was performed to determine the limit of detection (LoD), accuracy, specificity and sensitivity of both assays. Considering all tested reference dilutions and mutations the UMI assay performed best in terms of LoD (1% vs. 5%), sensitivity (95.8% vs. 81.3%), specificity (100% vs. 93.8%) and accuracy (96.9% vs. 84.4%). Comparing mutation status of diagnostic samples with both assays showed 81.3% concordance with primary mutation verifiable in 52% of cases. EGFR T790M was detected concordantly in 6/7 patients with allele frequencies from 0.1% to 27%. In one patient, the T790M mutation was exclusively detectable with the UMI assay. Our data demonstrate that both assays are applicable as multi-biomarker NGS tools enabling the simultaneous detection of primary EGFR driver and resistance mutations. However, for mutations with low allelic frequencies the use of NGS panels with UMI facilitates a more sensitive and reliable detection.

Validation and comparison of two NGS assays for the detection of EGFR T790M resistance mutation in liquid biopsies of NSCLC patients

PubMed Central

Vollbrecht, Claudia; Lehmann, Annika; Lenze, Dido; Hummel, Michael

2018-01-01

Analysis of circulating cell-free DNA (cfDNA) derived from peripheral blood (“liquid biopsy”) is an attractive alternative to identify non-small cell lung cancer (NSCLC) patients with the EGFR T790M mutation eligible for 3rd generation tyrosine kinase inhibitor therapy. We evaluated two PCR-based next generation sequencing (NGS) approaches, one including unique molecular identifiers (UMI), with focus on highly sensitive EGFR T790M mutation detection. Therefore, we extracted and sequenced cfDNA from synthetic plasma samples spiked with mutated DNA at decreasing allele frequencies and from 21 diagnostic NSCLC patients. Data evaluation was performed to determine the limit of detection (LoD), accuracy, specificity and sensitivity of both assays. Considering all tested reference dilutions and mutations the UMI assay performed best in terms of LoD (1% vs. 5%), sensitivity (95.8% vs. 81.3%), specificity (100% vs. 93.8%) and accuracy (96.9% vs. 84.4%). Comparing mutation status of diagnostic samples with both assays showed 81.3% concordance with primary mutation verifiable in 52% of cases. EGFR T790M was detected concordantly in 6/7 patients with allele frequencies from 0.1% to 27%. In one patient, the T790M mutation was exclusively detectable with the UMI assay. Our data demonstrate that both assays are applicable as multi-biomarker NGS tools enabling the simultaneous detection of primary EGFR driver and resistance mutations. However, for mutations with low allelic frequencies the use of NGS panels with UMI facilitates a more sensitive and reliable detection. PMID:29719623

Rapid and accurate detection of KRAS mutations in colorectal cancers using the isothermal-based optical sensor for companion diagnostics

PubMed Central

Koo, Bonhan; Lee, Tae Yoon; Lee, Jeong Hoon; Shin, Yong; Lim, Seok-Byung

2017-01-01

Although KRAS mutational status testing is becoming a companion diagnostic tool for managing patients with colorectal cancer (CRC), there are still several difficulties when analyzing KRAS mutations using the existing assays, particularly with regard to low sensitivity, its time-consuming, and the need for large instruments. We developed a rapid, sensitive, and specific mutation detection assay based on the bio-photonic sensor termed ISAD (isothermal solid-phase amplification/detection), and used it to analyze KRAS gene mutations in human clinical samples. To validate the ISAD-KRAS assay for use in clinical diagnostics, we examined for hotspot KRAS mutations (codon 12 and codon 13) in 70 CRC specimens using PCR and direct sequencing methods. In a serial dilution study, ISAD-KRAS could detect mutations in a sample containing only 1% of the mutant allele in a mixture of wild-type DNA, whereas both PCR and direct sequencing methods could detect mutations in a sample containing approximately 30% of mutant cells. The results of the ISAD-KRAS assay from 70 clinical samples matched those from PCR and direct sequencing, except in 5 cases, wherein ISAD-KRAS could detect mutations that were not detected by PCR and direct sequencing. We also found that the sensitivity and specificity of ISAD-KRAS were 100% within 30 min. The ISAD-KRAS assay provides a rapid, highly sensitive, and label-free method for KRAS mutation testing, and can serve as a robust and near patient testing approach for the rapid detection of patients most likely to respond to anti-EGFR drugs. PMID:29137388

Development of a PCR/LDR/capillary electrophoresis assay with potential for the detection of a beta-thalassemia fetal mutation in maternal plasma.

PubMed

Yi, Ping; Chen, Zhuqin; Yu, Lili; Zheng, Yingru; Liu, Guodong; Xie, Haichang; Zhou, Yuanguo; Zheng, Xiuhui; Han, Jian; Li, Li

2010-08-01

Analysis of fetal DNA in maternal plasma has recently been introduced for non-invasive prenatal diagnosis. We have now investigated the feasibility of polymerase chain reaction (PCR)/ligase detection reaction (LDR)/capillary electrophoresis for the detection of fetal point mutations, such as the beta-thalassemia mutation, IVS2 654(C --> T), in maternal plasma DNA. The sensitivity of LDR/capillary electrophoresis was examined by quantifying the mutant PCR products in the presence of a vast excess of non-mutant competitor template, a situation that mimics the detection of rare fetal mutations in the presence of excess maternal DNA. PCR/LDR/capillary electrophoresis was applied to detect the mutation, IVS2 654(C --> T), in an experimental model at different sensitivity levels and from 10 maternal plasma samples. Our results demonstrated that this approach to detect a low abundance IVS2 654(C --> T) mutation achieved a sensitivity of approximately 1:10,000. The approach was applied to maternal plasma DNA to detect the paternally inherited fetal IVS2 654(C --> T) mutation, and the results were equivalent to those obtained by PCR/reverse dot blot of amniotic fluid cell DNA. PCR/LDR/capillary electrophoresis has a very high sensitivity that can distinguish low abundance single nucleotide differences and can detect paternally inherited fetal point mutations in maternal plasma.

Limited copy number - high resolution melting (LCN-HRM) enables the detection and identification by sequencing of low level mutations in cancer biopsies

PubMed Central

Do, Hongdo; Dobrovic, Alexander

2009-01-01

Background Mutation detection in clinical tumour samples is challenging when the proportion of tumour cells, and thus mutant alleles, is low. The limited sensitivity of conventional sequencing necessitates the adoption of more sensitive approaches. High resolution melting (HRM) is more sensitive than sequencing but identification of the mutation is desirable, particularly when it is important to discriminate false positives due to PCR errors or template degradation from true mutations. We thus developed limited copy number - high resolution melting (LCN-HRM) which applies limiting dilution to HRM. Multiple replicate reactions with a limited number of target sequences per reaction allow low level mutations to be detected. The dilutions used (based on Ct values) are chosen such that mutations, if present, can be detected by the direct sequencing of amplicons with aberrant melting patterns. Results Using cell lines heterozygous for mutations, we found that the mutations were not readily detected when they comprised 10% of total alleles (20% tumour cells) by sequencing, whereas they were readily detectable at 5% total alleles by standard HRM. LCN-HRM allowed these mutations to be identified by direct sequencing of those positive reactions. LCN-HRM was then used to review formalin-fixed paraffin-embedded (FFPE) clinical samples showing discordant findings between sequencing and HRM for KRAS exon 2 and EGFR exons 19 and 21. Both true mutations present at low levels and sequence changes due to artefacts were detected by LCN-HRM. The use of high fidelity polymerases showed that the majority of the artefacts were derived from the damaged template rather than replication errors during amplification. Conclusion LCN-HRM bridges the sensitivity gap between HRM and sequencing and is effective in distinguishing between artefacts and true mutations. PMID:19811662

Limited copy number-high resolution melting (LCN-HRM) enables the detection and identification by sequencing of low level mutations in cancer biopsies.

PubMed

Do, Hongdo; Dobrovic, Alexander

2009-10-08

Mutation detection in clinical tumour samples is challenging when the proportion of tumour cells, and thus mutant alleles, is low. The limited sensitivity of conventional sequencing necessitates the adoption of more sensitive approaches. High resolution melting (HRM) is more sensitive than sequencing but identification of the mutation is desirable, particularly when it is important to discriminate false positives due to PCR errors or template degradation from true mutations.We thus developed limited copy number - high resolution melting (LCN-HRM) which applies limiting dilution to HRM. Multiple replicate reactions with a limited number of target sequences per reaction allow low level mutations to be detected. The dilutions used (based on Ct values) are chosen such that mutations, if present, can be detected by the direct sequencing of amplicons with aberrant melting patterns. Using cell lines heterozygous for mutations, we found that the mutations were not readily detected when they comprised 10% of total alleles (20% tumour cells) by sequencing, whereas they were readily detectable at 5% total alleles by standard HRM. LCN-HRM allowed these mutations to be identified by direct sequencing of those positive reactions.LCN-HRM was then used to review formalin-fixed paraffin-embedded (FFPE) clinical samples showing discordant findings between sequencing and HRM for KRAS exon 2 and EGFR exons 19 and 21. Both true mutations present at low levels and sequence changes due to artefacts were detected by LCN-HRM. The use of high fidelity polymerases showed that the majority of the artefacts were derived from the damaged template rather than replication errors during amplification. LCN-HRM bridges the sensitivity gap between HRM and sequencing and is effective in distinguishing between artefacts and true mutations.

Detection of K-ras gene mutations in feces by magnetic nanoprobe in patients with pancreatic cancer: A preliminary study.

PubMed

Wang, Xiaoguang; Wang, Jingshuai; Chen, Fei; Zhong, Zhengxiang; Qi, Lifeng

2018-01-01

The present study aimed to investigate the feasibility and effectiveness of detecting K-ras mutation by using magnetic nanoparticles in fecal samples of patients with pancreatic cancer at different stages. The novel methodology of K-ras mutation detection was compared to the existing methodology of cancer antigen (CA)19-9 examination. Patients with pancreatic cancer (n=88), pancreatic benign diseases who displayed chronic pancreatitis (n=35), pancreatic mucinous cyst neoplasms (n=10) and pancreatic serous cyst (n=9) admitted to the Department of Surgery, Jiaxing Second Hospital were enrolled in the present study. Fecal samples were collected from all patients, DNA was extracted and magnetic nanoprobe was then used to detect K-ras mutation. The results obtained using the novel magnetic nanoprobe detection technique showed a K-ras mutation rate of 81.8% (72/88) in the patients with pancreatic cancer and 18.5% (10/54) in patients with pancreatic benign diseases. In patients with pancreatic cancer, the K-ras mutation rate was comparable in stages I + IIA and IIB + III + IV (78.9 vs. 84.0%; P>0.05). The sensitivity and specificity of K-ras mutation for detection of pancreatic cancer was 81.8 and 81.5%, respectively. Sixty-eight pancreatic cancer patients had >37 U/ml CA99 with a sensitivity and specificity for pancreatic cancer detection of 77.3 and 77.8%, which was not significantly lower than detection by the fecal K-ras mutations (P>0.05). Combinational detection of fecal K-ras mutations and serum CA19-9 significantly increased the sensitivity regarding pancreatic cancer detection to 97.7% (P<0.05), while the specificity was not enhanced (80.9%; P>0.05) compared with fecal K-ras mutations or CA19-9 alone. The findings showed that the magnetic nanoprobe is able to detect fecal K-ras mutations in different stages of pancreatic cancer, with comparable sensitivity and specificity to CA19-9 examination for differentiating pancreatic cancer. Furthermore, combined detection of CA19-9 and K-ras mutations has enhanced sensitivity compared with CA19-9 alone.

Thermal biology, population fluctuations and implications of temperature extremes for the management of two globally significant insect pests.

PubMed

Nyamukondiwa, Casper; Weldon, Christopher W; Chown, Steven L; le Roux, Peter C; Terblanche, John S

2013-12-01

The link between environmental temperature, physiological processes and population fluctuations is a significant aspect of insect pest management. Here, we explore how thermal biology affects the population abundance of two globally significant pest fruit fly species, Ceratitis capitata (medfly) and C. rosa (Natal fruit fly), including irradiated individuals and those expressing a temperature sensitive lethal (tsl) mutation that are used in the sterile insect technique. Results show that upper and lower lethal temperatures are seldom encountered at the field sites, while critical minimum temperatures for activity and lower developmental thresholds are crossed more frequently. Estimates of abundance revealed that C. capitata are active year-round, but abundance declines markedly during winter. Temporal autocorrelation of average fortnightly trap captures and of development time, estimated from an integrated model to calculate available degree days, show similar seasonal lags suggesting that population increases in early spring occur after sufficient degree-days have accumulated. By contrast, population collapses coincide tightly with increasing frequency of low temperature events that fall below critical minimum temperatures for activity. Individuals of C. capitata expressing the tsl mutation show greater critical thermal maxima and greater longevity under field conditions than reference individuals. Taken together, this evidence suggests that low temperatures limit populations in the Western Cape, South Africa and likely do so elsewhere. Increasing temperature extremes and warming climates generally may extend the season over which these species are active, and could increase abundance. The sterile insect technique may prove profitable as climates change given that laboratory-reared tsl flies have an advantage under warmer conditions. Copyright © 2013 Elsevier Ltd. All rights reserved.

ON A CHANGE IN THE SPECTRUM OF SOMATIC MUTATIONS IN EPHESTIA KUEHNIELLA Z. BY TEMPERATURE TREATMENT BEFORE IRRADITION

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

Loebbecke, E.; Oltmanns, O.

1961-01-01

Pupae were maintained at different temperatures (-7, doses of x rays. The incidence of 4 types of scale mutations (ES 1, ES 2, ES 3, ES 4) in the butterflies was studied. It was found to vary significantly according to temperature. The ratio of ES 1/ES 2 mutations was lowest (5: 1) at -7 un. Concent 85% and highest (11: 1) at 35 un. Concent 85% . The ES 1 mutant showed highest frequency at 25 un. Concent 85% and fell to the lowest value after preincubation at 40 un. Concent 85% . The ES 2 mutant reached its lowestmore » incidence at 35 un. Concent 85% . The ES 3 mutant varied inconsistentiy and ES 4 frequency was oniy slightly dependent on temperature. The preincubation temperature of 40 un. Concent 85% , the lethal limit for the species, generally depressed mutation frequency. The precise reason for the effect of temperature on mutation frequency is unknown but it was previously found that chromosome fragmentation and translocation were reduced at elevated temperatures. (H.H.D.)« less

Secondary Somatic Mutations Restoring RAD51C and RAD51D Associated with Acquired Resistance to the PARP Inhibitor Rucaparib in High-Grade Ovarian Carcinoma

PubMed Central

Kondrashova, Olga; Nguyen, Minh; Shield-Artin, Kristy; Tinker, Anna V.; Teng, Nelson N.H.; Harrell, Maria I.; Kuiper, Michael J.; Ho, Gwo-Yaw; Barker, Holly; Jasin, Maria; Prakash, Rohit; Kass, Elizabeth M.; Sullivan, Meghan R.; Brunette, Gregory J.; Bernstein, Kara A.; Coleman, Robert L.; Floquet, Anne; Friedlander, Michael; Kichenadasse, Ganessan; O'Malley, David M.; Oza, Amit; Sun, James; Robillard, Liliane; Maloney, Lara; Giordano, Heidi; Wakefield, Matthew J.; Kaufmann, Scott H.; Simmons, Andrew D.; Harding, Thomas C.; Raponi, Mitch; McNeish, Iain A.; Swisher, Elizabeth M.; Lin, Kevin K.; Scott, Clare L.

2017-01-01

High-grade epithelial ovarian carcinomas containing mutated BRCA1 or BRCA2 (BRCA1/2) homologous recombination (HR) genes are sensitive to platinum-based chemotherapy and PARP inhibitors (PARPi), while restoration of HR function due to secondary mutations in BRCA1/2 has been recognized as an important resistance mechanism. We sequenced core HR pathway genes in 12 pairs of pretreatment and postprogression tumor biopsy samples collected from patients in ARIEL2 Part 1, a phase II study of the PARPi rucaparib as treatment for platinum-sensitive, relapsed ovarian carcinoma. In 6 of 12 pretreatment biopsies, a truncation mutation in BRCA1, RAD51C, or RAD51D was identified. In five of six paired postprogression biopsies, one or more secondary mutations restored the open reading frame. Four distinct secondary mutations and spatial heterogeneity were observed for RAD51C. In vitro complementation assays and a patient-derived xenograft, as well as predictive molecular modeling, confirmed that resistance to rucaparib was associated with secondary mutations. Significance Analyses of primary and secondary mutations in RAD51C and RAD51D provide evidence for these primary mutations in conferring PARPi sensitivity and secondary mutations as a mechanism of acquired PARPi resistance. PARPi resistance due to secondary mutations underpins the need for early delivery of PARPi therapy and for combination strategies. PMID:28588062

Monoallelic mutation analysis (MAMA) for identifying germline mutations.

PubMed

Papadopoulos, N; Leach, F S; Kinzler, K W; Vogelstein, B

1995-09-01

Dissection of germline mutations in a sensitive and specific manner presents a continuing challenge. In dominantly inherited diseases, mutations occur in only one allele and are often masked by the normal allele. Here we report the development of a sensitive and specific diagnostic strategy based on somatic cell hybridization termed MAMA (monoallelic mutation analysis). We have demonstrated the utility of this strategy in two different hereditary colorectal cancer syndromes, one caused by a defective tumour suppressor gene on chromosome 5 (familial adenomatous polyposis, FAP) and the other caused by a defective mismatch repair gene on chromosome 2 (hereditary non-polyposis colorectal cancer, HNPCC).

Multiplex picodroplet digital PCR to detect KRAS mutations in circulating DNA from the plasma of colorectal cancer patients.

PubMed

Taly, Valerie; Pekin, Deniz; Benhaim, Leonor; Kotsopoulos, Steve K; Le Corre, Delphine; Li, Xinyu; Atochin, Ivan; Link, Darren R; Griffiths, Andrew D; Pallier, Karine; Blons, Hélène; Bouché, Olivier; Landi, Bruno; Hutchison, J Brian; Laurent-Puig, Pierre

2013-12-01

Multiplex digital PCR (dPCR) enables noninvasive and sensitive detection of circulating tumor DNA with performance unachievable by current molecular-detection approaches. Furthermore, picodroplet dPCR facilitates simultaneous screening for multiple mutations from the same sample. We investigated the utility of multiplex dPCR to screen for the 7 most common mutations in codons 12 and 13 of the KRAS (Kirsten rat sarcoma viral oncogene homolog) oncogene from plasma samples of patients with metastatic colorectal cancer. Fifty plasma samples were tested from patients for whom the primary tumor biopsy tissue DNA had been characterized by quantitative PCR. Tumor characterization revealed that 19 patient tumors had KRAS mutations. Multiplex dPCR analysis of the plasma DNA prepared from these samples identified 14 samples that matched the mutation identified in the tumor, 1 sample contained a different KRAS mutation, and 4 samples had no detectable mutation. Among the tumor samples that were wild type for KRAS, 2 KRAS mutations were identified in the corresponding plasma samples. Duplex dPCR (i.e., wild-type and single-mutation assay) was also used to analyze plasma samples from patients with KRAS-mutated tumors and 5 samples expected to contain the BRAF (v-raf murine sarcoma viral oncogene homolog B) V600E mutation. The results for the duplex analysis matched those for the multiplex analysis for KRAS-mutated samples and, owing to its higher sensitivity, enabled detection of 2 additional samples with low levels of KRAS-mutated DNA. All 5 samples with BRAF mutations were detected. This work demonstrates the clinical utility of multiplex dPCR to screen for multiple mutations simultaneously with a sensitivity sufficient to detect mutations in circulating DNA obtained by noninvasive blood collection.

Diagnostic markers for the detection of ovarian cancer in BRCA1 mutation carriers

PubMed Central

Weingartshofer, Sigrid; Rappaport-Fürhauser, Christine; Zeilinger, Robert; Pils, Dietmar; Muhr, Daniela; Braicu, Elena I.; Kastner, Marie-Therese; Tan, Yen Y.; Semmler, Lorenz; Sehouli, Jalid; Singer, Christian F.

2017-01-01

Background Screening for ovarian cancer (OC) in women at high risk consists of a combination of carbohydrate antigen 125 (CA125) and transvaginal ultrasound, despite their low sensitivity and specificity. This could be improved by the combination of several biomarkers, which has been shown in average risk patients but has not been investigated until now in female BRCA mutation carriers. Methods Using a multiplex, bead-based, immunoassay system, we analyzed the concentrations of leptin, prolactin, osteopontin, insulin-like growth factor II, macrophage inhibitory factor, CA125 and human epididymis antigen 4 in 26 healthy wild type women, 26 healthy BRCA1 mutation carriers, 28 wildtype OC patients and 26 OC patients with BRCA1 mutation. Results Using the ROC analysis, we found a high overall sensitivity of 94.3% in differentiating healthy controls from OC patients with comparable results in the wildtype subgroup (sensitivity 92.8%, AUC = 0.988; p = 5.2e-14) as well as in BRCA1 mutation carriers (sensitivity 95.2%, AUC = 0.978; p = 1.7e-15) at an overall specificity of 92.3%. The used algorithm also allowed to identify healthy BRCA1 mutation carriers when compared to healthy wildtype women (sensitivity 88.4%, specificity 80.7%, AUC = 0.895; p = 6e-08), while this was less pronounced in patients with OC (sensitivity 66.7%, specificity 67.8%, AUC = 0.724; p = 0.00065). Conclusion We have developed an algorithm, which can differentiate between healthy women and OC patients and have for the first time shown, that such an algorithm can also be used in BRCA mutation carriers. To clarify a suggested benefit to the existing early detection program, large prospective trials with mainly early stage OC cases are warranted. PMID:29244844

Unc-45 Mutations in Caenorhabditis elegans Implicate a CRO1/She4p-like Domain in Myosin Assembly

PubMed Central

Barral, José M.; Bauer, Christopher C.; Ortiz, Irving; Epstein, Henry F.

1998-01-01

The Caenorhabditis elegans unc-45 locus has been proposed to encode a protein machine for myosin assembly. The UNC-45 protein is predicted to contain an NH2-terminal domain with three tetratricopeptide repeat motifs, a unique central region, and a COOH-terminal domain homologous to CRO1 and She4p. CRO1 and She4p are fungal proteins required for the segregation of other molecules in budding, endocytosis, and septation. Three mutations that lead to temperature-sensitive (ts) alleles have been localized to conserved residues within the CRO1/She4p-like domain, and two lethal alleles were found to result from stop codon mutations in the central region that would prevent translation of the COOH-terminal domain. Electron microscopy shows that thick filament accumulation in vivo is decreased by ∼50% in the CB286 ts mutant grown at the restrictive temperature. The thick filaments that assemble have abnormal structure. Immunofluorescence and immunoelectron microscopy show that myosins A and B are scrambled, in contrast to their assembly into distinct regions at the permissive temperature and in wild type. This abnormal structure correlates with the high degree of instability of the filaments in vitro as reflected by their extremely low yields and shortened lengths upon isolation. These results implicate the UNC-45 CRO1/She4p-like region in the assembly of myosin isoforms in C. elegans and suggest a possible common mechanism for the function of this UCS (UNC-45/CRO1/She4p) protein family. PMID:9832550

Novel insertion mutation of ABCB1 gene in an ivermectin-sensitive Border Collie.

PubMed

Han, Jae-Ik; Son, Hyoung-Won; Park, Seung-Cheol; Na, Ki-Jeong

2010-12-01

P-glycoprotein (P-gp) is encoded by the ABCB1 gene and acts as an efflux pump for xenobiotics. In the Border Collie, a nonsense mutation caused by a 4-base pair deletion in the ABCB1 gene is associated with a premature stop to P-gp synthesis. In this study, we examined the full-length coding sequence of the ABCB1 gene in an ivermectin-sensitive Border Collie that lacked the aforementioned deletion mutation. The sequence was compared to the corresponding sequences of a wild-type Beagle and seven ivermectin-tolerant family members of the Border Collie. When compared to the wild-type Beagle sequence, that of the ivermectin-sensitive Border Collie was found to have one insertion mutation and eight single nucleotide polymorphisms (SNPs) in the coding sequence of the ABCB1 gene. While the eight SNPs were also found in the family members' sequences, the insertion mutation was found only in the ivermectin-sensitive dog. These results suggest the possibility that the SNPs are species-specific features of the ABCB1 gene in Border Collies, and that the insertion mutation may be related to ivermectin intolerance.

Novel insertion mutation of ABCB1 gene in an ivermectin-sensitive Border Collie

PubMed Central

Han, Jae-Ik; Son, Hyoung-Won; Park, Seung-Cheol

2010-01-01

P-glycoprotein (P-gp) is encoded by the ABCB1 gene and acts as an efflux pump for xenobiotics. In the Border Collie, a nonsense mutation caused by a 4-base pair deletion in the ABCB1 gene is associated with a premature stop to P-gp synthesis. In this study, we examined the full-length coding sequence of the ABCB1 gene in an ivermectin-sensitive Border Collie that lacked the aforementioned deletion mutation. The sequence was compared to the corresponding sequences of a wild-type Beagle and seven ivermectin-tolerant family members of the Border Collie. When compared to the wild-type Beagle sequence, that of the ivermectin-sensitive Border Collie was found to have one insertion mutation and eight single nucleotide polymorphisms (SNPs) in the coding sequence of the ABCB1 gene. While the eight SNPs were also found in the family members' sequences, the insertion mutation was found only in the ivermectin-sensitive dog. These results suggest the possibility that the SNPs are species-specific features of the ABCB1 gene in Border Collies, and that the insertion mutation may be related to ivermectin intolerance. PMID:21113104

Investigating DNA Binding and Conformational Variation in Temperature Sensitive p53 Cancer Mutants Using QM-MM Simulations

PubMed Central

Koulgi, Shruti; Achalere, Archana; Sonavane, Uddhavesh; Joshi, Rajendra

2015-01-01

The tp53 gene is found to be mutated in 50% of all the cancers. The p53 protein, a product of tp53 gene, is a multi-domain protein. It consists of a core DNA binding domain (DBD) which is responsible for its binding and transcription of downstream target genes. The mutations in p53 protein are responsible for creating cancerous conditions and are found to be occurring at a high frequency in the DBD region of p53. Some of these mutations are also known to be temperature sensitive (ts) in nature. They are known to exhibit partial or strong binding with DNA in the temperature range (298–306 K). Whereas, at 310 K and above they show complete loss in binding. We have analyzed the changes in binding and conformational behavior at 300 K and 310 K for three of the ts-mutants viz., V143A, R249S and R175H. QM-MM simulations have been performed on the wild type and the above mentioned ts-mutants for 30 ns each. The optimal estimate of free energy of binding for a particular number of interface hydrogen bonds was calculated using the maximum likelihood method as described by Chodera et. al (2007). This parameter has been observed to be able to mimic the binding affinity of the p53 ts-mutants at 300 K and 310 K. Thus the correlation between MM-GBSA free energy of binding and hydrogen bonds formed by the interface residues between p53 and DNA has revealed the temperature dependent nature of these mutants. The role of main chain dihedrals was obtained by performing dihedral principal component analysis (PCA). This analysis, suggests that the conformational variations in the main chain dihedrals (ϕ and ψ) of the p53 ts-mutants may have caused reduction in the overall stability of the protein. The solvent exposure of the side chains of the interface residues were found to hamper the binding of the p53 to the DNA. Solvent Accessible Surface Area (SASA) also proved to be a crucial property in distinguishing the conformers obtained at 300 K and 310 K for the three ts-mutants from the wild type at 300 K. PMID:26579714

MethylMeter(®): bisulfite-free quantitative and sensitive DNA methylation profiling and mutation detection in FFPE samples.

PubMed

McCarthy, David; Pulverer, Walter; Weinhaeusel, Andreas; Diago, Oscar R; Hogan, Daniel J; Ostertag, Derek; Hanna, Michelle M

2016-06-01

Development of a sensitive method for DNA methylation profiling and associated mutation detection in clinical samples. Formalin-fixed and paraffin-embedded tumors received by clinical laboratories often contain insufficient DNA for analysis with bisulfite or methylation sensitive restriction enzymes-based methods. To increase sensitivity, methyl-CpG DNA capture and Coupled Abscription PCR Signaling detection were combined in a new assay, MethylMeter(®). Gliomas were analyzed for MGMT methylation, glioma CpG island methylator phenotype and IDH1 R132H. MethylMeter had 100% assay success rate measuring all five biomarkers in formalin-fixed and paraffin-embedded tissue. MGMT methylation results were supported by survival and mRNA expression data. MethylMeter is a sensitive and quantitative method for multitarget DNA methylation profiling and associated mutation detection. The MethylMeter-based GliomaSTRAT assay measures methylation of four targets and one mutation to simultaneously grade gliomas and predict their response to temozolomide. This information is clinically valuable in management of gliomas.

Label-free and high-sensitive detection for genetic point mutation based on hyperspectral interferometry

NASA Astrophysics Data System (ADS)

Fu, Rongxin; Li, Qi; Zhang, Junqi; Wang, Ruliang; Lin, Xue; Xue, Ning; Su, Ya; Jiang, Kai; Huang, Guoliang

2016-10-01

Label free point mutation detection is particularly momentous in the area of biomedical research and clinical diagnosis since gene mutations naturally occur and bring about highly fatal diseases. In this paper, a label free and high sensitive approach is proposed for point mutation detection based on hyperspectral interferometry. A hybridization strategy is designed to discriminate a single-base substitution with sequence-specific DNA ligase. Double-strand structures will take place only if added oligonucleotides are perfectly paired to the probe sequence. The proposed approach takes full use of the inherent conformation of double-strand DNA molecules on the substrate and a spectrum analysis method is established to point out the sub-nanoscale thickness variation, which benefits to high sensitive mutation detection. The limit of detection reach 4pg/mm2 according to the experimental result. A lung cancer gene point mutation was demonstrated, proving the high selectivity and multiplex analysis capability of the proposed biosensor.

The clinical features of squamous cell lung carcinoma with sensitive EGFR mutations.

PubMed

Taniguchi, Yuri; Matsumoto, Yoko; Furukawa, Ryutaro; Ohara, Sayaka; Usui, Kazuhiro

2018-06-01

The process of selecting patients on the basis of epidermal growth factor receptor (EGFR) mutations would likely result in a patient population with greater sensitivity to EGFR tyrosine kinase inhibitors (EGFR-TKIs). However, EGFR mutation status is not routinely examined in patients with squamous cell lung cancer (Sq) because of the low incidence of EGFR mutations and the poor clinical response to EGFR-TKIs. We retrospectively reviewed the clinical features of patients at our hospital with Sq who carried EGFR-TKI-sensitive EGFR mutations and assessed their responses to EGFR-TKIs. EGFR mutation status was tested in 23 of 441 patients with Sq (5.2%) admitted to our hospital during the study period. An EGFR mutation (exon 19 deletion 3, L858R 2) was identified in five of the 23 patients (21.7%), all of whom were female never-smokers. Of these five patients, four (4/9; 44.4%) were in the normal lung group, one (1/12; 8.3%) was in the emphysematous lung group, and none (0/2; 0%) in the fibrotic lung group. Two of these five patients with the EGFR mutation received gefitinib and two received afatinib. Although the two patients who were treated with gefitinib did not respond well to treatment (stable disease, 1 patient; progressive disease, 1 patient), the two patients who were treated with afatinib showed a good response (partial response, 2 patients). The administration of afatinib to Sq patients after selecting patients using the EGFR mutation test based on their underlying pulmonary disease and smoking status would likely result in a population with a greater sensitivity to afatinib.

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

Mutational profiling of non-small-cell lung cancer patients resistant to first-generation EGFR tyrosine kinase inhibitors using next generation sequencing

PubMed Central

Jin, Ying; Shao, Yang; Shi, Xun; Lou, Guangyuan; Zhang, Yiping; Wu, Xue; Tong, Xiaoling; Yu, Xinmin

2016-01-01

Patients with advanced non-small-cell lung cancer (NSCLC) harboring sensitive epithelial growth factor receptor (EGFR) mutations invariably develop acquired resistance to EGFR tyrosine kinase inhibitors (TKIs). Identification of actionable genetic alterations conferring drug-resistance can be helpful for guiding the subsequent treatment decision. One of the major resistant mechanisms is secondary EGFR-T790M mutation. Other mechanisms, such as HER2 and MET amplifications, and PIK3CA mutations, were also reported. However, the mechanisms in the remaining patients are still unknown. In this study, we performed mutational profiling in a cohort of 83 NSCLC patients with TKI-sensitizing EGFR mutations at diagnosis and acquired resistance to three different first-generation EGFR TKIs using targeted next generation sequencing (NGS) of 416 cancer-related genes. In total, we identified 322 genetic alterations with a median of 3 mutations per patient. 61% of patients still exhibit TKI-sensitizing EGFR mutations, and 36% of patients acquired EGFR-T790M. Besides other known resistance mechanisms, we identified TET2 mutations in 12% of patients. Interestingly, we also observed SOX2 amplification in EGFR-T790M negative patients, which are restricted to Icotinib treatment resistance, a drug widely used in Chinese NSCLC patients. Our study uncovered mutational profiles of NSCLC patients with first-generation EGFR TKIs resistance with potential therapeutic implications. PMID:27528220

Differential sensitivity of melanoma cell lines with BRAFV600E mutation to the specific Raf inhibitor PLX4032

PubMed Central

2010-01-01

Blocking oncogenic signaling induced by the BRAFV600E mutation is a promising approach for melanoma treatment. We tested the anti-tumor effects of a specific inhibitor of Raf protein kinases, PLX4032/RG7204, in melanoma cell lines. PLX4032 decreased signaling through the MAPK pathway only in cell lines with the BRAFV600E mutation. Seven out of 10 BRAFV600E mutant cell lines displayed sensitivity based on cell viability assays and three were resistant at concentrations up to 10 μM. Among the sensitive cell lines, four were highly sensitive with IC50 values below 1 μM, and three were moderately sensitive with IC50 values between 1 and 10 μM. There was evidence of MAPK pathway inhibition and cell cycle arrest in both sensitive and resistant cell lines. Genomic analysis by sequencing, genotyping of close to 400 oncogeninc mutations by mass spectrometry, and SNP arrays demonstrated no major differences in BRAF locus amplification or in other oncogenic events between sensitive and resistant cell lines. However, metabolic tracer uptake studies demonstrated that sensitive cell lines had a more profound inhibition of FDG uptake upon exposure to PLX4032 than resistant cell lines. In conclusion, BRAFV600E mutant melanoma cell lines displayed a range of sensitivities to PLX4032 and metabolic imaging using PET probes can be used to assess sensitivity. PMID:20406486

MDS1, a dosage suppressor of an mck1 mutant, encodes a putative yeast homolog of glycogen synthase kinase 3.

PubMed Central

Puziss, J W; Hardy, T A; Johnson, R B; Roach, P J; Hieter, P

1994-01-01

The yeast gene MCK1 encodes a serine/threonine protein kinase that is thought to function in regulating kinetochore activity and entry into meiosis. Disruption of MCK1 confers a cold-sensitive phenotype, a temperature-sensitive phenotype, and sensitivity to the microtubule-destabilizing drug benomyl and leads to loss of chromosomes during growth on benomyl. A dosage suppression selection was used to identify genes that, when present at high copy number, could suppress the cold-sensitive phenotype of mck1::HIS3 mutant cells. Several unique classes of clones were identified, and one of these, designated MDS1, has been characterized in some detail. Nucleotide sequence data reveal that MDS1 encodes a serine/threonine protein kinase that is highly homologous to the shaggy/zw3 kinase in Drosophila melanogaster and its functional homolog, glycogen synthase kinase 3, in rats. The presence of MDS1 in high copy number rescues both the cold-sensitive and the temperature-sensitive phenotypes, but not the benomyl-sensitive phenotype, associated with the disruption of MCK1. Analysis of strains harboring an mds1 null mutation demonstrates that MDS1 is not essential during normal vegetative growth but appears to be required for meiosis. Finally, in vitro experiments indicate that the proteins encoded by both MCK1 and MDS1 possess protein kinase activity with substrate specificity similar to that of mammalian glycogen synthase kinase 3. Images PMID:8264650

Total DNA input is a crucial determinant of the sensitivity of plasma cell-free DNA EGFR mutation detection using droplet digital PCR

PubMed Central

Zhao, Jing; Chen, Minjiang; Zhang, Li; Li, Longyun; Wang, Mengzhao

2017-01-01

We evaluated the use of droplet digital PCR (ddPCR) to detect plasma cell-free DNA (cfDNA) epidermal growth factor receptor (EGFR) mutations in advanced non-small cell lung cancer (NSCLC) patients. Compared with tumor-tissue-based detection, the sensitivity of ddPCR for detecting plasma cfDNA tyrosine kinase inhibitor (TKI)-sensitizing EGFR mutations was 61.3%, the specificity was 96.7%, and the consistency rate was 81.4% (?=0.605, 95% confidence interval: 0.501-0.706, p <0.0001). The sensitivity declined from 82.6% to 46.7% with decreasing cfDNA inputs (p=0.028). The plasma cfDNA concentration correlated with gender (males vs.females =11.69 ng/mL vs. 9.508 ng/mL; p=0.044), EGFR mutation status (tumor-tissue EGFR mutation-positive (EGFR M+) vs. EGFR mutation-negative (EGFR M-) = 9.61 ng/mL vs. 12.82 ng/mL; p =0.049) and specimen collection time (=2 years vs. >2 years=13.83 ng/mL vs. 6.575 ng/mL; p <0.001), and was greater in tumor-tissue EGFR M+ / plasma EGFR M+ patients than in tumor-tissue EGFR M+/plasma EGFR M- patients (11.61 vs. 7.73 ng/mL, respectively; p=0.003). Thus total cfDNA input crucially influences the sensitivity of plasma cfDNA EGFR mutation testing with ddPCR. Such analysis could be an effective supplemental test for advanced NSCLC patients. PMID:28052016

454 next generation-sequencing outperforms allele-specific PCR, Sanger sequencing, and pyrosequencing for routine KRAS mutation analysis of formalin-fixed, paraffin-embedded samples

PubMed Central

Altimari, Annalisa; de Biase, Dario; De Maglio, Giovanna; Gruppioni, Elisa; Capizzi, Elisa; Degiovanni, Alessio; D’Errico, Antonia; Pession, Annalisa; Pizzolitto, Stefano; Fiorentino, Michelangelo; Tallini, Giovanni

2013-01-01

Detection of KRAS mutations in archival pathology samples is critical for therapeutic appropriateness of anti-EGFR monoclonal antibodies in colorectal cancer. We compared the sensitivity, specificity, and accuracy of Sanger sequencing, ARMS-Scorpion (TheraScreen®) real-time polymerase chain reaction (PCR), pyrosequencing, chip array hybridization, and 454 next-generation sequencing to assess KRAS codon 12 and 13 mutations in 60 nonconsecutive selected cases of colorectal cancer. Twenty of the 60 cases were detected as wild-type KRAS by all methods with 100% specificity. Among the 40 mutated cases, 13 were discrepant with at least one method. The sensitivity was 85%, 90%, 93%, and 92%, and the accuracy was 90%, 93%, 95%, and 95% for Sanger sequencing, TheraScreen real-time PCR, pyrosequencing, and chip array hybridization, respectively. The main limitation of Sanger sequencing was its low analytical sensitivity, whereas TheraScreen real-time PCR, pyrosequencing, and chip array hybridization showed higher sensitivity but suffered from the limitations of predesigned assays. Concordance between the methods was k = 0.79 for Sanger sequencing and k > 0.85 for the other techniques. Tumor cell enrichment correlated significantly with the abundance of KRAS-mutated deoxyribonucleic acid (DNA), evaluated as ΔCt for TheraScreen real-time PCR (P = 0.03), percentage of mutation for pyrosequencing (P = 0.001), ratio for chip array hybridization (P = 0.003), and percentage of mutation for 454 next-generation sequencing (P = 0.004). Also, 454 next-generation sequencing showed the best cross correlation for quantification of mutation abundance compared with all the other methods (P < 0.001). Our comparison showed the superiority of next-generation sequencing over the other techniques in terms of sensitivity and specificity. Next-generation sequencing will replace Sanger sequencing as the reference technique for diagnostic detection of KRAS mutation in archival tumor tissues. PMID:23950653

Combining isothermal rolling circle amplification and electrochemiluminescence for highly sensitive point mutation detection

NASA Astrophysics Data System (ADS)

Su, Qiang; Zhou, Xiaoming

2008-12-01

Many pathogenic and genetic diseases are associated with changes in the sequence of particular genes. We describe here a rapid and highly efficient assay for the detection of point mutation. This method is a combination of isothermal rolling circle amplification (RCA) and high sensitive electrochemluminescence (ECL) detection. In the design, a circular template generated by ligation upon the recognition of a point mutation on DNA targets was amplified isothermally by the Phi29 polymerase using a biotinylated primer. The elongation products were hybridized with tris (bipyridine) ruthenium (TBR)-tagged probes and detected in a magnetic bead based ECL platform, indicating the mutation occurrence. P53 was chosen as a model for the identification of this method. The method allowed sensitive determination of the P53 mutation from wild-type and mutant samples. The main advantage of RCA-ECL is that it can be performed under isothermal conditions and avoids the generation of false-positive results. Furthermore, ECL provides a faster, more sensitive, and economical option to currently available electrophoresis-based methods.

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.

The yeast cytoplasmic LsmI/Pat1p complex protects mRNA 3' termini from partial degradation.

PubMed Central

He, W; Parker, R

2001-01-01

A key aspect of understanding eukaryotic gene regulation will be the identification and analysis of proteins that bind mRNAs and control their function. Recently, a complex of seven Lsm proteins and the Pat1p have been shown to interact with yeast mRNAs and promote mRNA decapping. In this study we present several observations to indicate that the LsmI/Pat1 complex has a second distinct function in protecting the 3'-UTR of mRNAs from trimming. First, mutations in the LSM1 to LSM7, as well as PAT1, genes led to the accumulation of MFA2pG and PGK1pG transcripts that had been shortened by 10-20 nucleotides at their 3' ends (referred to as trimming). Second, the trimming of these mRNAs was more severe at the high temperature, correlating with the inability of these mutant strains to grow at high temperature. In contrast, trimming did not occur in a dcp1 Delta strain, wherein the decapping enzyme is lacking. This indicates that trimming is not simply a consequence of the inhibition of mRNA decapping. Third, the temperature-sensitive growth of lsm and pat1 mutants was suppressed by mutations in the exosome or the functionally related Ski proteins, which are required for efficient 3' to 5' mRNA degradation of mRNA. Moreover, in lsm ski double mutants, higher levels of the trimmed mRNAs accumulated, indicating that exosome function is not required for mRNA trimming but that the exosome does degrade the trimmed mRNAs. These results raise the possibility that the temperature-sensitive growth of the lsm1-7 and pat1 mutants is at least partially due to mRNA trimming, which either inactivates the function of the mRNAs or makes them available for premature 3' to 5' degradation by the exosome. PMID:11514438

Functional mutation analysis of EGFR family genes and corresponding lymph node metastases in head and neck squamous cell carcinoma.

PubMed

Hama, Takanori; Yuza, Yuki; Suda, Toshihito; Saito, Yoshimichi; Norizoe, Chihiro; Kato, Takakuni; Moriyama, Hiroshi; Urashima, Mitsuyoshi

2012-01-01

Tumors with certain mutations in the epidermal growth factor receptor (EGFR) family genes dramatically respond to EGFR inhibitors. Therefore, these mutations are important factors that influence disease progression and patient survival. We previously studied the mutation status of EGFR in patients with head and neck squamous cell carcinoma (HNSCC). However, the mutation status of lymph node metastases and the frequency of mutations in EGFR family genes have not been extensively studied. In this study, we sequenced the catalytic domains of the three other members of the EGFR family, HER2, HER3, and HER4 in 92 clinical samples of HNSCC. We identified a HER2 mutation (K716E) in one sample but no mutations were found in HER3 or HER4. Next to investigate the relationship between EGFR mutations and tumor metastasis, we compared the DNA sequences of the EGFR gene between the primary tumor and the lymph node metastasis in 31 clinical samples. Only one of the patients with an EGFR mutation in the primary HNSCC carried the same mutation (L858R) in the lymph node metastasis. Finally, we explored the tumorigenic potential of the EGFR mutations that we had previously identified and their sensitivity to two different EGFR tyrosine kinase inhibitors (CL-387785, OSI-420). Ba/F3 cells transformed with mutant EGFR genes were sensitive to treatment with lower concentrations of CL-387785 than of OSI-420. These results contribute to our understanding of the genetic basis of drug sensitivity and will help design drugs that specifically target different subtypes of HNSCC.

Systematic exploration of essential yeast gene function with temperature-sensitive mutants

PubMed Central

Li, Zhijian; Vizeacoumar, Franco J; Bahr, Sondra; Li, Jingjing; Warringer, Jonas; Vizeacoumar, Frederick S; Min, Renqiang; VanderSluis, Benjamin; Bellay, Jeremy; DeVit, Michael; Fleming, James A; Stephens, Andrew; Haase, Julian; Lin, Zhen-Yuan; Baryshnikova, Anastasia; Lu, Hong; Yan, Zhun; Jin, Ke; Barker, Sarah; Datti, Alessandro; Giaever, Guri; Nislow, Corey; Bulawa, Chris; Myers, Chad L; Costanzo, Michael; Gingras, Anne-Claude; Zhang, Zhaolei; Blomberg, Anders; Bloom, Kerry; Andrews, Brenda; Boone, Charles

2012-01-01

Conditional temperature-sensitive (ts) mutations are valuable reagents for studying essential genes in the yeast Saccharomyces cerevisiae. We constructed 787 ts strains, covering 497 (~45%) of the 1,101 essential yeast genes, with ~30% of the genes represented by multiple alleles. All of the alleles are integrated into their native genomic locus in the S288C common reference strain and are linked to a kanMX selectable marker, allowing further genetic manipulation by synthetic genetic array (SGA)–based, high-throughput methods. We show two such manipulations: barcoding of 440 strains, which enables chemical-genetic suppression analysis, and the construction of arrays of strains carrying different fluorescent markers of subcellular structure, which enables quantitative analysis of phenotypes using high-content screening. Quantitative analysis of a GFP-tubulin marker identified roles for cohesin and condensin genes in spindle disassembly. This mutant collection should facilitate a wide range of systematic studies aimed at understanding the functions of essential genes. PMID:21441928

Cohort Removal Induces Changes in Body Temperature, Pain Sensitivity, and Anxiety-Like Behavior

PubMed Central

Takao, Keizo; Shoji, Hirotaka; Hattori, Satoko; Miyakawa, Tsuyoshi

2016-01-01

Mouse behavior is analyzed to elucidate the effects of various experimental manipulations, including gene mutation and drug administration. When the effect of a factor of interest is assessed, other factors, such as age, sex, temperature, apparatus, and housing, are controlled in experiments by matching, counterbalancing, and/or randomizing. One such factor that has not attracted much attention is the effect of sequential removal of animals from a common cage (cohort removal). Here we evaluated the effects of cohort removal on rectal temperature, pain sensitivity, and anxiety-like behavior by analyzing the combined data of a large number of C57BL/6J mice that we collected using a comprehensive behavioral test battery. Rectal temperature increased in a stepwise manner according to the position of sequential removal from the cage, consistent with previous reports. In the hot plate test, the mice that were removed first from the cage had a significantly longer latency to show the first paw response than the mice removed later. In the elevated plus maze, the mice removed first spent significantly less time on the open arms compared to the mice removed later. The results of the present study demonstrated that cohort removal induces changes in body temperature, pain sensitivity, and anxiety-like behavior in mice. Cohort removal also increased the plasma corticosterone concentration in mice. Thus, the ordinal position in the sequence of removal from the cage should be carefully counterbalanced between groups when the effect of experimental manipulations, including gene manipulation and drug administration, are examined using behavioral tests. PMID:27375443

Compound-Specific Effects of Mutations at Val787 in DII-S6 of Nav1.4 Sodium Channels on the Action of Sodium Channel Inhibitor Insecticides

PubMed Central

von Stein, Richard T.; Soderlund, David M.

2012-01-01

Sodium channel inhibitor (SCI) insecticides are hypothesized to inhibit voltage-gated sodium channels by binding selectively to the slow-inactivated state. Replacement of valine at position 787 in the S6 segment of homology domain II of the rat Nav1.4 sodium channel by lysine (V787K) enchances slow inactivation of this channel whereas replacement by alanine or cysteine (V787A, V787C) inhibits slow inactivation. To test the hypothesis that SCI insecticides bind selectively to the slow-inactivated state, we constructed mutated Nav1.4/V787A, Nav1.4/V787C, and Nav1.4/V787K cDNAs, expressed wildtype and mutated channels with the auxiliary β1 subunit in Xenopus oocytes, and used the two-electrode voltage clamp technique to examine the effects of these mutations on channel inhibition by four SCI insecticides (indoxacarb, its bioactivated metabolite DCJW, metaflumizone, and RH3421). Mutations at Val787 affected SCI insecticide sensitivity in a manner that was independent of mutation-induced changes in slow inactivation gating. Sensitivity to inhibition by 10 μM indoxacarb was significantly increased in all three mutated channels, whereas sensitivity to inhibition by 10 μM metaflumizone was significantly reduced in Nav1.4/V787A channels and completely abolished in Nav1.4/V787K channels. The effects of Val787 mutations on metaflumizone were correlated with the hydrophobicity of the substituted amino acid rather than the extent of slow inactivation. None of the mutations at Val787 significantly affected the sensitivity to inhibition by DCJW or RH3421. These results demonstrate that the impact of mutations at Val787 on sodium channel inhibition by SCI insecticides depends on the specific insecticide examined and is independent of mutation-induced changes in slow inactivation gating. We propose that Val787 may be a unique determinant of metaflumizone binding. PMID:22983119

Effects of helix and fingertip mutations on the thermostability of xyn11A investigated by molecular dynamics simulations and enzyme activity assays.

PubMed

Sutthibutpong, Thana; Rattanarojpong, Triwit; Khunrae, Pongsak

2017-12-04

Local conformational changes and global unfolding pathways of wildtype xyn11A recombinant and its mutated structures were studied through a series of atomistic molecular dynamics (MD) simulations, along with enzyme activity assays at three incubation temperatures to investigate the effects of mutations at three different sites to the thermostability. The first mutation was to replace an unstable negatively charged residue at a surface beta turn near the active site (D32G) by a hydrophobic residue. The second mutation was to create a disulphide bond (S100C/N147C) establishing a strong connection between an alpha helix and a distal beta hairpin associated with the thermally sensitive Thumb loop, and the third mutation add an extra hydrogen bond (A155S) to the same alpha helix. From the MD simulations performed, MM/PBSA energy calculations of the unfolding energy were in a good agreement with the enzyme activities measured from the experiment, as all mutated structures demonstrated the improved thermostability, especially the S100C/N147C proved to be the most stable mutant both by the simulations and the experiment. Local conformational analysis at the catalytic sites and the xylan access region also suggested that mutated xyn11A structures could accommodate xylan binding. However, the analysis of global unfolding pathways showed that structural disruptions at the beta sheet regions near the N-terminal were still imminent. These findings could provide the insight on the molecular mechanisms underlying the enhanced thermostability due to mutagenesis and changes in the protein unfolding pathways for further protein engineering of the GH11 family xylanase enzymes.

Thioredoxin is required for filamentous phage assembly.

PubMed Central

Russel, M; Model, P

1985-01-01

Sequence comparisons show that the fip gene product of Escherichia coli, which is required for filamentous phage assembly, is thioredoxin. Thioredoxin serves as a cofactor for reductive processes in many cell types and is a constituent of phage T7 DNA polymerase. The fip-1 mutation makes filamentous phage and T7 growth temperature sensitive in cells that carry it. The lesion lies within a highly conserved thioredoxin active site. Thioredoxin reductase (NADPH), as well as thioredoxin, is required for efficient filamentous phage production. Mutant phages defective in phage gene I are particularly sensitive to perturbations in the fip-thioredoxin system. A speculative model is presented in which thioredoxin reductase, thioredoxin, and the gene I protein interact to drive an engine for filamentous phage assembly. Images PMID:3881756

Whole exome sequencing is an efficient, sensitive and specific method of mutation detection in osteogenesis imperfecta and Marfan syndrome

PubMed Central

McInerney-Leo, Aideen M; Marshall, Mhairi S; Gardiner, Brooke; Coucke, Paul J; Van Laer, Lut; Loeys, Bart L; Summers, Kim M; Symoens, Sofie; West, Jennifer A; West, Malcolm J; Paul Wordsworth, B; Zankl, Andreas; Leo, Paul J; Brown, Matthew A; Duncan, Emma L

2013-01-01

Osteogenesis imperfecta (OI) and Marfan syndrome (MFS) are common Mendelian disorders. Both conditions are usually diagnosed clinically, as genetic testing is expensive due to the size and number of potentially causative genes and mutations. However, genetic testing may benefit patients, at-risk family members and individuals with borderline phenotypes, as well as improving genetic counseling and allowing critical differential diagnoses. We assessed whether whole exome sequencing (WES) is a sensitive method for mutation detection in OI and MFS. WES was performed on genomic DNA from 13 participants with OI and 10 participants with MFS who had known mutations, with exome capture followed by massive parallel sequencing of multiplexed samples. Single nucleotide polymorphisms (SNPs) and small indels were called using Genome Analysis Toolkit (GATK) and annotated with ANNOVAR. CREST, exomeCopy and exomeDepth were used for large deletion detection. Results were compared with the previous data. Specificity was calculated by screening WES data from a control population of 487 individuals for mutations in COL1A1, COL1A2 and FBN1. The target capture of five exome capture platforms was compared. All 13 mutations in the OI cohort and 9/10 in the MFS cohort were detected (sensitivity=95.6%) including non-synonymous SNPs, small indels (<10 bp), and a large UTR5/exon 1 deletion. One mutation was not detected by GATK due to strand bias. Specificity was 99.5%. Capture platforms and analysis programs differed considerably in their ability to detect mutations. Consumable costs for WES were low. WES is an efficient, sensitive, specific and cost-effective method for mutation detection in patients with OI and MFS. Careful selection of platform and analysis programs is necessary to maximize success. PMID:24501682

Improved EGFR mutation detection using combined exosomal RNA and circulating tumor DNA in NSCLC patient plasma

PubMed Central

Krug, A K; Enderle, D; Karlovich, C; Priewasser, T; Bentink, S; Spiel, A; Brinkmann, K; Emenegger, J; Grimm, D G; Castellanos-Rizaldos, E; Goldman, J W; Sequist, L V; Soria, J -C; Camidge, D R; Gadgeel, S M; Wakelee, H A; Raponi, M; Noerholm, M; Skog, J

2018-01-01

Abstract Background A major limitation of circulating tumor DNA (ctDNA) for somatic mutation detection has been the low level of ctDNA found in a subset of cancer patients. We investigated whether using a combined isolation of exosomal RNA (exoRNA) and cell-free DNA (cfDNA) could improve blood-based liquid biopsy for EGFR mutation detection in non-small-cell lung cancer (NSCLC) patients. Patients and methods Matched pretreatment tumor and plasma were collected from 84 patients enrolled in TIGER-X (NCT01526928), a phase 1/2 study of rociletinib in mutant EGFR NSCLC patients. The combined isolated exoRNA and cfDNA (exoNA) was analyzed blinded for mutations using a targeted next-generation sequencing panel (EXO1000) and compared with existing data from the same samples using analysis of ctDNA by BEAMing. Results For exoNA, the sensitivity was 98% for detection of activating EGFR mutations and 90% for EGFR T790M. The corresponding sensitivities for ctDNA by BEAMing were 82% for activating mutations and 84% for T790M. In a subgroup of patients with intrathoracic metastatic disease (M0/M1a; n = 21), the sensitivity increased from 26% to 74% for activating mutations (P = 0.003) and from 19% to 31% for T790M (P = 0.5) when using exoNA for detection. Conclusions Combining exoRNA and ctDNA increased the sensitivity for EGFR mutation detection in plasma, with the largest improvement seen in the subgroup of M0/M1a disease patients known to have low levels of ctDNA and poses challenges for mutation detection on ctDNA alone. Clinical Trials NCT01526928 PMID:29216356

Genetic Studies of the Prp17 Gene of Saccharomyces Cerevisiae: A Domain Essential for Function Maps to a Nonconserved Region of the Protein

PubMed Central

Seshadri, V.; Vaidya, V. C.; Vijayraghavan, U.

1996-01-01

The PRP17 gene product is required for the second step of pre-mRNA splicing reactions. The C-terminal half of this protein bears four repeat units with homology to the β transducin repeat. Missense mutations in three temperature-sensitive prp17 mutants map to a region in the N-terminal half of the protein. We have generated, in vitro, 11 missense alleles at the β transducin repeat units and find that only one affects function in vivo. A phenotypically silent missense allele at the fourth repeat unit enhances the slow-growing phenotype conferred by an allele at the third repeat, suggesting an interaction between these domains. Although many missense mutations in highly conserved amino acids lack phenotypic effects, deletion analysis suggests an essential role for these units. Only mutations in the N-terminal nonconserved domain of PRP17 are synthetically lethal in combination with mutations in PRP16 and PRP18, two other gene products required for the second splicing reaction. A mutually allele-specific interaction between prp17 and snr7, with mutations in U5 snRNA, was observed. We therefore suggest that the functional region of Prp17p that interacts with Prp18p, Prp16p, and U5 snRNA is in the N terminal region of the protein. PMID:8722761

High-sensitivity assay for monitoring ESR1 mutations in circulating cell-free DNA of breast cancer patients receiving endocrine therapy.

PubMed

Lupini, Laura; Moretti, Anna; Bassi, Cristian; Schirone, Alessio; Pedriali, Massimo; Querzoli, Patrizia; Roncarati, Roberta; Frassoldati, Antonio; Negrini, Massimo

2018-03-12

Approximately 70% of breast cancers (BCs) express estrogen receptor alpha (ERα) and are treated with endocrine therapy. However, the effectiveness of this therapy is limited by innate or acquired resistance in approximately one-third of patients. Activating mutations in the ESR1 gene that encodes ERα promote critical resistance mechanisms. Here, we developed a high sensitivity approach based on enhanced-ice-COLD-PCR for detecting ESR1 mutations. The method produced an enrichment up to 100-fold and allowed the unambiguous detection of ESR1 mutations even when they consisted of only 0.01% of the total ESR1 allelic fraction. After COLD-PCR enrichment, methods based on next-generation sequencing or droplet-digital PCR were employed to detect and quantify ESR1 mutations. We applied the method to detect ESR1 mutations in circulating free DNA from the plasma of 56 patients with metastatic ER-positive BC. Fifteen of these patients were found to have ESR1 mutations at codons 536-538. This study demonstrates the utility of the enhanced-ice-COLD-PCR approach for simplifying and improving the detection of ESR1 tumor mutations in liquid biopsies. Because of its high sensitivity, the approach may potentially be applicable to patients with non-metastatic disease.

Detection of MPL mutations by a novel allele-specific PCR-based strategy.

PubMed

Furtado, Larissa V; Weigelin, Helmut C; Elenitoba-Johnson, Kojo S J; Betz, Bryan L

2013-11-01

MPL mutation testing is recommended in patients with suspected primary myelofibrosis or essential thrombocythemia who lack the JAK2 V617F mutation. MPL mutations can occur at allelic levels below 15%, which may escape detection by commonly used mutation screening methods such as Sanger sequencing. We developed a novel multiplexed allele-specific PCR assay capable of detecting most recurrent MPL exon 10 mutations associated with primary myelofibrosis and essential thrombocythemia (W515L, W515K, W515A, and S505N) down to a sensitivity of 2.5% mutant allele. Test results were reviewed from 15 reference cases and 1380 consecutive specimens referred to our laboratory for testing. Assay performance was compared to Sanger sequencing across a series of 58 specimens with MPL mutations. Positive cases consisted of 45 with W515L, 6 with S505N, 5 with W515K, 1 with W515A, and 1 with both W515L and S505N. Seven cases had mutations below 5% that were undetected by Sanger sequencing. Ten additional cases had mutation levels between 5% and 15% that were not consistently detected by sequencing. All results were easily interpreted in the allele-specific test. This assay offers a sensitive and reliable solution for MPL mutation testing. Sanger sequencing appears insufficiently sensitive for robust MPL mutation detection. Our data also suggest the relative frequency of S505N mutations may be underestimated, highlighting the necessity for inclusion of this mutation in MPL test platforms. Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Sensitivity of the ViroSeq HIV-1 Genotyping System for Detection of the K103N Resistance Mutation in HIV-1 Subtypes A, C, and D

PubMed Central

Church, Jessica D.; Jones, Dana; Flys, Tamara; Hoover, Donald; Marlowe, Natalia; Chen, Shu; Shi, Chanjuan; Eshleman, James R.; Guay, Laura A.; Jackson, J. Brooks; Kumwenda, Newton; Taha, Taha E.; Eshleman, Susan H.

2006-01-01

The US Food and Drug Administration-cleared ViroSeq HIV-1 Genotyping System (ViroSeq) and other population sequencing-based human immunodeficiency virus type 1 (HIV-1) genotyping methods detect antiretroviral drug resistance mutations present in the major viral population of a test sample. These assays also detect some mutations in viral variants that are present as mixtures. We compared detection of the K103N nevirapine resistance mutation using ViroSeq and a sensitive, quantitative point mutation assay, LigAmp. The LigAmp assay measured the percentage of K103N-containing variants in the viral population (percentage of K103N). We analyzed 305 samples with HIV-1 subtypes A, C, and D collected from African women after nevirapine administration. ViroSeq detected K103N in 100% of samples with >20% K103N, 77.8% of samples with 10 to 20% K103N, 71.4% of samples with 5 to 10% K103N, and 16.9% of samples with 1 to 5% K103N. The sensitivity of ViroSeq for detection of K103N was similar for subtypes A, C, and D. These data indicate that the ViroSeq system reliably detects the K103N mutation at levels above 20% and frequently detects the mutation at lower levels. Further studies are needed to compare the sensitivity of different assays for detection of HIV-1 drug resistance mutations and to determine the clinical relevance of HIV-1 minority variants. PMID:16931582

Mutation-induced protein interaction kinetics changes affect apoptotic network dynamic properties and facilitate oncogenesis

PubMed Central

Zhao, Linjie; Sun, Tanlin; Pei, Jianfeng; Ouyang, Qi

2015-01-01

It has been a consensus in cancer research that cancer is a disease caused primarily by genomic alterations, especially somatic mutations. However, the mechanism of mutation-induced oncogenesis is not fully understood. Here, we used the mitochondrial apoptotic pathway as a case study and performed a systematic analysis of integrating pathway dynamics with protein interaction kinetics to quantitatively investigate the causal molecular mechanism of mutation-induced oncogenesis. A mathematical model of the regulatory network was constructed to establish the functional role of dynamic bifurcation in the apoptotic process. The oncogenic mutation enrichment of each of the protein functional domains involved was found strongly correlated with the parameter sensitivity of the bifurcation point. We further dissected the causal mechanism underlying this correlation by evaluating the mutational influence on protein interaction kinetics using molecular dynamics simulation. We analyzed 29 matched mutant–wild-type and 16 matched SNP—wild-type protein systems. We found that the binding kinetics changes reflected by the changes of free energy changes induced by protein interaction mutations, which induce variations in the sensitive parameters of the bifurcation point, were a major cause of apoptosis pathway dysfunction, and mutations involved in sensitive interaction domains show high oncogenic potential. Our analysis provided a molecular basis for connecting protein mutations, protein interaction kinetics, network dynamics properties, and physiological function of a regulatory network. These insights provide a framework for coupling mutation genotype to tumorigenesis phenotype and help elucidate the logic of cancer initiation. PMID:26170328

MethylMeter®: bisulfite-free quantitative and sensitive DNA methylation profiling and mutation detection in FFPE samples

PubMed Central

McCarthy, David; Pulverer, Walter; Weinhaeusel, Andreas; Diago, Oscar R; Hogan, Daniel J; Ostertag, Derek; Hanna, Michelle M

2016-01-01

Aim: Development of a sensitive method for DNA methylation profiling and associated mutation detection in clinical samples. Materials & methods: Formalin-fixed and paraffin-embedded tumors received by clinical laboratories often contain insufficient DNA for analysis with bisulfite or methylation sensitive restriction enzymes-based methods. To increase sensitivity, methyl-CpG DNA capture and Coupled Abscription PCR Signaling detection were combined in a new assay, MethylMeter®. Gliomas were analyzed for MGMT methylation, glioma CpG island methylator phenotype and IDH1 R132H. Results: MethylMeter had 100% assay success rate measuring all five biomarkers in formalin-fixed and paraffin-embedded tissue. MGMT methylation results were supported by survival and mRNA expression data. Conclusion: MethylMeter is a sensitive and quantitative method for multitarget DNA methylation profiling and associated mutation detection. The MethylMeter-based GliomaSTRAT assay measures methylation of four targets and one mutation to simultaneously grade gliomas and predict their response to temozolomide. This information is clinically valuable in management of gliomas. PMID:27337298

[Presence of the double pfmdr1 mutation 86Tyr and 1246 Tyr in clones of a chloroquine-resistant west African isolate of Plasmodium falciparum].

PubMed

Pinheiro, L; Franco, S; Adagu, I S; Rosa, R; Rosário, V E; Warhurst, D C

2003-01-01

Isolates of Plasmodium falciparum from three areas of West Africa were recovered from cryopreservation and their chloroquine-sensitivity were determined in vitro. Of the 90 samples studied, 60 were from Guinea-Bissau (30Resistant/30Sensitive), 15 were from S. Tomé and Príncipe (11Resistant/4Sensitive) and 15 were from Angola (11Resistant/4Sensitive). All the isolates were sensitive to mefloquine. Using the polymerase chain reaction/restriction fragment length polymorphism technique (PCR/RFLP) it was possible to detect two mutations in the pfmdr1 gene, often associated with chloroquine-resistance. 66% of the samples from Guiné-Bissau showed a correlation with chloroquine-resistance while 73% of the samples from São Tomé and Angola altogether had the 86Tyr mutation. The present study on West African isolates and clones showed, for the first time, the presence of a double point mutation in the pfmdr1 gene one being found, up to now, only in South America isolates of Plasmodium falciparum.

Distinct signatures for mutator sensitivity of lacZ reversions and for the spectrum of lacI/lacO forward mutations on the chromosome of nondividing Escherichia coli.

PubMed Central

Bharatan, Shanti M; Reddy, Manjula; Gowrishankar, J

2004-01-01

A conditional lethal galE(Ts)-based strategy was employed in Escherichia coli, first to eliminate all growth-associated chromosomal reversions in lacZ or forward mutations in lacI/lacO by incubation at the restrictive temperature and subsequently to recover (as papillae) spontaneous mutations that had arisen in the population of nondividing cells after shift to the permissive temperature. Data from lacZ reversion studies in mutator strains indicated that the products of all genes for mismatch repair (mutHLS, dam, uvrD), of some for oxidative damage repair (mutMT), and of that for polymerase proofreading (dnaQ) are required in dividing cells; some others for oxidative damage repair (mutY, nth nei) are required in both dividing and nondividing cells; and those for alkylation damage repair (ada ogt) are required in nondividing cells. The spectrum of lacI/lacO mutations in nondividing cells was distinguished both by lower frequencies of deletions and IS1 insertions and by the unique occurrence of GC-to-AT transitions at lacO +5. In the second approach to study mutations that had occurred in nondividing cells, lacI/lacO mutants were selected as late-arising papillae from the lawn of a galE+ strain; once again, transitions at lacO +5 were detected among the mutants that had been obtained from populations initially grown on poor carbon sources such as acetate, palmitate, or succinate. Our results indicate that the lacO +5 site is mutable only in nondividing cells, one possible mechanism for which might be that random endogenous alkylation (or oxidative) damage to DNA in these cells is efficiently corrected by the Ada Ogt (or Nth Nei) repair enzymes at most sites but not at lacO +5. Furthermore, the late-arising papillae from the second approach were composed almost exclusively of dominant lacI/lacO mutants. This finding lends support to "instantaneous gratification" models in which a spontaneous lesion, occurring at a random site in DNA of a nondividing cell, is most likely to be fixed as a mutation if it allows the cell to immediately exit the nondividing state. PMID:15020459

Familial hypertrophic cardiomyopathy: functional effects of myosin mutation R723G in cardiomyocytes.

PubMed

Kraft, Theresia; Witjas-Paalberends, E Rosalie; Boontje, Nicky M; Tripathi, Snigdha; Brandis, Almuth; Montag, Judith; Hodgkinson, Julie L; Francino, Antonio; Navarro-Lopez, Francisco; Brenner, Bernhard; Stienen, Ger J M; van der Velden, Jolanda

2013-04-01

Familial Hypertrophic Cardiomyopathy (FHC) is frequently caused by mutations in the β-cardiac myosin heavy chain (β-MyHC). To identify changes in sarcomeric function triggered by such mutations, distinguishing mutation effects from other functional alterations of the myocardium is essential. We previously identified a direct effect of mutation R723G (MyHC723) on myosin function in slow Musculus soleus fibers. Here we investigate contractile features of left ventricular cardiomyocytes of FHC-patients with the same MyHC723-mutation and compare these to the soleus data. In mechanically isolated, triton-permeabilized MyHC723-cardiomyocytes, maximum force was significantly lower but calcium-sensitivity was unchanged compared to donor. Conversely, MyHC723-soleus fibers showed significantly higher maximum force and reduced calcium-sensitivity compared to controls. Protein phosphorylation, a potential myocardium specific modifying mechanism, might account for differences compared to soleus fibers. Analysis revealed reduced phosphorylation of troponin I and T, myosin-binding-protein C, and myosin-light-chain 2 in MyHC723-myocardium compared to donor. Saturation of protein-kinaseA phospho-sites led to comparable, i.e., reduced MyHC723-calcium-sensitivity in cardiomyocytes as in M. soleus fibers, while maximum force remained reduced. Myofibrillar disarray and lower density of myofibrils, however, largely account for reduced maximum force in MyHC723-cardiomyocytes. The changes seen when phosphorylation of sarcomeric proteins in myocardium of affected patients is matched to control tissue suggest that the R723G mutation causes reduced Ca(++)-sensitivity in both cardiomyocytes and M. soleus fibers. In MyHC723-myocardium, however, hypophosphorylation can compensate for the reduced calcium-sensitivity, while maximum force generation, lowered by myofibrillar deficiency and disarray, remains impaired, and may only be compensated by hypertrophy. Copyright © 2013 Elsevier Ltd. All rights reserved.

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.

Rapid regulation of nuclear proteins by rapamycin-induced translocation in fission yeast

PubMed Central

Ding, Lin; Laor, Dana; Weisman, Ronit; Forsburg, Susan L

2014-01-01

Genetic analysis of protein function requires a rapid means of inactivating the gene under study. Typically this exploits temperature sensitive mutations, or promoter shut-off techniques. We report the adaptation to Schizosaccharomyces pombe of the Anchor Away technique, originally designed in budding yeast (Haruki et al., 2008a). This method relies on a rapamycin-mediated interaction between the FRB and FKBP12 binding domains, to relocalize nuclear proteins of interest to the cytoplasm. We demonstrate a rapid nuclear depletion of abundant proteins as proof-of-principle. PMID:24733494

Correlation of mutations and recombination with growth kinetics of poliovirus vaccine strains.

PubMed

Pliaka, V; Kyriakopoulou, Z; Tsakogiannis, D; Ruether, I G A; Gartzonika, C; Levidiotou-Stefanou, S; Krikelis, A; Markoulatos, P

2010-12-01

Attenuated strains of Sabin poliovirus vaccine replicate in the human gut and, in rare cases, may cause vaccine-associated paralytic poliomyelitis (VAPP). The genetic instability of Sabin strains constitutes one of the main causes of VAPP, a disease that is most frequently associated with type 3 and type 2 Sabin strains, and more rarely with type 1 Sabin strains. In the present study, the growth phenotype of eight oral poliovirus vaccine (OPV) isolates (two non-recombinants and six recombinants), as well as of Sabin vaccine strains, was evaluated using two different assays, the reproductive capacity at different temperatures (Rct) test and the one-step growth curve test in Hep-2 cells at two different temperatures (37°C and 40°C). The growth phenotype of isolates was correlated with genomic modifications in order to identify the determinants and mechanisms of reversion towards neurovirulence. All of the recombinant OPV isolates showed a thermoresistant phenotype in the Rct test. Moreover, both recombinant Sabin-3 isolates showed significantly higher viral yield than Sabin 3 vaccine strain at 37°C and 40°C in the one-step growth curve test. All of the OPV isolates displayed mutations at specific sites of the viral genome, which are associated with the attenuated and temperature-sensitive phenotype of Sabin strains. The results showed that both mutations and recombination events could affect the phenotype traits of Sabin derivatives and may lead to the reversion of vaccinal strains to neurovirulent ones. The use of phenotypic markers along with the genomic analysis may shed additional light on the molecular determinants of the reversed neurovirulent phenotype of Sabin derivatives.

Rearrangement of competing U2 RNA helices within the spliceosome promotes multiple steps in splicing

PubMed Central

Perriman, Rhonda J.; Ares, Manuel

2007-01-01

Nuclear pre-messenger RNA (pre-mRNA) splicing requires multiple spliceosomal small nuclear RNA (snRNA) and pre-mRNA rearrangements. Here we reveal a new snRNA conformational switch in which successive roles for two competing U2 helices, stem IIa and stem IIc, promote distinct splicing steps. When stem IIa is stabilized by loss of stem IIc, rapid ATP-independent and Cus2p-insensitive prespliceosome formation occurs. In contrast, hyperstabilized stem IIc improves the first splicing step on aberrant branchpoint pre-mRNAs and rescues temperature-sensitive U6–U57C, a U6 mutation that also suppresses first-step splicing defects of branchpoint mutations. A second, later role for stem IIa is revealed by its suppression of a cold-sensitive allele of the second-step splicing factor PRP16. Our data expose a spliceosomal progression cycle of U2 stem IIa formation, disruption by stem IIc, and then reformation of stem IIa before the second catalytic step. We propose that the competing stem IIa and stem IIc helices are key spliceosomal RNA elements that optimize juxtaposition of the proper reactive sites during splicing. PMID:17403781

An Escherichia coli strain, PGB01, isolated from feral pigeon Faeces, thermally fit to survive in pigeon, shows high level resistance to trimethoprim.

PubMed

Kumar, Arvind; Tiwary, Bipransh Kumar; Kachhap, Sangita; Nanda, Ashis Kumar; Chakraborty, Ranadhir

2015-01-01

In this study, of the hundred Escherichia coli strains isolated from feral Pigeon faeces, eighty five strains were resistant to one or more antibiotics and fifteen sensitive to all the antibiotics tested. The only strain (among all antibiotic-resistant E. coli isolates) that possessed class 1 integron was PGB01. The dihydrofolate reductase gene of the said integron was cloned, sequenced and expressed in E. coli JM109. Since PGB01 was native to pigeon's gut, we have compared the growth of PGB01 at two different temperatures, 42°C (normal body temperature of pigeon) and 37°C (optimal growth temperature of E. coli; also the human body temperature), with E. coli K12. It was found that PGB01 grew better than the laboratory strain E. coli K12 at 37°C as well as at 42°C. In the thermal fitness assay, it was observed that the cells of PGB01 were better adapted to 42°C, resembling the average body temperature of pigeon. The strain PGB01 also sustained more microwave mediated thermal stress than E. coli K12 cells. The NMR spectra of the whole cells of PGB01 varied from E. coli K12 in several spectral peaks relating some metabolic adaptation to thermotolerance. On elevating the growth temperature from 37°C to 42°C, susceptibility to kanamycin (both strains were sensitive to it) of E. coli K12 was increased, but in case of PGB01 no change in susceptibility took place. We have also attempted to reveal the basis of trimethoprim resistance phenotype conferred by the dfrA7 gene homologue of PGB01. Molecular Dynamics (MD) simulation study of docked complexes, PGB01-DfrA7 and E. coli TMP-sensitive-Dfr with trimethoprim (TMP) showed loss of some of the hydrogen and hydrophobic interaction between TMP and mutated residues in PGB01-DfrA7-TMP complex compared to TMP-sensitive-Dfr-TMP complex. This loss of interaction entails decrease in affinity of TMP for PGB01-DfrA7 compared to TMP-sensitive-Dfr.

Presence of novel compound BCR-ABL mutations in late chronic and advanced phase imatinib sensitive CML patients indicates their possible role in CML progression.

PubMed

Akram, Afia Muhammad; Iqbal, Zafar; Akhtar, Tanveer; Khalid, Ahmed Mukhtar; Sabar, Muhammad Farooq; Qazi, Mahmood Hussain; Aziz, Zeba; Sajid, Nadia; Aleem, Aamer; Rasool, Mahmood; Asif, Muhammad; Aloraibi, Saleh; Aljamaan, Khaled; Iqbal, Mudassar

2017-04-03

BCR-ABL kinase domain (K D ) mutations are well known for causing resistance against tyrosine kinase inhibitors (TKIs) and disease progression in chronic myeloid leukemia (CML). In recent years, compound BCR-ABL mutations have emerged as a new threat to CML patients by causing higher degrees of resistance involving multiple TKIs, including ponatinib. However, there are limited reports about association of compound BCR-ABL mutations with disease progression in imatinib (IM) sensitive CML patients. Therefore, we investigated presence of ABL-K D mutations in chronic phase (n = 41), late chronic phase (n = 33) and accelerated phase (n = 16) imatinib responders. Direct sequencing analysis was used for this purpose. Eleven patients (12.22%) in late-CP CML were detected having total 24 types of point mutations, out of which 8 (72.72%) harbored compound mutated sites. SH2 contact site mutations were dominant in our study cohort, with E355G (3.33%) being the most prevalent. Five patients (45%) all having compound mutated sites, progressed to advanced phases of disease during follow up studies. Two novel silent mutations G208G and E292E/E were detected in combination with other mutants, indicating limited tolerance for BCR-ABL1 kinase domain for missense mutations. However, no patient in early CP of disease manifested mutated ABL-K D . Occurrence of mutations was found associated with elevated platelet count (p = 0.037) and patients of male sex (p = 0.049). The median overall survival and event free survival of CML patients (n = 90) was 6.98 and 5.8 y respectively. The compound missense mutations in BCR-ABL kinase domain responsible to elicit disease progression, drug resistance or disease relapse in CML, can be present in yet Imatinib sensitive patients. Disease progression observed here, emphasizes the need of ABL-K D mutation screening in late chronic phase CML patients for improved clinical management of disease.

Hyperinsulinism–hyperammonaemia syndrome: novel mutations in the GLUD1 gene and genotype–phenotype correlations

PubMed Central

Kapoor, Ritika R; Flanagan, Sarah E; Fulton, Piers; Chakrapani, Anupam; Chadefaux, Bernadette; Ben-Omran, Tawfeg; Banerjee, Indraneel; Shield, Julian P; Ellard, Sian; Hussain, Khalid

2009-01-01

Background Activating mutations in the GLUD1 gene (which encodes for the intra-mitochondrial enzyme glutamate dehydrogenase, GDH) cause the hyperinsulinism–hyperammonaemia (HI/HA) syndrome. Patients present with HA and leucine-sensitive hypoglycaemia. GDH is regulated by another intra-mitochondrial enzyme sirtuin 4 (SIRT4). Sirt4 knockout mice demonstrate activation of GDH with increased amino acid-stimulated insulin secretion. Objectives To study the genotype–phenotype correlations in patients with GLUD1 mutations. To report the phenotype and functional analysis of a novel mutation (P436L) in the GLUD1 gene associated with the absence of HA. Patients and methods Twenty patients with HI from 16 families had mutational analysis of the GLUD1 gene in view of HA (n=19) or leucine sensitivity (n=1). Patients negative for a GLUD1 mutation had sequence analysis of the SIRT4 gene. Functional analysis of the novel P436L GLUD1 mutation was performed. Results Heterozygous missense mutations were detected in 15 patients with HI/HA, 2 of which are novel (N410D and D451V). In addition, a patient with a normal serum ammonia concentration (21 μmol/l) was heterozygous for a novel missense mutation P436L. Functional analysis of this mutation confirms that it is associated with a loss of GTP inhibition. Seizure disorder was common (43%) in our cohort of patients with a GLUD1 mutation. No mutations in the SIRT4 gene were identified. Conclusion Patients with HI due to mutations in the GLUD1 gene may have normal serum ammonia concentrations. Hence, GLUD1 mutational analysis may be indicated in patients with leucine sensitivity; even in the absence of HA. A high frequency of epilepsy (43%) was observed in our patients with GLUD1 mutations. PMID:19690084

Characterization of IDH1 p.R132H Mutant Clones Using Mutation-specific Antibody in Myeloid Neoplasms.

PubMed

Kurt, Habibe; Bueso-Ramos, Carlos E; Khoury, Joseph D; Routbort, Mark J; Kanagal-Shamanna, Rashmi; Patel, Umang V; Jorgensen, Jeffrey L; Wang, Sa A; Ravandi, Farhad; DiNardo, Courtney; Luthra, Rajyalakshmi; Medeiros, L Jeffrey; Patel, Keyur P

2018-05-01

Isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations occur in a variety of myeloid neoplasms. Immunohistochemistry (IHC)-based direct visualization of mutant clones of hematopoietic cells can be useful for rapid diagnostic screening and for monitoring treatment response. In this study, we first evaluated the sensitivity and specificity of the IDH1 p.R132H mutation-specific antibody by IHC. All IDH1 wild type cases (n=11) and IDH1 mutant cases with a non-p.R132H mutation (n=30) were negative by IHC, demonstrating 100% antibody specificity. All the initial diagnostic specimens with IDH1 p.R132H mutation including acute myeloid leukemia (n=30), myelodysplastic syndromes (MDS) (n=10), MDS/myeloproliferative neoplasms (MPN) (n=4), and MPN (n=5) were positive by IHC, demonstrating 100% antibody sensitivity. Both immature and mature myeloid cells showed immunoreactivity. Erythroid precursors, lymphoid cells, endothelial cells, and osteoblasts were consistently negative by IHC. We then evaluated the follow-up specimens with a known IDH1 mutation status including acute myeloid leukemia (n=23), MDS (n=2), MDS/MPN (n=2), and MPN (n=2). Thirty-three IDH1 p.R132H mutant cases were positive by IHC and 12 IDH1 mutation negative cases were negative by IHC. However, IHC reactivity in up to 25% of bone marrow cells was noted in 8 of 20 polymerase chain reaction-negative cases, all from patients with a known history of IDH1 p.R132H mutation indicating sampling error or a sensitivity issue with molecular tests. These data indicate that IHC is a highly specific and sensitive tool to detect IDH1 p.R132H mutation in bone marrow involved by myeloid neoplasms. In addition, IDH1 p.R132H IHC also allows localization and assessment of the maturation stage of the clones carrying the mutation.

Specific, sensitive, and rapid assay for human immunodeficiency virus type 1 pol mutations associated with resistance to zidovudine and didanosine.

PubMed Central

Frenkel, L M; Wagner, L E; Atwood, S M; Cummins, T J; Dewhurst, S

1995-01-01

The effectiveness of antiretroviral therapy may be limited by the development of human immunodeficiency virus type 1 (HIV-1) resistance. Monitoring for resistance will perhaps allow changes in therapy prior to deterioration in the patient's clinical or immunologic status. Our objective was to develop a rapid, specific, and sensitive genotypic assay for HIV-1 resistance to zidovudine (ZDV) and didanosine (ddI) which is simple to perform. In our assay the DNA of HIV-1 pol was amplified by PCR using two sets of nested oligonucleotide primers. Mutations of reverse transcriptase (RT) encoding amino acids (aa) 74 and 41, 70, and 215 which have been associated with HIV-1 resistance to ddI and ZDV, respectively, were detected with a ligase detection reaction (LDR) and indicated colorimetrically. The RT genotypes of 35 patient specimens (140 codons) blindly assessed for these mutations were in agreement by PCR-LDR and by dideoxynucleotide sequencing. To evaluate the limits of the assay, other specimens with mutations close to the ligation site were evaluated by PCR-LDR. The assay was sensitive and specific for all specimens except when mutations occurred within 2 bases on either side of the ligation site. In summary, this PCR-LDR assay specifically, sensitively, and rapidly detected pol mutations (RT aa 74, 41, 70, and 215) associated with HIV-1 resistance to ddI and ZDV. PMID:7714190

Genotypic, Phenotypic and Clinical Validation of GeneXpert in Extra-Pulmonary and Pulmonary Tuberculosis in India

PubMed Central

Singh, Urvashi B.; Pandey, Pooja; Mehta, Girija; Bhatnagar, Anuj K.; Mohan, Anant; Goyal, Vinay; Ahuja, Vineet; Ramachandran, Ranjani; Sachdeva, Kuldeep S.; Samantaray, Jyotish C.

2016-01-01

Background Newer molecular diagnostics have brought paradigm shift in early diagnosis of tuberculosis [TB]. WHO recommended use of GeneXpert MTB/RIF [Xpert] for Extra-pulmonary [EP] TB; critics have since questioned its efficiency. Methods The present study was designed to assess the performance of GeneXpert in 761 extra-pulmonary and 384 pulmonary specimens from patients clinically suspected of TB and compare with Phenotypic, Genotypic and Composite reference standards [CRS]. Results Comparison of GeneXpert results to CRS, demonstrated sensitivity of 100% and 90.68%, specificity of 100% and 99.62% for pulmonary and extra-pulmonary samples. On comparison with culture, sensitivity for Rifampicin [Rif] resistance detection was 87.5% and 81.82% respectively, while specificity was 100% for both pulmonary and extra-pulmonary TB. On comparison to sequencing of rpoB gene [Rif resistance determining region, RRDR], sensitivity was respectively 93.33% and 90% while specificity was 100% in both pulmonary and extra-pulmonary TB. GeneXpert assay missed 533CCG mutation in one sputum and dual mutation [517 & 519] in one pus sample, detected by sequencing. Sequencing picked dual mutation [529, 530] in a sputum sample sensitive to Rif, demonstrating, not all RRDR mutations lead to resistance. Conclusions Current study reports observations in a patient care setting in a high burden region, from a large collection of pulmonary and extra-pulmonary samples and puts to rest questions regarding sensitivity, specificity, detection of infrequent mutations and mutations responsible for low-level Rif resistance by GeneXpert. Improvements in the assay could offer further improvement in sensitivity of detection in different patient samples; nevertheless it may be difficult to improve sensitivity of Rif resistance detection if only one gene is targeted. Assay specificity was high both for TB detection and Rif resistance detection. Despite a few misses, the assay offers major boost to early diagnosis of TB and MDR-TB, in difficult to diagnose pauci-bacillary TB. PMID:26894283

Cost-effectiveness analysis of EGFR mutation testing in patients with non-small cell lung cancer (NSCLC) with gefitinib or carboplatin-paclitaxel.

PubMed

Arrieta, Oscar; Anaya, Pablo; Morales-Oyarvide, Vicente; Ramírez-Tirado, Laura Alejandra; Polanco, Ana C

2016-09-01

Assess the cost-effectiveness of an EGFR-mutation testing strategy for advanced NSCLC in first-line therapy with either gefitinib or carboplatin-paclitaxel in Mexican institutions. Cost-effectiveness analysis using a discrete event simulation (DES) model to simulate two therapeutic strategies in patients with advanced NSCLC. Strategy one included patients tested for EGFR-mutation and therapy given accordingly. Strategy two included chemotherapy for all patients without testing. All results are presented in 2014 US dollars. The analysis was made with data from the Mexican frequency of EGFR-mutation. A univariate sensitivity analysis was conducted on EGFR prevalence. Progression-free survival (PFS) transition probabilities were estimated on data from the IPASS and simulated with a Weibull distribution, run with parallel trials to calculate a probabilistic sensitivity analysis. PFS of patients in the testing strategy was 6.76 months (95 % CI 6.10-7.44) vs 5.85 months (95 % CI 5.43-6.29) in the non-testing group. The one-way sensitivity analysis showed that PFS has a direct relationship with EGFR-mutation prevalence, while the ICER and testing cost have an inverse relationship with EGFR-mutation prevalence. The probabilistic sensitivity analysis showed that all iterations had incremental costs and incremental PFS for strategy 1 in comparison with strategy 2. There is a direct relationship between the ICER and the cost of EGFR testing, with an inverse relationship with the prevalence of EGFR-mutation. When prevalence is >10 % ICER remains constant. This study could impact Mexican and Latin American health policies regarding mutation detection testing and treatment for advanced NSCLC.

Newly emerging mutations in the matrix genes of the human influenza A(H1N1)pdm09 and A(H3N2) viruses reduce the detection sensitivity of real-time reverse transcription-PCR.

PubMed

Yang, Ji-Rong; Kuo, Chuan-Yi; Huang, Hsiang-Yi; Wu, Fu-Ting; Huang, Yi-Lung; Cheng, Chieh-Yu; Su, Yu-Ting; Chang, Feng-Yee; Wu, Ho-Sheng; Liu, Ming-Tsan

2014-01-01

New variants of the influenza A(H1N1)pdm09 and A(H3N2) viruses were detected in Taiwan between 2012 and 2013. Some of these variants were not detected in clinical specimens using a common real-time reverse transcription-PCR (RT-PCR) assay that targeted the conserved regions of the viral matrix (M) genes. An analysis of the M gene sequences of the new variants revealed that several newly emerging mutations were located in the regions where the primers or probes of the real-time RT-PCR assay bind; these included three mutations (G225A, T228C, and G238A) in the A(H1N1)pdm09 virus, as well as one mutation (C163T) in the A(H3N2) virus. These accumulated mismatch mutations, together with the previously identified C154T mutation of the A(H1N1)pdm09 virus and the C153T and G189T mutations of the A(H3N2) virus, result in a reduced detection sensitivity for the real-time RT-PCR assay. To overcome the loss of assay sensitivity due to mismatch mutations, we established a real-time RT-PCR assay using degenerate nucleotide bases in both the primers and probe and successfully increased the sensitivity of the assay to detect circulating variants of the human influenza A viruses. Our observations highlight the importance of the simultaneous use of different gene-targeting real-time RT-PCR assays for the clinical diagnosis of influenza.

Glioma-derived mutations in isocitrate dehydrogenase 2 beneficial to traditional chemotherapy

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

Fu, Yuejun, E-mail: yjfu@sxu.edu.cn; Huang, Rui; Zheng, Yali

2011-07-01

Highlights: {yields} IDH1 and IDH2 mutations are not detected in the rat C6 glioma cell line model. {yields} IDH2 mutations are not required for the tumorigenesis of glioma. {yields} IDH2{sup R172G} can sensitize glioma sensitivity to chemotherapy through NADPH levels. {yields} IDH2{sup R172G} can give a benefit to traditional chemotherapy of glioma. {yields} This finding serves as an important complement to existing research on this topic. -- Abstract: Heterozygous mutations in either the R132 residue of isocitrate dehydrogenase I (IDH1) or the R172 residue of IDH2 in human gliomas were recently highlighted. In the present study, we report that mutationsmore » of IDH1 and IDH2 are not detected in the rat C6 glioma cell line model, which suggests that these mutations are not required for the development of glioblastoma induced by N,N'-nitroso-methylurea. The effects of IDH2 and IDH2{sup R172G} on C6 cells proliferation and sensitivity to chemotherapy and the possible mechanism are analyzed at the cellular level. IDH1 and IDH2 mutations lead to simultaneous loss and gain of activities in the production of {alpha}-ketoglutarate ({alpha}-KG) and 2-hydroxyglutarate (2HG), respectively, and result in lowering NADPH levels even further. The low NADPH levels can sensitize tumors to chemotherapy, and account for the prolonged survival of patients harboring the mutations. Our data extrapolate potential importance of the in vitro rat C6 glioma cell model, show that the IDH2{sup R172G} mutation in gliomas may give a benefit to traditional chemotherapy of this cancer and serve as an important complement to existing research on this topic.« less

Highly sensitive detection of the PIK3CA (H1047R) mutation in colorectal cancer using a novel PCR-RFLP method.

PubMed

Li, Wan-Ming; Hu, Ting-Ting; Zhou, Lin-Lin; Feng, Yi-Ming; Wang, Yun-Yi; Fang, Jin

2016-07-12

The PIK3CA (H1047R) mutation is considered to be a potential predictive biomarker for EGFR-targeted therapies. In this study, we developed a novel PCR-PFLP approach to detect the PIK3CA (H1047R) mutation in high effectiveness. A 126-bp fragment of PIK3CA exon-20 was amplified by PCR, digested with FspI restriction endonuclease and separated by 3 % agarose gel electrophoresis for the PCR-RFLP analysis. The mutant sequence of the PIK3CA (H1047R) was spiked into the corresponding wild-type sequence in decreasing ratios for sensitivity analysis. Eight-six cases of formalin-fixed paraffin-embedded colorectal cancer (CRC) specimens were subjected to PCR-RFLP to evaluate the applicability of the method. The PCR-RFLP method had a capability to detect as litter as 0.4 % of mutation, and revealed 16.3 % of the PIK3CA (H1047R) mutation in 86 CRC tissues, which was significantly higher than that discovered by DNA sequencing (9.3 %). A positive association between the PIK3CA (H1047R) mutation and the patients' age was first found, except for the negative relationship with the degree of tumor differentiation. In addition, the highly sensitive detection of a combinatorial mutation of PIK3CA, KRAS and BRAF was achieved using individual PCR-RFLP methods. We developed a sensitive, simple and rapid approach to detect the low-abundance PIK3CA (H1047R) mutation in real CRC specimens, providing an effective tool for guiding cancer targeted therapy.

DNA repair properties of Escherichia coli tif-1, recAo281 and lexA1 strains deficient in single-strand DNA binding protein.

PubMed

Whittier, R F; Chase, J W

1983-01-01

Mutations affecting single-strand DNA binding protein (SSB) impair induction of mutagenic (SOS) repair. To further investigate the role of SSB in SOS induction and DNA repair, isogenic strains were constructed combining the ssb+, ssb-1 or ssb-113 alleles with one or more mutations known to alter regulation of damage inducible functions. As is true in ssb+ strains tif-1 (recA441) was found to allow thermal induction of prophage lambda + and Weigle reactivation in ssb-1 and ssb-113 strains. Furthermore, tif-1 decreased the UV sensitivity of the ssb-113 strain slightly and permitted UV induction of prophage lambda + at 30 degrees C. Strains carrying the recAo281 allele were also constructed. This mutation causes high constitutive levels of RecA protein synthesis and relieves much of the UV sensitivity conferred by lexA- alleles without restoring SOS (error-prone) repair. In contrast, the recAo281 allele failed to alleviate the UV sensitivity associated with either ssb- mutation. In a lexA1 recAo281 background the ssb-1 mutation increased the extent of postirradiation DNA degradation and concommitantly increased UV sensitivity 20-fold to the level exhibited by a recA1 strain. The ssb-113 mutation also increased UV sensitivity markedly in this background but did so without greatly increasing postirradiation DNA degradation. These results suggest a direct role for SSB in recombinational repair apart from and in addition to its role in facilitating induction of the recA-lexA regulon.

ALK F1174V mutation confers sensitivity while ALK I1171 mutation confers resistance to alectinib. The importance of serial biopsy post progression.

PubMed

Ou, Sai-Hong; Milliken, Jeffrey C; Azada, Michele C; Miller, Vincent A; Ali, Siraj M; Klempner, Samuel J

2016-01-01

Many acquired resistant mutations to the anaplastic lymphoma kinase (ALK) gene have been identified during treatment of ALK-rearranged non-small cell lung cancer (NSCLC) patients with crizotinib, ceritinib, and alectinib. These various acquired resistant ALK mutations confer differential sensitivities to various ALK inhibitors and may provide guidance on how to sequence the use of many of the second generation ALK inhibitors. We described a patient who developed an acquired ALK F1174V resistant mutation on progression from crizotinib that responded to alectinib for 18 months but then developed an acquired ALK I1171S mutation to alectinib. Both tumor samples had essentially the same genomic profile by comprehensive genomic profiling otherwise. This is the first patient report that demonstrates ALK F1174V mutation is sensitive to alectinib and further confirms missense acquired ALK I1171 mutation is resistant to alectinib. Sequential tumor re-biopsy for comprehensive genomic profiling (CGP) is important to appreciate the selective pressure during treatment with various ALK inhibitors underpinning the evolution of the disease course of ALK+NSCLC patients while on treatment with the various ALK inhibitors. This approach will likely help inform the optimal sequencing strategy as more ALK inhibitors become available. This case report also validates the importance of developing structurally distinct ALK inhibitors for clinical use to overcome non-cross resistant ALK mutations. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

High and low temperatures have unequal reinforcing properties in Drosophila spatial learning.

PubMed

Zars, Melissa; Zars, Troy

2006-07-01

Small insects regulate their body temperature solely through behavior. Thus, sensing environmental temperature and implementing an appropriate behavioral strategy can be critical for survival. The fly Drosophila melanogaster prefers 24 degrees C, avoiding higher and lower temperatures when tested on a temperature gradient. Furthermore, temperatures above 24 degrees C have negative reinforcing properties. In contrast, we found that flies have a preference in operant learning experiments for a low-temperature-associated position rather than the 24 degrees C alternative in the heat-box. Two additional differences between high- and low-temperature reinforcement, i.e., temperatures above and below 24 degrees C, were found. Temperatures equally above and below 24 degrees C did not reinforce equally and only high temperatures supported increased memory performance with reversal conditioning. Finally, low- and high-temperature reinforced memories are similarly sensitive to two genetic mutations. Together these results indicate the qualitative meaning of temperatures below 24 degrees C depends on the dynamics of the temperatures encountered and that the reinforcing effects of these temperatures depend on at least some common genetic components. Conceptualizing these results using the Wolf-Heisenberg model of operant conditioning, we propose the maximum difference in experienced temperatures determines the magnitude of the reinforcement input to a conditioning circuit.

Clinical Sensitivity of Cystic Fibrosis Mutation Panels in a Diverse Population.

PubMed

Hughes, Erin E; Stevens, Colleen F; Saavedra-Matiz, Carlos A; Tavakoli, Norma P; Krein, Lea M; Parker, April; Zhang, Zhen; Maloney, Breanne; Vogel, Beth; DeCelie-Germana, Joan; Kier, Catherine; Anbar, Ran D; Berdella, Maria N; Comber, Paul G; Dozor, Allen J; Goetz, Danielle M; Guida, Louis; Kattan, Meyer; Ting, Andrew; Voter, Karen Z; van Roey, Patrick; Caggana, Michele; Kay, Denise M

2016-02-01

Infants are screened for cystic fibrosis (CF) in New York State (NYS) using an IRT-DNA algorithm. The purpose of this study was to validate and assess clinical validity of the US FDA-cleared Illumina MiSeqDx CF 139-Variant Assay (139-VA) in the diverse NYS CF population. The study included 439 infants with CF identified via newborn screening (NBS) from 2002 to 2012. All had been screened using the Abbott Molecular CF Genotyping Assay or the Hologic InPlex CF Molecular Test. All with CF and zero or one mutation were tested using the 139-VA. DNA extracted from dried blood spots was reliably and accurately genotyped using the 139-VA. Sixty-three additional mutations were identified. Clinical sensitivity of three panels ranged from 76.2% (23 mutations recommended for screening by ACMG/ACOG) to 79.7% (current NYS 39-mutation InPlex panel), up to 86.0% for the 139-VA. For all, sensitivity was highest in Whites and lowest in the Black population. Although the sample size was small, there was a nearly 20% increase in sensitivity for the Black CF population using the 139-VA (68.2%) over the ACMG/ACOG and InPlex panels (both 50.0%). Overall, the 139-VA is more sensitive than other commercially available panels, and could be considered for NBS, clinical, or research laboratories conducting CF screening. © 2015 WILEY PERIODICALS, INC.

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

PubMed

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

2016-10-01

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

Genotypic evaluation of etravirine sensitivity of clinical human immunodeficiency virus type 1 (HIV-1) isolates carrying resistance mutations to nevirapine and efavirenz.

PubMed

Oumar, A A; Jnaoui, K; Kabamba-Mukadi, B; Yombi, J C; Vandercam, B; Goubau, P; Ruelle, J

2010-01-01

Etravirine is a second-generation non-nucleoside reverse transcriptase inhibitor (NNRTI) with a pattern of resistance mutations quite distinct from the current NNRTIs. We collected all routine samples of HIV-1 patients followed in the AIDS reference laboratory of UCLouvain (in 2006 and 2007) carrying resistance-associated mutations to nevirapine (NVP) or efavirenz (EFV). The sensitivity to Etravirine was estimated using three different drug resistance algorithms: ANRS (July 2008), IAS (December 2008) and Stanford (November 2008). We also verified whether the mutations described as resistance mutations are not due to virus polymorphisms by the study of 58 genotypes of NNRTI-naive patients. Sixty one samples harboured resistance to NVP and EFV: 41/61 had at least one resistance mutation to Etravirine according to ANRS-IAS algorithms; 42/61 samples had at least one resistance mutation to Etravirine according to the Stanford algorithm. 48 and 53 cases were fully sensitive to Etravirine according to ANRS-IAS and Stanford algorithms, respectively. Three cases harboured more than three mutations and presented a pattern of high-degree resistance to Etravirine according to ANRS-IAS algorithm, while one case harboured more than three mutations and presented high degree resistance to Etravirine according to the Stanford algorithm. The V1061 and V179D mutations were more frequent in the ARV-naive group than in the NNRTI-experienced one. According to the currently available algorithms, Etravirine can still be used in the majority of patients with virus showing resistance to NVP and/or EFV, if a combination of other active drugs is included.

A new sensitive PCR assay for one-step detection of 12 IDH1/2 mutations in glioma.

PubMed

Catteau, Aurélie; Girardi, Hélène; Monville, Florence; Poggionovo, Cécile; Carpentier, Sabrina; Frayssinet, Véronique; Voss, Jesse; Jenkins, Robert; Boisselier, Blandine; Mokhtari, Karima; Sanson, Marc; Peyro-Saint-Paul, Hélène; Giannini, Caterina

2014-06-02

Mutations in isocitrate dehydrogenase genes IDH1 or IDH2 are frequent in glioma, and IDH mutation status is a strong diagnostic and prognostic marker. Current IDH mutation screening is performed with an immunohistochemistry (IHC) assay specific for IDH1 R132H, the most common mutation. Sequencing is recommended as a second-step test for IHC-negative or -equivocal cases. We developed and validated a new real-time quantitative polymerase chain reaction (PCR) assay for single-step detection of IDH1 R132H and 11 rare IDH1/2 mutations in formalin-fixed paraffin-embedded (FFPE) glioma samples. Performance of the IDH1/2 PCR assay was compared to IHC and Sanger sequencing. The IDH1/2 PCR assay combines PCR clamping for detection of 7 IDH1 and 5 IDH2 mutations, and Amplification Refractory Mutation System technology for specific identification of the 3 most common mutations (IDH1 R132H, IDH1 R132C, IDH2 R172K). Analytical sensitivity of the PCR assay for mutation detection was <5% for 11/12 mutations (mean: 3.3%), and sensitivity for mutation identification was very high (0.8% for IDH1 R132H; 1.2% for IDH1 R132C; 0.6% for IDH2 R172K). Assay performance was further validated on 171 clinical glioma FFPE samples; of these, 147 samples met the selection criteria and 146 DNA samples were successfully extracted. IDH1/2 status was successfully obtained in 91% of cases. All but one positive IDH1 R132H-IHC cases were concordantly detected by PCR and 3 were not detected by sequencing. Among the IHC-negative cases (n = 72), PCR detected 12 additional rare mutations (10 IDH1, 2 IDH2). All mutations detected by sequencing (n = 67) were concordantly detected by PCR and 5/66 sequencing-negative cases were PCR-positive (overall concordance: 96%). Analysis of synthetic samples representative of the 11 rare IDH1/2 mutations detected by the assay produced 100% correct results. The new IDH1/2 PCR assay has a high technical success rate and is more sensitive than Sanger sequencing. Positive concordance was 98% with IHC for IDH1 R132H detection and 100% with sequencing. The PCR assay can reliably be performed on FFPE samples and has a faster turnaround time than current IDH mutation detection algorithms. The assay should facilitate implementation of a comprehensive IDH1/2 testing protocol in routine clinical practice.

Interrelations of secondary structure stability and DNA-binding affinity in the bacteriophage SPO1-encoded type II DNA-binding protein TF1.

PubMed

Andera, L; Spangler, C J; Galeone, A; Mayol, L; Geiduschek, E P

1994-02-11

TF1, a homodimeric DNA-binding and -bending protein with a preference for hydroxymethyluracil-containing DNA is the Bacillus subtilis-encoded homolog of the bacterial HU proteins and of the E. coli integration host factor. A temperature-sensitive mutation at amino acid 25 of TF1 (L25-->A) and two intragenic second site revertants at amino acids 15 (E15-->G) and 32 (L32-->I) were previously identified and their effects on virus development were examined. The DNA-binding properties of these proteins and the thermal stability of their secondary structures have now been analyzed. Amino acids 15 and 32 are far removed from the putative DNA-binding domains of TF1 but changes there exert striking effects on DNA affinity that correlate with effects on structure. The double mutant protein TF1-G15I32 binds to a preferred site in hydroxymethyluracil-containing DNA 40 times more tightly, denatures at higher temperature (delta tm = 21 degrees C), and also exchanges subunits much more slowly than does the wild-type protein. The L25-->A mutation makes TF1 secondary structure and DNA-binding highly salt concentration-dependent. The E15-->G mutation partly suppresses this effect: secondary structure of TF1-A25G15 is restored at 21 degrees C by 1 M NaCl or, at low NaCl concentration, by binding to DNA.

Characterization of an Escherichia coli mutant (radB101) sensitive to. gamma. and uv radiation, and methyl methanesulfonate

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

Sargentini, N.J.; Smith, K.C.

1983-03-01

After N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis of Escherichia coli K-12 (xthA14), an X-ray-sensitive mutant was isolated. This sensitivity is due to a mutation, radB101, which is located at 56.5 min on the E.coli K-12 linkage map. The radB101 mutation sensitized wild-type cells to ..gamma.. and uv radiation, and to methyl methanesulfonate. When known DNA repair-deficient mutants were ranked for their ..gamma..-radiation sensitivity relative to their uv-radiation sensitivity, their order was (starting with the most selectively ..gamma..-radiation-sensitive strain): recB21, radB101, wild type, polA1, recF143, lexA101, recA56, uvrD3, and uvrA6. The radB mutant was normal for ..gamma..- and uv-radiation mutagenesis, it showed only a slightmore » enhancement of ..gamma..- and uv-radiation-induced DNA degradation, and it was approx. 60% deficient in recombination ability. The radB gene is suggested to play a role in the recA gene-dependent (Type III) repair of DNA single-strand breaks after ..gamma.. irradiation and in postreplication repair after uv irradiation for the following reasons: the radB strain was normal for the host-cell reactivation of ..gamma..- and uv-irradiated bacteriophage lambda; the radB mutation did not sensitize a recA strain, but did sensitize a polA strain to ..gamma.. and uv radiation; the radB mutation sensitized a uvrB strain to uv radiation.« less

Ultra-sensitive Sequencing Identifies High Prevalence of Clonal Hematopoiesis-Associated Mutations throughout Adult Life.

PubMed

Acuna-Hidalgo, Rocio; Sengul, Hilal; Steehouwer, Marloes; van de Vorst, Maartje; Vermeulen, Sita H; Kiemeney, Lambertus A L M; Veltman, Joris A; Gilissen, Christian; Hoischen, Alexander

2017-07-06

Clonal hematopoiesis results from somatic mutations in hematopoietic stem cells, which give an advantage to mutant cells, driving their clonal expansion and potentially leading to leukemia. The acquisition of clonal hematopoiesis-driver mutations (CHDMs) occurs with normal aging and these mutations have been detected in more than 10% of individuals ≥65 years. We aimed to examine the prevalence and characteristics of CHDMs throughout adult life. We developed a targeted re-sequencing assay combining high-throughput with ultra-high sensitivity based on single-molecule molecular inversion probes (smMIPs). Using smMIPs, we screened more than 100 loci for CHDMs in more than 2,000 blood DNA samples from population controls between 20 and 69 years of age. Loci screened included 40 regions known to drive clonal hematopoiesis when mutated and 64 novel candidate loci. We identified 224 somatic mutations throughout our cohort, of which 216 were coding mutations in known driver genes (DNMT3A, JAK2, GNAS, TET2, and ASXL1), including 196 point mutations and 20 indels. Our assay's improved sensitivity allowed us to detect mutations with variant allele frequencies as low as 0.001. CHDMs were identified in more than 20% of individuals 60 to 69 years of age and in 3% of individuals 20 to 29 years of age, approximately double the previously reported prevalence despite screening a limited set of loci. Our findings support the occurrence of clonal hematopoiesis-associated mutations as a widespread mechanism linked with aging, suggesting that mosaicism as a result of clonal evolution of cells harboring somatic mutations is a universal mechanism occurring at all ages in healthy humans. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Heterozygous ABCC8 mutations are a cause of MODY.

PubMed

Bowman, P; Flanagan, S E; Edghill, E L; Damhuis, A; Shepherd, M H; Paisey, R; Hattersley, A T; Ellard, S

2012-01-01

The ABCC8 gene encodes the sulfonylurea receptor 1 (SUR1) subunit of the pancreatic beta cell ATP-sensitive potassium (K(ATP)) channel. Inactivating mutations cause congenital hyperinsulinism (CHI) and activating mutations cause transient neonatal diabetes (TNDM) or permanent neonatal diabetes (PNDM) that can usually be treated with sulfonylureas. Sulfonylurea sensitivity is also a feature of HNF1A and HNF4A MODY, but patients referred for genetic testing with clinical features of these types of diabetes do not always have mutations in the HNF1A/4A genes. Our aim was to establish whether mutations in the ABCC8 gene cause MODY that is responsive to sulfonylurea therapy. We sequenced the ABCC8 gene in 85 patients with a BMI <30 kg/m², no family history of neonatal diabetes and who were deemed sensitive to sulfonylureas by the referring clinician or were sulfonylurea-treated. All had tested negative for mutations in the HNF1A and HNF4A genes. ABCC8 mutations were found in seven of the 85 (8%) probands. Four patients were heterozygous for previously reported mutations and four novel mutations, E100K, G214R, Q485R and N1245D, were identified. Only four probands fulfilled MODY criteria, with two diagnosed after 25 years and one patient, who had no family history of diabetes, as a result of a proven de novo mutation. ABCC8 mutations can cause MODY in patients whose clinical features are similar to those with HNF1A/4A MODY. Therefore, sequencing of ABCC8 in addition to the known MODY genes should be considered if such features are present, to facilitate optimal clinical management of these patients.

An immuno-wall microdevice exhibits rapid and sensitive detection of IDH1-R132H mutation specific to grade II and III gliomas

NASA Astrophysics Data System (ADS)

Yamamichi, Akane; Kasama, Toshihiro; Ohka, Fumiharu; Suzuki, Hiromichi; Kato, Akira; Motomura, Kazuya; Hirano, Masaki; Ranjit, Melissa; Chalise, Lushun; Kurimoto, Michihiro; Kondo, Goro; Aoki, Kosuke; Kaji, Noritada; Tokeshi, Manabu; Matsubara, Toshio; Senga, Takeshi; Kaneko, Mika K.; Suzuki, Hidenori; Hara, Masahito; Wakabayashi, Toshihiko; Baba, Yoshinobu; Kato, Yukinari; Natsume, Atsushi

2016-01-01

World Health Organization grade II and III gliomas most frequently occur in the central nervous system (CNS) in adults. Gliomas are not circumscribed; tumor edges are irregular and consist of tumor cells, normal brain tissue, and hyperplastic reactive glial cells. Therefore, the tumors are not fully resectable, resulting in recurrence, malignant progression, and eventual death. Approximately 69-80% of grade II and III gliomas harbor mutations in the isocitrate dehydrogenase 1 gene (IDH1), of which 83-90% are found to be the IDH1-R132H mutation. Detection of the IDH1-R132H mutation should help in the differential diagnosis of grade II and III gliomas from other types of CNS tumors and help determine the boundary between the tumor and normal brain tissue. In this study, we established a highly sensitive antibody-based device, referred to as the immuno-wall, to detect the IDH1-R132H mutation in gliomas. The immuno-wall causes an immunoreaction in microchannels fabricated using a photo-polymerizing polymer. This microdevice enables the analysis of the IDH1 status with a small sample within 15 min with substantially high sensitivity. Our results suggested that 10% content of the IDH1-R132H mutation in a sample of 0.33 μl volume, with 500 ng protein, or from 500 cells is theoretically sufficient for the analysis. The immuno-wall device will enable the rapid and highly sensitive detection of the IDH1-R132H mutation in routine clinical practice.

An immuno-wall microdevice exhibits rapid and sensitive detection of IDH1-R132H mutation specific to grade II and III gliomas.

PubMed

Yamamichi, Akane; Kasama, Toshihiro; Ohka, Fumiharu; Suzuki, Hiromichi; Kato, Akira; Motomura, Kazuya; Hirano, Masaki; Ranjit, Melissa; Chalise, Lushun; Kurimoto, Michihiro; Kondo, Goro; Aoki, Kosuke; Kaji, Noritada; Tokeshi, Manabu; Matsubara, Toshio; Senga, Takeshi; Kaneko, Mika K; Suzuki, Hidenori; Hara, Masahito; Wakabayashi, Toshihiko; Baba, Yoshinobu; Kato, Yukinari; Natsume, Atsushi

2016-01-01

World Health Organization grade II and III gliomas most frequently occur in the central nervous system (CNS) in adults. Gliomas are not circumscribed; tumor edges are irregular and consist of tumor cells, normal brain tissue, and hyperplastic reactive glial cells. Therefore, the tumors are not fully resectable, resulting in recurrence, malignant progression, and eventual death. Approximately 69-80% of grade II and III gliomas harbor mutations in the isocitrate dehydrogenase 1 gene ( IDH1 ), of which 83-90% are found to be the IDH1-R132H mutation. Detection of the IDH1-R132H mutation should help in the differential diagnosis of grade II and III gliomas from other types of CNS tumors and help determine the boundary between the tumor and normal brain tissue. In this study, we established a highly sensitive antibody-based device, referred to as the immuno-wall, to detect the IDH1-R132H mutation in gliomas. The immuno-wall causes an immunoreaction in microchannels fabricated using a photo-polymerizing polymer. This microdevice enables the analysis of the IDH1 status with a small sample within 15 min with substantially high sensitivity. Our results suggested that 10% content of the IDH1-R132H mutation in a sample of 0.33 μl volume, with 500 ng protein, or from 500 cells is theoretically sufficient for the analysis. The immuno-wall device will enable the rapid and highly sensitive detection of the IDH1-R132H mutation in routine clinical practice.

MIP-MAP: High-Throughput Mapping of Caenorhabditis elegans Temperature-Sensitive Mutants via Molecular Inversion Probes.

PubMed

Mok, Calvin A; Au, Vinci; Thompson, Owen A; Edgley, Mark L; Gevirtzman, Louis; Yochem, John; Lowry, Joshua; Memar, Nadin; Wallenfang, Matthew R; Rasoloson, Dominique; Bowerman, Bruce; Schnabel, Ralf; Seydoux, Geraldine; Moerman, Donald G; Waterston, Robert H

2017-10-01

Mutants remain a powerful means for dissecting gene function in model organisms such as Caenorhabditis elegans Massively parallel sequencing has simplified the detection of variants after mutagenesis but determining precisely which change is responsible for phenotypic perturbation remains a key step. Genetic mapping paradigms in C . elegans rely on bulk segregant populations produced by crosses with the problematic Hawaiian wild isolate and an excess of redundant information from whole-genome sequencing (WGS). To increase the repertoire of available mutants and to simplify identification of the causal change, we performed WGS on 173 temperature-sensitive (TS) lethal mutants and devised a novel mapping method. The mapping method uses molecular inversion probes (MIP-MAP) in a targeted sequencing approach to genetic mapping, and replaces the Hawaiian strain with a Million Mutation Project strain with high genomic and phenotypic similarity to the laboratory wild-type strain N2 We validated MIP-MAP on a subset of the TS mutants using a competitive selection approach to produce TS candidate mapping intervals with a mean size < 3 Mb. MIP-MAP successfully uses a non-Hawaiian mapping strain and multiplexed libraries are sequenced at a fraction of the cost of WGS mapping approaches. Our mapping results suggest that the collection of TS mutants contains a diverse library of TS alleles for genes essential to development and reproduction. MIP-MAP is a robust method to genetically map mutations in both viable and essential genes and should be adaptable to other organisms. It may also simplify tracking of individual genotypes within population mixtures. Copyright © 2017 by the Genetics Society of America.

MIP-MAP: High-Throughput Mapping of Caenorhabditis elegans Temperature-Sensitive Mutants via Molecular Inversion Probes

PubMed Central

Mok, Calvin A.; Au, Vinci; Thompson, Owen A.; Edgley, Mark L.; Gevirtzman, Louis; Yochem, John; Lowry, Joshua; Memar, Nadin; Wallenfang, Matthew R.; Rasoloson, Dominique; Bowerman, Bruce; Schnabel, Ralf; Seydoux, Geraldine; Moerman, Donald G.; Waterston, Robert H.

2017-01-01

Mutants remain a powerful means for dissecting gene function in model organisms such as Caenorhabditis elegans. Massively parallel sequencing has simplified the detection of variants after mutagenesis but determining precisely which change is responsible for phenotypic perturbation remains a key step. Genetic mapping paradigms in C. elegans rely on bulk segregant populations produced by crosses with the problematic Hawaiian wild isolate and an excess of redundant information from whole-genome sequencing (WGS). To increase the repertoire of available mutants and to simplify identification of the causal change, we performed WGS on 173 temperature-sensitive (TS) lethal mutants and devised a novel mapping method. The mapping method uses molecular inversion probes (MIP-MAP) in a targeted sequencing approach to genetic mapping, and replaces the Hawaiian strain with a Million Mutation Project strain with high genomic and phenotypic similarity to the laboratory wild-type strain N2. We validated MIP-MAP on a subset of the TS mutants using a competitive selection approach to produce TS candidate mapping intervals with a mean size < 3 Mb. MIP-MAP successfully uses a non-Hawaiian mapping strain and multiplexed libraries are sequenced at a fraction of the cost of WGS mapping approaches. Our mapping results suggest that the collection of TS mutants contains a diverse library of TS alleles for genes essential to development and reproduction. MIP-MAP is a robust method to genetically map mutations in both viable and essential genes and should be adaptable to other organisms. It may also simplify tracking of individual genotypes within population mixtures. PMID:28827289

Low-cost functional plasticity of TRPV1 supports heat tolerance in squirrels and camels.

PubMed

Laursen, Willem J; Schneider, Eve R; Merriman, Dana K; Bagriantsev, Sviatoslav N; Gracheva, Elena O

2016-10-04

The ability to sense heat is crucial for survival. Increased heat tolerance may prove beneficial by conferring the ability to inhabit otherwise prohibitive ecological niches. This phenomenon is widespread and is found in both large and small animals. For example, ground squirrels and camels can tolerate temperatures more than 40 °C better than many other mammalian species, yet a molecular mechanism subserving this ability is unclear. Transient receptor potential vanilloid 1 (TRPV1) is a polymodal ion channel involved in the detection of noxious thermal and chemical stimuli by primary afferents of the somatosensory system. Here, we show that thirteen-lined ground squirrels (Ictidomys tridecemlineatus) and Bactrian camels (Camelus ferus) express TRPV1 orthologs with dramatically reduced temperature sensitivity. The loss of sensitivity is restricted to temperature and does not affect capsaicin or acid responses, thereby maintaining a role for TRPV1 as a detector of noxious chemical cues. We show that heat sensitivity can be reengineered in both TRPV1 orthologs by a single amino acid substitution in the N-terminal ankyrin-repeat domain. Conversely, reciprocal mutations suppress heat sensitivity of rat TRPV1, supporting functional conservation of the residues. Our studies suggest that squirrels and camels co-opt a common molecular strategy to adapt to hot environments by suppressing the efficiency of TRPV1-mediated heat detection at the level of somatosensory neurons. Such adaptation is possible because of the remarkable functional flexibility of the TRPV1 molecule, which can undergo profound tuning at the minimal cost of a single amino acid change.

Low-cost functional plasticity of TRPV1 supports heat tolerance in squirrels and camels

PubMed Central

Laursen, Willem J.; Merriman, Dana K.; Bagriantsev, Sviatoslav N.; Gracheva, Elena O.

2016-01-01

The ability to sense heat is crucial for survival. Increased heat tolerance may prove beneficial by conferring the ability to inhabit otherwise prohibitive ecological niches. This phenomenon is widespread and is found in both large and small animals. For example, ground squirrels and camels can tolerate temperatures more than 40 °C better than many other mammalian species, yet a molecular mechanism subserving this ability is unclear. Transient receptor potential vanilloid 1 (TRPV1) is a polymodal ion channel involved in the detection of noxious thermal and chemical stimuli by primary afferents of the somatosensory system. Here, we show that thirteen-lined ground squirrels (Ictidomys tridecemlineatus) and Bactrian camels (Camelus ferus) express TRPV1 orthologs with dramatically reduced temperature sensitivity. The loss of sensitivity is restricted to temperature and does not affect capsaicin or acid responses, thereby maintaining a role for TRPV1 as a detector of noxious chemical cues. We show that heat sensitivity can be reengineered in both TRPV1 orthologs by a single amino acid substitution in the N-terminal ankyrin-repeat domain. Conversely, reciprocal mutations suppress heat sensitivity of rat TRPV1, supporting functional conservation of the residues. Our studies suggest that squirrels and camels co-opt a common molecular strategy to adapt to hot environments by suppressing the efficiency of TRPV1-mediated heat detection at the level of somatosensory neurons. Such adaptation is possible because of the remarkable functional flexibility of the TRPV1 molecule, which can undergo profound tuning at the minimal cost of a single amino acid change. PMID:27638213

Rapid polymerase chain reaction screening of Helicobacter pylori chromosomal point mutations.

PubMed

Ge, Z; Taylor, D E

1997-09-01

Microdiversity (within individual genes) in the genomes of different Helicobacter pylori strains has been demonstrated to be more frequent than that seen in other prokaryotes. Point mutations in some genes, such as the vacA and 23S ribosomal RNA genes could result in the alteration of pathogenicity or antibiotic susceptibility of individual H. pylori strains. Development of a simple, rapid, and reliable screening method would be useful in the molecular characterization of genetic variation among different H. pylori strains. The copP gene from H. pylori UA802 was used as a model for developing a mutation screening method. Four point mutations were introduced into the copP gene by in vitro site-directed mutagenesis and were verified by DNA sequencing. The mutated copP gene replaced the wild-type locus by natural transformation and homologous recombination. The site-specific mutants were screened by polymerase chain reaction (PCR) using 3'-end mismatched primers. The origins of the PCR fragments were demonstrated by Southern hybridization with the copP-derived DNA probe. Three of these four mutations were characterized by PCR with the specific primers that contained the 3'-terminal nucleotide complementary only to the mutated nucleotide on both plasmid and chromosomal DNA templates. One mutation was able to be identified with the foregoing primer containing an additional wild-type nucleotide at its 3'-end. Point mutant screening with these specific primers offers 100% sensitivity in the aforementioned conditions. To achieve optimal screening, the concentration of magnesium and the annealing temperature have to be adjusted. The procedure reported in this study is a simple, economical, rapid, and efficient approach in the identification of site-specific mutations on both plasmids and chromosomal DNA. Although the method was developed by using a specified H. pylori gene, it can be extended easily to other genes of interest in H. pylori or other organisms.

Human COQ9 Rescues a coq9 Yeast Mutant by Enhancing Coenzyme Q Biosynthesis from 4-Hydroxybenzoic Acid and Stabilizing the CoQ-Synthome

PubMed Central

He, Cuiwen H.; Black, Dylan S.; Allan, Christopher M.; Meunier, Brigitte; Rahman, Shamima; Clarke, Catherine F.

2017-01-01

Coq9 is required for the stability of a mitochondrial multi-subunit complex, termed the CoQ-synthome, and the deamination step of Q intermediates that derive from para-aminobenzoic acid (pABA) in yeast. In human, mutations in the COQ9 gene cause neonatal-onset primary Q10 deficiency. In this study, we determined whether expression of human COQ9 could complement yeast coq9 point or null mutants. We found that expression of human COQ9 rescues the growth of the temperature-sensitive yeast mutant, coq9-ts19, on a non-fermentable carbon source and increases the content of Q6, by enhancing Q biosynthesis from 4-hydroxybenzoic acid (4HB). To study the mechanism for the rescue by human COQ9, we determined the steady-state levels of yeast Coq polypeptides in the mitochondria of the temperature-sensitive yeast coq9 mutant expressing human COQ9. We show that the expression of human COQ9 significantly increased steady-state levels of yeast Coq4, Coq6, Coq7, and Coq9 at permissive temperature. Human COQ9 polypeptide levels persisted at non-permissive temperature. A small amount of the human COQ9 co-purified with tagged Coq6, Coq6-CNAP, indicating that human COQ9 interacts with the yeast Q-biosynthetic complex. These findings suggest that human COQ9 rescues the yeast coq9 temperature-sensitive mutant by stabilizing the CoQ-synthome and increasing Q biosynthesis from 4HB. This finding provides a powerful approach to studying the function of human COQ9 using yeast as a model. PMID:28736527

An Arabidopsis mutation in translation elongation factor 2 causes superinduction of CBF/DREB1 transcription factor genes but blocks the induction of their downstream targets under low temperatures.

PubMed

Guo, Yan; Xiong, Liming; Ishitani, Manabu; Zhu, Jian-Kang

2002-05-28

Low temperature regulates gene expression in bacteria, yeast, and animals as well as in plants. However, the signal transduction cascades mediating the low temperature responses are not well understood in any organism. To identify components in low temperature signaling genetically, we isolated Arabidopsis thaliana mutants in which cold-responsive genes are no longer induced by low temperatures. One of these mutations, los1-1, specifically blocks low temperature-induced transcription of cold-responsive genes. Surprisingly, cold-induced expression of the early response transcriptional activators, C-repeat/dehydration responsive element binding factors (CBF/DREB1s), is enhanced by the los1-1 mutation. The los1-1 mutation also reduces the capacity of plants to develop freezing tolerance but does not impair the vernalization response. Genetic analysis indicated that los1-1 is a recessive mutation in a single nuclear gene. The LOS1 gene encodes a translation elongation factor 2-like protein. Protein labeling studies show that new protein synthesis is blocked in los1-1 mutant plants specifically in the cold. These results reveal a critical role of new protein synthesis in the proper transduction of low temperature signals. Our results also suggest that cold-induced transcription of CBF/DREB1s is feedback inhibited by their gene products or by products of their downstream target genes.

Increased Sensitivity to Thermal Pain and Reduced Subcutaneous Lidocaine Efficacy in Redheads

PubMed Central

Liem, Edwin B.; Joiner, Teresa V.; Tsueda, Kentaro; Sessler, Daniel I.

2005-01-01

Background: Anesthetic requirement in redheads is exaggerated, suggesting that redheads may be especially sensitive to pain. We therefore tested the hypotheses that women with natural red hair are more sensitive to pain, and that redheads are resistant to topical and subcutaneous lidocaine. Methods: We evaluated pain sensitivity in red-haired (n=30) or dark-haired (n=30) women by determining the electrical current perception threshold, pain perception, and maximum pain tolerance with a Neurometer CPT/C (Neurotron, Inc., Baltimore, MD). We evaluated the analogous warm and cold temperature thresholds with the TSA-II Neurosensory Analyzer (Medoc Ltd., Minneapolis, MN). Volunteers were tested with both devices at baseline and with the Neurometer after 1-hour exposure to 4% liposomal lidocaine and after subcutaneous injection of 1% lidocaine. Data are presented as medians [interquartile ranges]. Results: Current perception, pain perception, and pain tolerance thresholds were similar in the red-haired and dark-haired women at 2000, 250, and 5 Hz. In contrast, redheads were more sensitive to cold pain perception (22.6°C [15.1, 26.1] vs. 12.6°C [0, 20], P=0.004), cold pain tolerance (6.0°C [0, 9.7] vs. 0.0°C [0.0, 2.0], P=0.001), and heat pain (46.3°C [45.7, 47.5] vs. 47.7°C [46.6, 48.7], P=0.009). Subcutaneous, lidocaine was significantly less effective in redheads, e.g., pain tolerance threshold at 2000 Hz stimulation in redheads was 11.0 mA [8.5, 16.5] vs. >20.0 mA [14.5, >20] in others, P=0.005). Conclusion: Red hair is the phenotype for mutations of the melanocortin 1 receptor. Our results indicate that redheads are more sensitive to thermal pain and are resistant to the analgesic effects of subcutaneous lidocaine. Mutations of the melanocortin 1 receptor, or a consequence thereof, thus modulate pain sensitivity. PMID:15731586

The Colony-Stimulating Factor 3 Receptor T640N Mutation Is Oncogenic, Sensitive to JAK Inhibition, and Mimics T618I.

PubMed

Maxson, Julia E; Luty, Samuel B; MacManiman, Jason D; Paik, Jason C; Gotlib, Jason; Greenberg, Peter; Bahamadi, Swaleh; Savage, Samantha L; Abel, Melissa L; Eide, Christopher A; Loriaux, Marc M; Stevens, Emily A; Tyner, Jeffrey W

2016-02-01

Colony-stimulating factor 3 receptor (CSF3R) mutations have been identified in the majority of chronic neutrophilic leukemia (CNL) and a smaller percentage of atypical chronic myeloid leukemia (aCML) cases. Although CSF3R point mutations (e.g., T618I) are emerging as key players in CNL/aCML, the significance of rarer CSF3R mutations is unknown. In this study, we assess the importance of the CSF3R T640N mutation as a marker of CNL/aCML and potential therapeutic target. Sanger sequencing of leukemia samples was performed to identify CSF3R mutations in CNL and aCML. The oncogenicity of the CSF3R T640N mutation relative to the T618I mutation was assessed by cytokine independent growth assays and by mouse bone marrow transplant. Receptor dimerization and O-glycosylation of the mutants was assessed by Western blot, and JAK inhibitor sensitivity was assessed by colony assay. Here, we identify a CSF3R T640N mutation in two patients with CNL/aCML, one of whom was originally diagnosed with MDS and acquired the T640N mutation upon evolution of disease to aCML. The T640N mutation is oncogenic in cellular transformation assays and an in vivo mouse bone marrow transplantation model. It exhibits many similar phenotypic features to T618I, including ligand independence and altered patterns of O-glycosylation--despite the transmembrane location of T640 preventing access by GalNAc transferase enzymes. Cells transformed by the T640N mutation are sensitive to JAK kinase inhibition to a similar degree as cells transformed by CSF3R T618I. Because of its similarities to CSF3R T618I, the T640N mutation likely has diagnostic and therapeutic relevance in CNL/aCML. ©2015 American Association for Cancer Research.

Determination of EGFR and KRAS mutational status in Greek non-small-cell lung cancer patients

PubMed Central

PAPADOPOULOU, EIRINI; TSOULOS, NIKOLAOS; TSIRIGOTI, ANGELIKI; APESSOS, ANGELA; AGIANNITOPOULOS, KONSTANTINOS; METAXA-MARIATOU, VASILIKI; ZAROGOULIDIS, KONSTANTINOS; ZAROGOULIDIS, PAVLOS; KASARAKIS, DIMITRIOS; KAKOLYRIS, STYLIANOS; DAHABREH, JUBRAIL; VLASTOS, FOTIS; ZOUBLIOS, CHARALAMPOS; RAPTI, AGGELIKI; PAPAGEORGIOU, NIKI GEORGATOU; VELDEKIS, DIMITRIOS; GAGA, MINA; ARAVANTINOS, GERASIMOS; KARAVASILIS, VASILEIOS; KARAGIANNIDIS, NAPOLEON; NASIOULAS, GEORGE

2015-01-01

It has been reported that certain patients with non-small-cell lung cancer (NSCLC) that harbor activating somatic mutations within the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene may be effectively treated using targeted therapy. The use of EGFR inhibitors in patient therapy has been demonstrated to improve response and survival rates; therefore, it was suggested that clinical screening for EGFR mutations should be performed for all patients. Numerous clinicopathological factors have been associated with EGFR and Kirsten-rat sarcoma oncogene homolog (KRAS) mutational status including gender, smoking history and histology. In addition, it was reported that EGFR mutation frequency in NSCLC patients was ethnicity-dependent, with an incidence rate of ~30% in Asian populations and ~15% in Caucasian populations. However, limited data has been reported on intra-ethnic differences throughout Europe. The present study aimed to investigate the frequency and spectrum of EGFR mutations in 1,472 Greek NSCLC patients. In addition, KRAS mutation analysis was performed in patients with known smoking history in order to determine the correlation of type and mutation frequency with smoking. High-resolution melting curve (HRM) analysis followed by Sanger sequencing was used to identify mutations in exons 18–21 of the EGFR gene and in exon 2 of the KRAS gene. A sensitive next-generation sequencing (NGS) technology was also employed to classify samples with equivocal results. The use of sensitive mutation detection techniques in a large study population of Greek NSCLC patients in routine diagnostic practice revealed an overall EGFR mutation frequency of 15.83%. This mutation frequency was comparable to that previously reported in other European populations. Of note, there was a 99.8% concordance between the HRM method and Sanger sequencing. NGS was found to be the most sensitive method. In addition, female non-smokers demonstrated a high prevalence of EGFR mutations. Furthermore, KRAS mutation analysis in patients with a known smoking history revealed no difference in mutation frequency according to smoking status; however, a different mutation spectrum was observed. PMID:26622815

Opposing effects of cancer-type-specific SPOP mutants on BET protein degradation and sensitivity to BET inhibitors.

PubMed

Janouskova, Hana; El Tekle, Geniver; Bellini, Elisa; Udeshi, Namrata D; Rinaldi, Anna; Ulbricht, Anna; Bernasocchi, Tiziano; Civenni, Gianluca; Losa, Marco; Svinkina, Tanya; Bielski, Craig M; Kryukov, Gregory V; Cascione, Luciano; Napoli, Sara; Enchev, Radoslav I; Mutch, David G; Carney, Michael E; Berchuck, Andrew; Winterhoff, Boris J N; Broaddus, Russell R; Schraml, Peter; Moch, Holger; Bertoni, Francesco; Catapano, Carlo V; Peter, Matthias; Carr, Steven A; Garraway, Levi A; Wild, Peter J; Theurillat, Jean-Philippe P

2017-09-01

It is generally assumed that recurrent mutations within a given cancer driver gene elicit similar drug responses. Cancer genome studies have identified recurrent but divergent missense mutations affecting the substrate-recognition domain of the ubiquitin ligase adaptor SPOP in endometrial and prostate cancers. The therapeutic implications of these mutations remain incompletely understood. Here we analyzed changes in the ubiquitin landscape induced by endometrial cancer-associated SPOP mutations and identified BRD2, BRD3 and BRD4 proteins (BETs) as SPOP-CUL3 substrates that are preferentially degraded by endometrial cancer-associated SPOP mutants. The resulting reduction of BET protein levels sensitized cancer cells to BET inhibitors. Conversely, prostate cancer-specific SPOP mutations resulted in impaired degradation of BETs, promoting their resistance to pharmacologic inhibition. These results uncover an oncogenomics paradox, whereby mutations mapping to the same domain evoke opposing drug susceptibilities. Specifically, we provide a molecular rationale for the use of BET inhibitors to treat patients with endometrial but not prostate cancer who harbor SPOP mutations.

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

PubMed

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

2018-05-07

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

Pain thresholds, supra-threshold pain and lidocaine sensitivity in patients with erythromelalgia, including the I848Tmutation in NaV 1.7.

PubMed

Helås, T; Sagafos, D; Kleggetveit, I P; Quiding, H; Jönsson, B; Segerdahl, M; Zhang, Z; Salter, H; Schmelz, M; Jørum, E

2017-09-01

Nociceptive thresholds and supra-threshold pain ratings as well as their reduction upon local injection with lidocaine were compared between healthy subjects and patients with erythromelalgia (EM). Lidocaine (0.25, 0.50, 1.0 or 10 mg/mL) or placebo (saline) was injected intradermally in non-painful areas of the lower arm, in a randomized, double-blind manner, to test the effect on dynamic and static mechanical sensitivity, mechanical pain sensitivity, thermal thresholds and supra-threshold heat pain sensitivity. Heat pain thresholds and pain ratings to supra-threshold heat stimulation did not differ between EM-patients (n = 27) and controls (n = 25), neither did the dose-response curves for lidocaine. Only the subgroup of EM-patients with mutations in sodium channel subunits Na V 1.7, 1.8 or 1.9 (n = 8) had increased lidocaine sensitivity for supra-threshold heat stimuli, contrasting lower sensitivity to strong mechanical stimuli. This pattern was particularly clear in the two patients carrying the Na V 1.7 I848T mutations in whom lidocaine's hyperalgesic effect on mechanical pain sensitivity contrasted more effective heat analgesia. Heat pain thresholds are not sensitized in EM patients, even in those with gain-of-function mutations in Na V 1.7. Differential lidocaine sensitivity was overt only for noxious stimuli in the supra-threshold range suggesting that sensitized supra-threshold encoding is important for the clinical pain phenotype in EM in addition to lower activation threshold. Intracutaneous lidocaine dose-dependently blocked nociceptive sensations, but we did not identify EM patients with particular high lidocaine sensitivity that could have provided valuable therapeutic guidance. Acute pain thresholds and supra-threshold heat pain in controls and patients with erythromelalgia do not differ and have the same lidocaine sensitivity. Acute heat pain thresholds even in EM patients with the Na V 1.7 I848T mutation are normal and only nociceptor sensitivity to intradermal lidocaine is changed. Only in EM patients with mutations in Na V 1.7, 1.8 or 1.9 supra-threshold heat and mechanical pain shows differential lidocaine sensitivity as compared to controls. © 2017 European Pain Federation - EFIC®.

Detection of BRAF mutations from solid tumors using Tumorplex™ technology

PubMed Central

Yo, Jacob; Hay, Katie S.L.; Vinayagamoorthy, Dilanthi; Maryanski, Danielle; Carter, Mark; Wiegel, Joseph; Vinayagamoorthy, Thuraiayah

2015-01-01

Allele specific multiplex sequencing (Tumorplex™) is a new molecular platform for the detection of single base mutation in tumor biopsies with high sensitivity for clinical testing. Tumorplex™ is a novel modification of Sanger sequencing technology that generates both mutant and wild type nucleotide sequences simultaneously in the same electropherogram. The molecular weight of the two sequencing primers are different such that the two sequences generated are separated, thus eliminating possible suppression of mutant signal by the more abundant wild type signal. Tumorplex™ platform technology was tested using BRAF mutation V600E. These studies were performed with cloned BRAF mutations and genomic DNA extracted from tumor cells carrying 50% mutant allele. The lower limit of detection for BRAF V600E was found to be 20 genome equivalents (GE) using genomic DNA extracted from mutation specific cell lines. Sensitivity of the assay was tested by challenging the mutant allele with wild type allele at 20 GE, and was able to detect BRAF mutant signal at a GE ration of 20:1 × 107 (mutant to wild-type). This level of sensitivity can detect low abundance of clonal mutations in tumor biopsies and eliminate the need for cell enrichment. • Tumorplex™ is a single tube assay that permits the recognition of mutant allele without suppression by wildtype signal. • Tumorplex™ provides a high level of sensitivity. • Tumorplex™ can be used with small sample size with mixed population of cells carrying heterogeneous gDNA. PMID:26258049

Analysis of mutagenic DNA repair in a thermoconditional mutant of Saccharomyces cerevisiae. III. Dose-response pattern of mutation induction in UV-irradiated rev2ts cells.

PubMed

Siede, W; Eckardt, F

1986-01-01

Recent studies regarding the influence of cycloheximide on the temperature-dependent increase in survival and mutation frequencies of a thermoconditional rev2 mutant lead to the suggestion that the REV2-coded mutagenic repair function is UV-inducible. In the present study we show that stationary-phase rev2ts cells are characterized by a biphasic linear-quadratic dose-dependence of mutation induction ("mutation kinetics") of ochre alleles at 23 degrees C (permissive temperature) but linear kinetics at the restrictive temperature of 36 degrees C. Mathematical analysis using a model based on Poisson statistics and a further mathematical procedure, the calculation of "apparent survival", support the assumption that the quadratic component of the reverse mutation kinetics investigated can be attributed to a UV-inducible component of mutagenic DNA repair controlled by the REV2 gene.

PPARγ2Pro12Ala Polymorphism and Human Health

PubMed Central

He, Weimin

2009-01-01

The nuclear hormone receptor peroxisome proliferator activated receptor gamma (PPARγ) is an important transcription factor regulating adipocyte differentiation, lipid and glucose homeostasis, and insulin sensitivity. Numerous genetic mutations of PPARγ have been identified and these mutations positively or negatively regulate insulin sensitivity. Among these, a relatively common polymorphism of PPARγ, Pro12Ala of PPARγ2, the isoform expressed only in adipose tissue has been shown to be associated with lower body mass index, enhanced insulin sensitivity, and resistance to the risk of type 2 diabetes in human subjects carrying this mutation. Subsequent studies in different ethnic populations, however, have revealed conflicting results, suggesting a complex interaction between the PPARγ2 Pro12Ala polymorphism and environmental factors such as the ratio of dietary unsaturated fatty acids to saturated fatty acids and/or between the PPARγ2 Pro12Ala polymorphism and genetic factors such as polymorphic mutations in other genes. In addition, this polymorphic mutation in PPARγ2 is associated with other aspects of human diseases, including cancers, polycystic ovary syndrome, Alzheimer disease and aging. This review will highlight findings from recent studies. PMID:19390629

PSO4: a novel gene involved in error-prone repair in Saccharomyces cerevisiae.

PubMed

Henriques, J A; Vicente, E J; Leandro da Silva, K V; Schenberg, A C

1989-09-01

The haploid xs9 mutant, originally selected for on the basis of a slight sensitivity to the lethal effect of X-rays, was found to be extremely sensitive to inactivation by 8-methoxypsoralen (8MOP) photoaddition, especially when cells are treated in the G2 phase of the cell cycle. As the xs9 mutation showed no allelism with any of the 3 known pso mutations, it was now given the name of pso4-1. Regarding inactivation, the pso4-1 mutant is also sensitive to mono- (HN1) or bi-functional (HN2) nitrogen mustards, it is slightly sensitive to 254 nm UV radiation (UV), and shows nearly normal sensitivity to 3-carbethoxypsoralen (3-CPs) photoaddition or methyl methanesulfonate (MMS). Regarding mutagenesis, the pso4-1 mutation completely blocks reverse and forward mutations induced by either 8MOP or 3CPs photoaddition, or by gamma-rays. In the cases of UV, HN1, HN2 or MMS treatments, while reversion induction is still completely abolished, forward mutagenesis is only partially inhibited for UV, HN1, or MMS, and it is unaffected for HN2. Besides severely inhibiting induced mutagenesis, the pso4-1 mutation was found to be semi-dominant, to block sporulation, to abolish the diploid resistance effect, and to block induced mitotic recombination, which indicates that the PSO4 gene is involved in a recombinational pathway of error-prone repair, comparable to the E. coli SOS repair pathway.

The Leaderless Bacteriocin Enterocin K1 Is Highly Potent against Enterococcus faecium: A Study on Structure, Target Spectrum and Receptor.

PubMed

Ovchinnikov, Kirill V; Kristiansen, Per Eugen; Straume, Daniel; Jensen, Marianne S; Aleksandrzak-Piekarczyk, Tamara; Nes, Ingolf F; Diep, Dzung B

2017-01-01

Enterocin K1 (EntK1), enterocin EJ97 (EntEJ97), and LsbB are three sequence related leaderless bacteriocins. Yet LsbB kills only lactococci while EntK1 and EntEJ97 target wider spectra with EntK1 being particularly active against Enterococcus faecium , including nosocomial multidrug resistant isolates. NMR study of EntK1 showed that it had a structure very similar to LsbB - both having an amphiphilic N-terminal α-helix and an unstructured C-terminus. The α-helix in EntK1 is, however, about 3-4 residues longer than that of LsbB. Enterococcal mutants highly resistant to EntEJ97 and EntK1 were found to have mutations within rseP , a gene encoding a stress response membrane-bound Zn-dependent protease. Heterologous expression of the enterococcal rseP rendered resistant cells of Streptococcus pneumoniae sensitive to EntK1 and EntEJ97, suggesting that RseP likely serves as the receptor for EntK1 and EntEJ97. It was also shown that the conserved proteolytic active site in E. faecalis RseP is partly required for EntK1 and EntEJ97 activity, since alanine substitutions of its conserved residues (HExxH) reduced the sensitivity of the clones to the bacteriocins. RseP is known to be involved in bacterial stress response. As expected, the growth of resistant mutants with mutations within rseP was severely affected when they were exposed to higher (stressing) growth temperatures, e.g., at 45°C, at which wild type cells still grew well. These findings allow us to design a hurdle strategy with a combination of the bacteriocin(s) and higher temperature that effectively kills bacteriocin sensitive bacteria and prevents the development of resistant cells.

Clinical mutational profiling of 1006 lung cancers by next generation sequencing

PubMed Central

Illei, Peter B.; Belchis, Deborah; Tseng, Li-Hui; Nguyen, Doreen; De Marchi, Federico; Haley, Lisa; Riel, Stacy; Beierl, Katie; Zheng, Gang; Brahmer, Julie R.; Askin, Frederic B.; Gocke, Christopher D.; Eshleman, James R.; Forde, Patrick M.; Lin, Ming-Tseh

2017-01-01

Analysis of lung adenocarcinomas for actionable mutations has become standard of care. Here, we report our experience using next generation sequencing (NGS) to examine AKT1, BRAF, EGFR, ERBB2, KRAS, NRAS, and PIK3CA genes in 1006 non-small cell lung cancers in a clinical diagnostic setting. NGS demonstrated high sensitivity. Among 760 mutations detected, the variant allele frequency (VAF) was 2–5% in 33 (4.3%) mutations and 2–10% in 101 (13%) mutations. A single bioinformatics pipeline using Torrent Variant Caller, however, missed a variety of EGFR mutations. Mutations were detected in KRAS (36% of tumors), EGFR (19%) including 8 (0.8%) within the extracellular domain (4 at codons 108 and 4 at codon 289), BRAF (6.3%), and PIK3CA (3.7%). With a broader reportable range, exon 19 deletion and p.L858R accounted for only 36% and 26% of EGFR mutations and p.V600E accounted for only 24% of BRAF mutations. NGS provided accurate sequencing of complex mutations seen in 19% of EGFR exon 19 deletion mutations. Doublet (compound) EGFR mutations were observed in 29 (16%) of 187 EGFR-mutated tumors, including 69% with two non-p.L858R missense mutations and 24% with p.L858 and non-p.L858R missense mutations. Concordant VAFs suggests doublet EGFR mutations were present in a dominant clone and cooperated in oncogenesis. Mutants with predicted impaired kinase, observed in 25% of BRAF-mutated tumors, were associated with a higher incidence of concomitant activating KRAS mutations. NGS demonstrates high analytic sensitivity, broad reportable range, quantitative VAF measurement, single molecule sequencing to resolve complex deletion mutations, and simultaneous detection of concomitant mutations. PMID:29228562

Mutations in repeating structural motifs of tropomyosin cause gain of function in skeletal muscle myopathy patients

PubMed Central

Marston, Steven; Memo, Massimiliano; Messer, Andrew; Papadaki, Maria; Nowak, Kristen; McNamara, Elyshia; Ong, Royston; El-Mezgueldi, Mohammed; Li, Xiaochuan; Lehman, William

2013-01-01

The congenital myopathies include a wide spectrum of clinically, histologically and genetically variable neuromuscular disorders many of which are caused by mutations in genes for sarcomeric proteins. Some congenital myopathy patients have a hypercontractile phenotype. Recent functional studies demonstrated that ACTA1 K326N and TPM2 ΔK7 mutations were associated with hypercontractility that could be explained by increased myofibrillar Ca2+ sensitivity. A recent structure of the complex of actin and tropomyosin in the relaxed state showed that both these mutations are located in the actin–tropomyosin interface. Tropomyosin is an elongated molecule with a 7-fold repeated motif of around 40 amino acids corresponding to the 7 actin monomers it interacts with. Actin binds to tropomyosin electrostatically at two points, through Asp25 and through a cluster of amino acids that includes Lys326, mutated in the gain-of-function mutation. Asp25 interacts with tropomyosin K6, next to K7 that was mutated in the other gain-of-function mutation. We identified four tropomyosin motifs interacting with Asp25 (K6-K7, K48-K49, R90-R91 and R167-K168) and three E-E/D-K/R motifs interacting with Lys326 (E139, E181 and E218), and we predicted that the known skeletal myopathy mutations ΔK7, ΔK49, R91G, ΔE139, K168E and E181K would cause a gain of function. Tests by an in vitro motility assay confirmed that these mutations increased Ca2+ sensitivity, while mutations not in these motifs (R167H, R244G) decreased Ca2+ sensitivity. The work reported here explains the molecular mechanism for 6 out of 49 known disease-causing mutations in the TPM2 and TPM3 genes, derived from structural data of the actin–tropomyosin interface. PMID:23886664

Polymerase ε (POLE) ultra-mutation in uterine tumors correlates with T lymphocyte infiltration and increased resistance to platinum-based chemotherapy in vitro

PubMed Central

Bellone, Stefania; Eliana, Bignotti; Lonardi, Silvia; Ferrari, Francesca; Centritto, Floriana; Masserdotti, Alice; Pettinella, Francesca; Black, Jonathan; Menderes, Gulden; Altwerger, Gary; Hui, Pei; Lopez, Salvatore; de Haydu, Christopher; Bonazzoli, Elena; Predolini, Federica; Zammataro, Luca; Cocco, Emiliano; Ferrari, Federico; Ravaggi, Antonella; Romani, Chiara; Facchettie, Fabio; Sartori, Enrico; Odicino, Franco E.; Silasi, Dan-Arin; Litkouhi, Babak; Ratner, Elena; Azodi, Masoud; Schwartz, Peter E.; Santin, Alessandro D.

2016-01-01

Objective Up to 12 % of all endometrial-carcinomas (EC) harbor DNA-polymerase-ε-(POLE) mutations. It is currently unknown whether the favorable prognosis of POLE-mutated EC is derived from their low metastatic capability, extraordinary number of somatic mutations thus imparting immunogenicity, or a high sensitivity to chemotherapy. Methods Polymerase-chain-reaction-amplification and Sanger-sequencing were used to test for POLE exonuclease-domain-mutations (exons 9–14) 131 EC. Infiltration of CD4+ and CD8+ T-lymphocytes (TIL) and PD-1-expression in POLE-mutated vs POLE wild-type EC was studied by immunohistochemistry (IHC) and the correlations between survival and molecular features were investigated. Finally, primary POLE-mutated and POLE-wild-type EC cell lines were established and compared in-vitro for their sensitivity to chemotherapy. Results Eleven POLE-mutated EC (8.5%) were identified. POLE-mutated tumors were associated with improved progression-free-survival (P<0.05) and displayed increased numbers of CD4+ (44.5 vs 21.8; P = .001) and CD8+ (32.8 vs 13.5; P < .001) TILs when compared to wild-type POLE EC. PD-1 receptor was overexpressed in TILs from POLE-mutated vs wild-type-tumors (81% vs 28%; P < .001). Primary POLE tumor cell lines were significantly more resistant to platinum-chemotherapy in-vitro when compared to POLE-wild-type tumors (P < 0.004). Conclusions POLE ultra-mutated EC are heavily infiltrated with CD4+/CD8+ TIL, overexpress PD-1 immune-check-point (i.e., features consistent with chronic antigen-exposure), and have a better prognosis when compared to other molecular subtypes of EC patients. POLE-mutated tumor-cell lines are resistant to platinum-chemotherapy in-vitro suggesting that the better prognosis of POLE-patients is not secondary to a higher sensitivity to chemotherapy but likely linked to enhanced immunogenicity. PMID:27894751

Significance of combined detection of JAK2V617F, MPL and CALR gene mutations in patients with essential thrombocythemia

PubMed Central

Ji, Liying; Qian, Mengyao; Wu, Nana; Wu, Jianmin

2017-01-01

The aim of this study was to analyze the mutation rate of JAK2V617F, MPLW515L/K and CALR genes in adult patients with essential thrombocythemia (ET) and the accuracy of the combined detection by the receiver operating curve. Three hundred and forty-two cases with high-platelets (≥300×109/l) were consecutively selected. The patients were analyzed for routine blood examination, bone marrow biopsy and genetic testing. One hundred and fifty-four cases (45.03%) were diagnosed with ET and 188 cases of secondary thrombocythemia according to the hematopoietic and lymphoid tissue tumor classification standards of 2008. It was found that the mutant type of three genes showed three bands, whereas only one band for wild-type. The JAK2V617F and MPL mutations did not cause a change in the open reading frame and the CALR mutation resulted in its change. The mutation rate of JAK2V617F and CALR in ET group was significantly higher than that in the secondary thrombocythemia group (p<0.05). The positive mutation rate of MPL was only 4.55%. JAK2V617F-positive mutation alone was used to diagnose with ET. The area under the curve (AUC) was 0.721. The sensitivity was 72.4%, the specificity was 79.5% and the cut-off value was 0.25. When CALR-positive mutation alone was used to diagnose ET, the AUC, sensitivity, specificity and cut-off value were 0.664, 68.4, 82.4 and 0.09%, respectively. JAK2V617F combined with CALR mutation were used for diagnosis of ET. The AUC was 0.862, the sensitivity was 85.9%, the specificity was 87.8%, and the cut-off values were 0.21 and 0.07. In conclusion, the positive mutation rate of JAK2V617F and CALR in ET was higher, and the sensitivity, specificity and accuracy of the diagnosis of ET were significantly improved using the detection of JAK2V617F and CALR. PMID:28450924

Significance of combined detection of JAK2V617F, MPL and CALR gene mutations in patients with essential thrombocythemia.

PubMed

Ji, Liying; Qian, Mengyao; Wu, Nana; Wu, Jianmin

2017-03-01

The aim of this study was to analyze the mutation rate of JAK2V617F, MPLW515L/K and CALR genes in adult patients with essential thrombocythemia (ET) and the accuracy of the combined detection by the receiver operating curve. Three hundred and forty-two cases with high-platelets (≥300×10 9 /l) were consecutively selected. The patients were analyzed for routine blood examination, bone marrow biopsy and genetic testing. One hundred and fifty-four cases (45.03%) were diagnosed with ET and 188 cases of secondary thrombocythemia according to the hematopoietic and lymphoid tissue tumor classification standards of 2008. It was found that the mutant type of three genes showed three bands, whereas only one band for wild-type. The JAK2V617F and MPL mutations did not cause a change in the open reading frame and the CALR mutation resulted in its change. The mutation rate of JAK2V617F and CALR in ET group was significantly higher than that in the secondary thrombocythemia group (p<0.05). The positive mutation rate of MPL was only 4.55%. JAK2V617F-positive mutation alone was used to diagnose with ET. The area under the curve (AUC) was 0.721. The sensitivity was 72.4%, the specificity was 79.5% and the cut-off value was 0.25. When CALR-positive mutation alone was used to diagnose ET, the AUC, sensitivity, specificity and cut-off value were 0.664, 68.4, 82.4 and 0.09%, respectively. JAK2V617F combined with CALR mutation were used for diagnosis of ET. The AUC was 0.862, the sensitivity was 85.9%, the specificity was 87.8%, and the cut-off values were 0.21 and 0.07. In conclusion, the positive mutation rate of JAK2V617F and CALR in ET was higher, and the sensitivity, specificity and accuracy of the diagnosis of ET were significantly improved using the detection of JAK2V617F and CALR.

TERT promoter mutations contribute to IDH mutations in predicting differential responses to adjuvant therapies in WHO grade II and III diffuse gliomas

PubMed Central

Ding, Xiao-Jie; Qin, Zhi-Yong; Hong, Christopher S.; Chen, Ling-Chao; Zhang, Xin; Zhao, Fang-Ping; Wang, Yin; Wang, Yang; Zhou, Liang-Fu; Zhuang, Zhengping; Ng, Ho-Keung; Yan, Hai; Yao, Yu; Mao, Ying

2015-01-01

IDH mutations frequently occur in WHO grade II and III diffuse gliomas and have favorable prognosis compared to wild-type tumors. However, whether IDH mutations in WHO grade II and II diffuse gliomas predict enhanced sensitivity to adjuvant radiation (RT) or chemotherapy (CHT) is still being debated. Recent studies have identified recurrent mutations in the promoter region of telomerase reverse transcriptase (TERT) in gliomas. We previously demonstrated that TERT promoter mutations may be promising biomarkers in glioma survival prognostication when combined with IDH mutations. This study analyzed IDH and TERT promoter mutations in 295 WHO grade II and III diffuse gliomas treated with or without adjuvant therapies to explore their impact on the sensitivity of tumors to genotoxic therapies. IDH mutations were found in 216 (73.2%) patients and TERT promoter mutations were found in 112 (38%) patients. In multivariate analysis, IDH mutations (p < 0.001) were independent prognostic factors for PFS and OS in patients receiving genotoxic therapies while TERT promoter mutations were not. In univariate analysis, IDH and TERT promoter mutations were not significant prognostic factors in patients who did not receive genotoxic therapies. Adjuvant RT and CHT were factors independently impacting PFS (RT p = 0.001, CHT p = 0.026) in IDH mutated WHO grade II and III diffuse gliomas but not in IDH wild-type group. Univariate and multivariate analyses demonstrated TERT promoter mutations further stratified IDH wild-type WHO grade II and III diffuse gliomas into two subgroups with different responses to genotoxic therapies. Adjuvant RT and CHT were significant parameters influencing PFS in the IDH wt/TERT mut subgroup (RT p = 0.015, CHT p = 0.015) but not in the IDH wt/TERT wt subgroup. Our data demonstrated that IDH mutated WHO grade II and III diffuse gliomas had better PFS and OS than their IDH wild-type counterparts when genotoxic therapies were administered after surgery. Importantly, we also found that TERT promoter mutations further stratify IDH wild-type WHO grade II and III diffuse gliomas into two subgroups with different responses to adjuvant therapies. Taken together, TERT promoter mutations may predict enhanced sensitivity to genotoxic therapies in IDH wild-type WHO grade II and III diffuse gliomas and may justify intensified treatment in this subgroup. PMID:26314843

[Expression of epidermal growth factor receptor mutation specific antibodies in lung adenocarcinoma: evaluation of sensitivity, specificity and relationship to histologic subtypes].

PubMed

Lai, Y M; Feng, Q; Sun, Y; Wang, P; Shi, Y F; Zhao, M; Wu, Q; Li, X H

2016-09-08

To evaluate the expression of epidermal growth factor receptor (EGFR) mutation specific antibodies in invasive lung adenocarcinomas, and their sensitivity, specificity, as well as relationship to histological subtypes. Immunostaining with EGFR mutation-specific antibodies, del E746-A750 in exon 19 and L858R in exon 21, was performed in tissue microarrays of 884 cases of resection specimens to study the relationship between the immunophenotypes and morphologic subtypes. The sensitivity and specificity of the stains were compared with gene mutations detected by amplified refractory mutation system-polymerase chain reaction (ARMS-PCR). Of the 884 cases, the expression of del E746-A750 in exon 19 was 3+ , 2+ , 1+ and 0 in 7 cases (0.79%), 38 cases (4.30%), 129 cases (14.59%) and 710 cases (80.32%), respectively. For L858R in exon 21, 3+ , 2+ , 1+ and 0 staining were seen in 82 cases (9.28%), 93 cases (10.52%), 82 cases (9.28%) and 627 cases (70.93%), respectively. For both antibodies, positive expression (1+ or more) was mainly observed in lepidic, acinar and papillary predominant subtypes, and rarely seen in solid subtype or invasive mucinous adenocarcinoma (P=0.014 and 0.016). If 1+ to 3+ expression was set as positive, the specificity of exon 19/exon 21 reached 98.59%/92.98%, while the sensitivity was relatively lower (62.86%/88.89%). If 2+ to 3+ expression was read as positive, the specificity and sensitivity were 99.30%/97.37% and 25.71%/74.60% for exon 19/exon 21. If only 3+ expression was considered positive, the specificity was 100.0% for both antibodies, with a low sensitivity (8.57% for exon 19 and 34.92% for exon 21). Of the 18 cases with E746-A750 del in exon 19 based on molecular detection, the sensitivity of immunohistochemistry for exon 19 was 88.89% if a positive cutoff value ≥1+ was used; in contrast, of the 8 cases harboring other deletions in exon 19, only two cases were positive as 1+ . Both the EGFR mutation specific antibodies del E746-A750 in exon 19 and L858R in exon 21 demonstrate high specificity and relatively low sensitivity, and are mostly expressed in lepidic, acinar and papillary predominant subtypes, but rarely in solid subtype or invasive mucinous adenocarcinoma. For cases with 3+ expression, a mutational statue for EGFR is likely. For the 2+ positive cases, the accuracy to predict mutation almost reaches 90%, but molecular detection for confirmation is desirable. For the 1+ and negative cases, DNA-based test is essential to avoid false negativity.

Molecular Genetic Characterization of Mutagenesis Using a Highly Sensitive Single-Stranded DNA Reporter System in Budding Yeast.

PubMed

Chan, Kin

2018-01-01

Mutations are permanent alterations to the coding content of DNA. They are starting material for the Darwinian evolution of species by natural selection, which has yielded an amazing diversity of life on Earth. Mutations can also be the fundamental basis of serious human maladies, most notably cancers. In this chapter, I describe a highly sensitive reporter system for the molecular genetic analysis of mutagenesis, featuring controlled generation of long stretches of single-stranded DNA in budding yeast cells. This system is ~100- to ~1000-fold more susceptible to mutation than conventional double-stranded DNA reporters, and is well suited for generating large mutational datasets to investigate the properties of mutagens.

An immuno-wall microdevice exhibits rapid and sensitive detection of IDH1-R132H mutation specific to grade II and III gliomas

PubMed Central

Yamamichi, Akane; Kasama, Toshihiro; Ohka, Fumiharu; Suzuki, Hiromichi; Kato, Akira; Motomura, Kazuya; Hirano, Masaki; Ranjit, Melissa; Chalise, Lushun; Kurimoto, Michihiro; Kondo, Goro; Aoki, Kosuke; Kaji, Noritada; Tokeshi, Manabu; Matsubara, Toshio; Senga, Takeshi; Kaneko, Mika K.; Suzuki, Hidenori; Hara, Masahito; Wakabayashi, Toshihiko; Baba, Yoshinobu; Kato, Yukinari; Natsume, Atsushi

2016-01-01

Abstract World Health Organization grade II and III gliomas most frequently occur in the central nervous system (CNS) in adults. Gliomas are not circumscribed; tumor edges are irregular and consist of tumor cells, normal brain tissue, and hyperplastic reactive glial cells. Therefore, the tumors are not fully resectable, resulting in recurrence, malignant progression, and eventual death. Approximately 69–80% of grade II and III gliomas harbor mutations in the isocitrate dehydrogenase 1 gene (IDH1), of which 83–90% are found to be the IDH1-R132H mutation. Detection of the IDH1-R132H mutation should help in the differential diagnosis of grade II and III gliomas from other types of CNS tumors and help determine the boundary between the tumor and normal brain tissue. In this study, we established a highly sensitive antibody-based device, referred to as the immuno-wall, to detect the IDH1-R132H mutation in gliomas. The immuno-wall causes an immunoreaction in microchannels fabricated using a photo-polymerizing polymer. This microdevice enables the analysis of the IDH1 status with a small sample within 15 min with substantially high sensitivity. Our results suggested that 10% content of the IDH1-R132H mutation in a sample of 0.33 μl volume, with 500 ng protein, or from 500 cells is theoretically sufficient for the analysis. The immuno-wall device will enable the rapid and highly sensitive detection of the IDH1-R132H mutation in routine clinical practice. PMID:27877908

Comprehensive profiling and quantitation of oncogenic mutations in non small-cell lung carcinoma using single molecule amplification and re-sequencing technology

PubMed Central

Jiang, Hong; Wang, Limin; Xu, Rujun; Shi, Yanbin; Zhang, Jianguang; Xu, Mengnan; Cram, David S.; Ma, Shenglin

2016-01-01

Activating and resistance mutations in the tyrosine kinase domain of several oncogenes are frequently associated with non-small cell lung carcinoma (NSCLC). In this study we assessed the frequency, type and abundance of EGFR, KRAS, BRAF, TP53 and ALK mutations in tumour specimens from 184 patients with early and late stage disease using single molecule amplification and re-sequencing technology (SMART). Based on modelling of EGFR mutations, the detection sensitivity of the SMART assay was at least 0.1%. Benchmarking EGFR mutation detection against the gold standard ARMS-PCR assay, SMART assay had a sensitivity and specificity of 98.7% and 99.0%. Amongst the 184 samples, EGFR mutations were the most prevalent (59.9%), followed by KRAS (16.9%), TP53 (12.7%), EML4-ALK fusions (6.3%) and BRAF (4.2%) mutations. The abundance and types of mutations in tumour specimens were extremely heterogeneous, involving either monoclonal (51.6%) or polyclonal (12.6%) mutation events. At the clinical level, although the spectrum of tumour mutation(s) was unique to each patient, the overall patterns in early or advanced stage disease were relatively similar. Based on these findings, we propose that personalized profiling and quantitation of clinically significant oncogenic mutations will allow better classification of patients according to tumour characteristics and provide clinicians with important ancillary information for treatment decision-making. PMID:27409166

Comprehensive profiling and quantitation of oncogenic mutations in non small-cell lung carcinoma using single molecule amplification and re-sequencing technology.

PubMed

Zhang, Shirong; Xia, Bing; Jiang, Hong; Wang, Limin; Xu, Rujun; Shi, Yanbin; Zhang, Jianguang; Xu, Mengnan; Cram, David S; Ma, Shenglin

2016-08-02

Activating and resistance mutations in the tyrosine kinase domain of several oncogenes are frequently associated with non-small cell lung carcinoma (NSCLC). In this study we assessed the frequency, type and abundance of EGFR, KRAS, BRAF, TP53 and ALK mutations in tumour specimens from 184 patients with early and late stage disease using single molecule amplification and re-sequencing technology (SMART). Based on modelling of EGFR mutations, the detection sensitivity of the SMART assay was at least 0.1%. Benchmarking EGFR mutation detection against the gold standard ARMS-PCR assay, SMART assay had a sensitivity and specificity of 98.7% and 99.0%. Amongst the 184 samples, EGFR mutations were the most prevalent (59.9%), followed by KRAS (16.9%), TP53 (12.7%), EML4-ALK fusions (6.3%) and BRAF (4.2%) mutations. The abundance and types of mutations in tumour specimens were extremely heterogeneous, involving either monoclonal (51.6%) or polyclonal (12.6%) mutation events. At the clinical level, although the spectrum of tumour mutation(s) was unique to each patient, the overall patterns in early or advanced stage disease were relatively similar. Based on these findings, we propose that personalized profiling and quantitation of clinically significant oncogenic mutations will allow better classification of patients according to tumour characteristics and provide clinicians with important ancillary information for treatment decision-making.

An Arabidopsis mutation in translation elongation factor 2 causes superinduction of CBF/DREB1 transcription factor genes but blocks the induction of their downstream targets under low temperatures

PubMed Central

Guo, Yan; Xiong, Liming; Ishitani, Manabu; Zhu, Jian-Kang

2002-01-01

Low temperature regulates gene expression in bacteria, yeast, and animals as well as in plants. However, the signal transduction cascades mediating the low temperature responses are not well understood in any organism. To identify components in low temperature signaling genetically, we isolated Arabidopsis thaliana mutants in which cold-responsive genes are no longer induced by low temperatures. One of these mutations, los1–1, specifically blocks low temperature-induced transcription of cold-responsive genes. Surprisingly, cold-induced expression of the early response transcriptional activators, C-repeat/dehydration responsive element binding factors (CBF/DREB1s), is enhanced by the los1–1 mutation. The los1–1 mutation also reduces the capacity of plants to develop freezing tolerance but does not impair the vernalization response. Genetic analysis indicated that los1–1 is a recessive mutation in a single nuclear gene. The LOS1 gene encodes a translation elongation factor 2-like protein. Protein labeling studies show that new protein synthesis is blocked in los1–1 mutant plants specifically in the cold. These results reveal a critical role of new protein synthesis in the proper transduction of low temperature signals. Our results also suggest that cold-induced transcription of CBF/DREB1s is feedback inhibited by their gene products or by products of their downstream target genes. PMID:12032361

Adaptive mutation of Acetobacter pasteurianus SKU1108 enhances acetic acid fermentation ability at high temperature.

PubMed

Matsutani, Minenosuke; Nishikura, Mitsuteru; Saichana, Natsaran; Hatano, Tomoyuki; Masud-Tippayasak, Uraiwan; Theergool, Gunjana; Yakushi, Toshiharu; Matsushita, Kazunobu

2013-05-20

In vitro adaptation is one of the most challenging subjects in biology to understand adaptive evolution. Microbial adaptation to temperature is not only interesting in terms of understanding the adaptation mechanism, but also useful for industrial applications. In this study, we attempted the in vitro adaptation of Acetobacter pasteurianus SKU1108 by repeating its cultivation under high-temperature acetic acid fermentation conditions. As a result, thermo-adapted strains having the higher fermentation ability than the wild-type strain were obtained. Mutations and/or disruptions in several proteins of the adapted strains were detected with NGS sequencing technology. In particular, two different adapted strains had mutations or disruptions in three specific genes in common, suggesting that these genes are essential for thermotolerance or fermentation at higher temperature. In order to clarify their involvement in thermotolerance, two of the three genes were disrupted and their phenotype was examined. The results showed that mutations of the two proteins, MarR and an amino acid transporter, are partly responsible for higher fermentation ability and/or thermotolerance. Thus, it was suggested that these elevated abilities of the adapted strains are acquired by assembling several single gene mutations including the above two mutations. Copyright © 2013 Elsevier B.V. All rights reserved.

[Hydrologic variability and sensitivity based on Hurst coefficient and Bartels statistic].

PubMed

Lei, Xu; Xie, Ping; Wu, Zi Yi; Sang, Yan Fang; Zhao, Jiang Yan; Li, Bin Bin

2018-04-01

Due to the global climate change and frequent human activities in recent years, the pure stochastic components of hydrological sequence is mixed with one or several of the variation ingredients, including jump, trend, period and dependency. It is urgently needed to clarify which indices should be used to quantify the degree of their variability. In this study, we defined the hydrological variability based on Hurst coefficient and Bartels statistic, and used Monte Carlo statistical tests to test and analyze their sensitivity to different variants. When the hydrological sequence had jump or trend variation, both Hurst coefficient and Bartels statistic could reflect the variation, with the Hurst coefficient being more sensitive to weak jump or trend variation. When the sequence had period, only the Bartels statistic could detect the mutation of the sequence. When the sequence had a dependency, both the Hurst coefficient and the Bartels statistics could reflect the variation, with the latter could detect weaker dependent variations. For the four variations, both the Hurst variability and Bartels variability increased with the increases of variation range. Thus, they could be used to measure the variation intensity of the hydrological sequence. We analyzed the temperature series of different weather stations in the Lancang River basin. Results showed that the temperature of all stations showed the upward trend or jump, indicating that the entire basin had experienced warming in recent years and the temperature variability in the upper and lower reaches was much higher. This case study showed the practicability of the proposed method.

A novel suicide shuttle plasmid for Streptococcus suis serotype 2 and Streptococcus equi ssp. zooepidemicus gene mutation

PubMed Central

Liu, Rui; Zhang, Ping; Su, Yiqi; Lin, Huixing; Zhang, Hui; Yu, Lei; Ma, Zhe; Fan, Hongjie

2016-01-01

The mariner-based Himar1 system has been utilized for creating mutant libraries of many Gram-positive bacteria. Streptococcus suis serotype 2 (SS2) and Streptococcus equi ssp. zooepidemicus (SEZ) are primary pathogens of swine that threaten the swine industry in China. To provide a forward-genetics technology for finding virulent phenotype-related genes in these two pathogens, we constructed a novel temperature-sensitive suicide shuttle plasmid, pMar4s, which contains the Himar1 system transposon, TnYLB-1, and the Himar1 C9 transposase from pMarA and the repTAs temperature-sensitive fragment from pSET4s. The kanamycin (Kan) resistance gene was in the TnYLB-1 transposon. Temperature sensitivity and Kan resistance allowed the selection of mutant strains and construction of the mutant library. The SS2 and SEZ mutant libraries were successfully constructed using the pMar4s plasmid. Inverse-Polymerase Chain Reaction (Inverse-PCR) results revealed large variability in transposon insertion sites and that the library could be used for phenotype alteration screening. The thiamine biosynthesis gene apbE was screened for its influence on SS2 anti-phagocytosis; likewise, the sagF gene was identified to be a hemolytic activity-related gene in SEZ. pMar4s was suitable for mutant library construction, providing more information regarding SS2 and SEZ virulence factors and illustrating the pathogenesis of swine streptococcosis. PMID:27256117

The induction of mutation and recombination following UV irradiation during meiosis in Saccharomyces cerevisiae.

PubMed

Kelly, S L; Parry, J M

1983-03-01

Irradiation of yeast cultures with ultraviolet light at discrete stages during meiosis produces cyclic variations in sensitivity, i.e. cells are more sensitive to the lethal effects of UV light prior to entry into the meiotic DNA synthesis, and this corresponds to a peak of induction of point mutation. Cells become more resistant to both induced point mutation and lethality as they enter meiotic DNA synthesis, but become more sensitive again during spore formation. The induced level of intragenic recombination rises during the period of commitment to recombination to a level indistinguishable from the full meiotic level of spontaneous intragenic recombination. Induced reciprocal recombination remains above the spontaneous level up to the point of commitment to sporulation.

Remarkable stabilization of a psychrotrophic RNase HI by a combination of thermostabilizing mutations identified by the suppressor mutation method.

PubMed

Tadokoro, Takashi; Matsushita, Kyoko; Abe, Yumi; Rohman, Muhammad Saifur; Koga, Yuichi; Takano, Kazufumi; Kanaya, Shigenori

2008-08-05

Ribonuclease HI from the psychrotrophic bacterium Shewanella oneidensis MR-1 (So-RNase HI) is much less stable than Escherichia coli RNase HI (Ec-RNase HI) by 22.4 degrees C in T m and 12.5 kJ mol (-1) in Delta G(H 2O), despite their high degrees of structural and functional similarity. To examine whether the stability of So-RNase HI increases to a level similar to that of Ec-RNase HI via introduction of several mutations, the mutations that stabilize So-RNase HI were identified by the suppressor mutation method and combined. So-RNase HI and its variant with a C-terminal four-residue truncation (154-RNase HI) complemented the RNase H-dependent temperature-sensitive (ts) growth phenotype of E. coli strain MIC3001, while 153-RNase HI with a five-residue truncation could not. Analyses of the activity and stability of these truncated proteins suggest that 153-RNase HI is nonfunctional in vivo because of a great decrease in stability. Random mutagenesis of 153-RNase HI using error-prone PCR, followed by screening for the revertants, allowed us to identify six single suppressor mutations that make 153-RNase HI functional in vivo. Four of them markedly increased the stability of the wild-type protein by 3.6-6.7 degrees C in T m and 1.7-5.2 kJ mol (-1) in Delta G(H 2O). The effects of these mutations were nearly additive, and combination of these mutations increased protein stability by 18.7 degrees C in T m and 12.2 kJ mol (-1) in Delta G(H 2O). These results suggest that several residues are not optimal for the stability of So-RNase HI, and their replacement with other residues strikingly increases it to a level similar to that of the mesophilic counterpart.

DPY-17 and MUA-3 Interact for Connective Tissue-Like Tissue Integrity in Caenorhabditis elegans: A Model for Marfan Syndrome.

PubMed

Fotopoulos, Pauline; Kim, Jeongho; Hyun, Moonjung; Qamari, Waiss; Lee, Inhwan; You, Young-Jai

2015-04-27

mua-3 is a Caenorhabditis elegans homolog of the mammalian fibrillin1, a monogenic cause of Marfan syndrome. We identified a new mutation of mua-3 that carries an in-frame deletion of 131 amino acids in the extracellular domain, which allows the mutants to survive in a temperature-dependent manner; at the permissive temperature, the mutants grow normally without obvious phenotypes, but at the nonpermissive temperature, more than 90% die during the L4 molt due to internal organ detachment. Using the temperature-sensitive lethality, we performed unbiased genetic screens to isolate suppressors to find genetic interactors of MUA-3. From two independent screens, we isolated mutations in dpy-17 as a suppressor. RNAi of dpy-17 in mua-3 rescued the lethality, confirming dpy-17 is a suppressor. dpy-17 encodes a collagen known to genetically interact with dpy-31, a BMP-1/Tolloid-like metalloprotease required for TGFβ activation in mammals. Human fibrillin1 mutants fail to sequester TGFβ2 leading to excess TGFβ signaling, which in turn contributes to Marfan syndrome or Marfan-related syndrome. Consistent with that, RNAi of dbl-1, a TGFβ homolog, modestly rescued the lethality of mua-3 mutants, suggesting a potentially conserved interaction between MUA-3 and a TGFβ pathway in C. elegans. Our work provides genetic evidence of the interaction between TGFβ and a fibrillin homolog, and thus provides a simple yet powerful genetic model to study TGFβ function in development of Marfan pathology. Copyright © 2015 Fotopoulos et al.

Variability in response to nicotine in the LSxSS RI strains: potential role of polymorphisms in alpha4 and alpha6 nicotinic receptor genes.

PubMed

Tritto, Theresa; Stitzel, Jerry A; Marks, Michael J; Romm, Elena; Collins, Allan C

2002-04-01

Several studies have shown that genetic factors influence the effects of nicotine on respiration, acoustic startle, Y-maze crosses and rears, heart rate and body temperature in the mouse. Recently, we identified restriction fragment length polymorphisms (RFLPs) associated with the alpha4 (Chrna4) and alpha6 (Chrna6) nicotinic cholinergic receptor genes in the recombinant inbred (RI) strains derived from the Long-Sleep (LS) and Short-Sleep (SS) mouse lines. The alpha4 polymorphism has been identified as a point-mutation at position 529 (threonine to alanine) and the alpha6 polymorphism has not yet been identified. The studies described here evaluated the potential role of these polymorphisms in regulating sensitivity to nicotine by constructing dose-response curves for the effects of nicotine on six responses in the LSxSS RI strains. The results obtained suggest that both of the polymorphisms may play a role in regulating variability in sensitivity to nicotine. Those RI strains carrying the LS-like alpha4 RFLP were significantly more sensitive to the effects of nicotine on Y-maze crosses and rears, temperature and respiration and were less sensitive to the effects of nicotine on acoustic startle than those strains carrying the SS-like alpha4 RFLP. Those RI strains carrying the LS-like alpha6 RFLP were more sensitive to the effects of nicotine on respiration and acoustic startle, and less sensitive to the effects of nicotine on Y-maze crosses than those strains carrying the SS-like alpha6 RFLP. These results suggest that genetically determined differences in sensitivity to nicotine may be explained, in part, by variability associated with at least two of the neuronal nicotinic receptor genes, alpha4 and alpha6.

Random mutagenesis screening indicates the absence of a separate H(+)-sensor in the pH-sensitive Kir channels.

PubMed

Paynter, Jennifer J; Shang, Lijun; Bollepalli, Murali K; Baukrowitz, Thomas; Tucker, Stephen J

2010-01-01

Several inwardly-rectifying (Kir) potassium channels (Kir1.1, Kir4.1 and Kir4.2) are characterised by their sensitivity to inhibition by intracellular H(+) within the physiological range. The mechanism by which these channels are regulated by intracellular pH has been the subject of intense scrutiny for over a decade, yet the molecular identity of the titratable pH-sensor remains elusive. In this study we have taken advantage of the acidic intracellular environment of S. cerevisiae and used a K(+) -auxotrophic strain to screen for mutants of Kir1.1 with impaired pH-sensitivity. In addition to the previously identified K80M mutation, this unbiased screening approach identified a novel mutation (S172T) in the second transmembrane domain (TM2) that also produces a marked reduction in pH-sensitivity through destabilization of the closed-state. However, despite this extensive mutagenic approach, no mutations could be identified which removed channel pH-sensitivity or which were likely to act as a separate H(+) -sensor unique to the pH-sensitive Kir channels. In order to explain these results we propose a model in which the pH-sensing mechanism is part of an intrinsic gating mechanism common to all Kir channels, not just the pH-sensitive Kir channels. In this model, mutations which disrupt this pH-sensor would result in an increase, not reduction, in pH-sensitivity. This has major implications for any future studies of Kir channel pH-sensitivity and explains why formal identification of these pH-sensing residues still represents a major challenge.

Effects of temperature and surface orientation on migration behaviours of helium atoms near tungsten surfaces

NASA Astrophysics Data System (ADS)

Wang, Xiaoshuang; Wu, Zhangwen; Hou, Qing

2015-10-01

Molecular dynamics simulations were performed to study the dependence of migration behaviours of single helium atoms near tungsten surfaces on the surface orientation and temperature. For W{100} and W{110} surfaces, He atoms can quickly escape out near the surface without accumulation even at a temperature of 400 K. The behaviours of helium atoms can be well-described by the theory of continuous diffusion of particles in a semi-infinite medium. For a W{111} surface, the situation is complex. Different types of trap mutations occur within the neighbouring region of the W{111} surface. The trap mutations hinder the escape of He atoms, resulting in their accumulation. The probability of a He atom escaping into vacuum from a trap mutation depends on the type of the trap mutation, and the occurrence probabilities of the different types of trap mutations are dependent on the temperature. This finding suggests that the escape rate of He atoms on the W{111} surface does not show a monotonic dependence on temperature. For instance, the escape rate at T = 1500 K is lower than the rate at T = 1100 K. Our results are useful for understanding the structural evolution and He release on tungsten surfaces and for designing models in other simulation methods beyond molecular dynamics.

Cytokine receptor signaling is required for the survival of ALK− anaplastic large cell lymphoma, even in the presence of JAK1/STAT3 mutations

PubMed Central

Chen, Jing; Zhang, Yong; Petrus, Michael N.; Xiao, Wenming; Nicolae, Alina; Raffeld, Mark; Pittaluga, Stefania; Bamford, Richard N.; Nakagawa, Masao; Ouyang, Sunny Tianyi; Epstein, Alan L.; Kadin, Marshall E.; Del Mistro, Annarose; Woessner, Richard; Jaffe, Elaine S.; Waldmann, Thomas A.

2017-01-01

Activating Janus kinase (JAK) and signal transducer and activator of transcription (STAT) mutations have been discovered in many T-cell malignancies, including anaplastic lymphoma kinase (ALK)− anaplastic large cell lymphomas (ALCLs). However, such mutations occur in a minority of patients. To investigate the clinical application of targeting JAK for ALK− ALCL, we treated ALK− cell lines of various histological origins with JAK inhibitors. Interestingly, most exogenous cytokine-independent cell lines responded to JAK inhibition regardless of JAK mutation status. JAK inhibitor sensitivity correlated with the STAT3 phosphorylation status of tumor cells. Using retroviral shRNA knockdown, we have demonstrated that these JAK inhibitor-sensitive cells are dependent on both JAK1 and STAT3 for survival. JAK1 and STAT3 gain-of-function mutations were found in some, but not all, JAK inhibitor-sensitive cells. Moreover, the mutations alone cannot explain the JAK1/STAT3 dependency, given that wild-type JAK1 or STAT3 was sufficient to promote cell survival in the cells that had either JAK1or STAT3 mutations. To investigate whether other mechanisms were involved, we knocked down upstream receptors GP130 or IL-2Rγ. Knockdown of GP130 or IL-2Rγ induced cell death in selected JAK inhibitor-sensitive cells. High expression levels of cytokines, including IL-6, were demonstrated in cell lines as well as in primary ALK− ALCL tumors. Finally, ruxolitinib, a JAK1/2 inhibitor, was effective in vivo in a xenograft ALK− ALCL model. Our data suggest that cytokine receptor signaling is required for tumor cell survival in diverse forms of ALK− ALCL, even in the presence of JAK1/STAT3 mutations. Therefore, JAK inhibitor therapy might benefit patients with ALK− ALCL who are phosphorylated STAT3+. PMID:28356514

Metastatic site location influences the diagnostic accuracy of ctDNA EGFR- mutation testing in NSCLC patients: a pooled analysis.

PubMed

Passiglia, Francesco; Rizzo, Sergio; Rolfo, Christian; Galvano, Antonio; Bronte, Enrico; Incorvaia, Lorena; Listi, Angela; Barraco, Nadia; Castiglia, Marta; Calo, Valentina; Bazan, Viviana; Russo, Antonio

2018-03-08

Recent studies evaluated the diagnostic accuracy of circulating tumor DNA (ctDNA) in the detection of epidermal growth factor receptor (EGFR) mutations from plasma of NSCLC patients, overall showing a high concordance as compared to standard tissue genotyping. However it is less clear if the location of metastatic site may influence the ability to identify EGFR mutations in plasma. This pooled analysis aims to evaluate the association between the metastatic site location and the sensitivity of ctDNA analysis in detecting EGFR mutations in NSCLC patients. Data from all published studies, evaluating the sensitivity of plasma-based EGFR-mutation testing, stratified by metastatic site location (extrathoracic (M1b) vs intrathoracic (M1a)) were collected by searching in PubMed, Cochrane Library, American Society of Clinical Oncology, and World Conference of Lung Cancer, meeting proceedings. Pooled Odds ratio (OR) and 95% confidence intervals (95% CIs) were calculated for the ctDNA analysis sensitivity, according to metastatic site location. A total of ten studies, with 1425 patients, were eligible. Pooled analysis showed that the sensitivity of ctDNA-based EGFR-mutation testing is significantly higher in patients with M1b vs M1a disease (OR: 5.09; 95% CIs: 2.93 - 8.84). A significant association was observed for both EGFR-activating (OR: 4.30, 95% CI: 2.35-7.88) and resistant T790M mutations (OR: 11.89, 95% CI: 1.45-97.22), regardless of the use of digital-PCR (OR: 5.85, 95% CI: 3.56-9.60) or non-digital PCR technologies (OR: 2.96, 95% CI: 2.24-3.91). These data suggest that the location of metastatic sites significantly influences the diagnostic accuracy of ctDNA analysis in detecting EGFR mutations in NSCLC patients. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Response of Head and Neck Squamous Cell Carcinoma Cells Carrying PIK3CA Mutations to Select Targeted Therapies

PubMed Central

Wirtz, Eric D; Hoshino, Daisuke; Maldonado, Anthony T; Tyson, Darren R; Weaver, Alissa M

2015-01-01

Importance The PIK3CA mutation is one of the most common mutations in Head and Neck Squamous Cell Carcinoma (HNSCC). Through this research we attempt to elicit the role of oncogene dependence and effects of targeted therapy on this PIK3CA mutation. Objectives 1) To determine the role of oncogene dependence on one of the more common and targetable oncogenes in HNSCC – PIK3CA; 2) To evaluate the consequence of this oncogene on the effectiveness of newly developed targeted therapies. Study Design In vitro study. Setting Academic research laboratory. Participants Cell culture based study assessing the viability of PIK3CA mutated head and neck cell lines when treated with targeted therapy. Exposures PIK3CA mutated head and neck cell lines were treated with 17-AAG, GDC-0941, trametinib, and BEZ-235. Main Outcome and Measures Assessment of cell viability of HNSCC cell lines characterized for PIK3CA mutations or SCC25 cells engineered to express the PIK3CA hotspot mutations E545K or H1047R Results Surprisingly, in engineered cell lines, the hotspot E545K and H1047R mutations conferred decreased, rather than increased, sensitivity as measured by IC50 when treated with the respective HSP90, PI3K, and MEK inhibitors, 17-AAG, GDC-0941, and trametinib, compared to the SCC25 control cell lines. When treated with BEZ-235, H1047R-expressing cell lines showed increased sensitivity to inhibition compared to control while those expressing E545K showed slightly increased sensitivity of unclear significance. Conclusions and Relevance 1) The PIK3CA mutations within our engineered cell model did not lead to enhanced oncogene-dependent cell death when treated with direct inhibition of the PI3K enzyme yet did show increased sensitivity compared to control with dual PI3K/mTOR inhibition. 2) Oncogene addiction to PIK3CA hot spot mutations, if it occurs, is likely to evolve in vivo molecular changes that remain to be identified. Additional study is required to develop new model systems and approaches to determine the role of targeted therapy in the treatment of PI3K-overactive HNSCC tumors. PMID:25855885

Cycling temperature capillary electrophoresis: A quantitative, fast and inexpensive method to detect mutations in mixed populations of human mitochondrial DNA.

PubMed

Refinetti, Paulo; Morgenthaler, Stephan; Ekstrøm, Per O

2016-07-01

Cycling temperature capillary electrophoresis has been optimised for mutation detection in 76% of the mitochondrial genome. The method was tested on a mixed sample and compared to mutation detection by next generation sequencing. Out of 152 fragments 90 were concordant, 51 discordant and in 11 were semi-concordant. Dilution experiments show that cycling capillary electrophoresis has a detection limit of 1-3%. The detection limit of routine next generation sequencing was in the ranges of 15 to 30%. Cycling temperature capillary electrophoresis detect and accurate quantify mutations at a fraction of the cost and time required to perform a next generation sequencing analysis. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Activating HER2 mutations in HER2 gene amplification negative breast cancer.

PubMed

Bose, Ron; Kavuri, Shyam M; Searleman, Adam C; Shen, Wei; Shen, Dong; Koboldt, Daniel C; Monsey, John; Goel, Nicholas; Aronson, Adam B; Li, Shunqiang; Ma, Cynthia X; Ding, Li; Mardis, Elaine R; Ellis, Matthew J

2013-02-01

Data from 8 breast cancer genome-sequencing projects identified 25 patients with HER2 somatic mutations in cancers lacking HER2 gene amplification. To determine the phenotype of these mutations, we functionally characterized 13 HER2 mutations using in vitro kinase assays, protein structure analysis, cell culture, and xenograft experiments. Seven of these mutations are activating mutations, including G309A, D769H, D769Y, V777L, P780ins, V842I, and R896C. HER2 in-frame deletion 755-759, which is homologous to EGF receptor (EGFR) exon 19 in-frame deletions, had a neomorphic phenotype with increased phosphorylation of EGFR or HER3. L755S produced lapatinib resistance, but was not an activating mutation in our experimental systems. All of these mutations were sensitive to the irreversible kinase inhibitor, neratinib. These findings show that HER2 somatic mutation is an alternative mechanism to activate HER2 in breast cancer and they validate HER2 somatic mutations as drug targets for breast cancer treatment. We show that the majority of HER2 somatic mutations in breast cancer patients are activating mutations that likely drive tumorigenesis. Several patients had mutations that are resistant to the reversible HER2 inhibitor lapatinib, but are sensitive to the irreversible HER2 inhibitor, neratinib. Our results suggest that patients with HER2 mutation–positive breast cancers could benefit from existing HER2-targeted drugs.

Tumour MLH1 promoter region methylation testing is an effective pre-screen for Lynch Syndrome (HNPCC)

PubMed Central

Newton, K; Jorgensen, NM; Wallace, AJ; Buchanan, DD; Lalloo, F; McMahon, RFT; Hill, J; Evans, DG

2016-01-01

Background & Aims Lynch syndrome patients have DNA mismatch repair deficiency and up to 80% life-time risk of colorectal cancer. Screening of mutation carriers reduces colorectal cancer incidence and mortality. Selection for constitutional mutation testing relies on family history (Amsterdam and Bethesda Guidelines) and tumour derived biomarkers. Initial biomarker analysis uses mismatch repair protein immunohistochemistry and microsatellite instability. Abnormalities in either identify mismatch repair deficiency but do not differentiate sporadic epigenetic defects, due to MLH1 promoter region methylation (13% of CRCs) from Lynch Syndrome (4% of CRCs). A diagnostic biomarker capable of making this distinction would be valuable. This study compared two biomarkers in tumours with mismatch repair deficiency; quantification of methylation of the MLH1 promoter region using a novel assay and BRAF c.1799T>A, p.(Val600Glu) mutation status in the identification of constitutional mutations. Methods Tumour DNA was extracted (FFPE tissue) and pyrosequencing used to test for MLH1 promoter methylation and presence of the BRAF c.1799T>A, p.(Val600Glu) mutation 71 CRCs from individuals with pathogenic MLH1 mutations and 73 CRCs with sporadic MLH1 loss. Specificity and sensitivity was compared. Findings Unmethylated MLH1 promoter: sensitivity 94.4% (95% CI 86.2–98.4%), specificity 87.7% (95% CI 77.9–94.2%), Wild-type BRAF (codon 600): sensitivity 65.8% (95% CI 53.7–76.5%), specificity 98.6% (95% CI 92.4–100.0%) for the identification of those with pathogenic MLH1 mutations. Conclusions Quantitative MLH1 promoter region methylation using pyrosequencing is superior to BRAF codon 600 mutation status in identifying constitutional mutations in mismatch repair deficient tumours. PMID:25280751

Detection of the KIT D816V mutation in peripheral blood of systemic mastocytosis: diagnostic implications.

PubMed

Jara-Acevedo, Maria; Teodosio, Cristina; Sanchez-Muñoz, Laura; Álvarez-Twose, Ivan; Mayado, Andrea; Caldas, Carolina; Matito, Almudena; Morgado, José M; Muñoz-González, Javier I; Escribano, Luis; Garcia-Montero, Andrés C; Orfao, Alberto

2015-08-01

Recent studies have found the KIT D816V mutation in peripheral blood of virtually all adult systemic mastocytosis patients once highly sensitive PCR techniques were used; thus, detection of the KIT D816V mutation in peripheral blood has been proposed to be included in the diagnostic work-up of systemic mastocytosis algorithms. However, the precise frequency of the mutation, the biological significance of peripheral blood-mutated cells and their potential association with involvement of bone marrow hematopoietic cells other than mast cells still remain to be investigated. Here, we determined the frequency of peripheral blood involvement by the KIT D816V mutation, as assessed by two highly sensitive PCR methods, and investigated its relationship with multilineage involvement of bone marrow hematopoiesis. Overall, our results confirmed the presence of the KIT D816V mutation in peripheral blood of most systemic mastocytosis cases (161/190; 85%)--with an increasing frequency from indolent systemic mastocytosis without skin lesions (29/44; 66%) to indolent systemic mastocytosis with skin involvement (124/135; 92%), and more aggressive disease subtypes (11/11; 100%)--as assessed by the allele-specific oligonucleotide-qPCR method, which was more sensitive (P<.0001) than the peptide nucleic acid-mediated PCR approach (84/190; 44%). Although the presence of the KIT mutation in peripheral blood, as assessed by the allele-specific oligonucleotide-qPCR technique, did not accurately predict for multilineage bone marrow involvement of hematopoiesis, the allele-specific oligonucleotide-qPCR allele burden and the peptide nucleic acid-mediated-PCR approach did. These results suggest that both methods provide clinically useful and complementary information through the identification and/or quantification of the KIT D816V mutation in peripheral blood of patients suspected of systemic mastocytosis.

Digital PCR Improves Mutation Analysis in Pancreas Fine Needle Aspiration Biopsy Specimens.

PubMed

Sho, Shonan; Court, Colin M; Kim, Stephen; Braxton, David R; Hou, Shuang; Muthusamy, V Raman; Watson, Rabindra R; Sedarat, Alireza; Tseng, Hsian-Rong; Tomlinson, James S

2017-01-01

Applications of precision oncology strategies rely on accurate tumor genotyping from clinically available specimens. Fine needle aspirations (FNA) are frequently obtained in cancer management and often represent the only source of tumor tissues for patients with metastatic or locally advanced diseases. However, FNAs obtained from pancreas ductal adenocarcinoma (PDAC) are often limited in cellularity and/or tumor cell purity, precluding accurate tumor genotyping in many cases. Digital PCR (dPCR) is a technology with exceptional sensitivity and low DNA template requirement, characteristics that are necessary for analyzing PDAC FNA samples. In the current study, we sought to evaluate dPCR as a mutation analysis tool for pancreas FNA specimens. To this end, we analyzed alterations in the KRAS gene in pancreas FNAs using dPCR. The sensitivity of dPCR mutation analysis was first determined using serial dilution cell spiking studies. Single-cell laser-microdissection (LMD) was then utilized to identify the minimal number of tumor cells needed for mutation detection. Lastly, dPCR mutation analysis was performed on 44 pancreas FNAs (34 formalin-fixed paraffin-embedded (FFPE) and 10 fresh (non-fixed)), including samples highly limited in cellularity (100 cells) and tumor cell purity (1%). We found dPCR to detect mutations with allele frequencies as low as 0.17%. Additionally, a single tumor cell could be detected within an abundance of normal cells. Using clinical FNA samples, dPCR mutation analysis was successful in all preoperative FNA biopsies tested, and its accuracy was confirmed via comparison with resected tumor specimens. Moreover, dPCR revealed additional KRAS mutations representing minor subclones within a tumor that were not detected by the current clinical gold standard method of Sanger sequencing. In conclusion, dPCR performs sensitive and accurate mutation analysis in pancreas FNAs, detecting not only the dominant mutation subtype, but also the additional rare mutation subtypes representing tumor heterogeneity.

Digital PCR Improves Mutation Analysis in Pancreas Fine Needle Aspiration Biopsy Specimens

PubMed Central

Court, Colin M.; Kim, Stephen; Braxton, David R.; Hou, Shuang; Muthusamy, V. Raman; Watson, Rabindra R.; Sedarat, Alireza; Tseng, Hsian-Rong; Tomlinson, James S.

2017-01-01

Applications of precision oncology strategies rely on accurate tumor genotyping from clinically available specimens. Fine needle aspirations (FNA) are frequently obtained in cancer management and often represent the only source of tumor tissues for patients with metastatic or locally advanced diseases. However, FNAs obtained from pancreas ductal adenocarcinoma (PDAC) are often limited in cellularity and/or tumor cell purity, precluding accurate tumor genotyping in many cases. Digital PCR (dPCR) is a technology with exceptional sensitivity and low DNA template requirement, characteristics that are necessary for analyzing PDAC FNA samples. In the current study, we sought to evaluate dPCR as a mutation analysis tool for pancreas FNA specimens. To this end, we analyzed alterations in the KRAS gene in pancreas FNAs using dPCR. The sensitivity of dPCR mutation analysis was first determined using serial dilution cell spiking studies. Single-cell laser-microdissection (LMD) was then utilized to identify the minimal number of tumor cells needed for mutation detection. Lastly, dPCR mutation analysis was performed on 44 pancreas FNAs (34 formalin-fixed paraffin-embedded (FFPE) and 10 fresh (non-fixed)), including samples highly limited in cellularity (100 cells) and tumor cell purity (1%). We found dPCR to detect mutations with allele frequencies as low as 0.17%. Additionally, a single tumor cell could be detected within an abundance of normal cells. Using clinical FNA samples, dPCR mutation analysis was successful in all preoperative FNA biopsies tested, and its accuracy was confirmed via comparison with resected tumor specimens. Moreover, dPCR revealed additional KRAS mutations representing minor subclones within a tumor that were not detected by the current clinical gold standard method of Sanger sequencing. In conclusion, dPCR performs sensitive and accurate mutation analysis in pancreas FNAs, detecting not only the dominant mutation subtype, but also the additional rare mutation subtypes representing tumor heterogeneity. PMID:28125707

Validation of Version 3.0 of the Breast Cancer Genetics Referral Screening Tool (B-RST™).

PubMed

Bellcross, Cecelia; Hermstad, April; Tallo, Christine; Stanislaw, Christine

2018-05-08

Despite increased awareness of hereditary breast and ovarian cancer among clinicians and the public, many BRCA1/2 mutation carriers remain unaware of their risk status. The Breast Cancer Genetics Referral Screening Tool (B-RST™) was created and validated to easily identify individuals at increased risk for hereditary breast and ovarian cancer for referral to cancer genetics services. The purpose of this study was to revise B-RST™ to maximize sensitivity against BRCA1/2 mutation status. We analyzed pedigrees of 277 individuals who had undergone BRCA1/2 testing to determine modifications to the B-RST™ 2.0 algorithm that would maximize sensitivity for mutations, while maintaining simplicity. We used McNemar's chi-square test to compare validation measures between the revised version (3.0) and the 2.0 version. Algorithmic changes made to B-RST™ 2.0 increased the sensitivity against BRCA1/2 mutation analysis from 71.1 to 94.0% (P < 0.0001). While specificity decreased, all screen-positive individuals were appropriate for cancer genetics referral, the primary purpose of the tool. Despite calls for BRCA1/2 population screening, there remains a critical need to identify those most at risk who should receive cancer genetics services. B-RST™ version 3.0 demonstrates high sensitivity for BRCA1/2 mutations, yet remains a simple and quick screening tool for at-risk individuals.

Highly sensitive and quantitative evaluation of the EGFR T790M mutation by nanofluidic digital PCR.

PubMed

Iwama, Eiji; Takayama, Koichi; Harada, Taishi; Okamoto, Isamu; Ookubo, Fumihiko; Kishimoto, Junji; Baba, Eishi; Oda, Yoshinao; Nakanishi, Yoichi

2015-08-21

The mutation of T790M in EGFR is a major mechanism of resistance to treatment with EGFR-TKIs. Only qualitative detection (presence or absence) of T790M has been described to date, however. Digital PCR (dPCR) analysis has recently been applied to the quantitative detection of target molecules in cancer with high sensitivity. In the present study, 25 tumor samples (13 obtained before and 12 after EGFR-TKI treatment) from 18 NSCLC patients with activating EGFR mutations were evaluated for T790M with dPCR. The ratio of the number of T790M alleles to that of activating mutation alleles (T/A) was determined. dPCR detected T790M in all 25 samples. Although T790M was present in all pre-TKI samples from 13 patients, 10 of these patients had a low T/A ratio and manifested substantial tumor shrinkage during treatment with EGFR-TKIs. In six of seven patients for whom both pre- and post-TKI samples were available, the T/A ratio increased markedly during EGFR-TKI treatment. Highly sensitive dPCR thus detected T790M in all NSCLC patients harboring activating EGFR mutations whether or not they had received EGFR-TKI treatment. Not only highly sensitive but also quantitative detection of T790M is important for evaluation of the contribution of T790M to EGFR-TKI resistance.

Metastatic melanoma cells with BRAF G469A mutation: nab-paclitaxel better than vemurafenib?

PubMed

Porcelli, Letizia; Guida, Gabriella; Tommasi, Stefania; Guida, Michele; Azzariti, Amalia

2015-08-01

BRAF G469A is a missense mutation within exon 11 of the BRAF gene resulting in a constitutively activated enzyme frequently associated with MAP kinase cascade signaling activation. No evidence currently exists about its role in determining sensitivity/resistance to BRAF inhibitors, utilized in the treatment of patients carrying BRAF V600 mutations, and to chemotherapy. The newly established metastatic melanoma (MM) cell line MO-1 was characterized for its sensitivity to vemurafenib and nab-paclitaxel, both already utilized for the treatment of MM. All analyses were carried out by comparing results with those found in MM cells wild type for BRAF or mutated in V600. In addition, cellular effectors were investigated by ELISA kits, western blotting and flow cytometry. The exposure to vemurafenib inhibited MO-1 cell proliferation at concentrations similar to those obtained in vemurafenib-resistant melanoma models, and an explanation of this sensitivity is the strong activation of Erk1/2 and the low expression of MITF. Nab-paclitaxel strongly reduced proliferation of MO-1 cells perhaps for the very low expression level of PMEL17, transcriptionally regulated by MITF and negatively involved in determining sensitivity to taxanes. Thus, the mutation BRAF G469A in MM might be related to a weak effectiveness of therapy with BRAF inhibitors and a promising therapeutic approach may be with nab-paclitaxel.

Enhanced sensitivity for detection of low-level germline mosaic RB1 mutations in sporadic retinoblastoma cases using deep semiconductor sequencing.

PubMed

Chen, Zhao; Moran, Kimberly; Richards-Yutz, Jennifer; Toorens, Erik; Gerhart, Daniel; Ganguly, Tapan; Shields, Carol L; Ganguly, Arupa

2014-03-01

Sporadic retinoblastoma (RB) is caused by de novo mutations in the RB1 gene. Often, these mutations are present as mosaic mutations that cannot be detected by Sanger sequencing. Next-generation deep sequencing allows unambiguous detection of the mosaic mutations in lymphocyte DNA. Deep sequencing of the RB1 gene on lymphocyte DNA from 20 bilateral and 70 unilateral RB cases was performed, where Sanger sequencing excluded the presence of mutations. The individual exons of the RB1 gene from each sample were amplified, pooled, ligated to barcoded adapters, and sequenced using semiconductor sequencing on an Ion Torrent Personal Genome Machine. Six low-level mosaic mutations were identified in bilateral RB and four in unilateral RB cases. The incidence of low-level mosaic mutation was estimated to be 30% and 6%, respectively, in sporadic bilateral and unilateral RB cases, previously classified as mutation negative. The frequency of point mutations detectable in lymphocyte DNA increased from 96% to 97% for bilateral RB and from 13% to 18% for unilateral RB. The use of deep sequencing technology increased the sensitivity of the detection of low-level germline mosaic mutations in the RB1 gene. This finding has significant implications for improved clinical diagnosis, genetic counseling, surveillance, and management of RB. © 2013 WILEY PERIODICALS, INC.

The detectability of the pretreatment EGFR T790M mutations in lung adenocarcinoma using CAST-PCR and digital PCR

PubMed Central

Tatematsu, Tsutomu; Suzuki, Ayumi; Oda, Risa; Sakane, Tadashi; Kawano, Osamu; Haneda, Hiroshi; Moriyama, Satoru; Sasaki, Hidefumi; Nakanishi, Ryoichi

2017-01-01

Background A gatekeeper T790M mutation is thought to cause resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) treatment. The detection of a 2nd mutation is important for planning the next therapy when patients acquire resistance to the first line EGFR-TKI. Methods We used a competitive allele-specific polymerase chain reaction (CAST-PCR) to analyze the incidence and clinical significance of T790M mutations in 153 lung adenocarcinomas with EGFR-activating mutations. To increase the sensitivity and specificity of the detection of T790M mutations, we subjected 20 of the 153 cases to a digital PCR. The genomic DNAs were extracted from frozen, surgically resected tumor tissue specimens. Results The CAST-PCR detected T790M mutations in 45 (29.4%) of the 153 cases. The analytical sensitivity in the detection T790M mutations was 0.13–2.65% (average 0.27%, median 0.20%). In contrast, the digital PCR, detected T790M mutations in 8 (40%) out of 20 cases. Conclusions Our study shows that the pretreatment incidence of T790M mutation was less than that reported in previous studies. In order to clinically use pretreatment EGFR T790M mutation identification method, we should clarify the adequate methods and tissue preserved status. PMID:28932544

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

PubMed

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

2018-01-01

We aimed to evaluate the distribution of individual epidermal growth factor receptor ( EGFR ) mutation subtypes found in routine cytological specimens. A retrospective audit was performed on EGFR testing results of 1,874 consecutive cytological samples of newly diagnosed or treatment-naïve Indonesian lung cancer patients (years 2015-2016). Testing was performed by ISO15189 accredited central laboratory. Overall test failure rate was 5.1%, with the highest failure (7.1%) observed in pleural effusion and lowest (1.6%) in needle aspiration samples. EGFR mutation frequency was 44.4%. Tyrosine kinase inhibitor (TKI)-sensitive common EGFR mutations (ins/dels exon 19, L858R) and uncommon mutations (G719X, T790M, L861Q) contributed 57.1% and 29%, respectively. Approximately 13.9% of mutation-positive patients carried a mixture of common and uncommon mutations. Women had higher EGFR mutation rate (52.9%) vs men (39.1%; p <0.05). In contrast, uncommon mutations conferring either TKI responsive (G719X, L861Q) or TKI resistance (T790M, exon 20 insertions) were consistently more frequent in men than in women (67.3% vs 32.7% or 69.4% vs 30.6%; p <0.05). Up to 10% EGFR mutation-positive patients had baseline single mutation T790M, exon 20 insertion, or in coexistence with TKI-sensitive mutations. Up to 9% patients had complex or multiple EGFR mutations, whereby 48.7% patients harbored TKI-resistant mutations. One patient presented third-generation TKI-resistant mutation L792F simultaneously with T790M. Routine diagnostic cytological techniques yielded similar success rate to detect EGFR mutations. Uncommon EGFR mutations were frequent events in Indonesian lung cancer patients.

A sensitive detection method for MPLW515L or MPLW515K mutation in chronic myeloproliferative disorders with locked nucleic acid-modified probes and real-time polymerase chain reaction.

PubMed

Pancrazzi, Alessandro; Guglielmelli, Paola; Ponziani, Vanessa; Bergamaschi, Gaetano; Bosi, Alberto; Barosi, Giovanni; Vannucchi, Alessandro M

2008-09-01

Acquired mutations in the juxtamembrane region of MPL (W515K or W515L), the receptor for thrombopoietin, have been described in patients with primary myelofibrosis or essential thrombocythemia, which are chronic myeloproliferative disorders. We have developed a real-time polymerase chain reaction assay for the detection and quantification of MPL mutations that is based on locked nucleic acid fluorescent probes. Mutational analysis was performed using DNA from granulocytes. Reference curves were obtained using cloned fragments of MPL containing either the wild-type or mutated sequence; the predicted sensitivity level was at least 0.1% mutant allele in a wild-type background. None of the 60 control subjects presented with a MPLW515L/K mutation. Of 217 patients with myelofibrosis, 19 (8.7%) harbored the MPLW515 mutation, 10 (52.6%) with the W515L allele. In one case, both the W515L and W515K alleles were detected by real-time polymerase chain reaction. By comparing results obtained with conventional sequencing, no erroneous genotype attribution using real-time polymerase chain reaction was found, whereas one patient considered wild type according to sequence analysis actually harbored a low W515L allele burden. This is a simple, sensitive, and cost-effective procedure for large-scale screening of the MPLW515L/K mutation in patients suspected to have a myeloproliferative disorder. It can also provide a quantitative estimate of mutant allele burden that might be useful for both patient prognosis and monitoring response to therapy.

Molecular Evolution of the Tissue-nonspecific Alkaline Phosphatase Allows Prediction and Validation of Missense Mutations Responsible for Hypophosphatasia*

PubMed Central

Silvent, Jérémie; Gasse, Barbara; Mornet, Etienne; Sire, Jean-Yves

2014-01-01

ALPL encodes the tissue nonspecific alkaline phosphatase (TNSALP), which removes phosphate groups from various substrates. Its function is essential for bone and tooth mineralization. In humans, ALPL mutations lead to hypophosphatasia, a genetic disorder characterized by defective bone and/or tooth mineralization. To date, 275 ALPL mutations have been reported to cause hypophosphatasia, of which 204 were simple missense mutations. Molecular evolutionary analysis has proved to be an efficient method to highlight residues important for the protein function and to predict or validate sensitive positions for genetic disease. Here we analyzed 58 mammalian TNSALP to identify amino acids unchanged, or only substituted by residues sharing similar properties, through 220 millions years of mammalian evolution. We found 469 sensitive positions of the 524 residues of human TNSALP, which indicates a highly constrained protein. Any substitution occurring at one of these positions is predicted to lead to hypophosphatasia. We tested the 204 missense mutations resulting in hypophosphatasia against our predictive chart, and validated 99% of them. Most sensitive positions were located in functionally important regions of TNSALP (active site, homodimeric interface, crown domain, calcium site, …). However, some important positions are located in regions, the structure and/or biological function of which are still unknown. Our chart of sensitive positions in human TNSALP (i) enables to validate or invalidate at low cost any ALPL mutation, which would be suspected to be responsible for hypophosphatasia, by contrast with time consuming and expensive functional tests, and (ii) displays higher predictive power than in silico models of prediction. PMID:25023282

Biochip-Based Detection of KRAS Mutation in Non-Small Cell Lung Cancer

PubMed Central

Kriegshäuser, Gernot; Fabjani, Gerhild; Ziegler, Barbara; Zöchbauer-Müller, Sabine; End, Adelheid; Zeillinger, Robert

2011-01-01

This study is aimed at evaluating the potential of a biochip assay to sensitively detect KRAS mutation in DNA from non-small cell lung cancer (NSCLC) tissue samples. The assay covers 10 mutations in codons 12 and 13 of the KRAS gene, and is based on mutant-enriched PCR followed by reverse-hybridization of biotinylated amplification products to an array of sequence-specific probes immobilized on the tip of a rectangular plastic stick (biochip). Biochip hybridization identified 17 (21%) samples to carry a KRAS mutation of which 16 (33%) were adenocarcinomas and 1 (3%) was a squamous cell carcinoma. All mutations were confirmed by DNA sequencing. Using 10 ng of starting DNA, the biochip assay demonstrated a detection limit of 1% mutant sequence in a background of wild-type DNA. Our results suggest that the biochip assay is a sensitive alternative to protocols currently in use for KRAS mutation testing on limited quantity samples. PMID:22272089

Detection of BRAF mutation in Chinese tumor patients using a highly sensitive antibody immunohistochemistry assay

NASA Astrophysics Data System (ADS)

Qiu, Tian; Lu, Haizhen; Guo, Lei; Huang, Wenting; Ling, Yun; Shan, Ling; Li, Wenbin; Ying, Jianming; Lv, Ning

2015-03-01

BRAF mutations can be found in various solid tumors. But accurate and reliable screening for BRAF mutation that is compatible for clinical application is not yet available. In this study, we used an automated immunohistochemistry (IHC) staining coupled with mouse monoclonal anti-BRAF V600E (VE1) primary antibody to screen the BRAF V600E mutation in 779 tumor cases, including 611 colorectal carcinomas (CRC), 127 papillary thyroid carcinomas (PTC) and 41 malignant melanomas. Among the 779 cases, 150 cases were positive for BRAF (V600E) staining, including 38 (of 611, 6%) CRCs, 102 (of 127, 80%) PTCs and 10 (of 41, 24%) malignant melanomas. Sanger sequencing and real-time PCR confirmed the sensitivity and specificity of IHC staining for the V600E mutation are 100% and 99%, respectively. Therefore, our study demonstrates that the fully automated IHC is a reliable tool to determine BRAF mutation status in CRC, PTC and melanoma and can be used for routine clinical screen.

An Escherichia coli Strain, PGB01, Isolated from Feral Pigeon Faeces, Thermally Fit to Survive in Pigeon, Shows High Level Resistance to Trimethoprim

PubMed Central

Kachhap, Sangita; Nanda, Ashis Kumar; Chakraborty, Ranadhir

2015-01-01

In this study, of the hundred Escherichia coli strains isolated from feral Pigeon faeces, eighty five strains were resistant to one or more antibiotics and fifteen sensitive to all the antibiotics tested. The only strain (among all antibiotic-resistant E. coli isolates) that possessed class 1 integron was PGB01. The dihydrofolate reductase gene of the said integron was cloned, sequenced and expressed in E. coli JM109. Since PGB01 was native to pigeon’s gut, we have compared the growth of PGB01 at two different temperatures, 42°C (normal body temperature of pigeon) and 37°C (optimal growth temperature of E. coli; also the human body temperature), with E. coli K12. It was found that PGB01 grew better than the laboratory strain E. coli K12 at 37°C as well as at 42°C. In the thermal fitness assay, it was observed that the cells of PGB01 were better adapted to 42°C, resembling the average body temperature of pigeon. The strain PGB01 also sustained more microwave mediated thermal stress than E. coli K12 cells. The NMR spectra of the whole cells of PGB01 varied from E. coli K12 in several spectral peaks relating some metabolic adaptation to thermotolerance. On elevating the growth temperature from 37°C to 42°C, susceptibility to kanamycin (both strains were sensitive to it) of E. coli K12 was increased, but in case of PGB01 no change in susceptibility took place. We have also attempted to reveal the basis of trimethoprim resistance phenotype conferred by the dfrA7 gene homologue of PGB01. Molecular Dynamics (MD) simulation study of docked complexes, PGB01-DfrA7 and E. coli TMP-sensitive-Dfr with trimethoprim (TMP) showed loss of some of the hydrogen and hydrophobic interaction between TMP and mutated residues in PGB01-DfrA7-TMP complex compared to TMP-sensitive-Dfr-TMP complex. This loss of interaction entails decrease in affinity of TMP for PGB01-DfrA7 compared to TMP-sensitive-Dfr. PMID:25750990

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

PubMed

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

2013-03-01

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

Sensitive Detection of Mono- and Polyclonal ESR1 Mutations in Primary Tumors, Metastatic Lesions, and Cell-Free DNA of Breast Cancer Patients.

PubMed

Wang, Peilu; Bahreini, Amir; Gyanchandani, Rekha; Lucas, Peter C; Hartmaier, Ryan J; Watters, Rebecca J; Jonnalagadda, Amruth R; Trejo Bittar, Humberto E; Berg, Aaron; Hamilton, Ronald L; Kurland, Brenda F; Weiss, Kurt R; Mathew, Aju; Leone, Jose Pablo; Davidson, Nancy E; Nikiforova, Marina N; Brufsky, Adam M; Ambros, Tadeu F; Stern, Andrew M; Puhalla, Shannon L; Lee, Adrian V; Oesterreich, Steffi

2016-03-01

Given the clinical relevance of ESR1 mutations as potential drivers of resistance to endocrine therapy, this study used sensitive detection methods to determine the frequency of ESR1 mutations in primary and metastatic breast cancer, and in cell-free DNA (cfDNA). Six ESR1 mutations (K303R, S463P, Y537C, Y537N, Y537S, D538G) were assessed by digital droplet PCR (ddPCR), with lower limits of detection of 0.05% to 0.16%, in primary tumors (n = 43), bone (n = 12) and brain metastases (n = 38), and cfDNA (n = 29). Correlations between ESR1 mutations in metastatic lesions and single (1 patient) or serial blood draws (4 patients) were assessed. ESR1 mutations were detected for D538G (n = 13), Y537S (n = 3), and Y537C (n = 1), and not for K303R, S463P, or Y537N. Mutation rates were 7.0% (3/43 primary tumors), 9.1% (1/11 bone metastases), 12.5% (3/24 brain metastases), and 24.1% (7/29 cfDNA). Two patients showed polyclonal disease with more than one ESR1 mutation. Mutation allele frequencies were 0.07% to 0.2% in primary tumors, 1.4% in bone metastases, 34.3% to 44.9% in brain metastases, and 0.2% to 13.7% in cfDNA. In cases with both cfDNA and metastatic samples (n = 5), mutations were detected in both (n = 3) or in cfDNA only (n = 2). Treatment was associated with changes in ESR1 mutation detection and allele frequency. ESR1 mutations were detected at very low allele frequencies in some primary breast cancers, and at high allele frequency in metastases, suggesting that in some tumors rare ESR1-mutant clones are enriched by endocrine therapy. Further studies should address whether sensitive detection of ESR1 mutations in primary breast cancer and in serial blood draws may be predictive for development of resistant disease. See related commentary by Gu and Fuqua, p. 1034. ©2015 American Association for Cancer Research.

Optimized p53 immunohistochemistry is an accurate predictor of TP53 mutation in ovarian carcinoma.

PubMed

Köbel, Martin; Piskorz, Anna M; Lee, Sandra; Lui, Shuhong; LePage, Cecile; Marass, Francesco; Rosenfeld, Nitzan; Mes Masson, Anne-Marie; Brenton, James D

2016-10-01

TP53 mutations are ubiquitous in high-grade serous ovarian carcinomas (HGSOC), and the presence of TP53 mutation discriminates between high and low-grade serous carcinomas and is now an important biomarker for clinical trials targeting mutant p53. p53 immunohistochemistry (IHC) is widely used as a surrogate for TP53 mutation but its accuracy has not been established. The objective of this study was to test whether improved methods for p53 IHC could reliably predict TP53 mutations independently identified by next generation sequencing (NGS). Four clinical p53 IHC assays and tagged-amplicon NGS for TP53 were performed on 171 HGSOC and 80 endometrioid carcinomas (EC). p53 expression was scored as overexpression (OE), complete absence (CA), cytoplasmic (CY) or wild type (WT). p53 IHC was evaluated as a binary classifier where any abnormal staining predicted deleterious TP53 mutation and as a ternary classifier where OE, CA or WT staining predicted gain-of-function (GOF or nonsynonymous), loss-of-function (LOF including stopgain, indel, splicing) or no detectable TP53 mutations (NDM), respectively. Deleterious TP53 mutations were detected in 169/171 (99%) HGSOC and 7/80 (8.8%) EC. The overall accuracy for the best performing IHC assay for binary and ternary prediction was 0.94 and 0.91 respectively, which improved to 0.97 (sensitivity 0.96, specificity 1.00) and 0.95 after secondary analysis of discordant cases. The sensitivity for predicting LOF mutations was lower at 0.76 because p53 IHC detected mutant p53 protein in 13 HGSOC with LOF mutations. CY staining associated with LOF was seen in 4 (2.3%) of HGSOC. Optimized p53 IHC can approach 100% specificity for the presence of TP53 mutation and its high negative predictive value is clinically useful as it can exclude the possibility of a low-grade serous tumour. 4.1% of HGSOC cases have detectable WT staining while harboring a TP53 LOF mutation, which limits sensitivity for binary prediction of mutation to 96%.

Optimized p53 immunohistochemistry is an accurate predictor of TP53 mutation in ovarian carcinoma

PubMed Central

Köbel, Martin; Piskorz, Anna M; Lee, Sandra; Lui, Shuhong; LePage, Cecile; Marass, Francesco; Rosenfeld, Nitzan; Mes Masson, Anne‐Marie

2016-01-01

Abstract TP53 mutations are ubiquitous in high‐grade serous ovarian carcinomas (HGSOC), and the presence of TP53 mutation discriminates between high and low‐grade serous carcinomas and is now an important biomarker for clinical trials targeting mutant p53. p53 immunohistochemistry (IHC) is widely used as a surrogate for TP53 mutation but its accuracy has not been established. The objective of this study was to test whether improved methods for p53 IHC could reliably predict TP53 mutations independently identified by next generation sequencing (NGS). Four clinical p53 IHC assays and tagged‐amplicon NGS for TP53 were performed on 171 HGSOC and 80 endometrioid carcinomas (EC). p53 expression was scored as overexpression (OE), complete absence (CA), cytoplasmic (CY) or wild type (WT). p53 IHC was evaluated as a binary classifier where any abnormal staining predicted deleterious TP53 mutation and as a ternary classifier where OE, CA or WT staining predicted gain‐of‐function (GOF or nonsynonymous), loss‐of‐function (LOF including stopgain, indel, splicing) or no detectable TP53 mutations (NDM), respectively. Deleterious TP53 mutations were detected in 169/171 (99%) HGSOC and 7/80 (8.8%) EC. The overall accuracy for the best performing IHC assay for binary and ternary prediction was 0.94 and 0.91 respectively, which improved to 0.97 (sensitivity 0.96, specificity 1.00) and 0.95 after secondary analysis of discordant cases. The sensitivity for predicting LOF mutations was lower at 0.76 because p53 IHC detected mutant p53 protein in 13 HGSOC with LOF mutations. CY staining associated with LOF was seen in 4 (2.3%) of HGSOC. Optimized p53 IHC can approach 100% specificity for the presence of TP53 mutation and its high negative predictive value is clinically useful as it can exclude the possibility of a low‐grade serous tumour. 4.1% of HGSOC cases have detectable WT staining while harboring a TP53 LOF mutation, which limits sensitivity for binary prediction of mutation to 96%. PMID:27840695

Detection of somatic mutations by high-resolution DNA melting (HRM) analysis in multiple cancers.

PubMed

Gonzalez-Bosquet, Jesus; Calcei, Jacob; Wei, Jun S; Garcia-Closas, Montserrat; Sherman, Mark E; Hewitt, Stephen; Vockley, Joseph; Lissowska, Jolanta; Yang, Hannah P; Khan, Javed; Chanock, Stephen

2011-01-17

Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM) curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each). HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples.

Detection of Somatic Mutations by High-Resolution DNA Melting (HRM) Analysis in Multiple Cancers

PubMed Central

Gonzalez-Bosquet, Jesus; Calcei, Jacob; Wei, Jun S.; Garcia-Closas, Montserrat; Sherman, Mark E.; Hewitt, Stephen; Vockley, Joseph; Lissowska, Jolanta; Yang, Hannah P.; Khan, Javed; Chanock, Stephen

2011-01-01

Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM) curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each). HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples. PMID:21264207

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

PubMed Central

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

2018-01-01

Purpose We aimed to evaluate the distribution of individual epidermal growth factor receptor (EGFR) mutation subtypes found in routine cytological specimens. Patients and methods A retrospective audit was performed on EGFR testing results of 1,874 consecutive cytological samples of newly diagnosed or treatment-naïve Indonesian lung cancer patients (years 2015–2016). Testing was performed by ISO15189 accredited central laboratory. Results Overall test failure rate was 5.1%, with the highest failure (7.1%) observed in pleural effusion and lowest (1.6%) in needle aspiration samples. EGFR mutation frequency was 44.4%. Tyrosine kinase inhibitor (TKI)-sensitive common EGFR mutations (ins/dels exon 19, L858R) and uncommon mutations (G719X, T790M, L861Q) contributed 57.1% and 29%, respectively. Approximately 13.9% of mutation-positive patients carried a mixture of common and uncommon mutations. Women had higher EGFR mutation rate (52.9%) vs men (39.1%; p<0.05). In contrast, uncommon mutations conferring either TKI responsive (G719X, L861Q) or TKI resistance (T790M, exon 20 insertions) were consistently more frequent in men than in women (67.3% vs 32.7% or 69.4% vs 30.6%; p<0.05). Up to 10% EGFR mutation–positive patients had baseline single mutation T790M, exon 20 insertion, or in coexistence with TKI-sensitive mutations. Up to 9% patients had complex or multiple EGFR mutations, whereby 48.7% patients harbored TKI-resistant mutations. One patient presented third-generation TKI-resistant mutation L792F simultaneously with T790M. Conclusion Routine diagnostic cytological techniques yielded similar success rate to detect EGFR mutations. Uncommon EGFR mutations were frequent events in Indonesian lung cancer patients. PMID:29615847

ssaD1, a suppressor of secA51(Ts) that renders growth of Escherichia coli cold sensitive, is an early amber mutation in the transcription factor gene nusB.

PubMed Central

Rajapandi, T.; Oliver, D.

1994-01-01

Complementation analysis of the ssaD1 mutation, isolated as a suppressor of the secA51(Ts) mutation that renders growth of Escherichia coli cold sensitive, was used to show that ssaD corresponds to nusB, a gene known to be important in transcription antitermination. DNA sequence analysis of the ssaD1 allele showed that it creates an amber mutation in the 15th codon of nusB. Analysis of the effect of different levels of NusB protein on secA transcription and translation suggested that NusB plays little or no role in the control of secA expression. Accordingly, mechanisms by which nusB inactivation can lead to suppression of secA51(Ts) and secY24(Ts) mutations without affecting secA expression need to be considered. PMID:8021230

Multiplex Ultrasensitive Genotyping of Patients with Non-Small Cell Lung Cancer for Epidermal Growth Factor Receptor (EGFR) Mutations by Means of Picodroplet Digital PCR.

PubMed

Watanabe, Masaru; Kawaguchi, Tomoya; Isa, Shun-Ichi; Ando, Masahiko; Tamiya, Akihiro; Kubo, Akihito; Saka, Hideo; Takeo, Sadanori; Adachi, Hirofumi; Tagawa, Tsutomu; Kawashima, Osamu; Yamashita, Motohiro; Kataoka, Kazuhiko; Ichinose, Yukito; Takeuchi, Yukiyasu; Watanabe, Katsuya; Matsumura, Akihide; Koh, Yasuhiro

2017-07-01

Epidermal growth factor receptor (EGFR) mutations have been used as the strongest predictor of effectiveness of treatment with EGFR tyrosine kinase inhibitors (TKIs). Three most common EGFR mutations (L858R, exon 19 deletion, and T790M) are known to be major selection markers for EGFR-TKIs therapy. Here, we developed a multiplex picodroplet digital PCR (ddPCR) assay to detect 3 common EGFR mutations in 1 reaction. Serial-dilution experiments with genomic DNA harboring EGFR mutations revealed linear performance, with analytical sensitivity ~0.01% for each mutation. All 33 EGFR-activating mutations detected in formalin-fixed paraffin-embedded (FFPE) tissue samples by the conventional method were also detected by this multiplex assay. Owing to the higher sensitivity, an additional mutation (T790M; including an ultra-low-level mutation, <0.1%) was detected in the same reaction. Regression analysis of the duplex assay and multiplex assay showed a correlation coefficient (R 2 ) of 0.9986 for L858R, 0.9844 for an exon 19 deletion, and 0.9959 for T790M. Using ddPCR, we designed a multiplex ultrasensitive genotyping platform for 3 common EGFR mutations. Results of this proof-of-principle study on clinical samples indicate clinical utility of multiplex ddPCR for screening for multiple EGFR mutations concurrently with an ultra-rare pretreatment mutation (T790M). Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Comparison of ALS functionality and plant growth in ALS-inhibitor susceptible and resistant Myosoton aquaticum L.

PubMed

Liu, Weitang; Bai, Shuang; Jia, Sisi; Guo, Wenlei; Zhang, Lele; Li, Wei; Wang, Jinxin

2017-10-01

Herbicide target-site resistance mutations may cause pleiotropic effects on plant ecology and physiology. The effect of several known (Pro197Ser, Pro197Leu Pro197Ala, and Pro197Glu) target-site resistance mutations of the ALS gene on both ALS functionality and plant vegetative growth of weed Myosoton aquaticum L. (water chickweed) have been investigated here. The enzyme kinetics of ALS from four purified water chickweed populations that each homozygous for the specific target-site resistance-endowing mutations were characterized and the effect of these mutations on plant growth was assessed via relative growth rate (RGR) analysis. Plants homozygous for Pro197Ser and Pro197Leu exhibited higher extractable ALS activity than susceptible (S) plants, while all ALS mutations with no negative change in ALS kinetics. The Pro197Leu mutation increased ALS sensitivity to isoleucine and valine, and Pro197Glu mutation slightly increased ALS sensitivity to isoleucine. RGR results indicated that none of these ALS resistance mutations impose negative pleiotropic effects on relative growth rate. However, resistant (R) seeds had a lowed germination rate than S seeds. This study provides baseline information on ALS functionality and plant growth characteristics associated with ALS inhibitor resistance-endowing mutations in water chickweed. Copyright © 2017. Published by Elsevier Inc.

A novel functional domain of Cdc15 kinase is required for its interaction with Tem1 GTPase in Saccharomyces cerevisiae.

PubMed Central

Asakawa, K; Yoshida, S; Otake, F; Toh-e, A

2001-01-01

Exit from mitosis requires the inactivation of cyclin-dependent kinase (CDK) activity. In the budding yeast Saccharomyces cerevisiae, a number of gene products have been identified as components of the signal transduction network regulating inactivation of CDK (called the MEN, for the mitotic exit network). Cdc15, one of such components of the MEN, is an essential protein kinase. By the two-hybrid screening, we identified Cdc15 as a binding protein of Tem1 GTPase, another essential regulator of the MEN. Coprecipitation experiments revealed that Tem1 binds to Cdc15 in vivo. By deletion analysis, we found that the Tem1-binding domain resides near the conserved kinase domain of Cdc15. The cdc15-LF mutation, which was introduced into the Tem1-binding domain, reduced the interaction with Cdc15 and Tem1 and caused temperature-sensitive growth.The kinase activity of Cdc15 was not so much affected by the cdc15-LF mutation. However, Cdc15-LF failed to localize to the SPB at the restrictive temperature. Our data show that the interaction with Tem1 is important for the function of Cdc15 and that Cdc15 and Tem1 function in a complex to direct the exit from mitosis. PMID:11290702

Mediator independently orchestrates multiple steps of preinitiation complex assembly in vivo

PubMed Central

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

2015-01-01

Mediator is a large multiprotein complex conserved in all eukaryotes, which has a crucial coregulator function in transcription by RNA polymerase II (Pol II). However, the molecular mechanisms of its action in vivo remain to be understood. Med17 is an essential and central component of the Mediator head module. In this work, we utilised our large collection of conditional temperature-sensitive med17 mutants to investigate Mediator's role in coordinating preinitiation complex (PIC) formation in vivo at the genome level after a transfer to a non-permissive temperature for 45 minutes. The effect of a yeast mutation proposed to be equivalent to the human Med17-L371P responsible for infantile cerebral atrophy was also analyzed. The ChIP-seq results demonstrate that med17 mutations differentially affected the global presence of several PIC components including Mediator, TBP, TFIIH modules and Pol II. Our data show that Mediator stabilizes TFIIK kinase and TFIIH core modules independently, suggesting that the recruitment or the stability of TFIIH modules is regulated independently on yeast genome. We demonstrate that Mediator selectively contributes to TBP recruitment or stabilization to chromatin. This study provides an extensive genome-wide view of Mediator's role in PIC formation, suggesting that Mediator coordinates multiple steps of a PIC assembly pathway. PMID:26240385

IBR5 Modulates Temperature-Dependent, R Protein CHS3-Mediated Defense Responses in Arabidopsis.

PubMed

Liu, Jingyan; Yang, Haibian; Bao, Fei; Ao, Kevin; Zhang, Xiaoyan; Zhang, Yuelin; Yang, Shuhua

2015-10-01

Plant responses to low temperature are tightly associated with defense responses. We previously characterized the chilling-sensitive mutant chs3-1 resulting from the activation of the Toll and interleukin 1 receptor-nucleotide binding-leucine-rich repeat (TIR-NB-LRR)-type resistance (R) protein harboring a C-terminal LIM (Lin-11, Isl-1 and Mec-3 domains) domain. Here we report the identification of a suppressor of chs3, ibr5-7 (indole-3-butyric acid response 5), which largely suppresses chilling-activated defense responses. IBR5 encodes a putative dual-specificity protein phosphatase. The accumulation of CHS3 protein at chilling temperatures is inhibited by the IBR5 mutation. Moreover, chs3-conferred defense phenotypes were synergistically suppressed by mutations in HSP90 and IBR5. Further analysis showed that IBR5, with holdase activity, physically associates with CHS3, HSP90 and SGT1b (Suppressor of the G2 allele of skp1) to form a complex that protects CHS3. In addition to the positive role of IBR5 in regulating CHS3, IBR5 is also involved in defense responses mediated by R genes, including SNC1 (Suppressor of npr1-1, Constitutive 1), RPS4 (Resistance to P. syringae 4) and RPM1 (Resistance to Pseudomonas syringae pv. maculicola 1). Thus, the results of the present study reveal a role for IBR5 in the regulation of multiple R protein-mediated defense responses.

Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated by Cetuximab plus chemotherapy

PubMed Central

Di Fiore, F; Blanchard, F; Charbonnier, F; Le Pessot, F; Lamy, A; Galais, M P; Bastit, L; Killian, A; Sesboüé, R; Tuech, J J; Queuniet, A M; Paillot, B; Sabourin, J C; Michot, F; Michel, P; Frebourg, T

2007-01-01

The predictive value of KRAS mutation in metastatic colorectal cancer (MCRC) patients treated with cetuximab plus chemotherapy has recently been suggested. In our study, 59 patients with a chemotherapy-refractory MCRC treated with cetuximab plus chemotherapy were included and clinical response was evaluated according to response evaluation criteria in solid tumours (RECIST). Tumours were screened for KRAS mutations using first direct sequencing, then two sensitive methods based on SNaPshot and PCR-ligase chain reaction (LCR) assays. Clinical response was evaluated according to gene mutations using the Fisher exact test. Times to progression (TTP) were calculated using the Kaplan–Meier method and compared with log-rank test. A KRAS mutation was detected in 22 out of 59 tumours and, in six cases, was missed by sequencing analysis but detected using the SNaPshot and PCR-LCR assays. Remarkably, no KRAS mutation was found in the 12 patients with clinical response. KRAS mutation was associated with disease progression (P=0.0005) and TTP was significantly decreased in mutated KRAS patients (3 vs 5.5 months, P=0.015). Our study confirms that KRAS mutation is highly predictive of a non-response to cetuximab plus chemotherapy in MCRC and highlights the need to use sensitive molecular methods, such as SNaPshot or PCR-LCR assays, to ensure an efficient mutation detection. PMID:17375050

Why individual thermo sensation and pain perception varies? Clue of disruptive mutations in TRPVs from 2504 human genome data.

PubMed

Ghosh, Arijit; Kaur, Navneet; Kumar, Abhishek; Goswami, Chandan

2016-09-02

Every individual varies in character and so do their sensory functions and perceptions. The molecular mechanism and the molecular candidates involved in these processes are assumed to be similar if not same. So far several molecular factors have been identified which are fairly conserved across the phylogenetic tree and are involved in these complex sensory functions. Among all, members belonging to Transient Receptor Potential (TRP) channels have been widely characterized for their involvement in thermo-sensation. These include TRPV1 to TRPV4 channels which reveal complex thermo-gating behavior in response to changes in temperature. The molecular evolution of these channels is highly correlative with the thermal response of different species. However, recent 2504 human genome data suggest that these thermo-sensitive TRPV channels are highly variable and carry possible deleterious mutations in human population. These unexpected findings may explain the individual differences in terms of complex sensory functions.

The modest beginnings of one genome project.

PubMed

Kaback, David B

2013-06-01

One of the top things on a geneticist's wish list has to be a set of mutants for every gene in their particular organism. Such a set was produced for the yeast, Saccharomyces cerevisiae near the end of the 20th century by a consortium of yeast geneticists. However, the functional genomic analysis of one chromosome, its smallest, had already begun more than 25 years earlier as a project that was designed to define most or all of that chromosome's essential genes by temperature-sensitive lethal mutations. When far fewer than expected genes were uncovered, the relatively new field of molecular cloning enabled us and indeed, the entire community of yeast researchers to approach this problem more definitively. These studies ultimately led to cloning, genomic sequencing, and the production and phenotypic analysis of the entire set of knockout mutations for this model organism as well as a better concept of what defines an essential function, a wish fulfilled that enables this model eukaryote to continue at the forefront of research in modern biology.

Rsp5-Bul1/2 complex is necessary for the HSE-mediated gene expression in budding yeast.

PubMed

Kaida, Daisuke; Toh-e, Akio; Kikuchi, Yoshiko

2003-07-11

Rsp5 is an essential ubiquitin ligase in Saccharomyces cerevisiae and is concerned with many functions such as endocytosis and transcription through ubiquitination of various substrates. Bul1 or its homologue Bul2 binds to Rsp5 through the PY-motif and the bul1 bul2 double mutant is sensitive to various stresses. We demonstrate here that heat shock element (HSE)-mediated gene expression was defective in both rsp5-101 and bul1 bul2 mutants under high temperature condition. The bul1 gene containing mutations in the PY motif region did not recover this defective gene expression of the bul1 bul2 mutant. The protein level and phosphorylation state of the HSE-binding transcription factor, Hsf1, was not affected by these mutations. Thus, the Rsp5-Bul1/2 complex has a new function for the HSE-mediated gene expression and may regulate it through other factors than Hsf1.

IDH1 R132H Mutation Enhances Cell Migration by Activating AKT-mTOR Signaling Pathway, but Sensitizes Cells to 5-FU Treatment as NADPH and GSH Are Reduced.

PubMed

Zhu, Huixia; Zhang, Ye; Chen, Jianfeng; Qiu, Jiangdong; Huang, Keting; Wu, Mindan; Xia, Chunlin

2017-01-01

Mutations of isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) gene were recently discovered in vast majority of World Health Organization (WHO) grade II/III gliomas. This study is to understand the effects of IDH1 R132H mutation in gliomagenesis and to develop new strategies to treat glioma with IDH1 R132H mutation. Over expression of IDH1 R132H in U87MG cells was done by transfecting cells with IDH1 R132H plasmid. MTT assay, scratch repair assay and western blot were performed to study effects of IDH1 R132H mutation on cell proliferation, migration, regulating AKT-mTOR signaling pathway and cell death respectively. NADP+/NADPH and GSH quantification assays were performed to evaluate effects of IDH1 R132H mutation on the production of antioxidant NADPH and GSH. We found that over expression of IDH1 R132H mutation decreased cell proliferation consistent with previous reports; however, it increased cell migration and enhanced AKT-mTOR signaling pathway activation. Mutations in isocitrate dehydrogenase (IDH) 1 also change the function of the enzymes and cause them to produce 2-hydroxyglutarate and not produce NADPH. We tested the level of NADPH and GSH and demonstrated that IDH1 R132H mutant stable cells had significantly low NADPH and GSH level compared to control or IDH1 wild type stable cells. The reduced antioxidants (NADPH and GSH) sensitized U87MG cells with IDH R132H mutant to 5-FU treatment. Our study highlights the important role of IHD1 R132H mutant in up- regulating AKT-mTOR signaling pathway and enhancing cell migration. Furthermore, we demonstrate that IDH1 R132H mutation affects cellular redox status and sensitizes gliomas cells with IDH1 R132H mutation to 5FU treatment.

Introduction of the hybcell-based compact sequencing technology and comparison to state-of-the-art methodologies for KRAS mutation detection.

PubMed

Zopf, Agnes; Raim, Roman; Danzer, Martin; Niklas, Norbert; Spilka, Rita; Pröll, Johannes; Gabriel, Christian; Nechansky, Andreas; Roucka, Markus

2015-03-01

The detection of KRAS mutations in codons 12 and 13 is critical for anti-EGFR therapy strategies; however, only those methodologies with high sensitivity, specificity, and accuracy as well as the best cost and turnaround balance are suitable for routine daily testing. Here we compared the performance of compact sequencing using the novel hybcell technology with 454 next-generation sequencing (454-NGS), Sanger sequencing, and pyrosequencing, using an evaluation panel of 35 specimens. A total of 32 mutations and 10 wild-type cases were reported using 454-NGS as the reference method. Specificity ranged from 100% for Sanger sequencing to 80% for pyrosequencing. Sanger sequencing and hybcell-based compact sequencing achieved a sensitivity of 96%, whereas pyrosequencing had a sensitivity of 88%. Accuracy was 97% for Sanger sequencing, 85% for pyrosequencing, and 94% for hybcell-based compact sequencing. Quantitative results were obtained for 454-NGS and hybcell-based compact sequencing data, resulting in a significant correlation (r = 0.914). Whereas pyrosequencing and Sanger sequencing were not able to detect multiple mutated cell clones within one tumor specimen, 454-NGS and the hybcell-based compact sequencing detected multiple mutations in two specimens. Our comparison shows that the hybcell-based compact sequencing is a valuable alternative to state-of-the-art methodologies used for detection of clinically relevant point mutations.

A mutation in the Gardos channel is associated with hereditary xerocytosis.

PubMed

Rapetti-Mauss, Raphael; Lacoste, Caroline; Picard, Véronique; Guitton, Corinne; Lombard, Elise; Loosveld, Marie; Nivaggioni, Vanessa; Dasilva, Nathalie; Salgado, David; Desvignes, Jean-Pierre; Béroud, Christophe; Viout, Patrick; Bernard, Monique; Soriani, Olivier; Vinti, Henri; Lacroze, Valérie; Feneant-Thibault, Madeleine; Thuret, Isabelle; Guizouarn, Hélène; Badens, Catherine

2015-09-10

The Gardos channel is a Ca(2+)-sensitive, intermediate conductance, potassium selective channel expressed in several tissues including erythrocytes and pancreas. In normal erythrocytes, it is involved in cell volume modification. Here, we report the identification of a dominantly inherited mutation in the Gardos channel in 2 unrelated families and its association with chronic hemolysis and dehydrated cells, also referred to as hereditary xerocytosis (HX). The affected individuals present chronic anemia that varies in severity. Their red cells exhibit a panel of various shape abnormalities such as elliptocytes, hemighosts, schizocytes, and very rare stomatocytic cells. The missense mutation concerns a highly conserved residue among species, located in the region interacting with Calmodulin and responsible for the channel opening and the K(+) efflux. Using 2-microelectrode experiments on Xenopus oocytes and patch-clamp electrophysiology on HEK293 cells, we demonstrated that the mutated channel exhibits a higher activity and a higher Ca(2+) sensitivity compared with the wild-type (WT) channel. The mutated channel remains sensitive to inhibition suggesting that treatment of this type of HX by a specific inhibitor of the Gardos channel could be considered. The identification of a KCNN4 mutation associated with chronic hemolysis constitutes the first report of a human disease caused by a defect of the Gardos channel. © 2015 by The American Society of Hematology.

Drosophila type IV collagen mutation associates with immune system activation and intestinal dysfunction.

PubMed

Kiss, Márton; Kiss, András A; Radics, Monika; Popovics, Nikoletta; Hermesz, Edit; Csiszár, Katalin; Mink, Mátyás

2016-01-01

The basal lamina (BM) contains numerous components with a predominance of type IV collagens. Clinical manifestations associated with mutations of the human COL4A1 gene include perinatal cerebral hemorrhage and porencephaly, hereditary angiopathy, nephropathy, aneurysms and muscle cramps (HANAC), ocular dysgenesis, myopathy, Walker–Warburg syndrome and systemic tissue degeneration. In Drosophila, the phenotype associated with dominant temperature sensitive mutations of col4a1 include severe myopathy resulting from massive degradation of striated muscle fibers, and in the gut, degeneration of circular visceral muscle cells and epithelial cells following detachment from the BM. In order to determine the consequences of altered BMfunctions due to aberrant COL4A1 protein, we have carried out a series of tests using Drosophila DTS-L3 mutants from our allelic series of col4a1 mutations with confirmed degeneration of various cell types and lowest survival rate among the col4a1 mutant lines at restrictive temperature. Results demonstrated epithelial cell degeneration in the gut, shortened gut, enlarged midgut with multiple diverticulae, intestinal dysfunction and shortened life span. Midgut immunohistochemistry analyses confirmed altered expression and distribution of BM components integrin PSI and PSII alpha subunits, laminin gamma 1, and COL4A1 both in larvae and adults. Global gene expression analysis revealed activation of the effector AMP genes of the primary innate immune system including Metchnikowin, Diptericin, Diptericin B, and edin that preceded morphological changes. Attacin::GFP midgut expression pattern further supported these changes. An increase in ROS production and changes in gut bacterial flora were also noted and may have further enhanced an immune response. The phenotypic features of Drosophila col4a1 mutants confirmed an essential role for type IV collagen in maintaining epithelial integrity, gut morphology and intestinal function and suggest that aberrant structure and function of the COL4A1 protein may also be a significant factor in modulating immunity.

Kdr mutations in Triatoma infestans from the Gran Chaco are distributed in two differentiated foci: Implications for pyrethroid resistance management.

PubMed

Sierra, Ivana; Capriotti, Natalia; Fronza, Georgina; Mougabure-Cueto, Gastón; Ons, Sheila

2016-06-01

Point mutations in the voltage-gated sodium channel, the primary target of pyrethroid insecticides, have been associated with the resistance in Triatoma infestans, an important vector of Chagas' disease. Hence, the sustainability of vector control programs requires the implementation of resistance management strategies. We determined the sensitivity of the molecular assays previously designed for early resistance detection to be used in pooled samples from a wide area of the endemic region, and validated them for their routine use in control campaigns for the monitoring of insecticide resistance in T. infestans. Consequently, we used these methods to examine the distribution of resistance-associated mutations in the sodium channel gene in populations of T. infestans from the Argentinean and Bolivian Gran Chaco. The PASA and REA assays tested proved sensitive enough to detect kdr SNPs in pooled samples, indicating these assays are suitable for routine screening in insecticide resistance surveillance. Two geographically differentiated foci were detected in T. infestans populations from the Argentinean and Bolivian Gran Chaco, with populations on the Bolivian-Argentinean border carrying L1014F mutation, and those from the Argentinean Chaco carrying L925I mutation. In all highly resistant populations analyzed, one of both kdr mutations was present, and toxicological assays determined that all pyrethroid resistant populations analyzed herein were sensitive to fenitrothion. The principal cause of pyrethroid resistance in T. infestans from the Gran Chaco ecoregion is kdr mutations in the sodium channel. Different levels of resistance occur in different populations carrying identical mutation, suggesting the existence of contributory mechanisms. Copyright © 2016 Elsevier B.V. All rights reserved.

Crizotinib-Resistant ROS1 Mutations Reveal a Predictive Kinase Inhibitor Sensitivity Model for ROS1- and ALK-Rearranged Lung Cancers.

PubMed

Facchinetti, Francesco; Loriot, Yohann; Kuo, Mei-Shiue; Mahjoubi, Linda; Lacroix, Ludovic; Planchard, David; Besse, Benjamin; Farace, Françoise; Auger, Nathalie; Remon, Jordi; Scoazec, Jean-Yves; André, Fabrice; Soria, Jean-Charles; Friboulet, Luc

2016-12-15

The identification of molecular mechanisms conferring resistance to tyrosine kinase inhibitor (TKI) is a key step to improve therapeutic results for patients with oncogene addiction. Several alterations leading to EGFR and anaplastic lymphoma kinase (ALK) resistance to TKI therapy have been described in non-small cell lung cancer (NSCLC). Only two mutations in the ROS1 kinase domain responsible for crizotinib resistance have been described in patients thus far. A patient suffering from a metastatic NSCLC harboring an ezrin (EZR)-ROS1 fusion gene developed acquired resistance to the ALK/ROS1 inhibitor crizotinib. Molecular analysis (whole-exome sequencing, CGH) and functional studies were undertaken to elucidate the mechanism of resistance. Based on this case, we took advantage of the structural homology of ROS1 and ALK to build a predictive model for drug sensitivity regarding future ROS1 mutations. Sequencing revealed a dual mutation, S1986Y and S1986F, in the ROS1 kinase domain. Functional in vitro studies demonstrated that ROS1 harboring either the S1986Y or the S1986F mutation, while conferring resistance to crizotinib and ceritinib, was inhibited by lorlatinib (PF-06463922). The patient's clinical response confirmed the potency of lorlatinib against S1986Y/F mutations. The ROS1 S1986Y/F and ALK C1156Y mutations are homologous and displayed similar sensitivity patterns to ALK/ROS1 TKIs. We extended this analogy to build a model predicting TKI efficacy against potential ROS1 mutations. Clinical evidence, in vitro validation, and homology-based prediction provide guidance for treatment decision making for patients with ROS1-rearranged NSCLC who progressed on crizotinib. Clin Cancer Res; 22(24); 5983-91. ©2016 AACR. ©2016 American Association for Cancer Research.

A Sensitive Detection Method for MPLW515L or MPLW515K Mutation in Chronic Myeloproliferative Disorders with Locked Nucleic Acid-Modified Probes and Real-Time Polymerase Chain Reaction

PubMed Central

Pancrazzi, Alessandro; Guglielmelli, Paola; Ponziani, Vanessa; Bergamaschi, Gaetano; Bosi, Alberto; Barosi, Giovanni; Vannucchi, Alessandro M.

2008-01-01

Acquired mutations in the juxtamembrane region of MPL (W515K or W515L), the receptor for thrombopoietin, have been described in patients with primary myelofibrosis or essential thrombocythemia, which are chronic myeloproliferative disorders. We have developed a real-time polymerase chain reaction assay for the detection and quantification of MPL mutations that is based on locked nucleic acid fluorescent probes. Mutational analysis was performed using DNA from granulocytes. Reference curves were obtained using cloned fragments of MPL containing either the wild-type or mutated sequence; the predicted sensitivity level was at least 0.1% mutant allele in a wild-type background. None of the 60 control subjects presented with a MPLW515L/K mutation. Of 217 patients with myelofibrosis, 19 (8.7%) harbored the MPLW515 mutation, 10 (52.6%) with the W515L allele. In one case, both the W515L and W515K alleles were detected by real-time polymerase chain reaction. By comparing results obtained with conventional sequencing, no erroneous genotype attribution using real-time polymerase chain reaction was found, whereas one patient considered wild type according to sequence analysis actually harbored a low W515L allele burden. This is a simple, sensitive, and cost-effective procedure for large-scale screening of the MPLW515L/K mutation in patients suspected to have a myeloproliferative disorder. It can also provide a quantitative estimate of mutant allele burden that might be useful for both patient prognosis and monitoring response to therapy. PMID:18669880

Assessment of cellularity, genomic DNA yields, and technical platforms for BRAF mutational testing in thyroid fine-needle aspirate samples.

PubMed

Dyhdalo, Kathryn; Macnamara, Stephen; Brainard, Jennifer; Underwood, Dawn; Tubbs, Raymond; Yang, Bin

2014-02-01

BRAF mutation V600E (substitution Val600Glu) is a molecular signature for papillary thyroid carcinoma (PTC). Testing for BRAF mutation is clinically useful in providing prognostic prediction and facilitating accurate diagnosis of PTC in thyroid fine-needle aspirate (FNA) samples. This study assessed the correlation of cellularity with DNA yield and compared 2 technical platforms with different sensitivities in detection of BRAF mutation in cytologic specimens. Cellularity was evaluated based on groups of 10+ cells on a ThinPrep slide: 1+ (1-5 groups), 2+ (6-10 groups), 3+ (11-20 groups), and 4+ (> 20 groups). Genomic DNA was extracted from residual materials of thyroid FNAs after cytologic diagnosis. Approximately 49% of thyroid FNA samples had low cellularity (1-2+). DNA yield is proportionate with increased cellularity and increased nearly 4-fold from 1+ to 4+ cellularity in cytologic samples. When applied to BRAF mutational assay, using a cutoff of 6 groups of follicular cells with 10+ cells per group, 96.7% of cases yielded enough DNA for at least one testing for BRAF mutation. Five specimens (11.6%) with lower cellularity did not yield sufficient DNA for duplicate testing. Comparison of Sanger sequencing to allele-specific polymerase chain reaction methods shows the latter confers better sensitivity in detection of BRAF mutation, especially in limited cytologic specimens with a lower percentage of malignant cells. This study demonstrates that by using 6 groups of 10+ follicular cells as a cutoff, nearly 97% of thyroid FNA samples contain enough DNA for BRAF mutational assay. Careful selection of a molecular testing system with high sensitivity facilitates the successful conduction of molecular testing in limited cytologic specimens. Cancer (Cancer Cytopathol) 2014;122:114-22 © 2013 American Cancer Society. © 2013 American Cancer Society.

Extreme assay sensitivity in molecular diagnostics further unveils intratumour heterogeneity in metastatic colorectal cancer as well as artifactual low-frequency mutations in the KRAS gene.

PubMed

Mariani, Sara; Bertero, Luca; Osella-Abate, Simona; Di Bello, Cristiana; Francia di Celle, Paola; Coppola, Vittoria; Sapino, Anna; Cassoni, Paola; Marchiò, Caterina

2017-07-25

Gene mutations in the RAS family rule out metastatic colorectal carcinomas (mCRCs) from anti-EGFR therapies. We report a retrospective analysis by Sequenom Massarray and fast COLD-PCR followed by Sanger sequencing on 240 mCRCs. By Sequenom, KRAS and NRAS exons 2-3-4 were mutated in 52.9% (127/240) of tumours, while BRAF codon 600 mutations reached 5% (12/240). Fast COLD-PCR found extra mutations at KRAS exon 2 in 15/166 (9%) of samples, previously diagnosed by Sequenom as wild-type or mutated at RAS (exons 3-4) or BRAF genes. After UDG digestion results were reproduced in 2/12 analysable subclonally mutated samples leading to a frequency of true subclonal KRAS mutations of 1.2% (2.1% of the previous Sequenom wild-type subgroup). In 10 out of 12 samples, the subclonal KRAS mutations disappeared (9 out of 12) or turned to a different sequence variant (1 out of 12). mCRC can harbour coexisting multiple gene mutations. High sensitivity assays allow the detection of a small subset of patients harbouring true subclonal KRAS mutations. However, DNA changes with mutant allele frequencies <3% detected in formalin-fixed paraffin-embedded samples may be artifactual in a non-negligible fraction of cases. UDG pre-treatment of DNA is mandatory to identify true DNA changes in archival samples and avoid misinterpretation due to artifacts.

Extreme assay sensitivity in molecular diagnostics further unveils intratumour heterogeneity in metastatic colorectal cancer as well as artifactual low-frequency mutations in the KRAS gene

PubMed Central

Mariani, Sara; Bertero, Luca; Osella-Abate, Simona; Di Bello, Cristiana; Francia di Celle, Paola; Coppola, Vittoria; Sapino, Anna; Cassoni, Paola; Marchiò, Caterina

2017-01-01

Background: Gene mutations in the RAS family rule out metastatic colorectal carcinomas (mCRCs) from anti-EGFR therapies. Methods: We report a retrospective analysis by Sequenom Massarray and fast COLD-PCR followed by Sanger sequencing on 240 mCRCs. Results: By Sequenom, KRAS and NRAS exons 2-3-4 were mutated in 52.9% (127/240) of tumours, while BRAF codon 600 mutations reached 5% (12/240). Fast COLD-PCR found extra mutations at KRAS exon 2 in 15/166 (9%) of samples, previously diagnosed by Sequenom as wild-type or mutated at RAS (exons 3-4) or BRAF genes. After UDG digestion results were reproduced in 2/12 analysable subclonally mutated samples leading to a frequency of true subclonal KRAS mutations of 1.2% (2.1% of the previous Sequenom wild-type subgroup). In 10 out of 12 samples, the subclonal KRAS mutations disappeared (9 out of 12) or turned to a different sequence variant (1 out of 12). Conclusions: mCRC can harbour coexisting multiple gene mutations. High sensitivity assays allow the detection of a small subset of patients harbouring true subclonal KRAS mutations. However, DNA changes with mutant allele frequencies <3% detected in formalin-fixed paraffin-embedded samples may be artifactual in a non-negligible fraction of cases. UDG pre-treatment of DNA is mandatory to identify true DNA changes in archival samples and avoid misinterpretation due to artifacts. PMID:28618430

[Analysis of EML4-ALK gene fusion mutation in patients 
with non-small cell lung cancer].

PubMed

Wang, Xuzhou; Chen, Weisheng; Yu, Yinghao

2015-02-01

Non-small cell lung cancer (NSCLC) is the main type of lung cancer, and the related locus mutation detection research has become a hot direction of molecular targeted therapy, studying on gene mutation status of echinodem microtubule associated protein like 4-Anaplastic lymphoma kinase (EML4-ALK) and epidermal growth factor receptor (EGFR), detecting the sensitivity of EML4-ALK gene fusion and gene mutation of EGFR. EML4-ALK gene fusion in 85 cases of paraffin embedded tumor tissue and adjacent lung tissue was detected with the application of immunohistochemistry (IHC), Scorpions amplification refractory mutation system (Scorpions ARMS) fluorescence quantitative PCR and fluorescence in situ hybridization (FISH) technology, and EGFR gene in 18, 19, 20 and 21 exon mutation status was detected with the application of ARMS method. In 115 cases of NSCLC, IHC showed 32 cases with ALK (D5F3) expression, the expression rate was 27.8%; ARMS showed 27 cases with EML4-ALK fusion gene mutation, the mutation detection rate was 23.5%; 53 cases were detected with EGFR mutation, the mutation rate was 46%. While FISH showed 23 cases with EML4-ALK fusion gene mutation, the detection rate was 20%, slightly lower than the ARMS detection results, suggesting that ARMS more sensitive. The application of IHC, ARMS fluorescence quantitative PCR and FISH technology can make a rapid and accurate evaluation of EML4-ALK gene fusion.

Simple, Sensitive and Accurate Multiplex Detection of Clinically Important Melanoma DNA Mutations in Circulating Tumour DNA with SERS Nanotags

PubMed Central

Wee, Eugene J.H.; Wang, Yuling; Tsao, Simon Chang-Hao; Trau, Matt

2016-01-01

Sensitive and accurate identification of specific DNA mutations can influence clinical decisions. However accurate diagnosis from limiting samples such as circulating tumour DNA (ctDNA) is challenging. Current approaches based on fluorescence such as quantitative PCR (qPCR) and more recently, droplet digital PCR (ddPCR) have limitations in multiplex detection, sensitivity and the need for expensive specialized equipment. Herein we describe an assay capitalizing on the multiplexing and sensitivity benefits of surface-enhanced Raman spectroscopy (SERS) with the simplicity of standard PCR to address the limitations of current approaches. This proof-of-concept method could reproducibly detect as few as 0.1% (10 copies, CV < 9%) of target sequences thus demonstrating the high sensitivity of the method. The method was then applied to specifically detect three important melanoma mutations in multiplex. Finally, the PCR/SERS assay was used to genotype cell lines and ctDNA from serum samples where results subsequently validated with ddPCR. With ddPCR-like sensitivity and accuracy yet at the convenience of standard PCR, we believe this multiplex PCR/SERS method could find wide applications in both diagnostics and research. PMID:27446486

Simple, Sensitive and Accurate Multiplex Detection of Clinically Important Melanoma DNA Mutations in Circulating Tumour DNA with SERS Nanotags.

PubMed

Wee, Eugene J H; Wang, Yuling; Tsao, Simon Chang-Hao; Trau, Matt

2016-01-01

Sensitive and accurate identification of specific DNA mutations can influence clinical decisions. However accurate diagnosis from limiting samples such as circulating tumour DNA (ctDNA) is challenging. Current approaches based on fluorescence such as quantitative PCR (qPCR) and more recently, droplet digital PCR (ddPCR) have limitations in multiplex detection, sensitivity and the need for expensive specialized equipment. Herein we describe an assay capitalizing on the multiplexing and sensitivity benefits of surface-enhanced Raman spectroscopy (SERS) with the simplicity of standard PCR to address the limitations of current approaches. This proof-of-concept method could reproducibly detect as few as 0.1% (10 copies, CV < 9%) of target sequences thus demonstrating the high sensitivity of the method. The method was then applied to specifically detect three important melanoma mutations in multiplex. Finally, the PCR/SERS assay was used to genotype cell lines and ctDNA from serum samples where results subsequently validated with ddPCR. With ddPCR-like sensitivity and accuracy yet at the convenience of standard PCR, we believe this multiplex PCR/SERS method could find wide applications in both diagnostics and research.

Impact of increased mutagenesis on adaptation to high temperature in bacteriophage Qβ.

PubMed

Arribas, María; Cabanillas, Laura; Kubota, Kirina; Lázaro, Ester

2016-10-01

RNA viruses replicate with very high error rates, which makes them more sensitive to additional increases in this parameter. This fact has inspired an antiviral strategy named lethal mutagenesis, which is based on the artificial increase of the error rate above a threshold incompatible with virus infectivity. A relevant issue concerning lethal mutagenesis is whether incomplete treatments might enhance the adaptive possibilities of viruses. We have addressed this question by subjecting an RNA virus, the bacteriophage Qβ, to different transmission regimes in the presence or the absence of sublethal concentrations of the mutagenic nucleoside analogue 5-azacytidine (AZC). Populations obtained were subsequently exposed to a non-optimal temperature and analyzed to determine their consensus sequences. Our results show that previously mutagenized populations rapidly fixed a specific set of mutations upon propagation at the new temperature, suggesting that the expansion of the mutant spectrum caused by AZC has an influence on later evolutionary behavior. Copyright © 2016 Elsevier Inc. All rights reserved.

Evolution of Escherichia coli to 42 °C and subsequent genetic engineering reveals adaptive mechanisms and novel mutations.

PubMed

Sandberg, Troy E; Pedersen, Margit; LaCroix, Ryan A; Ebrahim, Ali; Bonde, Mads; Herrgard, Markus J; Palsson, Bernhard O; Sommer, Morten; Feist, Adam M

2014-10-01

Adaptive laboratory evolution (ALE) has emerged as a valuable method by which to investigate microbial adaptation to a desired environment. Here, we performed ALE to 42 °C of ten parallel populations of Escherichia coli K-12 MG1655 grown in glucose minimal media. Tightly controlled experimental conditions allowed selection based on exponential-phase growth rate, yielding strains that uniformly converged toward a similar phenotype along distinct genetic paths. Adapted strains possessed as few as 6 and as many as 55 mutations, and of the 144 genes that mutated in total, 14 arose independently across two or more strains. This mutational recurrence pointed to the key genetic targets underlying the evolved fitness increase. Genome engineering was used to introduce the novel ALE-acquired alleles in random combinations into the ancestral strain, and competition between these engineered strains reaffirmed the impact of the key mutations on the growth rate at 42 °C. Interestingly, most of the identified key gene targets differed significantly from those found in similar temperature adaptation studies, highlighting the sensitivity of genetic evolution to experimental conditions and ancestral genotype. Additionally, transcriptomic analysis of the ancestral and evolved strains revealed a general trend for restoration of the global expression state back toward preheat stressed levels. This restorative effect was previously documented following evolution to metabolic perturbations, and thus may represent a general feature of ALE experiments. The widespread evolved expression shifts were enabled by a comparatively scant number of regulatory mutations, providing a net fitness benefit but causing suboptimal expression levels for certain genes, such as those governing flagellar formation, which then became targets for additional ameliorating mutations. Overall, the results of this study provide insight into the adaptation process and yield lessons important for the future implementation of ALE as a tool for scientific research and engineering. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Genetic stability of Rift Valley fever virus MP-12 vaccine during serial passages in culture cells.

PubMed

Lokugamage, Nandadeva; Ikegami, Tetsuro

2017-01-01

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa which affects both ruminants and humans. RVF causes serious damage to the livestock industry and is also a threat to public health. The Rift Valley fever virus has a segmented negative-stranded RNA genome consisting of Large (L)-, Medium (M)-, and Small (S)-segments. The live-attenuated MP-12 vaccine is immunogenic in livestock and humans, and is conditionally licensed for veterinary use in the U.S. The MP-12 strain encodes 23 mutations (nine amino acid substitutions) and is attenuated through a combination of mutations in the L-, M-, and S-segments. Among them, the M-U795C, M-A3564G, and L-G3104A mutations contribute to viral attenuation through the L- and M-segments. The M-U795C, M-A3564G, L-U533C, and L-G3750A mutations are also independently responsible for temperature-sensitive (ts) phenotype. We hypothesized that a serial passage of the MP-12 vaccine in culture cells causes reversions of the MP-12 genome. The MP-12 vaccine and recombinant rMP12-ΔNSs16/198 were serially passaged 25 times. Droplet digital PCR analysis revealed that the reversion occurred at L-G3750A during passages of MP-12 in Vero or MRC-5 cells. The reversion also occurred at M-A3564G and L-U533C of rMP12-ΔNSs16/198 in Vero cells. Reversion mutations were not found in MP-12 or the variant, rMP12-TOSNSs, in the brains of mice with encephalitis. This study characterized genetic stability of the MP-12 vaccine and the potential risk of reversion mutation at the L-G3750A ts mutation after excessive viral passages in culture cells.

Highly sensitive and quantitative evaluation of the EGFR T790M mutation by nanofluidic digital PCR

PubMed Central

Iwama, Eiji; Takayama, Koichi; Harada, Taishi; Okamoto, Isamu; Ookubo, Fumihiko; Kishimoto, Junji; Baba, Eishi; Oda, Yoshinao; Nakanishi, Yoichi

2015-01-01

The mutation of T790M in EGFR is a major mechanism of resistance to treatment with EGFR-TKIs. Only qualitative detection (presence or absence) of T790M has been described to date, however. Digital PCR (dPCR) analysis has recently been applied to the quantitative detection of target molecules in cancer with high sensitivity. In the present study, 25 tumor samples (13 obtained before and 12 after EGFR-TKI treatment) from 18 NSCLC patients with activating EGFR mutations were evaluated for T790M with dPCR. The ratio of the number of T790M alleles to that of activating mutation alleles (T/A) was determined. dPCR detected T790M in all 25 samples. Although T790M was present in all pre-TKI samples from 13 patients, 10 of these patients had a low T/A ratio and manifested substantial tumor shrinkage during treatment with EGFR-TKIs. In six of seven patients for whom both pre- and post-TKI samples were available, the T/A ratio increased markedly during EGFR-TKI treatment. Highly sensitive dPCR thus detected T790M in all NSCLC patients harboring activating EGFR mutations whether or not they had received EGFR-TKI treatment. Not only highly sensitive but also quantitative detection of T790M is important for evaluation of the contribution of T790M to EGFR-TKI resistance. PMID:26015401

The directed migration of gonadal distal tip cells in Caenorhabditis elegans requires NGAT-1, a ß1,4-N-acetylgalactosaminyltransferase enzyme

PubMed Central

Veyhl, Joseph; Dunn, Robert J.; Johnston, Wendy L.; Bennett, Alexa; Zhang, Lijia W.; Dennis, James W.; Schachter, Harry

2017-01-01

Glycoproteins such as growth factor receptors and extracellular matrix have well-known functions in development and cancer progression, however, the glycans at sites of modification are often heterogeneous molecular populations which makes their functional characterization challenging. Here we provide evidence for a specific, discrete, well-defined glycan modification and regulation of a stage-specific cell migration in Caenorhabditis elegans. We show that a chain-terminating, putative null mutation in the gene encoding a predicted β1,4-N-acetylgalactosaminyltransferase, named ngat-1, causes a maternally rescued temperature sensitive (ts) defect in the second phase of the three phase migration pattern of the posterior, but not the anterior, hermaphrodite Distal Tip Cell (DTC). An amino-terminal partial deletion of ngat-1 causes a similar but lower penetrance ts phenotype. The existence of multiple ts alleles with distinctly different molecular DNA lesions, neither of which is likely to encode a ts protein, indicates that NGAT-1 normally prevents innate temperature sensitivity for phase 2 DTC pathfinding. Temperature shift analyses indicate that the ts period for the ngat-1 mutant defect ends by the beginning of post-embryonic development–nearly 3 full larval stages prior to the defective phase 2 migration affected by ngat-1 mutations. NGAT-1 homologs generate glycan-terminal GalNAc-β1-4GlcNAc, referred to as LacdiNAc modifications, on glycoproteins and glycolipids. We also found that the absence of the GnT1/Mgat1 activity [UDP-N-acetyl-D-glucosamine:α-3-D-mannoside β-1,2-N-acetylglucosaminyltransferase 1 (encoded by C. elegans gly-12, gly-13, and gly-14 and homologous to vertebrate GnT1/Mgat1)], causes a similar spectrum of DTC phenotypes as ngat-1 mutations–primarily affecting posterior DTC phase 2 migration and preventing manifestation of the same innate ts period as ngat-1. GnT1/Mgat1 is a medial Golgi enzyme known to modify mannose residues and initiate N-glycan branching, an essential step in the biosynthesis of hybrid, paucimannose and complex-type N-glycans. Quadruple mutant animals bearing putative null mutations in ngat-1 and the three GnT genes (gly-12, gly-13, gly-14) were not enhanced for DTC migration defects, suggesting NGAT-1 and GnT1 act in the same pathway. These findings suggest that GnTI generates an N-glycan substrate for NGAT-1 modification, which is required at restrictive temperature (25°C) to prevent, stabilize, reverse or compensate a perinatal thermo-labile process (or structure) causing late larval stage DTC phase 2 migration errors. PMID:28817611

A yeast gene essential for regulation of spindle pole duplication.

PubMed Central

Baum, P; Yip, C; Goetsch, L; Byers, B

1988-01-01

In eucaryotic cells, duplication of spindle poles must be coordinated with other cell cycle functions. We report here the identification in Saccharomyces cerevisiae of a temperature-sensitive lethal mutation, esp1, that deregulates spindle pole duplication. Mutant cells transferred to the nonpermissive temperature became unable to continue DNA synthesis and cell division but displayed repeated duplication of their spindle pole bodies. Although entry into this state after transient challenge by the nonpermissive temperature was largely lethal, rare survivors were recovered and found to have become increased in ploidy. If the mutant cells were held in G0 or G1 during exposure to the elevated temperature, they remained viable and maintained normal numbers of spindle poles. These results suggest dual regulation of spindle pole duplication, including a mechanism that promotes duplication as cells enter the division cycle and a negative regulatory mechanism, controlled by ESP1, that limits duplication to a single occurrence in each cell division cycle. Tetrad analysis has revealed that ESP1 resides at a previously undescribed locus on the right arm of chromosome VII. Images PMID:3072479

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

PubMed

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

2012-01-01

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

Mechanism behind Resistance against the Organophosphate Azamethiphos in Salmon Lice (Lepeophtheirus salmonis)

PubMed Central

Kaur, Kiranpreet; Helgesen, Kari Olli; Bakke, Marit Jørgensen; Horsberg, Tor Einar

2015-01-01

Acetylcholinesterase (AChE) is the primary target for organophosphates (OP). Several mutations have been reported in AChE to be associated with the reduced sensitivity against OP in various arthropods. However, to the best of our knowledge, no such reports are available for Lepeophtheirus salmonis. Hence, in the present study, we aimed to determine the association of AChE(s) gene(s) with resistance against OP. We screened the AChE genes (L. salmonis ace1a and ace1b) in two salmon lice populations: one sensitive (n=5) and the other resistant (n=5) for azamethiphos, a commonly used OP in salmon farming. The screening led to the identification of a missense mutation Phe362Tyr in L. salmonis ace1a, (corresponding to Phe331 in Torpedo californica AChE) in all the samples of the resistant population. We confirmed the potential role of the mutation, with reduced sensitivity against azamethiphos in L. salmonis, by screening for Phe362Tyr in 2 sensitive and 5 resistant strains. The significantly higher frequency of the mutant allele (362Tyr) in the resistant strains clearly indicated the possible association of Phe362Tyr mutation in L. salmonis ace1a with resistance towards azamethiphos. The 3D modelling, short term survival experiments and enzymatic assays further supported the imperative role of Phe362Tyr in reduced sensitivity of L. salmonis for azamethiphos. Based on all these observations, the present study, for the first time, presents the mechanism of resistance in L. salmonis against azamethiphos. In addition, we developed a rapid diagnostic tool for the high throughput screening of Phe362Tyr mutation using High Resolution Melt analysis. PMID:25893248

Imaging work-up for screening of paraganglioma and pheochromocytoma in SDHx mutation carriers: a multicenter prospective study from the PGL.EVA Investigators.

PubMed

Gimenez-Roqueplo, Anne-Paule; Caumont-Prim, Aurore; Houzard, Claire; Hignette, Chantal; Hernigou, Anne; Halimi, Philippe; Niccoli, Patricia; Leboulleux, Sophie; Amar, Laurence; Borson-Chazot, Françoise; Cardot-Bauters, Catherine; Delemer, Brigitte; Chabolle, Frédéric; Coupier, Isabelle; Libé, Rossella; Peitzsch, Mirko; Peyrard, Séverine; Tenenbaum, Florence; Plouin, Pierre-François; Chatellier, Gilles; Rohmer, Vincent

2013-01-01

Recommendations have not been established concerning imaging to screen SDHx mutation carriers for paraganglioma and pheochromocytoma. Our objective was to compare the performance of gadolinium-enhanced magnetic resonance angiography, contrast-enhanced computed tomography, and [(123)I]metaiodo-benzylguanidine and somatostatin receptor scintigraphies for detecting head and neck and thoracic-abdominal-pelvic paragangliomas in SDHx mutation carriers. We conducted a prospective, multicenter study from June 2005 to December 2009 at 23 French medical centers. A total of 238 index cases or relatives carrying mutations in SDHD, SDHB, or SDHC genes were included. Images obtained by each technique were analyzed blind, without knowledge of results from other tests, first in each local center and then centrally. We evaluated sensitivity, specificity, and likelihood ratios for individual and combinations of tests, the gold standard being the consensus of an expert committee. Two hundred two tumors were diagnosed in 96 subjects. At local assessment, the sensitivity of anatomical imaging for detecting all tumors was higher (85.7%) than that of both scintigraphic techniques (42.7% for [(123)I]metaiodo-benzylguanidine and 69.5% for somatostatin receptor scintigraphy), except for thoracic localizations where somatostatin receptor scintigraphy was more sensitive (61.5 vs. 46.2% for anatomical imaging and 30.8% for [(123)I]metaiodo-benzylguanidine scintigraphy). The best diagnostic performance during local assessment was obtained by combining anatomical imaging tests and somatostatin receptor scintigraphy (sensitivity 91.7%). Central assessment significantly increased the sensitivity (98.6%) of tests in combination. In routine practice, the imaging work-up for screening SDHx mutation carriers should include thoraco-abdomino-pelvic computed tomography, head and neck magnetic angiography, and somatostatin receptor scintigraphy. Expert centralized image assessment is recommended.

Baseline clinical predictors of antitumor response to the PARP inhibitor olaparib in germline BRCA1/2 mutated patients with advanced ovarian cancer.

PubMed

Rafii, Saeed; Gourley, Charlie; Kumar, Rajiv; Geuna, Elena; Ern Ang, Joo; Rye, Tzyvia; Chen, Lee-May; Shapira-Frommer, Ronnie; Friedlander, Michael; Matulonis, Ursula; De Greve, Jacques; Oza, Amit M; Banerjee, Susana; Molife, L Rhoda; Gore, Martin E; Kaye, Stan B; Yap, Timothy A

2017-07-18

The PARP inhibitor olaparib was recently granted Food and Drug Administration (FDA) accelerated approval in patients with advanced BRCA1/2 mutation ovarian cancer. However, antitumor responses are observed in only approximately 40% of patients and the impact of baseline clinical factors on response to treatment remains unclear. Although platinum sensitivity has been suggested as a marker of response to PARP inhibitors, patients with platinum-resistant disease still respond to olaparib. 108 patients with advanced BRCA1/2 mutation ovarian cancers were included. The interval between the end of the most recent platinum chemotherapy and PARPi (PTPI) was used to predict response to olaparib independent of conventional definition of platinum sensitivity. RECIST complete response (CR) and partial response (PR) rates were 35% in patients with platinum-sensitive versus 13% in platinum-resistant (p<0.005). Independent of platinum sensitivity status, the RECIST CR/PR rates were 42% in patients with PTPI greater than 52 weeks and 18% in patients with PTPI less than 52 weeks (p=0.016). No association was found between baseline clinical factors such as FIGO staging, debulking surgery, BRCA1 versus BRCA2 mutations, prior history of breast cancer and prior chemotherapy for breast cancer, and the response to olaparib. We conducted an international multicenter retrospective study to investigate the association between baseline clinical characteristics of patients with advanced BRCA1/2 mutation ovarian cancers from eight different cancer centers and their antitumor response to olaparib. PTPI may be used to refine the prediction of response to PARP inhibition based on the conventional categorization of platinum sensitivity.

Using high-sensitivity sequencing for the detection of mutations in BTK and PLCγ2 genes in cellular and cell-free DNA and correlation with progression in patients treated with BTK inhibitors.

PubMed

Albitar, Adam; Ma, Wanlong; DeDios, Ivan; Estella, Jeffrey; Ahn, Inhye; Farooqui, Mohammed; Wiestner, Adrian; Albitar, Maher

2017-03-14

Patients with chronic lymphocytic leukemia (CLL) that develop resistance to Bruton tyrosine kinase (BTK) inhibitors are typically positive for mutations in BTK or phospholipase c gamma 2 (PLCγ2). We developed a high sensitivity (HS) assay utilizing wild-type blocking polymerase chain reaction achieved via bridged and locked nucleic acids. We used this high sensitivity assay in combination with Sanger sequencing and next generation sequencing (NGS) and tested cellular DNA and cell-free DNA (cfDNA) from patients with CLL treated with the BTK inhibitor, ibrutinib. We also tested ibrutinib-naïve patients with CLL. HS testing achieved 100x greater sensitivity than Sanger. HS Sanger sequencing was capable of detecting < 1 mutant allele in background of 1000 wild-type alleles (1:1000). Similar sensitivity was achieved with HS NGS. No BTK or PLCγ2 mutations were detected in any of the 44 ibrutinib-naïve CLL patients. We demonstrate that without the HS testing 56% of positive samples would have been missed for BTK and 85% of PLCγ2 would have been missed. With the use of HS, we were able to detect multiple mutant clones in the same sample in 37.5% of patients; most would have been missed without HS testing. We also demonstrate that with HS sequencing, plasma cfDNA is more reliable than cellular DNA in detecting mutations. Our studies indicate that wild-type blocking and HS sequencing is necessary for proper and early detection of BTK or PLCγ2 mutations in monitoring patients treated with BTK inhibitors. Furthermore, cfDNA from plasma is very reliable sample-type for testing.

Neonatal Diabetes Caused by Mutations in Sulfonylurea Receptor 1: Interplay between Expression and Mg-Nucleotide Gating Defects of ATP-Sensitive Potassium Channels

PubMed Central

Zhou, Qing; Garin, Intza; Castaño, Luis; Argente, Jesús; Muñoz-Calvo, Ma. Teresa; Perez de Nanclares, Guiomar; Shyng, Show-Ling

2010-01-01

Context: ATP-sensitive potassium (KATP) channels regulate insulin secretion by coupling glucose metabolism to β-cell membrane potential. Gain-of-function mutations in the sulfonylurea receptor 1 (SUR1) or Kir6.2 channel subunit underlie neonatal diabetes. Objective: The objective of the study was to determine the mechanisms by which two SUR1 mutations, E208K and V324M, associated with transient neonatal diabetes affect KATP channel function. Design: E208K or V324M mutant SUR1 was coexpressed with Kir6.2 in COS cells, and expression and gating properties of the resulting channels were assessed biochemically and electrophysiologically. Results: Both E208K and V324M augment channel response to MgADP stimulation without altering sensitivity to ATP4− or sulfonylureas. Surprisingly, whereas E208K causes only a small increase in MgADP response consistent with the mild transient diabetes phenotype, V324M causes a severe activating gating defect. Unlike E208K, V324M also impairs channel expression at the cell surface, which is expected to dampen its functional impact on β-cells. When either mutation was combined with a mutation in the second nucleotide binding domain of SUR1 previously shown to abolish Mg-nucleotide response, the activating effect of E208K and V324M was also abolished. Moreover, combination of E208K and V324M results in channels with Mg-nucleotide sensitivity greater than that seen in individual mutations alone. Conclusion: The results demonstrate that E208K and V324M, located in distinct domains of SUR1, enhance transduction of Mg-nucleotide stimulation from the SUR1 nucleotide binding folds to Kir6.2. Furthermore, they suggest that diabetes severity is determined by interplay between effects of a mutation on channel expression and channel gating. PMID:20810569

Inherited macular degeneration-associated mutations in CNGB3 increase the ligand sensitivity and spontaneous open probability of cone cyclic nucleotide-gated channels

PubMed Central

Meighan, Peter C.; Peng, Changhong; Varnum, Michael D.

2015-01-01

Cyclic nucleotide gated (CNG) channels are a critical component of the visual transduction cascade in the vertebrate retina. Mutations in the genes encoding these channels have been associated with a spectrum of inherited retinal disorders. To gain insight into their pathophysiological mechanisms, we have investigated the functional consequences of several CNGB3 mutations, previously associated with macular degeneration (Y469D and L595F) or complete achromatopsia (S156F, P309L, and G558C), by expressing these subunits in combination with wild-type CNGA3 in Xenopus oocytes and characterizing them using patch-clamp recordings in the inside-out configuration. These mutations did not prevent the formation of functional heteromeric channels, as indicated by sensitivity to block by L-cis-diltiazem. With the exception of S156F, each of the mutant channels displayed electrophysiological properties reflecting enhanced channel activity at physiological concentrations of cGMP (i.e., a gain-of-function phenotype). The increased channel activity produced by these mutations resulted from either increased functional expression levels, or increased sensitivity to cyclic nucleotides. Furthermore, L595F increased the spontaneous open probability in the absence of activating ligand, signifying a ligand independent gain-of-function change. In addition to the CNGB3 disease-associate mutations, we characterized the effects of several common CNGB3 and CNGA3 single-nucleotide polymorphisms (SNPs) on heteromeric CNGA3+CNGB3 channel function. Two of the SNPs examined (A3-T153M, and B3-W234C) produced decreased ligand sensitivity for heteromeric CNG channels. These changes may contribute to background disease susceptibility when combined with other genetic or non-genetic factors. Together, these studies help to define the underlying molecular phenotype for mutations relating to CNG channel disease pathogenesis. PMID:26106334

Evolving ecological networks and the emergence of biodiversity patterns across temperature gradients.

PubMed

Stegen, James C; Ferriere, Regis; Enquist, Brian J

2012-03-22

In ectothermic organisms, it is hypothesized that metabolic rates mediate influences of temperature on the ecological and evolutionary processes governing biodiversity. However, it is unclear how and to what extent the influence of temperature on metabolism scales up to shape large-scale diversity patterns. In order to clarify the roles of temperature and metabolism, new theory is needed. Here, we establish such theory and model eco-evolutionary dynamics of trophic networks along a broad temperature gradient. In the model temperature can influence, via metabolism, resource supply, consumers' vital rates and mutation rate. Mutation causes heritable variation in consumer body size, which diversifies and governs consumer function in the ecological network. The model predicts diversity to increase with temperature if resource supply is temperature-dependent, whereas temperature-dependent consumer vital rates cause diversity to decrease with increasing temperature. When combining both thermal dependencies, a unimodal temperature-diversity pattern evolves, which is reinforced by temperature-dependent mutation rate. Studying coexistence criteria for two consumers showed that these outcomes are owing to temperature effects on mutual invasibility and facilitation. Our theory shows how and why metabolism can influence diversity, generates predictions useful for understanding biodiversity gradients and represents an extendable framework that could include factors such as colonization history and niche conservatism.

KRAS mutation testing in colorectal cancer: comparison of the results obtained using 3 different methods for the analysis of codons G12 and G13.

PubMed

Bihl, Michel P; Hoeller, Sylvia; Andreozzi, Maria Carla; Foerster, Anja; Rufle, Alexander; Tornillo, Luigi; Terracciano, Luigi

2012-03-01

Targeting the epidermal growth factor receptor (EGFR) is a new therapeutic option for patients with metastatic colorectal or lung carcinoma. However, the therapy efficiency highly depends on the KRAS mutation status in the given tumour. Therefore a reliable and secure KRAS mutation testing is crucial. Here we investigated 100 colorectal carcinoma samples with known KRAS mutation status (62 mutated cases and 38 wild type cases) in a comparative manner with three different KRAS mutation testing techniques (Pyrosequencing, Dideoxysequencing and INFINITI) in order to test their reliability and sensitivity. For the large majority of samples (96/100, 96%), the KRAS mutation status obtained by all three methods was the same. Only two cases with clear discrepancies were observed. One case was reported as wild type by the INFINITI method while the two other methods detected a G13C mutation. In the second case the mutation could be detected by the Pyrosequencing and INFINITI method (15% and 15%), while no signal for mutation could be observed with the Dideoxysequencing method. Additional two unclear results were due to a detection of a G12V with the INFINITI method, which was below cut-off when repeated and which was not detectable by the other two methods and very weak signals in a G12V mutated case with the Dideoxy- and Pyroseqencing method compared to the INFINITI method, respectively. In summary all three methods are reliable and robust methods in detecting KRAS mutations. INFINITI, however seems to be slightly more sensitive compared to Dideoxy- and Pyrosequencing.

The genetic spectrum and the evaluation of CADASIL screening scale in Chinese patients with NOTCH3 mutations.

PubMed

Liu, Xiao; Zuo, Yuehuan; Sun, Wei; Zhang, Wei; Lv, He; Huang, Yining; Xiao, Jiangxi; Yuan, Yun; Wang, Zhaoxia

2015-07-15

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited small artery disease caused by NOTCH3 gene mutation. Here we report clinical, pathological and genetic profiles of 29 newly-diagnosed CADASIL patients, evaluation of the CADASIL scale in Chinese CADASIL patients, and reanalysis of all reported mainland Chinese patients with identified NOTCH3 gene mutation. We found two novel mutations (p.C134G and p.C291Y) and 13 reported NOTCH3 mutations in the newly-diagnosed group. CADASIL scale score was less than the cutoff score in 19 of 53 Chinese patients with NOTCH3 mutation, generating only a sensitivity of 64.1%. At the time of study, the total number of genetically confirmed CADASIL cases reached 158 from 97 unrelated mainland Chinese families, with 9/97 (9.3%) sporadic patients. The NOTCH3 gene mutation profile showed 43 mutations, with hotspots in exon 4, followed by exon 3. The considerable variability in onset age and CADASIL scale score in patients carrying the same NOTCH3 missense mutation suggested no obvious phenotype-genotype correlation. In conclusion, we report two novel mutations which expand the NOTCH3 mutational spectrum. Exons 4 and 3 are hotspots in mainland Chinese patients with NOTCH3 mutation. The low sensitivity of CADASIL scale in our patients group indicated that the CADASIL scale should be refined according to the clinical characteristics of Chinese CADASIL patients when used in Chinese populations. Copyright © 2015. Published by Elsevier B.V.

A rapid, highly accurate method for quantifying CALR mutant allele burden in persons with myeloproliferative neoplasms.

PubMed

Yao, Qiu-Mei; Zhou, Jiao; Gale, Robert Peter; Li, Jin-Lan; Li, Ling-Di; Li, Ning; Chen, Shan-Shan; Ruan, Guo-Rui

2015-10-01

Calreticulin (CALR) mutations were recently identified in a substantial proportion of persons with essential thrombocythemia (ET) and with primary myelofibrosis (PMF) without JAK2(V617F). Consequently rapid, sensitive, and specific methods to detect and quantify these mutations are needed. We studied samples from 1088 persons with myeloproliferative neoplasms (MPNs) including 421 JAK2(V617F) negative subjects with ET, PMF, polycythemia vera (PV), chronic myeloid leukemia (CML) and hyper-eosinophilic syndrome (HES). Detection of CALR exon 9 mutations was done by PCR amplification followed by fragment length analysis and direct sequencing. Dilution assays were used to determine CALR mutant allele burden. We detected CALR mutations in blood and bone marrow samples from 152 subjects with ET and with PMF but not in samples from normal or persons with PV, CML, or HES. CALR mutant peaks were distinct from wild-type peaks and dilution experiments indicated a sensitivity level of 0.5-5% for a CALR mutant allele in a wild-type background. Diverse types of mutations were detected including deletions, insertions, and complex indels. All mutations were confirmed by direct sequencing. We also used dilution experiments to quantify mutant allele burden. We were able to reproducibly detect mutant allele levels as low 5% (0.5-5%) in a wild-type background. PCR amplification followed by fragment length analysis is a rapid, sensitive, and specific method for screening persons with MPNs for CALR mutations, especially those with ET and PMF and for estimating mutant allele burden.

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

PubMed

Eckardt, F; Haynes, R H

1977-02-01

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

Peptide Nucleic Acid Array for Detection of Point Mutations in Hepatitis B Virus Associated with Antiviral Resistance ▿ †

PubMed Central

Jang, Hyunjung; Kim, Jihyun; Choi, Jae-jin; Son, Yeojin; Park, Heekyung

2010-01-01

The detection of antiviral-resistant hepatitis B virus (HBV) mutations is important for monitoring the response to treatment and for effective treatment decisions. We have developed an array using peptide nucleic acid (PNA) probes to detect point mutations in HBV associated with antiviral resistance. PNA probes were designed to detect mutations associated with resistance to lamivudine, adefovir, and entecavir. The PNA array assay was sensitive enough to detect 102 copies/ml. The PNA array assay was able to detect mutants present in more than 5% of the virus population when the total HBV DNA concentration was greater than 104 copies/ml. We analyzed a total of 68 clinical samples by this assay and validated its usefulness by comparing results to those of the sequencing method. The PNA array correctly identified viral mutants and has high concordance (98.3%) with direct sequencing in detecting antiviral-resistant mutations. Our results showed that the PNA array is a rapid, sensitive, and easily applicable assay for the detection of antiviral-resistant mutation in HBV. Thus, the PNA array is a useful and powerful diagnostic tool for the detection of point mutations or polymorphisms. PMID:20573874

Hotspot Mutations in KIT Receptor Differentially Modulate Its Allosterically Coupled Conformational Dynamics: Impact on Activation and Drug Sensitivity

PubMed Central

Chauvot de Beauchêne, Isaure; Allain, Ariane; Panel, Nicolas; Laine, Elodie; Trouvé, Alain; Dubreuil, Patrice; Tchertanov, Luba

2014-01-01

Receptor tyrosine kinase KIT controls many signal transduction pathways and represents a typical allosterically regulated protein. The mutation-induced deregulation of KIT activity impairs cellular physiological functions and causes serious human diseases. The impact of hotspots mutations (D816H/Y/N/V and V560G/D) localized in crucial regulatory segments, the juxtamembrane region (JMR) and the activation (A-) loop, on KIT internal dynamics was systematically studied by molecular dynamics simulations. The mutational outcomes predicted in silico were correlated with in vitro and in vivo activation rates and drug sensitivities of KIT mutants. The allosteric regulation of KIT in the native and mutated forms is described in terms of communication between the two remote segments, JMR and A-loop. A strong correlation between the communication profile and the structural and dynamical features of KIT in the native and mutated forms was established. Our results provide new insight on the determinants of receptor KIT constitutive activation by mutations and resistance of KIT mutants to inhibitors. Depiction of an intra-molecular component of the communication network constitutes a first step towards an integrated description of vast communication pathways established by KIT in physiopathological contexts. PMID:25079768

KRAS and BRAF Mutation Detection: Is Immunohistochemistry a Possible Alternative to Molecular Biology in Colorectal Cancer?

PubMed Central

Borrini, Francesco; Bolognese, Antonio; Lamy, Aude; Sabourin, Jean-Christophe

2015-01-01

KRAS genotyping is mandatory in metastatic colorectal cancer treatment prior to undertaking antiepidermal growth factor receptor (EGFR) monoclonal antibody therapy. BRAF V600E mutation is often present in colorectal carcinoma with CpG island methylator phenotype and microsatellite instability. Currently, KRAS and BRAF evaluation is based on molecular biology techniques such as SNaPshot or Sanger sequencing. As molecular testing is performed on formalin-fixed paraffin-embedded (FFPE) samples, immunodetection would appear to be an attractive alternative for detecting mutations. Thus, our objective was to assess the validity of KRAS and BRAF immunodetection of mutations compared with the genotyping reference method in colorectal adenocarcinoma. KRAS and BRAF genotyping was assessed by SNaPshot. A rabbit anti-human KRAS polyclonal antibody was tested on 33 FFPE colorectal tumor samples with known KRAS status. Additionally, a mouse anti-human BRAF monoclonal antibody was tested on 30 FFPE tumor samples with known BRAF status. KRAS immunostaining demonstrated both poor sensitivity (27%) and specificity (64%) in detecting KRAS mutation. Conversely, BRAF immunohistochemistry showed perfect sensitivity (100%) and specificity (100%) in detecting V600E mutation. Although molecular biology remains the reference method for detecting KRAS mutation, immunohistochemistry could be an attractive method for detecting BRAF V600E mutation in colorectal cancer. PMID:25983749

Aspergillus sensitization or carriage in cystic fibrosis patients.

PubMed

Fillaux, Judith; Brémont, François; Murris, Marlène; Cassaing, Sophie; Tétu, Laurent; Segonds, Christine; Pipy, Bernard; Magnaval, Jean-François

2014-07-01

Aspergillus fumigatus (Af) sensitization and persistent carriage are deleterious to lung function, but no consensus has been reached defining these medical entities. This work aimed to identify possible predictive factors for patients who become sensitized to Af, compared with a control group of non-sensitized Af carriers. Between 1995 and 2007, 117 pediatric patients were evaluated. Demographic data, CFTR gene mutations, body mass index and FEV1 were recorded. The presence of Af in sputum, the levels of Af-precipitin, total IgE (t-IgE) and specific IgE to Af (Af-IgE) were determined. Patients were divided into 2 groups: (1) "sensitization": level of Af-IgE > 0.35 IU/mL with t-IgE level < 500 IU/mL and (2) "persistent or transient carriage": Af-IgE level ≤ 0.35 IU/mL with either an Af transient or persistent positive culture. A survival analysis was performed with the appearance of Af-IgE in serum as an outcome variable. Severe mutation (hazard ratio = 3.2), FEV1 baseline over 70% of theoretical value (hazard ratio = 4.9), absence of Pa colonization, catalase activity and previous azithromycin administration (hazard ratio = 9.8, 4.1 and 1.9, respectively) were predictive factors for sensitization. We propose a timeline of the biological events and a tree diagram for risk calculation. Two profiles of cystic fibrosis patients can be envisaged: (1) patients with nonsevere mutation but low FEV1 baselines are becoming colonized with Af or (2) patients with high FEV1 baselines who present with severe mutation are more susceptible to the Af sensitization and then to the presentation of an allergic bronchopulmonary aspergillosis event.

Specific and straightforward molecular investigation of β-thalassemia mutations in the Malaysian Malays and Chinese using direct TaqMan genotyping assays.

PubMed

Kho, S L; Chua, K H; George, E; Tan, J A M A

2013-07-15

Beta-thalassemia is a life-threatening inherited blood disorder. Rapid characterization of β-globin gene mutations is necessary because of the high frequency of Malaysian β-thalassemia carriers. A combination real-time polymerase chain reaction genotyping assay using TaqMan probes was developed to confirm β-globin gene mutations. In this study, primers and probes were designed to specifically identify 8 common β-thalassemia mutations in the Malaysian Malay and Chinese ethnic groups using the Primer Express software. "Blind tests" using DNA samples from healthy individuals and β-thalassemia patients with different genotypes were performed to determine the specificity and sensitivity of this newly designed assay. Our results showed 100% sensitivity and specificity for this novel assay. In conclusion, the TaqMan genotyping assay is a straightforward assay that allows detection of β-globin gene mutations in less than 40 min. The simplicity and reproducibility of the TaqMan genotyping assay permit its use in laboratories as a rapid and cost-effective diagnostic tool for confirmation of common β-thalassemia mutations in Malaysia.

Elucidation of the molecular mechanism of heat shock proteins and its correlation with K722Q mutations in Lon protease.

PubMed

Bhattacharjee, Sanchari; Dasgupta, Rakhi; Bagchi, Angshuman

2017-09-01

Cells withstand the effects of temperature change with the help of small heat shock proteins IbpA and IbpB. The IbpAB protein complex interacts with Lon protease in their free form and gets degraded at physiological temperature when there is no temperature stress. However, the proteolytic degradation of IbpAB is diminished when Lon is mutated. The mutation K722Q in Lon brings about some structural changes so that the proteolytic interactions between the heat shock proteins with that of the mutated Lon protease are lost. However, the detailed molecular aspects of the interactions are not yet fully understood. In the present, we made an attempt to analyze the biochemical aspects of the interactions between the small heat shock proteins IbpAB with wild type and mutant Lon protease. We for the first time deciphered the molecular details of the mechanism of interaction of small heat shock proteins with Lon protease bearing K722Q mutation i.e. the interaction pattern of heat shock proteins with mutant Lon protease at physiological temperature in absence of proteolytic machinery. Our study may therefore be useful to elucidate the mechanistic details of the correlation with IbpA, IbpB and Lon protease. Copyright © 2017 Elsevier B.V. All rights reserved.

Response of head and neck squamous cell carcinoma cells carrying PIK3CA mutations to selected targeted therapies.

PubMed

Wirtz, Eric D; Hoshino, Daisuke; Maldonado, Anthony T; Tyson, Darren R; Weaver, Alissa M

2015-06-01

The PIK3CA mutation is one of the most common mutations in head and neck squamous cell carcinoma (HNSCC). Through this research we attempt to elicit the role of oncogene dependence and effects of targeted therapy on this PIK3CA mutation. (1) To determine the role of oncogene dependence on PIK3CA-one of the more common and targetable oncogenes in HNSCC, and (2) to evaluate the consequence of this oncogene on the effectiveness of newly developed targeted therapies. This was a cell culture-based, in vitro study performed at an academic research laboratory assessing the viability of PIK3CA-mutated head and neck cell lines when treated with targeted therapy. PIK3CA-mutated head and neck cell lines were treated with 17-AAG, GDC-0941, trametinib, and BEZ-235. Assessment of cell viability of HNSCC cell lines characterized for PIK3CA mutations or SCC25 cells engineered to express the PIK3CA hotspot mutations E545K or H1047R. Surprisingly, in engineered cell lines, the hotspot E545K and H1047R mutations conferred increased, rather than reduced, IC50 assay measurements when treated with the respective HSP90, PI3K, and MEK inhibitors, 17-AAG, GDC-0941, and trametinib, compared with the SCC25 control cell lines. When treated with BEZ-235, H1047R-expressing cell lines showed increased sensitivity to inhibition compared with control, whereas those expressing E545K showed slightly increased sensitivity of unclear significance. (1) The PIK3CA mutations within our engineered cell model did not lead to enhanced oncogene-dependent cell death when treated with direct inhibition of the PI3K enzyme yet did show increased sensitivity compared with control with dual PI3K/mTOR inhibition. (2) Oncogene addiction to PIK3CA hotspot mutations, if it occurs, is likely to evolve in vivo in the context of additional molecular changes that remain to be identified. Additional study is required to develop new model systems and approaches to determine the role of targeted therapy in the treatment of PI3K-overactive HNSCC tumors.

Detection of sdhB Gene Mutations in SDHI-Resistant Isolates of Botrytis cinerea Using High Resolution Melting (HRM) Analysis.

PubMed

Samaras, Anastasios; Madesis, Panagiotis; Karaoglanidis, George S

2016-01-01

Botrytis cinerea , is a high risk pathogen for fungicide resistance development. Pathogen' resistance to SDHIs is associated with several mutations in sdh gene. The diversity of mutations and their differential effect on cross-resistance patterns among SDHIs and the fitness of resistant strains necessitate the availability of a tool for their rapid identification. This study was initiated to develop and validate a high-resolution melting (HRM) analysis for the identification of P225H/F/L//T, N230I, and H272L/R/Y mutations. Based on the sequence of sdh B subunit of resistant and sensitive isolates, a universal primer pair was designed. The specificity of the HRM analysis primers was verified to ensure against the cross-reaction with other fungal species and its sensitivity was evaluated using concentrations of known amounts of mutant's DNA. The melting curve analysis generated nine distinct curve profiles, enabling the discrimination of all the four mutations located at codon 225, the N230I mutation, the three mutations located in codon 272, and the non-mutated isolates (isolates of wild-type sensitivity). Similar results were obtained when DNA was extracted directly from artificially inoculated strawberry fruit. The method was validated by monitoring the presence of sdh B mutations in samples of naturally infected strawberry fruits and stone fruit rootstock seedling plants showing damping-off symptoms. HRM analysis data were compared with a standard PIRA-PCR technique and an absolute agreement was observed suggesting that in both populations the H272R mutation was the predominant one, while H272Y, N230I, and P225H were detected in lower frequencies. The results of the study suggest that HRM analysis can be a useful tool for sensate, accurate, and rapid identification of several sdh B mutations in B. cinerea and it is expected to contribute in routine fungicide resistance monitoring or assessments of the effectiveness of anti-resistance strategies implemented in crops heavily treated with botryticides.

Detection of sdhB Gene Mutations in SDHI-Resistant Isolates of Botrytis cinerea Using High Resolution Melting (HRM) Analysis

PubMed Central

Samaras, Anastasios; Madesis, Panagiotis; Karaoglanidis, George S.

2016-01-01

Botrytis cinerea, is a high risk pathogen for fungicide resistance development. Pathogen’ resistance to SDHIs is associated with several mutations in sdh gene. The diversity of mutations and their differential effect on cross-resistance patterns among SDHIs and the fitness of resistant strains necessitate the availability of a tool for their rapid identification. This study was initiated to develop and validate a high-resolution melting (HRM) analysis for the identification of P225H/F/L//T, N230I, and H272L/R/Y mutations. Based on the sequence of sdhB subunit of resistant and sensitive isolates, a universal primer pair was designed. The specificity of the HRM analysis primers was verified to ensure against the cross-reaction with other fungal species and its sensitivity was evaluated using concentrations of known amounts of mutant’s DNA. The melting curve analysis generated nine distinct curve profiles, enabling the discrimination of all the four mutations located at codon 225, the N230I mutation, the three mutations located in codon 272, and the non-mutated isolates (isolates of wild-type sensitivity). Similar results were obtained when DNA was extracted directly from artificially inoculated strawberry fruit. The method was validated by monitoring the presence of sdhB mutations in samples of naturally infected strawberry fruits and stone fruit rootstock seedling plants showing damping-off symptoms. HRM analysis data were compared with a standard PIRA–PCR technique and an absolute agreement was observed suggesting that in both populations the H272R mutation was the predominant one, while H272Y, N230I, and P225H were detected in lower frequencies. The results of the study suggest that HRM analysis can be a useful tool for sensate, accurate, and rapid identification of several sdhB mutations in B. cinerea and it is expected to contribute in routine fungicide resistance monitoring or assessments of the effectiveness of anti-resistance strategies implemented in crops heavily treated with botryticides. PMID:27895633

Identifying Determinants of PARP Inhibitor Sensitivity in Ovarian Cancer

DTIC Science & Technology

2016-10-01

inhibitors. Ovarian cancer patients that harbored germ- line BRCA1 mutations treated with PARP inhibitors exhibited meaningful responses in early phase...hypothesized that a range of common ovarian cancer predisposing germ- line BRCA1 gene mutations produce semi-functional proteins that are capable of...we have started our work examining exome sequences and gene expression in PARPi sensitive and resistance cancer cell lines . I attended and presented

Efficacy of icotinib versus traditional chemotherapy as first-line treatment for preventing brain metastasis from advanced lung adenocarcinoma in patients with epidermal growth factor receptor-sensitive mutation.

PubMed

Zhao, Xiao; Zhu, Guangqin; Chen, Huoming; Yang, Ping; Li, Fang; Du, Nan

2016-01-01

This study aimed to investigate the potential use of icotinib as first-line treatment to prevent brain metastasis from advanced lung adenocarcinoma. This investigation was designed as a retrospective nonrandomized controlled study. Enrolled patients received either icotinib or traditional chemotherapy as their first-line treatment. The therapeutic efficacy was compared among patients with advanced. (stages IIIB and IV) lung adenocarcinoma with epidermal growth factor receptor (EGFR)-sensitive mutation. The primary endpoint was the cumulative incidence of brain metastasis, whereas, the secondary endpoint was overall survival(OS). Death without brain metastasis was considered a competitive risk to calculate the cumulative risk of brain metastasis. Survival analysis was conducted using the Kaplan-Meier method and statistical significance was determined using the log-rank test. The present study included 396 patients with 131 in the icotinib group and 265 in the chemotherapy group. Among those with EGFR-sensitive mutation, the cumulative risk of brain metastasis was lower in the icotinib group than in the chemotherapy group. However, no significant difference in OS was observed between the two groups. Icotinib can effectively reduce the incidence of brain metastasis and therefore improve prognosis in advanced lung adenocarcinoma patients with EGFR.sensitive mutation.

Efficacy of icotinib versus traditional chemotherapy as first-line treatment for preventing brain metastasis from advanced lung adenocarcinoma in patients with epidermal growth factor receptor-sensitive mutation.

PubMed

Zhao, Xiao; Zhu, Guangqin; Chen, Huoming; Yang, Ping; Li, Fang; Du, Nan

2014-11-01

This study aimed to investigate the potential use of icotinib as first-line treatment to prevent brain metastasis from advanced lung adenocarcinoma. This investigation was designed as a retrospective nonrandomized controlled study. Enrolled patients received either icotinib or traditional chemotherapy as their first-line treatment. The therapeutic efficacy was compared among patients with advanced (stages IIIB and IV) lung adenocarcinoma with epidermal growth factor receptor (EGFR)-sensitive mutation. The primary endpoint was the cumulative incidence of brain metastasis, whereas the secondary endpoint was overall survival (OS). Death without brain metastasis was considered a competitive risk to calculate the cumulative risk of brain metastasis. Survival analysis was conducted using the Kaplan-Meier method and statistical significance were determined using the log-rank test. The present study included 396 patients with 131 in the icotinib group and 265 in the chemotherapy group. Among those with EGFR-sensitive mutation, the cumulative risk of brain metastasis was lower in the icotinib group than in the chemotherapy group. However, no significant difference in OS was observed between the two groups. Icotinib can effectively reduce the incidence of brain metastasis and therefore improve prognosis in advanced lung adenocarcinoma patients with EGFR-sensitive mutation.

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

PubMed

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

2016-06-23

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

A high-sensitive HMab-2 specifically detects IDH1-R132H, the most common IDH mutation in gliomas.

PubMed

Fujii, Yuki; Ogasawara, Satoshi; Oki, Hiroharu; Liu, Xing; Kaneko, Mika K; Takano, Shingo; Kato, Yukinari

2015-10-30

Isocitrate dehydrogenase 1 (IDH1) mutations have been detected in gliomas and other tumors. Although IDH1 catalyzes the oxidative carboxylation of isocitrate to α-ketoglutarate (α-KG) in cytosol, mutated IDH1 proteins possess the ability to change α-KG into the oncometabolite D-2-hydroxyglutarate (D-2HG). Several monoclonal antibodies (mAbs) specific for IDH1 mutations have been established, such as H09, IMab-1, and HMab-1 against IDH1-R132H, which is the most frequent IDH1 mutation in gliomas. In this study, we established a novel high-sensitive mAb HMab-2, which reacts with IDH1-R132H but not with wild type IDH1 in ELISA. HMab-2 reacted only with IDH1-R132H, not with wild type IDH1/2 and other IDH1/2 mutants in Western-blot analysis. Furthermore, HMab-2 recognized IDH1-R132H more sensitively compared with our previously established HMab-1. HMab-2 detected endogenous IDH1-R132H protein expressed in glioblastoma in immunohistochemical analysis. HMab-2 is expected to be useful for the diagnosis of IDH1-R132H-bearing tumors. Copyright © 2015 Elsevier Inc. All rights reserved.

WW domains of Rsp5p define different functions: determination of roles in fluid phase and uracil permease endocytosis in Saccharomyces cerevisiae.

PubMed

Gajewska, B; Kamińska, J; Jesionowska, A; Martin, N C; Hopper, A K; Zoładek, T

2001-01-01

Rsp5p, ubiquitin-protein ligase, an enzyme of the ubiquitination pathway, contains three WW domains that mediate protein-protein interactions. To determine if these domains adapt Rsp5p to a subset of substrates involved in numerous cellular processes, we generated mutations in individual or combinations of the WW domains. The rsp5-w1, rsp5-w2, and rsp5-w3 mutant alleles complement RSP5 deletions at 30 degrees. Thus, individual WW domains are not essential. Each rsp5-w mutation caused temperature-sensitive growth. Among variants with mutations in multiple WW domains, only rsp5-w1w2 complemented the deletion. Thus, the WW3 domain is sufficient for Rsp5p essential functions. To determine whether rsp5-w mutations affect endocytosis, fluid phase and uracil permease (Fur4p) endocytosis was examined. The WW3 domain is important for both processes. WW2 appears not to be important for fluid phase endocytosis whereas it is important for Fur4p endocytosis. In contrast, the WW1 domain affects fluid phase endocytosis, but it does not appear to function in Fur4p endocytosis. Thus, various WW domains play different roles in the endocytosis of these two substrates. Rsp5p is located in the cytoplasm in a punctate pattern that does not change during the cell cycle. Altering WW domains does not change the location of Rsp5p.

WW domains of Rsp5p define different functions: determination of roles in fluid phase and uracil permease endocytosis in Saccharomyces cerevisiae.

PubMed Central

Gajewska, B; Kamińska, J; Jesionowska, A; Martin, N C; Hopper, A K; Zoładek, T

2001-01-01

Rsp5p, ubiquitin-protein ligase, an enzyme of the ubiquitination pathway, contains three WW domains that mediate protein-protein interactions. To determine if these domains adapt Rsp5p to a subset of substrates involved in numerous cellular processes, we generated mutations in individual or combinations of the WW domains. The rsp5-w1, rsp5-w2, and rsp5-w3 mutant alleles complement RSP5 deletions at 30 degrees. Thus, individual WW domains are not essential. Each rsp5-w mutation caused temperature-sensitive growth. Among variants with mutations in multiple WW domains, only rsp5-w1w2 complemented the deletion. Thus, the WW3 domain is sufficient for Rsp5p essential functions. To determine whether rsp5-w mutations affect endocytosis, fluid phase and uracil permease (Fur4p) endocytosis was examined. The WW3 domain is important for both processes. WW2 appears not to be important for fluid phase endocytosis whereas it is important for Fur4p endocytosis. In contrast, the WW1 domain affects fluid phase endocytosis, but it does not appear to function in Fur4p endocytosis. Thus, various WW domains play different roles in the endocytosis of these two substrates. Rsp5p is located in the cytoplasm in a punctate pattern that does not change during the cell cycle. Altering WW domains does not change the location of Rsp5p. PMID:11139494

Collagen Gly missense mutations: Effect of residue identity on collagen structure and integrin binding.

PubMed

Qiu, Yimin; Mekkat, Arya; Yu, Hongtao; Yigit, Sezin; Hamaia, Samir; Farndale, Richard W; Kaplan, David L; Lin, Yu-Shan; Brodsky, Barbara

2018-05-11

Gly missense mutations in type I collagen, which replace a conserved Gly in the repeating (Gly-Xaa-Yaa) n sequence with a larger residue, are known to cause Osteogenesis Imperfecta (OI). The clinical consequences of such mutations range from mild to lethal, with more serious clinical severity associated with larger Gly replacement residues. Here, we investigate the influence of the identity of the residue replacing Gly within and adjacent to the integrin binding 502 GFPGER 507 sequence on triple-helix structure, stability and integrin binding using a recombinant bacterial collagen system. Recombinant collagens were constructed with Gly substituted by Ala, Ser or Val at four positions within the integrin binding region. All constructs formed a stable triple-helix structure with a small decrease in melting temperature. Trypsin was used to probe local disruption of the triple helix, and Gly to Val replacements made the triple helix trypsin sensitive at three of the four sites. Any mutation at Gly505, eliminated integrin binding, while decreased integrin binding affinity was observed in the replacement of Gly residues at Gly502 following the order Val > Ser > Ala. Molecular dynamics simulations indicated that all Gly replacements led to transient disruption of triple-helix interchain hydrogen bonds in the region of the Gly replacement. These computational and experimental results lend insight into the complex molecular basis of the varying clinical severity of OI. Copyright © 2018. Published by Elsevier Inc.

False-negative BRAF V600E mutation results on fine-needle aspiration cytology of papillary thyroid carcinoma.

PubMed

Paek, Se Hyun; Kim, Byung Seup; Kang, Kyung Ho; Kim, Hee Sung

2017-11-13

The BRAF V600E mutation is highly specific for papillary thyroid carcinoma (PTC). A test for this mutation can increase the diagnostic accuracy of fine-needle aspiration cytology (FNAC), but a considerably high false-negative rate for the BRAF V600E mutation on FNAC has been reported. In this study, we investigated the risk factors associated with false-negative BRAF V600E mutation results on FNAC. BRAF V600E mutation results of 221 PTC nodules between December 2011 and June 2013 were retrospectively reviewed. BRAF V600E mutation results on both preoperative FNAC and postoperative formalin-fixed, paraffin-embedded (FFPE) samples were compared. We investigated the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of BRAF V600E mutation results on FNAC. And, we identified the risk factors associated with false-negative results. Of 221 PTC nodules, 150 (67.9%) on FNAC and 185 (83.7%) on FFPE samples were BRAF V600E mutation positive. The sensitivity, specificity, PPV, and NPV for BRAF V600E mutation testing with FNAC were 80.5, 97.2, 99.3, and 49.3%, respectively. Thirty-six (16.3%) BRAF V600E mutation-negative nodules on FNAC were mutation positive on FFPE sample analysis. Risk factors for these false-negative results were age, indeterminate FNAC results (nondiagnostic, atypia of undetermined significance (AUS), and findings suspicious for PTC), and PTC subtype. False-negative rate of BRAF mutation testing with FNAC for thyroid nodules is increased in cases of old age, indeterminate FNAC pathology results, and certain PTC subtypes. Therapeutic surgery can be considered for these cases. A well-designed prospective study with informed consent of patients will be essential for more informative results.

Tumour MLH1 promoter region methylation testing is an effective prescreen for Lynch Syndrome (HNPCC).

PubMed

Newton, K; Jorgensen, N M; Wallace, A J; Buchanan, D D; Lalloo, F; McMahon, R F T; Hill, J; Evans, D G

2014-12-01

Lynch syndrome (LS) patients have DNA mismatch repair deficiency and up to 80% lifetime risk of colorectal cancer (CRC). Screening of mutation carriers reduces CRC incidence and mortality. Selection for constitutional mutation testing relies on family history (Amsterdam and Bethesda Guidelines) and tumour-derived biomarkers. Initial biomarker analysis uses mismatch repair protein immunohistochemistry and microsatellite instability. Abnormalities in either identify mismatch repair deficiency but do not differentiate sporadic epigenetic defects, due to MLH1 promoter region methylation (13% of CRCs) from LS (4% of CRCs). A diagnostic biomarker capable of making this distinction would be valuable. This study compared two biomarkers in tumours with mismatch repair deficiency; quantification of methylation of the MLH1 promoter region using a novel assay and BRAF c.1799T>A, p.(Val600Glu) mutation status in the identification of constitutional mutations. Tumour DNA was extracted (formalin fixed, paraffin embedded, FFPE tissue) and pyrosequencing used to test for MLH1 promoter methylation and presence of the BRAF c.1799T>A, p.(Val600Glu) mutation 71 CRCs from individuals with pathogenic MLH1 mutations and 73 CRCs with sporadic MLH1 loss. Specificity and sensitivity was compared. Unmethylated MLH1 promoter: sensitivity 94.4% (95% CI 86.2% to 98.4%), specificity 87.7% (95% CI 77.9% to 94.2%), Wild-type BRAF (codon 600): sensitivity 65.8% (95% CI 53.7% to 76.5%), specificity 98.6% (95% CI 92.4% to 100.0%) for the identification of those with pathogenic MLH1 mutations. Quantitative MLH1 promoter region methylation using pyrosequencing is superior to BRAF codon 600 mutation status in identifying constitutional mutations in mismatch repair deficient tumours. 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.

Detection of BRAF-V600E and V600K in melanoma circulating tumour cells by droplet digital PCR.

PubMed

Reid, Anna L; Freeman, James B; Millward, Michael; Ziman, Melanie; Gray, Elin S

2015-10-01

Defining the BRAF mutation status in metastatic melanoma patients is critical to selecting patients for therapeutic treatment with targeted therapies. Circulating tumour cells (CTCs) can provide an alternative source of contemporaneous tumour genetic material. However methodologies to analyse the presence of rare mutations in a background of wild-type DNA requires a detailed assessment. Here we evaluate the sensitivity of two technologies for cancer mutation detection and the suitability of whole genome amplified DNA as a template for the detection of BRAF-V600 mutations. Serial dilutions of mutant BRAF-V600E DNA in wild-type DNA were tested using both competitive allele-specific PCR (castPCR) and droplet digital PCR (ddPCR), with and without previous whole genome amplification (WGA). Using immunomagnetic beads, we partially enriched CTCs from blood obtained from metastatic melanoma patients with confirmed BRAF mutation positive tumours and extracted RNA and DNA from the CTCs. We used RT-PCR of RNA to confirm the presence of melanoma cells in the CTC fraction then the DNAs of CTC positive fractions were WGA and tested for BRAF V600E or V600K mutations by ddPCRs. WGA DNA produced lower than expected fractional abundances by castPCR analysis but not by ddPCR. Moreover, ddPCR was found to be 200 times more sensitive than castPCR and in combination with WGA produced the most concordant results, with a limit of detection of 0.0005%. BRAF-V600E or V600K mutated DNA was detected in 77% and 44%, respectively, of enriched CTC fractions from metastatic melanoma patients carrying the corresponding mutations. Our results demonstrate that using ddPCR in combination with WGA DNA allows the detection with high sensitivity of cancer mutations in partially enriched CTC fractions. Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Heat Increases the Editing Efficiency of Human Papillomavirus E2 Gene by Inducing Upregulation of APOBEC3A and 3G.

PubMed

Yang, Yang; Wang, Hexiao; Zhang, Xinrui; Huo, Wei; Qi, Ruiqun; Gao, Yali; Zhang, Gaofeng; Song, Bing; Chen, Hongduo; Gao, Xinghua

2017-04-01

Apolipoprotein B mRNA-editing catalytic polypeptide (APOBEC) 3 proteins have been identified as potent viral DNA mutators and have broad antiviral activity. In this study, we demonstrated that apolipoprotein B mRNA-editing catalytic polypeptide 3A (A3A) and A3G expression levels were significantly upregulated in human papillomavirus (HPV)-infected cell lines and tissues. Heat treatment resulted in elevated expression of A3A and A3G in a temperature-dependent manner in HPV-infected cells. Correspondingly, HPV-infected cells heat-treated at 44 °C showed accumulated G-to-A or C-to-T mutation in HPV E2 gene. Knockdown of A3A or A3G could promote cell viability, along with the lower frequency of A/T in HPV E2 gene. In addition, regressing genital viral warts also harbored high G-to-A or C-to-T mutation in HPV E2 gene. Taken together, we demonstrate that apolipoprotein B mRNA-editing catalytic polypeptide 3 expression and editing function was heat sensitive to a certain degree, partly explaining the mechanism of action of local hyperthermia to treat viral warts. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Phenotypic and genomic analysis of serotype 3 Sabin poliovirus vaccine produced in MRC-5 cell substrate.

PubMed

Alirezaie, Behnam; Taqavian, Mohammad; Aghaiypour, Khosrow; Esna-Ashari, Fatemeh; Shafyi, Abbas

2011-05-01

The cell substrate has a pivotal role in live virus vaccines production. It is necessary to evaluate the effects of the cell substrate on the properties of the propagated viruses, especially in the case of viruses which are unstable genetically such as polioviruses, by monitoring the molecular and phenotypical characteristics of harvested viruses. To investigate the presence/absence of mutation(s), the near full-length genomic sequence of different harvests of the type 3 Sabin strain of poliovirus propagated in MRC-5 cells were determined. The sequences were compared with genomic sequences of different virus seeds, vaccines, and OPV-like isolates. Nearly complete genomic sequencing results, however, revealed no detectable mutations throughout the genome RNA-plaque purified (RSO)-derived monopool of type 3 OPVs manufactured in MRC-5. Thirty-six years of experience in OPV production, trend analysis, and vaccine surveillance also suggest that: (i) different monopools of serotype 3 OPV produced in MRC-5 retained their phenotypic characteristics (temperature sensitivity and neuroattenuation), (ii) MRC-5 cells support the production of acceptable virus yields, (iii) OPV replicated in the MRC-5 cell substrate is a highly efficient and safe vaccine. These results confirm previous reports that MRC-5 is a desirable cell substrate for the production of OPV. Copyright © 2011 Wiley-Liss, Inc.

The influence of the loop between residues 223-235 in beetle luciferase bioluminescence spectra: a solvent gate for the active site of pH-sensitive luciferases.

PubMed

Viviani, Vadim R; Silva Neto, Antonio J; Arnoldi, Frederico G C; Barbosa, João A R G; Ohmiya, Yoshihiro

2008-01-01

Beetle luciferases emit a wide range of bioluminescence colors, ranging from green to red. Firefly luciferases can shift the spectrum to red in response to pH and temperature changes, whereas click beetle and railroadworm luciferases do not. Despite many studies on firefly luciferases, the origin of pH-sensitivity is far from being understood. Through comparative site-directed mutagenesis and modeling studies, using the pH-sensitive luciferases (Macrolampis and Cratomorphus distinctus fireflies) and the pH-insensitive luciferases (Pyrearinus termitilluminans, Phrixotrix viviani and Phrixotrix hirtus) cloned by our group, here we show that substitutions dramatically affecting bioluminescence colors in both groups of luciferases are clustered in the loop between residues 223-235 (Photinus pyralis sequence). The substitutions at positions 227, 228 and 229 (P. pyralis sequence) cause dramatic redshift and temporal shift in both groups of luciferases, indicating their involvement in labile interactions. Modeling studies showed that the residues Y227 and N229 are buried in the protein core, fixing the loop to other structural elements participating at the bottom of the luciferin binding site. Changes in pH and temperature (in firefly luciferases), as well as point mutations in this loop, may disrupt the interactions of these structural elements exposing the active site and modulating bioluminescence colors.

An Updated Collection of Sequence Barcoded Temperature-Sensitive Alleles of Yeast Essential Genes

PubMed Central

Kofoed, Megan; Milbury, Karissa L.; Chiang, Jennifer H.; Sinha, Sunita; Ben-Aroya, Shay; Giaever, Guri; Nislow, Corey; Hieter, Philip; Stirling, Peter C.

2015-01-01

Systematic analyses of essential gene function using mutant collections in Saccharomyces cerevisiae have been conducted using collections of heterozygous diploids, promoter shut-off alleles, through alleles with destabilized mRNA, destabilized protein, or bearing mutations that lead to a temperature-sensitive (ts) phenotype. We previously described a method for construction of barcoded ts alleles in a systematic fashion. Here we report the completion of this collection of alleles covering 600 essential yeast genes. This resource covers a larger gene repertoire than previous collections and provides a complementary set of strains suitable for single gene and genomic analyses. We use deep sequencing to characterize the amino acid changes leading to the ts phenotype in half of the alleles. We also use high-throughput approaches to describe the relative ts behavior of the alleles. Finally, we demonstrate the experimental usefulness of the collection in a high-content, functional genomic screen for ts alleles that increase spontaneous P-body formation. By increasing the number of alleles and improving the annotation, this ts collection will serve as a community resource for probing new aspects of biology for essential yeast genes. PMID:26175450

An Updated Collection of Sequence Barcoded Temperature-Sensitive Alleles of Yeast Essential Genes.

PubMed

Kofoed, Megan; Milbury, Karissa L; Chiang, Jennifer H; Sinha, Sunita; Ben-Aroya, Shay; Giaever, Guri; Nislow, Corey; Hieter, Philip; Stirling, Peter C

2015-07-14

Systematic analyses of essential gene function using mutant collections in Saccharomyces cerevisiae have been conducted using collections of heterozygous diploids, promoter shut-off alleles, through alleles with destabilized mRNA, destabilized protein, or bearing mutations that lead to a temperature-sensitive (ts) phenotype. We previously described a method for construction of barcoded ts alleles in a systematic fashion. Here we report the completion of this collection of alleles covering 600 essential yeast genes. This resource covers a larger gene repertoire than previous collections and provides a complementary set of strains suitable for single gene and genomic analyses. We use deep sequencing to characterize the amino acid changes leading to the ts phenotype in half of the alleles. We also use high-throughput approaches to describe the relative ts behavior of the alleles. Finally, we demonstrate the experimental usefulness of the collection in a high-content, functional genomic screen for ts alleles that increase spontaneous P-body formation. By increasing the number of alleles and improving the annotation, this ts collection will serve as a community resource for probing new aspects of biology for essential yeast genes. Copyright © 2015 Kofoed et al.

5'-adenosine monophosphate mediated cooling treatment enhances ΔF508-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) stability in vivo.

PubMed

Zhang, Yueqiang; O'Brien, William G; Zhao, Zhaoyang; Lee, Cheng Chi

2015-09-04

Gene mutations that produce misprocessed proteins are linked to many human disorders. Interestingly, some misprocessed proteins retained their biological function when stabilized by low temperature treatment of cultured cells in vitro. Here we investigate whether low temperature treatment in vivo can rescue misfolded proteins by applying 5'-AMP mediated whole body cooling to a Cystic Fibrosis (CF) mouse model carrying a mutant cystic fibrosis transmembrane conductance regulator (CFTR) with a deletion of the phenylalanine residue in position 508 (ΔF508-CFTR). Low temperature treatment of cultured cells was previously shown to be able to alleviate the processing defect of ΔF508-CFTR, enhancing its plasma membrane localization and its function in mediating chloride ion transport. Here, we report that whole body cooling enhanced the retention of ΔF508-CFTR in intestinal epithelial cells. Functional analysis based on β-adrenergic dependent salivary secretion and post-natal mortality rate revealed a moderate but significant improvement in treated compared with untreated CF mice. Our findings demonstrate that temperature sensitive processing of mutant proteins can be responsive to low temperature treatment in vivo.

Larval rearing of zebrafish at suboptimal temperatures.

PubMed

Delomas, Thomas A; Dabrowski, Konrad

2018-05-01

Temperature-sensitive mutants have been widely utilized in single-cell and invertebrate model systems, particularly to study the function of essential genes. Few temperature-sensitive mutants have been identified in zebrafish, likely due to the difficulty of raising zebrafish at low temperatures. We describe a novel rearing protocol that allows rapid growth of larval and juvenile zebrafish at 23 °C compared to previous data in the literature. Embryos collected from four breeding pairs were maintained at 28.5 ± 0.5 °C until 5 days post-fertilization (dpf) - the onset of exogenous feeding. Larvae were then divided to six tanks and three tanks were cooled to 23 ± 0.2 °C. Fish were fed a live diet (marine rotifers Brachionus plicatilis and Artemia nauplii) and maintained under a set of environmental parameters shown to increase growth rate: continuous light, low salinity (3ppt), and algal turbidity. Mean total length and weight of fish at 21dpf were 12.7 ± 0.3 mm and 20.5 ± 1.5 mg for the 23 °C treatment and 18.5 ± 0.4 mm and 67.3 ± 3.4 mg for the 28.5 °C control. By 35 dpf, the fish raised at 23 °C had reached a mean length and weight of 18.9 ± 0.7 mm and 76.4 ± 6.7 mg, approximately the size control fish reached at 21 dpf. At 35 dpf, water temperature was raised to 28 °C and fish were reared to maturity (75 dpf) under standard conditions (freshwater, 13 L:11D photoperiod, dry diet, no added algal turbidity). Sex ratio and fertility were assessed and compared between temperature groups. There were no significant differences in sex ratio, fertilization rate, embryo viability at 1 dpf, clutch size, or relative fecundity. This rearing protocol will allow for efficient utilization of temperature-sensitive mutations in the zebrafish model system. Copyright © 2018 Elsevier Ltd. All rights reserved.

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

Using the MCF10A/MCF10CA1a Breast Cancer Progression Cell Line Model to Investigate the Effect of Active, Mutant Forms of EGFR in Breast Cancer Development and Treatment Using Gefitinib

PubMed Central

Bessette, Darrell C.; Tilch, Erik; Seidens, Tatjana; Quinn, Michael C. J.; Wiegmans, Adrian P.; Shi, Wei; Cocciardi, Sibylle; McCart-Reed, Amy; Saunus, Jodi M.; Simpson, Peter T.; Grimmond, Sean M.; Lakhani, Sunil R.; Khanna, Kum Kum; Waddell, Nic; Al-Ejeh, Fares; Chenevix-Trench, Georgia

2015-01-01

Background Basal-like and triple negative breast cancer (TNBC) share common molecular features, poor prognosis and a propensity for metastasis to the brain. Amplification of epidermal growth factor receptor (EGFR) occurs in ~50% of basal-like breast cancer, and mutations in the epidermal growth factor receptor (EGFR) have been reported in up to ~ 10% of Asian TNBC patients. In non-small cell lung cancer several different mutations in the EGFR tyrosine kinase domain confer sensitivity to receptor tyrosine kinase inhibitors, but the tumourigenic potential of EGFR mutations in breast cells and their potential for targeted therapy is unknown. Materials and Methods Constructs containing wild type, G719S or E746-A750 deletion mutant forms of EGFR were transfected into the MCF10A breast cells and their tumorigenic derivative, MCF10CA1a. The effects of EGFR over-expression and mutation on proliferation, migration, invasion, response to gefitinib, and tumour formation in vivo was investigated. Copy number analysis and whole exome sequencing of the MCF10A and MCF10CA1a cell lines were also performed. Results Mutant EGFR increased MCF10A and MCF10CA1a proliferation and MCF10A gefitinib sensitivity. The EGFR-E746-A750 deletion increased MCF10CA1a cell migration and invasion, and greatly increased MCF10CA1a xenograft tumour formation and growth. Compared to MCF10A cells, MCF10CA1a cells exhibited large regions of gain on chromosomes 3 and 9, deletion on chromosome 7, and mutations in many genes implicated in cancer. Conclusions Mutant EGFR enhances the oncogenic properties of MCF10A cell line, and increases sensitivity to gefitinib. Although the addition of EGFR E746-A750 renders the MCF10CA1a cells more tumourigenic in vivo it is not accompanied by increased gefitinib sensitivity, perhaps due to additional mutations, including the PIK3CA H1047R mutation, that the MCF10CA1a cell line has acquired. Screening TNBC/basal-like breast cancer for EGFR mutations may prove useful for directing therapy but, as in non-small cell lung cancer, accompanying mutations in PIK3CA may confer gefitinib resistance. PMID:25969993

PIK3CA dependence and sensitivity to therapeutic targeting in urothelial carcinoma.

PubMed

Ross, R L; McPherson, H R; Kettlewell, L; Shnyder, S D; Hurst, C D; Alder, O; Knowles, M A

2016-07-28

Many urothelial carcinomas (UC) contain activating PIK3CA mutations. In telomerase-immortalized normal urothelial cells (TERT-NHUC), ectopic expression of mutant PIK3CA induces PI3K pathway activation, cell proliferation and cell migration. However, it is not clear whether advanced UC tumors are PIK3CA-dependent and whether PI3K pathway inhibition is a good therapeutic option in such cases. We used retrovirus-mediated delivery of shRNA to knock down mutant PIK3CA in UC cell lines and assessed effects on pathway activation, cell proliferation, migration and tumorigenicity. The effect of the class I PI3K inhibitor GDC-0941 was assessed in a panel of UC cell lines with a range of known molecular alterations in the PI3K pathway. Specific knockdown of PIK3CA inhibited proliferation, migration, anchorage-independent growth and in vivo tumor growth of cells with PIK3CA mutations. Sensitivity to GDC-0941 was dependent on hotspot PIK3CA mutation status. Cells with rare PIK3CA mutations and co-occurring TSC1 or PTEN mutations were less sensitive. Furthermore, downstream PI3K pathway alterations in TSC1 or PTEN or co-occurring AKT1 and RAS gene mutations were associated with GDC-0941 resistance. Mutant PIK3CA is a potent oncogenic driver in many UC cell lines and may represent a valuable therapeutic target in advanced bladder cancer.

Primary and acquired EGFR T790M-mutant NSCLC patients identified by routine mutation testing show different characteristics but may both respond to osimertinib treatment.

PubMed

Li, Weihua; Qiu, Tian; Guo, Lei; Ling, Yun; Gao, Yibo; Ying, Jianming; He, Jie

2018-06-01

Primary EGFR T790M mutation is occasionally identified by routine mutation testing in tyrosine kinase inhibitor (TKI)-naive patients with non-small cell lung cancer (NSCLC). We herein aimed to compare the characteristics of primary and acquired T790M mutations in NSCLC patients, and their response to osimertinib. Using amplification refractory mutation system (ARMS) detection, primary T790M was identified in 0.5% (46/8723) of TKI-naive patients, whereas acquired T790M was detected in 49.7% (71/143) of TKI-relapsed patients. T790M always coexisted with a sensitizing EGFR mutation. Primary T790M more commonly coexisted with L858R, whereas acquired T790M was more likely to coexist with exon 19 deletions. Moreover, next-generation sequencing (NGS) showed that concomitant sensitizing EGFR and primary T790M mutant allele frequencies (MAFs) were highly concordant, but acquired T790M MAFs were significantly lower than the sensitizing EGFR MAFs. Sixteen acquired T790M-mutant patients received osimertinib. The median progression-free survival (PFS) was 8.1 months. Four primary T790M-mutant patients received osimertinib and the median PFS was 8.0 months. Together, our study demonstrates that primary and acquired T790M-mutant patients show distinct differences in some clinical and molecular characteristics, but may both respond to osimertinib treatment. Copyright © 2018 Elsevier B.V. All rights reserved.

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

PubMed

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

2011-09-01

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

Neurotoxic effects of ivermectin administration in genetically engineered mice with targeted insertion of the mutated canine ABCB1 gene.

PubMed

Orzechowski, Krystyna L; Swain, Marla D; Robl, Martin G; Tinaza, Constante A; Swaim, Heidi L; Jones, Yolanda L; Myers, Michael J; Yancy, Haile F

2012-09-01

To develop in genetically engineered mice an alternative screening method for evaluation of P-glycoprotein substrate toxicosis in ivermectin-sensitive Collies. 14 wild-type C57BL/6J mice (controls) and 21 genetically engineered mice in which the abcb1a and abcb1b genes were disrupted and the mutated canine ABCB1 gene was inserted. Mice were allocated to receive 10 mg of ivermectin/kg via SC injection (n = 30) or a vehicle-only formulation of propylene glycol and glycerol formal (5). Each was observed for clinical signs of toxic effects from 0 to 7 hours following drug administration. After ivermectin administration, considerable differences were observed in drug sensitivity between the 2 types of mice. The genetically engineered mice with the mutated canine ABCB1 gene had signs of severe sensitivity to ivermectin, characterized by progressive lethargy, ataxia, and tremors, whereas the wild-type control mice developed no remarkable effects related to the ivermectin. The ivermectin sensitivity modeled in the transgenic mice closely resembled the lethargy, stupor, disorientation, and loss of coordination observed in ivermectin-sensitive Collies with the ABCB1-1Δ mutation. As such, the model has the potential to facilitate toxicity assessments of certain drugs for dogs that are P-glycoprotein substrates, and it may serve to reduce the use of dogs in avermectin derivative safety studies that are part of the new animal drug approval process.

Detection of ultra-low levels of DNA changes by drinking water: epidemiologically important finding.

PubMed

Kumari, Parmila; Kamiseki, Meiko; Biyani, Manish; Suzuki, Miho; Nemoto, Naoto; Aita, Takuyo; Nishigaki, Koichi

2015-02-01

The safety of drinking water is essential to our health. In this context, the mutagenicity of water needs to be checked strictly. However, from the methodological limit, the lower concentration (less than parts per million) of mutagenicity could not be detected, though there have been of interest in the effect of less concentration mutagens. Here, we describe a highly sensitive mutation assay that detects mutagens at the ppb level, termed genome profiling-based mutation assay (GPMA). This consists of two steps; (i) Escherichia coli culture in the medium with/without mutagens and (ii) Genome profiling (GP) method (an integrated method of random PCR, temperature gradient gel electrophoresis and computer-aided normalization). Owing to high sensitivity of this method, very low concentration of mutagens in tap water could be directly detected without introducing burdensome concentration processes, enabling rapid measurement of low concentration samples. Less expectedly, all of the tap waters tested (22 samples) were shown to be significantly mutagenic while mineral waters were not. Resultantly, this article informs two facts that the GPMA method is competent to measure the mutagenicity of waters directly and the experimental results supported the former reports that the city tap waters contain very low level of mutagenicity reagent trihalomethanes. © The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

PAQR-2 Regulates Fatty Acid Desaturation during Cold Adaptation in C. elegans

PubMed Central

Svensk, Emma; Ståhlman, Marcus; Andersson, Carl-Henrik; Johansson, Maja; Borén, Jan; Pilon, Marc

2013-01-01

C. elegans PAQR-2 is homologous to the insulin-sensitizing adiponectin receptors in mammals, and essential for adaptation to growth at 15°C, a low but usually acceptable temperature for this organism. By screening for novel paqr-2 suppressors, we identified mutations in genes involved in phosphatidylcholine synthesis (cept-1, pcyt-1 and sams-1) and fatty acid metabolism (ech-7, hacd-1, mdt-15, nhr-49 and sbp-1). We then show genetic evidence that paqr-2, phosphatidylcholines, sbp-1 and Δ9-desaturases form a cold adaptation pathway that regulates the increase in unsaturated fatty acids necessary to retain membrane fluidity at low temperatures. This model is supported by the observations that the paqr-2 suppressors normalize the levels of saturated fatty acids, and that low concentrations of detergents that increase membrane fluidity can rescue the paqr-2 mutant. PMID:24068966

PAQR-2 regulates fatty acid desaturation during cold adaptation in C. elegans.

PubMed

Svensk, Emma; Ståhlman, Marcus; Andersson, Carl-Henrik; Johansson, Maja; Borén, Jan; Pilon, Marc

2013-01-01

C. elegans PAQR-2 is homologous to the insulin-sensitizing adiponectin receptors in mammals, and essential for adaptation to growth at 15°C, a low but usually acceptable temperature for this organism. By screening for novel paqr-2 suppressors, we identified mutations in genes involved in phosphatidylcholine synthesis (cept-1, pcyt-1 and sams-1) and fatty acid metabolism (ech-7, hacd-1, mdt-15, nhr-49 and sbp-1). We then show genetic evidence that paqr-2, phosphatidylcholines, sbp-1 and Δ9-desaturases form a cold adaptation pathway that regulates the increase in unsaturated fatty acids necessary to retain membrane fluidity at low temperatures. This model is supported by the observations that the paqr-2 suppressors normalize the levels of saturated fatty acids, and that low concentrations of detergents that increase membrane fluidity can rescue the paqr-2 mutant.

Molecular characterization, fitness and mycotoxin production of Fusarium graminearum laboratory strains resistant to benzimidazoles.

PubMed

Sevastos, A; Markoglou, A; Labrou, N E; Flouri, F; Malandrakis, A

2016-03-01

Six benzimidazole (BMZ)-resistant Fusarium graminearum strains were obtained after UV mutagenesis and selection on carbendazim (MBC)-amended medium. In vitro bioassays resulted in the identification of two resistant phenotypes that were highly HR (Rf: 40-170, based on EC50) and moderately MR (Rf: 10-20) resistant to carbendazim. Cross resistance studies with other fungicides showed that all mutant strains tested were also resistant to other BMZs, such as benomyl and thiabendazole, but retained their parental sensitivity to fungicides belonging to other chemical groups. A point mutation at codon 6 (His6Asn) was found in the β2-tubulin gene of MR isolates while another mutation at codon 200 (Phe200Tyr) was present in one MR and one HR isolates. Interestingly, low temperatures suppressed MBC-resistance in all isolates bearing the H6N mutation. The three-dimensional homology model of the wild-type and mutants of β-tubulins were constructed, and the possible carbendazim binding site was analyzed. Studies on fitness parameters showed that the mutation(s) for resistance to BMZs did not affect the mycelial growth rate whereas adverse effects were found in sporulation and conidial germination in most of the resistant mutants. Pathogenicity tests on corn cobs revealed that mutants were less or equally aggressive to the wild-type strain but expressed their BMZ-resistance after inoculation on maize cobs treated with MBC. Analysis of mycotoxin production by high performance liquid chromatography revealed that only two HR strains produced zearalenone (ZEA) at concentrations similar to that of the wild-type strain, while no ZEA levels were detected in the rest of the mutants. Copyright © 2015 Elsevier Inc. All rights reserved.

Primary hyperoxaluria type 1 in the Canary Islands: A conformational disease due to I244T mutation in the P11L-containing alanine:glyoxylate aminotransferase

PubMed Central

Santana, A.; Salido, E.; Torres, A.; Shapiro, L. J.

2003-01-01

Primary hyperoxaluria type 1 (PH1) is an inborn error of metabolism resulting from a deficiency of alanine:glyoxylate aminotransferase (AGXT; EC 2.6.1.44). Most of the PH1 alleles detected in the Canary Islands carry the Ile-244 → Thr (I244T) mutation in the AGXT gene, with 14 of 16 patients homozygous for this mutation. Four polymorphisms within AGXT and regional microsatellites also were shared in their haplotypes (AGXT*LTM), consistent with a founder effect. The consequences of these amino acid changes were investigated. Although I244T alone did not affect AGXT activity or subcellular localization, when present in the same protein molecule as Leu-11 → Pro (L11P), it resulted in loss of enzymatic activity in soluble cell extracts. Like its normal counterpart, the AGXT*LTM protein was present in the peroxisomes but it was insoluble in detergent-free buffers. The polymorphism L11P behaved as an intragenic modifier of the I244T mutation, with the resulting protein undergoing stable interaction with molecular chaperones and aggregation. This aggregation was temperature-sensitive. AGXT*LTM expressed in Escherichia coli, as a GST-fusion protein, and in insect cells could be purified and retained enzymatic activity. Among various chemical chaperones tested in cell culture, betaine substantially improved the solubility of the mutant protein and the enzymatic activity in cell lysates. In summary, I244T, the second most common mutation responsible for PH1, is a protein conformational disease that may benefit from new therapies with pharmacological chaperones or small molecules to minimize protein aggregation. PMID:12777626

Primary hyperoxaluria type 1 in the Canary Islands: a conformational disease due to I244T mutation in the P11L-containing alanine:glyoxylate aminotransferase.

PubMed

Santana, A; Salido, E; Torres, A; Shapiro, L J

2003-06-10

Primary hyperoxaluria type 1 (PH1) is an inborn error of metabolism resulting from a deficiency of alanine:glyoxylate aminotransferase (AGXT; EC 2.6.1.44). Most of the PH1 alleles detected in the Canary Islands carry the Ile-244 --> Thr (I244T) mutation in the AGXT gene, with 14 of 16 patients homozygous for this mutation. Four polymorphisms within AGXT and regional microsatellites also were shared in their haplotypes (AGXT*LTM), consistent with a founder effect. The consequences of these amino acid changes were investigated. Although I244T alone did not affect AGXT activity or subcellular localization, when present in the same protein molecule as Leu-11 --> Pro (L11P), it resulted in loss of enzymatic activity in soluble cell extracts. Like its normal counterpart, the AGXT*LTM protein was present in the peroxisomes but it was insoluble in detergent-free buffers. The polymorphism L11P behaved as an intragenic modifier of the I244T mutation, with the resulting protein undergoing stable interaction with molecular chaperones and aggregation. This aggregation was temperature-sensitive. AGXT*LTM expressed in Escherichia coli, as a GST-fusion protein, and in insect cells could be purified and retained enzymatic activity. Among various chemical chaperones tested in cell culture, betaine substantially improved the solubility of the mutant protein and the enzymatic activity in cell lysates. In summary, I244T, the second most common mutation responsible for PH1, is a protein conformational disease that may benefit from new therapies with pharmacological chaperones or small molecules to minimize protein aggregation.

Mutational Analysis of Influenza A Virus Nucleoprotein: Identification of Mutations That Affect RNA Replication

PubMed Central

Mena, Ignacio; Jambrina, Enrique; Albo, Carmen; Perales, Beatriz; Ortín, Juan; Arrese, Marta; Vallejo, Dolores; Portela, Agustín

1999-01-01

The influenza A virus nucleoprotein (NP) is a multifunctional polypeptide which plays a pivotal role in virus replication. To get information on the domains and specific residues involved in the different NP activities, we describe here the preparation and characterization of 20 influenza A virus mutant NPs. The mutations, mostly single-amino-acid substitutions, were introduced in a cDNA copy of the A/Victoria/3/75 NP gene and, in most cases, affected residues located in regions that were highly conserved across the NPs of influenza A, B, and C viruses. The mutant NPs were characterized (i) in vivo (cell culture) by analyzing their intracellular localization and their functionality in replication, transcription, and expression of model RNA templates; and (ii) in vitro by analyzing their RNA-binding and sedimentation properties. The results obtained allowed us to identify both a mutant protein that accumulated in the cytoplasm and mutations that altered the functionality and/or the oligomerization state of the NP polypeptide. Among the mutations that reduced the NP capability to express chloramphenicol acetyltransferase protein from a model viral RNA (vRNA) template, some displayed a temperature-sensitive phenotype. Interestingly, four mutant NPs, which showed a reduced functionality in synthesizing cRNA molecules from a vRNA template, were fully competent to reconstitute complementary ribonucleoproteins (cRNPs) capable of synthesizing vRNAs, which in turn yielded mRNA molecules. Based on the phenotype of these mutants and on previously published observations, it is proposed that these mutant NPs have a reduced capability to interact with the polymerase complex and that this NP-polymerase interaction is responsible for making vRNPs switch from mRNA to cRNA synthesis. PMID:9882320

Novel gyrA point mutation in a strain of Escherichia coli resistant to fluoroquinolones but not to nalidixic acid.

PubMed

Cambau, E; Bordon, F; Collatz, E; Gutmann, L

1993-06-01

We have previously described a clinical isolate of Escherichia coli (Q2) that is highly resistant to fluoroquinolones (MIC of ciprofloxacin, 16 micrograms/ml) but susceptible to nalidixic acid (MIC of nalidixic acid, 4 micrograms/ml) (N. Moniot-Ville, J. Guibert, N. Moreau, J.F. Acar, E. Collatz, and L. Gutmann, Antimicrob. Agents Chemother. 35:519-523, 1991). Transformation of strain Q2 with a plasmid carrying the wild-type gyrA gene from E. coli K-12(pAFF801) resulted in a 32-fold decrease in the MIC of ciprofloxacin, suggesting that at least one mutation in gyrA was involved in the resistance of Q2. Intragenic gyrA fragments of 668 and 2,500 bp from strain Q2 were amplified by the polymerase chain reaction. We sequenced the 668-bp fragment and identified a single novel point mutation (transition from G to A at position 242), leading to an amino acid substitution (Gly-81 to Asp) in the gyrase A subunit. We constructed hybrid plasmids by substituting either the 668-bp fragment or the 2,500-bp fragment from Q2 DNA, both of which contained the gyrA point mutation, for the corresponding fragments in wild-type gyrA (2,625 bp) of E. coli K-12. When introduced into E. coli KNK453 (gyrA temperature sensitive), both plasmids conferred an eightfold increase in the MIC of ciprofloxacin, but only a twofold increase in the MIC of nalidixic acid. When introduced into E. coli Q2, neither plasmid conferred any change in the MICs of ciprofloxacin or nalidixic acid, suggesting that only the point mutation found in gyrA was involved in the resistance that we observed.

Sensitivity of the Pig-a assay for detecting gene mutation in rats exposed acutely to strong clastogens

PubMed Central

Dobrovolsky, Vasily N.

2013-01-01

Clastogens are potential human carcinogens whose detection by genotoxicity assays is important for safety assessment. Although some endogenous genes are sensitive to the mutagenicity of clastogens, many genes that are used as reporters for in vivo mutation (e.g. transgenes) are not. In this study, we have compared responses in the erythrocyte Pig-a gene mutation assay with responses in a gene mutation assay that is relatively sensitive to clastogens, the lymphocyte Hprt assay, and in the reticulocyte micronucleus (MN) assay, which provides a direct measurement of clastogenicity. Male F344 rats were treated acutely with X-rays, cyclophosphamide (CP) and Cis-platin (Cis-Pt), and the frequency of micronucleated reticulocytes (MN RETs) in peripheral blood was measured 1 or 2 days later. The frequencies of CD59-deficient Pig-a mutant erythrocytes and 6-thioguanine-resistant Hprt mutant T-lymphocytes were measured at several times up to 16 weeks after the exposure. All three clastogens induced strong increases in the frequency of MN RETs, with X-rays and Cis-Pt producing near linear dose responses. The three agents also were positive in the two gene mutation assays although the assays detected them with different efficiencies. The Pig-a assay was more efficient in detecting the effect of Cis-Pt treatment, whereas the Hprt assay was more efficient for X-rays and CP. The results indicate that the erythrocyte Pig-a assay can detect the in vivo mutagenicity of clastogens although its sensitivity is variable in comparison with the lymphocyte Hprt assay. PMID:23677247

A combination of immunohistochemistry and molecular approaches improves highly sensitive detection of BRAF mutations in papillary thyroid cancer.

PubMed

Martinuzzi, Claudia; Pastorino, Lorenza; Andreotti, Virginia; Garuti, Anna; Minuto, Michele; Fiocca, Roberto; Bianchi-Scarrà, Giovanna; Ghiorzo, Paola; Grillo, Federica; Mastracci, Luca

2016-09-01

The optimal method for BRAF mutation detection remains to be determined despite advances in molecular detection techniques. The aim of this study was to compare, against classical Sanger sequencing, the diagnostic performance of two of the most recently developed, highly sensitive methods: BRAF V600E immunohistochemistry (IHC) and peptide nucleic-acid (PNA)-clamp qPCR. BRAF exon 15 mutations were searched in formalin-fixed paraffin-embedded tissues from 86 papillary thyroid carcinoma using the three methods. The limits of detection of Sanger sequencing in borderline or discordant cases were quantified by next generation sequencing. BRAF mutations were found in 74.4 % of cases by PNA, in 71 % of cases by IHC, and in 64 % of cases by Sanger sequencing. Complete concordance for the three methods was observed in 80 % of samples. Better concordance was observed with the combination of two methods, particularly PNA and IHC (59/64) (92 %), while the combination of PNA and Sanger was concordant in 55 cases (86 %). Sensitivity of the three methods was 99 % for PNA, 94.2 % for IHC, and 89.5 % for Sanger. Our data show that IHC could be used as a cost-effective, first-line method for BRAF V600E detection in daily practice, followed by PNA analysis in negative or uninterpretable cases, as the most efficient method. PNA-clamp quantitative PCR is highly sensitive and complementary to IHC as it also recognizes other mutations besides V600E and it is suitable for diagnostic purposes.

SNaPshot and StripAssay as Valuable Alternatives to Direct Sequencing for KRAS Mutation Detection in Colon Cancer Routine Diagnostics

PubMed Central

Fariña Sarasqueta, Arantza; Moerland, Elna; de Bruyne, Hanneke; de Graaf, Henk; Vrancken, Tamara; van Lijnschoten, Gesina; van den Brule, Adriaan J.C.

2011-01-01

Although direct sequencing is the gold standard for KRAS mutation detection in routine diagnostics, it remains laborious, time consuming, and not very sensitive. Our objective was to evaluate SNaPshot and the KRAS StripAssay as alternatives to sequencing for KRAS mutation detection in daily practice. KRAS exon 2–specific PCR followed by sequencing or by a SNaPshot reaction was performed. For the StripAssay, a mutant-enriched PCR was followed by hybridization to KRAS-specific probes bound to a nitrocellulose strip. To test sensitivities, dilution series of mutated DNA in wild-type DNA were made. Additionally, direct sequencing and SNaPshot were evaluated in 296 colon cancer samples. Detection limits of direct sequencing, SNaPshot, and StripAssay were 20%, 10%, and 1% tumor cells, respectively. Direct sequencing and SNaPshot can detect all 12 mutations in KRAS codons 12 and 13, whereas the StripAssay detects 10 of the most frequent ones. Workload and time to results are comparable for SNaPshot and direct sequencing. SNaPshot is flexible and easy to multiplex. The StripAssay is less time consuming for daily laboratory practice. SNaPshot is more flexible and slightly more sensitive than direct sequencing. The clinical evaluation showed comparable performances between direct sequencing and SNaPshot. The StripAssay is rapid and an extremely sensitive assay that could be considered when few tumor cells are available. However, found mutants should be confirmed to avoid risk of false positives. PMID:21354055

Adaptations of an RNA virus to increasing thermal stress

PubMed Central

Singhal, Sonia; Leon Guerrero, Cierra M.; Whang, Stella G.; McClure, Erin M.; Busch, Hannah G.; Kerr, Benjamin

2017-01-01

Environments can change in incremental fashions, where a shift from one state to another occurs over multiple organismal generations. The rate of the environmental change is expected to influence how and how well populations adapt to the final environmental state. We used a model system, the lytic RNA bacteriophage Φ6, to investigate this question empirically. We evolved viruses for thermostability by exposing them to heat shocks that increased to a maximum temperature at different rates. We observed increases in the ability of many heat-shocked populations to survive high temperature heat shocks. On their first exposure to the highest temperature, populations that experienced a gradual increase in temperature had higher average survival than populations that experienced a rapid temperature increase. However, at the end of the experiment, neither the survival of populations at the highest temperature nor the number of mutations per population varied significantly according to the rate of thermal change. We also evaluated mutations from the endpoint populations for their effects on viral thermostability and growth. As expected, some mutations did increase viral thermostability. However, other mutations decreased thermostability but increased growth rate, suggesting that benefits of an increased replication rate may have sometimes outweighed the benefits of enhanced thermostability. Our study highlights the importance of considering the effects of multiple selective pressures, even in environments where a single factor changes. PMID:29267297

Neonatal diabetes caused by a homozygous KCNJ11 mutation demonstrates that tiny changes in ATP sensitivity markedly affect diabetes risk.

PubMed

Vedovato, Natascia; Cliff, Edward; Proks, Peter; Poovazhagi, Varadarajan; Flanagan, Sarah E; Ellard, Sian; Hattersley, Andrew T; Ashcroft, Frances M

2016-07-01

The pancreatic ATP-sensitive potassium (KATP) channel plays a pivotal role in linking beta cell metabolism to insulin secretion. Mutations in KATP channel genes can result in hypo- or hypersecretion of insulin, as in neonatal diabetes mellitus and congenital hyperinsulinism, respectively. To date, all patients affected by neonatal diabetes due to a mutation in the pore-forming subunit of the channel (Kir6.2, KCNJ11) are heterozygous for the mutation. Here, we report the first clinical case of neonatal diabetes caused by a homozygous KCNJ11 mutation. A male patient was diagnosed with diabetes shortly after birth. At 5 months of age, genetic testing revealed he carried a homozygous KCNJ11 mutation, G324R, (Kir6.2-G324R) and he was successfully transferred to sulfonylurea therapy (0.2 mg kg(-1) day(-1)). Neither heterozygous parent was affected. Functional properties of wild-type, heterozygous and homozygous mutant KATP channels were examined after heterologous expression in Xenopus oocytes. Functional studies indicated that the Kir6.2-G324R mutation reduces the channel ATP sensitivity but that the difference in ATP inhibition between homozygous and heterozygous channels is remarkably small. Nevertheless, the homozygous patient developed neonatal diabetes, whereas the heterozygous parents were, and remain, unaffected. Kir6.2-G324R channels were fully shut by the sulfonylurea tolbutamide, which explains why the patient's diabetes was well controlled by sulfonylurea therapy. The data demonstrate that tiny changes in KATP channel activity can alter beta cell electrical activity and insulin secretion sufficiently to cause diabetes. They also aid our understanding of how the Kir6.2-E23K variant predisposes to type 2 diabetes.

HIV-1 Clinical Isolates Resistant to CCR5 Antagonists Exhibit Delayed Entry Kinetics That Are Corrected in the Presence of Drug

PubMed Central

Putcharoen, Opass; Lee, Sun Hee; Henrich, Timothy J.; Hu, Zixin; Vanichanan, Jakapat; Coakley, Eoin; Greaves, Wayne; Gulick, Roy M.; Kuritzkes, Daniel R.

2012-01-01

HIV CCR5 antagonists select for env gene mutations that enable virus entry via drug-bound coreceptor. To investigate the mechanisms responsible for viral adaptation to drug-bound coreceptor-mediated entry, we studied viral isolates from three participants who developed CCR5 antagonist resistance during treatment with vicriviroc (VCV), an investigational small-molecule CCR5 antagonist. VCV-sensitive and -resistant viruses were isolated from one HIV subtype C- and two subtype B-infected participants; VCV-resistant isolates had mutations in the V3 loop of gp120 and were cross-resistant to TAK-779, an investigational antagonist, and maraviroc (MVC). All three resistant isolates contained a 306P mutation but had variable mutations elsewhere in the V3 stem. We used a virus-cell β-lactamase (BlaM) fusion assay to determine the entry kinetics of recombinant viruses that incorporated full-length VCV-sensitive and -resistant envelopes. VCV-resistant isolates exhibited delayed entry rates in the absence of drug, relative to pretherapy VCV-sensitive isolates. The addition of drug corrected these delays. These findings were generalizable across target cell types with a range of CD4 and CCR5 surface densities and were observed when either population-derived or clonal envelopes were used to construct recombinant viruses. V3 loop mutations alone were sufficient to restore virus entry in the presence of drug, and the accumulation of V3 mutations during VCV therapy led to progressively higher rates of viral entry. We propose that the restoration of pre-CCR5 antagonist therapy HIV entry kinetics drives the selection of V3 loop mutations and may represent a common mechanism that underlies the emergence of CCR5 antagonist resistance. PMID:22090117

Bias-Corrected Targeted Next-Generation Sequencing for Rapid, Multiplexed Detection of Actionable Alterations in Cell-Free DNA from Advanced Lung Cancer Patients.

PubMed

Paweletz, Cloud P; Sacher, Adrian G; Raymond, Chris K; Alden, Ryan S; O'Connell, Allison; Mach, Stacy L; Kuang, Yanan; Gandhi, Leena; Kirschmeier, Paul; English, Jessie M; Lim, Lee P; Jänne, Pasi A; Oxnard, Geoffrey R

2016-02-15

Tumor genotyping is a powerful tool for guiding non-small cell lung cancer (NSCLC) care; however, comprehensive tumor genotyping can be logistically cumbersome. To facilitate genotyping, we developed a next-generation sequencing (NGS) assay using a desktop sequencer to detect actionable mutations and rearrangements in cell-free plasma DNA (cfDNA). An NGS panel was developed targeting 11 driver oncogenes found in NSCLC. Targeted NGS was performed using a novel methodology that maximizes on-target reads, and minimizes artifact, and was validated on DNA dilutions derived from cell lines. Plasma NGS was then blindly performed on 48 patients with advanced, progressive NSCLC and a known tumor genotype, and explored in two patients with incomplete tumor genotyping. NGS could identify mutations present in DNA dilutions at ≥ 0.4% allelic frequency with 100% sensitivity/specificity. Plasma NGS detected a broad range of driver and resistance mutations, including ALK, ROS1, and RET rearrangements, HER2 insertions, and MET amplification, with 100% specificity. Sensitivity was 77% across 62 known driver and resistance mutations from the 48 cases; in 29 cases with common EGFR and KRAS mutations, sensitivity was similar to droplet digital PCR. In two cases with incomplete tumor genotyping, plasma NGS rapidly identified a novel EGFR exon 19 deletion and a missed case of MET amplification. Blinded to tumor genotype, this plasma NGS approach detected a broad range of targetable genomic alterations in NSCLC with no false positives including complex mutations like rearrangements and unexpected resistance mutations such as EGFR C797S. Through use of widely available vacutainers and a desktop sequencing platform, this assay has the potential to be implemented broadly for patient care and translational research. ©2015 American Association for Cancer Research.

Bias-corrected targeted next-generation sequencing for rapid, multiplexed detection of actionable alterations in cell-free DNA from advanced lung cancer patients

PubMed Central

Paweletz, Cloud P.; Sacher, Adrian G.; Raymond, Chris K.; Alden, Ryan S.; O'Connell, Allison; Mach, Stacy L.; Kuang, Yanan; Gandhi, Leena; Kirschmeier, Paul; English, Jessie M.; Lim, Lee P.; Jänne, Pasi A.; Oxnard, Geoffrey R.

2015-01-01

Purpose Tumor genotyping is a powerful tool for guiding non-small cell lung cancer (NSCLC) care, however comprehensive tumor genotyping can be logistically cumbersome. To facilitate genotyping, we developed a next-generation sequencing (NGS) assay using a desktop sequencer to detect actionable mutations and rearrangements in cell-free plasma DNA (cfDNA). Experimental Design An NGS panel was developed targeting 11 driver oncogenes found in NSCLC. Targeted NGS was performed using a novel methodology that maximizes on-target reads, and minimizes artifact, and was validated on DNA dilutions derived from cell lines. Plasma NGS was then blindly performed on 48 patients with advanced, progressive NSCLC and a known tumor genotype, and explored in two patients with incomplete tumor genotyping. Results NGS could identify mutations present in DNA dilutions at ≥0.4% allelic frequency with 100% sensitivity/specificity. Plasma NGS detected a broad range of driver and resistance mutations, including ALK, ROS1, and RET rearrangements, HER2 insertions, and MET amplification, with 100% specificity. Sensitivity was 77% across 62 known driver and resistance mutations from the 48 cases; in 29 cases with common EGFR and KRAS mutations, sensitivity was similar to droplet digital PCR. In two cases with incomplete tumor genotyping, plasma NGS rapidly identified a novel EGFR exon 19 deletion and a missed case of MET amplification. Conclusion Blinded to tumor genotype, this plasma NGS approach detected a broad range of targetable genomic alterations in NSCLC with no false positives including complex mutations like rearrangements and unexpected resistance mutations such as EGFR C797S. Through use of widely available vacutainers and a desktop sequencing platform, this assay has the potential to be implemented broadly for patient care and translational research. PMID:26459174

Evaluation of gefitinib efficacy according to body mass index, body surface area, and body weight in patients with EGFR-mutated advanced non-small cell lung cancer.

PubMed

Imai, Hisao; Kuwako, Tomohito; Kaira, Kyoichi; Masuda, Tomomi; Miura, Yosuke; Seki, Kaori; Sakurai, Reiko; Utsugi, Mitsuyoshi; Shimizu, Kimihiro; Sunaga, Noriaki; Tomizawa, Yoshio; Ishihara, Shinichi; Ishizuka, Takao; Mogi, Akira; Hisada, Takeshi; Minato, Koichi; Takise, Atsushi; Saito, Ryusei; Yamada, Masanobu

2017-03-01

In patients with epidermal growth factor receptor (EGFR)-mutated, advanced, non-small cell lung cancer (NSCLC), common gefitinib-sensitive EGFR mutations that predict a greater response to therapy include the exon 19 deletion and L858R point mutation. The objective of this study was to evaluate whether body surface area (BSA), body weight (BW), and body mass index (BMI) affect gefitinib efficacy in such patients. The medical charts of 138 consecutive patients with advanced NSCLC harboring sensitive EGFR mutations, who underwent gefitinib treatment, were reviewed. The median BSA and BW were used as cutoff values to evaluate their impact on gefitinib efficacy. BMI was categorized as underweight (<18.5 kg/m 2 ), normal (18.5-25 kg/m 2 ), and overweight (≥25 kg/m 2 ). The median BSA and BW were 1.48 m 2 and 53 kg, respectively. The overall response rate, progression-free survival (PFS), and overall survival (OS) were 65.2%, 12.2, and 24.2 months, respectively. There were no significant differences in clinical outcomes according to BSA, BW, or BMI alone. Subgroup analysis based on the mutation type and BSA revealed no significant differences in PFS between the groups; however, the median OS in those with exon 19 deletion combined with low BSA was significantly favorable compared with the other groups. Gefitinib efficacy in patients with NSCLC harboring sensitive EGFR mutations did not differ according to BSA, BW, and BMI. However, OS was superior in patients with both the exon 19 deletion and low BSA.

Hypertrophic cardiomyopathy-linked mutation in troponin T causes myofibrillar disarray and pro-arrhythmic action potential changes in human iPSC cardiomyocytes.

PubMed

Wang, Lili; Kim, Kyungsoo; Parikh, Shan; Cadar, Adrian Gabriel; Bersell, Kevin R; He, Huan; Pinto, Jose R; Kryshtal, Dmytro O; Knollmann, Bjorn C

2018-01-01

Mutations in cardiac troponin T (TnT) are linked to increased risk of ventricular arrhythmia and sudden death despite causing little to no cardiac hypertrophy. Studies in mice suggest that the hypertrophic cardiomyopathy (HCM)-associated TnT-I79N mutation increases myofilament Ca sensitivity and is arrhythmogenic, but whether findings from mice translate to human cardiomyocyte electrophysiology is not known. To study the effects of the TnT-I79N mutation in human cardiomyocytes. Using CRISPR/Cas9, the TnT-I79N mutation was introduced into human induced pluripotent stem cells (hiPSCs). We then used the matrigel mattress method to generate single rod-shaped cardiomyocytes (CMs) and studied contractility, Ca handling and electrophysiology. Compared to isogenic control hiPSC-CMs, TnT-I79N hiPSC-CMs exhibited sarcomere disorganization, increased systolic function and impaired relaxation. The Ca-dependence of contractility was leftward shifted in mutation containing cardiomyocytes, demonstrating increased myofilament Ca sensitivity. In voltage-clamped hiPSC-CMs, TnT-I79N reduced intracellular Ca transients by enhancing cytosolic Ca buffering. These changes in Ca handling resulted in beat-to-beat instability and triangulation of the cardiac action potential, which are predictors of arrhythmia risk. The myofilament Ca sensitizer EMD57033 produced similar action potential triangulation in control hiPSC-CMs. The TnT-I79N hiPSC-CM model not only reproduces key cellular features of TnT-linked HCM such as myofilament disarray, hypercontractility and diastolic dysfunction, but also suggests that this TnT mutation causes pro-arrhythmic changes of the human ventricular action potential. Copyright © 2017 Elsevier Ltd. All rights reserved.

A novel XPD mutation in a compound heterozygote; the mutation in the second allele is present in three homozygous patients with mild sun sensitivity.

PubMed

Falik-Zaccai, Tzipora C; Erel-Segal, Reut; Horev, Liran; Bitterman-Deutsch, Ora; Koka, Sivan; Chaim, Sara; Keren, Zohar; Kalfon, Limor; Gross, Bella; Segal, Zvi; Orgal, Shlomi; Shoval, Yishay; Slor, Hanoch; Spivak, Graciela; Hanawalt, Philip C

2012-08-01

The XPD protein plays a pivotal role in basal transcription and in nucleotide excision repair (NER) as one of the ten known components of the transcription factor TFIIH. Mutations in XPD can result in the DNA repair-deficient diseases xeroderma pigmentosum (XP), trichothiodystrophy (TTD), cerebro-oculo-facial-skeletal syndrome, and in combined phenotypes such as XP/Cockayne syndrome and XP/TTD. We describe here an 18-year-old individual with mild sun sensitivity, no neurological abnormalities and no tumors, who carries a p.R683Q mutation in one allele, and the novel p.R616Q mutation in the other allele of the XPD gene. We also describe four patients from one family, homozygous for the identical p.R683Q mutation in XPD, who exhibit mild skin pigmentation and loss of tendon reflexes. Three homozygous patients presented with late-onset skin tumors, and two with features of premature aging and moderate cognitive decline. Cells from the compound heterozygous individual and from one of the patients homozygous for p.R683Q exhibited similar responses to UV irradiation: reduced viability and defective overall removal of UV-induced cyclobutane pyrimidine dimers, implying deficient global genomic NER. Cells from the compound heterozygous subject also failed to recover RNA synthesis after UV, indicating defective transcription-coupled NER. Mutations affecting codon 616 in XPD generally result in functionally null proteins; we hypothesize that the phenotype of the heterozygous patient results solely from expression of the p.R683Q allele. This study illustrates the importance of detailed follow up with sun sensitive individuals, to ensure appropriate prophylaxis and to understand the mechanistic basis of the implicated hereditary disease. Copyright © 2012 Wiley Periodicals, Inc.

Comparative analyses of fungicide sensitivity and SSR marker variations indicate a low risk of developing azoxystrobin resistance in Phytophthora infestans

PubMed Central

Qin, Chun-Fang; He, Meng-Han; Chen, Feng-Ping; Zhu, Wen; Yang, Li-Na; Wu, E-Jiao; Guo, Zheng-Liang; Shang, Li-Ping; Zhan, Jiasui

2016-01-01

Knowledge of the evolution of fungicide resistance is important in securing sustainable disease management in agricultural systems. In this study, we analyzed and compared the spatial distribution of genetic variation in azoxystrobin sensitivity and SSR markers in 140 Phytophthora infestans isolates sampled from seven geographic locations in China. Sensitivity to azoxystrobin and its genetic variation in the pathogen populations was measured by the relative growth rate (RGR) at four fungicide concentrations and determination of the effective concentration for 50% inhibition (EC50). We found that all isolates in the current study were sensitive to azoxystrobin and their EC50 was similar to that detected from a European population about 20 years ago, suggesting the risk of developing azoxystrobin resistance in P. infestans populations is low. Further analyses indicate that reduced genetic variation and high fitness cost in resistant mutations are the likely causes for the low evolutionary likelihood of developing azoxystrobin resistance in the pathogen. We also found a negative correlation between azoxystrobin tolerance in P. infestans populations and the mean annual temperature of collection sites, suggesting that global warming may increase the efficiency of using the fungicide to control the late blight. PMID:26853908

Comparative analyses of fungicide sensitivity and SSR marker variations indicate a low risk of developing azoxystrobin resistance in Phytophthora infestans.

PubMed

Qin, Chun-Fang; He, Meng-Han; Chen, Feng-Ping; Zhu, Wen; Yang, Li-Na; Wu, E-Jiao; Guo, Zheng-Liang; Shang, Li-Ping; Zhan, Jiasui

2016-02-08

Knowledge of the evolution of fungicide resistance is important in securing sustainable disease management in agricultural systems. In this study, we analyzed and compared the spatial distribution of genetic variation in azoxystrobin sensitivity and SSR markers in 140 Phytophthora infestans isolates sampled from seven geographic locations in China. Sensitivity to azoxystrobin and its genetic variation in the pathogen populations was measured by the relative growth rate (RGR) at four fungicide concentrations and determination of the effective concentration for 50% inhibition (EC50). We found that all isolates in the current study were sensitive to azoxystrobin and their EC50 was similar to that detected from a European population about 20 years ago, suggesting the risk of developing azoxystrobin resistance in P. infestans populations is low. Further analyses indicate that reduced genetic variation and high fitness cost in resistant mutations are the likely causes for the low evolutionary likelihood of developing azoxystrobin resistance in the pathogen. We also found a negative correlation between azoxystrobin tolerance in P. infestans populations and the mean annual temperature of collection sites, suggesting that global warming may increase the efficiency of using the fungicide to control the late blight.

Protein Surface Softness Is the Origin of Enzyme Cold-Adaptation of Trypsin

PubMed Central

Isaksen, Geir Villy; Åqvist, Johan; Brandsdal, Bjørn Olav

2014-01-01

Life has effectively colonized most of our planet and extremophilic organisms require specialized enzymes to survive under harsh conditions. Cold-loving organisms (psychrophiles) express heat-labile enzymes that possess a high specific activity and catalytic efficiency at low temperatures. A remarkable universal characteristic of cold-active enzymes is that they show a reduction both in activation enthalpy and entropy, compared to mesophilic orthologs, which makes their reaction rates less sensitive to falling temperature. Despite significant efforts since the early 1970s, the important question of the origin of this effect still largely remains unanswered. Here we use cold- and warm-active trypsins as model systems to investigate the temperature dependence of the reaction rates with extensive molecular dynamics free energy simulations. The calculations quantitatively reproduce the catalytic rates of the two enzymes and further yield high-precision Arrhenius plots, which show the characteristic trends in activation enthalpy and entropy. Detailed structural analysis indicates that the relationship between these parameters and the 3D structure is reflected by significantly different internal protein energy changes during the reaction. The origin of this effect is not localized to the active site, but is found in the outer regions of the protein, where the cold-active enzyme has a higher degree of softness. Several structural mechanisms for softening the protein surface are identified, together with key mutations responsible for this effect. Our simulations further show that single point-mutations can significantly affect the thermodynamic activation parameters, indicating how these can be optimized by evolution. PMID:25165981

IDH1 R132H Mutation Enhances Cell Migration by Activating AKT-mTOR Signaling Pathway, but Sensitizes Cells to 5-FU Treatment as NADPH and GSH Are Reduced

PubMed Central

Qiu, Jiangdong; Huang, Keting; Wu, Mindan; Xia, Chunlin

2017-01-01

Aim of study Mutations of isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) gene were recently discovered in vast majority of World Health Organization (WHO) grade II/III gliomas. This study is to understand the effects of IDH1 R132H mutation in gliomagenesis and to develop new strategies to treat glioma with IDH1 R132H mutation. Materials and methods Over expression of IDH1 R132H in U87MG cells was done by transfecting cells with IDH1 R132H plasmid. MTT assay, scratch repair assay and western blot were performed to study effects of IDH1 R132H mutation on cell proliferation, migration, regulating AKT-mTOR signaling pathway and cell death respectively. NADP+/NADPH and GSH quantification assays were performed to evaluate effects of IDH1 R132H mutation on the production of antioxidant NADPH and GSH. Results We found that over expression of IDH1 R132H mutation decreased cell proliferation consistent with previous reports; however, it increased cell migration and enhanced AKT-mTOR signaling pathway activation. Mutations in isocitrate dehydrogenase (IDH) 1 also change the function of the enzymes and cause them to produce 2-hydroxyglutarate and not produce NADPH. We tested the level of NADPH and GSH and demonstrated that IDH1 R132H mutant stable cells had significantly low NADPH and GSH level compared to control or IDH1 wild type stable cells. The reduced antioxidants (NADPH and GSH) sensitized U87MG cells with IDH R132H mutant to 5-FU treatment. Conclusion Our study highlights the important role of IHD1 R132H mutant in up- regulating AKT-mTOR signaling pathway and enhancing cell migration. Furthermore, we demonstrate that IDH1 R132H mutation affects cellular redox status and sensitizes gliomas cells with IDH1 R132H mutation to 5FU treatment. PMID:28052098

Gene encoding a deubiquitinating enzyme is mutated in artesunate- and chloroquine-resistant rodent malaria parasites.

PubMed

Hunt, Paul; Afonso, Ana; Creasey, Alison; Culleton, Richard; Sidhu, Amar Bir Singh; Logan, John; Valderramos, Stephanie G; McNae, Iain; Cheesman, Sandra; do Rosario, Virgilio; Carter, Richard; Fidock, David A; Cravo, Pedro

2007-07-01

Artemisinin- and artesunate-resistant Plasmodium chabaudi mutants, AS-ART and AS-ATN, were previously selected from chloroquine-resistant clones AS-30CQ and AS-15CQ respectively. Now, a genetic cross between AS-ART and the artemisinin-sensitive clone AJ has been analysed by Linkage Group Selection. A genetic linkage group on chromosome 2 was selected under artemisinin treatment. Within this locus, we identified two different mutations in a gene encoding a deubiquitinating enzyme. A distinct mutation occurred in each of the clones AS-30CQ and AS-ATN, relative to their respective progenitors in the AS lineage. The mutations occurred independently in different clones under drug selection with chloroquine (high concentration) or artesunate. Each mutation maps to a critical residue in a homologous human deubiquitinating protein structure. Although one mutation could theoretically account for the resistance of AS-ATN to artemisinin derivates, the other cannot account solely for the resistance of AS-ART, relative to the responses of its sensitive progenitor AS-30CQ. Two lines of Plasmodium falciparum with decreased susceptibility to artemisinin were also selected. Their drug-response phenotype was not genetically stable. No mutations in the UBP-1 gene encoding the P. falciparum orthologue of the deubiquitinating enzyme were observed. The possible significance of these mutations in parasite responses to chloroquine or artemisinin is discussed.

Gene encoding a deubiquitinating enzyme is mutated in artesunate- and chloroquine-resistant rodent malaria parasites§

PubMed Central

Hunt, Paul; Afonso, Ana; Creasey, Alison; Culleton, Richard; Sidhu, Amar Bir Singh; Logan, John; Valderramos, Stephanie G; McNae, Iain; Cheesman, Sandra; do Rosario, Virgilio; Carter, Richard; Fidock, David A; Cravo, Pedro

2007-01-01

Artemisinin- and artesunate-resistant Plasmodium chabaudi mutants, AS-ART and AS-ATN, were previously selected from chloroquine-resistant clones AS-30CQ and AS-15CQ respectively. Now, a genetic cross between AS-ART and the artemisinin-sensitive clone AJ has been analysed by Linkage Group Selection. A genetic linkage group on chromosome 2 was selected under artemisinin treatment. Within this locus, we identified two different mutations in a gene encoding a deubiquitinating enzyme. A distinct mutation occurred in each of the clones AS-30CQ and AS-ATN, relative to their respective progenitors in the AS lineage. The mutations occurred independently in different clones under drug selection with chloroquine (high concentration) or artesunate. Each mutation maps to a critical residue in a homologous human deubiquitinating protein structure. Although one mutation could theoretically account for the resistance of AS-ATN to artemisinin derivates, the other cannot account solely for the resistance of AS-ART, relative to the responses of its sensitive progenitor AS-30CQ. Two lines of Plasmodium falciparum with decreased susceptibility to artemisinin were also selected. Their drug-response phenotype was not genetically stable. No mutations in the UBP-1 gene encoding the P. falciparum orthologue of the deubiquitinating enzyme were observed. The possible significance of these mutations in parasite responses to chloroquine or artemisinin is discussed. PMID:17581118

Tissue or blood: which is more suitable for detection of EGFR mutations in non-small cell lung cancer?

PubMed

Biaoxue, Rong; Shuanying, Yang

2018-01-01

Many studies have evaluated the accuracy of EGFR mutation status in blood against that in tumor tissues as the reference. We conducted this systematic review and meta-analysis to assess whether blood can be used as a substitute for tumor tissue in detecting EGFR mutations. Investigations that provided data on EGFR mutation status in blood were searched in the databases of Medline, Embase, Ovid Technologies and Web of Science. The detect efficiency of EGFR mutations in paired blood and tissues was compared using a random-effects model of meta-analysis. Pooled sensitivity and specificity and diagnostic accuracy were calculated by receiver operating characteristic curve. A total of 19 studies with 2,922 individuals were involved in this meta-analysis. The pooled results showed the positive detection rate of EGFR mutations in lung cancer tissues was remarkably higher than that of paired blood samples (odds ratio [OR] = 1.47, p<0.001). The pooled sensitivity and specificity of blood were 0.65 and 0.91, respectively, and the area under the receiver operating characteristic curve was 0.89. Although blood had a better specificity for detecting EGFR mutations, the absence of blood positivity should not necessarily be construed as confirmed negativity. Patients with negative results for blood should decidedly undergo further biopsies to ascertain EGFR mutations.

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

PubMed

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

2014-01-01

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

High resolution melting analysis for epidermal growth factor receptor mutations in formalin-fixed paraffin-embedded tissue and plasma free DNA from non-small cell lung cancer patients.

PubMed

Jing, Chang-Wen; Wang, Zhuo; Cao, Hai-Xia; Ma, Rong; Wu, Jian-Zhong

2014-01-01

The aim of the research was to explore a cost effective, fast, easy to perform, and sensitive method for epidermal growth factor receptor (EGFR) mutation testing. High resolution melting analysis (HRM) was introduced to evaluate the efficacy of the analysis for dectecting EGFR mutations in exons 18 to 21 using formalin-fixed paraffin-embedded (FFPE) tissues and plasma free DNA from 120 patients. The total EGFR mutation rate was 37.5% (45/120) detected by direct sequencing. There were 48 mutations in 120 FFPE tissues assessed by HRM. For plasma free DNA, the EGFR mutation rate was 25.8% (31/120). The sensitivity of HRM assays in FFPE samples was 100% by HRM. There was a low false-positive mutation rate but a high false-negative rate in plasma free DNA detected by HRM. Our results show that HRM analysis has the advantage of small tumor sample need. HRM applied with plasma free DNA showed a high false-negative rate but a low false-positive rate. Further research into appropriate methods and analysis needs to be performed before HRM for plasma free DNA could be accepted as an option in diagnostic or screening settings.

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

PubMed

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

2015-07-01

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

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

PubMed

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

2015-10-30

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

Tuning the free-energy landscape of a WW domain by temperature, mutation, and truncation

PubMed Central

Nguyen, Houbi; Jäger, Marcus; Moretto, Alessandro; Gruebele, Martin; Kelly, Jeffery W.

2003-01-01

The equilibrium unfolding of the Formin binding protein 28 (FBP) WW domain, a stable three-stranded β-sheet protein, can be described as reversible apparent two-state folding. Kinetics studied by laser temperature jump reveal a third state at temperatures below the midpoint of unfolding. The FBP free-energy surface can be tuned between three-state and two-state kinetics by changing the temperature, by truncation of the C terminus, or by selected point mutations. FBP WW domain is the smallest three-state folder studied to date and the only one that can be freely tuned between three-state and apparent two-state folding by several methods (temperature, truncation, and mutation). Its small size (28–37 residues), the availability of a quantitative reaction coordinate (φT), the fast folding time scale (10s of μs), and the tunability of the folding routes by small temperature or sequence changes make this system the ideal prototype for studying more subtle features of the folding free-energy landscape by simulations or analytical theory. PMID:12651955

Tuning the free-energy landscape of a WW domain by temperature, mutation, and truncation.

PubMed

Nguyen, Houbi; Jager, Marcus; Moretto, Alessandro; Gruebele, Martin; Kelly, Jeffery W

2003-04-01

The equilibrium unfolding of the Formin binding protein 28 (FBP) WW domain, a stable three-stranded beta-sheet protein, can be described as reversible apparent two-state folding. Kinetics studied by laser temperature jump reveal a third state at temperatures below the midpoint of unfolding. The FBP free-energy surface can be tuned between three-state and two-state kinetics by changing the temperature, by truncation of the C terminus, or by selected point mutations. FBP WW domain is the smallest three-state folder studied to date and the only one that can be freely tuned between three-state and apparent two-state folding by several methods (temperature, truncation, and mutation). Its small size (28-37 residues), the availability of a quantitative reaction coordinate (phi(T)), the fast folding time scale (10s of micros), and the tunability of the folding routes by small temperature or sequence changes make this system the ideal prototype for studying more subtle features of the folding free-energy landscape by simulations or analytical theory.

Next-Generation Genotyping by Digital PCR to Detect and Quantify the BRAF V600E Mutation in Melanoma Biopsies.

PubMed

Lamy, Pierre-Jean; Castan, Florence; Lozano, Nicolas; Montélion, Cécile; Audran, Patricia; Bibeau, Frédéric; Roques, Sylvie; Montels, Frédéric; Laberenne, Anne-Claire

2015-07-01

The detection of the BRAF V600E mutation in melanoma samples is used to select patients who should respond to BRAF inhibitors. Different techniques are routinely used to determine BRAF status in clinical samples. However, low tumor cellularity and tumor heterogeneity can affect the sensitivity of somatic mutation detection. Digital PCR (dPCR) is a next-generation genotyping method that clonally amplifies nucleic acids and allows the detection and quantification of rare mutations. Our aim was to evaluate the clinical routine performance of a new dPCR-based test to detect and quantify BRAF mutation load in 47 paraffin-embedded cutaneous melanoma biopsies. We compared the results obtained by dPCR with high-resolution melting curve analysis and pyrosequencing or with one of the allele-specific PCR methods available on the market. dPCR showed the lowest limit of detection. dPCR and allele-specific amplification detected the highest number of mutated samples. For the BRAF mutation load quantification both dPCR and pyrosequencing gave similar results with strong disparities in allele frequencies in the 47 tumor samples under study (from 0.7% to 79% of BRAF V600E mutations/sample). In conclusion, the four methods showed a high degree of concordance. dPCR was the more-sensitive method to reliably and easily detect mutations. Both pyrosequencing and dPCR could quantify the mutation load in heterogeneous tumor samples. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

High-resolution melting (HRM) assay for the detection of recurrent BRCA1/BRCA2 germline mutations in Tunisian breast/ovarian cancer families.

PubMed

Riahi, Aouatef; Kharrat, Maher; Lariani, Imen; Chaabouni-Bouhamed, Habiba

2014-12-01

Germline deleterious mutations in the BRCA1/BRCA2 genes are associated with an increased risk for the development of breast and ovarian cancer. Given the large size of these genes the detection of such mutations represents a considerable technical challenge. Therefore, the development of cost-effective and rapid methods to identify these mutations became a necessity. High resolution melting analysis (HRM) is a rapid and efficient technique extensively employed as high-throughput mutation scanning method. The purpose of our study was to assess the specificity and sensitivity of HRM for BRCA1 and BRCA2 genes scanning. As a first step we estimate the ability of HRM for detection mutations in a set of 21 heterozygous samples harboring 8 different known BRCA1/BRCA2 variations, all samples had been preliminarily investigated by direct sequencing, and then we performed a blinded analysis by HRM in a set of 68 further sporadic samples of unknown genotype. All tested heterozygous BRCA1/BRCA2 variants were easily identified. However the HRM assay revealed further alteration that we initially had not searched (one unclassified variant). Furthermore, sequencing confirmed all the HRM detected mutations in the set of unknown samples, including homozygous changes, indicating that in this cohort, with the optimized assays, the mutations detections sensitivity and specificity were 100 %. HRM is a simple, rapid and efficient scanning method for known and unknown BRCA1/BRCA2 germline mutations. Consequently the method will allow for the economical screening of recurrent mutations in Tunisian population.

Determination of ubiquitin fitness landscapes under different chemical stresses in a classroom setting.

PubMed

Mavor, David; Barlow, Kyle; Thompson, Samuel; Barad, Benjamin A; Bonny, Alain R; Cario, Clinton L; Gaskins, Garrett; Liu, Zairan; Deming, Laura; Axen, Seth D; Caceres, Elena; Chen, Weilin; Cuesta, Adolfo; Gate, Rachel E; Green, Evan M; Hulce, Kaitlin R; Ji, Weiyue; Kenner, Lillian R; Mensa, Bruk; Morinishi, Leanna S; Moss, Steven M; Mravic, Marco; Muir, Ryan K; Niekamp, Stefan; Nnadi, Chimno I; Palovcak, Eugene; Poss, Erin M; Ross, Tyler D; Salcedo, Eugenia C; See, Stephanie K; Subramaniam, Meena; Wong, Allison W; Li, Jennifer; Thorn, Kurt S; Conchúir, Shane Ó; Roscoe, Benjamin P; Chow, Eric D; DeRisi, Joseph L; Kortemme, Tanja; Bolon, Daniel N; Fraser, James S

2016-04-25

Ubiquitin is essential for eukaryotic life and varies in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies indicate that ubiquitin is highly tolerant to single mutations. We hypothesized that this tolerance would be reduced by chemically induced physiologic perturbations. To test this hypothesis, a class of first year UCSF graduate students employed deep mutational scanning to determine the fitness landscape of all possible single residue mutations in the presence of five different small molecule perturbations. These perturbations uncover 'shared sensitized positions' localized to areas around the hydrophobic patch and the C-terminus. In addition, we identified perturbation specific effects such as a sensitization of His68 in HU and a tolerance to mutation at Lys63 in DTT. Our data show how chemical stresses can reduce buffering effects in the ubiquitin proteasome system. Finally, this study demonstrates the potential of lab-based interdisciplinary graduate curriculum.

IDH1(R132H) mutation increases U87 glioma cell sensitivity to radiation therapy in hypoxia.

PubMed

Wang, Xiao-Wei; Labussière, Marianne; Valable, Samuel; Pérès, Elodie A; Guillamo, Jean-Sébastien; Bernaudin, Myriam; Sanson, Marc

2014-01-01

IDH1 codon 132 mutation (mostly Arg132His) is frequently found in gliomas and is associated with longer survival. However, it is still unclear whether IDH1 mutation renders the cell more vulnerable to current treatment, radio- and chemotherapy. We transduced U87 with wild type IDH1 or IDH1 (R132H) expressing lentivirus and analyzed the radiosensitivity (dose ranging 0 to 10 Gy) under normoxia (20% O2) and moderate hypoxia (1% O2). We observed that IDH1 (R132H) U87 cells grow faster in hypoxia and were more sensitive to radiotherapy (in terms of cell mortality and colony formation assay) compared to nontransduced U87 and IDH1 (wt) cells. This effect was not observed in normoxia. These data suggest that IDH1 (R132H) mutation increases radiosensitivity in mild hypoxic conditions.

Detection of AGXT bgene mutations by denaturing high-performance liquid chromatography for diagnosis of hyperoxaluria type 1.

PubMed

Pirulli, D; Giordano, M; Lessi, M; Spanò, A; Puzzer, D; Zezlina, S; Boniotto, M; Crovella, S; Florian, F; Marangella, M; Momigliano-Richiardi, P; Savoldi, S; Amoroso, A

2001-06-01

Primary hyperoxaluria type 1 is an autosomal recessive disorder of glyoxylate metabolism, caused by a deficiency of alanine:glyoxylate aminotransferase, which is encoded by a single copy gene (AGXT. The aim of this research was to standardize denaturing high-performance liquid chromatography, a new, sensitive, relatively inexpensive, and automated technique, for the detection of AGXT mutation. Denaturing high-performance liquid chromatography was used to analyze in blind the AGXT gene in 20 unrelated Italian patients with primary hyperoxaluria type I previously studied by other standard methods (single-strand conformation polymorphism analysis and direct sequencing) and 50 controls. Denaturing high-performance liquid chromatography allowed us to identify 13 mutations and the polymorphism at position 154 in exon I of the AGXT gene. Hence the method is more sensitive and less time consuming than single-strand conformation polymorphism analysis for the detection of AGXT mutations, thus representing a useful and reliable tool for detecting the mutations responsible for primary hyperoxaluria type 1. The new technology could also be helpful in the search for healthy carriers of AGXT mutations amongst family members and their partners, and for screening of AGXT polymorphisms in patients with nephrolithiasis and healthy populations.

The STAT3 HIES mutation is a gain-of-function mutation that activates genes via AGG-element carrying promoters.

PubMed

Xu, Li; Ji, Jin-Jun; Le, Wangping; Xu, Yan S; Dou, Dandan; Pan, Jieli; Jiao, Yifeng; Zhong, Tianfei; Wu, Dehong; Wang, Yumei; Wen, Chengping; Xie, Guan-Qun; Yao, Feng; Zhao, Heng; Fan, Yong-Sheng; Chin, Y Eugene

2015-10-15

Cytokine or growth factor activated STAT3 undergoes multiple post-translational modifications, dimerization and translocation into nuclei, where it binds to serum-inducible element (SIE, 'TTC(N3)GAA')-bearing promoters to activate transcription. The STAT3 DNA binding domain (DBD, 320-494) mutation in hyper immunoglobulin E syndrome (HIES), called the HIES mutation (R382Q, R382W or V463Δ), which elevates IgE synthesis, inhibits SIE binding activity and sensitizes genes such as TNF-α for expression. However, the mechanism by which the HIES mutation sensitizes STAT3 in gene induction remains elusive. Here, we report that STAT3 binds directly to the AGG-element with the consensus sequence 'AGG(N3)AGG'. Surprisingly, the helical N-terminal region (1-355), rather than the canonical STAT3 DBD, is responsible for AGG-element binding. The HIES mutation markedly enhances STAT3 AGG-element binding and AGG-promoter activation activity. Thus, STAT3 is a dual specificity transcription factor that promotes gene expression not only via SIE- but also AGG-promoter activity. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

The anisotropy1 D604N Mutation in the Arabidopsis Cellulose Synthase1 Catalytic Domain Reduces Cell Wall Crystallinity and the Velocity of Cellulose Synthase Complexes1[W][OA

PubMed Central

Fujita, Miki; Himmelspach, Regina; Ward, Juliet; Whittington, Angela; Hasenbein, Nortrud; Liu, Christine; Truong, Thy T.; Galway, Moira E.; Mansfield, Shawn D.; Hocart, Charles H.; Wasteneys, Geoffrey O.

2013-01-01

Multiple cellulose synthase (CesA) subunits assemble into plasma membrane complexes responsible for cellulose production. In the Arabidopsis (Arabidopsis thaliana) model system, we identified a novel D604N missense mutation, designated anisotropy1 (any1), in the essential primary cell wall CesA1. Most previously identified CesA1 mutants show severe constitutive or conditional phenotypes such as embryo lethality or arrest of cellulose production but any1 plants are viable and produce seeds, thus permitting the study of CesA1 function. The dwarf mutants have reduced anisotropic growth of roots, aerial organs, and trichomes. Interestingly, cellulose microfibrils were disordered only in the epidermal cells of the any1 inflorescence stem, whereas they were transverse to the growth axis in other tissues of the stem and in all elongated cell types of roots and dark-grown hypocotyls. Overall cellulose content was not altered but both cell wall crystallinity and the velocity of cellulose synthase complexes were reduced in any1. We crossed any1 with the temperature-sensitive radial swelling1-1 (rsw1-1) CesA1 mutant and observed partial complementation of the any1 phenotype in the transheterozygotes at rsw1-1’s permissive temperature (21°C) and full complementation by any1 of the conditional rsw1-1 root swelling phenotype at the restrictive temperature (29°C). In rsw1-1 homozygotes at restrictive temperature, a striking dissociation of cellulose synthase complexes from the plasma membrane was accompanied by greatly diminished motility of intracellular cellulose synthase-containing compartments. Neither phenomenon was observed in the any1 rsw1-1 transheterozygotes, suggesting that the proteins encoded by the any1 allele replace those encoded by rsw1-1 at restrictive temperature. PMID:23532584

The anisotropy1 D604N mutation in the Arabidopsis cellulose synthase1 catalytic domain reduces cell wall crystallinity and the velocity of cellulose synthase complexes.

PubMed

Fujita, Miki; Himmelspach, Regina; Ward, Juliet; Whittington, Angela; Hasenbein, Nortrud; Liu, Christine; Truong, Thy T; Galway, Moira E; Mansfield, Shawn D; Hocart, Charles H; Wasteneys, Geoffrey O

2013-05-01

Multiple cellulose synthase (CesA) subunits assemble into plasma membrane complexes responsible for cellulose production. In the Arabidopsis (Arabidopsis thaliana) model system, we identified a novel D604N missense mutation, designated anisotropy1 (any1), in the essential primary cell wall CesA1. Most previously identified CesA1 mutants show severe constitutive or conditional phenotypes such as embryo lethality or arrest of cellulose production but any1 plants are viable and produce seeds, thus permitting the study of CesA1 function. The dwarf mutants have reduced anisotropic growth of roots, aerial organs, and trichomes. Interestingly, cellulose microfibrils were disordered only in the epidermal cells of the any1 inflorescence stem, whereas they were transverse to the growth axis in other tissues of the stem and in all elongated cell types of roots and dark-grown hypocotyls. Overall cellulose content was not altered but both cell wall crystallinity and the velocity of cellulose synthase complexes were reduced in any1. We crossed any1 with the temperature-sensitive radial swelling1-1 (rsw1-1) CesA1 mutant and observed partial complementation of the any1 phenotype in the transheterozygotes at rsw1-1's permissive temperature (21°C) and full complementation by any1 of the conditional rsw1-1 root swelling phenotype at the restrictive temperature (29°C). In rsw1-1 homozygotes at restrictive temperature, a striking dissociation of cellulose synthase complexes from the plasma membrane was accompanied by greatly diminished motility of intracellular cellulose synthase-containing compartments. Neither phenomenon was observed in the any1 rsw1-1 transheterozygotes, suggesting that the proteins encoded by the any1 allele replace those encoded by rsw1-1 at restrictive temperature.

Flexible Lab-Tailored Cut-Offs for Suitability of Formalin-Fixed Tumor Samples for Diagnostic Mutational Analyses

PubMed Central

Mariani, Sara; Tondat, Fabrizio; Pacchioni, Donatella; Molinaro, Luca; Barreca, Antonella; Macrì, Luigia; Chiusa, Luigi; di Celle, Paola Francia; Cassoni, Paola; Sapino, Anna

2015-01-01

The selection of proper tissues from formalin-fixed and paraffin-embedded tumors before diagnostic molecular testing is responsibility of the pathologist and represents a crucial step to produce reliable test results. The international guidelines suggest two cut-offs, one for the percentage and one for the number of tumor cells, in order to enrich the tumor content before DNA extraction. The aim of the present work was two-fold: to evaluate to what extent a low percentage or absolute number of tumor cells can be qualified for somatic mutation testing; and to determine how assay sensitivities can guide pathologists towards a better definition of morphology-based adequacy cut-offs. We tested 1797 tumor specimens from melanomas, colorectal and lung adenocarcinomas. Respectively, their BRAF, K-RAS and EGFR genes were analyzed at specific exons by mutation-enriched PCR, pyrosequencing, direct sequencing and real-time PCR methods. We demonstrate that poorly cellular specimens do not modify the frequency distribution of either mutated or wild-type DNA samples nor that of specific mutations. This observation suggests that currently recommended cut-offs for adequacy of specimens to be processed for molecular assays seem to be too much stringent in a laboratory context that performs highly sensitive routine analytical methods. In conclusion, new cut-offs are needed based on test sensitivities and documented tumor heterogeneity. PMID:25844806

Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations.

PubMed

Zonta, Eleonora; Garlan, Fanny; Pécuchet, Nicolas; Perez-Toralla, Karla; Caen, Ouriel; Milbury, Coren; Didelot, Audrey; Fabre, Elizabeth; Blons, Hélène; Laurent-Puig, Pierre; Taly, Valérie

2016-01-01

In cancer research, the accuracy of the technology used for biomarkers detection is remarkably important. In this context, digital PCR represents a highly sensitive and reproducible method that could serve as an appropriate tool for tumor mutational status analysis. In particular, droplet-based digital PCR approaches have been developed for detection of tumor-specific mutated alleles within plasmatic circulating DNA. Such an approach calls for the development and validation of a very significant quantity of assays, which can be extremely costly and time consuming. Herein, we evaluated assays for the detection and quantification of various mutations occurring in three genes often misregulated in cancers: the epidermal growth factor receptor (EGFR), the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and the Tumoral Protein p53 (TP53) genes. In particular, commercial competitive allele-specific TaqMan® PCR (castPCR™) technology, as well as TaqMan® and ZEN™ assays, have been evaluated for EGFR p.L858R, p.T790M, p.L861Q point mutations and in-frame deletions Del19. Specificity and sensitivity have been determined on cell lines DNA, plasmatic circulating DNA of lung cancer patients or Horizon Diagnostics Reference Standards. To show the multiplexing capabilities of this technology, several multiplex panels for EGFR (several three- and four-plexes) have been developed, offering new "ready-to-use" tests for lung cancer patients.

Whole exome sequencing is an efficient, sensitive and specific method for determining the genetic cause of short-rib thoracic dystrophies.

PubMed

McInerney-Leo, A M; Harris, J E; Leo, P J; Marshall, M S; Gardiner, B; Kinning, E; Leong, H Y; McKenzie, F; Ong, W P; Vodopiutz, J; Wicking, C; Brown, M A; Zankl, A; Duncan, E L

2015-12-01

Short-rib thoracic dystrophies (SRTDs) are congenital disorders due to defects in primary cilium function. SRTDs are recessively inherited with mutations identified in 14 genes to date (comprising 398 exons). Conventional mutation detection (usually by iterative Sanger sequencing) is inefficient and expensive, and often not undertaken. Whole exome massive parallel sequencing has been used to identify new genes for SRTD (WDR34, WDR60 and IFT172); however, the clinical utility of whole exome sequencing (WES) has not been established. WES was performed in 11 individuals with SRTDs. Compound heterozygous or homozygous mutations were identified in six confirmed SRTD genes in 10 individuals (IFT172, DYNC2H1, TTC21B, WDR60, WDR34 and NEK1), giving overall sensitivity of 90.9%. WES data from 993 unaffected individuals sequenced using similar technology showed two individuals with rare (minor allele frequency <0.005) compound heterozygous variants of unknown significance in SRTD genes (specificity >99%). Costs for consumables, laboratory processing and bioinformatic analysis were

Abscisic Acid Deficiency Antagonizes High-Temperature Inhibition of Disease Resistance through Enhancing Nuclear Accumulation of Resistance Proteins SNC1 and RPS4 in Arabidopsis[C][W

PubMed Central

Mang, Hyung-Gon; Qian, Weiqiang; Zhu, Ying; Qian, Jun; Kang, Hong-Gu; Klessig, Daniel F.; Hua, Jian

2012-01-01

Plant defense responses to pathogens are influenced by abiotic factors, including temperature. Elevated temperatures often inhibit the activities of disease resistance proteins and the defense responses they mediate. A mutant screen with an Arabidopsis thaliana temperature-sensitive autoimmune mutant bonzai1 revealed that the abscisic acid (ABA)–deficient mutant aba2 enhances resistance mediated by the resistance (R) gene SUPPRESSOR OF npr1-1 CONSTITUTIVE1 (SNC1) at high temperature. ABA deficiency promoted nuclear accumulation of SNC1, which was essential for it to function at low and high temperatures. Furthermore, the effect of ABA deficiency on SNC1 protein accumulation is independent of salicylic acid, whose effects are often antagonized by ABA. ABA deficiency also promotes the activity and nuclear localization of R protein RESISTANCE TO PSEUDOMONAS SYRINGAE4 at higher temperature, suggesting that the effect of ABA on R protein localization and nuclear activity is rather broad. By contrast, mutations that confer ABA insensitivity did not promote defense responses at high temperature, suggesting either tissue specificity of ABA signaling or a role of ABA in defense regulation independent of the core ABA signaling machinery. Taken together, this study reveals a new intersection between ABA and disease resistance through R protein localization and provides further evidence of antagonism between abiotic and biotic responses. PMID:22454454

Ultra-deep sequencing confirms immunohistochemistry as a highly sensitive and specific method for detecting BRAF V600E mutations in colorectal carcinoma.

PubMed

Rössle, Matthias; Sigg, Michèle; Rüschoff, Jan H; Wild, Peter J; Moch, Holger; Weber, Achim; Rechsteiner, Markus P

2013-11-01

The activating BRAF (V600) mutation is a well-established negative prognostic biomarker in metastatic colorectal carcinoma (CRC). A recently developed monoclonal mouse antibody (clone VE1) has been shown to detect reliably BRAF (V600E) mutated protein by immunohistochemistry (IHC). In this study, we aimed to compare the detection of BRAF (V600E) mutations by IHC, Sanger sequencing (SaS), and ultra-deep sequencing (UDS) in CRC. VE1-IHC was established in a cohort of 68 KRAS wild-type CRCs. The VE1-IHC was only positive in the three patients with a known BRAF (V600E) mutation as assessed by SaS and UDS. The test cohort consisted of 265 non-selected, consecutive CRC samples. Thirty-nine out of 265 cases (14.7%) were positive by VE1-IHC. SaS of 20 randomly selected IHC negative tumors showed BRAF wild-type (20/20). Twenty-four IHC-positive cases were confirmed by SaS (24/39; 61.5%) and 15 IHC-positive cases (15/39; 38.5%) showed a BRAF wild-type by SaS. UDS detected a BRAF (V600E) mutation in 13 of these 15 discordant cases. In one tumor, the mutation frequency was below our threshold for UDS positivity, while in another case, UDS could not be performed due to low DNA amount. Statistical analysis showed sensitivities of 100% and 63% and specificities of 95 and 100% for VE1-IHC and SaS, respectively, compared to combined results of SaS and UDS. Our data suggests that there is high concordance between UDS and IHC using the anti-BRAF(V600E) (VE1) antibody. Thus, VE1 immunohistochemistry is a highly sensitive and specific method in detecting BRAF (V600E) mutations in colorectal carcinoma.

PEST domain mutations in Notch receptors comprise an oncogenic driver segment in triple-negative breast cancer sensitive to a γ-secretase inhibitor.

PubMed

Wang, Kai; Zhang, Qin; Li, Danan; Ching, Keith; Zhang, Cathy; Zheng, Xianxian; Ozeck, Mark; Shi, Stephanie; Li, Xiaorong; Wang, Hui; Rejto, Paul; Christensen, James; Olson, Peter

2015-03-15

To identify and characterize novel, activating mutations in Notch receptors in breast cancer and to determine response to the gamma secretase inhibitor (GSI) PF-03084014. We used several computational approaches, including novel algorithms, to analyze next-generation sequencing data and related omic datasets from The Cancer Genome Atlas (TCGA) breast cancer cohort. Patient-derived xenograft (PDX) models were sequenced, and Notch-mutant models were treated with PF-03084014. Gene-expression and functional analyses were performed to study the mechanism of activation through mutation and inhibition by PF-03084014. We identified mutations within and upstream of the PEST domains of NOTCH1, NOTCH2, and NOTCH3 in the TCGA dataset. Mutations occurred via several genetic mechanisms and compromised the function of the PEST domain, a negative regulatory domain commonly mutated in other cancers. Focal amplifications of NOTCH2 and NOTCH3 were also observed, as were heterodimerization or extracellular domain mutations at lower incidence. Mutations and amplifications often activated the Notch pathway as evidenced by increased expression of canonical Notch target genes, and functional mutations were significantly enriched in the triple-negative breast cancer subtype (TNBC). PDX models were also identified that harbored PEST domain mutations, and these models were highly sensitive to PF-03084014. This work suggests that Notch-altered breast cancer constitutes a bona fide oncogenic driver segment with the most common alteration being PEST domain mutations present in multiple Notch receptors. Importantly, functional studies suggest that this newly identified class can be targeted with Notch inhibitors, including GSIs. ©2015 American Association for Cancer Research.

Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues.

PubMed

Chat-Uthai, Nunthawut; Vejvisithsakul, Pichpisith; Udommethaporn, Sutthirat; Meesiri, Puttarakun; Danthanawanit, Chetiya; Wongchai, Yannawan; Teerapakpinyo, Chinachote; Shuangshoti, Shanop; Poungvarin, Naravat

2018-01-01

The protein kinase BRAF is one of the key players in regulating cellular responses to extracellular signals. Somatic mutations of the BRAF gene, causing constitutive activation of BRAF, have been found in various types of human cancers such as malignant melanoma, and colorectal cancer. BRAF V600E and V600K, most commonly observed mutations in these cancers, may predict response to targeted therapies. Many techniques suffer from a lack of diagnostic sensitivity in mutation analysis in clinical samples with a low cancer cell percentage or poor-quality fragmented DNA. Here we present allele-specific real-time PCR assay for amplifying 35- to 45-base target sequences in BRAF gene. Forward primer designed for BRAF V600E detection is capable of recognizing both types of BRAF V600E mutation, i.e. V600E1 (c.1799T>A) and V600E2 (c.1799_1800delTGinsAA), as well as complex tandem mutation caused by nucleotide changes in codons 600 and 601. We utilized this assay to analyze Thai formalin-fixed paraffin-embedded tissues. Forty-eight percent of 178 Thai colorectal cancer tissues has KRAS mutation detected by highly sensitive commercial assays. Although these DNA samples contain low overall yield of amplifiable DNA, our newly-developed assay successfully revealed BRAF V600 mutations in 6 of 93 formalin-fixed paraffin-embedded colorectal cancer tissues which KRAS mutation was not detected. Ultra-short PCR assay with forward mutation-specific primers is potentially useful to detect BRAF V600 mutations in highly fragmented DNA specimens from cancer patients.

Detection of Rare Mutations in EGFR-ARMS-PCR-Negative Lung Adenocarcinoma by Sanger Sequencing.

PubMed

Liang, Chaoyue; Wu, Zhuolin; Gan, Xiaohong; Liu, Yuanbin; You, You; Liu, Chenxian; Zhou, Chengzhi; Liang, Ying; Mo, Haiyun; Chen, Allen M; Zhang, Jiexia

2018-01-01

This study aimed to identify potential epidermal growth factor receptor (EGFR) gene mutations in non-small cell lung cancer that went undetected by amplification refractory mutation system-Scorpion real-time PCR (ARMS-PCR). A total of 200 specimens were obtained from the First Affiliated Hospital of Guangzhou Medical University from August 2014 to August 2015. In total, 100 ARMS-negative and 100 ARMS-positive specimens were evaluated for EGFR gene mutations by Sanger sequencing. The methodology and sensitivity of each method and the outcomes of EGFR-tyrosine kinase inhibitor (TKI) therapy were analyzed. Among the 100 ARMS-PCR-positive samples, 90 were positive by Sanger sequencing, while 10 cases were considered negative, because the mutation abundance was less than 10%. Among the 100 negative cases, three were positive for a rare EGFR mutation by Sanger sequencing. In the curative effect analysis of EGFR-TKIs, the progression-free survival (PFS) analysis based on ARMS and Sanger sequencing results showed no difference. However, the PFS of patients with a high abundance of EGFR mutation was 12.4 months [95% confidence interval (CI), 11.6-12.4 months], which was significantly higher than that of patients with a low abundance of mutations detected by Sanger sequencing (95% CI, 10.7-11.3 months) (p<0.001). The ARMS method demonstrated higher sensitivity than Sanger sequencing, but was prone to missing mutations due to primer design. Sanger sequencing was able to detect rare EGFR mutations and deemed applicable for confirming EGFR status. A clinical trial evaluating the efficacy of EGFR-TKIs in patients with rare EGFR mutations is needed. © Copyright: Yonsei University College of Medicine 2018

High-resolution melting analysis for noninvasive prenatal diagnosis of IVS-II-I (G-A) fetal DNA in minor beta-thalassemia mothers.

PubMed

Zafari, Mandana; Gill, Pooria; Kowsaryan, Mehrnoush; Alipour, Abbass; Banihashemi, Ali

2016-10-01

The high-resolution melting (HRM) technique is fast, effective and successful method for mutation detection. The aim of this study was to determine the sensitivity and specificity of the HRM method for detection of a paternally inherited mutation in a fetus as a noninvasive prenatal diagnosis of β-thalassemia. Genomic DNAs were prepared from 50 β-thalassemia minor couples whose pregnancy was at risk for homozygous β-thalassemia. Ten milliliters of the maternal blood from each pregnant woman were collected and after separating plasma stored at -80 °C until analysis. The extracted DNAs were analyzed by HRM real-time PCR for detection of IVS-II-I (G-A) as a paternally inherited mutation. The gold standard was the result of a chorionic villus sampling by a standard reverse dot blotting test. The sensitivity and specificity of HRM real-time PCR were 92.6% and 82.6%, respectively. Also, the positive and negative predictive values were 86.2% and 90.47%, respectively. HRM real-time PCR was a sensitive and specific method for determining the paternally inherited mutation in the fetus at risk with thalassemia major.

Poly ADP-ribose polymerase-1 as a potential therapeutic target in Merkel cell carcinoma.

PubMed

Ferrarotto, Renata; Cardnell, Robert; Su, Shirley; Diao, Lixia; Eterovic, A Karina; Prieto, Victor; Morrisson, William H; Wang, Jing; Kies, Merrill S; Glisson, Bonnie S; Byers, Lauren Averett; Bell, Diana

2018-03-23

Patients with metastatic Merkel cell carcinoma are treated similarly to small cell lung cancer (SCLC). Poly ADP-ribose polymerase-1 (PARP1) is overexpressed in SCLC and response to PARP inhibitors have been reported in patients with SCLC. Our study explores PARP as a therapeutic target in Merkel cell carcinoma. We evaluated PARP1 expression and Merkel cell polyomavirus (MCPyV) in 19 patients with Merkel cell carcinoma. Target exome-sequencing was performed in 14 samples. Sensitivity to olaparib was tested in 4 Merkel cell carcinoma cell lines. Most Merkel cell carcinomas (74%) express PARP1 at high levels. Mutations in DNA-damage repair genes were identified in 9 samples (64%), occurred exclusively in head neck primaries, and correlated with TP53/RB1 mutations. The TP53/RB1 mutations were more frequent in MCPyV-negative tumors. Sensitivity to olaparib was seen in the Merkel cell carcinoma line with highest PARP1 expression. Based on PARP1 overexpression, DNA-damage repair gene mutations, platinum sensitivity, and activity of olaparib in a Merkel cell carcinoma line, clinical trials with PARP inhibitors are warranted in Merkel cell carcinoma. © 2018 Wiley Periodicals, Inc.

Performance Comparison of Bench-Top Next Generation Sequencers Using Microdroplet PCR-Based Enrichment for Targeted Sequencing in Patients with Autism Spectrum Disorder

PubMed Central

Okamoto, Nobuhiko; Nakashima, Mitsuko; Tsurusaki, Yoshinori; Miyake, Noriko; Saitsu, Hirotomo; Matsumoto, Naomichi

2013-01-01

Next-generation sequencing (NGS) combined with enrichment of target genes enables highly efficient and low-cost sequencing of multiple genes for genetic diseases. The aim of this study was to validate the accuracy and sensitivity of our method for comprehensive mutation detection in autism spectrum disorder (ASD). We assessed the performance of the bench-top Ion Torrent PGM and Illumina MiSeq platforms as optimized solutions for mutation detection, using microdroplet PCR-based enrichment of 62 ASD associated genes. Ten patients with known mutations were sequenced using NGS to validate the sensitivity of our method. The overall read quality was better with MiSeq, largely because of the increased indel-related error associated with PGM. The sensitivity of SNV detection was similar between the two platforms, suggesting they are both suitable for SNV detection in the human genome. Next, we used these methods to analyze 28 patients with ASD, and identified 22 novel variants in genes associated with ASD, with one mutation detected by MiSeq only. Thus, our results support the combination of target gene enrichment and NGS as a valuable molecular method for investigating rare variants in ASD. PMID:24066114

Molecular mechanism of a COOH-terminal gating determinant in the ROMK channel revealed by a Bartter's disease mutation

PubMed Central

Flagg, Thomas P; Yoo, Dana; Sciortino, Christopher M; Tate, Margaret; Romero, Michael F; Welling, Paul A

2002-01-01

The ROMK subtypes of inward-rectifier K+ channels mediate potassium secretion and regulate NaCl reabsorption in the kidney. Loss-of-function mutations in this pH-sensitive K+ channel cause Bartter's disease, a familial salt wasting nephropathy. One disease-causing mutation truncates the extreme COOH-terminus and induces a closed gating conformation. Here we identify a region within the deleted domain that plays an important role in pH-dependent gating. The domain contains a structural element that functionally interacts with the pH sensor in the cytoplasmic NH2-terminus to set a physiological range of pH sensitivity. Removal of the domain shifts the pKa towards alkaline pH values, causing channel inactivation under physiological conditions. Suppressor mutations within the pH sensor rescued channel gating and trans addition of the cognate peptide restored pH sensitivity. A specific interdomain interaction was revealed in an in vitro protein-protein binding assay between the NH2- and COOH-terminal cytoplasmic domains expressed as bacterial fusion proteins. These results provide new insights into the molecular mechanisms underlying Kir channel regulation and channel gating defects that are associated with Bartter's disease. PMID:12381810

What to do with thyroid nodules showing benign cytology and BRAF(V600E) mutation? A study based on clinical and radiologic features using a highly sensitive analytic method.

PubMed

Kim, Soo-Yeon; Kim, Eun-Kyung; Kwak, Jin Young; Moon, Hee Jung; Yoon, Jung Hyun

2015-02-01

BRAF(V600E) mutation analysis has been used as a complementary diagnostic tool to ultrasonography-guided, fine-needle aspiration (US-FNA) in the diagnosis of thyroid nodule with high specificity reported up to 100%. When highly sensitive analytic methods are used, however, false-positive results of BRAF(V600E) mutation analysis have been reported. In this study, we investigated the clinical, US features, and outcome of patients with thyroid nodules with benign cytology but positive BRAF(V600E) mutation using highly sensitive analytic methods from US-FNA. This study included 22 nodules in 22 patients (3 men, 19 women; mean age, 53 years) with benign cytology but positive BRAF(V600E) mutation from US-FNA. US features were categorized according to the internal components, echogenicity, margin, calcifications, and shape. Suspicious US features included markedly hypoechogenicity, noncircumscribed margins, micro or mixed calcifications, and nonparallel shape. Nodules were considered to have either concordant or discordant US features to benign cytology. Medical records and imaging studies were reviewed for final cytopathology results and outcomes during follow-up. Among the 22 nodules, 17 nodules were reviewed. Fifteen of 17 nodules were malignant, and 2 were benign. The benign nodules were confirmed as adenomatous hyperplasia with underlying lymphocytic thyroiditis and a fibrotic nodule with dense calcification. Thirteen of the 15 malignant nodules had 2 or more suspicious US features, and all 15 nodules were considered to have discordant cytology considering suspicious US features. Five nodules had been followed with US or US-FNA without resection, and did not show change in size or US features on follow-up US examinations. BRAF(V600E) mutation analysis is a highly sensitive diagnostic tool in the diagnosis of papillary thyroid carcinomas. In the management of thyroid nodules with benign cytology but positive BRAF(V600E) mutation, thyroidectomy should be considered in nodules which have 2 or more suspicious US features and are considered discordant on image-cytology correlation. Copyright © 2015 Elsevier Inc. All rights reserved.

The effect of icotinib combined with chemotherapy in untreated non-small-cell lung cancer that harbored EGFR-sensitive mutations in a real-life setting: a retrospective analysis.

PubMed

Wang, Lulu; Li, Yan; Li, Luchun; Wu, Zhijuan; Yang, Dan; Ma, Huiwen; Wang, Donglin

2018-01-01

This study was conducted to compare the efficacy of a combination of icotinib and chemotherapy with icotinib or chemotherapy alone in untreated non-small cell lung cancer (NSCLC) patients harboring epidermal growth factor receptor (EGFR)-sensitive mutations and to analyze the curative effect of different treatments on different genetic mutations (EGFR 19 exon deletion and L858R mutation) in a real-life setting. One hundred ninety-one patients were studied in this retrospective analysis from January 2013 to December 2015. The baseline characteristics, curative effects and adverse events of patients were analyzed. The primary endpoint was progression free survival (PFS). Longer PFS and overall survival (OS), and better objective response rate (ORR) were observed in the combination group compared to icotinib or chemotherapy along. For patients with an EGFR 19 exon deletion, the PFS, OS, and ORR in the combination group were superior to those in the icotinib or chemotherapy group. For the patients with the EGFR L858R mutation, better PFS and ORR were observed in the combination group, but OS was not obviously prolonged. Grade 3 or 4 adverse events were most commonly reported with combination therapy or chemotherapy alone. No possible drug-related interstitial lung disease or of drug related deaths occurred. The combination of icotinib and chemotherapy in patients with untreated NSCLC harboring sensitive EGFR mutations resulted in improved PFS and OS, especially in those who harbored the EGFR exon 19 deletion.

G2019S LRRK2 mutant fibroblasts from Parkinson's disease patients show increased sensitivity to neurotoxin 1-methyl-4-phenylpyridinium dependent of autophagy.

PubMed

Yakhine-Diop, Sokhna M S; Bravo-San Pedro, José M; Gómez-Sánchez, Rubén; Pizarro-Estrella, Elisa; Rodríguez-Arribas, Mario; Climent, Vicente; Aiastui, Ana; López de Munain, Adolfo; Fuentes, José M; González-Polo, Rosa A

2014-10-03

Parkinson's disease (PD) is a neurodegenerative disorder of unknown etiology. It is considered as a multifactorial disease dependent on environmental and genetic factors. Deregulation in cell degradation has been related with a significant increase in cell damage, becoming a target for studies on the PD etiology. In the present study, we have characterized the parkinsonian toxin 1-methyl-4-phenylpyridinium ion (MPP(+))-induced damage in fibroblasts from Parkinson's patients with the mutation G2019S in leucine-rich repeat kinase 2 protein (LRRK2) and control individuals without this mutation. The results reveal that MPP(+) induces mTOR-dependent autophagy in fibroblasts. Moreover, the effects of caspase-dependent cell death to MPP(+) were higher in cells with the G2019S LRRK2 mutation, which showed basal levels of autophagy due to the G2019S LRRK2 mutation (mTOR-independent). The inhibition of autophagy by 3-methyladenine (3-MA) treatment reduces these sensitivity differences between both cell types, however, the inhibition of autophagosome-lysosome fusion by bafilomycin A1 (Baf A1) increases these differences. This data confirm the importance of the combination of genetic and environmental factors in the PD etiology. Thereby, the sensitivity to the same damage may be different in function of a genetic predisposition, reason why individuals with certain mutations can develop some early-onset diseases, such as individuals with G2019S LRRK2 mutation and PD. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Crizotinib Synergizes with Chemotherapy in Preclinical Models of Neuroblastoma

PubMed Central

Krytska, Kateryna; Ryles, Hannah T.; Sano, Renata; Raman, Pichai; Infarinato, Nicole R.; Hansel, Theodore D.; Makena, Monish R.; Song, Michael M.; Reynolds, C. Patrick; Mossé, Yael P.

2015-01-01

Purpose The presence of an ALK aberration correlates with inferior survival for patients with high-risk neuroblastoma. The emergence of ALK inhibitors such as crizotinib has provided novel treatment opportunities. However, certain ALK mutations result in de novo crizotinib resistance, and a phase I trial of crizotinib showed a lack of response in patients harboring those ALK mutations. Thus, understanding mechanisms of resistance and defining circumvention strategies for the clinic is critical. Experimental Design The sensitivity of human neuroblastoma-derived cell lines, cell line-derived and patient-derived xenograft (PDX) models with varying ALK statuses to crizotinib combined with topotecan and cyclophosphamide (topo/cyclo) was examined. Cultured cells and xenografts were evaluated for effects of these drugs on proliferation, signaling, and cell death, and assessment of synergy. Results In neuroblastoma murine xenografts harboring the most common ALK mutations, including those mutations associated with resistance to crizotinib (but not in those with wild-type ALK), crizotinib combined with topo/cyclo enhanced tumor responses and mouse event-free-survival. Crizotinib + topo/cyclo showed synergistic cytotoxicity and higher caspase-dependent apoptosis than crizotinib or topo/cyclo alone in neuroblastoma cell lines with ALK aberrations (mutation or amplification). Conclusions Combining crizotinib with chemotherapeutic agents commonly used in treating newly diagnosed patients with high-risk neuroblastoma restores sensitivity in preclinical models harboring both sensitive ALK aberrations and de novo resistant ALK mutations. These data support clinical testing of crizotinib and conventional chemotherapy with the goal of integrating ALK inhibition into multi-agent therapy for ALK-aberrant neuroblastoma patients. PMID:26438783

The effect of icotinib combined with chemotherapy in untreated non-small-cell lung cancer that harbored EGFR-sensitive mutations in a real-life setting: a retrospective analysis

PubMed Central

Wang, Lulu; Li, Yan; Li, Luchun; Wu, Zhijuan; Yang, Dan; Ma, Huiwen; Wang, Donglin

2018-01-01

Purpose This study was conducted to compare the efficacy of a combination of icotinib and chemotherapy with icotinib or chemotherapy alone in untreated non-small cell lung cancer (NSCLC) patients harboring epidermal growth factor receptor (EGFR)-sensitive mutations and to analyze the curative effect of different treatments on different genetic mutations (EGFR 19 exon deletion and L858R mutation) in a real-life setting. Patients and methods One hundred ninety-one patients were studied in this retrospective analysis from January 2013 to December 2015. The baseline characteristics, curative effects and adverse events of patients were analyzed. The primary endpoint was progression free survival (PFS). Results Longer PFS and overall survival (OS), and better objective response rate (ORR) were observed in the combination group compared to icotinib or chemotherapy along. For patients with an EGFR 19 exon deletion, the PFS, OS, and ORR in the combination group were superior to those in the icotinib or chemotherapy group. For the patients with the EGFR L858R mutation, better PFS and ORR were observed in the combination group, but OS was not obviously prolonged. Grade 3 or 4 adverse events were most commonly reported with combination therapy or chemotherapy alone. No possible drug-related interstitial lung disease or of drug related deaths occurred. Conclusion The combination of icotinib and chemotherapy in patients with untreated NSCLC harboring sensitive EGFR mutations resulted in improved PFS and OS, especially in those who harbored the EGFR exon 19 deletion. PMID:29731642

WNT activation by lithium abrogates TP53 mutation associated radiation resistance in medulloblastoma.

PubMed

Zhukova, Nataliya; Ramaswamy, Vijay; Remke, Marc; Martin, Dianna C; Castelo-Branco, Pedro; Zhang, Cindy H; Fraser, Michael; Tse, Ken; Poon, Raymond; Shih, David J H; Baskin, Berivan; Ray, Peter N; Bouffet, Eric; Dirks, Peter; von Bueren, Andre O; Pfaff, Elke; Korshunov, Andrey; Jones, David T W; Northcott, Paul A; Kool, Marcel; Pugh, Trevor J; Pomeroy, Scott L; Cho, Yoon-Jae; Pietsch, Torsten; Gessi, Marco; Rutkowski, Stefan; Bognár, Laszlo; Cho, Byung-Kyu; Eberhart, Charles G; Conter, Cecile Faure; Fouladi, Maryam; French, Pim J; Grajkowska, Wieslawa A; Gupta, Nalin; Hauser, Peter; Jabado, Nada; Vasiljevic, Alexandre; Jung, Shin; Kim, Seung-Ki; Klekner, Almos; Kumabe, Toshihiro; Lach, Boleslaw; Leonard, Jeffrey R; Liau, Linda M; Massimi, Luca; Pollack, Ian F; Ra, Young Shin; Rubin, Joshua B; Van Meir, Erwin G; Wang, Kyu-Chang; Weiss, William A; Zitterbart, Karel; Bristow, Robert G; Alman, Benjamin; Hawkins, Cynthia E; Malkin, David; Clifford, Steven C; Pfister, Stefan M; Taylor, Michael D; Tabori, Uri

2014-12-24

TP53 mutations confer subgroup specific poor survival for children with medulloblastoma. We hypothesized that WNT activation which is associated with improved survival for such children abrogates TP53 related radioresistance and can be used to sensitize TP53 mutant tumors for radiation. We examined the subgroup-specific role of TP53 mutations in a cohort of 314 patients treated with radiation. TP53 wild-type or mutant human medulloblastoma cell-lines and normal neural stem cells were used to test radioresistance of TP53 mutations and the radiosensitizing effect of WNT activation on tumors and the developing brain. Children with WNT/TP53 mutant medulloblastoma had higher 5-year survival than those with SHH/TP53 mutant tumours (100% and 36.6%±8.7%, respectively (p<0.001)). Introduction of TP53 mutation into medulloblastoma cells induced radioresistance (survival fractions at 2Gy (SF2) of 89%±2% vs. 57.4%±1.8% (p<0.01)). In contrast, β-catenin mutation sensitized TP53 mutant cells to radiation (p<0.05). Lithium, an activator of the WNT pathway, sensitized TP53 mutant medulloblastoma to radiation (SF2 of 43.5%±1.5% in lithium treated cells vs. 56.6±3% (p<0.01)) accompanied by increased number of γH2AX foci. Normal neural stem cells were protected from lithium induced radiation damage (SF2 of 33%±8% for lithium treated cells vs. 27%±3% for untreated controls (p=0.05). Poor survival of patients with TP53 mutant medulloblastoma may be related to radiation resistance. Since constitutive activation of the WNT pathway by lithium sensitizes TP53 mutant medulloblastoma cells and protect normal neural stem cells from radiation, this oral drug may represent an attractive novel therapy for high-risk medulloblastomas.

A compromised yeast RNA polymerase II enhances UV sensitivity in the absence of global genome nucleotide excision repair.

PubMed

Wong, J M; Ingles, C J

2001-02-01

Nucleotide excision repair is the major pathway responsible for removing UV-induced DNA damage, and is therefore essential for cell survival following exposure to UV radiation. In this report, we have assessed the contributions of some components of the RNA polymerase II (Pol II) transcription machinery to UV resistance in Saccharomyces cerevisiae. Deletion of the gene encoding the Pol II elongation factor TFIIS (SII) resulted in enhanced UV sensitivity, but only in the absence of global genome repair dependent on the RAD7 and RAD16 genes, a result seen previously with deletions of RAD26 and RAD28, yeast homologs of the human Cockayne syndrome genes CSB and CSA, respectively. A RAD7/16-dependent reduction in survival after UV irradiation was also seen in the presence of mutations in RNA Pol II that confer a defect in its response to SII, as well as with other mutations which reside in regions of the largest subunit of Pol II not involved in SII interactions. Indeed, an increase in UV sensitivity was achieved by simply decreasing the steadystate level of RNA Pol II. Truncation of the C-terminal domain and other RNA Pol II mutations conferred sensitivity to the ribonucleotide reductase inhibitor hydroxyurea and induction of RNR1 and RNR2 mRNAs after UV irradiation was attenuated in these mutant cells. That UV sensitivity can be a consequence of mutations in the RNA Pol II machinery in yeast cells suggests that alterations in transcriptional programs could underlie some of the pathophysiological defects seen in the human disease Cockayne syndrome.

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.

Knock-in mice harboring a Ca(2+) desensitizing mutation in cardiac troponin C develop early onset dilated cardiomyopathy.

PubMed

McConnell, Bradley K; Singh, Sonal; Fan, Qiying; Hernandez, Adriana; Portillo, Jesus P; Reiser, Peter J; Tikunova, Svetlana B

2015-01-01

The physiological consequences of aberrant Ca(2+) binding and exchange with cardiac myofilaments are not clearly understood. In order to examine the effect of decreasing Ca(2+) sensitivity of cTnC on cardiac function, we generated knock-in mice carrying a D73N mutation (not known to be associated with heart disease in human patients) in cTnC. The D73N mutation was engineered into the regulatory N-domain of cTnC in order to reduce Ca(2+) sensitivity of reconstituted thin filaments by increasing the rate of Ca(2+) dissociation. In addition, the D73N mutation drastically blunted the extent of Ca(2+) desensitization of reconstituted thin filaments induced by cTnI pseudo-phosphorylation. Compared to wild-type mice, heterozygous knock-in mice carrying the D73N mutation exhibited a substantially decreased Ca(2+) sensitivity of force development in skinned ventricular trabeculae. Kaplan-Meier survival analysis revealed that median survival time for knock-in mice was 12 weeks. Echocardiographic analysis revealed that knock-in mice exhibited increased left ventricular dimensions with thinner walls. Echocardiographic analysis also revealed that measures of systolic function, such as ejection fraction (EF) and fractional shortening (FS), were dramatically reduced in knock-in mice. In addition, knock-in mice displayed electrophysiological abnormalities, namely prolonged QRS and QT intervals. Furthermore, ventricular myocytes isolated from knock-in mice did not respond to β-adrenergic stimulation. Thus, knock-in mice developed pathological features similar to those observed in human patients with dilated cardiomyopathy (DCM). In conclusion, our results suggest that decreasing Ca(2+) sensitivity of the regulatory N-domain of cTnC is sufficient to trigger the development of DCM.

Inhibition of PI3K-AKT-mTOR pathway sensitizes endometrial cancer cell lines to PARP inhibitors.

PubMed

Philip, Charles-André; Laskov, Ido; Beauchamp, Marie-Claude; Marques, Maud; Amin, Oreekha; Bitharas, Joanna; Kessous, Roy; Kogan, Liron; Baloch, Tahira; Gotlieb, Walter H; Yasmeen, Amber

2017-09-08

Phosphatase and Tensin homolog (PTEN) is a tumor suppressor gene. Loss of its function is the most frequent genetic alteration in endometrioid endometrial cancers (70-80%) and high grade tumors (90%). We assessed the sensitivity of endometrial cancer cell lines to PARP inhibitors (olaparib and BMN-673) and a PI3K inhibitor (BKM-120), alone or in combination, in the context of their PTEN mutation status. We also highlighted a direct pathway linking PTEN to DNA repair. Using endometrial cancer cellular models with known PTEN status, we evaluated their homologous recombination (HR) functionality by RAD51 foci formation assay. The 50% Inhibitory concentration (IC50) of PI3K and PARP inhibitors in these cells was assessed, and western blotting was performed to determine the expression of proteins involved in the PI3K/mTOR pathway. Moreover, we explored the interaction between RAD51 and PI3K/mTOR by immunofluorescence. Next, the combination effect of PI3K and PARP inhibitors on cell proliferation was evaluated by a clonogenic assay. Cells with mutated PTEN showed over-activation of the PI3K/mTOR pathway. These cells were more sensitive to PARP inhibition compared to PTEN wild-type cells. In addition, PI3K inhibitor treatment reduced RAD51 foci formation in PTEN mutated cells, and sensitized these cells to PARP inhibitor. Targeting both PARP and PI3K might lead to improved personalized therapeutic approaches in endometrial cancer patients with PTEN mutations. Understanding the complex interaction of PTEN mutations with DNA repair in endometrial cancer will help to better select patients that are likely to respond to some of the new and costly targeted therapies.

Role of DNA protection and repair in resistance of Bacillus subtilis spores to ultrahigh shock pressures simulating hypervelocity impacts.

PubMed

Moeller, Ralf; Horneck, Gerda; Rabbow, Elke; Reitz, Günther; Meyer, Cornelia; Hornemann, Ulrich; Stöffler, Dieter

2008-11-01

Impact-induced ejections of rocks from planetary surfaces are frequent events in the early history of the terrestrial planets and have been considered as a possible first step in the potential interplanetary transfer of microorganisms. Spores of Bacillus subtilis were used as a model system to study the effects of a simulated impact-caused ejection on rock-colonizing microorganisms using a high-explosive plane wave setup. Embedded in different types of rock material, spores were subjected to extremely high shock pressures (5 to 50 GPa) lasting for fractions of microseconds to seconds. Nearly exponential pressure response curves were obtained for spore survival and linear dependency for the induction of sporulation-defective mutants. Spores of strains defective in major small, acid-soluble spore proteins (SASP) (alpha/beta-type SASP) that largely protect the spore DNA and spores of strains deficient in nonhomologous-end-joining DNA repair were significantly more sensitive to the applied shock pressure than were wild-type spores. These results indicate that DNA may be the sensitive target of spores exposed to ultrahigh shock pressures. To assess the nature of the critical physical parameter responsible for spore inactivation by ultrahigh shock pressures, the resulting peak temperature was varied by lowering the preshock temperature, changing the rock composition and porosity, or increasing the water content of the samples. Increased peak temperatures led to increased spore inactivation and reduced mutation rates. The data suggested that besides the potential mechanical stress exerted by the shock pressure, the accompanying high peak temperatures were a critical stress parameter that spores had to cope with.

Mutational analysis of the gag-pol junction of Moloney murine leukemia virus: requirements for expression of the gag-pol fusion protein.

PubMed Central

Felsenstein, K M; Goff, S P

1992-01-01

The gag-pol polyprotein of the murine and feline leukemia viruses is expressed by translational readthrough of a UAG terminator codon at the 3' end of the gag gene. To explore the cis-acting sequence requirements for the readthrough event in vivo, we generated a library of mutants of the Moloney murine leukemia virus with point mutations near the terminator codon and tested the mutant viral DNAs for the ability to direct synthesis of the gag-pol fusion protein and formation of infectious virus. The analysis showed that sequences 3' to the terminator are necessary and sufficient for the process. The results do not support a role for one proposed stem-loop structure that includes the terminator but are consistent with the involvement of another stem-loop 3' to the terminator. One mutant, containing two compensatory changes in this stem structure, was temperature sensitive for replication and for formation of the gag-pol protein. The results suggest that RNA sequence and structure are critical determinants of translational readthrough in vivo. Images PMID:1404606

HSP90 Shapes the Consequences of Human Genetic Variation.

PubMed

Karras, Georgios I; Yi, Song; Sahni, Nidhi; Fischer, Máté; Xie, Jenny; Vidal, Marc; D'Andrea, Alan D; Whitesell, Luke; Lindquist, Susan

2017-02-23

HSP90 acts as a protein-folding buffer that shapes the manifestations of genetic variation in model organisms. Whether HSP90 influences the consequences of mutations in humans, potentially modifying the clinical course of genetic diseases, remains unknown. By mining data for >1,500 disease-causing mutants, we found a strong correlation between reduced phenotypic severity and a dominant (HSP90 ≥ HSP70) increase in mutant engagement by HSP90. Examining the cancer predisposition syndrome Fanconi anemia in depth revealed that mutant FANCA proteins engaged predominantly by HSP70 had severely compromised function. In contrast, the function of less severe mutants was preserved by a dominant increase in HSP90 binding. Reducing HSP90's buffering capacity with inhibitors or febrile temperatures destabilized HSP90-buffered mutants, exacerbating FA-related chemosensitivities. Strikingly, a compensatory FANCA somatic mutation from an "experiment of nature" in monozygotic twins both prevented anemia and reduced HSP90 binding. These findings provide one plausible mechanism for the variable expressivity and environmental sensitivity of genetic diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

Sequence-specific "gene signatures" can be obtained by PCR with single specific primers at low stringency.

PubMed Central

Pena, S D; Barreto, G; Vago, A R; De Marco, L; Reinach, F C; Dias Neto, E; Simpson, A J

1994-01-01

Low-stringency single specific primer PCR (LSSP-PCR) is an extremely simple PCR-based technique that detects single or multiple mutations in gene-sized DNA fragments. A purified DNA fragment is subjected to PCR using high concentrations of a single specific oligonucleotide primer, large amounts of Taq polymerase, and a very low annealing temperature. Under these conditions the primer hybridizes specifically to its complementary region and nonspecifically to multiple sites within the fragment, in a sequence-dependent manner, producing a heterogeneous set of reaction products resolvable by electrophoresis. The complex banding pattern obtained is significantly altered by even a single-base change and thus constitutes a unique "gene signature." Therefore LSSP-PCR will have almost unlimited application in all fields of genetics and molecular medicine where rapid and sensitive detection of mutations and sequence variations is important. The usefulness of LSSP-PCR is illustrated by applications in the study of mutants of smooth muscle myosin light chain, analysis of a family with X-linked nephrogenic diabetes insipidus, and identity testing using human mitochondrial DNA. Images PMID:8127912

THE APPLICATION OF NUCLEAR ENERGY TO AGRICULTURE. Annual Report

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

Moh, C.C.

1963-07-01

Progress is reported in basic and applied agricultural research using nuclear energy as a tool. Emphasis was placed on tropical agriculture and student training. Results are reported on studies of mutations induced by irradiation of seeds or plant parts of plant species for food crops or local industrial uses. Seeds of 7 pine species that grow at altitudes of 1600 to over 3000 meters, papaya, beans, cassava branches with nodes, and young mahogany plants were irradiated and progenies were examined for mutation frequency and morphology, disease resistance, sensitivity to low temperatures, and other characteristics. Tracer studies of plant physiology includedmore » sulfur metabolism in photosynthetic bacteria and plants, iron metabolism in plants, and the effects of iron on growth of coffee and cacao seediings. Results are included from tracer studies on dispersion and longevity of the Mediterranean fruit fly under natural conditions and the effects of radiosterilization at various developmental stages on eradication of these flies. A list of publications during the period covered by this report is included. (C.H.)« less

Ten1 functions in telomere end protection and length regulation in association with Stn1 and Cdc13

PubMed Central

Grandin, Nathalie; Damon, Christelle; Charbonneau, Michel

2001-01-01

In Saccharomyces cerevisiae, Cdc13 has been proposed to mediate telomerase recruitment at telomere ends. Stn1, which associates with Cdc13 by the two-hybrid interaction, has been implicated in telomere maintenance. Ten1, a previously uncharacterized protein, was found to associate physically with both Stn1 and Cdc13. A binding defect between Stn1-13 and Ten1 was responsible for the long telomere phenotype of stn1-13 mutant cells. Moreover, rescue of the cdc13-1 mutation by STN1 was much improved when TEN1 was simultaneously overexpressed. Several ten1 mutations were found to confer telomerase-dependent telomere lengthening. Other, temperature-sensitive, mutants of TEN1 arrested at G2/M via activation of the Rad9-dependent DNA damage checkpoint. These ten1 mutant cells were found to accumulate single-stranded DNA in telomeric regions of the chromosomes. We propose that Ten1 is required to regulate telomere length, as well as to prevent lethal damage to telomeric DNA. PMID:11230140

Importance of individual amino acids in the Switch I region in eEF2 studied by functional complementation in S. cerevisiae.

PubMed

Bartish, Galyna; Nygård, Odd

2008-05-01

Elongation factor 2 (eEF2) is a member of the G-protein super family. G-proteins undergo conformational changes associated with binding of the guanosine nucleotide and hydrolysis of the bound GTP. These structural rearrangements affects the Switch I region (also known as the Effector loop). We have studied the role of individual amino acids in the Switch I region (amino acids 25-73) of S. cerevisiae eEF2 using functional complementation in yeast. 21 point mutations in the Switch I region were created by site-directed mutagenesis. Mutants K49R, E52Q, A53G, F55Y, K60R, Q63A, T68S, I69M and A73G were functional while mutants R54H, F55N, D57A, D57E, D57S, R59K, R59M, Q63E, R65A, R65N, T68A and T68M were inactive. Expression of mutants K49R, A53G, Q63A, I69M and A73G was associated with markedly decreased growth rates and yeast cells expressing mutants A53G and I69M became temperature sensitive. The functional capacity of eEF2 in which the major part Switch I (amino acids T56 to I69) was converted into the homologous sequence found in EF-G from E. coli was also studied. This protein chimera could functionally replace yeast eEF2 in vivo. Yeast cells expressing this mutant grew extremely slowly, showed increased cell death and became temperature sensitive. The ability of the mutant to replace authentic eEF2 in vivo indicates that the structural rearrangement of Switch I necessary for eEF2 function is similar in eukaryotes and bacteria. The effect of two point mutations in the P-loop was also studied. Mutant A25G but not A25V could functionally replace yeast eEF2 even if cells expressing the mutant grew slowly. The A25G mutation converted the consensus sequences AXXXXGK[T/S] in eEF2 to the corresponding motif GXXXXGK[T/S] found in all other G-proteins, suggesting that the alanine found in the P-loop of peptidyltranslocases are not essential for function.

Noninvasive detection of activating estrogen receptor 1 (ESR1) mutations in estrogen receptor-positive metastatic breast cancer.

PubMed

Guttery, David S; Page, Karen; Hills, Allison; Woodley, Laura; Marchese, Stephanie D; Rghebi, Basma; Hastings, Robert K; Luo, Jinli; Pringle, J Howard; Stebbing, Justin; Coombes, R Charles; Ali, Simak; Shaw, Jacqueline A

2015-07-01

Activating mutations in the estrogen receptor 1 (ESR1) gene are acquired on treatment and can drive resistance to endocrine therapy. Because of the spatial and temporal limitations of needle core biopsies, our goal was to develop a highly sensitive, less invasive method of detecting activating ESR1 mutations via circulating cell-free DNA (cfDNA) and tumor cells as a "liquid biopsy." We developed a targeted 23-amplicon next-generation sequencing (NGS) panel for detection of hot-spot mutations in ESR1, phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA), tumor protein p53 (TP53), fibroblast growth factor receptor 1 (FGFR1), and fibroblast growth factor receptor 2 (FGFR2) in 48 patients with estrogen receptor-α-positive metastatic breast cancer who were receiving systemic therapy. Selected mutations were validated using droplet digital PCR (ddPCR). Nine baseline cfDNA samples had an ESR1 mutation. NGS detected 3 activating mutations in ESR1, and 3 hot-spot mutations in PIK3CA, and 3 in TP53 in baseline cfDNA, and the ESR1 p.D538G mutation in 1 matched circulating tumor cell sample. ddPCR analysis was more sensitive than NGS and identified 6 additional baseline cfDNA samples with the ESR1 p.D538G mutation at a frequency of <1%. In serial blood samples from 11 patients, 4 showed changes in cfDNA, 2 with emergence of a mutation in ESR1. We also detected a low frequency ESR1 mutation (1.3%) in cfDNA of 1 primary patient who was thought to have metastatic disease but was clear by scans. Early identification of ESR1 mutations by liquid biopsy might allow for cessation of ineffective endocrine therapies and switching to other treatments, without the need for tissue biopsy and before the emergence of metastatic disease. © 2015 American Association for Clinical Chemistry.

Immunostaining with EGFR mutation-specific antibodies: a reliable screening method for lung adenocarcinomas harboring EGFR mutation in biopsy and resection samples.

PubMed

Fan, Xiangshan; Liu, Biao; Xu, Haodong; Yu, Bo; Shi, Shanshan; Zhang, Jin; Wang, Xuan; Wang, Jiandong; Lu, Zhenfeng; Ma, Henghui; Zhou, Xiaojun

2013-08-01

Mutation analysis of epidermal growth factor receptor (EGFR) is essential in determining the therapeutic strategy for lung adenocarcinoma. Immunohistochemical (IHC) staining with EGFR mutation-specific antibodies of del E746-A750 in exon 19 and L858R in exon 21 has been evaluated in resection specimens in a few studies but rarely in biopsy samples. A total of 169 cases (78 biopsies and 91 resected specimens) of lung adenocarcinoma with EGFR mutation status predefined by direct DNA sequencing were histologically examined, and IHC was performed using EGFR mutation-specific antibodies of del E746-A750 and L858R. The cases with positive results by IHC but negative results by direct DNA sequencing were examined by amplified refractory mutation system. Our results showed that the frequency of EGFR mutations for both E746-A750 deletion and L858R mutation was 38.5% (65/169) by DNA sequencing or amplified refractory mutation system and 34.3% (58/169) by IHC in lung adenocarcinomas. Based on molecular test results, the overall sensitivity, specificity, positive predictive value, and negative predictive value of IHC using these 2 antibodies in all (biopsy/resection) cases were 87.7% (80%/94.3%), 99.0% (97.9%/100%), 98.3% (96%/100%), and 92.8% (88.7%/96.6%), respectively. Lung adenocarcinomas with a predominant acinar, papillary, lepidic, or solid growth pattern more often harbor EGFR mutation of del E746-A750 or L858R. In conclusion, the immunostaining with EGFR del E746-A750 and L858R mutation antibodies is a reliable screening method with high specificity and sensitivity for identifying the EGFR mutation in both resected and biopsied lung adenocarcinomas. Copyright © 2013 Elsevier Inc. All rights reserved.

Driven by Mutations: The Predictive Value of Mutation Subtype in EGFR-Mutated Non-Small Cell Lung Cancer.

PubMed

Castellanos, Emily; Feld, Emily; Horn, Leora

2017-04-01

EGFR-mutated NSCLC is a genetically heterogeneous disease that includes more than 200 distinct mutations. The implications of mutational subtype for both prognostic and predictive value are being increasingly understood. Although the most common EGFR mutations-exon 19 deletions or L858R mutations-predict sensitivity to EGFR tyrosine kinase inhibitors (TKIs), it is now being recognized that outcomes may be improved in patients with exon 19 deletions. Additionally, 10% of patients will have an uncommon EGFR mutation, and response to EGFR TKI therapy is highly variable depending on the mutation. Given the growing recognition of the genetic and clinical variation seen in this disease, the development of comprehensive bioinformatics-driven tools to both analyze response in uncommon mutation subtypes and inform clinical decision making will be increasingly important. Clinical trials of novel EGFR TKIs should prospectively account for the presence of uncommon mutation subtypes in study design. Copyright © 2016 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Characterization of the hyperrecombination phenotype of the pol3-t mutation of Saccharomyces cerevisiae.

PubMed

Galli, Alvaro; Cervelli, Tiziana; Schiestl, Robert H

2003-05-01

The DNA polymerase delta (Pol3p/Cdc2p) allele pol3-t of Saccharomyces cerevisiae has previously been shown to increase the frequency of deletions between short repeats (several base pairs), between homologous DNA sequences separated by long inverted repeats, and between distant short repeats, increasing the frequency of genomic deletions. We found that the pol3-t mutation increased intrachromosomal recombination events between direct DNA repeats up to 36-fold and interchromosomal recombination 14-fold. The hyperrecombination phenotype of pol3-t was partially dependent on the Rad52p function but much more so on Rad1p. However, in the double-mutant rad1 Delta rad52 Delta, the pol3-t mutation still increased spontaneous intrachromosomal recombination frequencies, suggesting that a Rad1p Rad52p-independent single-strand annealing pathway is involved. UV and gamma-rays were less potent inducers of recombination in the pol3-t mutant, indicating that Pol3p is partly involved in DNA-damage-induced recombination. In contrast, while UV- and gamma-ray-induced intrachromosomal recombination was almost completely abolished in the rad52 or the rad1 rad52 mutant, there was still good induction in those mutants in the pol3-t background, indicating channeling of lesions into the above-mentioned Rad1p Rad52p-independent pathway. Finally, a heterozygous pol3-t/POL3 mutant also showed an increased frequency of deletions and MMS sensitivity at the restrictive temperature, indicating that even a heterozygous polymerase delta mutation might increase the frequency of genetic instability.

The BRAF{sup T1799A} mutation confers sensitivity of thyroid cancer cells to the BRAF{sup V600E} inhibitor PLX4032 (RG7204)

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

Xing, Joanna; Liu, Ruixin; Xing, Mingzhao

2011-01-28

Research highlights: {yields} Exciting therapeutic potential has been recently reported for the BRAF{sup V600E} inhibitor PLX4032 in melanoma. {yields} We tested the effects of PLX4032 on the growth of thyroid cancer cells which often harbor the BRAF{sup V600E} mutation. {yields} We observed a potent BRAF{sup V600E}-dependent inhibition of thyroid cancer cells by PLX4032. {yields} We thus demonstrated an important therapeutic potential of PLX4032 for thyroid cancer. -- Abstract: Aberrant signaling of the Ras-Raf-MEK-ERK (MAP kinase) pathway driven by the mutant kinase BRAF{sup V600E}, as a result of the BRAF{sup T1799A} mutation, plays a fundamental role in thyroid tumorigenesis. This studymore » investigated the therapeutic potential of a BRAF{sup V600E}-selective inhibitor, PLX4032 (RG7204), for thyroid cancer by examining its effects on the MAP kinase signaling and proliferation of 10 thyroid cancer cell lines with wild-type BRAF or BRAF{sup T1799A} mutation. We found that PLX4032 could effectively inhibit the MAP kinase signaling, as reflected by the suppression of ERK phosphorylation, in cells harboring the BRAF{sup T1799A} mutation. PLX4032 also showed a potent and BRAF mutation-selective inhibition of cell proliferation in a concentration-dependent manner. PLX4032 displayed low IC{sub 50} values (0.115-1.156 {mu}M) in BRAF{sup V600E} mutant cells, in contrast with wild-type BRAF cells that showed resistance to the inhibitor with high IC{sub 50} values (56.674-1349.788 {mu}M). Interestingly, cells with Ras mutations were also sensitive to PLX4032, albeit moderately. Thus, this study has confirmed that the BRAF{sup T1799A} mutation confers cancer cells sensitivity to PLX4032 and demonstrated its specific potential as an effective and BRAF{sup T1799A} mutation-selective therapeutic agent for thyroid cancer.« less

Detection of epidermal growth factor receptor mutation in lung cancer by droplet digital polymerase chain reaction

PubMed Central

Xu, Qing; Zhu, Yazhen; Bai, Yali; Wei, Xiumin; Zheng, Xirun; Mao, Mao; Zheng, Guangjuan

2015-01-01

Background Two types of epidermal growth factor receptor (EGFR) mutations in exon 19 and exon 21 (ex19del and L858R) are prevalent in lung cancer patients and sensitive to targeted EGFR inhibition. A resistance mutation in exon 20 (T790M) has been found to accompany drug treatment when patients relapse. These three mutations are valuable companion diagnostic biomarkers for guiding personalized treatment. Quantitative polymerase chain reaction (qPCR)-based methods have been widely used in the clinic by physicians to guide treatment decisions. The aim of this study was to evaluate the technical and clinical sensitivity and specificity of the droplet digital polymerase chain reaction (ddPCR) method in detecting the three EGFR mutations in patients with lung cancer. Methods Genomic DNA from H1975 and PC-9 cells, as well as 92 normal human blood specimens, was used to determine the technical sensitivity and specificity of the ddPCR assays. Genomic DNA of formalin-fixed, paraffin-embedded specimens from 78 Chinese patients with lung adenocarcinoma were assayed using both qPCR and ddPCR. Results The three ddPCR assays had a limit of detection of 0.02% and a wide dynamic range from 1 to 20,000 copies measurement. The L858R and ex19del assays had a 0% background level in the technical and clinical settings. The T790M assay appeared to have a 0.03% technical background. The ddPCR assays were robust for correct determination of EGFR mutation status in patients, and the dynamic range appeared to be better than qPCR methods. The ddPCR assay for T790M could detect patient samples that the qPCR method failed to detect. About 49% of this patient cohort had EGFR mutations (L858R, 15.4%; ex19del, 29.5%; T790M, 6.4%). Two patients with the ex19del mutation also had a naïve T790M mutation. Conclusion These data suggest that the ddPCR method could be useful in the personalized treatment of patients with lung cancer. PMID:26124670

Multiplex KRASG12/G13 mutation testing of unamplified cell-free DNA from the plasma of patients with advanced cancers using droplet digital polymerase chain reaction.

PubMed

Janku, F; Huang, H J; Fujii, T; Shelton, D N; Madwani, K; Fu, S; Tsimberidou, A M; Piha-Paul, S A; Wheler, J J; Zinner, R G; Naing, A; Hong, D S; Karp, D D; Cabrilo, G; Kopetz, E S; Subbiah, V; Luthra, R; Kee, B K; Eng, C; Morris, V K; Karlin-Neumann, G A; Meric-Bernstam, F

2017-03-01

Cell-free DNA (cfDNA) from plasma offers easily obtainable material for KRAS mutation analysis. Novel, multiplex, and accurate diagnostic systems using small amounts of DNA are needed to further the use of plasma cfDNA testing in personalized therapy. Samples of 16 ng of unamplified plasma cfDNA from 121 patients with diverse progressing advanced cancers were tested with a KRASG12/G13 multiplex assay to detect the seven most common mutations in the hotspot of exon 2 using droplet digital polymerase chain reaction (ddPCR). The results were retrospectively compared to mutation analysis of archival primary or metastatic tumor tissue obtained at different points of clinical care. Eighty-eight patients (73%) had KRASG12/G13 mutations in archival tumor specimens collected on average 18.5 months before plasma analysis, and 78 patients (64%) had KRASG12/G13 mutations in plasma cfDNA samples. The two methods had initial overall agreement in 103 (85%) patients (kappa, 0.66; ddPCR sensitivity, 84%; ddPCR specificity, 88%). Of the 18 discordant cases, 12 (67%) were resolved by increasing the amount of cfDNA, using mutation-specific probes, or re-testing the tumor tissue, yielding overall agreement in 115 patients (95%; kappa 0.87; ddPCR sensitivity, 96%; ddPCR specificity, 94%). The presence of ≥ 6.2% of KRASG12/G13 cfDNA in the wild-type background was associated with shorter survival (P = 0.001). Multiplex detection of KRASG12/G13 mutations in a small amount of unamplified plasma cfDNA using ddPCR has good sensitivity and specificity and good concordance with conventional clinical mutation testing of archival specimens. A higher percentage of mutant KRASG12/G13 in cfDNA corresponded with shorter survival. © The Author 2016. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Impact of Weight Loss at Presentation on Survival in Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors (EGFR-TKI) Sensitive Mutant Advanced Non-small Cell Lung Cancer (NSCLC) Treated with First-line EGFR-TKI.

PubMed

Lin, Liping; Zhao, Juanjuan; Hu, Jiazhu; Huang, Fuxi; Han, Jianjun; He, Yan; Cao, Xiaolong

2018-01-01

Purpose The aim of this study is to evaluate the impact of weight loss at presentation on treatment outcomes of first-line EGFR-tyrosine kinase inhibitors (EGFR-TKI) in EGFR-TKI sensitive mutant NSCLC patients. Methods We retrospectively analyzed the clinical outcomes of 75 consecutive advanced NSCLC patients with EGFR-TKI sensitive mutations (exon 19 deletion or exon 21 L858R) received first-line gefitinib or erlotinib therapy according to weight loss status at presentation in our single center. Results Of 75 EGFR-TKI sensitive mutant NSCLC patients, 49 (65.3%) patients had no weight loss and 26 (34.7%) had weight loss at presentation, the objective response rate (ORR) to EGFR-TKI treatment were similar between the two groups (79.6% vs. 76.9%, p = 0.533). Patients without weight loss at presentation had significantly longer median progression free survival (PFS) (12.4 months vs. 7.6 months; hazard ratio [HR] 0.356, 95% confidence interval [CI] 0.212-0.596, p < 0.001) and overall survival (OS) (28.5 months vs. 20.7 months; HR 0.408, 95% CI 0.215-0.776, p = 0.006) than those with weight loss at presentation; moreover, the stratified analysis by EGFR-TKI sensitive mutation types also found similar trend between these two groups except for OS in EGFR exon 21 L858R mutation patients. Multivariate analysis identified weight loss at presentation and EGFR-TKI sensitive mutation types were independent predictive factors for PFS and OS. Conclusions Weight loss at presentation had a detrimental impact on PFS and OS in EGFR-TKI sensitive mutant advanced NSCLC patients treated with first-line EGFR-TKI. It should be considered as an important factor in the treatment decision or designing of EGFR-TKI clinical trials.

KRAS Mutation Is a Predictor of Oxaliplatin Sensitivity in Colon Cancer Cells

PubMed Central

Lin, Yu-Lin; Ou, Da-Liang; Lin, Liang-In; Tseng, Li-Hui; Chang, Yih-Leong; Yeh, Kun-Huei; Cheng, Ann-Lii

2012-01-01

Molecular biomarkers to determine the effectiveness of targeted therapies in cancer treatment have been widely adopted in colorectal cancer (CRC), but those to predict chemotherapy sensitivity remain poorly defined. We tested our hypothesis that KRAS mutation may be a predictor of oxaliplatin sensitivity in CRC. KRAS was knocked-down in KRAS-mutant CRC cells (DLD-1G13D and SW480G12V) by small interfering RNAs (siRNA) and overexpressed in KRAS-wild-type CRC cells (COLO320DM) by KRAS-mutant vectors to generate paired CRC cells. These paired CRC cells were tested by oxaliplatin, irinotecan and 5FU to determine the change in drug sensitivity by MTT assay and flow cytometry. Reasons for sensitivity alteration were further determined by western blot and real-time quantitative reverse transcriptase polymerase chain reaction (qRT -PCR). In KRAS-wild-type CRC cells (COLO320DM), KRAS overexpression by mutant vectors caused excision repair cross-complementation group 1 (ERCC1) downregulation in protein and mRNA levels, and enhanced oxaliplatin sensitivity. In contrast, in KRAS-mutant CRC cells (DLD-1G13D and SW480G12V), KRAS knocked-down by KRAS-siRNA led to ERCC1 upregulation and increased oxaliplatin resistance. The sensitivity of irinotecan and 5FU had not changed in the paired CRC cells. To validate ERCC1 as a predictor of sensitivity for oxaliplatin, ERCC1 was knocked-down by siRNA in KRAS-wild-type CRC cells, which restored oxaliplatin sensitivity. In contrast, ERCC1 was overexpressed by ERCC1-expressing vectors in KRAS-mutant CRC cells, and caused oxaliplatin resistance. Overall, our findings suggest that KRAS mutation is a predictor of oxaliplatin sensitivity in colon cancer cells by the mechanism of ERCC1 downregulation. PMID:23209813

Modest increased sensitivity to radiation oncogenesis in ATM heterozygous versus wild-type mammalian cells

NASA Technical Reports Server (NTRS)

Smilenov, L. B.; Brenner, D. J.; Hall, E. J.

2001-01-01

Subpopulations that are genetically predisposed to radiation-induced cancer could have significant public health consequences. Individuals homozygous for null mutations at the ataxia telangiectasia gene are indeed highly radiosensitive, but their numbers are very small. Ataxia Telangiectasia heterozygotes (1-2% of the population) have been associated with somewhat increased radiosensitivity for some end points, but none directly related to carcinogenesis. Here, intralitter comparisons between wild-type mouse embryo fibroblasts and mouse embryo fibroblasts carrying ataxia telangiectasia mutated (ATM) null mutation indicate that the heterozygous cells are more sensitive to radiation oncogenesis than their normal, litter-matched, counterparts. From these data we suggest that Ataxia Telangiectasia heterozygotes could indeed represent a societally-significant radiosensitive human subpopulation.

The heat-transfer method: a versatile low-cost, label-free, fast, and user-friendly readout platform for biosensor applications.

PubMed

van Grinsven, Bart; Eersels, Kasper; Peeters, Marloes; Losada-Pérez, Patricia; Vandenryt, Thijs; Cleij, Thomas J; Wagner, Patrick

2014-08-27

In recent years, biosensors have become increasingly important in various scientific domains including medicine, biology, and pharmacology, resulting in an increased demand for fast and effective readout techniques. In this Spotlight on Applications, we report on the recently developed heat-transfer method (HTM) and illustrate the use of the technique by zooming in on four established bio(mimetic) sensor applications: (i) mutation analysis in DNA sequences, (ii) cancer cell identification through surface-imprinted polymers, (iii) detection of neurotransmitters with molecularly imprinted polymers, and (iv) phase-transition analysis in lipid vesicle layers. The methodology is based on changes in heat-transfer resistance at a functionalized solid-liquid interface. To this extent, the device applies a temperature gradient over this interface and monitors the temperature underneath and above the functionalized chip in time. The heat-transfer resistance can be obtained by dividing this temperature gradient by the power needed to achieve a programmed temperature. The low-cost, fast, label-free and user-friendly nature of the technology in combination with a high degree of specificity, selectivity, and sensitivity makes HTM a promising sensor technology.

Simian virus 40 small t antigen is not required for the maintenance of transformation but may act as a promoter (cocarcinogen) during establishment of transformation in resting rat cells.

PubMed Central

Seif, R; Martin, R G

1979-01-01

Simian virus 40 deletion mutants affecting the 20,000-dalton (20K) t antigen and tsA mutants rendering the 90K T antigen temperature sensitive, as well as double mutants containing both mutations, induced host DNA synthesis in resting rat cells at the restrictive temperature. Nonetheless, the deletion mutants and double mutants did not induce transformation in resting cells even at the permissive temperature. On the other hand, the deletion mutants did induce full transformants when actively growing rat cells were infected; the transformants grew efficiently in agar and to high saturation densities on platic. The double mutants did not induce T-antigen-independent (temperature-insensitive) transformants which were shown previously to arise preferentially from resting cells. Thus, small t antigen was dispensable for the maintenance of the transformed phenotype in T-antigen-dependent rat transformants (transformants derived from growing cells) and may play a role in the establishment of T-antigen-independent transformants. We attempt to establish a parallel between transformation induced by chemical carcinogens and simian virus 40-induced transformation. Images PMID:229274

Simian virus 40 small t antigen is not required for the maintenance of transformation but may act as a promoter (cocarcinogen) during establishment of transformation in resting rat cells.

PubMed

Seif, R; Martin, R G

1979-12-01

Simian virus 40 deletion mutants affecting the 20,000-dalton (20K) t antigen and tsA mutants rendering the 90K T antigen temperature sensitive, as well as double mutants containing both mutations, induced host DNA synthesis in resting rat cells at the restrictive temperature. Nonetheless, the deletion mutants and double mutants did not induce transformation in resting cells even at the permissive temperature. On the other hand, the deletion mutants did induce full transformants when actively growing rat cells were infected; the transformants grew efficiently in agar and to high saturation densities on platic. The double mutants did not induce T-antigen-independent (temperature-insensitive) transformants which were shown previously to arise preferentially from resting cells. Thus, small t antigen was dispensable for the maintenance of the transformed phenotype in T-antigen-dependent rat transformants (transformants derived from growing cells) and may play a role in the establishment of T-antigen-independent transformants. We attempt to establish a parallel between transformation induced by chemical carcinogens and simian virus 40-induced transformation.

Temperature influences the level of glyphosate resistance in barnyardgrass (Echinochloa colona).

PubMed

Nguyen, Thai Hoan; Malone, Jenna M; Boutsalis, Peter; Shirley, Neil; Preston, Christopher

2016-05-01

Echinochloa colona is an important summer-growing weed species in cropping regions of northern Australia that has evolved resistance to glyphosate owing to intensive use of this herbicide in summer fallow. Pot trials conducted at 20 and 30 °C on six E. colona populations showed a significant increase in the level of glyphosate resistance in resistant populations at 30 °C compared with 20 °C. However, there was no influence of growth temperature on glyphosate susceptibility of the sensitive population. Sequencing of the target-site gene (EPSPS) of the six populations identified a mutation at position 106 leading to a change from proline to serine in the most resistant population A533.1 only. EPSPS gene amplification was not detected in any of the resistant populations examined. Examining (14) C-glyphosate uptake on two resistant and one susceptible population showed a twofold increase at 20 °C; however, few differences in glyphosate translocation occurred from the treated leaf to other plant parts between populations or temperatures. There is reduced efficacy of glyphosate at high temperatures on resistant E. colona populations, making these populations harder to control in summer. © 2015 Society of Chemical Industry.

Adaptation of cone pigments found in green rods for scotopic vision through a single amino acid mutation

PubMed Central

Kojima, Keiichi; Matsutani, Yuki; Yamashita, Takahiro; Yanagawa, Masataka; Imamoto, Yasushi; Yamano, Yumiko; Wada, Akimori; Hisatomi, Osamu; Nishikawa, Kanto; Sakurai, Keisuke; Shichida, Yoshinori

2017-01-01

Most vertebrate retinas contain a single type of rod for scotopic vision and multiple types of cones for photopic and color vision. The retinas of certain amphibian species uniquely contain two types of rods: red rods, which express rhodopsin, and green rods, which express a blue-sensitive cone pigment (M1/SWS2 group). Spontaneous activation of rhodopsin induced by thermal isomerization of the retinal chromophore has been suggested to contribute to the rod’s background noise, which limits the visual threshold for scotopic vision. Therefore, rhodopsin must exhibit low thermal isomerization rate compared with cone visual pigments to adapt to scotopic condition. In this study, we determined whether amphibian blue-sensitive cone pigments in green rods exhibit low thermal isomerization rates to act as rhodopsin-like pigments for scotopic vision. Anura blue-sensitive cone pigments exhibit low thermal isomerization rates similar to rhodopsin, whereas Urodela pigments exhibit high rates like other vertebrate cone pigments present in cones. Furthermore, by mutational analysis, we identified a key amino acid residue, Thr47, that is responsible for the low thermal isomerization rates of Anura blue-sensitive cone pigments. These results strongly suggest that, through this mutation, anurans acquired special blue-sensitive cone pigments in their green rods, which could form the molecular basis for scotopic color vision with normal red rods containing green-sensitive rhodopsin. PMID:28484015

A novel mutation of the high-temperature requirement A serine peptidase 1 (HTRA1) gene in a Chinese family with cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL).

PubMed

Chen, Yan; He, Zhiyi; Meng, Su; Li, Lei; Yang, Hua; Zhang, Xiaotang

2013-10-01

Mutations in the high-temperature requirement A serine peptidase 1 (HTRA1) gene were studied in a Chinese family with cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL). Exons 1-9 of the HTRA1 gene were amplified and bidirectionally sequenced in a Chinese family with CARASIL. Mutation effects were analysed by three-dimensional modelling of the serine protease HTRA1 protein. The proband was found to be homozygous for a novel missense mutation (c.854 C > T) identified in exon 4 of the HTRA1 gene; the parents of the proband were heterozygous for the same missense mutation. This c.854 C > T mutation resulted in a change from proline to leucine (p.P285L) in serine protease HTRA1, and was absent in 260 control chromosomes. Three-dimensional models showed that the change from proline to leucine (p.P285L) could attenuate the hydrogen bond between S284 and S287 residues, which might affect function of serine protease HTRA1. Discovery of a novel missense mutation (c.854C>T) associated with CARASIL expands the known CARASIL-related mutations in HTRA1.

KRAS mutation analysis of washing fluid from endoscopic ultrasound-guided fine needle aspiration improves cytologic diagnosis of pancreatic ductal adenocarcinoma.

PubMed

Park, Joo Kyung; Lee, Yoon Jung; Lee, Jong Kyun; Lee, Kyu Taek; Choi, Yoon-La; Lee, Kwang Hyuck

2017-01-10

EUS-FNA becomes one of the most important diagnostic modalities for PDACs. However, acquired tissue specimens were sometimes insufficient to make a definite cytological diagnosis. On the other hand, KRAS mutation is the most frequently acquired genetic alteration found more than 90% of PDACs. To investigate the way to improve diagnostic accuracy for PDACs using both cytological examination and KRAS mutation analysis would be a great help. Therefore, the aims of this study were to evaluate usefulness of conventional cytological examination combined with KRAS mutation analysis with modified PCR technology to improve the sensitivity and the accuracy. We enrolled 43 patients with solid pancreatic masses and 86 EUS-FNA specimens were obtained. During the EUS-FNA, the needle catheter was flushed with 2 cc of saline and the washed fluid was collected for KRAS mutation analysis for the first 2 passes; PNAClamp™ KRAS Mutation Detection Kit. There were 46 specimens from the 23 PDACs and 40 specimens from the 20 other pancreatic diseases. The sensitivity, specificity and accuracy were as follows; conventional cytopathologic examination: 63%, 100% and 80%; combination of cytopathologic examination and K-ras mutation analysis: 87%, 100% and 93%. Furthermore, KRAS mutation was detected 11 out of 17 PDAC samples whose cytopathology results were inconclusive. KRAS mutation analysis with PNAClamp™ technique using washing fluid from EUS-FNA along with cytological examination may not only improve the diagnostic accuracy of PDACs, but also establish the platform using genetic analysis which would be helpful as diagnostic modality for PDACs.

Whole exome sequencing for determination of tumor mutation load in liquid biopsy from advanced cancer patients.

PubMed

Koeppel, Florence; Blanchard, Steven; Jovelet, Cécile; Genin, Bérengère; Marcaillou, Charles; Martin, Emmanuel; Rouleau, Etienne; Solary, Eric; Soria, Jean-Charles; André, Fabrice; Lacroix, Ludovic

2017-01-01

Tumor mutation load (TML) has been proposed as a biomarker of patient response to immunotherapy in several studies. TML is usually determined by tumor biopsy DNA (tDNA) whole exome sequencing (WES), therefore TML evaluation is limited by informative biopsy availability. Circulating cell free DNA (cfDNA) provided by liquid biopsy is a surrogate specimen to biopsy for molecular profiling. Nevertheless performing WES on DNA from plasma is technically challenging and the ability to determine tumor mutation load from liquid biopsies remains to be demonstrated. In the current study, WES was performed on cfDNA from 32 metastatic patients of various cancer types included into MOSCATO 01 (NCT01566019) and/or MATCHR (NCT02517892) molecular triage trials. Results from targeted gene sequencing (TGS) and WES performed on cfDNA were compared to results from tumor tissue biopsy. In cfDNA samples, WES mutation detection sensitivity was 92% compared to targeted sequencing (TGS). When comparing cfDNA-WES to tDNA-WES, mutation detection sensitivity was 53%, consistent with previously published prospective study comparing cfDNA-TGS to tDNA-TGS. For samples in which presence of tumor DNA was confirmed in cfDNA, tumor mutation load from liquid biopsy was correlated with tumor biopsy. Taken together, this study demonstrated that liquid biopsy may be applied to determine tumor mutation load. Qualification of liquid biopsy for interpretation is a crucial point to use cfDNA for mutational load estimation.

Whole exome sequencing for determination of tumor mutation load in liquid biopsy from advanced cancer patients

PubMed Central

Blanchard, Steven; Jovelet, Cécile; Genin, Bérengère; Marcaillou, Charles; Martin, Emmanuel; Rouleau, Etienne; Solary, Eric; Soria, Jean-Charles; André, Fabrice; Lacroix, Ludovic

2017-01-01

Tumor mutation load (TML) has been proposed as a biomarker of patient response to immunotherapy in several studies. TML is usually determined by tumor biopsy DNA (tDNA) whole exome sequencing (WES), therefore TML evaluation is limited by informative biopsy availability. Circulating cell free DNA (cfDNA) provided by liquid biopsy is a surrogate specimen to biopsy for molecular profiling. Nevertheless performing WES on DNA from plasma is technically challenging and the ability to determine tumor mutation load from liquid biopsies remains to be demonstrated. In the current study, WES was performed on cfDNA from 32 metastatic patients of various cancer types included into MOSCATO 01 (NCT01566019) and/or MATCHR (NCT02517892) molecular triage trials. Results from targeted gene sequencing (TGS) and WES performed on cfDNA were compared to results from tumor tissue biopsy. In cfDNA samples, WES mutation detection sensitivity was 92% compared to targeted sequencing (TGS). When comparing cfDNA-WES to tDNA-WES, mutation detection sensitivity was 53%, consistent with previously published prospective study comparing cfDNA-TGS to tDNA-TGS. For samples in which presence of tumor DNA was confirmed in cfDNA, tumor mutation load from liquid biopsy was correlated with tumor biopsy. Taken together, this study demonstrated that liquid biopsy may be applied to determine tumor mutation load. Qualification of liquid biopsy for interpretation is a crucial point to use cfDNA for mutational load estimation. PMID:29161279

Crystal digital droplet PCR for detection and quantification of circulating EGFR sensitizing and resistance mutations in advanced non-small cell lung cancer

PubMed Central

Madic, Jordan; Remon, Jordi; Honoré, Aurélie; Girard, Romain; Rouleau, Etienne; André, Barbara; Besse, Benjamin; Droniou, Magali; Lacroix, Ludovic

2017-01-01

Over the past years, targeted therapies using tyrosine kinase inhibitors (TKI) have led to an increase in progression-free survival and response rate for a subgroup of non-small cell lung cancer (NSCLC) patients harbouring specific gene abnormalities compared with chemotherapy. However long-lasting tumor regression is rarely achieved, due to the development of resistant tumoral subclones, which requires alternative therapeutic approaches. Molecular profile at progressive disease is a challenge for making adaptive treatment decisions. The aim of this study was to monitor EGFR-mutant tumors over time based on the quantity of mutant DNA circulating in plasma (ctDNA), comparing two different methods, Crystal™ Digital™ PCR and Massive Parallel Sequencing (MPS). In plasma circulating cell free DNA (cfDNA) of 61 advanced NSCLC patients we found an overall correlation of 78% between mutated allelic fraction measured by Crystal Digital PCR and MPS. 7 additional samples with sensitizing mutations and 4 additional samples with the resistance mutation were detected with Crystal Digital PCR, but not with MPS. Monitoring levels of both mutation types over time showed a correlation between levels and trends of mutated ctDNA detected and clinical assessment of disease for the 6 patients tested. In conclusion, Crystal Digital PCR exhibited good performance for monitoring mutational status in plasma cfDNA, and also appeared as better suited to the detection of known mutations than MPS in terms of features such as time to results. PMID:28829811

Next-generation sequencing-based method shows increased mutation detection sensitivity in an Indian retinoblastoma cohort

PubMed Central

Singh, Jaya; Mishra, Avshesh; Pandian, Arunachalam Jayamuruga; Mallipatna, Ashwin C.; Khetan, Vikas; Sripriya, S.; Kapoor, Suman; Agarwal, Smita; Sankaran, Satish; Katragadda, Shanmukh; Veeramachaneni, Vamsi; Hariharan, Ramesh; Subramanian, Kalyanasundaram

2016-01-01

Purpose Retinoblastoma (Rb) is the most common primary intraocular cancer of childhood and one of the major causes of blindness in children. India has the highest number of patients with Rb in the world. Mutations in the RB1 gene are the primary cause of Rb, and heterogeneous mutations are distributed throughout the entire length of the gene. Therefore, genetic testing requires screening of the entire gene, which by conventional sequencing is time consuming and expensive. Methods In this study, we screened the RB1 gene in the DNA isolated from blood or saliva samples of 50 unrelated patients with Rb using the TruSight Cancer panel. Next-generation sequencing (NGS) was done on the Illumina MiSeq platform. Genetic variations were identified using the Strand NGS software and interpreted using the StrandOmics platform. Results We were able to detect germline pathogenic mutations in 66% (33/50) of the cases, 12 of which were novel. We were able to detect all types of mutations, including missense, nonsense, splice site, indel, and structural variants. When we considered bilateral Rb cases only, the mutation detection rate increased to 100% (22/22). In unilateral Rb cases, the mutation detection rate was 30% (6/20). Conclusions Our study suggests that NGS-based approaches increase the sensitivity of mutation detection in the RB1 gene, making it fast and cost-effective compared to the conventional tests performed in a reflex-testing mode. PMID:27582626

Molecular and Proteomic Analysis of Levofloxacin and Metronidazole Resistant Helicobacter pylori.

PubMed

Hanafi, Aimi; Lee, Woon Ching; Loke, Mun Fai; Teh, Xinsheng; Shaari, Ain; Dinarvand, Mojdeh; Lehours, Philippe; Mégraud, Francis; Leow, Alex Hwong Ruey; Vadivelu, Jamuna; Goh, Khean Lee

2016-01-01

Antibiotic resistance in bacteria incurs fitness cost, but compensatory mechanisms may ameliorate the cost and sustain the resistance even under antibiotics-free conditions. The aim of this study was to determine compensatory mechanisms of antibiotic resistance in H. pylori . Five strains of levofloxacin-sensitive H. pylori were induced in vitro to develop resistance. In addition, four pairs of metronidazole-sensitive and -resistant H. pylori strains were isolated from patients carrying dual H. pylori populations that consist of both sensitive and resistant phenotypes. Growth rate, virulence and biofilm-forming ability of the sensitive and resistant strains were compared to determine effects of compensatory response. Proteome profiles of paired sensitive and resistant strains were analyzed by liquid chromatography/mass spectrophotometry (LC/MS). Although there were no significant differences in growth rate between sensitive and resistant pairs, bacterial virulence (in terms of abilities to induce apoptosis and form biofilm) differs from pair to pair. These findings demonstrate the complex and strain-specific phenotypic changes in compensation for antibiotics resistance. Compensation for in vitro induced levofloxacin resistance involving mutations of gyrA and gyrB was functionally random. Furthermore, higher protein translation and non-functional protein degradation capabilities in naturally-occuring dual population metronidazole sensitive-resistant strains may be a possible alternative mechanism underlying resistance to metronidazole without mutations in rdxA and frxA . This may explain the lack of mutations in target genes in ~10% of metronidazole resistant strains.

Molecular and Proteomic Analysis of Levofloxacin and Metronidazole Resistant Helicobacter pylori

PubMed Central

Hanafi, Aimi; Lee, Woon Ching; Loke, Mun Fai; Teh, Xinsheng; Shaari, Ain; Dinarvand, Mojdeh; Lehours, Philippe; Mégraud, Francis; Leow, Alex Hwong Ruey; Vadivelu, Jamuna; Goh, Khean Lee

2016-01-01

Antibiotic resistance in bacteria incurs fitness cost, but compensatory mechanisms may ameliorate the cost and sustain the resistance even under antibiotics-free conditions. The aim of this study was to determine compensatory mechanisms of antibiotic resistance in H. pylori. Five strains of levofloxacin-sensitive H. pylori were induced in vitro to develop resistance. In addition, four pairs of metronidazole-sensitive and -resistant H. pylori strains were isolated from patients carrying dual H. pylori populations that consist of both sensitive and resistant phenotypes. Growth rate, virulence and biofilm-forming ability of the sensitive and resistant strains were compared to determine effects of compensatory response. Proteome profiles of paired sensitive and resistant strains were analyzed by liquid chromatography/mass spectrophotometry (LC/MS). Although there were no significant differences in growth rate between sensitive and resistant pairs, bacterial virulence (in terms of abilities to induce apoptosis and form biofilm) differs from pair to pair. These findings demonstrate the complex and strain-specific phenotypic changes in compensation for antibiotics resistance. Compensation for in vitro induced levofloxacin resistance involving mutations of gyrA and gyrB was functionally random. Furthermore, higher protein translation and non-functional protein degradation capabilities in naturally-occuring dual population metronidazole sensitive-resistant strains may be a possible alternative mechanism underlying resistance to metronidazole without mutations in rdxA and frxA. This may explain the lack of mutations in target genes in ~10% of metronidazole resistant strains. PMID:28018334

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

Development and inter-laboratory validation of unlabeled probe melting curve analysis for detection of JAK2 V617F mutation in polycythemia vera.

PubMed

Wu, Zhiyuan; Yuan, Hong; Zhang, Xinju; Liu, Weiwei; Xu, Jinhua; Zhang, Wei; Guan, Ming

2011-01-01

JAK2 V617F, a somatic point mutation that leads to constitutive JAK2 phosphorylation and kinase activation, has been incorporated into the WHO classification and diagnostic criteria of myeloid neoplasms. Although various approaches such as restriction fragment length polymorphism, amplification refractory mutation system and real-time PCR have been developed for its detection, a generic rapid closed-tube method, which can be utilized on routine genetic testing instruments with stability and cost-efficiency, has not been described. Asymmetric PCR for detection of JAK2 V617F with a 3'-blocked unlabeled probe, saturate dye and subsequent melting curve analysis was performed on a Rotor-Gene® Q real-time cycler to establish the methodology. We compared this method to the existing amplification refractory mutation systems and direct sequencing. Hereafter, the broad applicability of this unlabeled probe melting method was also validated on three diverse real-time systems (Roche LightCycler® 480, Applied Biosystems ABI® 7500 and Eppendorf Mastercycler® ep realplex) in two different laboratories. The unlabeled probe melting analysis could genotype JAK2 V617F mutation explicitly with a 3% mutation load detecting sensitivity. At level of 5% mutation load, the intra- and inter-assay CVs of probe-DNA heteroduplex (mutation/wild type) covered 3.14%/3.55% and 1.72%/1.29% respectively. The method could equally discriminate mutant from wild type samples on the other three real-time instruments. With a high detecting sensitivity, unlabeled probe melting curve analysis is more applicable to disclose JAK2 V617F mutation than conventional methodologies. Verified with the favorable inter- and intra-assay reproducibility, unlabeled probe melting analysis provided a generic mutation detecting alternative for real-time instruments.

Clinical significance of disease-specific MYD88 mutations in circulating DNA in primary central nervous system lymphoma.

PubMed

Hattori, Keiichiro; Sakata-Yanagimoto, Mamiko; Suehara, Yasuhito; Yokoyama, Yasuhisa; Kato, Takayasu; Kurita, Naoki; Nishikii, Hidekazu; Obara, Naoshi; Takano, Shingo; Ishikawa, Eiichi; Matsumura, Akira; Hasegawa, Yuichi; Chiba, Shigeru

2018-01-01

Recent sequencing studies demonstrated the MYD88 L265P mutation in more than 70% of primary central nervous system lymphomas (PCNSL), and the clinical significance of this mutation has been proposed as diagnostic and prognostic markers in PCNSL. In contrast, mutational analyses using cell-free DNAs have been reported in a variety of systemic lymphomas. To investigate how sensitively the MYD88 L265P mutation can be identified in cell-free DNA from PCNSL patients, we carried out droplet digital PCR (ddPCR) and targeted deep sequencing (TDS) in 14 consecutive PCNSL patients from whom paired tumor-derived DNA and cell-free DNA was available at diagnosis. The MYD88 L265P mutation was found in tumor-derived DNA from all 14 patients (14/14, 100%). In contrast, among 14 cell-free DNAs evaluated by ddPCR (14/14) and TDS (13/14), the MYD88 L265P mutation was detected in eight out of 14 (ddPCR) and in 0 out of 13 (TDS) samples, implying dependence on the detection method. After chemotherapy, the MYD88 L265P mutation in cell-free DNAs was traced in five patients; unexpectedly, the mutations disappeared after chemotherapy was given, and they remained undetectable in all patients. These observations suggest that ddPCR can sensitively detect the MYD88 L265P mutation in cell-free DNA and could be used as non-invasive diagnostics, but may not be applicable for monitoring minimal residual diseases in PCNSL. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Impact of Emergent Circulating Tumor DNA RAS Mutation in Panitumumab-Treated Chemoresistant Metastatic Colorectal Cancer.

PubMed

Kim, Tae Won; Peeters, Marc; Thomas, Anne L; Gibbs, Peter; Hool, Kristina; Zhang, Jianqi; Ang, Agnes; Bach, Bruce Allen; Price, Timothy

2018-06-13

The accumulation of emergent RAS mutations during anti-epidermal growth factor receptor (EGFR) therapy is of interest as a mechanism for acquired resistance to anti-EGFR treatment. Plasma analysis of circulating tumor (ct) DNA is a minimally invasive and highly sensitive method to determine RAS mutational status. This biomarker analysis of the global phase III ASPECCT study used next-generation sequencing to detect expanded RAS ctDNA mutations in panitumumab-treated patients. Plasma samples collected at baseline and posttreatment were analyzed categorically for the presence of RAS mutations by the Plasma Select -R™ 64-gene panel at 0.1% sensitivity. Among panitumumab-treated patients with evaluable plasma samples at baseline (n = 238), 188 (79%) were wild-type (WT) RAS, and 50 (21%) were mutant RAS Of the 188 patients with baseline ctDNA WT RAS status, 164 had evaluable posttreatment results with a 32% rate of emergent RAS mutations. The median overall survival (OS) for WT and RAS mutant status by ctDNA at baseline was 13.7 (95% confidence interval: 11.5-15.4) and 7.9 months (6.4-9.6), respectively ( P < 0.0001). Clinical outcomes were not significantly different between patients with and without emergent ctDNA RAS mutations. Although patients with baseline ctDNA RAS mutations had worse outcomes than patients who were WT RAS before initiating treatment, emergent ctDNA RAS mutations were not associated with less favorable patient outcomes in panitumumab-treated patients. Further research is needed to determine a clinically relevant threshold for baseline and emergent ctDNA RAS mutations. Copyright ©2018, American Association for Cancer Research.

Effect of Hypertrophic Cardiomyopathy-Linked Troponin C Mutations on the Response of Reconstituted Thin Filaments to Calcium upon Troponin I Phosphorylation†

PubMed Central

Albury, Acchia N. J.; Swindle, Nicholas; Swartz, Darl R.; Tikunova, Svetlana B.

2012-01-01

The objective of this work was to investigate the effect of hypertrophic cardiomyopathy-linked A8V and E134D mutations in cardiac troponin C (cTnC) on the response of reconstituted thin filaments to calcium upon phosphorylation of cardiac troponin I (cTnI) by protein kinase A. The phosphorylation of cTnI at protein kinase A sites was mimicked by S22D/S23D mutation in cTnI. Our results demonstrate that the A8V and E134D mutations had no effect on the extent of calcium desensitization of reconstituted thin filaments induced by cTnI pseudo-phosphorylation. However, the A8V mutation enhanced the effect of cTnI pseudo-phosphorylation on the rate of calcium dissociation from reconstituted thin filaments and on calcium dependence of actomyosin ATPase. Consequently, while the A8V mutation still led to a slower rate of calcium dissociation from reconstituted thin filaments upon pseudo-phosphorylation of cTnI, the ability of the A8V mutation to decrease the rate of calcium dissociation was diminished. In addition, the ability of the A8V mutation to sensitize actomyosin ATPase to calcium was diminished after cTnI was replaced by the phosphorylation mimetic of cTnI. Consistent with the hypothesis that the E134D mutation is benign, it exerted minor to no effect on the rate of calcium dissociation from reconstituted thin filaments, and on calcium sensitivity of actomyosin ATPase, regardless of cTnI phosphorylation status. In conclusion, our study enhances understanding of how cardiomyopathy-linked cTnC mutations affect the response of reconstituted thin filaments to calcium upon cTnI phosphorylation. PMID:22489623

Comparison of Immunohistochemistry and Direct Sanger Sequencing for Detection of the BRAFV600E Mutation in Thyroid Neoplasm

PubMed Central

Oh, Hye-Seon; Kwon, Hyemi; Park, Suyeon; Kim, Mijin; Jeon, Min Ji; Kim, Tae Yong; Shong, Young Kee; Kim, Won Bae; Choi, Jene

2018-01-01

Background The BRAFV600E mutation is the most common genetic alteration identified in papillary thyroid carcinoma (PTC). Because of its costs effectiveness and sensitivity, direct Sanger sequencing has several limitations. The aim of this study was to evaluate the efficiency of immunohistochemistry (IHC) as an alternative method to detect the BRAFV600E mutation in preoperative and postoperative tissue samples. Methods We evaluated 71 patients who underwent thyroid surgery with the result of direct sequencing of the BRAFV600E mutation. IHC staining of the BRAFV600E mutation was performed in 49 preoperative and 23 postoperative thyroid specimens. Results Sixty-two patients (87.3%) had PTC, and of these, BRAFV600E was confirmed by direct sequencing in 57 patients (91.9%). In 23 postoperative tissue samples, the BRAFV600E mutation was detected in 16 samples (70%) by direct sequencing and 18 samples (78%) by IHC. In 24 fine needle aspiration (FNA) samples, BRAFV600E was detected in 18 samples (75%) by direct sequencing and 16 samples (67%) by IHC. In 25 core needle biopsy (CNB) samples, the BRAFV600E mutation was detected in 15 samples (60%) by direct sequencing and 16 samples (64%) by IHC. The sensitivity and specificity of IHC for detecting the BRAFV600E mutation were 77.8% and 66.7% in FNA samples and 99.3% and 80.0% in CNB samples. Conclusion IHC could be an alternative method to direct Sanger sequencing for BRAFV600E mutation detection both in postoperative and preoperative samples. However, application of IHC to detect the BRAFV600E mutation in FNA samples is of limited value compared with direct sequencing. PMID:29388401

Activating mutations in the tyrosine kinase domain of the epidermal growth factor receptor are associated with improved survival in gefitinib-treated chemorefractory lung adenocarcinomas.

PubMed

Taron, Miguel; Ichinose, Yukito; Rosell, Rafael; Mok, Tony; Massuti, Bartomeu; Zamora, Lurdes; Mate, Jose Luis; Manegold, Christian; Ono, Mayumi; Queralt, Cristina; Jahan, Thierry; Sanchez, Jose Javier; Sanchez-Ronco, Maria; Hsue, Victor; Jablons, David; Sanchez, Jose Miguel; Moran, Teresa

2005-08-15

Activating mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) confer a strong sensitivity to gefitinib, a selective tyrosine kinase inhibitor of EGFR. We examined EGFR mutations at exons 18, 19, and 21 in tumor tissue from 68 gefitinib-treated, chemorefractory, advanced non-small cell lung cancer patients from the United States, Europe, and Asia and in a highly gefitinib-sensitive non-small cell lung cancer cell line and correlated their presence with response and survival. In addition, in a subgroup of 28 patients for whom the remaining tumor tissue was available, we examined the relationship among EGFR mutations, CA repeats in intron 1 of EGFR, EGFR and caveolin-1 mRNA levels, and increased EGFR gene copy numbers. Seventeen patients had EGFR mutations, all of which were in lung adenocarcinomas. Radiographic response was observed in 16 of 17 (94.1%) patients harboring EGFR mutations, in contrast with 6 of 51 (12.6%) with wild-type EGFR (P < 0.0001). Probability of response increased significantly in never smokers, patients receiving a greater number of prior chemotherapy regimens, Asians, and younger patients. Median survival was not reached for patients with EGFR mutations and was 9.9 months for those with wild-type EGFR (P = 0.001). EGFR mutations tended to be associated with increased numbers of CA repeats and increased EGFR gene copy numbers but not with EGFR and caveolin-1 mRNA overexpression (P = not significant). The presence of EGFR mutations is a major determinant of gefitinib response, and targeting EGFR should be considered in preference to chemotherapy as first-line treatment in lung adenocarcinomas that have demonstrable EGFR mutations.

Amplicon Resequencing Identified Parental Mosaicism for Approximately 10% of “de novo” SCN1A Mutations in Children with Dravet Syndrome

PubMed Central

Xu, Xiaojing; Yang, Xiaoxu; Wu, Qixi; Liu, Aijie; Yang, Xiaoling; Ye, Adam Yongxin; Huang, August Yue; Li, Jiarui; Wang, Meng; Yu, Zhe; Wang, Sheng; Zhang, Zhichao; Wu, Xiru

2015-01-01

ABSTRACT The majority of children with Dravet syndrome (DS) are caused by de novo SCN1A mutations. To investigate the origin of the mutations, we developed and applied a new method that combined deep amplicon resequencing with a Bayesian model to detect and quantify allelic fractions with improved sensitivity. Of 174 SCN1A mutations in DS probands which were considered “de novo” by Sanger sequencing, we identified 15 cases (8.6%) of parental mosaicism. We identified another five cases of parental mosaicism that were also detectable by Sanger sequencing. Fraction of mutant alleles in the 20 cases of parental mosaicism ranged from 1.1% to 32.6%. Thirteen (65% of 20) mutations originated paternally and seven (35% of 20) maternally. Twelve (60% of 20) mosaic parents did not have any epileptic symptoms. Their mutant allelic fractions were significantly lower than those in mosaic parents with epileptic symptoms (P = 0.016). We identified mosaicism with varied allelic fractions in blood, saliva, urine, hair follicle, oral epithelium, and semen, demonstrating that postzygotic mutations could affect multiple somatic cells as well as germ cells. Our results suggest that more sensitive tools for detecting low‐level mosaicism in parents of families with seemingly “de novo” mutations will allow for better informed genetic counseling. PMID:26096185

Pyrosequencing®-Based Identification of Low-Frequency Mutations Enriched Through Enhanced-ice-COLD-PCR.

PubMed

How-Kit, Alexandre; Tost, Jörg

2015-01-01

A number of molecular diagnostic assays have been developed in the last years for mutation detection. Although these methods have become increasingly sensitive, most of them are incompatible with a sequencing-based readout and require prior knowledge of the mutation present in the sample. Consequently, coamplification at low denaturation (COLD)-PCR-based methods have been developed and combine a high analytical sensitivity due to mutation enrichment in the sample with the identification of known or unknown mutations by downstream sequencing experiments. Among these methods, the recently developed Enhanced-ice-COLD-PCR appeared as the most powerful method as it outperformed the other COLD-PCR-based methods in terms of the mutation enrichment and due to the simplicity of the experimental setup of the assay. Indeed, E-ice-COLD-PCR is very versatile as it can be used on all types of PCR platforms and is applicable to different types of samples including fresh frozen, FFPE, and plasma samples. The technique relies on the incorporation of an LNA containing blocker probe in the PCR reaction followed by selective heteroduplex denaturation enabling amplification of the mutant allele while amplification of the wild-type allele is prevented. Combined with Pyrosequencing(®), which is a very quantitative high-resolution sequencing technology, E-ice-COLD-PCR can detect and identify mutations with a limit of detection down to 0.01 %.

Next-generation sequencing identifies a novel compound heterozygous mutation in MYO7A in a Chinese patient with Usher Syndrome 1B.

PubMed

Wei, Xiaoming; Sun, Yan; Xie, Jiansheng; Shi, Quan; Qu, Ning; Yang, Guanghui; Cai, Jun; Yang, Yi; Liang, Yu; Wang, Wei; Yi, Xin

2012-11-20

Targeted enrichment and next-generation sequencing (NGS) have been employed for detection of genetic diseases. The purpose of this study was to validate the accuracy and sensitivity of our method for comprehensive mutation detection of hereditary hearing loss, and identify inherited mutations involved in human deafness accurately and economically. To make genetic diagnosis of hereditary hearing loss simple and timesaving, we designed a 0.60 MB array-based chip containing 69 nuclear genes and mitochondrial genome responsible for human deafness and conducted NGS toward ten patients with five known mutations and a Chinese family with hearing loss (never genetically investigated). Ten patients with five known mutations were sequenced using next-generation sequencing to validate the sensitivity of the method. We identified four known mutations in two nuclear deafness causing genes (GJB2 and SLC26A4), one in mitochondrial DNA. We then performed this method to analyze the variants in a Chinese family with hearing loss and identified compound heterozygosity for two novel mutations in gene MYO7A. The compound heterozygosity identified in gene MYO7A causes Usher Syndrome 1B with severe phenotypes. The results support that the combination of enrichment of targeted genes and next-generation sequencing is a valuable molecular diagnostic tool for hereditary deafness and suitable for clinical application. Copyright © 2012 Elsevier B.V. All rights reserved.

A comparison of ARMS-Plus and droplet digital PCR for detecting EGFR activating mutations in plasma

PubMed Central

Zhang, Xinxin; Chang, Ning; Yang, Guohua; Zhang, Yong; Ye, Mingxiang; Cao, Jing; Xiong, Jie; Han, Zhiping; Wu, Shuo; Shang, Lei; Zhang, Jian

2017-01-01

In this study, we introduce a novel amplification refractory mutation system (ARMS)-based assay, namely ARMS-Plus, for the detection of epidermal growth factor receptor (EGFR) mutations in plasma samples. We evaluated the performance of ARMS-Plus in comparison with droplet digital PCR (ddPCR) and assessed the significance of plasma EGFR mutations in predicting efficacy of EGFR-tyrosine kinase inhibitor (TKI) regimen. A total of 122 advanced non-small cell lung cancer (NSCLC) patients were enrolled in this study. The tumor tissue samples from these patients were evaluated by conventional ARMS PCR method to confirm their EGFR mutation status. For the 116 plasma samples analyzed by ARMS-Plus, the sensitivity, specificity, and concordance rate were 77.27% (34/44), 97.22% (70/72), and 89.66% (104/116; κ=0.77, P<0.0001), respectively. Among the 71 plasma samples analyzed by both ARMS-Plus and ddPCR, ARMS-Plus showed a higher sensitivity than ddPCR (83.33% versus 70.83%). The presence of EGFR activating mutations in plasma was not associated with the response to EGFR-TKI, although further validation with a larger cohort is required to confirm the correlation. Collectively, the performance of ARMS-Plus and ddPCR are comparable. ARMS-Plus could be a potential alternative to tissue genotyping for the detection of plasma EGFR mutations in NSCLC patients. PMID:29340107

Quantification of the Mutant CALR Allelic Burden by Digital PCR: Application to Minimal Residual Disease Evaluation after Bone Marrow Transplantation.

PubMed

Mansier, Olivier; Migeon, Marina; Saint-Lézer, Arnaud; James, Chloé; Verger, Emmanuelle; Robin, Marie; Socié, Gérard; Bidet, Audrey; Mahon, François-Xavier; Cassinat, Bruno; Lippert, Eric

2016-01-01

With the recent discovery of CALR mutations, >80% of patients with myeloproliferative neoplasms carry a phenotype-driving mutation. For JAK2 V617F, the most frequent mutation in myeloproliferative neoplasms, accurate determination of mutational loads is of interest at diagnosis, for phenotypic and prognostic purposes, and during follow-up for minimal residual disease assessment. We developed a digital PCR technique that allowed the accurate determination of CALR allelic burdens for the main mutations (types 1 and 2). Compared with the commonly used fluorescent PCR product analysis, digital PCR is more precise, reproducible, and accurate. Furthermore, this method reached a very high sensitivity. We detected at least 0.025% CALR mutants. It can thus be used for patient characterization at diagnosis and for minimal residual disease monitoring. When applied to patients with primary myelofibrosis who underwent hematopoietic stem cell transplant, the digital PCR detected low levels of minimal residual disease. After negativation of the mutational load in all patients, the disease reappeared at a low level in one patient, preceding hematologic relapse. In conclusion, digital PCR adapted to type 1 and 2 CALR mutations is an inexpensive, highly precise, and sensitive technique suitable for evaluation of myeloproliferative neoplasm patients during follow-up. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Restraint of the G2/M Transition by the SR/RRM Family mRNA Shuttling Binding Protein SNXAHRB1 in Aspergillus nidulans

PubMed Central

James, Steven W.; Banta, Travis; Barra, James; Ciraku, Lorela; Coile, Clifford; Cuda, Zach; Day, Ryan; Dixit, Cheshil; Eastlack, Steven; Giang, Anh; Goode, James; Guice, Alexis; Huff, Yulon; Humbert, Sara; Kelliher, Christina; Kobie, Julie; Kohlbrenner, Emily; Mwambutsa, Faustin; Orzechowski, Amanda; Shingler, Kristin; Spell, Casey; Anglin, Sarah Lea

2014-01-01

Control of the eukaryotic G2/M transition by CDC2/CYCLINB is tightly regulated by protein–protein interactions, protein phosphorylations, and nuclear localization of CDC2/CYCLINB. We previously reported a screen, in Aspergillus nidulans, for extragenic suppressors of nimX2cdc2 that resulted in the identification of the cold-sensitive snxA1 mutation. We demonstrate here that snxA1 suppresses defects in regulators of the CDK1 mitotic induction pathway, including nimX2cdc2, nimE6cyclinB, and nimT23cdc25, but does not suppress G2-arresting nimA1/nimA5 mutations, the S-arresting nimE10cyclinB mutation, or three other G1/S phase mutations. snxA encodes the A. nidulans homolog of Saccharomyces cerevisiae Hrb1/Gbp2; nonessential shuttling messenger RNA (mRNA)-binding proteins belonging to the serine-arginine-rich (SR) and RNA recognition motif (RRM) protein family; and human heterogeneous ribonucleoprotein-M, a spliceosomal component involved in pre-mRNA processing and alternative splicing. snxAHrb1 is nonessential, its deletion phenocopies the snxA1 mutation, and its overexpression rescues snxA1 and ΔsnxA mutant phenotypes. snxA1 and a second allele isolated in this study, snxA2, are hypomorphic mutations that result from decreased transcript and protein levels, suggesting that snxA acts normally to restrain cell cycle progression. SNXAHRB1 is predominantly nuclear, but is not retained in the nucleus during the partially closed mitosis of A. nidulans. We show that the snxA1 mutation does not suppress nimX2 by altering NIMX2CDC2/NIMECYCLINB kinase activity and that snxA1 or ΔsnxA alter localization patterns of NIMECYCLINB at the restrictive temperatures for snxA1 and nimX2. Together, these findings suggest a novel and previously unreported role of an SR/RRM family protein in cell cycle regulation, specifically in control of the CDK1 mitotic induction pathway. PMID:25104516

Impact of the F508del mutation on ovine CFTR, a Cl− channel with enhanced conductance and ATP-dependent gating

PubMed Central

Cai, Zhiwei; Palmai-Pallag, Timea; Khuituan, Pissared; Mutolo, Michael J; Boinot, Clément; Liu, Beihui; Scott-Ward, Toby S; Callebaut, Isabelle; Harris, Ann; Sheppard, David N

2015-01-01

Cross-species comparative studies are a powerful approach to understanding the epithelial Cl− channel cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF). Here, we investigate the single-channel behaviour of ovine CFTR and the impact of the most common CF mutation, F508del-CFTR, using excised inside-out membrane patches from transiently transfected CHO cells. Like human CFTR, ovine CFTR formed a weakly inwardly rectifying Cl− channel regulated by PKA-dependent phosphorylation, inhibited by the open-channel blocker glibenclamide. However, for three reasons, ovine CFTR was noticeably more active than human CFTR. First, single-channel conductance was increased. Second, open probability was augmented because the frequency and duration of channel openings were increased. Third, with enhanced affinity and efficacy, ATP more strongly stimulated ovine CFTR channel gating. Consistent with these data, the CFTR modulator phloxine B failed to potentiate ovine CFTR Cl− currents. Similar to its impact on human CFTR, the F508del mutation caused a temperature-sensitive folding defect, which disrupted ovine CFTR protein processing and reduced membrane stability. However, the F508del mutation had reduced impact on ovine CFTR channel gating in contrast to its marked effects on human CFTR. We conclude that ovine CFTR forms a regulated Cl− channel with enhanced conductance and ATP-dependent channel gating. This phylogenetic analysis of CFTR structure and function demonstrates that subtle changes in structure have pronounced effects on channel function and the consequences of the CF mutation F508del. Key points Malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), a gated pathway for chloride movement, causes the common life-shortening genetic disease cystic fibrosis (CF). Towards the development of a sheep model of CF, we have investigated the function of sheep CFTR. We found that sheep CFTR was noticeably more active than human CFTR, while the most common CF mutation, F508del, had reduced impact on sheep CFTR function. Our results demonstrate that subtle changes in protein structure have marked effects on CFTR function and the consequences of the CF mutation F508del. PMID:25763566

Aggressive natural killer-cell leukemia mutational landscape and drug profiling highlight JAK-STAT signaling as therapeutic target.

PubMed

Dufva, Olli; Kankainen, Matti; Kelkka, Tiina; Sekiguchi, Nodoka; Awad, Shady Adnan; Eldfors, Samuli; Yadav, Bhagwan; Kuusanmäki, Heikki; Malani, Disha; Andersson, Emma I; Pietarinen, Paavo; Saikko, Leena; Kovanen, Panu E; Ojala, Teija; Lee, Dean A; Loughran, Thomas P; Nakazawa, Hideyuki; Suzumiya, Junji; Suzuki, Ritsuro; Ko, Young Hyeh; Kim, Won Seog; Chuang, Shih-Sung; Aittokallio, Tero; Chan, Wing C; Ohshima, Koichi; Ishida, Fumihiro; Mustjoki, Satu

2018-04-19

Aggressive natural killer-cell (NK-cell) leukemia (ANKL) is an extremely aggressive malignancy with dismal prognosis and lack of targeted therapies. Here, we elucidate the molecular pathogenesis of ANKL using a combination of genomic and drug sensitivity profiling. We study 14 ANKL patients using whole-exome sequencing (WES) and identify mutations in STAT3 (21%) and RAS-MAPK pathway genes (21%) as well as in DDX3X (29%) and epigenetic modifiers (50%). Additional alterations include JAK-STAT copy gains and tyrosine phosphatase mutations, which we show recurrent also in extranodal NK/T-cell lymphoma, nasal type (NKTCL) through integration of public genomic data. Drug sensitivity profiling further demonstrates the role of the JAK-STAT pathway in the pathogenesis of NK-cell malignancies, identifying NK cells to be highly sensitive to JAK and BCL2 inhibition compared to other hematopoietic cell lineages. Our results provide insight into ANKL genetics and a framework for application of targeted therapies in NK-cell malignancies.

Cross-talk between AMPK and EGFR dependent Signaling in Non-Small Cell Lung Cancer

NASA Astrophysics Data System (ADS)

Praveen, Paurush; Hülsmann, Helen; Sültmann, Holger; Kuner, Ruprecht; Fröhlich, Holger

2016-06-01

Lung cancers globally account for 12% of new cancer cases, 85% of these being Non Small Cell Lung Cancer (NSCLC). Therapies like erlotinib target the key player EGFR, which is mutated in about 10% of lung adenocarcinoma. However, drug insensitivity and resistance caused by second mutations in the EGFR or aberrant bypass signaling have evolved as a major challenge in controlling these tumors. Recently, AMPK activation was proposed to sensitize NSCLC cells against erlotinib treatment. However, the underlying mechanism is largely unknown. In this work we aim to unravel the interplay between 20 proteins that were previously associated with EGFR signaling and erlotinib drug sensitivity. The inferred network shows a high level of agreement with protein-protein interactions reported in STRING and HIPPIE databases. It is further experimentally validated with protein measurements. Moreover, predictions derived from our network model fairly agree with somatic mutations and gene expression data from primary lung adenocarcinoma. Altogether our results support the role of AMPK in EGFR signaling and drug sensitivity.

Cross-talk between AMPK and EGFR dependent Signaling in Non-Small Cell Lung Cancer

PubMed Central

Praveen, Paurush; Hülsmann, Helen; Sültmann, Holger; Kuner, Ruprecht; Fröhlich, Holger

2016-01-01

Lung cancers globally account for 12% of new cancer cases, 85% of these being Non Small Cell Lung Cancer (NSCLC). Therapies like erlotinib target the key player EGFR, which is mutated in about 10% of lung adenocarcinoma. However, drug insensitivity and resistance caused by second mutations in the EGFR or aberrant bypass signaling have evolved as a major challenge in controlling these tumors. Recently, AMPK activation was proposed to sensitize NSCLC cells against erlotinib treatment. However, the underlying mechanism is largely unknown. In this work we aim to unravel the interplay between 20 proteins that were previously associated with EGFR signaling and erlotinib drug sensitivity. The inferred network shows a high level of agreement with protein-protein interactions reported in STRING and HIPPIE databases. It is further experimentally validated with protein measurements. Moreover, predictions derived from our network model fairly agree with somatic mutations and gene expression data from primary lung adenocarcinoma. Altogether our results support the role of AMPK in EGFR signaling and drug sensitivity. PMID:27279498

Cross-talk between AMPK and EGFR dependent Signaling in Non-Small Cell Lung Cancer.

PubMed

Praveen, Paurush; Hülsmann, Helen; Sültmann, Holger; Kuner, Ruprecht; Fröhlich, Holger

2016-06-09

Lung cancers globally account for 12% of new cancer cases, 85% of these being Non Small Cell Lung Cancer (NSCLC). Therapies like erlotinib target the key player EGFR, which is mutated in about 10% of lung adenocarcinoma. However, drug insensitivity and resistance caused by second mutations in the EGFR or aberrant bypass signaling have evolved as a major challenge in controlling these tumors. Recently, AMPK activation was proposed to sensitize NSCLC cells against erlotinib treatment. However, the underlying mechanism is largely unknown. In this work we aim to unravel the interplay between 20 proteins that were previously associated with EGFR signaling and erlotinib drug sensitivity. The inferred network shows a high level of agreement with protein-protein interactions reported in STRING and HIPPIE databases. It is further experimentally validated with protein measurements. Moreover, predictions derived from our network model fairly agree with somatic mutations and gene expression data from primary lung adenocarcinoma. Altogether our results support the role of AMPK in EGFR signaling and drug sensitivity.

UCS protein Rng3p activates actin filament gliding by fission yeast myosin-II

PubMed Central

Lord, Matthew; Pollard, Thomas D.

2004-01-01

We purified native Myo2p/Cdc4p/Rlc1p (Myo2), the myosin-II motor required for cytokinesis by Schizosaccharomyces pombe. The Myo2p heavy chain associates with two light chains, Cdc4p and Rlc1p. Although crude Myo2 supported gliding motility of actin filaments in vitro, purified Myo2 lacked this activity in spite of retaining full Ca-ATPase activity and partial actin-activated Mg-ATPase activity. Unc45-/Cro1p-/She4p-related (UCS) protein Rng3p restored the full motility and actin-activated Mg-ATPase activity of purified Myo2. The COOH-terminal UCS domain of Rng3p alone restored motility to pure Myo2. Thus, Rng3p contributes directly to the motility activity of native Myo2. Consistent with a role in Myo2 activation, Rng3p colocalizes with Myo2p in the cytokinetic contractile ring. The absence of Rlc1p or mutations in the Myo2p head or Rng3p compromise the in vitro motility of Myo2 and explain the defects in cytokinesis associated with some of these mutations. In contrast, Myo2 with certain temperature-sensitive forms of Cdc4p has normal motility, so these mutations compromise other functions of Cdc4p required for cytokinesis. PMID:15504913

A novel class of Saccharomyces cerevisiae mutants specifically UV-sensitive to "petite" induction.

PubMed

Moustacchi, E; Perlman, P S; Mahler, H R

1976-11-17

A mutant of Saccharomyces cerevisiae has been isolated which, though exhibiting a normal response to nuclear genetic damage by ultraviolet light (UV), is more sensitive than its wild type specifically in the production of the cytoplasmic (rho-) mutation by this agent. Some of the features of this mutation which has been designated uvsrho 5 are: i) The mutation is recessive, it exhibits a Mendelian, and hence presumably nuclear, pattern of segregation, but manifests its effects specifically and pleiotropically on mitochondrial functions. ii) Mutant cells resemble their wild type parents in a) growth characteristics on glucose; b) in their UV induced dose response to lethality or nuclear mutation and c) the ability of their mitochondrial genome, upon mating with appropriate testers, of transmitting and recombining various markers, albeit with enhanced efficiency. Similarly, d) they are able to modulate the expression of mitochondrial mutagenesis by ethidium bromide. Thus their mitochondrial DNA appears genetically as competent as that of the wild type. iii) Mutant cells differ from their wild type parents in a) growth characteristics on glycerol; b) susceptibility to induction of the mitochondrial (rho-) mutation by various mutagens, in that the rate of spontaneous mutation is slightly and that by UV is significantly enhanced, whild that by ethidium bromide is greatly diminished. Conversely, c) modulating influences resulting in the repair of initial damage are diminished fro UV and stimulated in the case of Berenil. iv) The amount of mitochondrial DNA per cell appears elevated in the mutant, relative to wild type, and its rate of degradation subsequent to a mutagenic exposure to either UV or ethidium bromide is diminished. v) A self-consistent scheme to account for this and all other information so far available for the induction and modulation of the (rho-) mutation is presented. In a previous study it was shown that some nuclear mutants of Saccharomyces cerevisiae, more sensitive to lethal damage induced by ultraviolet light (rad) than their parent wild type (RAD), also exhibit a concomitant modification in sensitivity to both nuclear and cytoplasmic genetic damage (Moustacchi, 1971). However, another class of rad mutants respond to the induction of the cytoplasmic "petite" also designated as rho- (or rho-) mutation by UV in a manner indistinguishable from that of the RAD strain. One possible interpretation of this last observation is that some of the steps in the expression of the UV damage on mitochondrial (mt)DNA may be governed by other nuclear and cytoplasmic genetic determinants, the products of which may then act specifically on mitochondrial lesions. If this assumption is correct, it should be possible to find mutants with a normal response to nuclear damage but specifically UV-sensitive towards induction of (rho-)...

Sec34p, a Protein Required for Vesicle Tethering to the Yeast Golgi Apparatus, Is in a Complex with Sec35p

PubMed Central

VanRheenen, Susan M.; Cao, Xiaochun; Sapperstein, Stephanie K.; Chiang, Elbert C.; Lupashin, Vladimir V.; Barlowe, Charles; Waters, M. Gerard

1999-01-01

A screen for mutants of Saccharomyces cerevisiae secretory pathway components previously yielded sec34, a mutant that accumulates numerous vesicles and fails to transport proteins from the ER to the Golgi complex at the restrictive temperature (Wuestehube, L.J., R. Duden, A. Eun, S. Hamamoto, P. Korn, R. Ram, and R. Schekman. 1996. Genetics. 142:393–406). We find that SEC34 encodes a novel protein of 93-kD, peripherally associated with membranes. The temperature-sensitive phenotype of sec34-2 is suppressed by the rab GTPase Ypt1p that functions early in the secretory pathway, or by the dominant form of the ER to Golgi complex target-SNARE (soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor)–associated protein Sly1p, Sly1-20p. Weaker suppression is evident upon overexpression of genes encoding the vesicle tethering factor Uso1p or the vesicle-SNAREs Sec22p, Bet1p, or Ykt6p. This genetic suppression profile is similar to that of sec35-1, a mutant allele of a gene encoding an ER to Golgi vesicle tethering factor and, like Sec35p, Sec34p is required in vitro for vesicle tethering. sec34-2 and sec35-1 display a synthetic lethal interaction, a genetic result explained by the finding that Sec34p and Sec35p can interact by two-hybrid analysis. Fractionation of yeast cytosol indicates that Sec34p and Sec35p exist in an ∼750-kD protein complex. Finally, we describe RUD3, a novel gene identified through a genetic screen for multicopy suppressors of a mutation in USO1, which suppresses the sec34-2 mutation as well. PMID:10562277

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.

Differential Effects of CSF-1R D802V and KIT D816V Homologous Mutations on Receptor Tertiary Structure and Allosteric Communication

PubMed Central

Da Silva Figueiredo Celestino Gomes, Priscila; Panel, Nicolas; Laine, Elodie; Pascutti, Pedro Geraldo; Solary, Eric; Tchertanov, Luba

2014-01-01

The colony stimulating factor-1 receptor (CSF-1R) and the stem cell factor receptor KIT, type III receptor tyrosine kinases (RTKs), are important mediators of signal transduction. The normal functions of these receptors can be compromised by gain-of-function mutations associated with different physiopatological impacts. Whereas KIT D816V/H mutation is a well-characterized oncogenic event and principal cause of systemic mastocytosis, the homologous CSF-1R D802V has not been identified in human cancers. The KIT D816V oncogenic mutation triggers resistance to the RTK inhibitor Imatinib used as first line treatment against chronic myeloid leukemia and gastrointestinal tumors. CSF-1R is also sensitive to Imatinib and this sensitivity is altered by mutation D802V. Previous in silico characterization of the D816V mutation in KIT evidenced that the mutation caused a structure reorganization of the juxtamembrane region (JMR) and facilitated its departure from the kinase domain (KD). In this study, we showed that the equivalent CSF-1R D802V mutation does not promote such structural effects on the JMR despite of a reduction on some key H-bonds interactions controlling the JMR binding to the KD. In addition, this mutation disrupts the allosteric communication between two essential regulatory fragments of the receptors, the JMR and the A-loop. Nevertheless, the mutation-induced shift towards an active conformation observed in KIT D816V is not observed in CSF-1R D802V. The distinct impact of equivalent mutation in two homologous RTKs could be associated with the sequence difference between both receptors in the native form, particularly in the JMR region. A local mutation-induced perturbation on the A-loop structure observed in both receptors indicates the stabilization of an inactive non-inhibited form, which Imatinib cannot bind. PMID:24828813

The Diageotropica Gene Differentially Affects Auxin and Cytokinin Responses throughout Development in Tomato1

PubMed Central

Coenen, Catharina; Lomax, Terri L.

1998-01-01

The interactions between the plant hormones auxin and cytokinin throughout plant development are complex, and genetic investigations of the interdependency of auxin and cytokinin signaling have been limited. We have characterized the cytokinin sensitivity of the auxin-resistant diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) in a range of auxin- and cytokinin-regulated responses. Intact, etiolated dgt seedlings showed cross-resistance to cytokinin with respect to root elongation, but cytokinin effects on hypocotyl growth and ethylene synthesis in these seedlings were not impaired by the dgt mutation. Seven-week-old, green wild-type and dgt plants were also equally sensitive to cytokinin with respect to shoot growth and hypocotyl and internode elongation. The effects of cytokinin and the dgt mutation on these processes appeared additive. In tissue culture organ regeneration from dgt hypocotyl explants showed reduced sensitivity to auxin but normal sensitivity to cytokinin, and the effects of cytokinin and the mutation were again additive. However, although callus induction from dgt hypocotyl explants required auxin and cytokinin, dgt calli did not show the typical concentration-dependent stimulation of growth by either auxin or cytokinin observed in wild-type calli. Cross-resistance of the dgt mutant to cytokinin thus was found to be limited to a small subset of auxin- and cytokinin-regulated growth processes affected by the dgt mutation, indicating that auxin and cytokinin regulate plant growth through both shared and separate signaling pathways. PMID:9576775

A Genetic Basis for Mechanosensory Traits in Humans

PubMed Central

Frenzel, Henning; Bohlender, Jörg; Pinsker, Katrin; Wohlleben, Bärbel; Tank, Jens; Lechner, Stefan G.; Schiska, Daniela; Jaijo, Teresa; Rüschendorf, Franz; Saar, Kathrin; Jordan, Jens; Millán, José M.; Gross, Manfred; Lewin, Gary R.

2012-01-01

In all vertebrates hearing and touch represent two distinct sensory systems that both rely on the transformation of mechanical force into electrical signals. There is an extensive literature describing single gene mutations in humans that cause hearing impairment, but there are essentially none for touch. Here we first asked if touch sensitivity is a heritable trait and second whether there are common genes that influence different mechanosensory senses like hearing and touch in humans. Using a classical twin study design we demonstrate that touch sensitivity and touch acuity are highly heritable traits. Quantitative phenotypic measures of different mechanosensory systems revealed significant correlations between touch and hearing acuity in a healthy human population. Thus mutations in genes causing deafness genes could conceivably negatively influence touch sensitivity. In agreement with this hypothesis we found that a proportion of a cohort of congenitally deaf young adults display significantly impaired measures of touch sensitivity compared to controls. In contrast, blind individuals showed enhanced, not diminished touch acuity. Finally, by examining a cohort of patients with Usher syndrome, a genetically well-characterized deaf-blindness syndrome, we could show that recessive pathogenic mutations in the USH2A gene influence touch acuity. Control Usher syndrome cohorts lacking demonstrable pathogenic USH2A mutations showed no impairment in touch acuity. Our study thus provides comprehensive evidence that there are common genetic elements that contribute to touch and hearing and has identified one of these genes as USH2A. PMID:22563300

Mutation detection using ENDO1: application to disease diagnostics in humans and TILLING and Eco-TILLING in plants.

PubMed

Triques, Karine; Piednoir, Elodie; Dalmais, Marion; Schmidt, Julien; Le Signor, Christine; Sharkey, Mark; Caboche, Michel; Sturbois, Bénédicte; Bendahmane, Abdelhafid

2008-04-23

Most enzymatic mutation detection methods are based on the cleavage of heteroduplex DNA by a mismatch-specific endonuclease at mismatch sites and the analysis of the digestion product on a DNA sequencer. Important limitations of these methods are the availability of a mismatch-specific endonuclease, their sensitivity in detecting one allele in pool of DNA, the cost of the analysis and the ease by which the technique could be implemented in a standard molecular biology laboratory. The co-agroinfiltration of ENDO1 and p19 constructs into N. benthamiana leaves allowed high level of transient expression of a mismatch-specific and sensitive endonuclease, ENDO1 from Arabidopsis thaliana. We demonstrate the broad range of uses of the produced enzyme in detection of mutations. In human, we report the diagnosis of the G1691A mutation in Leiden factor-V gene associated with venous thrombosis and the fingerprinting of HIV-1 quasispecies in patients subjected to antiretroviral treatments. In plants, we report the use of ENDO1 system for detection of mutant alleles of Retinoblastoma-related gene by TILLING in Pisum sativum and discovery of natural sequence variations by Eco-TILLING in Arabidopsis thaliana. We introduce a cost-effective tool based on a simplified purification protocol of a mismatch-specific and sensitive endonuclease, ENDO1. Especially, we report the successful applications of ENDO1 in mutation diagnostics in humans, fingerprinting of complex population of viruses, and in TILLING and Eco-TILLING in plants.

Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations

PubMed Central

Zonta, Eleonora; Garlan, Fanny; Pécuchet, Nicolas; Perez-Toralla, Karla; Caen, Ouriel; Milbury, Coren; Didelot, Audrey; Fabre, Elizabeth; Blons, Hélène; Laurent-Puig, Pierre; Taly, Valérie

2016-01-01

In cancer research, the accuracy of the technology used for biomarkers detection is remarkably important. In this context, digital PCR represents a highly sensitive and reproducible method that could serve as an appropriate tool for tumor mutational status analysis. In particular, droplet-based digital PCR approaches have been developed for detection of tumor-specific mutated alleles within plasmatic circulating DNA. Such an approach calls for the development and validation of a very significant quantity of assays, which can be extremely costly and time consuming. Herein, we evaluated assays for the detection and quantification of various mutations occurring in three genes often misregulated in cancers: the epidermal growth factor receptor (EGFR), the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and the Tumoral Protein p53 (TP53) genes. In particular, commercial competitive allele-specific TaqMan® PCR (castPCR™) technology, as well as TaqMan® and ZEN™ assays, have been evaluated for EGFR p.L858R, p.T790M, p.L861Q point mutations and in-frame deletions Del19. Specificity and sensitivity have been determined on cell lines DNA, plasmatic circulating DNA of lung cancer patients or Horizon Diagnostics Reference Standards. To show the multiplexing capabilities of this technology, several multiplex panels for EGFR (several three- and four-plexes) have been developed, offering new "ready-to-use" tests for lung cancer patients. PMID:27416070

Development and validation of a whole-exome sequencing test for simultaneous detection of point mutations, indels and copy-number alterations for precision cancer care

PubMed Central

Rennert, Hanna; Eng, Kenneth; Zhang, Tuo; Tan, Adrian; Xiang, Jenny; Romanel, Alessandro; Kim, Robert; Tam, Wayne; Liu, Yen-Chun; Bhinder, Bhavneet; Cyrta, Joanna; Beltran, Himisha; Robinson, Brian; Mosquera, Juan Miguel; Fernandes, Helen; Demichelis, Francesca; Sboner, Andrea; Kluk, Michael; Rubin, Mark A; Elemento, Olivier

2016-01-01

We describe Exome Cancer Test v1.0 (EXaCT-1), the first New York State-Department of Health-approved whole-exome sequencing (WES)-based test for precision cancer care. EXaCT-1 uses HaloPlex (Agilent) target enrichment followed by next-generation sequencing (Illumina) of tumour and matched constitutional control DNA. We present a detailed clinical development and validation pipeline suitable for simultaneous detection of somatic point/indel mutations and copy-number alterations (CNAs). A computational framework for data analysis, reporting and sign-out is also presented. For the validation, we tested EXaCT-1 on 57 tumours covering five distinct clinically relevant mutations. Results demonstrated elevated and uniform coverage compatible with clinical testing as well as complete concordance in variant quality metrics between formalin-fixed paraffin embedded and fresh-frozen tumours. Extensive sensitivity studies identified limits of detection threshold for point/indel mutations and CNAs. Prospective analysis of 337 cancer cases revealed mutations in clinically relevant genes in 82% of tumours, demonstrating that EXaCT-1 is an accurate and sensitive method for identifying actionable mutations, with reasonable costs and time, greatly expanding its utility for advanced cancer care. PMID:28781886

Wee-1 Kinase Inhibition Overcomes Cisplatin Resistance Associated with High-Risk TP53 Mutations in Head and Neck Cancer through Mitotic Arrest Followed by Senescence

PubMed Central

Osman, Abdullah A.; Monroe, Marcus M.; Ortega Alves, Marcus V.; Patel, Ameeta A.; Katsonis, Panagiotis; Fitzgerald, Alison L.; Neskey, David M.; Frederick, Mitchell J.; Woo, Sang Hyeok; Caulin, Carlos; Hsu, Teng-Kuei; McDonald, Thomas O.; Kimmel, Marek; Meyn, Raymond E.; Lichtarge, Olivier; Myers, Jeffrey N.

2015-01-01

Although cisplatin has played a role in “standard-of-care” multimodality therapy for patients with advanced squamous cell carcinoma of the head and neck (HNSCC), the rate of treatment failure remains particularly high for patients receiving cisplatin whose tumors have mutations in the TP53 gene. We found that cisplatin treatment of HNSCC cells with mutant TP53 leads to arrest of cells in the G2 phase of the cell cycle, leading us to hypothesize that the wee-1 kinase inhibitor MK-1775 would abrogate the cisplatin-induced G2 block and thereby sensitize isogenic HNSCC cells with mutant TP53 or lacking p53 expression to cisplatin. We tested this hypothesis using clonogenic survival assays, flow cytometry, and in vivo tumor growth delay experiments with an orthotopic nude mouse model of oral tongue cancer. We also used a novel TP53 mutation classification scheme to identify which TP53 mutations are associated with limited tumor responses to cisplatin treatment. Clonogenic survival analyses indicate that nanomolar concentration of MK-1775 sensitizes HNSCC cells with high-risk mutant p53 to cisplatin. Consistent with its ability to chemosensitize, MK-1775 abrogated the cisplatin-induced G2 block in p53-defective cells leading to mitotic arrest associated with a senescence-like phenotype. Furthermore, MK-1775 enhanced the efficacy of cisplatin in vivo in tumors harboring TP53 mutations. These results indicate that HNSCC cells expressing high-risk p53 mutations are significantly sensitized to cisplatin therapy by the selective wee-1 kinase inhibitor, supporting the clinical evaluation of MK-1775 in combination with cisplatin for the treatment of patients with TP53 mutant HNSCC. PMID:25504633

PIF3 is a negative regulator of the CBF pathway and freezing tolerance in Arabidopsis

PubMed Central

Jiang, Bochen; Shi, Yiting; Zhang, Xiaoyan; Xin, Xiaoyun; Qi, Lijuan; Guo, Hongwei; Li, Jigang; Yang, Shuhua

2017-01-01

Light and temperature are major environmental factors that coordinately control plant growth and survival. However, how plants integrate light and temperature signals to better adapt to environmental stresses is poorly understood. PHYTOCHROME-INTERACTING FACTOR 3 (PIF3), a key transcription factor repressing photomorphogenesis, has been shown to play a pivotal role in mediating plants’ responses to various environmental signals. In this study, we found that PIF3 functions as a negative regulator of Arabidopsis freezing tolerance by directly binding to the promoters of C-REPEAT BINDING FACTOR (CBF) genes to down-regulate their expression. In addition, two F-box proteins, EIN3-BINDING F-BOX 1 (EBF1) and EBF2, directly target PIF3 for 26S proteasome-mediated degradation. Consistently, ebf1 and ebf2 mutants were more sensitive to freezing than were the wild type, and the pif3 mutation suppressed the freezing-sensitive phenotype of ebf1. Furthermore, cold treatment promoted the degradation of EBF1 and EBF2, leading to increased stability of the PIF3 protein and reduced expression of the CBF genes. Together, our study uncovers an important role of PIF3 in Arabidopsis freezing tolerance by negatively regulating the expression of genes in the CBF pathway. PMID:28739888

[Evaluation of Consistency in detection of epidermal growth factor receptor gene T790M mutation in plasma and tumor specimens of patients with lung adenocarcinoma].

PubMed

Du, J; Wang, Z; Yang, L; Di, J; Zhang, J G; Wang, T Y; Liu, D G

2018-01-23

Objective: To evaluate the consistency in detection of T790M mutation of epidermal growth factor receptor gene (EGFR) in plasma and tumor samples of patients with lung adenocarcinoma. Methods: The tumor tissues or cytological specimens of 12 patients with operable lung adenocarcinoma(stage Ⅰ-ⅢA) and 100 patients with advanced stage ⅢB-Ⅳ lung adenocarcinoma were collected, among which 11 patients showed acquired resistance for gefitinib (11/100). In the same period, peripheral blood samples were collected from all patients and 50 healthy volunteers. Amplification refractory mutation system (ARMS) was used to detect EGFR mutations in tumor specimens. Next Generation Sequencing(NGS) based circulating single-molecule amplification and resequencing technology (cSMART)was performed to quantitatively detect the EGFR mutations in circulating tumor DNA (ctDNA) from plasma specimens. Results: The sensitivity, specificity and concordance rate of EGFR T790M mutation between plasma and tissue specimens from 100 advanced stage patients were 50.0%, 72.9% and 72.0%, respectively. For L858R mutation and exon 19 deletion mutations, the above mentioned sensitivity, specificity and concordance rate were 91.7%, 100.0%, and 98.0%, as well as 79.2%, 100.0% and 95.0%, respectively. The L858R mutation and exon 19 deletion mutations were not detected in plasma of 50 healthy volunteers, whereasT790M mutation(1.0±0.0 copies) was found in 7 individuals(7/50, 14.0%). Similarly, in 12 resectable patients, 4 (4/12, 33.3%) T790M mutations were found in plasma (1.2±0.2 copies), but no L858R mutation and 19 exon deletion mutations. In comparison, 28.0% of patients with advanced lung adenocarcinoma (28/100)had detectable T790M mutation in plasma with copy numbers (34.0±22.7 copies). Furthermore, the copy numbers of T790M were 268.2±119.9 in plasma of 5 cases with acquired gefitinib-resistance. Conclusions: In patients with advanced stages of lung adenocarcinoma, the detection of T790M mutation in plasma and tumor specimens is low. The T790M mutation also exists in the plasma of some healthy controls, suggesting that T790M mutation participates in EGFR signaling pathway and it might function in healthy population.

Identification of two new genes, mukE and mukF, involved in chromosome partitioning in Escherichia coli.

PubMed

Yamanaka, K; Ogura, T; Niki, H; Hiraga, S

1996-02-25

We have previously reported that the MukB protein is essential for chromosome partitioning in Escherichia coli and that mukB mutants produce anucleate cells and are temperature-sensitive for colony formation. The mukB gene maps at 21 min on the E. coli chromosome and smtA-mukF-mukE-mukB genes might comprise an operon, which is transcribed in a clockwise direction. Here, we report that mukF and mukE null mutants are both temperature-sensitive for colony formation and produce anucleate cells even at the permissive temperature. These phenotypes are the same as those observed in the mukB null mutant. The primary sequence of MukF includes a leucine zipper structure and an acidic domain. Mutational analysis revealed that both are required for MukF function. When the MukF protein was overproduced in the wild-type strain, anucleate cells were produced. In contrast, overproduction of either MukE or MukB did not cause the defect. In null mutants for the mukF, mukE, and mukB genes, the synchronous initiation of chromosome replication was not affected. The mini-F plasmid was as stably maintained in these mutants as in the wild-type strain. These results indicate that the MukF, MukE, and MukB proteins are involved in the chromosome partitioning steps, but are not required for mini-F plasmid partitioning.

Accuracy of screening women at familial risk of breast cancer without a known gene mutation: Individual patient data meta-analysis.

PubMed

Phi, Xuan-Anh; Houssami, Nehmat; Hooning, Maartje J; Riedl, Christopher C; Leach, Martin O; Sardanelli, Francesco; Warner, Ellen; Trop, Isabelle; Saadatmand, Sepideh; Tilanus-Linthorst, Madeleine M A; Helbich, Thomas H; van den Heuvel, Edwin R; de Koning, Harry J; Obdeijn, Inge-Marie; de Bock, Geertruida H

2017-11-01

Women with a strong family history of breast cancer (BC) and without a known gene mutation have an increased risk of developing BC. We aimed to investigate the accuracy of screening using annual mammography with or without magnetic resonance imaging (MRI) for these women outside the general population screening program. An individual patient data (IPD) meta-analysis was conducted using IPD from six prospective screening trials that had included women at increased risk for BC: only women with a strong familial risk for BC and without a known gene mutation were included in this analysis. A generalised linear mixed model was applied to estimate and compare screening accuracy (sensitivity, specificity and predictive values) for annual mammography with or without MRI. There were 2226 women (median age: 41 years, interquartile range 35-47) with 7478 woman-years of follow-up, with a BC rate of 12 (95% confidence interval 9.3-14) in 1000 woman-years. Mammography screening had a sensitivity of 55% (standard error of mean [SE] 7.0) and a specificity of 94% (SE 1.3). Screening with MRI alone had a sensitivity of 89% (SE 4.6) and a specificity of 83% (SE 2.8). Adding MRI to mammography increased sensitivity to 98% (SE 1.8, P < 0.01 compared to mammography alone) but lowered specificity to 79% (SE 2.7, P < 0.01 compared with mammography alone). In this population of women with strong familial BC risk but without a known gene mutation, in whom BC incidence was high both before and after age 50, adding MRI to mammography substantially increased screening sensitivity but also decreased its specificity. Copyright © 2017 Elsevier Ltd. All rights reserved.

IDH1-mutant cancer cells are sensitive to cisplatin and an IDH1-mutant inhibitor counteracts this sensitivity.

PubMed

Khurshed, Mohammed; Aarnoudse, Niels; Hulsbos, Renske; Hira, Vashendriya V V; van Laarhoven, Hanneke W M; Wilmink, Johanna W; Molenaar, Remco J; van Noorden, Cornelis J F

2018-06-07

Isocitrate dehydrogenase ( IDH1)-1 is mutated in various types of human cancer, and the presence of this mutation is associated with improved responses to irradiation and chemotherapy in solid tumor cells. Mutated IDH1 (IDH1 MUT ) enzymes consume NADPH to produce d-2-hydroxyglutarate (d-2HG) resulting in the decreased reducing power needed for detoxification of reactive oxygen species (ROS), for example. The objective of the current study was to investigate the mechanism behind the chemosensitivity of the widely-used anticancer agent cisplatin in IDH1 MUT cancer cells. Oxidative stress, DNA damage, and mitochondrial dysfunction caused by cisplatin treatment were monitored in IDH1 MUT HCT116 colorectal cancer cells and U251 glioma cells. We found that exposure to cisplatin induced higher levels of ROS, DNA double-strand breaks (DSBs), and cell death in IDH1 MUT cancer cells, as compared with IDH1 wild-type ( IDH1 WT ) cells. Mechanistic investigations revealed that cisplatin treatment dose dependently reduced oxidative respiration in IDH1 MUT cells, which was accompanied by disturbed mitochondrial proteostasis, indicative of impaired mitochondrial activity. These effects were abolished by the IDH1 MUT inhibitor AGI-5198 and were restored by treatment with d-2HG. Thus, our study shows that altered oxidative stress responses and a vulnerable oxidative metabolism underlie the sensitivity of IDH1 MUT cancer cells to cisplatin.-Khurshed, M., Aarnoudse, N., Hulsbos, R., Hira, V. V. V., van Laarhoven, H. W. M., Wilmink, J. W., Molenaar, R. J., van Noorden, C. J. F. IDH1-mutated cancer cells are sensitive to cisplatin and an IDH1-mutant inhibitor counteracts this sensitivity.

A mutated dph3 gene causes sensitivity of Schizosaccharomyces pombe cells to cytotoxic agents.

PubMed

Villahermosa, Desirée; Knapp, Karen; Fleck, Oliver

2017-12-01

Dph3 is involved in diphthamide modification of the eukaryotic translation elongation factor eEF2 and in Elongator-mediated modifications of tRNAs, where a 5-methoxycarbonyl-methyl moiety is added to wobble uridines. Lack of such modifications affects protein synthesis due to inaccurate translation of mRNAs at ribosomes. We have discovered that integration of markers at the msh3 locus of Schizosaccharomyces pombe impaired the function of the nearby located dph3 gene. Such integrations rendered cells sensitive to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. We constructed dph3 and msh3 strains with mutated ATG start codons (ATGmut), which allowed investigating drug sensitivity without potential interference by marker insertions. The dph3-ATGmut and a dph3::loxP-ura4-loxM gene disruption strain, but not msh3-ATGmut, turned out to be sensitive to hydroxyurea and methyl methanesulfonate, likewise the strains with cassettes integrated at the msh3 locus. The fungicide sordarin, which inhibits diphthamide modified eEF2 of Saccharomyces cerevisiae, barely affected survival of wild type and msh3Δ S. pombe cells, while the dph3Δ mutant was sensitive. The msh3-ATG mutation, but not dph3Δ or the dph3-ATG mutation caused a defect in mating-type switching, indicating that the ura4 marker at the dph3 locus did not interfere with Msh3 function. We conclude that Dph3 is required for cellular resistance to the fungicide sordarin and to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. This is likely mediated by efficient translation of proteins in response to DNA damage and replication stress.

Mechanism for initiation of food allergy: Dependence on skin barrier mutations and environmental allergen costimulation.

PubMed

Walker, Matthew T; Green, Jeremy E; Ferrie, Ryan P; Queener, Ashley M; Kaplan, Mark H; Cook-Mills, Joan M

2018-05-01

Mechanisms for the development of food allergy in neonates are unknown but clearly linked in patient populations to a genetic predisposition to skin barrier defects. Whether skin barrier defects contribute functionally to development of food allergy is unknown. The purpose of the study was to determine whether skin barrier mutations, which are primarily heterozygous in patient populations, contribute to the development of food allergy. Mice heterozygous for the filaggrin (Flg) ft and Tmem79 ma mutations were skin sensitized with environmental and food allergens. After sensitization, mice received oral challenge with food allergen, and then inflammation, inflammatory mediators, and anaphylaxis were measured. We define development of inflammation, inflammatory mediators, and food allergen-induced anaphylaxis in neonatal mice with skin barrier mutations after brief concurrent cutaneous exposure to food and environmental allergens. Moreover, neonates of allergic mothers have increased responses to suboptimal sensitization with food allergens. Importantly, responses to food allergens by these neonatal mice were dependent on genetic defects in skin barrier function and on exposure to environmental allergens. ST2 blockade during skin sensitization inhibited the development of anaphylaxis, antigen-specific IgE, and inflammatory mediators. Neonatal anaphylactic responses and antigen-specific IgE were also inhibited by oral pre-exposure to food allergen, but interestingly, this was blunted by concurrent pre-exposure of the skin to environmental allergen. These studies uncover mechanisms for food allergy sensitization and anaphylaxis in neonatal mice that are consistent with features of human early-life exposures and genetics in patients with clinical food allergy and demonstrate that changes in barrier function drive development of anaphylaxis to food allergen. Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Cell-intrinsic determinants of ibrutinib-induced apoptosis in Chronic Lymphocytic Leukemia

PubMed Central

Amin, Nisar A.; Balasubramanian, Sriram; Saiya-Cork, Kamlai; Shedden, Kerby; Hu, Nan; Malek, Sami N.

2016-01-01

Purpose Ibrutinib, a Bruton’s tyrosine kinase (BTK) inhibitor, is approved for the treatment of relapsed CLL and CLL with del17p. Mechanistically, ibrutinib interferes with BCR signaling as well as multiple CLL cell to microenvironment interactions. Given the importance of ibrutinib in the management of CLL, a deeper understanding of factors governing sensitivity and resistance is warranted. Experimental Design We studied 48 longitudinally sampled paired CLL samples, 42 of which were procured before and after standard CLL chemotherapies, and characterized them for well-studied CLL molecular traits as well as by whole exome sequencing and SNP 6.0 array profiling. We exposed these samples to 0.25 μM – 5 μM of ibrutinib ex vivo and measured apoptosis fractions as well as BCR signaling by immunoblotting. We disrupted TP53 in HG3, PGA1 and PG-EBV cell lines and measured BCR signaling and ibrutinib responses. Results CLL samples demonstrated a surprisingly wide range of ex vivo sensitivities to ibrutinib with IC50 values ranging from 0.4 μM – 9.7 μM. Unmutated IGVH status, elevated ZAP70 expression and trisomy 12 were associated with heightened sensitivity to ibrutinib treatment. Five CLL samples were substantially more resistant to ibrutinib following relapse from chemotherapy; of these, three had acquired a del17p/TP53 mutated status. A validation sample of 15 CLL carrying TP53 mutations, of which 13 carried both del17p and a TP53 mutation confirmed substantially less sensitivity to ibrutinib-induced apoptosis. Conclusions This study identifies that CLL harboring del17p/TP53 mutated cells are substantially less sensitive to ibrutinib-induced apoptosis than del17p/TP53 wild type cells. PMID:27535981

Next-generation sequencing of circulating tumor DNA to predict recurrence in triple-negative breast cancer patients with residual disease after neoadjuvant chemotherapy.

PubMed

Chen, Yu-Hsiang; Hancock, Bradley A; Solzak, Jeffrey P; Brinza, Dumitru; Scafe, Charles; Miller, Kathy D; Radovich, Milan

2017-01-01

Next-generation sequencing to detect circulating tumor DNA is a minimally invasive method for tumor genotyping and monitoring therapeutic response. The majority of studies have focused on detecting circulating tumor DNA from patients with metastatic disease. Herein, we tested whether circulating tumor DNA could be used as a biomarker to predict relapse in triple-negative breast cancer patients with residual disease after neoadjuvant chemotherapy. In this study, we analyzed samples from 38 early-stage triple-negative breast cancer patients with matched tumor, blood, and plasma. Extracted DNA underwent library preparation and amplification using the Oncomine Research Panel consisting of 134 cancer genes, followed by high-coverage sequencing and bioinformatics. We detected high-quality somatic mutations from primary tumors in 33 of 38 patients. TP53 mutations were the most prevalent (82%) followed by PIK3CA (16%). Of the 33 patients who had a mutation identified in their primary tumor, we were able to detect circulating tumor DNA mutations in the plasma of four patients (three TP53 mutations, one AKT1 mutation, one CDKN2A mutation). All four patients had recurrence of their disease (100% specificity), but sensitivity was limited to detecting only 4 of 13 patients who clinically relapsed (31% sensitivity). Notably, all four patients had a rapid recurrence (0.3, 4.0, 5.3, and 8.9 months). Patients with detectable circulating tumor DNA had an inferior disease free survival ( p  < 0.0001; median disease-free survival: 4.6 mos. vs. not reached; hazard ratio = 12.6, 95% confidence interval: 3.06-52.2). Our study shows that next-generation circulating tumor DNA sequencing of triple-negative breast cancer patients with residual disease after neoadjuvant chemotherapy can predict recurrence with high specificity, but moderate sensitivity. For those patients where circulating tumor DNA is detected, recurrence is rapid.

Multiplex picoliter-droplet digital PCR for quantitative assessment of EGFR mutations in circulating cell-free DNA derived from advanced non-small cell lung cancer patients.

PubMed

Yu, Qian; Huang, Fei; Zhang, Meilin; Ji, Haiying; Wu, Shenchao; Zhao, Ying; Zhang, Chunyan; Wu, Jiong; Wang, Beili; Pan, Baisheng; Zhang, Xin; Guo, Wei

2017-08-01

To explore the possible diagnostic value of liquid biopsy, two multiplex panels using picoliter-droplet digital polymerase chain reaction (ddPCR) were established to quantitatively assess the epidermal growth factor receptor (EGFR) mutations in cell‑free DNA (cfDNA) extracted from the plasma of advanced non‑small cell lung cancer (NSCLC) patients. Plasma samples derived from 22 patients with stage IIIB/IV NSCLC harboring EGFR mutations in matched tumor tissues confirmed by amplification refractory mutation system (ARMS) analysis were subjected to two multiplex ddPCR panels to assess the abundance of tyrosine kinase inhibitor (TKI) ‑sensitive (19DEL, L858R) and TKI‑resistant (T790 M) mutations. Fluctuations in EGFR mutant abundance were monitored by either of the multiplex ddPCR panels for three patients undergoing EGFR‑TKI treatment, with serial plasma sample collections over 2 months. The multiplex ddPCR panels applied to plasma cfDNA from advanced NSCLC patients achieved a total concordance rate of 80% with the EGFR mutation profiles obtained by ARMS from matched biopsy tumor specimens (90% for 19DEL, 95% for L858R, 95% for T790M, respectively) and revealed additional mutant alleles in two subjects. The respective sensitivity and specificity were 90.9 and 88.9% for 19DEL, 87.5 and 100% for L858R, 100 and 93.8% for T790M. The fluctuations of EGFR mutant abundance in serial plasma cfDNA were in accordance with the changes in tumor size as assessed by imaging scans. The authors demonstrated the utility of multiplex ddPCR panels with ultra‑sensitivity for quantitative analysis of EGFR mutations in plasma cfDNA and obtained promising usefulness in EGFR‑TKI decision‑making for advanced NSCLC patients.

Predictive models for mutations in mismatch repair genes: implication for genetic counseling in developing countries.

PubMed

Monteiro Santos, Erika Maria; Valentin, Mev Dominguez; Carneiro, Felipe; de Oliveira, Ligia Petrolini; de Oliveira Ferreira, Fabio; Junior, Samuel Aguiar; Nakagawa, Wilson Toshihiko; Gomy, Israel; de Faria Ferraz, Victor Evangelista; da Silva Junior, Wilson Araujo; Carraro, Dirce Maria; Rossi, Benedito Mauro

2012-02-09

Lynch syndrome (LS) is the most common form of inherited predisposition to colorectal cancer (CRC), accounting for 2-5% of all CRC. LS is an autosomal dominant disease characterized by mutations in the mismatch repair genes mutL homolog 1 (MLH1), mutS homolog 2 (MSH2), postmeiotic segregation increased 1 (PMS1), post-meiotic segregation increased 2 (PMS2) and mutS homolog 6 (MSH6). Mutation risk prediction models can be incorporated into clinical practice, facilitating the decision-making process and identifying individuals for molecular investigation. This is extremely important in countries with limited economic resources. This study aims to evaluate sensitivity and specificity of five predictive models for germline mutations in repair genes in a sample of individuals with suspected Lynch syndrome. Blood samples from 88 patients were analyzed through sequencing MLH1, MSH2 and MSH6 genes. The probability of detecting a mutation was calculated using the PREMM, Barnetson, MMRpro, Wijnen and Myriad models. To evaluate the sensitivity and specificity of the models, receiver operating characteristic curves were constructed. Of the 88 patients included in this analysis, 31 mutations were identified: 16 were found in the MSH2 gene, 15 in the MLH1 gene and no pathogenic mutations were identified in the MSH6 gene. It was observed that the AUC for the PREMM (0.846), Barnetson (0.850), MMRpro (0.821) and Wijnen (0.807) models did not present significant statistical difference. The Myriad model presented lower AUC (0.704) than the four other models evaluated. Considering thresholds of ≥ 5%, the models sensitivity varied between 1 (Myriad) and 0.87 (Wijnen) and specificity ranged from 0 (Myriad) to 0.38 (Barnetson). The Barnetson, PREMM, MMRpro and Wijnen models present similar AUC. The AUC of the Myriad model is statistically inferior to the four other models.

Multiplex picoliter-droplet digital PCR for quantitative assessment of EGFR mutations in circulating cell-free DNA derived from advanced non-small cell lung cancer patients

PubMed Central

Yu, Qian; Huang, Fei; Zhang, Meilin; Ji, Haiying; Wu, Shenchao; Zhao, Ying; Zhang, Chunyan; Wu, Jiong; Wang, Beili; Pan, Baisheng; Zhang, Xin; Guo, Wei

2017-01-01

To explore the possible diagnostic value of liquid biopsy, two multiplex panels using picoliter-droplet digital polymerase chain reaction (ddPCR) were established to quantitatively assess the epidermal growth factor receptor (EGFR) mutations in cell-free DNA (cfDNA) extracted from the plasma of advanced non-small cell lung cancer (NSCLC) patients. Plasma samples derived from 22 patients with stage IIIB/IV NSCLC harboring EGFR mutations in matched tumor tissues confirmed by amplification refractory mutation system (ARMS) analysis were subjected to two multiplex ddPCR panels to assess the abundance of tyrosine kinase inhibitor (TKI) -sensitive (19DEL, L858R) and TKI-resistant (T790 M) mutations. Fluctuations in EGFR mutant abundance were monitored by either of the multiplex ddPCR panels for three patients undergoing EGFR-TKI treatment, with serial plasma sample collections over 2 months. The multiplex ddPCR panels applied to plasma cfDNA from advanced NSCLC patients achieved a total concordance rate of 80% with the EGFR mutation profiles obtained by ARMS from matched biopsy tumor specimens (90% for 19DEL, 95% for L858R, 95% for T790M, respectively) and revealed additional mutant alleles in two subjects. The respective sensitivity and specificity were 90.9 and 88.9% for 19DEL, 87.5 and 100% for L858R, 100 and 93.8% for T790M. The fluctuations of EGFR mutant abundance in serial plasma cfDNA were in accordance with the changes in tumor size as assessed by imaging scans. The authors demonstrated the utility of multiplex ddPCR panels with ultra-sensitivity for quantitative analysis of EGFR mutations in plasma cfDNA and obtained promising usefulness in EGFR-TKI decision-making for advanced NSCLC patients. PMID:29067441

Oncogenic PIK3CA gene mutations and HER2/neu gene amplifications determine the sensitivity of uterine serous carcinoma cell lines to GDC-0980, a selective inhibitor of Class I PI3 kinase and mTOR kinase (TORC1/2).

PubMed

English, Diana P; Bellone, Stefania; Cocco, Emiliano; Bortolomai, Ileana; Pecorelli, Sergio; Lopez, Salvatore; Silasi, Dan-Arin; Schwartz, Peter E; Rutherford, Thomas; Santin, Alessandro D

2013-11-01

To evaluate PIK3CA mutational status and c-erbB2 gene amplification in a series of primary uterine serous carcinomas (USC) cell lines. To assess the efficacy of GDC-0980, a potent inhibitor of Class I PI3 kinase and mTOR kinase (TORC1/2), against primary USC harboring HER2/neu gene amplification and/or PIK3CA mutations. Twenty-two primary USC cell lines were evaluated for c-erbB2 oncogene amplification by fluorescence in situ hybridization (FISH) assays and for PIK3CA gene mutations by direct DNA sequencing of exons 9 and 20. In vitro sensitivity to GDC-0980 was evaluated by flow-cytometry-based viability and proliferation assays. Downstream cellular responses to GDC-0980 were assessed by measuring phosphorylation of the 4-EBP1 protein by flow-cytometry. Five of 22 (22.7%) USC cell lines contained oncogenic PIK3CA mutations although 9 (40.9%) harbored c-erbB2 gene amplification by FISH. GDC-0980 caused a strong differential growth inhibition in FISH+ USC when compared with FISH- (GDC-0980 IC50 mean ± SEM = 0.29 ± 0.05 μM in FISH+ vs 1.09 ± 0.20 μM in FISH- tumors, P = .02). FISH+ USC harboring PIK3CA mutations were significantly more sensitive to GDC-0980 exposure when compared with USC cell lines harboring wild-type PIK3CA (P = .03). GDC-0980 growth-inhibition was associated with a significant and dose-dependent decline in phosphorylated 4-EBP1 levels. Oncogenic PIK3CA mutations and c-erbB2 gene amplification may represent biomarkers to identify patients harboring USC who may benefit most from the use of GDC-0980. Copyright © 2013 Mosby, Inc. All rights reserved.

Determination of ubiquitin fitness landscapes under different chemical stresses in a classroom setting

PubMed Central

Mavor, David; Barlow, Kyle; Thompson, Samuel; Barad, Benjamin A; Bonny, Alain R; Cario, Clinton L; Gaskins, Garrett; Liu, Zairan; Deming, Laura; Axen, Seth D; Caceres, Elena; Chen, Weilin; Cuesta, Adolfo; Gate, Rachel E; Green, Evan M; Hulce, Kaitlin R; Ji, Weiyue; Kenner, Lillian R; Mensa, Bruk; Morinishi, Leanna S; Moss, Steven M; Mravic, Marco; Muir, Ryan K; Niekamp, Stefan; Nnadi, Chimno I; Palovcak, Eugene; Poss, Erin M; Ross, Tyler D; Salcedo, Eugenia C; See, Stephanie K; Subramaniam, Meena; Wong, Allison W; Li, Jennifer; Thorn, Kurt S; Conchúir, Shane Ó; Roscoe, Benjamin P; Chow, Eric D; DeRisi, Joseph L; Kortemme, Tanja; Bolon, Daniel N; Fraser, James S

2016-01-01

Ubiquitin is essential for eukaryotic life and varies in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies indicate that ubiquitin is highly tolerant to single mutations. We hypothesized that this tolerance would be reduced by chemically induced physiologic perturbations. To test this hypothesis, a class of first year UCSF graduate students employed deep mutational scanning to determine the fitness landscape of all possible single residue mutations in the presence of five different small molecule perturbations. These perturbations uncover 'shared sensitized positions' localized to areas around the hydrophobic patch and the C-terminus. In addition, we identified perturbation specific effects such as a sensitization of His68 in HU and a tolerance to mutation at Lys63 in DTT. Our data show how chemical stresses can reduce buffering effects in the ubiquitin proteasome system. Finally, this study demonstrates the potential of lab-based interdisciplinary graduate curriculum. DOI: http://dx.doi.org/10.7554/eLife.15802.001 PMID:27111525

[Control levels of Sin3 histone deacetylase for spontaneous and UV-induced mutagenesis in yeasts Saccharomyces cerevisiae].

PubMed

Lebovka, I Iu; Kozhina, T N; Fedorova, I V; Peshekhonov, V T; Evstiukhina, T A; Chernenkov, A Iu; Korolev, V G

2014-01-01

SIN3 gene product operates as a repressor for a huge amount of genes in Saccharomyces cerevisiae. Sin3 protein with a mass of about 175 kDa is a member of the RPD3 protein complex with an assessed mass of greater than 2 million Da. It was previously shownthat RPD3 gene mutations influence recombination and repair processes in S. cerevisiae yeasts. We studied the impacts of the sin3 mutation on UV-light sensitivity and UV-induced mutagenesis in budding yeast cells. The deletion ofthe SIN3 gene causes weak UV-sensitivity of mutant budding cells as compared to the wild-type strain. These results show that the sin3 mutation decreases both spontaneous and UV-induced levels of levels. This fact is hypothetically related to themalfunction of ribonucleotide reductase activity regulation, which leads to a decrease in the dNTP pool and the inaccurate error-prone damage bypass postreplication repair pathway, which in turn provokes a reduction in the incidence of mutations.